Recombinant Protein Glutaminase from Probiotic Escherichia coli Nissle 1917 for Enhancing the Functional Properties of Caseins

Recombinant Protein Glutaminase from Probiotic Escherichia coli Nissle 1917 for Enhancing the Functional Properties of Caseins | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Recombinant Protein Glutaminase from Probiotic Escherichia coli Nissle 1917 for Enhancing the Functional Properties of Caseins Zheng Zhang, Lihui Zheng, Yuxi Li, Shuchao Jiao, Yelin Wu, Mingfei Jin, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3842060/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 3 You are reading this latest preprint version Abstract Protein glutaminase (PG; EC 3.5.1.44) is widely used in the food industry because it catalyzes the deamidation of peptide chain glutamine residues and enhances the functional properties of food proteins. Here, a strategy for PG production by probiotic Escherichia coli Nissle 1917 (EcN) is proposed. The yield of mature PG (mPG) was increased to 8.69 U/mL after testing a series of pSEVA vectors. The purified mPG showed significant deamidation activity against a wide range of protein substrates. Among these tested substrates, the functional properties of PG-modified casein were investigated. Deamidation of casein by PG was more effective at 60°C, pH = 7, and an enzyme-to-substrate ratio (E/S) of 5 U/g protein. Casein is deamidated up to 53.29%, which leads to a solubility of more than 90% for a 5% casein solution. Foam capacity can be nearly doubled. Emulsifiability, especially emulsification stability, is substantially improved. With increasing DD of casein, the α-helix and β-turn in the secondary structure of deamidated casein increased from 0–22.5%, and from 2.8–27.2% respectively, while β-fold and random decreased from 54.6–10.5%, and from 42.6–39.8% respectively. The enhancement of the absorbance values, endogenous fluorescence peaks, and surface hydrophobicity are due to the exposure of hydrophobic amino acids inside the tertiary structure of deamidated casein. Furthermore, deamidated casein particle size reduced while particle size homogeneity rose. After deamidation by PG, casein has achieved enhanced functional properties which improves its usability as a functional ingredient in the food industry. Deamidation PG-modified casein foaming capacity emulsification structural properties Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. INTRODUCTION PG, produced in Chryseobacterium proteolyticum , is a novel food enzyme addition in recent years. PG contains a Cys-His-Asp catalytic triplet that specifically hydrolyzes glutamine residues on proteins or peptide chains and produces glutamate residues and NH 4 + , without the hydrolytic and cross-linking activity of protease or glutamine aminotransferase(Hashizume, et al., 2011 ; Yamaguchi, et al., 2001 ). PG effectively reduces the content of amide groups in proteins and enhances the functional properties of proteins, such as solubility and emulsification of plant proteins. Thus, the potential application values of PG have attracted a lot of attention from the food industry(X. Liu, et al., 2022 ). It was shown that the solubility of PG-deamidated soy protein was enhanced under acidic and neutral conditions(Suppavorasatit, et al., 2011 ). The secondary structure of PG deamidated oat protein was more flexible and the protein solution was more homogeneous and stable(Jiang, et al., 2015 ). The natural PG-producing strain was discovered in C. proteolyticum 9670 T , isolated from the soil in 2000(Yamaguchi & Yokoe, 2000 ), which was proven to be food safe(Scheuplein, et al., 2007 ) and certified as a g enerally r ecognized a s s afe (GRAS) strain by US FDA. However, as of present, the recently reported yield of PG in C. proteolyticum is only 2.91 U/mL, which is far below the requirement for industrial applications in the food processing industry(Wang, et al., 2023 ). Heterologous expression is an alternative strategy to overcome this problem. Currently, The maximum PG enzyme activity of 0.175 U/mL was achieved in E. coli (Lu, et al., 2020 ). The PG enzyme activity of 7.07 U/mL and 26 U/mg were achieved in Bacillus subtilis (Ouyang, et al., 2021 ; Yin, et al., 2021 ) and Corynebacterium glutamicum (Kikuchi, et al., 2008 ; Qu, et al., 2022 ; Wang, et al., 2023 ), respectively. Unfortunately, the expression effect of E. coli , which is the preferred host for superior heterologous gene expression, seems to be not optimistic for PG production. E. coli , an excellent host for heterologous gene expression, is used in various production and research areas of biology due to its clear background, simple genetic manipulation, broad compatibility, and ability to perform high-density fermentation. Optimization of gene expression using various vectors containing different promoters was widely used to screen for appropriate or high yields of target proteins(Cheng, et al., 2018 ; Schwaneberg, et al., 2016 ). The Standard European Vector Architecture (SEVA) creates a set of plasmids with different replication systems for screening suitable hosts and genes(Martinez-Garcia, et al., 2015b ). In food application, a natural octapeptide was constructed in E. coli by genetic engineering method to verify the taste of fresh peptide(Zhang, et al., 2017 ), and an enzyme of alkaline Arxula adeninivorans urate oxidase expressed in E. coli reduced the uric acid level of food(R. Zhang, et al., 2019 ). In addition, the preparation of monoclonal antibodies against aflatoxin B1(Min, et al., 2011 ) and the production of d-pantothenic acid(B. Zhang, et al., 2019 ) were both realized in E. coli , in terms of antibody and metabolic engineering, respectively. All of these cases illustrate that E. coli is an excellent host for heterologous expression. However, as a Gram-negative bacterium, E. coli produces endotoxins that lead to increased downstream production processes and costs, which is another reason for the difficulty in industrial food production in E. coli . Fortunately, the non-pathogenic probiotic E. coli Nissle 1917 (EcN) of serotype O6:K5:H1, without virulence factors, has been discovered to prevent pathogenic bacteria from attacking the intestinal mucosa in the intestine and has a protective and repairing effect on the intestinal mucosal barrier(Jacobi & Malfertheiner, 2011 ). Based on the properties of EcN, recent studies have proposed a series of clinical ideas for heterologous gene expression using EcN as a favored chassis for disease treatment, including sustained heterologous expression of 3-hydroxybutyric acid for the treatment of colitis(Yan, et al., 2021 ), heterologous expression of uric enzyme for the treatment of hyperuricemia(He, et al., 2023 ), and design of near-infrared nano light for controlling gene expression for the treatment of tumors(Zhu, et al., 2023 ). EcN has been reviewed recently to be well employed in biomedical engineering for the treatment of infectious diseases, metabolic disorders, and inflammatory bowel diseases (IBDs) as well as cancers(Lynch, et al., 2022 ; Yu, et al., 2020 ). However, most of the studies on the heterologous expression of EcN have focused on biomedical engineering and neglected the application value of EcN in the food industry. Due to the probiotic and genetically manipulable properties of EcN, a recent study expressed the sulfotransferase domain of human N-deacetylase/N-sulfotransferase-1 (NDST-1) and the catalytic domain of mouse 3-O-sulfotransferase-1 (3-OST-1) in the engineered strain EcN::T7M with fed-batch fermentation, which brought the yield of NST to 0.21 g/L and the yield of heparosan to 0.85 g/L, respectively(Li, et al., 2021 ). In addition, EcN was used to engineer a probiotic to produce β-carotene in the gut and metabolize it to vitamin A to treat diarrheal diseases(Miller, et al., 2013 ). Therefore, the use of non-pathogenic EcN to establish recombinant expression of PG is a very safe and effective strategy in food applications. Milk is an important part of people's daily dietary structure for nutritional acquisition, especially for its amino acid content. Milk consists of a colloidal particle complex of associated proteins and calcium phosphate with an average size of 150–200 nm(Duerasch, et al., 2018 ). These conjoined colloids are known as casein micelles(Fox, 2003 ). Casein is by far the most essential and valuable component of milk, and many dairy products derive their textural, organoleptic, and nutritional properties primarily from casein, which has attracted people to use it for thousands of years. Casein consists of its αs 1 -casein, αs 2 -casein, β-casein, and κ-casein and inorganic material, which account for approximately 76–86% of milk(Swaisgood, 2003 ). However, the solubility of casein is less than 10% compared to natural casein in cow's milk, limiting the application scenarios of casein(Lee, et al., 2010 ). Chemical modification of casein has been expanded to improve the solubility of casein, including succinylation, acetylation, dephosphorylation, and neutralization(Sarah, et al., 2018 ; Wu, et al., 2020 ; Yang, et al., 2016 ). Sodium caseinate is prepared by fermentation, a process in which non-fat milk or Qula is fermented with lactic acid bacteria (LAB). Subsequently, the solid proteins are separated and adjusted to neutrality with NaOH, and finally, citric acid is added to form sodium caseinate(H. N. Liu, et al., 2013 ). Sodium caseinate is used in meat processing, bakery, ice cream, and other industries to increase the binder, emulsification, and insufficient nutrition of food products(Garcia, et al., 2004 ; Sudha, et al., 2014 ; Voronin, et al., 2021 ). However, there are some negative effects of chemical methods compared to enzymatic modification, such as loss of nutritional value, decreased emulsification and foaming, and the chemical process still contains a potential consumption threat. The modification of casein by enzymatic methods to improve its functional properties and broaden its application scenarios is still at a limited level of research. The development of a relatively cheap, efficient, and mild soluble casein production process without the intervention of exogenous chemicals is urgent to expand the application of casein in the food industry. In this study, an expression system for high PG yield was produced in probiotic EcN, which promotes the safe production of PG and simplifies the purifying procedure. The functional properties of casein were significantly improved by PG deamidation which improves its usability as a functional ingredient in the food industry. 2. MATERIAL AND METHODS 2.1 Strains, plasmids, and culture conditions. The strains, plasmids, and primers used in this study are listed in Table S1 , Table S2 , and Table S3 in the Supplemental information, respectively. E. coi l strains were grown at 37°C in LB broth supplemented with 100 µg/mL of ampicillin or 15 µg/mL of tetracycline, and 1mM isopropyl-D-thiogalatopyranoside (IPTG) when required. 2.2 SDS-PAGE. Protein samples were mixed in 5 x loading buffer and denatured and reduced. Subsequent electrophoresis and analysis were performed using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a protein marker (Thermo Fisher Scientific, USA, 26610) as standard. Gels were stained with Coomassie Brilliant Blue R250 and decolorized using a 10% acetic acid-ethanol solution, followed by gel imaging. 2.3 Deamidation degree. The method is referenced from Inthawoot et al. with a slight modification(Suppavorasatit, et al., 2011 ). Ammonia content from complete deamidation of casein: Take 500 µl of casein sample in a 1.5 mL EP tube, add an equal volume of 2 N sulfuric acid solution, heat in a water bath at 100 ℃ for 4 h, centrifuge at 12000 rpm for 10 min, and take an appropriate amount of supernatant to determine the NH 4 + in the supernatant using an ammonia assay kit (Sigma-Aldrich, USA). The ammonia content of casein deamidation catalyzed by PG: When casein was deamidated catalyzed by PG, it was centrifuged at 12000 rpm for 10 min, and an appropriate amount of supernatant was taken to determine NH 4 + in the supernatant using an ammonia assay kit. The DD is the percentage of ammonia produced by PG-catalyzed deamidation of casein over that produced by complete deamidation of casein 2.4 Hydrolysis degree . Determination of complete hydrolysis of casein: Take 500 µl of casein sample in 2 mL EP tube, add an equal volume of 2 N sulfuric acid solution, heat in water bath at 100 ℃ for 4 h, add 1 mL of 0.2 N trichloroacetic acid solution, centrifuge at 12000 rpm for 10 min, take 10 µl of supernatant in a 96-well microplate, and determine the total soluble protein in the supernatant by BCA protein concentration method. The total soluble protein content in the supernatant was measured by the BCA protein concentration method, i.e. the degree of complete hydrolysis of casein. Determination of hydrolysis degree of casein after deamidation reaction: When the casein was completed by PG-catalyzed deamidation reaction, the reaction was terminated by 80 ℃ water bath for 10 min, ice bath for 30 min, 1 mL of modified casein sample was taken in 2 mL EP tube, 1 mL of 0.2 N trichloroacetic acid solution was added, centrifuged at 12000 rpm for 10 min, 10 µl of supernatant was taken in a 96-well microtiter plate, and the protein concentration was measured by BCA. The protein concentration method was used to determine the soluble protein content in the supernatant after the casein deamidation reaction, i.e. the degree of hydrolysis after the casein deamidation reaction. The degree of hydrolysis is the percentage of hydrolysis of casein after the deamidation reaction over the complete hydrolysis of casein. 2.5 Solubility. Determination of protein content in unmodified casein solution by BCA measurement of protein concentration. The same concentration of modified casein solution was taken, and the protein content in the modified casein solution was determined by the BCA protein concentration method. The solubility is the percentage of protein content in the modified casein solution over the protein content in the unmodified casein solution. 2.6 Emulsifiability. With modified casein solution volume to soybean oil volume equal to 3:1, placed in a tissue homogenizer, homogenized the mixture at 20000 rpm for 2 min, immediately aspirated 50 µl of emulsion from the bottom of the solution in 5 mL of 0.1% SDS solution, mixed, OD500 nm to detect the absorbance value, recorded as A 0 , and calculated the emulsifiability (EAI) of the protein solution. EAI (m 2 /g) = 2×2.303×A 0 ÷ 0.25 (1) 2.7 Emulsification stability. The modified casein solution volume to soybean oil volume is equal to 3:1, placed in tissue homogenizer, homogenized the mixture at 20000 rpm for 2 min, immediately aspirated 50 µl of emulsion from the bottom of emulsion in 5 mL of 0.1% SDS solution, mixed, absorbance value detected at OD500 nm, recorded as A 0 , after standing at room temperature for 10 min, again aspirated from the bottom of emulsion After standing for 10 min at room temperature, 50 µl of emulsion was again drawn from the bottom of the emulsion in 5 mL of 0.1% SDS solution, mixed well, and the absorbance value was detected at OD500 and recorded as A 10 . The stability of emulsification (ESI) was calculated as: ESI (min) = A 0 ÷ (A 0 - A 10 ) × 10. (2) 2.8 Enzyme purification. Equilibrate the Ni-NTA column with 5 times the packing volume of the protein washing solution (20 mM Tris, 500 mM NaCl). Put the supernatant of the centrifuged bacterial culture on the column after centrifugation with a flow rate of 4 s/drop to fully bind the target protein to the Ni-NTA column and collect the penetration solution. Wash the Ni-NTA column with 4 times the volume of the pre-cooled protein washing solution to remove the non-specific binding protein and collect the washing solution. The target protein was eluted with 4 times the volume of pre-cooled protein eluent (20 mM Tris, 500 mM NaCl, imidazole concentration gradient of 30–500 mM), and the eluate was collected. A small amount of samples from each stage were subjected to SDS-PAGE, and the rest of the samples were stored at 4 ℃. According to the results of SDS-PAGE, the eluate containing the purer target protein was selected for dialysis at 4 ℃ (20 mM Tris), and the dialysate was changed every 4 h. The dialyzed protein solution was concentrated by ultrafiltration, centrifuged at 4000g to 3–5 mL at 4 ℃, filtered and de-bacterized in a sterile ultraclean table, and then frozen and stored in a -80 ℃ refrigerator for backup. 2.9 Particle size and Zeta potential. Caseins with different DD were prepared in ultrapure water to a final concentration of 1 mg/mL, and the Zeta potentials, particle sizes, and polydispersity of caseins with different DD were determined by using a Zetasizer Nano (Malvern NaNO-ZS ZEN3600). 2.10 UV-visible spectra and fluorescence spectra. For detection of UV-visible spectra (Shimadzu UV-3600 Plus), 10 mg of casein with different DD were dispersed into 5 mL of distilled water, vortexed, and mixed well, and the protein solution was scanned by a UV spectrophotometer at 200 ~ 700 nm with a spectral slit width of 1 nm. The UV-visible spectra of casein with different DD were obtained by using distilled water as the blank spectrum for baseline correction. For detection of fluorescence spectra (HORIBA FluoroMax-4), 10 mg of casein with different DD were dispersed into 5 mL of distilled water and vortexed to mix well. The fluorescence spectra of the protein solution were scanned under the following conditions: excitation wavelength of 285 nm, emission wavelengths of 290 ~ 500 nm, and spectral slit width of 1.5 nm. 2.11 Surface hydrophobicity. Casein solutions with different deamidation degrees of 0.1, 0.2, 0.3, and 0.4 mg/mL were prepared, and 8-Anilino-1-naphthalenesulfonic acid ammonium salt (ANS-NH4) was prepared to 8 mmol/L with distilled water. 20 µL of ANS-NH4 solution was added to 4 mL of protein solution for 30 min under dark conditions. 20 µL of ANS-NH4 solution was added to 4 mL of protein solution, and the reaction was carried out in the dark for 30 min, with the excitation wavelength at 360 nm, the emission wavelength at 370–700 nm, and the spectral slit width of 5 nm. Then, the fluorescence intensity of the sample was measured (HORIBA FluoroMax-4). The concentration of casein and the fluorescence intensity were plotted, and the initial slope of the curve was the surface hydrophobicity index of wheat alcohol-soluble protein with different degrees of deamidation. 2.12 Circular dichroism (CD). The secondary structure of casein was determined by the method described by Suppavorasatit et al.(Suppavorasatit, et al., 2011 ). Caseins were dispersed at a concentration of 10 mM in phosphate buffer (pH = 7) at 20 ℃. The CD spectra in the far UV region (190 ~ 260 nm) of each sample were determined using JASCO spectropolarimeter (Model J-1500, Tokyo, Japan). The samples were analyzed using a 1 cm path-length square quartz cuvette with a Teflon cap. The molar ellipticity values were calculated using the formula as shown in the equation: $${\left[\theta \right]}_{\text{m}\text{o}\text{l}\text{a}\text{r},{\lambda }}\left(\text{deg}{cm}^{2} {\text{d}\text{m}\text{o}\text{l}}^{-1}\right)=100\times \frac{{\theta }_{\lambda }}{m}\times d$$ 3 where θ λ is the observed ellipticity (degrees) at wavelength λ, m is the molar concentration of a solute, and d is the path length (cm). Prediction of the percent of protein secondary structure from CD spectra was obtained using software from a webserver: http://perry.freeshell.org/raussens.html , which uses the method of Raussens et al(Vincent, et al., 2003). 2.13 PG enzyme activity. PG enzyme activity assay was referenced to Qu et al. with slight modifications(Qu, et al., 2022 ). 10 µl solution containing PG was reacted with 100 µl of Cbz-Gln-Gly (10.11 g/L) of the experimental group or inactivated with 100 µl of 1 N TCA of the control group at 37°C for 30 min. The reaction was subsequently terminated with 100 µl of TCA and the system was made up of 100 µl of Cbz-Gln-Gly, respectively. After that, 12 µl of the reaction solution and different concentrations of ammonium chloride solution were taken as the standard curve for each sample, 60 µl of color development solution A (40.46 g/L phenol, 0.15 g/L sodium nitroprusside), 48 µl of distilled water, 30 µl of color development solution B (49.94 g/L potassium hydroxide) and 60 µl of color development solution C (200 g/L potassium carbonate, 8.37 mL/L sodium hypochlorite solution) were added. The reaction was carried out at 37 ℃ for 20 min, and the absorbance value was measured at OD630. The enzyme activity was calculated by the formula: PG Enzyme activity (U/mL)=(A 0 -A 1 )×21/17.03/30×k (4) In the formula, A 0 is the absorbance value of the experimental group; A 1 is the absorbance value of the control group; k is the slope of the ammonia standard curve. 2.14 Statistical analysis and reproducibility. The GraphPad Prism 8.0 was used for statistical analysis. The data are presented as means ± SEM. A two-tailed t-test was used to determine the significance between the two groups. One-way or two-way ANOVA with Bonferroni post-test was used to analyze multiple groups. For all the statistical tests, P values < 0.05 were considered to be statistically significant. ns: no significant, * : 0.01 ≤ P < 0.05, ** : 0.001 ≤ P < 0.01, *** : 0.0001 ≤ P < 0.001, **** : P < 0.0001. 3. RESULTS AND DISCUSSIONS 3.1 Establishment of expression system of PG in E. coli Nissle 1917(EcN) To simplify the food safety problems caused by endotoxin in industrial production, this study optimized the strategy of heterologous expression of PG in E. coli by using probiotic EcN as chassis cells. Since EcN is a non-engineered wild-type strain, to better serve as a platform for the expression of heterologous genes by chassis cells, a series of pSEVA vectors containing different replicons and replication proteins, suitable for Gram-negative bacteria(Martinez-Garcia, et al., 2015a ), were loaded with PG expression frames (Fig. 1 A). Compared with pET32a(+)/ P ro- P G (PP) expressed in E. coli BL21(DE3), the PG enzyme activity of pSEVA521/PP, pSEVA541/PP, pSEVA551/PP and pSEVA581/PP in EcN had significantly higher, except that of pSEVA531/PP in EcN was reduced, and the PG enzyme activity had the highest yield up to 8.69 U/mL after trypsin cleavage in pSEVA551/PP EcN strain (Fig. 1 B). PP bands detected by SDS-PAGE showed the same results ( Fig. 1 C ) . The above results indicate that the optimized expression of PG can be efficiently produced in the probiotic EcN with 8.69 U/mL. A series of pSEVA plasmids, containing different replication systems of RK2, pBBR1, pRO1600/ColE1, RSF1010, and pUC, were screened as expression vectors for PG(Jahn, et al., 2016 ). The results showed that the pSEVA551/PP plasmid containing the RSF1010 replication system expressed the highest recombinant PG enzyme activity in EcN up to 8.69 U/mL, which was 63.03% higher than the expression of recombinant PG by pET32a(+)/PP containing the pBR322 replication system in BL21(DE3). This result is in part due to differences in plasmid copy number, which in turn affects the variation in gene expression(Jahn, et al., 2016 ). In addition, even in the case of identical promoter and plasmid, differences in the open reading frame of genes can lead to differences in the yield of the target product(Xu, et al., 2021 ). To determine the activity of recombinant PG derived from EcN and used for protein modification, PP was purified by affinity lamination with NTA-Ni columns ( Fig. S1 ). The purified PP was cleaved by trypsin for 0-300 min, and the gradual disappearance of PP bands and a gradual increase of mPG bands could be both observed, demonstrating the validity of the event that PP was cleaved by trypsin to produce mPG ( Fig. S2A ), which is consistent with Ouyang et al. who used trypsin to cleave PP and produce mPG(Ouyang, et al., 2021 ). The amino acid composition between the pro-peptide and mPG as K114-L115 has been confirmed, which is located on the surface of the protein structure and cleaved by trypsin of the serine protease family(Yamaguchi, et al., 2001 ; Zheng, et al., 2022 ). PG enzyme activity assay on temporally incremental trypsin-cleaved PP similarly showed incremental activity ( Fig. S2B ). Further, mPG, recombinant PP expressed in EcN activated by trypsin, was purified ( Fig. S3 ), and different protein substrates were used for deamidation, demonstrating PG expressed by EcN effectively had deamidation activity against different substrates (Fig. 1 D). The above data indicates the feasibility of proper expression of PG in probiotic E. coli EcN. 3.2 Effect of temperature on casein modification. As shown in Fig. 2 A, the values of the DD increased in a temperature-dependent manner, indicating that casein was effectively deamidated by PG and that the elevated temperature was able to promote the deamidation reaction of PG more effectively. Similarly, the DD of coconut protein modified by PG increased with increasing temperature; however, the DD decreased when the modification of PG was performed at more than 60°C(Kunarayakul, et al., 2018 ). Higher temperatures were not further attempted because higher temperatures resulted in protein denaturation of PG and a rapid decrease in PG enzyme activity ( Fig. S4 ). Yamaguchi et al.(Yamaguchi, et al., 2001 ) and Lu et al.(Lu, et al., 2020 ) demonstrated a rapid decrease in PG enzyme activity at temperatures above 60°C. As the value of DD increased, the precipitation rate of casein obtained decreased, indicating an improved solubility of deamidated casein (Fig. 2 B). Analysis of the functional properties of the casein with different values of DD showed a significant improvement in foaming, emulsification, and emulsion stability, expanding the range of casein applications (Fig. 2 C, 2 D and 2 E). In addition, the values of the degree of hydrolysis (DH) results showed no significant hydrolysis of casein by PG, which is consistent with the previously reported properties of PG for EPSC protein(Hadidi, et al., 2021 ) (Fig. 2 F). To further visualize the effect of deamidated casein, it was imaged and showed a slight decrease in precipitation of deamidated casein at 30°C and 40°C, while the turbidity of the supernatant increased visually, compared to the control. The casein deamidated at 50°C showed a milky solution with good homogeneity and dispersion in aqueous solution without significant precipitation, while the casein deamidated at 60°C was more clarified and translucent, probably due to a further increase in the solubility of casein in aqueous solution, as well as the separation of casein micelles and the reduction of casein particles (Fig. 2 G). To better present the correlation between DD and solution turbidity, casein that was deamidated at different lengths of OD600 and DD was examined. As Fig. S5A and Fig. S5B showed, the turbidity of soluble casein solutions showed a trend of increasing and then decreasing with increasing DD, which was since at low DD of casein, most of the casein was in insoluble state, and soluble solutions were relatively clarified. At moderate DD, the aggregated casein micelles dissociate, and casein solubility increases, maintaining good dispersion without precipitation, which leads to an increase in turbidity. Miwa et al. modified skim milk by PG and showed a gradual decrease in turbidity as the DD increased. The reason for this difference may be that in this study the samples were centrifuged and only the turbidity of the non-precipitated supernatant was calculated(Miwa, et al., 2010 ). At high DD, the casein particles were reduced and completely dissolved to form a stable casein solution, leading to a decrease in turbidity and a state similar to that of sodium caseinate solution. Taken together, the above data demonstrate that EcN-derived PG can effectively deamidate casein and increase the protein functional properties of casein in a temperature-dependent manner. 3.3 Effect of pH on casein modification Since pH can affect the properties of proteins relevant to the application scene, PG was used for the deamidation of casein at different pH. Before that, we explored the effect of different pH on the PG enzyme activity itself, and the results showed that PG activity does not decrease significantly at pH = 4–8. At pH = 9, there is a small decrease in PG enzyme activity, while at pH higher than 9 or lower than 4, there is an almost complete loss of PG enzyme activity, which indicates the pH stability of PG and the range of applications of the modified protein ( Fig. S6 ). Similar to the result of the present study, Yamaguchi et al. and Lu et al. demonstrated that the relative enzyme activity of PG in pH = 4–7 environments was both above 70%(Lu, et al., 2020 ; Yamaguchi, et al., 2001 ). As shown in Fig. 4 A, at around neutral conditions, pH = 5, 6, and 7, the DD of casein was modified to a higher level. At alkaline conditions, pH = 9, the DD of casein decreased slightly due to a slight decrease in the activity of PG under that condition. In acidic conditions, with pH = 5, the DD of casein decreased significantly, due to the weak acidic conditions causing denaturation and precipitation of casein (Fig. 3 B and Fig. 3 G), even if the PG enzyme activity did not decrease significantly at pH = 5. Figure 3 B showed that the casein precipitation rate under weakly acidic conditions could reach 48.13%, indicating the poor solubility of casein under a weakly acidic environment, even though a certain DD had occurred. The foaming, emulsification, and emulsion stability of the deamidated casein at different pH decreased to some extent, compared to pH = 7, suggesting the best effect of PG on casein deamidation and application under neutral conditions, which may be related to the nature of the modified protein itself (Fig. 3 C, 3 D and 3 E). It is noteworthy that although the value of DD of casein was higher at pH = 6, it already suffered a certain degree of precipitation and behaved as a suspension, showing that the denaturation and precipitation of proteins seriously affected the emulsion stability (Fig. 3 G). In addition, at pH = 8 and 9, even though the protein did not denature and precipitate, the emulsion stability was still severely reduced. pH had no significant effect on the degree of hydrolysis when PG modified casein (Fig. 3 F). Although the effect of pH in the appropriate range on PG enzyme activity was not significant, the effect of pH on casein was greater, especially under acidic conditions where casein solubility was greatly restricted, even at similar DD. This is because casein has an isoelectric point (PI) of 4.6, which is acid-sensitive and will precipitate at a lower pH(Bertsch, et al., 2019 ). Similar to this result, α- zein showed low solubility under acidic conditions at pH = 3 with or without PG deamidation, due to the approaching of the isoelectric point of α- zein to acidic region(Yong, et al., 2006 ). These data indicate that the modification of casein under neutral conditions is more effective, while application under non-acidic conditions is more valuable. 3.4 Effect of enzyme/substrate (E/S) ratio on casein modification To investigate the ability of PG to modify different casein concentrations to suit the required additions for various applications, 5% casein was subjected to deamidation reactions, and the results showed that the values of DD of casein increased with increasing E/S ratio, with the highest DD being 51.25% at E/S ratio equal to 5 U/g (Fig. 4 A). The results of Miwa et al. showed that the DD and ammonia release from skimmed milk powder increased with increasing enzyme-to-substrate ratio and casein was the major protein in skimmed milk powder, which is consistent with the results of this study(Miwa, et al., 2010 ). The precipitation rates of casein modified by PG were substantially reduced, indicating that PG was able to greatly promote casein solubility (Fig. 4 B). The foaming, emulsification, and emulsion stability gradually increased with growing E/S ratio, and further, the functional properties of these caseins were again enhanced with the addition of PG, extending the application potential of the functional properties of deamidated casein (Fig. 4 C, 4 D and 4 E). Similarly, an improvement in the E/S ratio did not result in PG hydrolytic activity (Fig. 4 F), which is consistent with the result that EPSC and soy protein modified by PG do not affect the degree of hydrolysis(Hadidi, et al., 2021 ; Suppavorasatit, et al., 2011 ). The photographs show that PG can effectively promote solubilization, indicating the great potential of PG to promote casein solubilization (Fig. 4 G). The functional properties of modified casein were similarly enhanced with a gradual increase in the E/S ratio, which has been similarly demonstrated on evening primrose seed cake (EPSC) protein(Hadidi, et al., 2021 ). The above data showed that PG maintained a good deamidation ability and improved the functional properties of casein. 3.5 Primary and secondary structural properties of modified casein To investigate the mechanism of the improvement of the functional properties of deamidated casein, SDS-PAGE was performed on casein with different degrees of deamidation (Fig. 5 A). Casein with different degrees of deamidation showed no significant degradation of the bands, indicating no hydrolytic activity of PG(Hadidi, et al., 2021 ; Yong, et al., 2006 ). In addition, PG deamidation resulted in a slight upward migration of the casein bands, which could be attributed to the slight change in the SDS-PAGE pattern by PG is attributable to the inhibition of SDS binding to proteins because of the increase in the negative charge by deamidation(Miwa, et al., 2010 ). The “diffused” set of protein bands may be due to the aggregation of protein molecules, which is similar to the phenomenon studied by Yong et al.(Yong, et al., 2006 ). SDS-PAGE showed that the deamidation of casein by PG did not change the primary structure of casein and had no hydrolytic activity. Circular dichroism was used to reflect changes in the proportion of deamidated casein secondary structure. Figure 5 B shows the CD spectra of the soluble fractions of control and deamidated casein. The CD spectra of the untreated and deamidated casein samples showed a negative board band at around 200 ~ 220 nm. The percentage content of α-helices and β-turns increased and the content of β-sheets decreased in deamidated casein compared to un-deamidated casein, and there appeared to be no clear pattern of change in the content of random (Table 1 )(Kunarayakul, et al., 2018 ; Suppavorasatit, et al., 2011 ). The result that the content of β-sheets decreased with increasing degree of deamidation was similar to that of deamidated soy protein(Suppavorasatit, et al., 2011 ). These results suggest that the deamidation of casein by PG can lead to changes in the casein secondary structure. Table 1 Secondary structures a of non-deamidated and deamidated casein. Caseins with different DD Secondary structure (%) Control 17.5% 32.8% 40.8% α-Helix(%) 0 6.7 7.7 22.5 β-sheet(%) 54.6 44.4 46.1 10.5 β-turn(%) 2.8 10.2 8.8 27.2 Random(%) 42.6 38.6 37.4 39.8 Sum(%) 100 100 100 100 a Data were derived by analysis of CD spectra. 3.6 Tertiary structure properties of modified casein The characteristic absorption at 278 nm is mainly due to the presence of tyrosine (275 nm), tryptophan (279 nm), and phenylalanine (257 nm)(Grimsley & Pace, 2004 ). The absorbance values at around 275 nm were significantly increased in PG-deaminated casein compared to non-deaminated casein, suggesting that PG deamidation can alter the tertiary structure of casein (Fig. 6 A), exposing hydrophobic amino acids (especially tyrosine and tryptophan) in the internal structure(Jin, et al., 2015 ). Tryptophan, tyrosine, and phenylalanine residues in proteins, especially tryptophan residues, fluoresce in a manner that is dependent on protein folding, and thus they can serve as sensitive monitors of conformational changes in tertiary structures(Xiong, et al., 2018 ). This speculation is consistent with the results reflected by the fluorescence spectra, i.e., the endogenous fluorescence peaks at 340 nm were both significantly enhanced in the deamidated casein compared to the non-deamidated casein (Fig. 6 B). This is due to a change in the tertiary structure of PG-deamidated casein, which unfolds the protein conformation and leads to exposure of the tryptophan moiety, coupled with an increase in amounts of the fluorescence intensity(Jin, et al., 2015 ). Alterations in the tertiary structure of deamidated casein result in the exposure of internal hydrophobic amino acids, such as tryptophan and phenylalanine, enhancing the surface hydrophobicity of deamidated casein (Fig. 6 C). It was shown that surface hydrophobicity promotes protein foaming properties(Moro, et al., 2011 ), which is consistent with the results of enhanced foaming properties of deamidated casein. Casein particles are micelles of casein consisting of αs1-casein, αs2-casein, β-casein, and κ-casein, which may cause structural changes in the protein after deamidation by PG. Based on the above hypothesis, the particle size, polydispersity, and zeta potential of casein with different degrees of deamidation were examined. particle size light intensity distribution results showed that the particle size of non-deamidated casein had a wide range of particle sizes, and the homogeneity of particle size was poor. The results showed that the particle size distribution of casein with undeamidated degree was wide and the particle size uniformity was poor. As the degree of deamidation increased, the particle size distribution of casein gradually narrowed to about 200 nm, and the particle size homogeneity increased (Fig. 6 D). The results of PDI also proved that the degree of deamidation of casein was positively correlated with the particle size homogeneity (Fig. 6 E). Meanwhile, the zeta potential indicated that the stability of the casein dispersion system increased with increasing degree of deamidation and reached its maximum at a DD of 40.8% (Fig. 6 F). Particle size distribution analysis and transmission electron microscopy showed that smaller particle sizes were produced in skim milk with high deamidation. This indicates that the deamidation of PG induces the dissociation of casein micelles(Miwa, et al., 2010 ). The above results indicate that deamidation of casein with PG alters the secondary and tertiary structure of casein, which correlates with improved functional properties of deamidated casein. 4. CONCLUSION Based on the safety of food enzyme production, we optimized the expression of PG in the probiotic EcN to achieve a PG yield of 8.69 U/mL, which is 49.66 times higher than the highest yield reported in E. coli at present. In addition, EcN has improved safety and reduced production costs compared to other E. coli . Purified PG was deamidated to a variety of substrate proteins. Casein was deamidated by PG with improved functional properties, including solubility, foaming, emulsification, and emulsion stability. Although the primary structure of the deamidated casein did not undergo hydrolysis, the content of α-Helix and β-turn increased while the content of β-sheet and Random decreased in the secondary structure. Meanwhile, the alteration of the tertiary structure of deamidated casein led to the exposure of hydrophobic amino acids such as tyrosine and tryptophan, which caused the enhancement of absorbance values at 275 nm, endogenous fluorescence peaks at 340 nm, and surface hydrophobicity. In addition, depolymerization of casein micelles occurred after deamidation, and particle size, PDI, and zeta potential decreased. In short, this strategy for producing recombinant PG in EcN extends safe enzyme production, improves the functional properties of casein, and elucidates the structural properties of deamidated casein. These results advance the production of PG and the application of casein in the food industry. AUTHOR INFORMATION Declarations AUTHOR INFORMATION Corresponding Author Deming Jiang - School of Life Science, East China Normal University, Shanghai 200241, P. R. China; Phone: +86 021 54341957; Email: [email protected] Jing Huang - School of Life Science, East China Normal University, Shanghai 200241, P. R. China; Phone: +86 021 54341055; Email: [email protected] Authors Zheng Zhang - School of Life Science, East China Normal University, Shanghai 200241, P. R. China. ORCID: 0009-0003-4021-9349 Lihui Zheng - School of Life Science, East China Normal University, Shanghai 200241, P. R. China Yuxi Li - School of Life Science, East China Normal University, Shanghai 200241, P. R. China Shuchao Jiao - School of Life Science, East China Normal University, Shanghai 200241, P. R. China Yelin Wu - Tongji University Cancer Center, Shanghai Tenth People's Hospital, Tongji University School of Medicine, Shanghai 200072, P. R. China . ORCID:0000-0002-0746-8541 Mingfei Jin - School of Life Science, East China Normal University, Shanghai 200241, P. R. China Shuo Zhang - School of Life Science, East China Normal University, Shanghai 200241, P. R. China Hongliang Gao - School of Life Science, East China Normal University, Shanghai 200241, P. R. China Zhongyi Chang - School of Life Science, East China Normal University, Shanghai 200241, P. R. China AUTHOR CONTRIBUTIONS J.H. and D.J. conceived and supervised the study. Z.Z. designed and performed the experiments. L.Z. and Y.L. performed most molecular biological experiments. S.J. and H.G. assisted with experiments on the functional characterization of casein. Y.W. and Z. S. assisted with experiments on the structural characterization of casein. M.J. and Z.C. analyzed the data. J.H. and D.J. wrote and proofread the manuscript. All authors contributed to the review, revision and finalization of the manuscript. CONFLICTS OF INTEREST All authors report no conflict or declaration of interest. FUNDING The authors would greatly acknowledge the support by Instruments Sharing Platform of School of Life science, ECNU. ACKNOWLEDGEMENT The authors thank ECNU Instruments Sharing Platform of School of Life Sciences for technology support. The authors thank Y.W. for support with experimental techniques and data processing. Figure 1A created with BioRender.com. DATA AVAILABILITY The main data supporting the results of this study are available within the paper and its Supplementary Information. All data generated in this study are available from the corresponding authors. References Bertsch, P., Savorani, L., & Fischer, P. (2019). Rheology of Swiss Cheese Fondue. ACS Omega, 4 (1), 1103-1109.http://dx.doi.org/10.1021/acsomega.8b02424 Cheng, F., Xiang, C., Zhang, X. J., Liu, Z. Q., & Zheng, Y. G. (2018). ReToAd: simple method for the rapid replacement of promoters to improve protein production. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3842060","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":265835048,"identity":"bc098ceb-966e-4419-870b-0d723458c778","order_by":0,"name":"Zheng Zhang","email":"","orcid":"","institution":"East China Normal University","correspondingAuthor":false,"prefix":"","firstName":"Zheng","middleName":"","lastName":"Zhang","suffix":""},{"id":265835049,"identity":"56b9a012-1a6f-406a-98a7-3de218db3499","order_by":1,"name":"Lihui Zheng","email":"","orcid":"","institution":"East China Normal University","correspondingAuthor":false,"prefix":"","firstName":"Lihui","middleName":"","lastName":"Zheng","suffix":""},{"id":265835050,"identity":"cc402508-adcd-46e0-8391-1cb048d67236","order_by":2,"name":"Yuxi Li","email":"","orcid":"","institution":"East China Normal University","correspondingAuthor":false,"prefix":"","firstName":"Yuxi","middleName":"","lastName":"Li","suffix":""},{"id":265835051,"identity":"02865961-cb52-4dd1-97fa-499bb5ce907c","order_by":3,"name":"Shuchao Jiao","email":"","orcid":"","institution":"East China Normal University","correspondingAuthor":false,"prefix":"","firstName":"Shuchao","middleName":"","lastName":"Jiao","suffix":""},{"id":265835052,"identity":"1a8869d6-0b61-4a9b-b3be-7911269b2382","order_by":4,"name":"Yelin Wu","email":"","orcid":"","institution":"Tongji University Cancer Center, Tongji University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yelin","middleName":"","lastName":"Wu","suffix":""},{"id":265835053,"identity":"c63cfddd-91a6-49aa-8986-d326f3159b32","order_by":5,"name":"Mingfei Jin","email":"","orcid":"","institution":"East China Normal University","correspondingAuthor":false,"prefix":"","firstName":"Mingfei","middleName":"","lastName":"Jin","suffix":""},{"id":265835054,"identity":"8431569b-2cac-4a12-ad92-1ed3a987cfdf","order_by":6,"name":"Shuo Zhang","email":"","orcid":"","institution":"East China Normal University","correspondingAuthor":false,"prefix":"","firstName":"Shuo","middleName":"","lastName":"Zhang","suffix":""},{"id":265835055,"identity":"ec528d0e-7796-4e6c-aad7-144d7a1ac4d9","order_by":7,"name":"Hongliang Gao","email":"","orcid":"","institution":"East China Normal University","correspondingAuthor":false,"prefix":"","firstName":"Hongliang","middleName":"","lastName":"Gao","suffix":""},{"id":265835056,"identity":"50385e7e-82f3-4c81-9efa-5908cdb60348","order_by":8,"name":"Zhongyi Chang","email":"","orcid":"","institution":"East China Normal University","correspondingAuthor":false,"prefix":"","firstName":"Zhongyi","middleName":"","lastName":"Chang","suffix":""},{"id":265835057,"identity":"ab7d6877-ff88-48b4-8d6e-bd7d9ce9497b","order_by":9,"name":"Deming Jiang","email":"","orcid":"","institution":"East China Normal University","correspondingAuthor":false,"prefix":"","firstName":"Deming","middleName":"","lastName":"Jiang","suffix":""},{"id":265835058,"identity":"96a99f60-02bb-4b0f-971d-0eef704c3c47","order_by":10,"name":"Jing Huang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6klEQVRIiWNgGAWjYBACxhlgyiaBDSGWQJSWNBK0MEiAycPIyghoYZ7dY/i54Nf5PD72BsYHP3ccZuBnzzFg+LkDj8PmnDGWntl3u5iN5wCzYe+ZwwySPW8MGHvP4NEyI8dAmrfndmKbRAKbNGPbYQaDGzkGzIxteLUY/+btOQfSwv4bpMWeCC1m0jw/DoBtYQbbIkFQS1qZNW9DMtAvB5sle9vSeSTOPCs42ItHi+GM5M23ef7Y5cm3Nx/88LPNWo6/PXnjg5/4tDSArAIrYAQxGXhAxAHcGhgY5MHkH3xKRsEoGAWjYMQDAIvITf4f8RJGAAAAAElFTkSuQmCC","orcid":"","institution":"East China Normal University","correspondingAuthor":true,"prefix":"","firstName":"Jing","middleName":"","lastName":"Huang","suffix":""}],"badges":[],"createdAt":"2024-01-07 09:29:45","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3842060/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3842060/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49385307,"identity":"9c2f1a2c-1b87-4a9a-b08f-705faab0e41f","added_by":"auto","created_at":"2024-01-09 19:50:30","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":137135,"visible":true,"origin":"","legend":"\u003cp\u003ePP construction and expression in probiotic \u003cem\u003eE. coli\u003c/em\u003e Nissle 1917 (EcN):\u003cstrong\u003e A\u003c/strong\u003e construction and expression model of PP in EcN; \u003cstrong\u003eB\u003c/strong\u003ePG enzyme activity of intracellular soluble proteins of \u003cem\u003eE. coli\u003c/em\u003e after trypsin digestion, compared to pET32a(+)/PP; \u003cstrong\u003eC\u003c/strong\u003eSDS-PAGE of intracellular soluble proteins of \u003cem\u003eE. coli\u003c/em\u003e; \u003cstrong\u003eD\u003c/strong\u003e deamidation activity of PG for different substrates\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3842060/v1/41e5881546764c678f58b7bc.jpg"},{"id":49383192,"identity":"8e425f4f-c2f8-4b3f-bce4-6558e94cf9c9","added_by":"auto","created_at":"2024-01-09 19:42:30","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":95460,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of temperature on casein modification by the PG with\u003cstrong\u003e \u003c/strong\u003e5% casein suspension, E/S equal to 5 U/g protein, pH=7, 2 h reaction or no reaction:\u003cstrong\u003e A\u003c/strong\u003e delamination degree; \u003cstrong\u003eB\u003c/strong\u003e precipitation rate; \u003cstrong\u003eC\u003c/strong\u003e foaming capacity; \u003cstrong\u003eD\u003c/strong\u003e emulsifying activity index; \u003cstrong\u003eE\u003c/strong\u003e emulsifying stability index; \u003cstrong\u003eF\u003c/strong\u003e hydrolysis degree; \u003cstrong\u003eG\u003c/strong\u003e Photograph of deamidated casein\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3842060/v1/8bb320c36610eb22f21b7a1d.jpg"},{"id":49383193,"identity":"caad9a86-2f6c-47ca-9ccd-56c272ebb2a7","added_by":"auto","created_at":"2024-01-09 19:42:30","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":87378,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of pH on casein modification by the PG with 5% casein, E/S equal to 5 U/g protein, temperature 60 °C, reaction 2 h or no reaction: \u003cstrong\u003eA\u003c/strong\u003e delamination degree; \u003cstrong\u003eB\u003c/strong\u003eprecipitation rate; \u003cstrong\u003eC\u003c/strong\u003e foaming capacity; \u003cstrong\u003eD\u003c/strong\u003e emulsifying activity index; \u003cstrong\u003eE\u003c/strong\u003e emulsifying stability index; \u003cstrong\u003eF\u003c/strong\u003e hydrolysis degree; \u003cstrong\u003eG\u003c/strong\u003e Photograph of deamidated casein; all data analyses were compared to pH=7\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3842060/v1/df12b7f4711cc8654db70047.jpg"},{"id":49383194,"identity":"86fdf544-58da-45a6-a495-e1518c093e8c","added_by":"auto","created_at":"2024-01-09 19:42:30","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":99254,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of E/S on casein modification by the PG with 5% casein, E/S=0 - 5 U/g protein, temperature 60 °C, pH=7, reaction 2 h: \u003cstrong\u003eA\u003c/strong\u003edelamination degree; \u003cstrong\u003eB\u003c/strong\u003e precipitation rate; \u003cstrong\u003eC\u003c/strong\u003e foaming capacity; \u003cstrong\u003eD\u003c/strong\u003e emulsifying activity index; \u003cstrong\u003eE\u003c/strong\u003e emulsifying stability index; \u003cstrong\u003eF\u003c/strong\u003e hydrolysis degree; \u003cstrong\u003eG\u003c/strong\u003e Photograph of deamidated casein\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3842060/v1/3b5db0e4571fc023afebb0f2.jpg"},{"id":49383197,"identity":"58f6f085-613c-4312-997d-412775b9eace","added_by":"auto","created_at":"2024-01-09 19:42:30","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":71702,"visible":true,"origin":"","legend":"\u003cp\u003ePrimary and secondary structural properties of deamidated casein: \u003cstrong\u003eA\u003c/strong\u003e SDS-PAGE for primary structural characterization; \u003cstrong\u003eB\u003c/strong\u003e circular dichroism for secondary structural characteristics\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3842060/v1/cb5cc264a0fc91649958ded9.jpg"},{"id":49385308,"identity":"f9e4e49d-f2a8-4473-9458-8dd95646e693","added_by":"auto","created_at":"2024-01-09 19:50:30","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":102714,"visible":true,"origin":"","legend":"\u003cp\u003eTertiary structural properties of deamidated casein: \u003cstrong\u003eA\u003c/strong\u003e UV-visible spectra; \u003cstrong\u003eB\u003c/strong\u003e fluorescence spectra; \u003cstrong\u003eC\u003c/strong\u003e surface hydrophobicity; \u003cstrong\u003eD\u003c/strong\u003e particle size light intensity distributions; \u003cstrong\u003eE\u003c/strong\u003e polydispersity; \u003cstrong\u003eF\u003c/strong\u003e Zeta potential\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3842060/v1/6234a2b19ac4828afe64e287.jpg"},{"id":49386700,"identity":"82b63fa6-63d6-4cbc-aac7-03bfef37df9f","added_by":"auto","created_at":"2024-01-09 19:58:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":967218,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3842060/v1/0338dab5-c0f6-41a2-8d4d-6c9185d1f8ab.pdf"},{"id":49385309,"identity":"9bc2dcdc-50be-408b-b5b9-abe00eb70a55","added_by":"auto","created_at":"2024-01-09 19:50:30","extension":"rar","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":27022,"visible":true,"origin":"","legend":"","description":"","filename":"TableofSupplementaryinformation.rar","url":"https://assets-eu.researchsquare.com/files/rs-3842060/v1/caa43244cf7e38f6d01a6288.rar"},{"id":49383199,"identity":"89c63448-eb9a-466b-8fc2-cf10e1032b08","added_by":"auto","created_at":"2024-01-09 19:42:30","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":935653,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-3842060/v1/a4bcbd223a61fd66e0333853.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Recombinant Protein Glutaminase from Probiotic Escherichia coli Nissle 1917 for Enhancing the Functional Properties of Caseins","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003ePG, produced in \u003cem\u003eChryseobacterium proteolyticum\u003c/em\u003e, is a novel food enzyme addition in recent years. PG contains a Cys-His-Asp catalytic triplet that specifically hydrolyzes glutamine residues on proteins or peptide chains and produces glutamate residues and NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e, without the hydrolytic and cross-linking activity of protease or glutamine aminotransferase(Hashizume, et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Yamaguchi, et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). PG effectively reduces the content of amide groups in proteins and enhances the functional properties of proteins, such as solubility and emulsification of plant proteins. Thus, the potential application values of PG have attracted a lot of attention from the food industry(X. Liu, et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). It was shown that the solubility of PG-deamidated soy protein was enhanced under acidic and neutral conditions(Suppavorasatit, et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The secondary structure of PG deamidated oat protein was more flexible and the protein solution was more homogeneous and stable(Jiang, et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe natural PG-producing strain was discovered in \u003cem\u003eC. proteolyticum\u003c/em\u003e 9670\u003csup\u003eT\u003c/sup\u003e, isolated from the soil in 2000(Yamaguchi \u0026amp; Yokoe, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), which was proven to be food safe(Scheuplein, et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) and certified as a \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eg\u003c/span\u003eenerally \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003er\u003c/span\u003eecognized \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ea\u003c/span\u003es \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003es\u003c/span\u003eafe (GRAS) strain by US FDA. However, as of present, the recently reported yield of PG in \u003cem\u003eC. proteolyticum\u003c/em\u003e is only 2.91 U/mL, which is far below the requirement for industrial applications in the food processing industry(Wang, et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Heterologous expression is an alternative strategy to overcome this problem. Currently, The maximum PG enzyme activity of 0.175 U/mL was achieved in \u003cem\u003eE. coli\u003c/em\u003e(Lu, et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The PG enzyme activity of 7.07 U/mL and 26 U/mg were achieved in \u003cem\u003eBacillus subtilis\u003c/em\u003e(Ouyang, et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Yin, et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and \u003cem\u003eCorynebacterium glutamicum\u003c/em\u003e(Kikuchi, et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Qu, et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Wang, et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), respectively. Unfortunately, the expression effect of \u003cem\u003eE. coli\u003c/em\u003e, which is the preferred host for superior heterologous gene expression, seems to be not optimistic for PG production.\u003c/p\u003e \u003cp\u003e \u003cem\u003eE. coli\u003c/em\u003e, an excellent host for heterologous gene expression, is used in various production and research areas of biology due to its clear background, simple genetic manipulation, broad compatibility, and ability to perform high-density fermentation. Optimization of gene expression using various vectors containing different promoters was widely used to screen for appropriate or high yields of target proteins(Cheng, et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Schwaneberg, et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The Standard European Vector Architecture (SEVA) creates a set of plasmids with different replication systems for screening suitable hosts and genes(Martinez-Garcia, et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2015b\u003c/span\u003e). In food application, a natural octapeptide was constructed in \u003cem\u003eE. coli\u003c/em\u003e by genetic engineering method to verify the taste of fresh peptide(Zhang, et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), and an enzyme of alkaline Arxula adeninivorans urate oxidase expressed in \u003cem\u003eE. coli\u003c/em\u003e reduced the uric acid level of food(R. Zhang, et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In addition, the preparation of monoclonal antibodies against aflatoxin B1(Min, et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) and the production of d-pantothenic acid(B. Zhang, et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) were both realized in \u003cem\u003eE. coli\u003c/em\u003e, in terms of antibody and metabolic engineering, respectively. All of these cases illustrate that \u003cem\u003eE. coli\u003c/em\u003e is an excellent host for heterologous expression.\u003c/p\u003e \u003cp\u003eHowever, as a Gram-negative bacterium, \u003cem\u003eE. coli\u003c/em\u003e produces endotoxins that lead to increased downstream production processes and costs, which is another reason for the difficulty in industrial food production in \u003cem\u003eE. coli\u003c/em\u003e. Fortunately, the non-pathogenic probiotic \u003cem\u003eE. coli\u003c/em\u003e Nissle 1917 (EcN) of serotype O6:K5:H1, without virulence factors, has been discovered to prevent pathogenic bacteria from attacking the intestinal mucosa in the intestine and has a protective and repairing effect on the intestinal mucosal barrier(Jacobi \u0026amp; Malfertheiner, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Based on the properties of EcN, recent studies have proposed a series of clinical ideas for heterologous gene expression using EcN as a favored chassis for disease treatment, including sustained heterologous expression of 3-hydroxybutyric acid for the treatment of colitis(Yan, et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), heterologous expression of uric enzyme for the treatment of hyperuricemia(He, et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and design of near-infrared nano light for controlling gene expression for the treatment of tumors(Zhu, et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). EcN has been reviewed recently to be well employed in biomedical engineering for the treatment of infectious diseases, metabolic disorders, and inflammatory bowel diseases (IBDs) as well as cancers(Lynch, et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Yu, et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, most of the studies on the heterologous expression of EcN have focused on biomedical engineering and neglected the application value of EcN in the food industry. Due to the probiotic and genetically manipulable properties of EcN, a recent study expressed the sulfotransferase domain of human N-deacetylase/N-sulfotransferase-1 (NDST-1) and the catalytic domain of mouse 3-O-sulfotransferase-1 (3-OST-1) in the engineered strain EcN::T7M with fed-batch fermentation, which brought the yield of NST to 0.21 g/L and the yield of heparosan to 0.85 g/L, respectively(Li, et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In addition, EcN was used to engineer a probiotic to produce β-carotene in the gut and metabolize it to vitamin A to treat diarrheal diseases(Miller, et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Therefore, the use of non-pathogenic EcN to establish recombinant expression of PG is a very safe and effective strategy in food applications.\u003c/p\u003e \u003cp\u003eMilk is an important part of people's daily dietary structure for nutritional acquisition, especially for its amino acid content. Milk consists of a colloidal particle complex of associated proteins and calcium phosphate with an average size of 150\u0026ndash;200 nm(Duerasch, et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). These conjoined colloids are known as casein micelles(Fox, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Casein is by far the most essential and valuable component of milk, and many dairy products derive their textural, organoleptic, and nutritional properties primarily from casein, which has attracted people to use it for thousands of years. Casein consists of its αs\u003csub\u003e1\u003c/sub\u003e-casein, αs\u003csub\u003e2\u003c/sub\u003e-casein, β-casein, and κ-casein and inorganic material, which account for approximately 76\u0026ndash;86% of milk(Swaisgood, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). However, the solubility of casein is less than 10% compared to natural casein in cow's milk, limiting the application scenarios of casein(Lee, et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Chemical modification of casein has been expanded to improve the solubility of casein, including succinylation, acetylation, dephosphorylation, and neutralization(Sarah, et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Wu, et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Yang, et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Sodium caseinate is prepared by fermentation, a process in which non-fat milk or Qula is fermented with lactic acid bacteria (LAB). Subsequently, the solid proteins are separated and adjusted to neutrality with NaOH, and finally, citric acid is added to form sodium caseinate(H. N. Liu, et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Sodium caseinate is used in meat processing, bakery, ice cream, and other industries to increase the binder, emulsification, and insufficient nutrition of food products(Garcia, et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Sudha, et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Voronin, et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, there are some negative effects of chemical methods compared to enzymatic modification, such as loss of nutritional value, decreased emulsification and foaming, and the chemical process still contains a potential consumption threat. The modification of casein by enzymatic methods to improve its functional properties and broaden its application scenarios is still at a limited level of research. The development of a relatively cheap, efficient, and mild soluble casein production process without the intervention of exogenous chemicals is urgent to expand the application of casein in the food industry.\u003c/p\u003e \u003cp\u003eIn this study, an expression system for high PG yield was produced in probiotic EcN, which promotes the safe production of PG and simplifies the purifying procedure. The functional properties of casein were significantly improved by PG deamidation which improves its usability as a functional ingredient in the food industry.\u003c/p\u003e"},{"header":"2. MATERIAL AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Strains, plasmids, and culture conditions.\u003c/h2\u003e \u003cp\u003eThe strains, plasmids, and primers used in this study are listed in \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e, \u003cb\u003eTable S2\u003c/b\u003e, and \u003cb\u003eTable S3\u003c/b\u003e in the Supplemental information, respectively. \u003cem\u003eE. coi\u003c/em\u003el strains were grown at 37\u0026deg;C in LB broth supplemented with 100 \u0026micro;g/mL of ampicillin or 15 \u0026micro;g/mL of tetracycline, and 1mM isopropyl-D-thiogalatopyranoside (IPTG) when required.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 SDS-PAGE.\u003c/h2\u003e \u003cp\u003eProtein samples were mixed in 5 x loading buffer and denatured and reduced. Subsequent electrophoresis and analysis were performed using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a protein marker (Thermo Fisher Scientific, USA, 26610) as standard. Gels were stained with Coomassie Brilliant Blue R250 and decolorized using a 10% acetic acid-ethanol solution, followed by gel imaging.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Deamidation degree.\u003c/h2\u003e \u003cp\u003eThe method is referenced from Inthawoot et al. with a slight modification(Suppavorasatit, et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Ammonia content from complete deamidation of casein: Take 500 \u0026micro;l of casein sample in a 1.5 mL EP tube, add an equal volume of 2 N sulfuric acid solution, heat in a water bath at 100 ℃ for 4 h, centrifuge at 12000 rpm for 10 min, and take an appropriate amount of supernatant to determine the NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e in the supernatant using an ammonia assay kit (Sigma-Aldrich, USA). The ammonia content of casein deamidation catalyzed by PG: When casein was deamidated catalyzed by PG, it was centrifuged at 12000 rpm for 10 min, and an appropriate amount of supernatant was taken to determine NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e in the supernatant using an ammonia assay kit. The DD is the percentage of ammonia produced by PG-catalyzed deamidation of casein over that produced by complete deamidation of casein\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.4 Hydrolysis degree\u003c/b\u003e.\u003c/h2\u003e \u003cp\u003eDetermination of complete hydrolysis of casein: Take 500 \u0026micro;l of casein sample in 2 mL EP tube, add an equal volume of 2 N sulfuric acid solution, heat in water bath at 100 ℃ for 4 h, add 1 mL of 0.2 N trichloroacetic acid solution, centrifuge at 12000 rpm for 10 min, take 10 \u0026micro;l of supernatant in a 96-well microplate, and determine the total soluble protein in the supernatant by BCA protein concentration method. The total soluble protein content in the supernatant was measured by the BCA protein concentration method, i.e. the degree of complete hydrolysis of casein. Determination of hydrolysis degree of casein after deamidation reaction: When the casein was completed by PG-catalyzed deamidation reaction, the reaction was terminated by 80 ℃ water bath for 10 min, ice bath for 30 min, 1 mL of modified casein sample was taken in 2 mL EP tube, 1 mL of 0.2 N trichloroacetic acid solution was added, centrifuged at 12000 rpm for 10 min, 10 \u0026micro;l of supernatant was taken in a 96-well microtiter plate, and the protein concentration was measured by BCA. The protein concentration method was used to determine the soluble protein content in the supernatant after the casein deamidation reaction, i.e. the degree of hydrolysis after the casein deamidation reaction. The degree of hydrolysis is the percentage of hydrolysis of casein after the deamidation reaction over the complete hydrolysis of casein.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Solubility.\u003c/h2\u003e \u003cp\u003eDetermination of protein content in unmodified casein solution by BCA measurement of protein concentration. The same concentration of modified casein solution was taken, and the protein content in the modified casein solution was determined by the BCA protein concentration method. The solubility is the percentage of protein content in the modified casein solution over the protein content in the unmodified casein solution.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Emulsifiability.\u003c/h2\u003e \u003cp\u003eWith modified casein solution volume to soybean oil volume equal to 3:1, placed in a tissue homogenizer, homogenized the mixture at 20000 rpm for 2 min, immediately aspirated 50 \u0026micro;l of emulsion from the bottom of the solution in 5 mL of 0.1% SDS solution, mixed, OD500 nm to detect the absorbance value, recorded as A\u003csub\u003e0\u003c/sub\u003e, and calculated the emulsifiability (EAI) of the protein solution.\u003c/p\u003e \u003cp\u003eEAI (m\u003csup\u003e2\u003c/sup\u003e /g)\u0026thinsp;=\u0026thinsp;2\u0026times;2.303\u0026times;A\u003csub\u003e0\u003c/sub\u003e\u0026thinsp;\u0026divide;\u0026thinsp;0.25 (1)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Emulsification stability.\u003c/h2\u003e \u003cp\u003eThe modified casein solution volume to soybean oil volume is equal to 3:1, placed in tissue homogenizer, homogenized the mixture at 20000 rpm for 2 min, immediately aspirated 50 \u0026micro;l of emulsion from the bottom of emulsion in 5 mL of 0.1% SDS solution, mixed, absorbance value detected at OD500 nm, recorded as A\u003csub\u003e0\u003c/sub\u003e, after standing at room temperature for 10 min, again aspirated from the bottom of emulsion After standing for 10 min at room temperature, 50 \u0026micro;l of emulsion was again drawn from the bottom of the emulsion in 5 mL of 0.1% SDS solution, mixed well, and the absorbance value was detected at OD500 and recorded as A\u003csub\u003e10\u003c/sub\u003e. The stability of emulsification (ESI) was calculated as:\u003c/p\u003e \u003cp\u003eESI (min)\u0026thinsp;=\u0026thinsp;A\u003csub\u003e0\u003c/sub\u003e \u0026divide; (A\u003csub\u003e0\u003c/sub\u003e- A\u003csub\u003e10\u003c/sub\u003e) \u0026times; 10. (2)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Enzyme purification.\u003c/h2\u003e \u003cp\u003eEquilibrate the Ni-NTA column with 5 times the packing volume of the protein washing solution (20 mM Tris, 500 mM NaCl). Put the supernatant of the centrifuged bacterial culture on the column after centrifugation with a flow rate of 4 s/drop to fully bind the target protein to the Ni-NTA column and collect the penetration solution. Wash the Ni-NTA column with 4 times the volume of the pre-cooled protein washing solution to remove the non-specific binding protein and collect the washing solution. The target protein was eluted with 4 times the volume of pre-cooled protein eluent (20 mM Tris, 500 mM NaCl, imidazole concentration gradient of 30\u0026ndash;500 mM), and the eluate was collected. A small amount of samples from each stage were subjected to SDS-PAGE, and the rest of the samples were stored at 4 ℃. According to the results of SDS-PAGE, the eluate containing the purer target protein was selected for dialysis at 4 ℃ (20 mM Tris), and the dialysate was changed every 4 h. The dialyzed protein solution was concentrated by ultrafiltration, centrifuged at 4000g to 3\u0026ndash;5 mL at 4 ℃, filtered and de-bacterized in a sterile ultraclean table, and then frozen and stored in a -80 ℃ refrigerator for backup.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Particle size and Zeta potential.\u003c/h2\u003e \u003cp\u003eCaseins with different DD were prepared in ultrapure water to a final concentration of 1 mg/mL, and the Zeta potentials, particle sizes, and polydispersity of caseins with different DD were determined by using a Zetasizer Nano (Malvern NaNO-ZS ZEN3600).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 UV-visible spectra and fluorescence spectra.\u003c/h2\u003e \u003cp\u003eFor detection of UV-visible spectra (Shimadzu UV-3600 Plus), 10 mg of casein with different DD were dispersed into 5 mL of distilled water, vortexed, and mixed well, and the protein solution was scanned by a UV spectrophotometer at 200\u0026thinsp;~\u0026thinsp;700 nm with a spectral slit width of 1 nm. The UV-visible spectra of casein with different DD were obtained by using distilled water as the blank spectrum for baseline correction. For detection of fluorescence spectra (HORIBA FluoroMax-4), 10 mg of casein with different DD were dispersed into 5 mL of distilled water and vortexed to mix well. The fluorescence spectra of the protein solution were scanned under the following conditions: excitation wavelength of 285 nm, emission wavelengths of 290\u0026thinsp;~\u0026thinsp;500 nm, and spectral slit width of 1.5 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Surface hydrophobicity.\u003c/h2\u003e \u003cp\u003eCasein solutions with different deamidation degrees of 0.1, 0.2, 0.3, and 0.4 mg/mL were prepared, and 8-Anilino-1-naphthalenesulfonic acid ammonium salt (ANS-NH4) was prepared to 8 mmol/L with distilled water. 20 \u0026micro;L of ANS-NH4 solution was added to 4 mL of protein solution for 30 min under dark conditions. 20 \u0026micro;L of ANS-NH4 solution was added to 4 mL of protein solution, and the reaction was carried out in the dark for 30 min, with the excitation wavelength at 360 nm, the emission wavelength at 370\u0026ndash;700 nm, and the spectral slit width of 5 nm. Then, the fluorescence intensity of the sample was measured (HORIBA FluoroMax-4). The concentration of casein and the fluorescence intensity were plotted, and the initial slope of the curve was the surface hydrophobicity index of wheat alcohol-soluble protein with different degrees of deamidation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Circular dichroism (CD).\u003c/h2\u003e \u003cp\u003eThe secondary structure of casein was determined by the method described by Suppavorasatit et al.(Suppavorasatit, et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Caseins were dispersed at a concentration of 10 mM in phosphate buffer (pH\u0026thinsp;=\u0026thinsp;7) at 20 ℃. The CD spectra in the far UV region (190\u0026thinsp;~\u0026thinsp;260 nm) of each sample were determined using JASCO spectropolarimeter (Model J-1500, Tokyo, Japan). The samples were analyzed using a 1 cm path-length square quartz cuvette with a Teflon cap. The molar ellipticity values were calculated using the formula as shown in the equation:\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$${\\left[\\theta \\right]}_{\\text{m}\\text{o}\\text{l}\\text{a}\\text{r},{\\lambda }}\\left(\\text{deg}{cm}^{2} {\\text{d}\\text{m}\\text{o}\\text{l}}^{-1}\\right)=100\\times \\frac{{\\theta }_{\\lambda }}{m}\\times d$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e3\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere θ\u003csub\u003eλ\u003c/sub\u003e is the observed ellipticity (degrees) at wavelength λ, m is the molar concentration of a solute, and d is the path length (cm). Prediction of the percent of protein secondary structure from CD spectra was obtained using software from a webserver: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://perry.freeshell.org/raussens.html\u003c/span\u003e\u003cspan address=\"http://perry.freeshell.org/raussens.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, which uses the method of Raussens et al(Vincent, et al., 2003).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13 PG enzyme activity.\u003c/h2\u003e \u003cp\u003ePG enzyme activity assay was referenced to Qu et al. with slight modifications(Qu, et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). 10 \u0026micro;l solution containing PG was reacted with 100 \u0026micro;l of Cbz-Gln-Gly (10.11 g/L) of the experimental group or inactivated with 100 \u0026micro;l of 1 N TCA of the control group at 37\u0026deg;C for 30 min. The reaction was subsequently terminated with 100 \u0026micro;l of TCA and the system was made up of 100 \u0026micro;l of Cbz-Gln-Gly, respectively. After that, 12 \u0026micro;l of the reaction solution and different concentrations of ammonium chloride solution were taken as the standard curve for each sample, 60 \u0026micro;l of color development solution A (40.46 g/L phenol, 0.15 g/L sodium nitroprusside), 48 \u0026micro;l of distilled water, 30 \u0026micro;l of color development solution B (49.94 g/L potassium hydroxide) and 60 \u0026micro;l of color development solution C (200 g/L potassium carbonate, 8.37 mL/L sodium hypochlorite solution) were added. The reaction was carried out at 37 ℃ for 20 min, and the absorbance value was measured at OD630. The enzyme activity was calculated by the formula:\u003c/p\u003e \u003cp\u003ePG Enzyme activity (U/mL)=(A\u003csub\u003e0\u003c/sub\u003e-A\u003csub\u003e1\u003c/sub\u003e)\u0026times;21/17.03/30\u0026times;k (4)\u003c/p\u003e \u003cp\u003eIn the formula, A\u003csub\u003e0\u003c/sub\u003e is the absorbance value of the experimental group; A\u003csub\u003e1\u003c/sub\u003e is the absorbance value of the control group; k is the slope of the ammonia standard curve.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14 Statistical analysis and reproducibility.\u003c/h2\u003e \u003cp\u003eThe GraphPad Prism 8.0 was used for statistical analysis. The data are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. A two-tailed t-test was used to determine the significance between the two groups. One-way or two-way ANOVA with Bonferroni post-test was used to analyze multiple groups. For all the statistical tests, P values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered to be statistically significant. ns: no significant, \u003csup\u003e*\u003c/sup\u003e: 0.01\u0026thinsp;\u0026le;\u0026thinsp;P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e**\u003c/sup\u003e: 0.001\u0026thinsp;\u0026le;\u0026thinsp;P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, \u003csup\u003e***\u003c/sup\u003e: 0.0001\u0026thinsp;\u0026le;\u0026thinsp;P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, \u003csup\u003e****\u003c/sup\u003e: P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. RESULTS AND DISCUSSIONS","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Establishment of expression system of PG in \u003cem\u003eE. coli\u003c/em\u003e Nissle 1917(EcN)\u003c/h2\u003e \u003cp\u003eTo simplify the food safety problems caused by endotoxin in industrial production, this study optimized the strategy of heterologous expression of PG in \u003cem\u003eE. coli\u003c/em\u003e by using probiotic EcN as chassis cells. Since EcN is a non-engineered wild-type strain, to better serve as a platform for the expression of heterologous genes by chassis cells, a series of pSEVA vectors containing different replicons and replication proteins, suitable for Gram-negative bacteria(Martinez-Garcia, et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015a\u003c/span\u003e), were loaded with PG expression frames (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Compared with pET32a(+)/\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eP\u003c/span\u003ero-\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eP\u003c/span\u003eG (PP) expressed in \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3), the PG enzyme activity of pSEVA521/PP, pSEVA541/PP, pSEVA551/PP and pSEVA581/PP in EcN had significantly higher, except that of pSEVA531/PP in EcN was reduced, and the PG enzyme activity had the highest yield up to 8.69 U/mL after trypsin cleavage in pSEVA551/PP EcN strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). PP bands detected by SDS-PAGE showed the same results \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. The above results indicate that the optimized expression of PG can be efficiently produced in the probiotic EcN with 8.69 U/mL.\u003c/p\u003e \u003cp\u003eA series of pSEVA plasmids, containing different replication systems of RK2, pBBR1, pRO1600/ColE1, RSF1010, and pUC, were screened as expression vectors for PG(Jahn, et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The results showed that the pSEVA551/PP plasmid containing the RSF1010 replication system expressed the highest recombinant PG enzyme activity in EcN up to 8.69 U/mL, which was 63.03% higher than the expression of recombinant PG by pET32a(+)/PP containing the pBR322 replication system in BL21(DE3). This result is in part due to differences in plasmid copy number, which in turn affects the variation in gene expression(Jahn, et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In addition, even in the case of identical promoter and plasmid, differences in the open reading frame of genes can lead to differences in the yield of the target product(Xu, et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo determine the activity of recombinant PG derived from EcN and used for protein modification, PP was purified by affinity lamination with NTA-Ni columns (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). The purified PP was cleaved by trypsin for 0-300 min, and the gradual disappearance of PP bands and a gradual increase of mPG bands could be both observed, demonstrating the validity of the event that PP was cleaved by trypsin to produce mPG (\u003cb\u003eFig. S2A\u003c/b\u003e), which is consistent with Ouyang et al. who used trypsin to cleave PP and produce mPG(Ouyang, et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The amino acid composition between the pro-peptide and mPG as K114-L115 has been confirmed, which is located on the surface of the protein structure and cleaved by trypsin of the serine protease family(Yamaguchi, et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Zheng, et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). PG enzyme activity assay on temporally incremental trypsin-cleaved PP similarly showed incremental activity (\u003cb\u003eFig. S2B\u003c/b\u003e). Further, mPG, recombinant PP expressed in EcN activated by trypsin, was purified (\u003cb\u003eFig. S3\u003c/b\u003e), and different protein substrates were used for deamidation, demonstrating PG expressed by EcN effectively had deamidation activity against different substrates (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). The above data indicates the feasibility of proper expression of PG in probiotic \u003cem\u003eE. coli\u003c/em\u003e EcN.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Effect of temperature on casein modification.\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, the values of the DD increased in a temperature-dependent manner, indicating that casein was effectively deamidated by PG and that the elevated temperature was able to promote the deamidation reaction of PG more effectively. Similarly, the DD of coconut protein modified by PG increased with increasing temperature; however, the DD decreased when the modification of PG was performed at more than 60\u0026deg;C(Kunarayakul, et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Higher temperatures were not further attempted because higher temperatures resulted in protein denaturation of PG and a rapid decrease in PG enzyme activity (\u003cb\u003eFig. S4\u003c/b\u003e). Yamaguchi et al.(Yamaguchi, et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) and Lu et al.(Lu, et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) demonstrated a rapid decrease in PG enzyme activity at temperatures above 60\u0026deg;C. As the value of DD increased, the precipitation rate of casein obtained decreased, indicating an improved solubility of deamidated casein (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Analysis of the functional properties of the casein with different values of DD showed a significant improvement in foaming, emulsification, and emulsion stability, expanding the range of casein applications (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). In addition, the values of the degree of hydrolysis (DH) results showed no significant hydrolysis of casein by PG, which is consistent with the previously reported properties of PG for EPSC protein(Hadidi, et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eTo further visualize the effect of deamidated casein, it was imaged and showed a slight decrease in precipitation of deamidated casein at 30\u0026deg;C and 40\u0026deg;C, while the turbidity of the supernatant increased visually, compared to the control. The casein deamidated at 50\u0026deg;C showed a milky solution with good homogeneity and dispersion in aqueous solution without significant precipitation, while the casein deamidated at 60\u0026deg;C was more clarified and translucent, probably due to a further increase in the solubility of casein in aqueous solution, as well as the separation of casein micelles and the reduction of casein particles (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). To better present the correlation between DD and solution turbidity, casein that was deamidated at different lengths of OD600 and DD was examined. As \u003cb\u003eFig. S5A\u003c/b\u003e and \u003cb\u003eFig. S5B\u003c/b\u003e showed, the turbidity of soluble casein solutions showed a trend of increasing and then decreasing with increasing DD, which was since at low DD of casein, most of the casein was in insoluble state, and soluble solutions were relatively clarified. At moderate DD, the aggregated casein micelles dissociate, and casein solubility increases, maintaining good dispersion without precipitation, which leads to an increase in turbidity. Miwa et al. modified skim milk by PG and showed a gradual decrease in turbidity as the DD increased. The reason for this difference may be that in this study the samples were centrifuged and only the turbidity of the non-precipitated supernatant was calculated(Miwa, et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). At high DD, the casein particles were reduced and completely dissolved to form a stable casein solution, leading to a decrease in turbidity and a state similar to that of sodium caseinate solution. Taken together, the above data demonstrate that EcN-derived PG can effectively deamidate casein and increase the protein functional properties of casein in a temperature-dependent manner.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Effect of pH on casein modification\u003c/h2\u003e \u003cp\u003eSince pH can affect the properties of proteins relevant to the application scene, PG was used for the deamidation of casein at different pH. Before that, we explored the effect of different pH on the PG enzyme activity itself, and the results showed that PG activity does not decrease significantly at pH\u0026thinsp;=\u0026thinsp;4\u0026ndash;8. At pH\u0026thinsp;=\u0026thinsp;9, there is a small decrease in PG enzyme activity, while at pH higher than 9 or lower than 4, there is an almost complete loss of PG enzyme activity, which indicates the pH stability of PG and the range of applications of the modified protein (\u003cb\u003eFig. S6\u003c/b\u003e). Similar to the result of the present study, Yamaguchi et al. and Lu et al. demonstrated that the relative enzyme activity of PG in pH\u0026thinsp;=\u0026thinsp;4\u0026ndash;7 environments was both above 70%(Lu, et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Yamaguchi, et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, at around neutral conditions, pH\u0026thinsp;=\u0026thinsp;5, 6, and 7, the DD of casein was modified to a higher level. At alkaline conditions, pH\u0026thinsp;=\u0026thinsp;9, the DD of casein decreased slightly due to a slight decrease in the activity of PG under that condition. In acidic conditions, with pH\u0026thinsp;=\u0026thinsp;5, the DD of casein decreased significantly, due to the weak acidic conditions causing denaturation and precipitation of casein (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG), even if the PG enzyme activity did not decrease significantly at pH\u0026thinsp;=\u0026thinsp;5. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB showed that the casein precipitation rate under weakly acidic conditions could reach 48.13%, indicating the poor solubility of casein under a weakly acidic environment, even though a certain DD had occurred. The foaming, emulsification, and emulsion stability of the deamidated casein at different pH decreased to some extent, compared to pH\u0026thinsp;=\u0026thinsp;7, suggesting the best effect of PG on casein deamidation and application under neutral conditions, which may be related to the nature of the modified protein itself (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). It is noteworthy that although the value of DD of casein was higher at pH\u0026thinsp;=\u0026thinsp;6, it already suffered a certain degree of precipitation and behaved as a suspension, showing that the denaturation and precipitation of proteins seriously affected the emulsion stability (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). In addition, at pH\u0026thinsp;=\u0026thinsp;8 and 9, even though the protein did not denature and precipitate, the emulsion stability was still severely reduced. pH had no significant effect on the degree of hydrolysis when PG modified casein (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Although the effect of pH in the appropriate range on PG enzyme activity was not significant, the effect of pH on casein was greater, especially under acidic conditions where casein solubility was greatly restricted, even at similar DD. This is because casein has an isoelectric point (PI) of 4.6, which is acid-sensitive and will precipitate at a lower pH(Bertsch, et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Similar to this result, α- zein showed low solubility under acidic conditions at pH\u0026thinsp;=\u0026thinsp;3 with or without PG deamidation, due to the approaching of the isoelectric point of α- zein to acidic region(Yong, et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). These data indicate that the modification of casein under neutral conditions is more effective, while application under non-acidic conditions is more valuable.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Effect of enzyme/substrate (E/S) ratio on casein modification\u003c/h2\u003e \u003cp\u003eTo investigate the ability of PG to modify different casein concentrations to suit the required additions for various applications, 5% casein was subjected to deamidation reactions, and the results showed that the values of DD of casein increased with increasing E/S ratio, with the highest DD being 51.25% at E/S ratio equal to 5 U/g (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The results of Miwa et al. showed that the DD and ammonia release from skimmed milk powder increased with increasing enzyme-to-substrate ratio and casein was the major protein in skimmed milk powder, which is consistent with the results of this study(Miwa, et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The precipitation rates of casein modified by PG were substantially reduced, indicating that PG was able to greatly promote casein solubility (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The foaming, emulsification, and emulsion stability gradually increased with growing E/S ratio, and further, the functional properties of these caseins were again enhanced with the addition of PG, extending the application potential of the functional properties of deamidated casein (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Similarly, an improvement in the E/S ratio did not result in PG hydrolytic activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF), which is consistent with the result that EPSC and soy protein modified by PG do not affect the degree of hydrolysis(Hadidi, et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Suppavorasatit, et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The photographs show that PG can effectively promote solubilization, indicating the great potential of PG to promote casein solubilization (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). The functional properties of modified casein were similarly enhanced with a gradual increase in the E/S ratio, which has been similarly demonstrated on evening primrose seed cake (EPSC) protein(Hadidi, et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The above data showed that PG maintained a good deamidation ability and improved the functional properties of casein.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Primary and secondary structural properties of modified casein\u003c/h2\u003e \u003cp\u003eTo investigate the mechanism of the improvement of the functional properties of deamidated casein, SDS-PAGE was performed on casein with different degrees of deamidation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Casein with different degrees of deamidation showed no significant degradation of the bands, indicating no hydrolytic activity of PG(Hadidi, et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Yong, et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). In addition, PG deamidation resulted in a slight upward migration of the casein bands, which could be attributed to the slight change in the SDS-PAGE pattern by PG is attributable to the inhibition of SDS binding to proteins because of the increase in the negative charge by deamidation(Miwa, et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The \u0026ldquo;diffused\u0026rdquo; set of protein bands may be due to the aggregation of protein molecules, which is similar to the phenomenon studied by Yong et al.(Yong, et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). SDS-PAGE showed that the deamidation of casein by PG did not change the primary structure of casein and had no hydrolytic activity. Circular dichroism was used to reflect changes in the proportion of deamidated casein secondary structure. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB shows the CD spectra of the soluble fractions of control and deamidated casein. The CD spectra of the untreated and deamidated casein samples showed a negative board band at around 200\u0026thinsp;~\u0026thinsp;220 nm. The percentage content of α-helices and β-turns increased and the content of β-sheets decreased in deamidated casein compared to un-deamidated casein, and there appeared to be no clear pattern of change in the content of random (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e)(Kunarayakul, et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Suppavorasatit, et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The result that the content of β-sheets decreased with increasing degree of deamidation was similar to that of deamidated soy protein(Suppavorasatit, et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). These results suggest that the deamidation of casein by PG can lead to changes in the casein secondary structure.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSecondary structures\u003csup\u003ea\u003c/sup\u003e of non-deamidated and deamidated casein.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eCaseins with different DD\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSecondary structure (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17.5%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32.8%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e40.8%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eα-Helix(%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e22.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eβ-sheet(%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e54.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e44.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e46.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eβ-turn(%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e27.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRandom(%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e42.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e38.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e37.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e39.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSum(%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003ea\u003c/sup\u003e Data were derived by analysis of CD spectra.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Tertiary structure properties of modified casein\u003c/h2\u003e \u003cp\u003eThe characteristic absorption at 278 nm is mainly due to the presence of tyrosine (275 nm), tryptophan (279 nm), and phenylalanine (257 nm)(Grimsley \u0026amp; Pace, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). The absorbance values at around 275 nm were significantly increased in PG-deaminated casein compared to non-deaminated casein, suggesting that PG deamidation can alter the tertiary structure of casein (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), exposing hydrophobic amino acids (especially tyrosine and tryptophan) in the internal structure(Jin, et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Tryptophan, tyrosine, and phenylalanine residues in proteins, especially tryptophan residues, fluoresce in a manner that is dependent on protein folding, and thus they can serve as sensitive monitors of conformational changes in tertiary structures(Xiong, et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). This speculation is consistent with the results reflected by the fluorescence spectra, i.e., the endogenous fluorescence peaks at 340 nm were both significantly enhanced in the deamidated casein compared to the non-deamidated casein (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). This is due to a change in the tertiary structure of PG-deamidated casein, which unfolds the protein conformation and leads to exposure of the tryptophan moiety, coupled with an increase in amounts of the fluorescence intensity(Jin, et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Alterations in the tertiary structure of deamidated casein result in the exposure of internal hydrophobic amino acids, such as tryptophan and phenylalanine, enhancing the surface hydrophobicity of deamidated casein (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). It was shown that surface hydrophobicity promotes protein foaming properties(Moro, et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), which is consistent with the results of enhanced foaming properties of deamidated casein.\u003c/p\u003e \u003cp\u003eCasein particles are micelles of casein consisting of αs1-casein, αs2-casein, β-casein, and κ-casein, which may cause structural changes in the protein after deamidation by PG. Based on the above hypothesis, the particle size, polydispersity, and zeta potential of casein with different degrees of deamidation were examined. particle size light intensity distribution results showed that the particle size of non-deamidated casein had a wide range of particle sizes, and the homogeneity of particle size was poor. The results showed that the particle size distribution of casein with undeamidated degree was wide and the particle size uniformity was poor. As the degree of deamidation increased, the particle size distribution of casein gradually narrowed to about 200 nm, and the particle size homogeneity increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). The results of PDI also proved that the degree of deamidation of casein was positively correlated with the particle size homogeneity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Meanwhile, the zeta potential indicated that the stability of the casein dispersion system increased with increasing degree of deamidation and reached its maximum at a DD of 40.8% (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). Particle size distribution analysis and transmission electron microscopy showed that smaller particle sizes were produced in skim milk with high deamidation. This indicates that the deamidation of PG induces the dissociation of casein micelles(Miwa, et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The above results indicate that deamidation of casein with PG alters the secondary and tertiary structure of casein, which correlates with improved functional properties of deamidated casein.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. CONCLUSION","content":"\u003cp\u003eBased on the safety of food enzyme production, we optimized the expression of PG in the probiotic EcN to achieve a PG yield of 8.69 U/mL, which is 49.66 times higher than the highest yield reported in \u003cem\u003eE. coli\u003c/em\u003e at present. In addition, EcN has improved safety and reduced production costs compared to other \u003cem\u003eE. coli\u003c/em\u003e. Purified PG was deamidated to a variety of substrate proteins. Casein was deamidated by PG with improved functional properties, including solubility, foaming, emulsification, and emulsion stability. Although the primary structure of the deamidated casein did not undergo hydrolysis, the content of α-Helix and β-turn increased while the content of β-sheet and Random decreased in the secondary structure. Meanwhile, the alteration of the tertiary structure of deamidated casein led to the exposure of hydrophobic amino acids such as tyrosine and tryptophan, which caused the enhancement of absorbance values at 275 nm, endogenous fluorescence peaks at 340 nm, and surface hydrophobicity. In addition, depolymerization of casein micelles occurred after deamidation, and particle size, PDI, and zeta potential decreased. In short, this strategy for producing recombinant PG in EcN extends safe enzyme production, improves the functional properties of casein, and elucidates the structural properties of deamidated casein. These results advance the production of PG and the application of casein in the food industry.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAUTHOR INFORMATION\u003c/b\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAUTHOR INFORMATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding Author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeming Jiang\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e-\u003c/strong\u003e \u003cem\u003eSchool of Life Science, East China Normal University, Shanghai 200241, P. R. China;\u003c/em\u003e Phone: +86 021 54341957; Email: [email protected]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJing Huang\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e-\u003c/strong\u003e \u003cem\u003eSchool of Life Science, East China Normal University, Shanghai 200241, P. R. China;\u003c/em\u003e Phone: +86 021 54341055; Email:\u0026nbsp;[email protected]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eZheng Zhang\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e-\u003c/strong\u003e \u003cem\u003eSchool of Life Science, East China Normal University, Shanghai 200241, P. R. China.\u003c/em\u003e\u003cem\u003e\u0026nbsp;ORCID:\u003c/em\u003e \u003cem\u003e0009-0003-4021-9349\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLihui Zheng\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e-\u003c/strong\u003e \u003cem\u003eSchool of Life Science, East China Normal University, Shanghai 200241, P. R. China\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYuxi Li\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003eSchool of Life Science, East China Normal University, Shanghai 200241, P. R. China\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eShuchao Jiao\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e-\u003c/strong\u003e \u003cem\u003eSchool of Life Science, East China Normal University, Shanghai 200241, P. R. China\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYelin Wu\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e-\u0026nbsp;\u003c/strong\u003e\u003cem\u003eTongji University Cancer Center, Shanghai Tenth People\u0026apos;s Hospital, Tongji University School of Medicine, Shanghai 200072, P. R. China\u003c/em\u003e\u003cem\u003e. ORCID:0000-0002-0746-8541\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMingfei Jin\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e-\u003c/strong\u003e \u003cem\u003eSchool of Life Science, East China Normal University, Shanghai 200241, P. R. China\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eShuo Zhang\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e-\u003c/strong\u003e \u003cem\u003eSchool of Life Science, East China Normal University, Shanghai 200241, P. R. China\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHongliang Gao\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e-\u003c/strong\u003e \u003cem\u003eSchool of Life Science, East China Normal University, Shanghai 200241, P. R. China\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eZhongyi Chang\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e-\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cem\u003eSchool of Life Science, East China Normal University, Shanghai 200241, P. R. China\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ.H. and D.J. conceived and supervised the study. Z.Z. designed and performed the experiments. L.Z. and Y.L. performed most molecular biological experiments. S.J. and H.G. assisted with experiments on the functional characterization of casein. Y.W. and Z. S. assisted with experiments on the structural characterization of casein. M.J. and Z.C. analyzed the data. J.H. and D.J. wrote and proofread the manuscript. All authors contributed to the review, revision and finalization of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICTS OF INTEREST\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors report no conflict or declaration of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would greatly acknowledge the support by Instruments Sharing Platform of School of Life science, ECNU.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank ECNU Instruments Sharing Platform of School of Life Sciences for technology support. The authors thank Y.W. for support with experimental techniques and data processing. Figure 1A created with BioRender.com.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe main data supporting the results of this study are available within the paper and its Supplementary Information. All data generated in this study are available from the corresponding authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBertsch, P., Savorani, L., \u0026amp; Fischer, P. (2019). Rheology of Swiss Cheese Fondue. \u003cem\u003eACS Omega, 4\u003c/em\u003e(1), 1103-1109.http://dx.doi.org/10.1021/acsomega.8b02424\u003c/li\u003e\n\u003cli\u003eCheng, F., Xiang, C., Zhang, X. J., Liu, Z. Q., \u0026amp; Zheng, Y. G. (2018). 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Near-Infrared Nano-Optogenetic Activation of Cancer Immunotherapy via Engineered Bacteria. \u003cem\u003eAdvanced Materials, 35\u003c/em\u003e(8).http://dx.doi.org/10.1002/adma.202207198\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"food-and-bioprocess-technology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food and Bioprocess Technology](https://www.springer.com/journal/11947)","snPcode":"11947","submissionUrl":"https://submission.nature.com/new-submission/11947/3","title":"Food and Bioprocess Technology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Deamidation, PG-modified casein, foaming capacity, emulsification, structural properties","lastPublishedDoi":"10.21203/rs.3.rs-3842060/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3842060/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eProtein glutaminase (PG; EC 3.5.1.44) is widely used in the food industry because it catalyzes the deamidation of peptide chain glutamine residues and enhances the functional properties of food proteins. Here, a strategy for PG production by probiotic \u003cem\u003eEscherichia coli\u003c/em\u003e Nissle 1917 (EcN) is proposed. The yield of mature PG (mPG) was increased to 8.69 U/mL after testing a series of pSEVA vectors. The purified mPG showed significant deamidation activity against a wide range of protein substrates. Among these tested substrates, the functional properties of PG-modified casein were investigated. Deamidation of casein by PG was more effective at 60\u0026deg;C, pH\u0026thinsp;=\u0026thinsp;7, and an enzyme-to-substrate ratio (E/S) of 5 U/g protein. Casein is deamidated up to 53.29%, which leads to a solubility of more than 90% for a 5% casein solution. Foam capacity can be nearly doubled. Emulsifiability, especially emulsification stability, is substantially improved. With increasing DD of casein, the α-helix and β-turn in the secondary structure of deamidated casein increased from 0\u0026ndash;22.5%, and from 2.8\u0026ndash;27.2% respectively, while β-fold and random decreased from 54.6\u0026ndash;10.5%, and from 42.6\u0026ndash;39.8% respectively. The enhancement of the absorbance values, endogenous fluorescence peaks, and surface hydrophobicity are due to the exposure of hydrophobic amino acids inside the tertiary structure of deamidated casein. Furthermore, deamidated casein particle size reduced while particle size homogeneity rose. After deamidation by PG, casein has achieved enhanced functional properties which improves its usability as a functional ingredient in the food industry.\u003c/p\u003e","manuscriptTitle":"Recombinant Protein Glutaminase from Probiotic Escherichia coli Nissle 1917 for Enhancing the Functional Properties of Caseins","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-09 19:42:25","doi":"10.21203/rs.3.rs-3842060/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorAssigned","content":"","date":"2024-01-08T08:38:29+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-01-08T08:14:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"Food and Bioprocess Technology","date":"2024-01-07T09:24:09+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"food-and-bioprocess-technology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food and Bioprocess Technology](https://www.springer.com/journal/11947)","snPcode":"11947","submissionUrl":"https://submission.nature.com/new-submission/11947/3","title":"Food and Bioprocess Technology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d92bba05-53ca-4f85-8c16-ad55a625392f","owner":[],"postedDate":"January 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-01-09T19:42:25+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-09 19:42:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3842060","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3842060","identity":"rs-3842060","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}