Introduction of multiple disulfide bonds increases the thermostability of transglutaminase

Introduction of multiple disulfide bonds increases the thermostability of transglutaminase | 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 Article Introduction of multiple disulfide bonds increases the thermostability of transglutaminase Takuto Ono, Kazutoshi Takahashi, Yoshinori Hirao, Yasuhiro Mihara, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5776787/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract Microbial transglutaminase (MTG) is an enzyme that catalyzes the cross-linking of glutamine and lysine residues in proteins. Because of its ability to modify proteins, MTG has various applications in the medical and food industries. Most studies have aimed to enhance the thermal stability of MTG by focusing only on point mutations. Introducing a disulfide (S-S) bond in the N-terminal region has been found to be effective, whereas S-S bonds in other regions were considered ineffective. Therefore, this study aimed to evaluate the impact of introducing an additional S-S bond on the thermal stability of an MTG mutant. We found that adding S-S bonds to regions other than the N-terminal, in conjunction with the N-terminal S-S bond, significantly enhanced thermal stability. This finding demonstrates the importance of reinforcing the weakest part of the protein first, followed by strengthening other regions for optimal thermal stability. The MTG variant with two S-S bonds retained its catalytic activity and substrate specificity towards protein substrates, making it a promising candidate for industrial applications. Thus, introducing S-S bonds could be an effective strategy to increase thermal stability of MTG and other industrial enzymes, thereby contributing to their potential industrial applications. Biological sciences/Biochemistry/Enzymes Biological sciences/Biochemistry/Proteins Microbial Transglutaminase disulfide bond thermal stability mutant enzyme kinetics Figures Figure 1 Figure 2 Figure 3 Introduction Transglutaminase (TG: protein-glutamine γ-glutamyltransferases, EC 2.3.2.13) is a family of enzymes that catalyzes the formation of covalent bonds between the γ-carboxyamide group of the glutamine residue and the ε-amino group of the lysine residue in a peptide or protein, leading to the cross-linking of the ε-(γ-glutamyl) lysine bridge 1 . TG is widely distributed in various mammalian cells and tissues, and its physiological characteristics have been studied previously 2 . The enzymatic activity of TG is crucial in various biological processes, including blood clotting, skin formation, and wound healing 3 . TG in mammals is calcium-dependent; however, Streptomyces mobaraensis TG is calcium independent 4 , 5 . Compared to TG derived from mammalian cells, microbial TG (MTG) has a shorter amino acid sequence, lower homology, and a different three-dimensional structure 6 .MTG is expressed in a form where the pro-sequence is combined (Pro-MTG), which matures after being cleaved by a protease 7 . The pro-sequence reportedly promotes efficient protein folding and secretion, and suppresses its enzymatic activity 8 . Since MTG is easy to purify, it is being used in various research and industrial applications, replacing TG obtained from animal tissues and organs 9 . Specifically, MTG has significant applications in the food, pharmaceutical, and textile industries owing to their ability to modify protein structures and enhance the functional properties of proteins 10 – 13 . The thermal stability of an enzyme determines its applicability in industrial processes that often require high temperatures. Enzymes with higher thermal stability can maintain their activity over a broader range of temperatures, showing their versatility and cost-effectiveness for industrial applications. Several methods use protein engineering techniques to identify thermostable enzymes. Notably, various proteins have been stabilized through random mutagenesis, directed evolution, and rational mutagenesis using the three-dimensional structure. Studies have been conducted to enhance the thermal stability of MTG. For example, studies have reported multiple mutants obtained through random mutagenesis, DNA shuffling, and saturation mutagenesis 10 – 13 , 16 . In addition, rational modification of flexible regions (S2P-S23V-Y24N-E28T-S116A-S179L-S199A-A265P-A287P-K294L) has produced a thermostable MTG mutant, TGm2A 17 – 20 . Moreover, introducing disulfide (S-S) bonds in the N-terminal region can significantly enhance the thermal stability of proteins. For instance, the D3C/G283C or T7C/E58C mutant of MTG reportedly improved thermal stability compared to the wild-type (WT) 21 – 23 . However, the introduction of S-S bonds in other regions was considered ineffective 21 . By systematically introducing S-S bonds into different regions of MTG, we aim to identify the most effective strategies for enhancing its thermal stability. We hypothesize that introducing S-S bonds into regions of this enzyme that are inherently less thermally stable will significantly improve their overall thermal stability. Therefore, the current study aims to evaluate how the introduction of an additional S-S bond to the D3C/G283C MTG mutant will impact its thermal stability. Moreover, this strategy might provide insights into improving the thermal stability of other industrial enzymes. Results In silico mutant design The three-dimensional structure of MTG is formed from three domains, namely, the α-helix domain 1 starting from the N-terminus, β-sheet domain, and α-helix domain 2 (Fig. 1 A). Previous research has shown that the S2C/G283C, D3C/G283C, or T7C/E59C mutations introduced into the α-helix domain 1 improved thermal stability 21 , 22 . Conversely, it has been found that the E93C/V112C and A106C/D213C mutations introduced in the α-helix domain 2 did not improve thermal stability 21 . Our results identified α-helix 1 as the weakest region in MTG, emphasizing the need to strengthen this region. In other words, we suggest that strengthening α-helix 2 without strengthening α-helix 1 would not contribute to thermal stability because α-helix 1 would unfold. Furthermore, introducing two S-S bonds into α-helix 1 would have no effect because a single S-S bond in α-helix 1 sufficiently enhances thermal stability. Therefore, we first strengthened the α-helix domain 1 using D3C/G283C and introduced S-S bonds into the β-sheet domain or α-helix domain 2. To introduce S-S bonds into the protein, it is necessary to mutate the amino acid residue pairs to cysteines. These amino acid residue pairs were predicted using the crystal structure of MTG and a computer program and then verified experimentally. First, we calculated the relative positions of all the amino acid pairs in MTG and extracted those in which the distance between Cβ is within 2Å. We then removed the mutants that were expected to cause significant steric hindrance due to mutation to Cys residues based on the energy values. The introduction of S-S bonds to confer thermal stability was achieved by rigidifying the protein structure. Therefore, mutations to nearby residues and mutations that loop the base of the hairpin structure were removed from the candidate sequence. This occurred because we assumed that they only made the protein locally rigid, and the effect of thermal stability on the entire protein would not be significant. In addition, the hairpin, which contains residues 239–254 and forms a pocket for the active residue Cys64, has a high B-factor and is highly flexible. However, we assumed that fixing the catalytic pocket would reduce enzyme activity and narrow substrate recognition. Hence, we did not introduce S-S bonds to these sites. Finally, five additional mutations were each introduced into the D3C/G283C mutant and evaluated. These five mutants were A81C/V311C, E93C/V112C, A106C/D213C, E107C/Y217C, and A160C/G228C, and the positions of the mutations in the sequence and structure are shown in Fig. 1 B and Fig. 1 C. In this study, the S-S bonds that showed improved thermal stability are summarized in Table 1 . The distance between each S-S bond was 3.3 and 4.4 Å, and the dihedral angle values varied. The secondary structure was effective when introduced into α-helix, β-sheet, and coil; however, introducing it into α-helix/α-helix was better. The B-factor was high in some cases and low in others. Overall, it is assumed that introducing a mutation into a place with a high B-factor is better; however, it is not applicable when introducing a S-S bond into MTG. Table 1 Feature of S-S bond that provides thermostability Distance(Cβ)༈Å༉ dihedral(Ca-Cβ-Cβ-Ca)༈°༉ secondary structure B-factor(Cα)༈Å^2༉ A81C/V311C 4.2 -38.4 coil/beta-sheet 10.04/18.34 E93C/V112C 4.2 -163.8 alpha/alpha 35.86/19.07 A106C/D213C 3.6 -144.8 alpha/alpha 33.55/34.48 E107C/Y217C 4.4 -84.9 alpha/alpha 26.13/24.34 A160C/G228C 3.3 160.1 coil/coil 5.46/12.94 Evaluation of optimal temperature and thermostability of MTG S-S mutants Designed mutants were expressed, purified (Supplementary Fig. S1 ) and their enzyme activity was evaluated. To assess the thermostability of the WT MTG and various S-S mutants, the purified proteins were incubated at 37, 45, 55, 60, 65, 70, 75, and 80゜C for 10 min before analyzing their enzyme activity. MTG activity was measured using a hydroxamate assay. Among the S-S bond-introduced mutants in the WT, only the D3C/G283C mutant exhibited improved thermal stability (Fig. 2 A). Conversely, there was no change in the optimal temperature for the other S-S bond-introducing mutants (A81C/V311C, E93C/V112C, A106C/D213C, E107C/Y217C, and A160C/G228C). However, when additional S-S bonds were introduced into the D3C/G283C mutant rather than the WT, an improvement in thermal stability was observed compared to D3C/G283C alone. To evaluate the optimal temperature of the WT MTG and the various S-S mutants, MTG activity was measured using a hydroxamate assay. As predicted from the thermal stability results following the introduction of S-S bonds into the WT, only the D3C/G283C mutant exhibited an increase in the optimum temperature from 55 to 60°C. In contrast, no change in the optimum temperature was observed in other S-S bond-introduced mutants (A81C/V311C, E93C/V112C, A106C/D213C, E107C/Y217C, and A160C/G228C) (Fig. 2 B and 2 C). However, when additional S-S bonds were introduced into the D3C/G283C mutant, an improvement in activity under conditions above 65°C was observed compared to D3C/G283C alone. Characterization of the enzyme kinetics of S-S mutants The enzyme kinetics of the WT MTG and the various S-S mutants were measured by a glutamate dehydrogenase (GDH)-coupled enzyme assay. The enzymatic kinetics of each S-S mutant were similar to those of the WT MTG. A81C/V311C showed a decreased K m , D3C/G283C showed a slightly decreased K m , whereas E93C/V112C, A106C/D213C, E107C/Y217C, and A160C/G228C showed 5–21% higher K m than wild-type MTG, and thus 71–88% reduced k cat / K m values (Table 2 ). The effects on K m and k cat by combining S-S bonds were minimal. These results indicate that the introduction of S-S bonds makes it possible to acquire an enzyme with enhanced thermal stability while maintaining comparable catalytic activity. Table 2 Kinetic constant of MTG mutants for the GDH-coupled enzyme assay K m V max k cat k cat /K m mM µmol/min/mg s − 1 M − 1 s − 1 WT 1.7 ± 0.4 1.0 ± 0.1 0.7 ± 0 409 ± 69 A81C/V311C 1.0 ± 0 0.9 ± 0 0.5 ± 0 537 ± 7 E93C/V112C 2.0 ± 0.2 1.0 ± 0 0.6 ± 0 302 ± 21 A106C/D213C 1.8 ± 0.2 1.0 ± 0.1 0.6 ± 0 360 ± 28 E107C/Y217C 1.8 ± 0 0.9 ± 0 0.5 ± 0 294 ± 4 A160C/G228C 1.9 ± 0.1 0.9 ± 0 0.6 ± 0 292 ± 16 D3C/G283C 1.4 ± 0.2 0.9 ± 0 0.5 ± 0 382 ± 26 D3C/G283C/A81C/V311C 0.8 ± 0.1 0.8 ± 0 0.5 ± 0 659 ± 29 D3C/G283C/E93C/V112C 1.8 ± 0.1 1.1 ± 0 0.7 ± 0 385 ± 24 D3C/G283C/A106C/D213C 1.5 ± 0.1 1.1 ± 0 0.7 ± 0 466 ± 24 D3C/G283C/E107C/Y217C 1.5 ± 0 1.0 ± 0 0.6 ± 0 424 ± 8 D3C/G283C/A160C/G228C 1.4 ± 0.1 1.0 ± 0 0.6 ± 0 438 ± 36 Data are expressed as means ± SD (n = 3) Evaluation of protein substrate reactivity The reactivity of MTG and S-S mutants with various proteins was measured. To investigate the possibility of application to foods, the reactivity of MTG to sodium caseinate, fish gelatin, soy protein isolate, and α-lactalbumin was evaluated. We measured the amounts of ammonia released after MTG was added to 2% various protein solutions and incubated for 1 h at 37゜C. Although variations in reactivity were observed depending on the protein substrate type, no substantial differences in reactivity were noted between each mutant and the WT (Fig. 3 ). The introduction of S-S bonds did not affect the reactivity toward polymer substrates. This indicates that it is possible to create industrially useful enzymes with enhanced thermal stability, comparable catalytic activity, and unchanged reactivity towards polymer substrates. Selection of effective sites for introducing S-S bonds utilizing sequence information To verify the possibility of predicting effective sites for S-S bond introduction based on sequence information, we analyzed the evolutionary conservation of the sites where S-S bonds were introduced. Using 406 sequences of MTG homologs, the amino acid conservation at the mutation sites was analyzed. Regardless of the conservation level of the surrounding sequences, the results showed that mutants with improved and decreased thermal stability were observed (Supplementary Fig. S2). Discussion The introduction of S-S bonds is a simple and effective method for improving thermal stability without requiring multiple amino acid substitutions. In this study, we investigated further improvements in thermal stability using a MTG mutant by introducing the most important S-S bond (D3C/G283C). Although thermal stability improvements through random mutagenesis have been reported 23 , we successfully created a MTG mutant with a higher thermal stability than those in previous studies 17 , 18 , 21 – 23 . We achieved this by introducing additional S-S bonds as a new approach. It is difficult to predict the appropriate site for introducing S-S bonds from sequence information containing evolutionary information. Previously, it was essential to utilize experimental structural information; however, the introduction of AlphaFold2 or RoseTTAfold reduced this limitation 24 , 25 . From the S-S bonds mutants that has acquired thermal stability, it was found that the thermal stability was improved in the five mutants in which D3C/G283C on the α-helix domain 1 was strengthened and added to the β-sheet domain by S-S bonding. Furthermore, we confirmed that introducing a S-S bond to the β-sheet domain without introducing it to the α-helix domain 1 did not improve thermal stability. This indicates that the stability of the entire protein cannot be improved unless the weakest region of the protein is strengthened alongside others. Furthermore, the thermal stability of MTG was improved only when the S-S mutation was introduced into the β-sheet domain after the α-helix domain 1, which is the weakest part, was strengthened. This suggested that the β-sheet domain is the next weakest region of MTG. The activity of this heat-resistant enzyme did not reduce, showing its significance. It was generally believed that there was a trade-off between thermal stability and activity 26 , 27 . However, many unknowns exist, and recent reports have questioned this trade-off 28 . There was no trade-off between thermal stability and activity in the mutants obtained in the present study. The strategy of not including S-S in the active pocket has produced good results. The results of this study suggest that in order to improve thermal stability without reducing enzyme activity, it is important to introduce S-S bonds in the region that is the weakest in the enzyme and not too close to the active domain. Structural information is essential for this, and obtaining structural information has become much easier with tools such as AlphaFold 2. On the other hand, it is still laborious to investigate the heat-sensitive parts of a protein's structure using instrumental analysis, so it is probably easier to use in silico methods such as Molecular dynamics or to screen a certain number of mutants. Conclusion The thermal stability of MTG was improved by introducing S-S bonds. When heated at 65°C for 10 min, the wild-type MTG had no remaining activity. However, the D3C/G283C mutant, which had one S-S bond introduced, had approximately 40% of its activity retained. 21 – 23 In this study, a second S-S bond was introduced at a different site to the first S-S bond (A81C/V311C, E93C/V112C, A106C/D213C, E107C/Y217C, or A160C/G283C). These mutants improved their residual activity to approximately 80%. They maintained the same activity as the WT, and the substrate specificity for the protein was also maintained, it can be used in high-temperature range, which was difficult to do with WT MTG, thus enhancing their applicability in the food and medical industries. Methods Mutants Design The Schrödinger software (Schrödinger, Inc.) was used for in silico S-S and site prediction. The three-dimensional structure of MTG was obtained from Protein Data Bank (PDB code 1iu4) 6 . First, the missing side chains and protons were added using the Protein Preparation Program, and the structure was optimized 29 . The amino acid pairs that were in a position relationship and could form an S-S bond were extracted using the S-S prediction program 30 . We used a program’s weighted score of 1,000 or less to determine whether the distance and angle were suitable for an S-S bond. Furthermore, we selected five mutants by excluding those located near the catalytic pocket, those with amino acid sequences differing by ten or fewer residues, and those with mutations in the same domain as the D3C/G283C mutant. Plasmids and bacterial strains Corynebacterium glutamicum YDK010 was used as an expression host for MTGs 31 . pPSPTG1 is a plasmid for expressing pro-MTG. 32 Genes coding for MTG mutants were synthesized by optimizing codon usage for C. glutamicum (Genescript) and inserted at the KpnI and BamHI sites of pPSPTG1. Culture medium for protein production C. glutamicum was grown at 30°C in the medium (5 g/L glucose, 10 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl, and 0.2 g/L DL-methionine, pH 7.2) containing 25 mg/mL kanamycin (NACALAI TESQUE, INC.). To produce the pro-form MTG mutants, C. glutamicum was grown at 30°C in the medium (60 g/L glucose, 1 g/L MgSO 4 , 30 g/L (NH 4 ) 2 SO 4 , 1.5 g/L KH 2 PO 4 , 0.01 g/L FeSO 4 ·7H 2 O, 0.01 g/L MnSO 4 ·7H 2 O, 450 µg/L biotin, 0.15 g/L DL-methionine, and 50 g/L CaCO 3 , pH 7.5) containing 25 mg/mL kanamycin. To produce MTG mutants containing an S-S bond, 3 mM dithiothreitol was added 5 h after the culture ensued, as described previously 22 . Determination of protein concentration Reverse-phase HPLC was performed using Proteonavi C4 column (4.6 mm id ×15 cm, Osaka Soda) as described previously 22 . Purified WT MTG was used as a standard. It was purified from the Streptoverticillium spp. S-8112 supernatant culture, as described previously 4 . The concentration of the standard WT MTG was determined using the Bradford assay, with bovine serum albumin (Bio-Rad) serving as the protein standard. Purification of MTGs MTG was purified as described previously 22 . In summary, culture supernatant was obtained by removing bacterial cells from the culture solution through centrifugation (10,000 x g, 4゜C, 10 min) and filtration. The supernatant was subjected to buffer exchange using a Sephadex G-25 (M) (Cytiva) column equilibrated with 20 mM MES (pH 5.5). After adjusting the pH to 7.0 with sodium hydroxide, Pro-MTG was activated by protease from Bacillus licheniformis (Sigma-Aldrich) treatment, which cleaved and removed the pro-sequence. Then, 0.5-1.0% v/w of protease was added, and the MTG concentration was determined using UHPLC (LC-2040C 3D, SHIMADZU CORPORATION). After adjusting the pH to 5.5 with 10% acetic acid, the sample was loaded onto a cation-exchange column RESOURCE™ S 6 mL (Cytiva) equilibrated with 20 mM MES (pH 5.5). After the column was washed with 10-column volumes of the same buffer, MTG was eluted with a linear gradient of sodium chloride from 0 to 500 mM over 10-column volumes at a flow rate of 6 mL/min. Fractions at the top were collected and subjected to buffer exchange. For buffer exchange, samples were subjected to a gel filtration column HiPrep™ 26/10 Desalting (Cytiva) equilibrated with 20 mM sodium phosphate (pH 6.0). Colorimetric hydroxamate assay A colorimetric hydroxamate assay using N-carbobenzoxy-L-glutaminylglycine (Z-QG) was employed to measure the specific MTG activity, as described previously 33 . The reaction solution A comprised 30 mM Z-QG, 100 mM NH 2 OH, 10 mM reduced glutathione, and 50 mM MES (pH 6.0). Reactions were quenched by adding stop solution B (1 N HCl, 4% TCA, and 5% FeCl 3 ・6H 2 O). The assays were performed as follows: A 50 µL aliquot of the enzyme sample was added to 500 µL of the reaction solution A and incubated for 10 min at 37°C. The enzymatic reaction was quenched by adding 500 µL of stop solution B, and the absorbance was measured at 525 nm. One unit of enzyme activity was defined as the formation of 1 µmol of hydroxamic acid/min, using L-glutamic acid γ-monohydroxamate as the standard. Temperature characteristics of MTG The MTG solution (50 µL, 0.05 mg/mL) was added to 500 µL of hydroxamate assay reaction solution A. The solution obtained was reacted for 10 min at 37, 45, 55, 60, 65, 70, 75, and 80゜C. Then, 500 µL of hydroxamate assay stop solution B was added to the reaction mixture, and the amount of the product was measured by recording the absorbance at 525 nm using SpectraMax M2 Microplate Reader (Molecular Devices). To evaluate the thermal stability, the enzyme solution was dissolved in 20 mM MES buffer (pH 6.0) solution to a final concentration of 1.5 U/mL and incubated for 10 min at 50, 60, 65, 70, and 75゜C. After thermal incubation, the enzyme solution was quickly transferred to an ice, and the enzyme activity was subsequently measured. Kinetic measurement of MTG and their mutants by GDH-coupled enzyme assay Kinetic parameters of MTG were determined through a GDH-coupled enzyme assay 34 . The reaction solution consisted of 200 mM MOPS-NaOH buffer (pH 7.2), 10 mM α-ketoglutarate, 1 mM EDTA, 0.5 mM NADH, varying concentrations of Z-QG as the acyl-donor substrate (0, 0.63, 2.5, 10 and 40 mM), 10 mM PEG-NH 2 (SUNBRIGHT MEPA-20H, NOF Corporation, Tokyo, Japan) as an acyl-acceptor substrate, 20 U/mL of glutamate dehydrogenase (Sigma-Aldrich, G7882), and 10 µg/mL MTG. The assays were performed as follows: A 20 µL aliquot of purified enzyme sample was added to 180 µL of reaction solution. The enzyme assay was carried out at 37°C in a 96-well plate, and the decline in the absorbance at 340 nm by oxidation of NADH was monitored using SpectraMax M2 Microplate Reader (Molecular Devices). Substrate reactivity of the enzyme To measure the reactivity with various proteins, a 50 mM MES buffer (pH 6.0) was prepared containing 2% (w/v) of sodium caseinate (Sigma-Aldrich), α- lactalbumin (Sigma-Aldrich), fish gelatin (Sigma-Aldrich), and soy protein isolate (Merck). The MTG solution (10 µL, 2 U/mL) was added to 100 µL of each protein solution and incubated for 1 h at 37゜C. Reaction was quenched by adding 110 µL of 12% TCA, and the ammonia was measured using a LabAssay™ Ammonia (FUJIFILM Wako Pure Chemical Corporation). Abbreviations GDH Glutamate dehydrogenase MTG microbial transglutaminase TG transglutaminase S-S disulfide Declarations Declaration of Competing Interest K. Takahashi and T. Ono have competing interests related to this research. The details of the competing interests are as follows: WO/2022/071061. Declarations Competing Interests K. Takahashi and T. Ono have competing interests related to this research. The details of the competing interests are as follows: WO/2022/071061. Funding This work was supported by Ajinomoto Co., Inc. Author Contribution All authors contributed to the conception and design of the study. T.O. and K.T. designed the mutants, planned and performed the experiments. The manuscript was written by T.O. and K.T. All authors reviewed and approved the final manuscript. Acknowledgement We received a lot of advice from K. Yokoyama, T. Kashiwagi, N. Miwa and M. Date about the content of past research on MTG. We are grateful to Y. Matsui and K. Takei for technical assistance with the experiments. References Ikura, K. et al. Amino acid sequence of guinea pig liver transglutaminase from its cDNA sequence. Biochemistry 27 , 2898–2905. 10.1021/bi00408a035 (1988). Folk, J. E. & Transglutaminases Annu. Rev. Biochem. 49 , 517–531, doi: 10.1146/annurev.bi.49.070180.002505 (1980). Duarte, L., Matte, C. R., Bizarro, C. V. & Ayub, M. A. Z. 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M., Adzhigirey, M., Day, T., Annabhimoju, R. & Sherman, W. Protein and ligand preparation: parameters, protocols, and influence on virtual screening enrichments. J. Comput. Aided Mol. Des. 27 , 221–234. 10.1007/s10822-013-9644-8 (2013). Salam, N. K., Adzhigirey, M., Sherman, W. & Pearlman, D. A. Structure-based approach to the prediction of disulfide bonds in proteins. Protein Eng. Des. Sel. 27 , 365–374. 10.1093/protein/gzu017 (2014). Date, M., Yokoyama, K., Umezawa, Y., Matsui, H. & Kikuchi, Y. High level expression of Streptomyces mobaraensis transglutaminase in Corynebacterium glutamicum using a chimeric pro-region from Streptomyces cinnamoneus transglutaminase. J. Biotechnol. 110 , 219–226. 10.1016/j.jbiotec.2004.02.011 (2004). Kikuchi, Y., Date, M., Yokoyama, K., Umezawa, Y. & Matsui, H. Secretion of active-form Streptoverticillium mobaraense transglutaminase by Corynebacterium glutamicum: processing of the pro-transglutaminase by a cosecreted subtilisin-Like protease from Streptomyces albogriseolus. Appl. Environ. Microbiol. 69 , 358–366. 10.1128/AEM.69.1.358-366.2003 (2003). Yokoyama, K. et al. In vitro refolding process of urea-denatured microbial transglutaminase without pro-peptide sequence. Protein Expr Purif. 26 , 329–335. 10.1016/s1046-5928(02)00536-3 (2002). Oteng-Pabi, S. K. & Keillor, J. W. Continuous enzyme-coupled assay for microbial transglutaminase activity. Anal. Biochem. 441 , 169–173. 10.1016/j.ab.2013.07.014 (2013). Crooks, G. E., Hon, G., Chandonia, J. M. & Brenner, S. E. WebLogo: a sequence logo generator. Genome Res. 14 , 1188–1190. 10.1101/gr.849004 (2004). Additional Declarations Competing interest reported. K. Takahashi and T. Ono have competing interests related to this research. The details of the competing interests are as follows: WO/2022/071061. Other authors declare no competing interests. Supplementary Files SupplementaryInformation.docx SSTGRawData.xlsx FigS1rawdata.jpg Cite Share Download PDF Status: Published Journal Publication published 01 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 11 Apr, 2025 Reviews received at journal 06 Apr, 2025 Reviewers agreed at journal 03 Apr, 2025 Reviews received at journal 29 Jan, 2025 Reviewers agreed at journal 18 Jan, 2025 Reviewers agreed at journal 17 Jan, 2025 Reviewers invited by journal 17 Jan, 2025 Editor assigned by journal 17 Jan, 2025 Editor invited by journal 17 Jan, 2025 Submission checks completed at journal 17 Jan, 2025 First submitted to journal 06 Jan, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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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-5776787","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":408505446,"identity":"b58cfa2c-ded0-43f5-af98-8d59ee20e28a","order_by":0,"name":"Takuto Ono","email":"","orcid":"","institution":"Ajinomoto Co., Inc","correspondingAuthor":false,"prefix":"","firstName":"Takuto","middleName":"","lastName":"Ono","suffix":""},{"id":408505450,"identity":"4eca9211-de69-45b8-bba4-6829fcdcc5c8","order_by":1,"name":"Kazutoshi Takahashi","email":"data:image/png;base64,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","orcid":"","institution":"Ajinomoto Co., Inc","correspondingAuthor":true,"prefix":"","firstName":"Kazutoshi","middleName":"","lastName":"Takahashi","suffix":""},{"id":408505452,"identity":"1c6ecec5-a1ce-4e44-ae13-9d6240fe957b","order_by":2,"name":"Yoshinori Hirao","email":"","orcid":"","institution":"Ajinomoto Co., Inc","correspondingAuthor":false,"prefix":"","firstName":"Yoshinori","middleName":"","lastName":"Hirao","suffix":""},{"id":408505454,"identity":"a4026927-1e3a-4ed1-9d2b-7ab152cf3fbb","order_by":3,"name":"Yasuhiro Mihara","email":"","orcid":"","institution":"Ajinomoto Co., Inc","correspondingAuthor":false,"prefix":"","firstName":"Yasuhiro","middleName":"","lastName":"Mihara","suffix":""},{"id":408505456,"identity":"dc0c9f01-479f-4e92-bd3d-9a6a6bb308eb","order_by":4,"name":"Isao Abe","email":"","orcid":"","institution":"Ajinomoto Co., Inc","correspondingAuthor":false,"prefix":"","firstName":"Isao","middleName":"","lastName":"Abe","suffix":""},{"id":408505457,"identity":"f40d5c94-dc78-462a-a001-e61553c60870","order_by":5,"name":"Masayuki Sugiki","email":"","orcid":"","institution":"Ajinomoto Co., Inc","correspondingAuthor":false,"prefix":"","firstName":"Masayuki","middleName":"","lastName":"Sugiki","suffix":""}],"badges":[],"createdAt":"2025-01-07 00:38:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5776787/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5776787/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-07842-5","type":"published","date":"2025-07-01T15:57:42+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":75945423,"identity":"c118b7d1-464b-495c-bcd0-f0fb7d878709","added_by":"auto","created_at":"2025-02-10 20:26:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":366387,"visible":true,"origin":"","legend":"\u003cp\u003eSequence and structure of MTG mutants.\u003c/p\u003e\n\u003cp\u003eA) The structure of MTG. Left：α-helix domain 1 is shown in blue, β-sheet domain in green, and α-helix domain 2 in red. Right: Colored according to B-factor values. Warmer colors indicate higher B-factor, while cooler colors indicate lower B-factor.\u003c/p\u003e\n\u003cp\u003eB) The sequence of MTG. α-helix domain 1 is shown in blue, β-sheet domain in green, and α-helix domain 2 in red. The dashed lines indicate combinations of amino acid residues with S-S bonds introduced.\u003c/p\u003e\n\u003cp\u003eC) Structure models of the S-S mutant MTGs. Red color indicates the Cys residue introduced to form an S-S bond.\u003c/p\u003e","description":"","filename":"Screenshot20250210at3.23.45PM.png","url":"https://assets-eu.researchsquare.com/files/rs-5776787/v1/705d8374f6bb4bc2353cd3dd.png"},{"id":75945425,"identity":"60fa94e6-fa96-4f5a-b956-c410e93950a7","added_by":"auto","created_at":"2025-02-10 20:26:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":261832,"visible":true,"origin":"","legend":"\u003cp\u003eThermal stability and temperature dependence of MTG mutants.\u003c/p\u003e\n\u003cp\u003eA) Residual activity of each mutant after thermal treatment (n = 3). Error bars indicate standard errors.\u003c/p\u003e\n\u003cp\u003eB) Specific activity of each mutant measured at each temperature (n = 3). Error bars indicate standard errors.\u003c/p\u003e\n\u003cp\u003eC) Relative activity of each mutant measured at each temperature (n = 3). Error bars indicate standard errors.\u003c/p\u003e","description":"","filename":"Screenshot20250210at3.24.30PM.png","url":"https://assets-eu.researchsquare.com/files/rs-5776787/v1/c86025a79924529355050ff2.png"},{"id":75945578,"identity":"01e46c16-f486-49a7-97c8-09cde1c790ef","added_by":"auto","created_at":"2025-02-10 20:34:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":278566,"visible":true,"origin":"","legend":"\u003cp\u003eProtein substrate reactivity of MTG S-S mutants.\u003c/p\u003e\n\u003cp\u003eThe activity after incubation at 37°C for 60 min (n = 3). Error bars indicate standard errors.\u003c/p\u003e","description":"","filename":"Screenshot20250210at3.24.56PM.png","url":"https://assets-eu.researchsquare.com/files/rs-5776787/v1/cf5fd336813e26a3e14d5ff5.png"},{"id":86179026,"identity":"625339da-0e9c-41fe-b707-da31163f7e45","added_by":"auto","created_at":"2025-07-07 16:14:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1604353,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5776787/v1/57309ff6-7450-47f1-a4a1-73995f1f1c15.pdf"},{"id":75945426,"identity":"3cd794b3-47c3-4eda-809d-30a633f7b0a9","added_by":"auto","created_at":"2025-02-10 20:26:26","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":798480,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-5776787/v1/886076df751193b10baa66c4.docx"},{"id":75945434,"identity":"e020cde5-4b80-4064-b8ed-4343425950a9","added_by":"auto","created_at":"2025-02-10 20:26:26","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":341726,"visible":true,"origin":"","legend":"","description":"","filename":"SSTGRawData.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5776787/v1/4e9231483ed581e62f06cb2b.xlsx"},{"id":75945432,"identity":"bdbca717-6afe-4e8a-a66c-65e42dc35382","added_by":"auto","created_at":"2025-02-10 20:26:26","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":317059,"visible":true,"origin":"","legend":"","description":"","filename":"FigS1rawdata.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5776787/v1/dd88c5474daacb743f24d110.jpg"}],"financialInterests":"Competing interest reported. K. Takahashi and T. Ono have competing interests related to this research. The details of the competing interests are as follows: WO/2022/071061. Other authors declare no competing interests.","formattedTitle":"Introduction of multiple disulfide bonds increases the thermostability of transglutaminase","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTransglutaminase (TG: protein-glutamine γ-glutamyltransferases, EC 2.3.2.13) is a family of enzymes that catalyzes the formation of covalent bonds between the γ-carboxyamide group of the glutamine residue and the ε-amino group of the lysine residue in a peptide or protein, leading to the cross-linking of the ε-(γ-glutamyl) lysine bridge\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. TG is widely distributed in various mammalian cells and tissues, and its physiological characteristics have been studied previously\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The enzymatic activity of TG is crucial in various biological processes, including blood clotting, skin formation, and wound healing\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. TG in mammals is calcium-dependent; however, \u003cem\u003eStreptomyces mobaraensis\u003c/em\u003e TG is calcium independent\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Compared to TG derived from mammalian cells, microbial TG (MTG) has a shorter amino acid sequence, lower homology, and a different three-dimensional structure\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.MTG is expressed in a form where the pro-sequence is combined (Pro-MTG), which matures after being cleaved by a protease\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. The pro-sequence reportedly promotes efficient protein folding and secretion, and suppresses its enzymatic activity\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Since MTG is easy to purify, it is being used in various research and industrial applications, replacing TG obtained from animal tissues and organs\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Specifically, MTG has significant applications in the food, pharmaceutical, and textile industries owing to their ability to modify protein structures and enhance the functional properties of proteins\u003csup\u003e\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe thermal stability of an enzyme determines its applicability in industrial processes that often require high temperatures. Enzymes with higher thermal stability can maintain their activity over a broader range of temperatures, showing their versatility and cost-effectiveness for industrial applications. Several methods use protein engineering techniques to identify thermostable enzymes. Notably, various proteins have been stabilized through random mutagenesis, directed evolution, and rational mutagenesis using the three-dimensional structure. Studies have been conducted to enhance the thermal stability of MTG. For example, studies have reported multiple mutants obtained through random mutagenesis, DNA shuffling, and saturation mutagenesis\u003csup\u003e\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. In addition, rational modification of flexible regions (S2P-S23V-Y24N-E28T-S116A-S179L-S199A-A265P-A287P-K294L) has produced a thermostable MTG mutant, TGm2A\u003csup\u003e\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Moreover, introducing disulfide (S-S) bonds in the N-terminal region can significantly enhance the thermal stability of proteins. For instance, the D3C/G283C or T7C/E58C mutant of MTG reportedly improved thermal stability compared to the wild-type (WT)\u003csup\u003e\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. However, the introduction of S-S bonds in other regions was considered ineffective\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBy systematically introducing S-S bonds into different regions of MTG, we aim to identify the most effective strategies for enhancing its thermal stability. We hypothesize that introducing S-S bonds into regions of this enzyme that are inherently less thermally stable will significantly improve their overall thermal stability. Therefore, the current study aims to evaluate how the introduction of an additional S-S bond to the D3C/G283C MTG mutant will impact its thermal stability. Moreover, this strategy might provide insights into improving the thermal stability of other industrial enzymes.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eIn silico\u003c/span\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003emutant design\u003c/span\u003e\u003c/p\u003e \u003cp\u003eThe three-dimensional structure of MTG is formed from three domains, namely, the α-helix domain 1 starting from the N-terminus, β-sheet domain, and α-helix domain 2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Previous research has shown that the S2C/G283C, D3C/G283C, or T7C/E59C mutations introduced into the α-helix domain 1 improved thermal stability\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Conversely, it has been found that the E93C/V112C and A106C/D213C mutations introduced in the α-helix domain 2 did not improve thermal stability\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Our results identified α-helix 1 as the weakest region in MTG, emphasizing the need to strengthen this region. In other words, we suggest that strengthening α-helix 2 without strengthening α-helix 1 would not contribute to thermal stability because α-helix 1 would unfold. Furthermore, introducing two S-S bonds into α-helix 1 would have no effect because a single S-S bond in α-helix 1 sufficiently enhances thermal stability. Therefore, we first strengthened the α-helix domain 1 using D3C/G283C and introduced S-S bonds into the β-sheet domain or α-helix domain 2. To introduce S-S bonds into the protein, it is necessary to mutate the amino acid residue pairs to cysteines. These amino acid residue pairs were predicted using the crystal structure of MTG and a computer program and then verified experimentally. First, we calculated the relative positions of all the amino acid pairs in MTG and extracted those in which the distance between Cβ is within 2\u0026Aring;. We then removed the mutants that were expected to cause significant steric hindrance due to mutation to Cys residues based on the energy values. The introduction of S-S bonds to confer thermal stability was achieved by rigidifying the protein structure. Therefore, mutations to nearby residues and mutations that loop the base of the hairpin structure were removed from the candidate sequence. This occurred because we assumed that they only made the protein locally rigid, and the effect of thermal stability on the entire protein would not be significant. In addition, the hairpin, which contains residues 239\u0026ndash;254 and forms a pocket for the active residue Cys64, has a high B-factor and is highly flexible. However, we assumed that fixing the catalytic pocket would reduce enzyme activity and narrow substrate recognition. Hence, we did not introduce S-S bonds to these sites. Finally, five additional mutations were each introduced into the D3C/G283C mutant and evaluated. These five mutants were A81C/V311C, E93C/V112C, A106C/D213C, E107C/Y217C, and A160C/G228C, and the positions of the mutations in the sequence and structure are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC.\u003c/p\u003e \u003cp\u003eIn this study, the S-S bonds that showed improved thermal stability are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The distance between each S-S bond was 3.3 and 4.4 \u0026Aring;, and the dihedral angle values varied. The secondary structure was effective when introduced into α-helix, β-sheet, and coil; however, introducing it into α-helix/α-helix was better. The B-factor was high in some cases and low in others. Overall, it is assumed that introducing a mutation into a place with a high B-factor is better; however, it is not applicable when introducing a S-S bond into MTG.\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\u003eFeature of S-S bond that provides thermostability\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=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" 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=\"char\" char=\".\" 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\" colname=\"c2\"\u003e \u003cp\u003eDistance(Cβ)༈\u0026Aring;༉\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003edihedral(Ca-Cβ-Cβ-Ca)༈\u0026deg;༉\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003esecondary structure\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eB-factor(Cα)༈\u0026Aring;^2༉\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA81C/V311C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-38.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ecoil/beta-sheet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e10.04/18.34\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE93C/V112C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-163.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ealpha/alpha\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e35.86/19.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA106C/D213C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-144.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ealpha/alpha\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e33.55/34.48\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE107C/Y217C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-84.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ealpha/alpha\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e26.13/24.34\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA160C/G228C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e160.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ecoil/coil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.46/12.94\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of optimal temperature and thermostability of MTG S-S mutants\u003c/h2\u003e \u003cp\u003eDesigned mutants were expressed, purified (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) and their enzyme activity was evaluated. To assess the thermostability of the WT MTG and various S-S mutants, the purified proteins were incubated at 37, 45, 55, 60, 65, 70, 75, and 80゜C for 10 min before analyzing their enzyme activity. MTG activity was measured using a hydroxamate assay. Among the S-S bond-introduced mutants in the WT, only the D3C/G283C mutant exhibited improved thermal stability (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Conversely, there was no change in the optimal temperature for the other S-S bond-introducing mutants (A81C/V311C, E93C/V112C, A106C/D213C, E107C/Y217C, and A160C/G228C). However, when additional S-S bonds were introduced into the D3C/G283C mutant rather than the WT, an improvement in thermal stability was observed compared to D3C/G283C alone. To evaluate the optimal temperature of the WT MTG and the various S-S mutants, MTG activity was measured using a hydroxamate assay. As predicted from the thermal stability results following the introduction of S-S bonds into the WT, only the D3C/G283C mutant exhibited an increase in the optimum temperature from 55 to 60\u0026deg;C. In contrast, no change in the optimum temperature was observed in other S-S bond-introduced mutants (A81C/V311C, E93C/V112C, A106C/D213C, E107C/Y217C, and A160C/G228C) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). However, when additional S-S bonds were introduced into the D3C/G283C mutant, an improvement in activity under conditions above 65\u0026deg;C was observed compared to D3C/G283C alone.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCharacterization of the enzyme kinetics of S-S mutants\u003c/h3\u003e\n\u003cp\u003eThe enzyme kinetics of the WT MTG and the various S-S mutants were measured by a glutamate dehydrogenase (GDH)-coupled enzyme assay. The enzymatic kinetics of each S-S mutant were similar to those of the WT MTG. A81C/V311C showed a decreased \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e, D3C/G283C showed a slightly decreased \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e, whereas E93C/V112C, A106C/D213C, E107C/Y217C, and A160C/G228C showed 5\u0026ndash;21% higher \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e than wild-type MTG, and thus 71\u0026ndash;88% reduced \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ecat\u003c/em\u003e\u003c/sub\u003e/ \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e values (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The effects on \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ecat\u003c/em\u003e\u003c/sub\u003e by combining S-S bonds were minimal. These results indicate that the introduction of S-S bonds makes it possible to acquire an enzyme with enhanced thermal stability while maintaining comparable catalytic activity.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eKinetic constant of MTG mutants for the GDH-coupled enzyme assay\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\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ecat\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ecat\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e/K\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003emM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026micro;mol/min/mg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eM\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e409\u0026thinsp;\u0026plusmn;\u0026thinsp;69\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA81C/V311C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e537\u0026thinsp;\u0026plusmn;\u0026thinsp;7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE93C/V112C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e302\u0026thinsp;\u0026plusmn;\u0026thinsp;21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA106C/D213C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e360\u0026thinsp;\u0026plusmn;\u0026thinsp;28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE107C/Y217C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e294\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA160C/G228C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e292\u0026thinsp;\u0026plusmn;\u0026thinsp;16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD3C/G283C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e382\u0026thinsp;\u0026plusmn;\u0026thinsp;26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD3C/G283C/A81C/V311C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e659\u0026thinsp;\u0026plusmn;\u0026thinsp;29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD3C/G283C/E93C/V112C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e385\u0026thinsp;\u0026plusmn;\u0026thinsp;24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD3C/G283C/A106C/D213C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e466\u0026thinsp;\u0026plusmn;\u0026thinsp;24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD3C/G283C/E107C/Y217C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e424\u0026thinsp;\u0026plusmn;\u0026thinsp;8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD3C/G283C/A160C/G228C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e438\u0026thinsp;\u0026plusmn;\u0026thinsp;36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eData are expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (n\u0026thinsp;=\u0026thinsp;3)\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eEvaluation of protein substrate reactivity\u003c/h3\u003e\n\u003cp\u003eThe reactivity of MTG and S-S mutants with various proteins was measured. To investigate the possibility of application to foods, the reactivity of MTG to sodium caseinate, fish gelatin, soy protein isolate, and α-lactalbumin was evaluated. We measured the amounts of ammonia released after MTG was added to 2% various protein solutions and incubated for 1 h at 37゜C. Although variations in reactivity were observed depending on the protein substrate type, no substantial differences in reactivity were noted between each mutant and the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The introduction of S-S bonds did not affect the reactivity toward polymer substrates. This indicates that it is possible to create industrially useful enzymes with enhanced thermal stability, comparable catalytic activity, and unchanged reactivity towards polymer substrates.\u003c/p\u003e\n\u003ch3\u003eSelection of effective sites for introducing S-S bonds utilizing sequence information\u003c/h3\u003e\n\u003cp\u003eTo verify the possibility of predicting effective sites for S-S bond introduction based on sequence information, we analyzed the evolutionary conservation of the sites where S-S bonds were introduced. Using 406 sequences of MTG homologs, the amino acid conservation at the mutation sites was analyzed. Regardless of the conservation level of the surrounding sequences, the results showed that mutants with improved and decreased thermal stability were observed (Supplementary Fig. S2).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe introduction of S-S bonds is a simple and effective method for improving thermal stability without requiring multiple amino acid substitutions. In this study, we investigated further improvements in thermal stability using a MTG mutant by introducing the most important S-S bond (D3C/G283C). Although thermal stability improvements through random mutagenesis have been reported\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, we successfully created a MTG mutant with a higher thermal stability than those in previous studies\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. We achieved this by introducing additional S-S bonds as a new approach. It is difficult to predict the appropriate site for introducing S-S bonds from sequence information containing evolutionary information. Previously, it was essential to utilize experimental structural information; however, the introduction of AlphaFold2 or RoseTTAfold reduced this limitation\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFrom the S-S bonds mutants that has acquired thermal stability, it was found that the thermal stability was improved in the five mutants in which D3C/G283C on the α-helix domain 1 was strengthened and added to the β-sheet domain by S-S bonding. Furthermore, we confirmed that introducing a S-S bond to the β-sheet domain without introducing it to the α-helix domain 1 did not improve thermal stability. This indicates that the stability of the entire protein cannot be improved unless the weakest region of the protein is strengthened alongside others. Furthermore, the thermal stability of MTG was improved only when the S-S mutation was introduced into the β-sheet domain after the α-helix domain 1, which is the weakest part, was strengthened. This suggested that the β-sheet domain is the next weakest region of MTG. The activity of this heat-resistant enzyme did not reduce, showing its significance. It was generally believed that there was a trade-off between thermal stability and activity\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. However, many unknowns exist, and recent reports have questioned this trade-off\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. There was no trade-off between thermal stability and activity in the mutants obtained in the present study. The strategy of not including S-S in the active pocket has produced good results.\u003c/p\u003e \u003cp\u003eThe results of this study suggest that in order to improve thermal stability without reducing enzyme activity, it is important to introduce S-S bonds in the region that is the weakest in the enzyme and not too close to the active domain. Structural information is essential for this, and obtaining structural information has become much easier with tools such as AlphaFold 2. On the other hand, it is still laborious to investigate the heat-sensitive parts of a protein's structure using instrumental analysis, so it is probably easier to use in silico methods such as Molecular dynamics or to screen a certain number of mutants.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe thermal stability of MTG was improved by introducing S-S bonds. When heated at 65\u0026deg;C for 10 min, the wild-type MTG had no remaining activity. However, the D3C/G283C mutant, which had one S-S bond introduced, had approximately 40% of its activity retained.\u003csup\u003e\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e In this study, a second S-S bond was introduced at a different site to the first S-S bond (A81C/V311C, E93C/V112C, A106C/D213C, E107C/Y217C, or A160C/G283C). These mutants improved their residual activity to approximately 80%. They maintained the same activity as the WT, and the substrate specificity for the protein was also maintained, it can be used in high-temperature range, which was difficult to do with WT MTG, thus enhancing their applicability in the food and medical industries.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eMutants Design\u003c/h2\u003e \u003cp\u003eThe Schr\u0026ouml;dinger software (Schr\u0026ouml;dinger, Inc.) was used for \u003cem\u003ein silico\u003c/em\u003e S-S and site prediction. The three-dimensional structure of MTG was obtained from Protein Data Bank (PDB code 1iu4)\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. First, the missing side chains and protons were added using the Protein Preparation Program, and the structure was optimized\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. The amino acid pairs that were in a position relationship and could form an S-S bond were extracted using the S-S prediction program\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. We used a program\u0026rsquo;s weighted score of 1,000 or less to determine whether the distance and angle were suitable for an S-S bond. Furthermore, we selected five mutants by excluding those located near the catalytic pocket, those with amino acid sequences differing by ten or fewer residues, and those with mutations in the same domain as the D3C/G283C mutant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePlasmids and bacterial strains\u003c/h2\u003e \u003cp\u003e \u003cem\u003eCorynebacterium glutamicum\u003c/em\u003e YDK010 was used as an expression host for MTGs\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. pPSPTG1 is a plasmid for expressing pro-MTG.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e Genes coding for MTG mutants were synthesized by optimizing codon usage for \u003cem\u003eC. glutamicum\u003c/em\u003e (Genescript) and inserted at the KpnI and BamHI sites of pPSPTG1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCulture medium for protein production\u003c/h2\u003e \u003cp\u003e \u003cem\u003eC. glutamicum\u003c/em\u003e was grown at 30\u0026deg;C in the medium (5 g/L glucose, 10 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl, and 0.2 g/L DL-methionine, pH 7.2) containing 25 mg/mL kanamycin (NACALAI TESQUE, INC.). To produce the pro-form MTG mutants, \u003cem\u003eC. glutamicum\u003c/em\u003e was grown at 30\u0026deg;C in the medium (60 g/L glucose, 1 g/L MgSO\u003csub\u003e4\u003c/sub\u003e, 30 g/L (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, 1.5 g/L KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 0.01 g/L FeSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO, 0.01 g/L MnSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO, 450 \u0026micro;g/L biotin, 0.15 g/L DL-methionine, and 50 g/L CaCO\u003csub\u003e3\u003c/sub\u003e, pH 7.5) containing 25 mg/mL kanamycin. To produce MTG mutants containing an S-S bond, 3 mM dithiothreitol was added 5 h after the culture ensued, as described previously\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of protein concentration\u003c/h2\u003e \u003cp\u003eReverse-phase HPLC was performed using Proteonavi C4 column (4.6 mm id \u0026times;15 cm, Osaka Soda) as described previously\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Purified WT MTG was used as a standard. It was purified from the \u003cem\u003eStreptoverticillium\u003c/em\u003e spp. S-8112 supernatant culture, as described previously\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. The concentration of the standard WT MTG was determined using the Bradford assay, with bovine serum albumin (Bio-Rad) serving as the protein standard.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePurification of MTGs\u003c/h2\u003e \u003cp\u003eMTG was purified as described previously\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. In summary, culture supernatant was obtained by removing bacterial cells from the culture solution through centrifugation (10,000 x g, 4゜C, 10 min) and filtration. The supernatant was subjected to buffer exchange using a Sephadex G-25 (M) (Cytiva) column equilibrated with 20 mM MES (pH 5.5). After adjusting the pH to 7.0 with sodium hydroxide, Pro-MTG was activated by protease from \u003cem\u003eBacillus licheniformis\u003c/em\u003e (Sigma-Aldrich) treatment, which cleaved and removed the pro-sequence. Then, 0.5-1.0% v/w of protease was added, and the MTG concentration was determined using UHPLC (LC-2040C 3D, SHIMADZU CORPORATION). After adjusting the pH to 5.5 with 10% acetic acid, the sample was loaded onto a cation-exchange column RESOURCE\u0026trade; S 6 mL (Cytiva) equilibrated with 20 mM MES (pH 5.5). After the column was washed with 10-column volumes of the same buffer, MTG was eluted with a linear gradient of sodium chloride from 0 to 500 mM over 10-column volumes at a flow rate of 6 mL/min. Fractions at the top were collected and subjected to buffer exchange. For buffer exchange, samples were subjected to a gel filtration column HiPrep\u0026trade; 26/10 Desalting (Cytiva) equilibrated with 20 mM sodium phosphate (pH 6.0).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eColorimetric hydroxamate assay\u003c/h2\u003e \u003cp\u003eA colorimetric hydroxamate assay using N-carbobenzoxy-L-glutaminylglycine (Z-QG) was employed to measure the specific MTG activity, as described previously\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. The reaction solution A comprised 30 mM Z-QG, 100 mM NH\u003csub\u003e2\u003c/sub\u003eOH, 10 mM reduced glutathione, and 50 mM MES (pH 6.0). Reactions were quenched by adding stop solution B (1 N HCl, 4% TCA, and 5% FeCl\u003csub\u003e3\u003c/sub\u003e・6H\u003csub\u003e2\u003c/sub\u003eO). The assays were performed as follows: A 50 \u0026micro;L aliquot of the enzyme sample was added to 500 \u0026micro;L of the reaction solution A and incubated for 10 min at 37\u0026deg;C. The enzymatic reaction was quenched by adding 500 \u0026micro;L of stop solution B, and the absorbance was measured at 525 nm. One unit of enzyme activity was defined as the formation of 1 \u0026micro;mol of hydroxamic acid/min, using L-glutamic acid γ-monohydroxamate as the standard.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eTemperature characteristics of MTG\u003c/h2\u003e \u003cp\u003eThe MTG solution (50 \u0026micro;L, 0.05 mg/mL) was added to 500 \u0026micro;L of hydroxamate assay reaction solution A. The solution obtained was reacted for 10 min at 37, 45, 55, 60, 65, 70, 75, and 80゜C. Then, 500 \u0026micro;L of hydroxamate assay stop solution B was added to the reaction mixture, and the amount of the product was measured by recording the absorbance at 525 nm using SpectraMax M2 Microplate Reader (Molecular Devices).\u003c/p\u003e \u003cp\u003eTo evaluate the thermal stability, the enzyme solution was dissolved in 20 mM MES buffer (pH 6.0) solution to a final concentration of 1.5 U/mL and incubated for 10 min at 50, 60, 65, 70, and 75゜C. After thermal incubation, the enzyme solution was quickly transferred to an ice, and the enzyme activity was subsequently measured.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eKinetic measurement of MTG and their mutants by GDH-coupled enzyme assay\u003c/h2\u003e \u003cp\u003eKinetic parameters of MTG were determined through a GDH-coupled enzyme assay\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. The reaction solution consisted of 200 mM MOPS-NaOH buffer (pH 7.2), 10 mM α-ketoglutarate, 1 mM EDTA, 0.5 mM NADH, varying concentrations of Z-QG as the acyl-donor substrate (0, 0.63, 2.5, 10 and 40 mM), 10 mM PEG-NH\u003csub\u003e2\u003c/sub\u003e (SUNBRIGHT MEPA-20H, NOF Corporation, Tokyo, Japan) as an acyl-acceptor substrate, 20 U/mL of glutamate dehydrogenase (Sigma-Aldrich, G7882), and 10 \u0026micro;g/mL MTG. The assays were performed as follows: A 20 \u0026micro;L aliquot of purified enzyme sample was added to 180 \u0026micro;L of reaction solution. The enzyme assay was carried out at 37\u0026deg;C in a 96-well plate, and the decline in the absorbance at 340 nm by oxidation of NADH was monitored using SpectraMax M2 Microplate Reader (Molecular Devices).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eSubstrate reactivity of the enzyme\u003c/h2\u003e \u003cp\u003eTo measure the reactivity with various proteins, a 50 mM MES buffer (pH 6.0) was prepared containing 2% (w/v) of sodium caseinate (Sigma-Aldrich), α- lactalbumin (Sigma-Aldrich), fish gelatin (Sigma-Aldrich), and soy protein isolate (Merck). The MTG solution (10 \u0026micro;L, 2 U/mL) was added to 100 \u0026micro;L of each protein solution and incubated for 1 h at 37゜C. Reaction was quenched by adding 110 \u0026micro;L of 12% TCA, and the ammonia was measured using a LabAssay\u0026trade; Ammonia (FUJIFILM Wako Pure Chemical Corporation).\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGDH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGlutamate dehydrogenase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMTG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emicrobial transglutaminase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etransglutaminase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eS-S\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edisulfide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eDeclaration of Competing Interest\u003c/h2\u003e \u003cp\u003eK. Takahashi and T. Ono have competing interests related to this research. The details of the competing interests are as follows: WO/2022/071061.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eDeclarations\u003c/h2\u003e\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003cp\u003eK. Takahashi and T. Ono have competing interests related to this research. The details of the competing interests are as follows: WO/2022/071061.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by Ajinomoto Co., Inc.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors contributed to the conception and design of the study. T.O. and K.T. designed the mutants, planned and performed the experiments. The manuscript was written by T.O. and K.T. All authors reviewed and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe received a lot of advice from K. Yokoyama, T. Kashiwagi, N. Miwa and M. Date about the content of past research on MTG. We are grateful to Y. Matsui and K. Takei for technical assistance with the experiments.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eIkura, K. et al. Amino acid sequence of guinea pig liver transglutaminase from its cDNA sequence. \u003cem\u003eBiochemistry\u003c/em\u003e \u003cb\u003e27\u003c/b\u003e, 2898\u0026ndash;2905. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1021/bi00408a035\u003c/span\u003e\u003cspan address=\"10.1021/bi00408a035\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1988).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFolk, J. E. \u0026amp; Transglutaminases \u003cem\u003eAnnu. Rev. 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WebLogo: a sequence logo generator. \u003cem\u003eGenome Res.\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e, 1188\u0026ndash;1190. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1101/gr.849004\u003c/span\u003e\u003cspan address=\"10.1101/gr.849004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2004).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Microbial Transglutaminase, disulfide bond, thermal stability, mutant, enzyme kinetics","lastPublishedDoi":"10.21203/rs.3.rs-5776787/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5776787/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMicrobial transglutaminase (MTG) is an enzyme that catalyzes the cross-linking of glutamine and lysine residues in proteins. Because of its ability to modify proteins, MTG has various applications in the medical and food industries. Most studies have aimed to enhance the thermal stability of MTG by focusing only on point mutations. Introducing a disulfide (S-S) bond in the N-terminal region has been found to be effective, whereas S-S bonds in other regions were considered ineffective. Therefore, this study aimed to evaluate the impact of introducing an additional S-S bond on the thermal stability of an MTG mutant. We found that adding S-S bonds to regions other than the N-terminal, in conjunction with the N-terminal S-S bond, significantly enhanced thermal stability. This finding demonstrates the importance of reinforcing the weakest part of the protein first, followed by strengthening other regions for optimal thermal stability. The MTG variant with two S-S bonds retained its catalytic activity and substrate specificity towards protein substrates, making it a promising candidate for industrial applications. Thus, introducing S-S bonds could be an effective strategy to increase thermal stability of MTG and other industrial enzymes, thereby contributing to their potential industrial applications.\u003c/p\u003e","manuscriptTitle":"Introduction of multiple disulfide bonds increases the thermostability of transglutaminase","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-10 20:26:21","doi":"10.21203/rs.3.rs-5776787/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-11T07:30:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-07T02:13:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"24788120924371589253730780488740652655","date":"2025-04-03T23:00:39+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-29T07:46:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"257040963955784438743305802631303946371","date":"2025-01-18T06:25:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"304660326570035660561944222095230679601","date":"2025-01-18T02:15:35+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-01-17T20:16:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-01-17T20:12:59+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-01-17T20:08:50+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-01-17T06:21:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-01-07T00:29:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"baed7426-f52b-4016-a7c1-a7db49d66b36","owner":[],"postedDate":"February 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":43557120,"name":"Biological sciences/Biochemistry/Enzymes"},{"id":43557121,"name":"Biological sciences/Biochemistry/Proteins"}],"tags":[],"updatedAt":"2025-07-07T16:03:00+00:00","versionOfRecord":{"articleIdentity":"rs-5776787","link":"https://doi.org/10.1038/s41598-025-07842-5","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-07-01 15:57:42","publishedOnDateReadable":"July 1st, 2025"},"versionCreatedAt":"2025-02-10 20:26:21","video":"","vorDoi":"10.1038/s41598-025-07842-5","vorDoiUrl":"https://doi.org/10.1038/s41598-025-07842-5","workflowStages":[]},"version":"v1","identity":"rs-5776787","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5776787","identity":"rs-5776787","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}