Enhancing the Stress Resistance of Nitrile Hydratase from Rhodococcus ruber via SpyTag/SpyCatcher-mediated α- and β- subunits ligation

Enhancing the Stress Resistance of Nitrile Hydratase from Rhodococcus ruber via SpyTag/SpyCatcher-mediated α- and β- subunits ligation | 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 Short Report Enhancing the Stress Resistance of Nitrile Hydratase from Rhodococcus ruber via SpyTag/SpyCatcher-mediated α- and β- subunits ligation Miaomiao Wang, Huimin Yu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4315377/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Jul, 2024 Read the published version in Molecular Biology Reports → Version 1 posted 9 You are reading this latest preprint version Abstract Nitrile Hydratase (NHase) is one of the most important industrial enzyme widely used in the petroleum exploitation field. The enzyme, composed of two unrelated α- and β-subunits, catalyzes the conversion of acrylonitrile to acrylamide, releasing a significant amount of heat and generating the organic solvent product, acrylamide. Both the heat and acrylamide solvent have an impact on the structural stability of NHase and its catalytic activity. To improve the thermostability and acrylamide tolerance of NHase, the two subunits were fused in vivo using SpyTag and SpyCatcher, which were attached to the termini of each subunit in various combinations. Analysis of the engineered strains showed that the C-terminus of β-NHase is a better fusion site than the N-terminus, while the C-terminus of α-NHase is the most suitable site for fusion with a larger protein. Fusion of SpyTag and SpyCatcher to the C-terminus of β-NHase and α-NHase, respectively, led to improved acrylamide tolerance and a slight enhancement in the thermostability of one of the engineered strains, NBSt. These results indicate that in vivo ligation of different subunits using SpyTag/SpyCatcher is a valuable strategy for enhancing subunit interaction and improving stress tolerance. Nitrile Hydratase SpyTag/SpyCatcher R. ruber Thermostability Acrylamide resistance Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Nitrile Hydratase (NHase), comprised of α- and β-subuits, is extensively utilized in the industrial production of acrylamide (AM), which is the monomer of polyacrlamide has been sucessfully applied in the petroleum exploitation and water treatment fields [ 1 , 2 ]. In the biotransformation of acrylonitrile (AN) to AM, both AM and AN are harmful organic compounds that can netatively affect the integrity of NHase or NHase-containing biocatalysts [ 3 , 4 ]. Additionally, the enzyme reaction is highly exothermic, leading to momentary thermal stress in microenvionment [ 4 ]. Both high concentrations of toxic compounds and momentary thermal stress can induce conformational changes and/or amino acid damages to intracellular NHase. This inevitably affects the catalytic activity of the enzyme [ 3 , 5 ]. Therefore, improving the thermal stability and tolerance of NHase to toxic substances would be valuable in the petroleum industry. Various strategies, such as introducing salt-bridges into thermal-sensitive regions [ 6 ], assembling enzymes into ordered nanostructures [ 7 ], swapping specific fragment [ 8 ], introducing disulfide bonds [ 9 ] to stabilize the binding between subunits, and introducing isolated or phused chaperons [ 5 , 10 ], have been applied to improve the stability of NHase or to renature the denatured NHase to varying degrees. However, the AM manufacturing bio-industry requires further improved stability of NHase toward thermal or AM stress. The pair SpyTag and SpyCatcher from Streptococcus pyogenes was discovered by Proft et al in 2007 [ 11 ]. By forming an amide bond between the lysine of SpyTag and the asperate acid of SpyCatcher, SpyTag and SpyCatcher become irreversibly ligated [ 12 ], and the resulting ligation product exhibits high chemical stability and resistance to high temperatures [ 13 – 15 ]. The SpyTag/SpyCatcher strategy has been succesfully applied to enhance the mechanical behavior of proteins [ 12 ], obtain nonlinear macromolecules with varying molecular weight and topologies [ 16 ], assemble living biomaterial [ 17 ], and enhance the resilience of enzymes to stress [ 14 ]. Several reports have applied this strategy to improve enzyme tolerance by introducting SpyTag and SpyCatcher to the enzyme through fusion to the N- and C- termini, resulting in cyclization of the enzyme (e.g. β-lactamase [ 14 ], L-phenylserine aldolase [ 18 ], dihydrofolate reductase [ 19 ], firefly luciferase, and lichenase [ 20 ]). In this study, we demonstrated the in vivo ligation of the α- and β-subunits of NHase from Rhodococcus ruber by fusing SpyTag and SpyCatcher, resulting in the generation of several engineered enzymes and strains. These strains were then examined for the effects of the ligation on NHase thermo-stability and acrylamide tolerance. 2. Material and methods 2.1 Reagents The PCR for gene amplification, agarose electrophoresis, restriction enzyme digestion, DNA ligation and DNA transformation were performed following the standard protocols [ 21 ]. Polymerase and ligase were obtained from Vazyme (Nanjing, China). Restriction enzymes were purchased from Takara (Dalian, China). The kits used for genomic DNA isolation, plasmid extraction, and gene retrieval were purchased from Omega Bio-Tek. 2.2 Plasmids, strains and gene manipulation The strains and plasmids used in this study were listed in Table 1 . R. ruber TH3 [ 22 ] was constructed by knocking out the amidase gene from R. ruber TH and deposited in the China General Microbiological Culture Collection Center (CGMCC No. 2381). R. ruber TH8 [ 9 ] was contructed by overexpressing the mutant NHase gene using plasmid in R. ruber TH3. All the R. ruber strains were grown at 28 ℃ in fermentation medium in previously study [ 5 ]. E. coli TOP10 and the derivatives were routinely grown at 37 o C in Luria–Bertani (LB) medium. Antibiotics were added as the following concentrations: kanamycin, 30 µg/mL and tetracycline, 15 µg/mL. Table 1 The strains and plasmids used in this study Strains, plasmids Genotype or sequence Reference or source Strains E. coli TOP 10 Applied for harvesting plasmid Solarbio (Beijing, China) R. ruber TH3 amiE-mutant (amiE::pPHU281, Tc r ) [ 22 ] R. ruber TH8 R. ruber TH3 carrying plasmid pNV-Pa2-NHase M [ 9 ] NBSt R. ruber TH3 carrying plasmid pNV-Pa2-NBSt This study StNB R. ruber TH3 carrying plasmid pNV-Pa2-StNB This study NASt R. ruber TH3 carrying plasmid pNV-Pa2-NASt This study StNA R. ruber TH3 carrying plasmid pNV-Pa2-StNA This study Plasmids pNV-Pa2 ColE1 ori, Kana r , Pa2 promoter [ 23 ] pNV-Pa2-NBSt β-NHase-SpyTag and α-NHase-SpyCatcher fusion-expression This study pNV-Pa2-StNB SpyTag-β-NHase and α-NHase-SpyCatcher fusion-expression This study pNV-Pa2-NASt β-NHase-SpyCatcher and α-NHase-SpyTag fusion-expression This study pNV-Pa2-StNA β-NHase-SpyCatcher and SpyTag-α-NHase fusion-expression This study 2.3 Construction of the engineered R. ruber NBSt, StNB, NASt, StNA The primers used in this study were listed in Supplementary Table S1 . The plasmid pNV-Pa2 was devised from pNV18.1 by substituting the original promoter with Pa2 [ 23 ]. The original sequences of SpyCatcher and SpyTag were published on NCBI website ( https://www.ncbi.nlm.nih.gov/ ), the Genebank number of SpyCatcher is JQ478411, and the PDB number of SpyTag is 4MLI_B. The codon-optimized spycatcher gene and spytag gene were synthesized by GeneWiz (Suzhou, China). The mutant NHase gene (α-, β-NHase subunits) was amplied from the pNV-SBMDB contained in R. ruber TH8 [ 9 ] by the primers NB-F/R and NA-F/R, respectively, for the preparation of phusion products. The GS linker was added between spytag/spycatcher and α-,β- subunits of NHase. The overlapped fragments β-NHase-SpyTag (NBSt), α-NHase-SpyCatcher (NASc), SpyTag-β-NHase (StNB), β-NHase-SpyCatcher (NBSc), α-NHase-SpyTag (NASt)and SpyTag-α-NHase (StNA) were amplified using the primers listed in Supplementary Table S2, digested and ligated into pNV-Pa2 to construct the plasmids pNV-Pa2-NBSt, pNV-Pa2-StNB, pNV-Pa2-NASt, pNV-Pa2-StNA, respectively. Take the construction of pNV-Pa2-NBSt as example, this plasmid contains two phusion genes, NBSt and NASc. The spytag (St) fragment was fused to the C- terminus of β-NHase (NB) by PCR using the primers NBSt-F and NBSt-R to make the NBSt, and the sequence was added in the primer NBSt-R. The spycatcher (Sc) fragment was fused to the C- terminus of α-NHase (NA) by the common GS linker (GSGGSGG) via overlap PCR to make the fragment NASc. And then, the gene NBSt digested by Xba I/ Bam H I and NASc digested by Bam H I/ Xba I were ligated into the plasmid pNV-Pa2 digested by Xba I/ Kpn I to construct the plasmid pNV-Pa2-NBSt. Similarly, the plasmids pNV-Pa2-NBSt, pNV-Pa2-StNB, pNV-Pa2-NASt, pNV-Pa2-StNA were constructed. And then all these plasmids were elec-transformed into R. ruber TH3 and spread onto solid plates containing 30 ug/mL kanamycin to obtain the engineered R. ruber strains: NBSt, StNB, NASt and StNA (Fig. 1 B), respectively. Gene sequencing was confirmed by GeneWiz (Tianjin, China). 2.4 Protein SDS-PAGE The strains R. ruber TH8, NBSt, StNB, NASt and StNA were harvested after cultured in fermentation medium for 48 h, washed twice by 10 mM PBS, resuspended in PBS (final OD 400 = 30) and disrupted by ultrasonication (240 W, 6×4×180 times). The supernatant was collected by centrifugation (12,000 rpm, 10 min, 4 ℃), mixed with 5× protein loading buffer and boiled for 5 min. SDS-PAGE electrophoresis was performed with a 12% ExpressPlus™ page gel (GenScript, Piscataway, USA). After that, the gel was stained with coomassie brilliant blue. 2.5 NHase activity assay NHase activity of R. ruber strains were determined as previously described by Ma et al [ 22 ]. After cultured in fermentation medium for 48 h, the R. ruber cells were harvested by centrifugation (12,000 rpm, 5 min) and resuspended in 10 mM PBS buffer (final OD 460 = 50) to be measured. The NHase activity was determined in a 5 mL catalytic reaction contained 200 µL of acrylonitrile, 4.5 mL PBS buffer (10 mM) and 100 µL resuspended cells liquid. The mixture was incubated at 28℃ for 5 min, and the reaction was terminated with 200 µL 2.5 M HCl. The supernatant was collected by centrifugation (12,000 rpm, 10 min) and then analyzed by gas chromatography (GC). Experiments were done in triplicate. 2.6 Thermal stability The thermal stability of NHase was determined by comparing the NHase activity of NHase-expression strains with or without heat shocked (60 ℃, 10 min). Experiments were done in triplicate. 2.7 AM tolerance After cultured in fermentation medium for 48 h, the R. ruber cells were harvested by centrifugation (12,000 rpm, 5 min). The PBS and 60% AM solution were added to the collected cells to make the samples with the same cell density (OD 460 = 50) and different AM concentration, 30%, 40% and 50%. The volume of added PBS and 60% AM solution was shown in Supplementary Table S2. And then, the mixture was incubated at room temperature for 20 min. After that, the cells were immediately collected, washed, resuspended and measured the NHase activity by GC. Experiments were done in triplicate. 2.8 Determination of kinetic parameters Kinetic parameters of original and ligated NHase were estimated by measuring the initial activity of the recombinant cells at 28°C with a substrate concentration range of 0.05 ~ 0.6 M, respectively. K m and V max were calculated using the double reciprocal Lineweaver-Burk plot method. 3. Results and Discussion 3.1 Strategy of ligating the α- and β-NHase subunits by SpyTag/Spycatcher NHase, which catalyzes the formation of AM, is one of the most important industrial enzymes[ 1 , 22 ]. The stress, heat release, and solvent immerse present in this reaction can cause conformational changes or damage to the amino acids of NHase. Therefore, enhancing thermostability or solvent tolerance is crucial [ 5 , 10 ]. In this study, the mutant NHase gene [ 9 ], containing a disulfide bond to stabilize the binding between subunits and exhibiting better thermal stability and AM tolerance than the natural gene, was selected as the parent gene. To further enhance the interaction between the α- and β-NHase subunits, SpyTag and SpyCatcher were fused to the N- and C- termini of the α- and β-NHase subunits in different combinations. They were then overexpressed using the R. ruber - E.coli shuttle plasmid pNV-Pa2 [ 23 ] to obtain the engineered R. ruber strains NBSt, StNB, NASt and StNA (Fig. 1 ). NBSt and StNB were generated by fusing SpyCatcher to the C-terminus of α-NHase, and then SpyTag was fused to either the N- or C-terminus, respectively, of β-NHase. NASt and StNA were generated by fusing SpyCatcher to the C-terminus of β-NHase, and then SpyTag was fused to either the N- or C-terminus, respectively, of α-NHase. Meanwhile, R. ruber TH8 [ 9 ] engineered by overexpressing the template NHase gene in R. ruber TH3 [ 22 ], served as the control. 3.2 Verification of the ligation conformation of α- and β-NHase subunits Parallel cultures of the engineered R. ruber NBSt, StNB, NASt, StNA, and the control strain R. ruber TH8 were cultivated in shake flasks to assess the NHase expression via SDS-PAGE (Fig. 2 ). The protein sample from TH8 was separated, and the α- and β-NHase subunits were identified after SDS-PAGE; their known molecular weights are 22.8 and 26.4 kDa, respectively. The ligation of the α- and β-NHase subunits by SpyTag/SpyCatcher fusion in all engineered strains was successful, as indicated by the band at ~ 60 kDa, even after sample boiling. The molecular weights were determined using AAT Bioquest ( https://www.aatbio.com/tools/calculate-peptide-and-protein-molecular-weight-mw ); the molecular weights of SpyTag and SpyCatcher are 1.5 and 12.3 kDa, respectively. This experiment further demonstrated the successful application of SpyTag/SpyCatcher. It showed that the strategy of cyclizing two termini of one enzyme or binding two different subunits by introducing SpyTag/SpyCatcher is universal, as supported by other literature reports [ 12 , 15 , 18 , 20 , 24 ]. The percentage of total protein represented by ligated NHase was calculated using Gel-Pro Analyzer software and varied based on the ligation strategy: 9.5%, 9.6%, 6.4% and 8.2% for NBSt, StNB, NASt and StNA, respectively. Additionally, SDS-PAGE analysis revealed a single subunit NHase band in all engineered strains, attributed to the native NHase encoded by the genome of the host strain TH3 [ 22 ]. 3.3 Effect of introduced SpyTag/SpyCatcher on NHase activity The biomass and NHase activity of the engineered R. ruber strains NBSt, StNB, NASt, StNA, and the control R. ruber TH8 were measured and compared in Fig. 3 . SpyTag/SpyCatcher ligation did not reduce the biomass of NHase (Fig. 3 A), but it did reduce NHase activity (Fig. 3 B). Previous reports have also documented a reduction in enzyme activity caused by SpyTag/SpyCatcher-mediated cyclization of lichenase [ 20 ]. NHase activity decreased from 15.4–37.9% depending on the fusion strategy. R. ruber strain NBSt, engineered by fusing SpyTag to the C-terminus of β-NHase and fusing SpyCatcher to the C-terminus of α-NHase, exhibited the highest activity at 2801.7 U/mL, representing a 15.4% decrease compared to the TH8 control. The only difference in fusion strategy between NBSt and StNB was the β-NHase terminus fused to SpyTag; the C-terminus was fused in NBSt, whereas the N-terminus was fused in StNB. The different fusion termini led to similar ligated-NHase biomass, but NHase activity was further reduced to 2057 U/mL in StNB, a decrease of 37.9% compared to the control. This is in accordance with previous studies showing that decreasing NHase activity occurred by introducing salt bridges [ 1 , 6 ] or reducing NHase solubility by inserting assembly peptides [ 25 ] at the N-terminus of β-NHase. The fusion of SpyCatcher to the C-terminus of β-NHase in the NASt engineered strain also resulted in reduced NHase activity, 2154 U/mL, relative to the control. This activity was similar to that of StNA (2213 U/mL), in which SpyTag was fused to the N- terminus of α-NHase. Comparing NBSt and NASt, NHase activity was higher in NBSt, suggesting that the larger SpyCatcher protein should be fused to the α-NHase subunit rather than the β-subunit [ 6 , 10 ]. Based on these results, we conclude that the C-terminus of β-NHase is a better fusion site than the N-terminus, the C-terminus of α-NHase is more suitable for fusion with a larger protein. fusion of a peptide to the N- or C-terminus of α-NHase yields similar results, and the N-terminus of β-NHase is more sensitive to peptide fusion than the N-terminus of α-NHase. 3.4 Stress resistance of ligated-NHase The stress resistance (thermostability and solvent tolerant) of the different ligated-NHase strains and the original NHase was tested using TH8, NBSt, StNB, NASt, and StNA cells (Fig. 4 ). As shown in Fig. 4 A, the control TH8 cells retained 62.2% of their NHase activity after incubating at 60 ℃ for 10 min. The residual NHase activity of the engineered strains was 63.2%, 14.4%, 34.6%, and 20.3% in NBSt, StNB, NASt, and StNA, respectively. Among all the engineered strains, NBSt exhibited superior thermal-tolerance. The 9.5% ligated-NHase mediated by SpyTag/SpyCatcher in NBSt showed a 1% increase in heat shock resistance. By fusing SpyTag and SpyCatcher to the C-terminus of β- and α-NHase, respectively, NHase thermostability was improved, but the effect was not significant. The results obtained here show little enhanced thermostability achieved by introducing SpyTag/SpyCatcher-mediated enzyme ligation or cyclization, similar to the situation of cyclizated l-phenylalanine aldolase caused by SpyTag/SpyCatcher [ 18 ]. Additionally, a few studies have reported that introducing SpyTag/SpyCatcher cyclization did enhance the thermostability of Lichenase [ 20 ], Firefly Luciferase [ 26 ], β-lactamase [ 27 ], and phytase [ 28 ]. This suggests that the strategy of cyclizing one enzyme or ligating two subunits to fix the structure or conformation of the enzyme to enhance thermostability is not universal, and this needs to be examined in each specific case. Thermostability of all mutants assessedbefore and after heat shock treatment at 60 ℃ for 10 min(A), and the solvent tolerance of all mutants evaluated after immersion in 30, 40, and 50% acrylamide (AM) solutions for 20 min (B). Industrially, different applications require the production of 30%, 40% and 50% AM solutions. Thus, was assessed the effect of these AM concentrations on ligated-NHase activity by immersing the respective cells for 20 min (Fig. 4 B). NHase activity decreased as the AM concentration increased in both the control and all engineered strains. The engineered strain NBSt exhibited better AM tolerance than the other engineered strains. Upon immersion of the cells in 30%, 40%, and 50% AM solution, approximately 40.7%, 32.8% and 22.1% of the NHase activity remained in the control cells, while 48.2%, 38.6% and 24.1% remained in NBSt, respectively. The residual NHase activity of NBSt was higher than that of the control in all AM concentrations. This indicates that the fusion of SpyTag and SpyCatcher to the C-terminus of β- and α-NHase, respectively, in NBSt results in higher AM tolerance compared to the control NHase and other ligated-NHase strains. The improved AM tolerance achieved by stabilizing the enzyme structure through the introduction of a covalent bond between two subunits using SpyTag/SpyCatcher is similar to the results of our previous study, which focused on enhancing enzyme structure through the introduction of salt bridges [ 6 ] or a disulfide bond [ 9 ] between two subunits. All these results indicate that the SpyTag/SpyCatcher-mediated ligation between subunits of an enzyme is an effective strategy to enhance the toxic AM tolerance of NHase. To investigate the effect of introducing SpyTag/SpyCatcher on NHase, the native NHase gene in the genome of R. ruber TH8 and NBSt was knocked out using CRISPR/cas9 [ 29 ] to obtain the engineered R. ruber TH and TH-NBSt. The kinetic parameters of intracellular NHase produced by these two engineered strains were measured. The K m value of ligated-NHase mediated by introducing SpyTag/Spycatcher is 0.311 mM, which is higher than that of the control TH (0.204 mM). However, the V max of ligated-NHase (5.956 µmol/min/mg DCW) is lower than that of the control TH (1.034 µmol/min/mg DCW). This result indicates that the ligated NHase mediated by SpyTag and SpyCatcher decrease the substrate affinity but increases the structural stability of the enzyme stability of the enzyme to some extent. These results are not consistent with the application of SpyTag/SpyCatcher on L-phenylalanine aldolase and lichenase. Overall, introducing SpyTag/SpyCatcher to cyclize mono-subunit or ligate different subunits of the enzyme can improve the structural stability, but enhancing it is a case-by-case trial. Conclusion Enhancing the thermostability and/or acrylamide tolerance of NHase is of great industrial significance. In this work, we successfully increased the tolerance of NHase to industrial levels of acrylamide, a potentially toxic product of the NHase reaction. Additionally, we enhanced solvent tolerance by utilizing SpyTag/SpyCatcher to covalently link the α- and β-subunits of NHase, thereby stabilizing the enzyme’s structure in a fixed conformation. Further investigation is required to study the SpyTag/SpyCatcher-mediated ligation of NHase in R. ruber hosts where NHase is not encoded in the genome. Declarations Acknowlegement/Funding This work is supported by National Key R&D Program of China (2018YFA0901700), National Natural Science Foundation (No.21706145) of P. R. China and Beijing Municipal Science & Technology Commission (No.Z231100003723015). Conflicts of interest There are no conflicts of interest to declare. Consent to publish All the authors listed have seen the manuscript and approved its submission. Ethical Approval This work did not involve human or animal subjects, and thus, no ethical approval was required. Author Contribution Miaomiao Wang conceived the pivotal idea of the study, designed and performed the experiments, and wrote the manuscript. Huimin Yu conceived the pivotal idea of the study, and supervised the work References Jiao S, Li FL, Yu HM et al (2020) Advances in acrylamide bioproduction catalyzed with Rhodococcus cells harboring nitrile hydratase. Appl Microbiol Biotechnol 104:1001–1012. https://doi.org/10.1007/s00253-019-10284-5 Bhalla TC, Prasad S (2010) Nitrile hydratases (NHases): At the interface of academia and industry. 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Supplementary Files Supplementaryfiles.docx Cite Share Download PDF Status: Published Journal Publication published 16 Jul, 2024 Read the published version in Molecular Biology Reports → Version 1 posted Editorial decision: Revision requested 13 Jun, 2024 Reviews received at journal 27 May, 2024 Reviewers agreed at journal 16 May, 2024 Reviewers agreed at journal 16 May, 2024 Reviewers agreed at journal 16 May, 2024 Reviewers invited by journal 16 May, 2024 Editor assigned by journal 15 May, 2024 Submission checks completed at journal 15 May, 2024 First submitted to journal 24 Apr, 2024 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-4315377","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":303439810,"identity":"ded53ed6-957e-4710-aa84-7e66262e4434","order_by":0,"name":"Miaomiao Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYPACGyBmbiBJSxoQM5Km5TAJWgyOnz386mbOeTmD441tkj8q7jHwt3cn4NdyJi/NOnfbbWODMwfbJCTOFDNInDm7Aa8WswM5ZsZALYkzZyS2SRi2JTAYSOQS0HL+DUjLuXqwlsR/xGi5kWP8OHfbgQR+CaCWgw1EaLG/8caMOXdbsmE/z8Fmy4ZjCTwE/SLZn2P8OXebnTwbe/PBmz9qEuT423vxawECNgkogwXE4CGkHASYP6AzRsEoGAWjYBSgAACKvUqpETDMRAAAAABJRU5ErkJggg==","orcid":"","institution":"Beijing Evolyzer Co., Ltd","correspondingAuthor":true,"prefix":"","firstName":"Miaomiao","middleName":"","lastName":"Wang","suffix":""},{"id":303439811,"identity":"b5a8e798-80e0-4e4f-b7bc-eff6de64ee43","order_by":1,"name":"Huimin Yu","email":"","orcid":"","institution":"Tsinghua University","correspondingAuthor":false,"prefix":"","firstName":"Huimin","middleName":"","lastName":"Yu","suffix":""}],"badges":[],"createdAt":"2024-04-24 04:26:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4315377/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4315377/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11033-024-09760-7","type":"published","date":"2024-07-16T16:13:18+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":57084792,"identity":"32fb5352-0554-4f02-ae16-e4df193144a9","added_by":"auto","created_at":"2024-05-24 11:30:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":359817,"visible":true,"origin":"","legend":"\u003cp\u003ePrinciple of SpyTag/SpyCatcher-mediated ligation design\u003c/p\u003e\n\u003cp\u003e(A) Schematic of amide bond formation between SpyTag and SpyCatcher. (B) Tertiary structure of NHase marked C-, N- termini of α- and β-subunit. (C) Schematic map of the SpyTag/SpyCatcher-mediated ligation of NHase illustrating the different R. ruber strains generated. The strains are named based on the fusion orientation of SpyTag (St) relative to either subunit; TH8 served as the control strain. Pa2: promoter; NA: α-subunit of NHase; NB: β-subunit of NHase; St: SpyTag; Sc: SpyCatcher; T: terminator.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4315377/v1/341d38dcbe2b5be129250293.png"},{"id":57084794,"identity":"adbe49e8-2971-4f50-941b-92cf87109ac8","added_by":"auto","created_at":"2024-05-24 11:30:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":106832,"visible":true,"origin":"","legend":"\u003cp\u003eSDS-PAGE results of SpyTag/SpyCatcher-mediated ligation between α- and β-NHase\u003c/p\u003e\n\u003cp\u003eLanes: M, protein molecular marker; 1, TH8; 2, NBSt; 3, StNB; 4, NASt; 5, StNA. β-NHase, 26.4 kDa; α-NHase, 22.8 kDa; β-NHase+α-NHase+SpyCatcher+SpyTag,～60 kDa.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4315377/v1/efd7311298fe22a212a87ddc.png"},{"id":57084795,"identity":"903453d7-9279-4adc-ac51-b5d510d00acc","added_by":"auto","created_at":"2024-05-24 11:30:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":54472,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of SpyTag/SpyCatcher mediated ligation of the α- and β-NHase subunits on biomass (A) and NHase activity (B)\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4315377/v1/4cc66e970c2e71030bacf641.png"},{"id":57085458,"identity":"3b065036-c952-46a9-adee-3780e3184495","added_by":"auto","created_at":"2024-05-24 11:38:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":40737,"visible":true,"origin":"","legend":"\u003cp\u003eThe stress resistance of ligated NHase\u003c/p\u003e\n\u003cp\u003eThermostability of all mutants assessedbefore and after heat shock treatment at 60 ℃ for 10 min(A), and the solvent tolerance of all mutants evaluated after immersion in 30, 40, and 50% acrylamide (AM) solutions for 20 min (B).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4315377/v1/14c45e259cfdb1eab144f2c9.png"},{"id":61596458,"identity":"7f037a32-7e1e-49e1-b4ac-3ac5e1505391","added_by":"auto","created_at":"2024-08-01 17:27:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1073677,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4315377/v1/02a8c96c-ca44-4439-a0a8-c59bd5500cfa.pdf"},{"id":57084797,"identity":"c4ed980e-d662-4d20-b460-97b2a8d0c889","added_by":"auto","created_at":"2024-05-24 11:30:09","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":15532,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfiles.docx","url":"https://assets-eu.researchsquare.com/files/rs-4315377/v1/6068542ea699efa0753fa814.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Enhancing the Stress Resistance of Nitrile Hydratase from Rhodococcus ruber via SpyTag/SpyCatcher-mediated α- and β- subunits ligation","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eNitrile Hydratase (NHase), comprised of α- and β-subuits, is extensively utilized in the industrial production of acrylamide (AM), which is the monomer of polyacrlamide has been sucessfully applied in the petroleum exploitation and water treatment fields [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In the biotransformation of acrylonitrile (AN) to AM, both AM and AN are harmful organic compounds that can netatively affect the integrity of NHase or NHase-containing biocatalysts [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Additionally, the enzyme reaction is highly exothermic, leading to momentary thermal stress in microenvionment [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Both high concentrations of toxic compounds and momentary thermal stress can induce conformational changes and/or amino acid damages to intracellular NHase. This inevitably affects the catalytic activity of the enzyme [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Therefore, improving the thermal stability and tolerance of NHase to toxic substances would be valuable in the petroleum industry. Various strategies, such as introducing salt-bridges into thermal-sensitive regions [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], assembling enzymes into ordered nanostructures [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], swapping specific fragment [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], introducing disulfide bonds [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] to stabilize the binding between subunits, and introducing isolated or phused chaperons [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], have been applied to improve the stability of NHase or to renature the denatured NHase to varying degrees. However, the AM manufacturing bio-industry requires further improved stability of NHase toward thermal or AM stress.\u003c/p\u003e \u003cp\u003eThe pair SpyTag and SpyCatcher from \u003cem\u003eStreptococcus pyogenes\u003c/em\u003e was discovered by Proft et al in 2007 [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. By forming an amide bond between the lysine of SpyTag and the asperate acid of SpyCatcher, SpyTag and SpyCatcher become irreversibly ligated [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], and the resulting ligation product exhibits high chemical stability and resistance to high temperatures [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The SpyTag/SpyCatcher strategy has been succesfully applied to enhance the mechanical behavior of proteins [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], obtain nonlinear macromolecules with varying molecular weight and topologies [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], assemble living biomaterial [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], and enhance the resilience of enzymes to stress [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Several reports have applied this strategy to improve enzyme tolerance by introducting SpyTag and SpyCatcher to the enzyme through fusion to the N- and C- termini, resulting in cyclization of the enzyme (e.g. β-lactamase [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], L-phenylserine aldolase [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], dihydrofolate reductase [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], firefly luciferase, and lichenase [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]).\u003c/p\u003e \u003cp\u003eIn this study, we demonstrated the \u003cem\u003ein vivo\u003c/em\u003e ligation of the α- and β-subunits of NHase from \u003cem\u003eRhodococcus ruber\u003c/em\u003e by fusing SpyTag and SpyCatcher, resulting in the generation of several engineered enzymes and strains. These strains were then examined for the effects of the ligation on NHase thermo-stability and acrylamide tolerance.\u003c/p\u003e"},{"header":"2. Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Reagents\u003c/h2\u003e \u003cp\u003eThe PCR for gene amplification, agarose electrophoresis, restriction enzyme digestion, DNA ligation and DNA transformation were performed following the standard protocols [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Polymerase and ligase were obtained from Vazyme (Nanjing, China). Restriction enzymes were purchased from Takara (Dalian, China). The kits used for genomic DNA isolation, plasmid extraction, and gene retrieval were purchased from Omega Bio-Tek.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Plasmids, strains and gene manipulation\u003c/h2\u003e \u003cp\u003eThe strains and plasmids used in this study were listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. \u003cem\u003eR. ruber\u003c/em\u003e TH3 [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] was constructed by knocking out the amidase gene from \u003cem\u003eR. ruber\u003c/em\u003e TH and deposited in the China General Microbiological Culture Collection Center (CGMCC No. 2381). \u003cem\u003eR. ruber\u003c/em\u003e TH8 [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] was contructed by overexpressing the mutant NHase gene using plasmid in \u003cem\u003eR. ruber\u003c/em\u003e TH3. All the \u003cem\u003eR. ruber\u003c/em\u003e strains were grown at 28 ℃ in fermentation medium in previously study [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. \u003cem\u003eE. coli\u003c/em\u003e TOP10 and the derivatives were routinely grown at 37 \u003csup\u003eo\u003c/sup\u003eC in Luria\u0026ndash;Bertani (LB) medium. Antibiotics were added as the following concentrations: kanamycin, 30 \u0026micro;g/mL and tetracycline, 15 \u0026micro;g/mL.\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\u003eThe strains and plasmids used in this study\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrains, plasmids\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGenotype or sequence\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReference or source\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eStrains\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e TOP 10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eApplied for harvesting plasmid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSolarbio (Beijing, China)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eR. ruber\u003c/em\u003e TH3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eamiE-mutant (amiE::pPHU281, Tc\u003csup\u003er\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eR. ruber\u003c/em\u003e TH8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eR. ruber\u003c/em\u003e TH3 carrying plasmid pNV-Pa2-NHase\u003csup\u003eM\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNBSt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eR. ruber\u003c/em\u003e TH3 carrying plasmid pNV-Pa2-NBSt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStNB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eR. ruber\u003c/em\u003e TH3 carrying plasmid pNV-Pa2-StNB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNASt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eR. ruber\u003c/em\u003e TH3 carrying plasmid pNV-Pa2-NASt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eR. ruber\u003c/em\u003e TH3 carrying plasmid pNV-Pa2-StNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePlasmids\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epNV-Pa2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eColE1 ori, Kana\u003csup\u003er\u003c/sup\u003e, Pa2 promoter\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epNV-Pa2-NBSt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eβ-NHase-SpyTag and α-NHase-SpyCatcher fusion-expression\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epNV-Pa2-StNB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSpyTag-β-NHase and α-NHase-SpyCatcher fusion-expression\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epNV-Pa2-NASt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eβ-NHase-SpyCatcher and α-NHase-SpyTag fusion-expression\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epNV-Pa2-StNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eβ-NHase-SpyCatcher and SpyTag-α-NHase fusion-expression\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Construction of the engineered R. ruber NBSt, StNB, NASt, StNA\u003c/h2\u003e \u003cp\u003eThe primers used in this study were listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The plasmid pNV-Pa2 was devised from pNV18.1 by substituting the original promoter with Pa2 [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The original sequences of SpyCatcher and SpyTag were published on NCBI website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), the Genebank number of SpyCatcher is JQ478411, and the PDB number of SpyTag is 4MLI_B. The codon-optimized spycatcher gene and spytag gene were synthesized by GeneWiz (Suzhou, China). The mutant NHase gene (α-, β-NHase subunits) was amplied from the pNV-SBMDB contained in \u003cem\u003eR. ruber\u003c/em\u003e TH8 [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] by the primers NB-F/R and NA-F/R, respectively, for the preparation of phusion products. The GS linker was added between spytag/spycatcher and α-,β- subunits of NHase. The overlapped fragments β-NHase-SpyTag (NBSt), α-NHase-SpyCatcher (NASc), SpyTag-β-NHase (StNB), β-NHase-SpyCatcher (NBSc), α-NHase-SpyTag (NASt)and SpyTag-α-NHase (StNA) were amplified using the primers listed in Supplementary Table S2, digested and ligated into pNV-Pa2 to construct the plasmids pNV-Pa2-NBSt, pNV-Pa2-StNB, pNV-Pa2-NASt, pNV-Pa2-StNA, respectively. Take the construction of pNV-Pa2-NBSt as example, this plasmid contains two phusion genes, NBSt and NASc. The spytag (St) fragment was fused to the C- terminus of β-NHase (NB) by PCR using the primers NBSt-F and NBSt-R to make the NBSt, and the sequence was added in the primer NBSt-R. The spycatcher (Sc) fragment was fused to the C- terminus of α-NHase (NA) by the common GS linker (GSGGSGG) via overlap PCR to make the fragment NASc. And then, the gene NBSt digested by \u003cem\u003eXba\u003c/em\u003e I/\u003cem\u003eBam\u003c/em\u003eH I and NASc digested by \u003cem\u003eBam\u003c/em\u003eH I/\u003cem\u003eXba\u003c/em\u003e I were ligated into the plasmid pNV-Pa2 digested by \u003cem\u003eXba\u003c/em\u003e I/\u003cem\u003eKpn\u003c/em\u003e I to construct the plasmid pNV-Pa2-NBSt. Similarly, the plasmids pNV-Pa2-NBSt, pNV-Pa2-StNB, pNV-Pa2-NASt, pNV-Pa2-StNA were constructed. And then all these plasmids were elec-transformed into \u003cem\u003eR. ruber\u003c/em\u003e TH3 and spread onto solid plates containing 30 ug/mL kanamycin to obtain the engineered \u003cem\u003eR. ruber\u003c/em\u003e strains: NBSt, StNB, NASt and StNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), respectively. Gene sequencing was confirmed by GeneWiz (Tianjin, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Protein SDS-PAGE\u003c/h2\u003e \u003cp\u003eThe strains \u003cem\u003eR. ruber\u003c/em\u003e TH8, NBSt, StNB, NASt and StNA were harvested after cultured in fermentation medium for 48 h, washed twice by 10 mM PBS, resuspended in PBS (final OD\u003csub\u003e400\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;30) and disrupted by ultrasonication (240 W, 6\u0026times;4\u0026times;180 times). The supernatant was collected by centrifugation (12,000 rpm, 10 min, 4 ℃), mixed with 5\u0026times; protein loading buffer and boiled for 5 min. SDS-PAGE electrophoresis was performed with a 12% ExpressPlus\u0026trade; page gel (GenScript, Piscataway, USA). After that, the gel was stained with coomassie brilliant blue.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 NHase activity assay\u003c/h2\u003e \u003cp\u003eNHase activity of \u003cem\u003eR. ruber\u003c/em\u003e strains were determined as previously described by Ma et al [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. After cultured in fermentation medium for 48 h, the \u003cem\u003eR. ruber\u003c/em\u003e cells were harvested by centrifugation (12,000 rpm, 5 min) and resuspended in 10 mM PBS buffer (final OD\u003csub\u003e460\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;50) to be measured. The NHase activity was determined in a 5 mL catalytic reaction contained 200 \u0026micro;L of acrylonitrile, 4.5 mL PBS buffer (10 mM) and 100 \u0026micro;L resuspended cells liquid. The mixture was incubated at 28℃ for 5 min, and the reaction was terminated with 200 \u0026micro;L 2.5 M HCl. The supernatant was collected by centrifugation (12,000 rpm, 10 min) and then analyzed by gas chromatography (GC). Experiments were done in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Thermal stability\u003c/h2\u003e \u003cp\u003eThe thermal stability of NHase was determined by comparing the NHase activity of NHase-expression strains with or without heat shocked (60 ℃, 10 min). Experiments were done in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 AM tolerance\u003c/h2\u003e \u003cp\u003eAfter cultured in fermentation medium for 48 h, the \u003cem\u003eR. ruber\u003c/em\u003e cells were harvested by centrifugation (12,000 rpm, 5 min). The PBS and 60% AM solution were added to the collected cells to make the samples with the same cell density (OD\u003csub\u003e460\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;50) and different AM concentration, 30%, 40% and 50%. The volume of added PBS and 60% AM solution was shown in Supplementary Table S2. And then, the mixture was incubated at room temperature for 20 min. After that, the cells were immediately collected, washed, resuspended and measured the NHase activity by GC. Experiments were done in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Determination of kinetic parameters\u003c/h2\u003e \u003cp\u003eKinetic parameters of original and ligated NHase were estimated by measuring the initial activity of the recombinant cells at 28\u0026deg;C with a substrate concentration range of 0.05\u0026thinsp;~\u0026thinsp;0.6 M, respectively. K\u003csub\u003em\u003c/sub\u003e and V\u003csub\u003emax\u003c/sub\u003e were calculated using the double reciprocal Lineweaver-Burk plot method.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n\u003ch2\u003e3.1 Strategy of ligating the \u0026alpha;- and \u0026beta;-NHase subunits by SpyTag/Spycatcher\u003c/h2\u003e\n\u003cp\u003eNHase, which catalyzes the formation of AM, is one of the most important industrial enzymes[\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e]. The stress, heat release, and solvent immerse present in this reaction can cause conformational changes or damage to the amino acids of NHase. Therefore, enhancing thermostability or solvent tolerance is crucial [\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eIn this study, the mutant NHase gene [\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e], containing a disulfide bond to stabilize the binding between subunits and exhibiting better thermal stability and AM tolerance than the natural gene, was selected as the parent gene. To further enhance the interaction between the \u0026alpha;- and \u0026beta;-NHase subunits, SpyTag and SpyCatcher were fused to the N- and C- termini of the \u0026alpha;- and \u0026beta;-NHase subunits in different combinations. They were then overexpressed using the \u003cem\u003eR. ruber\u003c/em\u003e-\u003cem\u003eE.coli\u003c/em\u003e shuttle plasmid pNV-Pa2 [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e] to obtain the engineered \u003cem\u003eR. ruber\u003c/em\u003e strains NBSt, StNB, NASt and StNA (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). NBSt and StNB were generated by fusing SpyCatcher to the C-terminus of \u0026alpha;-NHase, and then SpyTag was fused to either the N- or C-terminus, respectively, of \u0026beta;-NHase. NASt and StNA were generated by fusing SpyCatcher to the C-terminus of \u0026beta;-NHase, and then SpyTag was fused to either the N- or C-terminus, respectively, of \u0026alpha;-NHase. Meanwhile, \u003cem\u003eR. ruber\u003c/em\u003e TH8 [\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e] engineered by overexpressing the template NHase gene in \u003cem\u003eR. ruber\u003c/em\u003e TH3 [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e], served as the control.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n\u003ch2\u003e3.2 Verification of the ligation conformation of \u0026alpha;- and \u0026beta;-NHase subunits\u003c/h2\u003e\n\u003cp\u003eParallel cultures of the engineered \u003cem\u003eR. ruber\u003c/em\u003e NBSt, StNB, NASt, StNA, and the control strain \u003cem\u003eR. ruber\u003c/em\u003e TH8 were cultivated in shake flasks to assess the NHase expression via SDS-PAGE (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). The protein sample from TH8 was separated, and the \u0026alpha;- and \u0026beta;-NHase subunits were identified after SDS-PAGE; their known molecular weights are 22.8 and 26.4 kDa, respectively. The ligation of the \u0026alpha;- and \u0026beta;-NHase subunits by SpyTag/SpyCatcher fusion in all engineered strains was successful, as indicated by the band at ~\u0026thinsp;60 kDa, even after sample boiling. The molecular weights were determined using AAT Bioquest (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.aatbio.com/tools/calculate-peptide-and-protein-molecular-weight-mw\u003c/span\u003e\u003c/span\u003e); the molecular weights of SpyTag and SpyCatcher are 1.5 and 12.3 kDa, respectively.\u003c/p\u003e\n\u003cp\u003eThis experiment further demonstrated the successful application of SpyTag/SpyCatcher. It showed that the strategy of cyclizing two termini of one enzyme or binding two different subunits by introducing SpyTag/SpyCatcher is universal, as supported by other literature reports [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e]. The percentage of total protein represented by ligated NHase was calculated using Gel-Pro Analyzer software and varied based on the ligation strategy: 9.5%, 9.6%, 6.4% and 8.2% for NBSt, StNB, NASt and StNA, respectively. Additionally, SDS-PAGE analysis revealed a single subunit NHase band in all engineered strains, attributed to the native NHase encoded by the genome of the host strain TH3 [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n\u003ch2\u003e3.3 Effect of introduced SpyTag/SpyCatcher on NHase activity\u003c/h2\u003e\n\u003cp\u003eThe biomass and NHase activity of the engineered \u003cem\u003eR. ruber\u003c/em\u003e strains NBSt, StNB, NASt, StNA, and the control \u003cem\u003eR. ruber\u003c/em\u003e TH8 were measured and compared in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. SpyTag/SpyCatcher ligation did not reduce the biomass of NHase (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA), but it did reduce NHase activity (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). Previous reports have also documented a reduction in enzyme activity caused by SpyTag/SpyCatcher-mediated cyclization of lichenase [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e]. NHase activity decreased from 15.4\u0026ndash;37.9% depending on the fusion strategy. \u003cem\u003eR. ruber\u003c/em\u003e strain NBSt, engineered by fusing SpyTag to the C-terminus of \u0026beta;-NHase and fusing SpyCatcher to the C-terminus of \u0026alpha;-NHase, exhibited the highest activity at 2801.7 U/mL, representing a 15.4% decrease compared to the TH8 control. The only difference in fusion strategy between NBSt and StNB was the \u0026beta;-NHase terminus fused to SpyTag; the C-terminus was fused in NBSt, whereas the N-terminus was fused in StNB. The different fusion termini led to similar ligated-NHase biomass, but NHase activity was further reduced to 2057 U/mL in StNB, a decrease of 37.9% compared to the control. This is in accordance with previous studies showing that decreasing NHase activity occurred by introducing salt bridges [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e] or reducing NHase solubility by inserting assembly peptides [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e] at the N-terminus of \u0026beta;-NHase.\u003c/p\u003e\n\u003cp\u003eThe fusion of SpyCatcher to the C-terminus of \u0026beta;-NHase in the NASt engineered strain also resulted in reduced NHase activity, 2154 U/mL, relative to the control. This activity was similar to that of StNA (2213 U/mL), in which SpyTag was fused to the N- terminus of \u0026alpha;-NHase. Comparing NBSt and NASt, NHase activity was higher in NBSt, suggesting that the larger SpyCatcher protein should be fused to the \u0026alpha;-NHase subunit rather than the \u0026beta;-subunit [\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e]. Based on these results, we conclude that the C-terminus of \u0026beta;-NHase is a better fusion site than the N-terminus, the C-terminus of \u0026alpha;-NHase is more suitable for fusion with a larger protein. fusion of a peptide to the N- or C-terminus of \u0026alpha;-NHase yields similar results, and the N-terminus of \u0026beta;-NHase is more sensitive to peptide fusion than the N-terminus of \u0026alpha;-NHase.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n\u003ch2\u003e3.4 Stress resistance of ligated-NHase\u003c/h2\u003e\n\u003cp\u003eThe stress resistance (thermostability and solvent tolerant) of the different ligated-NHase strains and the original NHase was tested using TH8, NBSt, StNB, NASt, and StNA cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA, the control TH8 cells retained 62.2% of their NHase activity after incubating at 60 ℃ for 10 min. The residual NHase activity of the engineered strains was 63.2%, 14.4%, 34.6%, and 20.3% in NBSt, StNB, NASt, and StNA, respectively. Among all the engineered strains, NBSt exhibited superior thermal-tolerance. The 9.5% ligated-NHase mediated by SpyTag/SpyCatcher in NBSt showed a 1% increase in heat shock resistance. By fusing SpyTag and SpyCatcher to the C-terminus of \u0026beta;- and \u0026alpha;-NHase, respectively, NHase thermostability was improved, but the effect was not significant. The results obtained here show little enhanced thermostability achieved by introducing SpyTag/SpyCatcher-mediated enzyme ligation or cyclization, similar to the situation of cyclizated l-phenylalanine aldolase caused by SpyTag/SpyCatcher [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e]. Additionally, a few studies have reported that introducing SpyTag/SpyCatcher cyclization did enhance the thermostability of Lichenase [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e], Firefly Luciferase [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e], \u0026beta;-lactamase [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e], and phytase [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]. This suggests that the strategy of cyclizing one enzyme or ligating two subunits to fix the structure or conformation of the enzyme to enhance thermostability is not universal, and this needs to be examined in each specific case.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThermostability of all mutants assessedbefore and after heat shock treatment at 60 ℃ for 10 min(A), and the solvent tolerance of all mutants evaluated after immersion in 30, 40, and 50% acrylamide (AM) solutions for 20 min (B).\u003c/p\u003e\n\u003cp\u003eIndustrially, different applications require the production of 30%, 40% and 50% AM solutions. Thus, was assessed the effect of these AM concentrations on ligated-NHase activity by immersing the respective cells for 20 min (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). NHase activity decreased as the AM concentration increased in both the control and all engineered strains. The engineered strain NBSt exhibited better AM tolerance than the other engineered strains. Upon immersion of the cells in 30%, 40%, and 50% AM solution, approximately 40.7%, 32.8% and 22.1% of the NHase activity remained in the control cells, while 48.2%, 38.6% and 24.1% remained in NBSt, respectively. The residual NHase activity of NBSt was higher than that of the control in all AM concentrations. This indicates that the fusion of SpyTag and SpyCatcher to the C-terminus of \u0026beta;- and \u0026alpha;-NHase, respectively, in NBSt results in higher AM tolerance compared to the control NHase and other ligated-NHase strains. The improved AM tolerance achieved by stabilizing the enzyme structure through the introduction of a covalent bond between two subunits using SpyTag/SpyCatcher is similar to the results of our previous study, which focused on enhancing enzyme structure through the introduction of salt bridges [\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e] or a disulfide bond [\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e] between two subunits. All these results indicate that the SpyTag/SpyCatcher-mediated ligation between subunits of an enzyme is an effective strategy to enhance the toxic AM tolerance of NHase.\u003c/p\u003e\n\u003cp\u003eTo investigate the effect of introducing SpyTag/SpyCatcher on NHase, the native NHase gene in the genome of \u003cem\u003eR. ruber\u003c/em\u003e TH8 and NBSt was knocked out using CRISPR/cas9 [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e] to obtain the engineered \u003cem\u003eR. ruber\u003c/em\u003e TH and TH-NBSt. The kinetic parameters of intracellular NHase produced by these two engineered strains were measured. The \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e value of ligated-NHase mediated by introducing SpyTag/Spycatcher is 0.311 mM, which is higher than that of the control TH (0.204 mM). However, the V\u003csub\u003emax\u003c/sub\u003e of ligated-NHase (5.956 \u0026micro;mol/min/mg DCW) is lower than that of the control TH (1.034 \u0026micro;mol/min/mg DCW). This result indicates that the ligated NHase mediated by SpyTag and SpyCatcher decrease the substrate affinity but increases the structural stability of the enzyme stability of the enzyme to some extent. These results are not consistent with the application of SpyTag/SpyCatcher on L-phenylalanine aldolase and lichenase. Overall, introducing SpyTag/SpyCatcher to cyclize mono-subunit or ligate different subunits of the enzyme can improve the structural stability, but enhancing it is a case-by-case trial.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eEnhancing the thermostability and/or acrylamide tolerance of NHase is of great industrial significance. In this work, we successfully increased the tolerance of NHase to industrial levels of acrylamide, a potentially toxic product of the NHase reaction. Additionally, we enhanced solvent tolerance by utilizing SpyTag/SpyCatcher to covalently link the \u0026alpha;- and \u0026beta;-subunits of NHase, thereby stabilizing the enzyme\u0026rsquo;s structure in a fixed conformation. Further investigation is required to study the SpyTag/SpyCatcher-mediated ligation of NHase in \u003cem\u003eR. ruber\u003c/em\u003e hosts where NHase is not encoded in the genome.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowlegement/Funding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work is supported by National Key R\u0026amp;D Program of China (2018YFA0901700), National Natural Science Foundation (No.21706145) of P. R. China and Beijing Municipal Science \u0026amp; Technology Commission (No.Z231100003723015).\u003c/p\u003e\u003cp\u003e \u003ch2\u003eConflicts of interest\u003c/h2\u003e \u003cp\u003eThere are no conflicts of interest to declare.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent to publish\u003c/strong\u003e \u003cp\u003eAll the authors listed have seen the manuscript and approved its submission.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eEthical Approval\u003c/strong\u003e \u003cp\u003eThis work did not involve human or animal subjects, and thus, no ethical approval was required.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eMiaomiao Wang conceived the pivotal idea of the study, designed and performed the experiments, and wrote the manuscript. Huimin Yu conceived the pivotal idea of the study, and supervised the work\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eJiao S, Li FL, Yu HM et al (2020) Advances in acrylamide bioproduction catalyzed with Rhodococcus cells harboring nitrile hydratase. Appl Microbiol Biotechnol 104:1001\u0026ndash;1012. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00253-019-10284-5\u003c/span\u003e\u003cspan address=\"10.1007/s00253-019-10284-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBhalla TC, Prasad S (2010) Nitrile hydratases (NHases): At the interface of academia and industry. 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Metab Eng 57:13\u0026ndash;22. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ymben.2019.10.003\u003c/span\u003e\u003cspan address=\"10.1016/j.ymben.2019.10.003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Nitrile Hydratase, SpyTag/SpyCatcher, R. ruber, Thermostability, Acrylamide resistance","lastPublishedDoi":"10.21203/rs.3.rs-4315377/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4315377/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNitrile Hydratase (NHase) is one of the most important industrial enzyme widely used in the petroleum exploitation field. The enzyme, composed of two unrelated α- and β-subunits, catalyzes the conversion of acrylonitrile to acrylamide, releasing a significant amount of heat and generating the organic solvent product, acrylamide. Both the heat and acrylamide solvent have an impact on the structural stability of NHase and its catalytic activity. To improve the thermostability and acrylamide tolerance of NHase, the two subunits were fused \u003cem\u003ein vivo\u003c/em\u003e using SpyTag and SpyCatcher, which were attached to the termini of each subunit in various combinations. Analysis of the engineered strains showed that the C-terminus of β-NHase is a better fusion site than the N-terminus, while the C-terminus of α-NHase is the most suitable site for fusion with a larger protein. Fusion of SpyTag and SpyCatcher to the C-terminus of β-NHase and α-NHase, respectively, led to improved acrylamide tolerance and a slight enhancement in the thermostability of one of the engineered strains, NBSt. These results indicate that \u003cem\u003ein vivo\u003c/em\u003e ligation of different subunits using SpyTag/SpyCatcher is a valuable strategy for enhancing subunit interaction and improving stress tolerance.\u003c/p\u003e","manuscriptTitle":"Enhancing the Stress Resistance of Nitrile Hydratase from Rhodococcus ruber via SpyTag/SpyCatcher-mediated α- and β- subunits ligation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-24 11:30:04","doi":"10.21203/rs.3.rs-4315377/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-06-13T10:33:12+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-27T05:49:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"217647507751412771713777510576097956538","date":"2024-05-17T00:15:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"45290455253188816273394656201788990747","date":"2024-05-16T12:16:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"13350484929009889658224209202427840759","date":"2024-05-16T11:08:44+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-16T10:38:26+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-15T15:00:34+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-15T15:00:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Biology Reports","date":"2024-04-24T04:14:02+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"8f9d701e-0fd4-48d4-b68d-cf3a0af936da","owner":[],"postedDate":"May 24th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-01T17:13:56+00:00","versionOfRecord":{"articleIdentity":"rs-4315377","link":"https://doi.org/10.1007/s11033-024-09760-7","journal":{"identity":"molecular-biology-reports","isVorOnly":false,"title":"Molecular Biology Reports"},"publishedOn":"2024-07-16 16:13:18","publishedOnDateReadable":"July 16th, 2024"},"versionCreatedAt":"2024-05-24 11:30:04","video":"","vorDoi":"10.1007/s11033-024-09760-7","vorDoiUrl":"https://doi.org/10.1007/s11033-024-09760-7","workflowStages":[]},"version":"v1","identity":"rs-4315377","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4315377","identity":"rs-4315377","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}