Streptomyces mobaraensis transglutaminase (TGase), with its ability to catalyze protein cross-linking, is widely applied in food processing.Previously, a thermostable variant FRAPD-TGm2 of S.mobaraensis TGase was constructed to improve its application performance under high temperatures.This study identified a TGase variant with further improved thermostability based on dynamic cross-correlation analysis.Firstly, based on molecular dynamics simulation and dynamic cross-correlation analysis, this study identified 48 residues exhibiting dynamic cross-correlation with the flexible region of the FRAPD-TGm2 substrate-binding pocket.Subsequently, virtual saturation mutagenesis on these residues employing Rosetta Cartesian_ddg resulted in 20 variants with a folding free energy change of less than -1 kcal/mol.These mutants were then expressed, purified, and subjected to enzymatic property characterization.Among them, the 60 ℃ half-life and 65 ℃ specific enzyme activity of the variant FRAPD-TGm2-Y34 W reached 100.5 min and 134.2 U/mg, respectively, which were 89.1% and 28.5% higher than that of FRAPD-TGm2.Furthermore, this study observed significant effects on enzyme activity and thermostability in FRAPD-TGm2 due to distal residues His201 and Asn32.The above results indicate that enzyme molecular redesign, based on dynamic cross-correlation and folding free energy analysis, can effectively enhance the thermostability of TGase.
YANG Penghui
,
QIU Wenxuan
,
YE Jiacai
,
DU Guocheng
,
LIU Song
. Thermostability modification of transglutaminase based on dynamic cross-correlation and folding free energy analysis[J]. Food and Fermentation Industries, 2024
, 50(24)
: 1
-8
.
DOI: 10.13995/j.cnki.11-1802/ts.038976
[1] AKBARI M, RAZAVI S H, KIELISZEK M.Recent advances in microbial transglutaminase biosynthesis and its application in the food industry[J].Trends in Food Science & Technology, 2021, 110:458-469.
[2] KASHIWAGI T, YOKOYAMA K I, ISHIKAWA K, et al.Crystal structure of microbial transglutaminase from Streptoverticillium mobaraense[J].Journal of Biological Chemistry, 2002, 277(46):44252-44260.
[3] FUCHSBAUER H L.Approaching transglutaminase from Streptomyces bacteria over three decades[J].The FEBS Journal, 2022, 289(16):4680-4703.
[4] KIELISZEK M, MISIEWICZ A.Microbial transglutaminase and its application in the food industry:A review[J].Folia Microbiologica, 2014, 59(3):241-250.
[5] BUETTNER K, HERTEL T C, PIETZSCH M.Increased thermostability of microbial transglutaminase by combination of several hot spots evolved by random and saturation mutagenesis[J].Amino Acids, 2012, 42(2-3):987-996.
[6] SORAPUKDEE S, TANGWATCHARIN P.Quality of steak restructured from beef trimmings containing microbial transglutaminase and impacted by freezing and grading by fat level[J].Asian-Australasian Journal of Animal Sciences, 2018, 31(1):129-137.
[7] MARX C K, HERTEL T C, PIETZSCH M.Random mutagenesis of a recombinant microbial transglutaminase for the generation of thermostable and heat-sensitive variants[J].Journal of Biotechnology, 2008, 136(3-4):156-162.
[8] YOKOYAMA K, UTSUMI H, NAKAMURA T, et al.Screening for improved activity of a transglutaminase from Streptomyces mobaraensis created by a novel rational mutagenesis and random mutagenesis[J].Applied Microbiology and Biotechnology, 2010, 87(6):2087-2096.
[9] WANG X L, DU J H, ZHAO B C, et al.Significantly improving the thermostability and catalytic efficiency of Streptomyces mobaraenesis transglutaminase through combined rational design[J].Journal of Agricultural and Food Chemistry, 2021, 69(50):15268-15278.
[10] XIE Y, AN J, YANG G Y, et al.Enhanced enzyme kinetic stability by increasing rigidity within the active site[J].Journal of Biological Chemistry, 2014, 289(11):7994-8006.
[11] ZENG W, LI X Q, YANG Y Y, et al.Substrate-binding mode of a thermophilic PET hydrolase and engineering the enzyme to enhance the hydrolytic efficacy[J].ACS Catalysis, 2022, 12(5):3033-3040.
[12] JOO S, CHO I J, SEO H, et al.Structural insight into molecular mechanism of poly(ethylene terephthalate) degradation[J].Nature Communications, 2018, 9(1):382.
[13] YU H R, DALBY P A.Exploiting correlated molecular-dynamics networks to counteract enzyme activity-stability trade-off[J].Proceedings of the National Academy of Sciences of the United States of America, 2018, 115(52):E12192-E12200.
[14] GU J, XU Y, NIE Y.Role of distal sites in enzyme engineering[J].Biotechnology Advances, 2023, 63:108094.
[15] THILTGEN G, GOLDSTEIN R A.Assessing predictors of changes in protein stability upon mutation using self-consistency[J].PLoS One, 2012, 7(10):e46084.
[16] DOLGIKH B, WOLDRING D.Site-wise diversification of combinatorial libraries using insights from structure-guided stability calculations[J].Methods in Molecular Biology, 2022, 2491:63-73.
[17] PARK H, BRADLEY P, GREISEN P Jr, et al.Simultaneous optimization of biomolecular energy functions on features from small molecules and macromolecules[J].Journal of Chemical Theory and Computation, 2016, 12(12):6201-6212.
[18] 杜坤, 周丽, 堵国成,等.内含肽介导谷氨酰胺转胺酶酶原的活化[J].食品科学, 2013, 34(9):90-94.
DU K, ZHOU L, DU G C, et al.Intein-mediated activation of transglutaminase from Streptomyces hygroscopicus[J].Food Science, 2013, 34(9):90-94.
[19] MIRDITA M, SCHÜTZE K, MORIWAKI Y, et al.ColabFold:Making protein folding accessible to all[J].Nature Methods, 2022, 19(6):679-682.
[20] CONWAY P, TYKA M D, DIMAIO F, et al.Relaxation of backbone bond geometry improves protein energy landscape modeling[J].Protein Science, 2014, 23(1):47-55.
[21] ABRAHAM M J, MURTOLA T, SCHULZ R, et al.GROMACS:High performance molecular simulations through multi-level parallelism from laptops to super computers[J].SoftwareX, 2015, 1-2:19-25.
[22] MAIER J A, MARTINEZ C, KASAVAJHALA K, et al.ff14SB:Improving the accuracy of protein side chain and backbone parameters from ff99SB[J].Journal of Chemical Theory and Computation, 2015, 11(8):3696-3713.
[23] YU H R, DALBY P A.A beginner’s guide to molecular dynamics simulations and the identification of cross-correlation networks for enzyme engineering[J].Methods in Enzymology, 2020, 643:15-49.
[24] DUARTE L, MATTE C R, BIZARRO C V, et al.Review transglutaminases:Part II-industrial applications in food, biotechnology, textiles and leather products[J].World Journal of Microbiology & Biotechnology, 2019, 36(1):11.
[25] BOMMARIUS A S, PAYE M F.Stabilizing biocatalysts[J].Chemical Society Reviews, 2013, 42(15):6534-6565.
