Food and Fermentation Industries >

2026 , Vol. 52 >Issue 2: 1 - 9

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.042634

Efficient production of a thermostable variant of transglutaminase in Streptomyces mobaraensis

  • ZHANG Ni ,
  • QIU Jieru ,
  • GUAN Xingzi ,
  • YE Jiacai ,
  • LIU Song
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  • 1(Science Center for Future Foods, Jiangnan University, Wuxi 214122, China)
    2(School of Biotechnology, Jiangnan University, Wuxi 214122, China)

Received date: 2024-03-07

  Revised date: 2024-05-26

  Online published: 2026-03-18

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Abstract

Transglutaminase (EC 2.3.2.13 TGase) is an important industrial enzyme capable of catalyzing protein cross-linking or modification by small molecules.To enhance the fermentation level of thermostable TGase, this study expressed the thermostable mutant TGm2A in the mutant strain smY2022Δtg of Streptomyces mobaraensis and analyzed its enzymatic properties and protein cross-linking ability under high-temperature conditions.Initially, the impact of six strong promoters on TGm2A expression was evaluated, revealing that the glyceraldehyde-3-phosphate dehydrogenase promoter (Pgapdh) achieved the highest yield of TGm2A at 31.48 U/mL.Subsequently, by tandemly linking the TGase promoter (PsmY2022-2) with Pgapdh to form the promoter PsmY2022-2/gapdh, the yield of TGm2A was further increased to 64.94 U/mL.Building on this, a double-copy TGm2A strain, smY2022-TGm2A-2C, was constructed, which achieved an enzyme activity of 93.62 U/mL after 84 h of fermentation, making it the highest-yielding TGase strain reported to date.Enzymatic characterization indicated that TGm2A has a half-life of 120.36 min at 60 ℃, which is 100 times that of the wild type.Additionally, TGm2A demonstrated significantly superior cross-linking ability with β-casein at 70 ℃ compared to the wild-type TGase.This study lays the foundation for the industrial production of TGase and its application in high-temperature processing.

Key words： Streptomyces mobaraensis; transglutaminase; secretory expression; promoter; copy number; catalytic property

Cite this article

ZHANG Ni , QIU Jieru , GUAN Xingzi , YE Jiacai , LIU Song . Efficient production of a thermostable variant of transglutaminase in Streptomyces mobaraensis[J]. Food and Fermentation Industries, 2026 , 52(2) : 1 -9 . DOI: 10.13995/j.cnki.11-1802/ts.042634

References

[1] SANTHI D, KALAIKANNAN A, MALAIRAJ P, et al.Application of microbial transglutaminase in meat foods:A review[J].Critical Reviews in Food Science and Nutrition 2017, 57(10):2071-2076.
[2] 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.
[3] 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.
[4] NISHIOKA R, IIDA R, MINAMIHATA K, et al.Transglutaminase-mediated proximity labeling of a specific Lys residue in a native IgG antibody[J].Chemical Communications 2024, 60(65):8545-8548.
[5] VASIĆ K, KNEZ Ž, LEITGEB M.Transglutaminase in foods and biotechnology[J].International Journal of Molecular Sciences, 2023, 24(15):12402.
[6] DA SILVA FREITAS D, MERO A, PASUT G.Chemical and enzymatic site specific PEGylation of hGH[J].Bioconjugate Chemistry, 2013, 24(3):456-463.
[7] DONDERO M, FIGUEROA V, MORALES X, et al.Transglutaminase effects on gelation capacity of thermally induced beef protein gels[J].Food Chemistry, 2006, 99(3):546-554.
[8] LIU J, YANG S R, TANG R Y, et al.Enhanced tofu quality through calcium sulfate and transglutaminase treatment:Physicochemical properties and digestibility analysis[J].International Journal of Food Science and Technology, 2024, 59(10):7610-7618.
[9] 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.
[10] 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.
[11] YANG P H, WANG X L, YE J C, et al.Enhanced thermostability and catalytic activity of Streptomyces mobaraenesis transglutaminase by rationally engineering its flexible regions[J].Journal of Agricultural and Food Chemistry, 2023, 71(16):6366-6375.
[12] NODA S, MIYAZAKI T, TANAKA T, et al.High-level production of mature active-form Streptomyces mobaraensis transglutaminase via pro-transglutaminase processing using Streptomyces lividans as a host[J].Biochemical Engineering Journal, 2013, 74:76-80.
[13] MA T G, LU J J, ZHU J, et al.The secretion of Streptomyces monbaraensis transglutaminase from Lactococcus lactis and immobilization on porous magnetic nanoparticles[J].Frontiers in Microbiology, 2019, 10:1675.
[14] WANG S T, YANG Z G, LI Z J, et al.Heterologous expression of recombinant transglutaminase in Bacillus subtilis SCK6 with optimized signal peptide and Codon, and its impact on gelatin properties[J].Journal of Microbiology and Biotechnology, 2020, 30(7):1082-1091.
[15] AQEEL S M, ABDULQADER A A, DU G C, et al.Integrated strategies for efficient production of Streptomyces mobaraensis transglutaminase in Komagataella phaffii[J].International Journal of Biological Macromolecules, 2024, 273:133113.
[16] ZOTZEL J, KELLER P, FUCHSBAUER H L.Transglutaminase from Streptomyces mobaraensis is activated by an endogenous metalloprotease[J].European Journal of Biochemistry, 2003, 270(15):3214-3222.
[17] JUETTNER N E, SCHMELZ S, ANDERL A, et al.The N-terminal peptide of the transglutaminase-activating metalloprotease inhibitor from Streptomyces mobaraensis accommodates both inhibition and glutamine cross-linking sites[J].The FEBS Journal, 2020, 287(4):708-720.
[18] YIN X Q, LI Y Y, ZHOU J W, et al.Enhanced production of transglutaminase in Streptomyces mobaraensis through random mutagenesis and site-directed genetic modification[J].Journal of Agricultural and Food Chemistry, 2021, 69(10):3144-3153.
[19] YE J C, YANG P H, ZHOU J W, et al.Efficient production of a thermostable mutant of transglutaminase by Streptomyces mobaraensis[J].Journal of Agricultural and Food Chemistry, 2024, 72(8):4207-4216.
[20] BAI C X, ZHANG Y, ZHAO X J, et al.Exploiting a precise design of universal synthetic modular regulatory elements to unlock the microbial natural products in Streptomyces[J].Proceedings of the National Academy of Sciences of the United States of America, 2015, 112(39):12181-12186.
[21] WANG Y, COBB R E, ZHAO H.High-efficiency genome editing of Streptomyces species by an engineered CRISPR/Cas system[J].Methods in Enzymology, 2016, 575:271-284.
[22] PASTERNACK R, DORSCH S, OTTERBACH J T, et al.Bacterial pro-transglutaminase from Streptoverticillium mobaraense:Purification, characterisation and sequence of the zymogen[J].European Journal of Biochemistry, 1998, 257(3):570-576.
[23] LIU S, WANG M, DU G C, et al.Improving the active expression of transglutaminase in Streptomyces lividans by promoter engineering and Codon optimization[J].BMC Biotechnology, 2016, 16(1):75.
[24] BIBB M J, JANSSEN G R, WARD J M.Cloning and analysis of the promoter region of the erythromycin resistance gene (ermE) of Streptomyces erythraeus[J].Gene, 1985, 38(1-3):215-226.
[25] MYRONOVSKYI M, LUZHETSKYY A.Native and engineered promoters in natural product discovery[J].Natural Product Reports, 2016, 33(8):1006-1019.
[26] SHAO Z Y, RAO G D, LI C, et al.Refactoring the silent spectinabilin gene cluster using a plug-and-play scaffold[J].ACS Synthetic Biology, 2013, 2(11):662-669.
[27] 叶佳才. 耐热谷氨酰胺转氨酶在茂原链霉菌中的表达研究[D].无锡:江南大学, 2024.
YE J C.Expression of thermostable transglutaminase in Streptomyces mobaraensis[D].Wuxi:Jiangnan University, 2024.
[28] YAO D B, HAN X D, GAO H H, et al.Enhanced extracellular production of raw starch-degrading α-amylase in Bacillus subtilis through expression regulatory element modification and fermentation optimization[J].Microbial Cell Factories, 2023, 22(1):118.
[29] LIU L Y, NING N Y, XU S M, et al.Double promoter and tandem gene strategy for efficiently expressing recombinant FGF21[J].Microbial Cell Factories, 2024, 23(1):171.
[30] WU Y J, FENG S, SUN Z A, et al.An outlook to sophisticated technologies and novel developments for metabolic regulation in the Saccharomyces cerevisiae expression system[J].Frontiers in Bioengineering and Biotechnology, 2023, 11:1249841.
[31] YADAV D, RANJAN B, MCHUNU N, et al.Enhancing the expression of recombinant small laccase in Pichia pastoris by a double promoter system and application in antibiotics degradation[J].Folia Microbiologica, 2021, 66(6):917-930.
[32] TAO H, ZHANG Y C, DENG Z X, et al.Strategies for enhancing the yield of the potent insecticide spinosad in Actinomycetes[J].Biotechnology Journal, 2019, 14(1):e1700769.
[33] SIOUD S, AIGLE B, KARRAY-REBAI I, et al.Integrative gene cloning and expression system for Streptomyces sp.US 24 and Streptomyces sp.TN 58 bioactive molecule producing strains[J].Journal of biomedicine & Biotechnology, 2009(1):464986.
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