Abstract
Microbial transglutaminase (MTG) from Streptomyces mobaraensis has been widely used for crosslinking proteins in order to acquire products with improved properties. To improve the yield and enable a facile and efficient purification process, recombinant vectors, harboring various heterologous signal peptide-encoding fragments fused to the mtg gene, were constructed in Escherichia coli and then expressed in Bacillus subtilis. Signal peptides of both WapA and AmyQ (SPwapA and SPamyQ) were able to direct the secretion of pre-pro-MTG into the medium. A constitutive promoter (PhpaII) was used for the expression of SPwapA-mtg, while an inducible promoter (Plac) was used for SPamyQ-mtg. After purification from the supernatant of the culture by immobilized metal affinity chromatography and proteolysis by trypsin, 63.0 ± 0.6 mg/L mature MTG was released, demonstrated to have 29.6 ± 0.9 U/mg enzymatic activity and shown to crosslink soy protein properly. This is the first report on secretion of S. mobaraensis MTG from B. subtilis, with similar enzymatic activities and yields to that produced from Escherichia coli, but enabling a much easier purification process.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Ando H, Adachi M, Umeda K, Matsuura A, Nonaka M, Uchio R, Tanaka H, Motoki M (1989) Purification and characteristics of a novel transglutaminase derived from microorganisms. Agric Biol Chem 53(10):2613–2617. https://doi.org/10.1080/00021369.1989.10869735
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254. https://doi.org/10.1016/0003-2697(76)90527-3
Cao G, Zhang X, Zhong L, Lu Z (2011) A modified electro-transformation method for Bacillus subtilis and its application in the production of antimicrobial lipopeptides. Biotechnol Lett 33(5):1047–1051. https://doi.org/10.1007/s10529-011-0531-x
Chung SI, Lewis MS, Folk JE (1974) Relationships of the catalytic properties of human plasma and platelet transglutaminases (activated blood coagulation factor XIII) to their subunit structures. J Biol Chem 249(3):940–950
de Ruyter PG, Kuipers OP, De Vos WM (1996) Controlled gene expression systems for Lactococcus lactis with the food-grade inducer nisin. Appl Environ Microbiol 62(10):3662–3667
Della MM, Caparrósruiz D, Claparols I, Serafinifracassini D, Rigau J (2004) AtPng1p. The first plant transglutaminase. Plant Physiol 135(4):2046–2054. https://doi.org/10.1104/pp.104.042549
Dower WJ, Miller JF, Ragsdale CW (1988) High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res 16(13):6127–6145. https://doi.org/10.1093/nar/16.13.6127
Feng Y, Liu S, Jiao Y, Gao H, Wang M, Du G, Chen J (2017) Enhanced extracellular production of L-asparaginase from Bacillus subtilis 168 by B. subtilis WB600 through a combined strategy. Appl Microbiol Biotechnol 101(4):1–12. https://doi.org/10.1007/s00253-016-7816-x
Folk JE, Cole PW (1966) Identification of a functional cysteine essential for the activity of guinea pig liver transglutaminase. J Biol Chem 241(13):3238–3240
Gaspar AL, de Góes-Favoni SP (2015) Action of microbial transglutaminase (MTGase) in the modification of food proteins: a review. Food Chem 171:315–322. https://doi.org/10.1016/j.foodchem.2014.09.019
Gorman JJ, Folk JE (1980) Structural features of glutamine substrates for human plasma factor XIIIa (activated blood coagulation factor XIII). J Biol Chem 255(2):419–427
Griffin M, Casadio R, Bergamini CM (2002) Transglutaminases: nature’s biological glues. Biochem J 368(Pt 2):377–396. https://doi.org/10.1042/BJ20021234
Grossowicz N, Wainfan E, Borek E, Waelsch H (1950) The enzymatic formation of hydroxamic acids from glutamine and asparagine. J Biol Chem 187(1):111–125
Guan C, Cui W, He X, Hu X, Xu J, Du G, Chen J, Zhou Z (2015) Construction and development of a novel expression system of Streptomyces. Protein Expr Purif 113:17–22. https://doi.org/10.1016/j.pep.2015.04.009
Gundersen MT, Keillor JW, Pelletier JN (2014) Microbial transglutaminase displays broad acyl-acceptor substrate specificity. Appl Microbiol Biotechnol 98(1):219–230. https://doi.org/10.1007/s00253-013-4886-x
Guo S, Tang JJ, Wei DZ, Wei W (2014) Construction of a shuttle vector for protein secretory expression in Bacillus subtilis and the application of the mannanase functional heterologous expression. J Microbiol Biotechnol 24(4):431–439. https://doi.org/10.4014/jmb.1311.11009
Harwood CR (1992) Bacillus subtilis and its relatives: molecular biological and industrial workhorses. Trends Biotechnol 10(7):247–256. https://doi.org/10.1016/0167-7799(92)90233-L
Heckman KL, Pease LR (2007) Gene splicing and mutagenesis by PCR-driven overlap extension. Nat Protoc 2(4):924–932. https://doi.org/10.1038/nprot.2007.132
Jaros D, Partschefeld C, Henle T, Rohm H (2006) Transglutaminase in dairy products: chemistry, physics, applications. J Texture Stud 37(2):113–155. https://doi.org/10.1111/j.1745-4603.2006.00042.x
Kieliszek M, Misiewicz A (2014) Microbial transglutaminase and its application in the food industry. A review. Folia Microbiol (Praha) 59(3):241–250. https://doi.org/10.1007/s12223-013-0287-x
Kikuchi Y, Date M, Yokoyama K, Umezawa Y, Matsui H (2003) 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(1):358–366. https://doi.org/10.1128/AEM.69.1.358-366.2003
Ling LF, Zi RX, Wei FL, Jiang BS, Ping L, Hu CX (2007) Protein secretion pathways in Bacillus subtilis : implication for optimization of heterologous protein secretion. Biotechnol Adv 25(1):1–12. https://doi.org/10.1016/j.biotechadv.2006.08.002
Liu L, Liu Y, Shin HD, Chen RR, Wang NS, Li J, Du G, Chen J (2013) Developing Bacillus spp. as a cell factory for production of microbial enzymes and industrially important biochemicals in the context of systems and synthetic biology. Appl Microbiol Biotechnol 97(14):6113–6127. https://doi.org/10.1007/s00253-013-4960-4
Maarten VDJ, Michael H (2013) Bacillus subtilis: from soil bacterium to super-secreting cell factory. Microb Cell Factories 12(1):3. https://doi.org/10.1186/1475-2859-12-3
Macedo JA, Sette LD, Sato HH (2011) Purification and characterization of a new transglutaminase from Streptomyces Sp. isolated in Brazilian soil. J Food Biochem 35(4):1361–1372. https://doi.org/10.1111/j.1745-4514.2010.00456.x
Marx CK, Hertel TC, Pietzsch M (2007) Soluble expression of a pro-transglutaminase from Streptomyces mobaraensis in Escherichia coli. Enzym Microb Technol 40(6):1543–1550. https://doi.org/10.1016/j.enzmictec.2006.10.036
Masayo, Yokoyama K i, Umezawa Y, Matsui H, Kikuchi Y (2004) High level expression of Streptomyces mobaraensis transglutaminase in Corynebacterium glutamicum using a chimeric pro-region from Streptomyces cinnamoneus transglutaminase. J Biotechnol 110(3):219–226. https://doi.org/10.1016/j.jbiotec.2004.02.011
Mu D, Montalbán-López M, Masuda Y, Kuipers OP (2013) Zirex: a novel zinc-regulated expression system for Lactococcus lactis. Appl Environ Microbiol 79(14):4503–4508. https://doi.org/10.1128/AEM.00866-13
Mu D, Lu J, Shu C, Li H, Li X, Cai J, Luo S, Yang P, Jiang S, Zheng Z (2018) Improvement of the activity and thermostability of microbial transglutaminase by multiple-site mutagenesis. Biosci Biotechnol Biochem 82(1):106–109. https://doi.org/10.1080/09168451.2017.1403881
Nguyen HD, Phan TTP, Schumann W (2007) Expression vectors for the rapid purification of recombinant proteins in Bacillus subtilis. Curr Microbiol 55(2):89–93. https://doi.org/10.1007/s00284-006-0419-5
Nielsen PM (1995) Reactions and potential industrial applications of transglutaminase. Review of literature and patents. Food Biotechnol 9(3):119–156. https://doi.org/10.1080/08905439509549889
Niwa Y, Matsumura M, Shiratori Y, Imamura M, Kato N, Shiina S, Okudaira T, Ikeda Y, Inoue T, Omata M (1996) Quantitation of α-fetoprotein and albumin messenger RNA in human hepatocellular carcinoma. Hepatology 23(6):1384–1392. https://doi.org/10.1053/jhep.1996.v23.pm0008675155
Phan TT, Nguyen HD, Schumann W (2006) Novel plasmid-based expression vectors for intra- and extracellular production of recombinant proteins in Bacillus subtilis. Protein Expr Purif 46(2):189–195. https://doi.org/10.1016/j.pep.2005.07.005
Salis B, Spinetti G, Scaramuzza S, Bossi M, Saccani Jotti G, Tonon G, Crobu D, Schrepfer R (2015) High-level expression of a recombinant active microbial transglutaminase in Escherichia coli. BMC Biotechnol 15:84. https://doi.org/10.1186/s12896-015-0202-4
Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
Song Y, Nikoloff JM, Zhang D (2015) Improving protein production on the level of regulation of both expression and secretion pathways in Bacillus subtilis. J Microbiol Biotechnol 25(7):963–977. https://doi.org/10.4014/jmb.1501.01028
Suzuki M, Taguchi S, Yamada S, Kojima S, Miura KI, Momose H (1997) A novel member of the subtilisin-like protease family from Streptomyces albogriseolus. J Bacteriol 179(2):430–438. https://doi.org/10.1128/jb.179.2.430-438.1997
Taguchi S, Arakawa K, Yokoyama K, Takehana S, Takagi H, Momose H (2002) Overexpression and purification of microbial pro-transglutaminase from Streptomyces cinnamoneum and in vitro processing by Streptomyces albogriseolus proteases. J Biosci Bioeng 94(5):478–481. https://doi.org/10.1016/S1389-1723(02)80228-6
Wu XC, Lee W, Tran L, Wong SL (1991) Engineering a Bacillus subtilis expression-secretion system with a strain deficient in six extracellular proteases. J Bacteriol 173(16):4952–4958. https://doi.org/10.1128/jb.173.16.4952-4958.1991
Yang M, Chang C, Wang JM, Wu TK, Wang Y, Chang C, Li TT (2011) Crystal structure and inhibition studies of transglutaminase from Streptomyces mobaraense. J Biol Chem 286(9):7301–7307. https://doi.org/10.1074/jbc.M110.203315
Yokoyama K, Nakamura N, Seguro K, Kubota K (2000) Overproduction of microbial transglutaminase in Escherichia coli, in vitro refolding, and characterization of the refolded form. Biosci Biotechnol Biochem 64(6):1263–1270. https://doi.org/10.1271/bbb.64.1263
Yokoyama K, Nio N, Kikuchi Y (2004) Properties and applications of microbial transglutaminase. Appl Microbiol Biotechnol 64(4):447–454. https://doi.org/10.1007/s00253-003-1539-5
Zhang M, Shi M, Zhou Z, Yang S, Yuan Z, Ye Q (2006) Production of Alcaligenes faecalis penicillin G acylase in Bacillus subtilis WB600 (pMA5) fed with partially hydrolyzed starch. Enzym Microb Technol 39(4):555–560. https://doi.org/10.1016/j.enzmictec.2006.01.011
Zhang J, Kang Z, Ling Z, Cao W, Liu L, Wang M, Du G, Chen J (2013) High-level extracellular production of alkaline polygalacturonate lyase in Bacillus subtilis with optimized regulatory elements. Bioresour Technol 146(146C):543–548. https://doi.org/10.1016/j.biortech.2013.07.129
Funding
This work was supported by Anhui Provincial Natural Science Foundation (grant number 1708085QC65); Fundamental Research Funds for the Central Universities, China (grant number JZ2016HGBZ0777); National High Technology Research and Development Program (“863” Program) of China (grant number 2013AA102201); Major Project of Science and Technology of Anhui Province, China (grant number 1301031031) and Natural Science Foundation of China (grant number 51702004).
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Ethical statement
This article does not contain any studies with human participants or animals performed by any of the authors.
Additional information
Dongdong Mu and Jiaojiao Lu Shared the first authors
Rights and permissions
About this article
Cite this article
Mu, D., Lu, J., Qiao, M. et al. Heterologous signal peptides-directing secretion of Streptomyces mobaraensis transglutaminase by Bacillus subtilis. Appl Microbiol Biotechnol 102, 5533–5543 (2018). https://doi.org/10.1007/s00253-018-9000-y
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-018-9000-y