High-level Expression And Thermostability Modification Of Streptomyces Mobaraensis Transglutaminase

Transglutaminase（Transglutaminase,EC2.3.2.13,TGase）can transfer acyl group from y-carboxamide group（acyl donor）of the glutamine residue to acyl acceptor.It has a wide range of applications in food processing,biomedicine,textile materials and leather processing.Streptomyces mobaraensis is the most important source of microbial TGase in industrial production nowadays,but the products are mostly concentrated as insoluble,and it is required to subsequent processing of the cleavage of zymogen by dispase.With the increasing demand for TGase in industries,it has become an urgent problem that how to achieve high-efficiency expression of pro-TGase extracellularly.In this study,the proM-TGase of S.mobaraensis was expressed in Escherichia coli BL21（DE3）.The extracellular high-efficiency expression of pro-TGase was achieved through strategies such as optimization of fermentation conditions,construction of high expression strains,and construction of autophagic strains.The thermostability of TGase is improved by forming disulfide bonds in protein molecules.There are main results:（1）The improvement of yield of pro-TGase by optimization of fermentation and genetic engineering in E.coli.The pro-TGase gene in S.mobaraensis was cloned into vector pET-22b to obtain the recombinant plasmid pET-22b-proM-TGase,which was transformed into E.coli BL21（DE3）.Recombinant pro-TGase is activated by dispase in vitro.The optimal expression condition for recombinant bacteria is to use TB medium with adding 0.5%ethanol,induce the cell when OD₆₀₀=1.00 and add 0.1 mM isopropyl-β-D-thiogalactoside under 20℃.Under this condition,the recombinant bacteria fermented for 36 h,and the total intracellular and extracellular enzyme activities reached 2.75 U mL^-1.In order to further increase the expression of TGase,the zymogen region of S.mobaraensis pro-TGase was replaced with proC（Streptomyces caniferus,Genebank:AM746294.1）、proF（Streptomyces fradiae,Genebank:AM746294.1）、proH（Streptomyces hygroscopicus,Genebank:EU477523.1）、proN（Streptomycesnetropsis,Genebank:EF195356.1）and prop（Streptomyces platensis,Genebank:AM746296.1）.The expression results showed that the expression of TGase fused with proC and proH zymogen regions was 1.43 and 1.13 times higher than that of S.mobaraensis pro-TGase,respectively.Through the optimization measures such as high expression,high stability site mutation,codon optimization and integration of solubilizing protein tags TrxA,MBP and GST,the yield of expression of pro-TGase of the recombinant plasmid pET-22b-TrxA-proC-MS-OP finally reached to 25.00 U mL^-1,and the extracellular yield was 0.50 mg mL^-1.（2）The construction of autolysis system to realize high-efficiency extracellular expression of pro-TGaseOn the basis of the efficient expression of the pro-TGase plasmid,the plasmid pRSFDuet-1-SRRz which can express the phage cleavage protein SRRz was introduced and co-expressed with pro-TGase.The temperature-sensitive promoter PL/PR induces the expression of lambda phage cleavage protein SRRz.In the middle and late stages of fermentation,the cells are lysed by heating,and the pro-TGase enriched in the cell is released to the outside of the cell.The extracellular TGase activity is 21.30 U mL^-1.（3）The construction of disulfide bonds to screen of high-stability mutant TGaseFirstly,the simulated structure of TGase thermostable mutant MS（S2P-S23V-Y24N-S199A-K294L）was constructed based on the crystal structure of wild-type TGase（PDB:liu4）.Secondly,the disulfide bond was predicted by disulfide by Design 2.0.According to the free energy of disulfide bond formation,12 disulfide bond mutants were selected and expressed in E.coli BL21（DE3）.Finally,the enzymatic properties of the purified mutants were characterized,and the stabilization mechanism was analyzed.The results showed that the residual enzyme activities of D118C-K121C,P244C-E249C and P22C-Q328C were 77.39%,71.58%and 91.06%higher than those of MS after treatment at 60℃ for 20 min,respectively.Compared to MS,the t1/2（60℃）and tm of MS-P22C-Q328C increased by 2.06 times and 1.06℃,respectively,but its specific activity was decreased by 13.20%.