Expression Of Streptomyces Mobaraensis Microbial Transglutaminase In Gram-positive Bacteria

Transglutaminase(EC 2.3.2.13,protein-glutamine gammaglutamyl transferase,TG)is an enzyme that catalyzes the acyl transfer reaction of a y-carboxamide group and an amide group between a peptide or a protein primary amine.The products crosslinked by transglutaminase have high stability and are highly resistant to protease degradation and chemical damage.Therefore,they are widely used in various fields such as food,medicine,textile,and material engineering.Microbial transglutaminase(MTG)has become a hot issue because of its wide range of substrates and independent of Ca2+,but it has problems of high production cost and complex purification process.In this paper,rmtg derived from Strepto,myces mobaraensis was used to construct MTG constitutive and inducible recombinant expression vectors in Bacillus subtilis and Lactococcus lactis,respectively.The recombinant strains are optimized for expression time and inducer concentration to obtain high-yield recombinant strains.MTG of high enzyme activity is obtained by zymogen activation and Ni column purification.Compared with commercial MTG,it can catalyze the cross-linking of soy protein isolate more efficiently.The main findings are as follows:(1)Using the genomic DNA of S.mobaraensis as a template,the mtg gene was amplified and fused behind the heterologous signal peptide SPwapA/amyQ,and the fusion fragment SPwapA/amyQ-mtg was ligated to the constitutive(pMA5)and inducible(pHT43)expression vectors.respectively.The recombinant vectors pMA5mtg and pHT43mtg were constructed in Escherichia coli and transformed into B.subtilis for heterologous secretion.SDS-PAGE protein electrophoresis showed that the signal peptide SPwapA could direct the secretion of MTG in B.subtilis.The amount of wild-type B.subtilis 168 produced was slightly higher than that of the six protease-deficient B.subtilis WB600,but there was no significant difference,indicating that MTG may not be degraded by six endogenous extracellular proteases/peptidases in B.subtilis.When the expression time is 48 h,recombinant B.subtilis 168 obtains the highest yield of MTG;signal peptide SPamyocould direct the secretion of MTG in B.subtilis,secretory expression was carried out in B.subtilis 168 with an expression time of 12 h and an inducer IPTG concentration of 80 ?M to obtain the highest enzyme production.After zymogen activation and purification by Ni column,SPwapA directed secretion of 63.0 ± 0.6 mg/L MTG,enzyme activity was 27.0 ± 0.4 U/mg;SPamyQ directed secretion of 54.1 ± 0.3 mg/L MTG,enzyme activity was 29.6 ± 0.9 U/mg.Compared with SPwapA,SPamyQ has a lower secretion of S.mobaraensis MTG from Bacillus subtilis,which may be due to the better transport ability of B.subtilis natural signal peptide or because the Sec pathway is not as good as the protein secretion by the Tat pathway.The mature MTG obtained after the activation of the zymogen was applied to the soy protein isolate cross-linking reaction,and it was found that MTG can effectively catalyze the formation of cross-linking of the soy protein isolate,and its catalytic effect is higher than that of the commercial MTG.(2)Using genomic DNA of S.mobaraensis as a template,the,mtg gene was amplified and fused with the heterologous signal peptide SPusp45,and the fusion fragment was ligated to the constitutive(pNZ8048-p5)and inducible(pNZ8048)expression vectors,respectively.The MTG recombinant expression vectors pNZ8048-Pp5-SPusp45(K2A)-pro,mtg and pNZ8048-SPusp45-promtg,which are transcribed by the constitutive promoter(Pp5)and the inducible promoter(PnisA),are heterologously secreted in L.lactis.SDS-PAGE protein electrophoresis showed that under the control of constitutive promoter Pp5,the signal peptide SPusp45(K2A)could direct the secretion of MTG in L.lactis,and the highest yield of MTG was obtained at 12 h.Under the control of PnisA,the signal peptide SPusp45 can direct the secretion of MTG in L.lactis.The expression time is optimized at 48 h,and the optimal inducer concentration of nisin is 5 ng/mL.After zymogen activation and purification by Ni column,SPusp45(K2A)was induced to secrete 70.5 ± 0.4 mg/L MTG with an enzyme activity of 30.6 ± 0.5 U/mg;SPusp45 was induced to secrete 65.2 ± 0.5 mg/L MTG with an enzyme activity of 29.4 ±0.3 U/mg.There was no significant difference between SPusp45(K2A)and SPusp45 in guiding the secretion of MTG,indicating that the mutation of the lysine at position 2 of the signal peptide SP,sp45 to alanine due to the introduction of the NcoI restriction site could not influence the secretion of heterologous MTG in L.lactis.The mature MTG obtained from the zymogen was applied to the soy protein isolate cross-linking reaction,and it was found that MTG can effectively catalyze the formation of cross-linking of the soy protein isolate,and its catalytic effect is higher than that of the commercial MTG.(3)Optimizing the expression yield of the MTG recombinant expression strain L.lactis(pNZ8048-SPusp45-promtg),and the mature mtg was cloned into L.lactis(pNZ804S-SPusp45-promtg)in an attempt to improve the growth of the recombinant strain L.lactis,thereby improving heterologous expression of MTG-6His.The growth curve of L.lactis(pNZ8048-SPusp45-pro,mtg-,mature-,mtg)and L.lactis(pNZ8048-SP,Sp45-promtg)were determined,and the results showed that L.lactis(pNZ8048-SPusp45-promtg-mature-,mtg)has a higher growth rate in the exponential growth phase.After reaching the stationary phase,the strain OD600 was stabled at about 3.2,indicating that the presence of mature MTG improved the growth of L.lactis strain.Under the same induction conditions,L.lactis(pNZ8048-SPusp45-promtg-mature-mtg)produced more pro-MTG-6His than L.lactis(pNZ8048-SPusp45-promtg),indicating that the presence of mature MTG may promote the growth of the L.lactis strain and then accelerates the synthesis of cellular material and enhances the secretion of heterologous pro-MTG-6His.