High-level Expression And Molecular Modification Of Streptomyces Mobaraense Transglutaminase In Yarrowia Lipolytica

Transglutaminase?EC 2.3.2.13,TGase?is an enzyme that catalyzes the covalent cross-ling of proteins or polypeptides.TGase is widely used in the field of food processing,pharmaceutical,textiles,leather processing and other fields.With the increasing demand of TGase,the high level expression of active TGase is becoming more and more important.The TGase gene derived from S.mobaraense was firstly expressed in recombinat Y.lipolytica Po1h in this study.Then,the yield of TGase was significantly improved by replacing the pro-region of transglutaminase.Next,the active TGase was successfully expressed by inserting the Kex2 recognition site between the pro-region and mature-region of transglutaminase,and co-expressing hpro-TGase and transglutaminase activating metalloprotease?TAMEP?.In addition,the specific activity and catalytic activity of TGase were increased by site-directed mutagenesis.The main results are as follows:?1?The mpro-TGase gene was expressed in recombinant Y.lipolytica Po1hp INA1297/mpro-TGase was obtained by cloning the S.mobaraensem TGase zymogen?mpro-TGase,mpro is the pro-region of S.mobaraense?into the multi-copy integrated plasmid p INA1297.Which was transformed into Y.lipolytica Po1h to obtain the recombinant Y.lipolytica Po1h/mpro-TGase,the activity of TGase in intracellular and extracellular was1.25 and 0.11 U�m L^-1,respectively.The recombinat Y.lipolytica Po1h/hpro-TGase was obtained by the replacement of S.mobaraense pro-region mpro with Streptomyces.hygroscopicus pro-region hpro.The activity of TGase in extracellular reached 11.69 U�m L^-1,which was 106 times higher than that of Y.lipolytica Po1h/mpro-TGase.Moreover,the activity reached 41.21 U�m L^-1 in 3 L tank batch fermentation.Obviously,the pro-region has an important influence on the expression and secretion of transglutaminase.And site-directed mutagenesis analysis showed that the first two amino acids at the N-terminus of the pro-region were the key amino acids that affected the expression of TGase in Y.lipolytica Po1h.?2?Expression of actived TGase in recombinant Y.lipolytica Po1hBased on p INA1297/hpro-TGase,by inserting the Kex2 protease recognition site in between hpro and TGase?strategy 1?and co-expressing hpro-TGase and TAMEP?strategy 2?,hpro-TGase was expressed in Y.lipolytica at first,then the pro-region of hpro-TGase was excised and the mature TGase was achieved.The results of shake flask fermentation showed that the TGase activity of recombinant strains constructed by strategy 1 and strategy 2 were5.26 U�mL^-1 and 6.77 U�mL^-1,respectively.Moreover,the enzymatic properties were further studied,the results showed that the specific activities,K_m and k_cat/K_m of recombinant TGase obtained by strategy 1 and strategy 2 were significantly higher than those of S.mobaraensis TGase.?3?Increasing the catalytic activity of TGase by site-directed mutagenesisIn order to enhance the catalytic activity of TGase,the amino acid sites that could affect the binding energy of S.mobaraense TGase and its substrate?-N-CBZ-GLN-GLY was predicted through Discovery Studio 2017,then mutants with reduced binding energy were constructed:Y24W,E300W and Y302R.Compared with wild TGase,the specific activity of E300W were increased by 31%;the of K_m,k_cat and k_cat/K_m vaule of E300W were increased by10%,42%and 29%respectively,Which showed that the increase of specific enzyme activity is mainly due to the increase of enzymatic conversion number.While the thermal stability of all the mutants decreased in varying degrees.And the structure analysis indicated that the hydrophobic interactions and ion-interactions were the main factors affecting the thermal stability of TGase.These results suggested that the strategy based on the analysis of binding free energy can rapidly identify the key amino acids that affect the catalytic activity of TGase,and further mutation may effectively improve its catalytic activity.