| Transglutaminase is a transferase that catalyzes the transfer of acyl group, resulting incrosslinking among the proteins. It can improve water-solubility, retentiveness and thermalstability of protein.We are focusing on the transglutaminase from Streptomyces mobaraensis. Thetransglutaminase gene was optimizied for codon usage and expressed in E. coli. Resultsdemonstrated that GC content of the optimized gene decreased and its expression level wasimproved by3.4-fold than that of the unoptimized gene. In order to improve the specificactivity of transglutaminase, the serine at the second codon of mature transglutaminaseregions was mutated to proline by site-directed mutagenesis technology. The specific activitywas improved by1.26-fold. The codon usage optimization and site-directed mutagenesistechnology could be utilized to improve the expression level of pro-transglutaminase in E.coli.Many proteases are applied to activating proMTG by the removal of the pro-peptide. Butthose proteases are not specific resulting digestion whole proMTG. To overcome thisdisadvantage, we introduce a highiy specific cleavage site(DDDDK) of enterokinase betweenthe pro-peptide and mature MTG and then expressed in E. coli. The enterokinase was appliedto activate proMTG with the highly specificity. Results suggested that we can removal of thepro-peptide using enterokinase without degrading the mature MTG.Due to no expression of proMTG with scretory manner in E.coli, we used Bacillussubtilis WB800as host strain alteratively. But the enterokinase can not be expressed inB.subtilis WB800. The protease SAMP45from Streptomyces albogriseolus,which canexpressed in B.subtilis, was used to activate proMTG, and it gene was integrated to thegenome of B.subtilis WB800, named B.subtilis WB800S. proMTG was also co-expressed inB.subtilis WB800S host strain. Results demonstrated that proMTG was expressed with thescretory manner and then activated specifically in culture media. After fermentation, theenzyme activity was up to4.6U/mL. |
