Abstract: The paper investigated the improvement of the production of microbial transglutaminase( MTGase,MTG)by recombinant Escherichia coli BL21( DE3) / mtg in 250 m L shake flasks.Using single factor experiment,the types and the concentrations of carbon sources and nitrogen sources of the media,the inoculum sizes,the medium volumes,the culture temperature,the initial p H,the inducer concentration,the adding time of inducer,the induction temperature and the induction time were optimized,respectively. Among the above factors,3 key factors,the culture temperature,the initial p H and the induction temperature,were selected by Plackett- Burman design.And a response surface model of the 3 factors and the specific activity of MTG were constructed by Box- Benhnken design.The optimal conditions were as follows,the media contained 5 g / L of glucose,28 g / L of yeast extract and14 g / L of tryptone,and the bacteria were cultured in 50 m L( 250 m L triangle bottle),p H7.0 optimized media at34 ℃ for 4 h with 5%( v / v) inocula and were induced by 0.6 mmol / L IPTG at 25 ℃ for 14 h. Under the optimal conditions,the maximum specific activity of MTG was 1.507 U / mg,which was 1.56 times larger than the MTG produced in initial conditions.