Fermentation Optimization And Control Of Microbial Urethanase Production By Lysinibacillus Fusiformis SC02

Ethyl carbamate or urethane(EC), a potential carcinogen, exists in most alcohol beverages and fermented foods. EC is a stable compound and is difficult to be eliminated once formed from its precursors. Thus, employment and development of enzymes for degrading EC is of great importance and helpful for controlling and reduction of EC in fermented foods.In this study, medium composition and fermentation conditions for urethanase production by L. fusiformis SC02 was optimized using single-factor analysis method. The optimal medium composition was confirmed by response surface method.Optimization of fermentation conditions was performed in the bioreactor for developing fermentation optimization strategies. The main results are:(1) The fermentation medium for urethanase production was optimized by using single-factor test. The optimal medium composition was determined to be: galactose 25 g×L-1, soy peptone 20 g×L-1, urea 4 g×L-1, Cu SO4 0.02 g×L-1, initial p H 7.0; the optimal conditions for enzyme-producing were: temperature 37 oC, inoculum size 3%, loading 20 ml medium in a 250 ml flask. The titre of urethanase was improved to 4500 U×L-1, which was 350% higher than the control.(2) Soy peptone and medium initial p H were determined to be critical factors for urethanase production by using Placket-Burman. Then the steepest climbing experiment and response surface analysis were applied to optimize these two factors. At last, the optimal fermentation medium components were determined as follows: galactose 25 g×L-1, soy peptone 25.43 g×L-1, urea 4 g×L-1, Cu SO4 0.02 g×L-1, initial p H 7.58. Urethanase production level of the strain was up to 6161.79 U×L-1 after being cultured for 12 h using the medium mentioned above, which was 37% higher than the one under the original production level 4500 U×L-1.(3) Fermentation of urethanase by L. fusiformis SC02 was optimized by employment of agitation control strategy and feeding strategyin a 3 L fermentor. The production of urethanase was increased to 8206.3 U×L-1 by a two-stage agitation control strategy, which was improved almost 82.32%. By using the feeding strategy, the yield of urethanase reached to 8570 U×L-1.