Overexpression Of Urethanase In Bacillus Subtilis And Fermentation Optimization

Ethyl carbamate or urethane (EC), a by-product of fermented foods and alcoholic beverages, was formed in the process of fermentation. It has potential carcinogenicity and genotoxicity to human beings. Enzyme degradation is an efficiency way to reduce EC in fermented products. Urethanase (UH) degrades EC into ethanol, ammonia and carbon dioxide, makes it a bright application prospects in ensuring fermented foods safety.An urethanase was heterologously overexpressed and the fermentation conditions were optimized in this study. The main results are as follows:(1) The gene encoding urethanase (UH) from Lysinibacillus fusiformis SC02 was cloned (Bacillus subtilis WB600/pMA5-uh) and overexpressed in Bacillus subtilis WB600.At the same time, Lactococcus lactis and Bacillus subtilis secretory expression system was constructed for secretory production of UH.(2) Production of UH was optimized based on single factor experiments at shake flask level. The optimal fermentation medium was determined as:starch, malt extract, trypone, MgS04 and K2HPO4,. The optimal fermentation temperature and inoculum size were 37℃ and 4% respectively. Thus resulted in 7.20 U·mL1 urethanase under the optimal condition.(3) Trypone and K2HPO4 were determined to be critical factors for urethanase production by using Placket-Burman. The optimal fermentation medium was determined as:starch 10 g·L-1, K2HPO49 g·L-1, malt extract 25 g·L-1, MgSO4 10 mmol·L-1 and trypone 55 g·L-1. Production of recombinant enzyme in a 3 L fermenter showed that enzyme activity reached the highest titer of 18.03 U·mL-1 after 16 h, which was 17 folds of the original yield.(4) The capability to ethanol-tolerance and acidic condition of UH was improved by addition of stabilizers. The effects of 11 different stabilizers on the ethanol-tolerance capability were studied, glycerol, sucrose, glucose were chosen for further experiment. The optimal stabilizers was determined to be:glycerol 80 g·L-1, sucrose 3 g·L-1, glucose 1 g·L-1. The ethanol-tolerance, pH stability of UH were significantly improved by adding combined stabilizers.