Characterization And Heterologous Expression Of Urethanase From Lysinibacillus Fusiformis

Ethyl carbamate or urethane (EC), a by-product of fermented foods and beverages, wasformed in the period of fermentation and storage. It is carcinogenic to animals and human,which may induce lung tumor, liver cancer, skin cancer and so on. Urethane can be degradedby urethanase yield nontoxic substance of ammonia, ethanol and carbon dioxide. Theapplication of urethanase is probably practical way for reduction the risk of cancer and toensure food safety.This study focused on screening and identification of microorganisms that could degradeurethane and characterization and heterologous expression of urethanase. The main results are:1. A Lysinibacillus fusiformis strain was isolated from gastrointestinal tracts of mice,which can degrade urethane.2. The protein with urethanase activity was purified to electrophoretic homogeneity byammonium sulfate precipitation, ion exchange chromatography, hydrophobic interactionchromatography and gelfiltration chromatography. It was purifed279-fold to the specifcactivity of81.7U·mg-1with recovery of7%. The monomeric molecular weight of thisurethanase was detected to be approximate50kDa by SDS-PAGE.3. Enzymatic properties analysis demonstrated that the purified enzyme was stable attemperature range of15-45℃, and the optimal reaction conditions were determined to be at35℃(pH7.0). The urethanase activity was inhibited by metal ions but EDTA had no effecton enzyme activity. The results showed that the urethanase was not an ion-associated enzyme.Using urethane as the substrate, the kinetic constants of Kmand Vmaxwere detected to be37.2mmol·L-1and7764.1μmol·min-1, respectively. In addition, the enzyme showed tolerance tolow concentration of ethanol and NaCl. The N-terminal sequence was determined to beTTDLHLKSVEEL.The specific primers were designed by the blast result of N-terminal sequence withNCBI database. Then, A1419bp DNA fragment was sequenced and cloned based on theurethanase N-terminal sequence. Expression of this DNA fragment confirmed urethanaseactivity in E. coli BL21(DE3).4. The solubility of the recombinant urethanase was increased by the means ofoptimizing fermentation medium, temperature, IPTG concentration, and fermentation time inE. coli BL21(DE3). The activity was increased by5.3times per unit cell while the activitywas increased by16-fold per unit volume.