Screening Of Urethanase And Its Molecular Modifications

Ethyl carbamate(EC) is a potential carcinogen to human that commonly exists in traditional fermented foods and beverages. This compound normally formed in the process of fermentation and storage. EC can be hydrolyzed by urethanase and decomposed into ethanol, carbon dioxide and ammonia. The employment of urethanase in food processing may confer great potential value to ensure food safety. Urethanase(UH) with fine substrate specificity and affinity, as well as acid and ethanol tolerance is not available. Therefore, looking for acidic urethanase with good stability and high substrate affinity is an important prerequisite for its industrial application.This study aimed on obtaining urethanase that meet the industrial requirements. Urethanase with good application properties can be obtained by screening strains that produce novel urethanase or selection of available enzymes with better properties. Molecular modification of urethanase will be performed to improve its application characteristics such as substrate affinity and resistance to ethanol and acid. The main results are:1. Three strains with urethanase activity were isolated from Daqu using high-throughput screening techniques. Among them the crude urethanase of Staphylococcus saprophyticus L1 showed better ethanol tolerance. Purification of this urethanase was done by ammonium sulfate precipitation, ion exchange chromatography, hydrophobic interaction chromatography and gelfiltration chromatography. Tolerance to ethanol of this purified enzyme was dramatically reduced. In addition, the ethanol tolerance of urethanase from S. saprophyticus L1 was worse than that of a previously characterized UH from L. fusiformis SC02. Therefore, UH of L. fusiformis SC02 was chosen for molecular modifications.2. Six self-assembling amphipathic peptides were fused at the N-terminal of L. fusiformis UH and were overexpressed in E.coli BL21. It was found that only SAP1-UH retained enzyme activity. The optimal reaction temperature of SAP1-UH was unchanged. However, SAP1-UH exhibited a slightly lower optimum pH than the wild type UH. The t1/2 of SAP1-UH at 40 oC was increased from 3.7 min to 15.4 min, which was 4.2 folds longer than that of the original enzyme. Substrate specific activity of SAP1-UH was increased by 93%.3. The three dimensional structure of L. fusiformis SC02 UH was built using homologous modelling. Then the RMSF value and B-factor of amino acids of UH were also predicted based on molecular dynamics and protein folding free energy change. Q328 and Q357 were characterized to be the unstable residue of L. fusiformis UH. The saturated mutants of Q328 site in unstable region were constructed and Q328 C, Q328 V, Q328 R, Q328 H with catalytic activity was obtained. The half-life of mutant Q328 C at 40 oC were detected to be 7.46 folds higher than that of the original enzyme. The tolerance of the mutant Q328 C to ethanol and acid also increased.4. To improve the application properties of UH from L. fusiformis, Q328 C derivatives with mutation on Q357 site were constructed. A mutant Q328C:Q357W with better stability was achieved. The t1/2 of Q328C:Q357W at 40 oC was increased to 89.4 min, which is 23.8 folds higher than the wild type UH. The optimum pH of Q328C:Q357W was changed from 7 to 6.5. Its relative activity at pH 6.0, 5.5 and 5.0 was increased by 30.2%, 21% and 7%, respectively. Moreover, Q328C:Q357W showed improved ethanol tolerance, the relative activity was increased by 29%、22.1% and 8.5%, respectively, when incubated in 2%, 5% and 10% ethanol. The K_m of Q328C:Q357W reduced 2 times compared with that of wild type UH.