脲酶基因挖掘及其在枯草芽孢杆菌中的重组表达

Gene Mining of Urease and Recombinant Expression in Bacillus subtilis

摘要:

氨基甲酸乙酯（ethyl carbamate，简称EC）是一种存在于黄酒中的潜在致癌物，酶法降解EC及其前体物质尿素因此备受关注。该研究通过基因挖掘获得了解淀粉芽孢杆菌IT-45的脲酶（Ba-urease）基因并在食品级系统枯草芽孢杆菌中实现了表达与应用。通过密码子优化、核糖体结合位点优化重构脲酶基因簇后，酶活力从6.85 U/mL提升至9.01 U/mL；通过调整基因ureC的位置，脲酶活力进一步提升至10.15 U/mL。研究发现，重组脲酶的最适反应温度为37 ℃；最适反应pH范围为6.5~7.0，为中性脲酶。摇瓶发酵的最佳培养条件为：TB培养基、接种体积分数5%、诱导温度28 ℃、Ni²⁺添加量4 mmol/L。在最佳发酵条件下，酶活力达到12.5 U/mL，在酶法降解黄酒中的尿素具有应用前景。

Abstract:

Ethyl carbamate (EC) is a potential carcinogenic chemical in yellow rice wine. Urease has attracted much attention because of its degradation of urea, the precursor of EC. To guarantee the food safety of rice wine, the gene encoding Bacillus amyloliquefaciens IT-45 urease (Ba-urease) was realized by gene mining and expressed in recombinant Bacillus subtilis. The enzyme activity of urease increased from 6.85 U/mL to 9.01 U/mL after the urease gene cluster reconstructed by the optimization of codon and ribosome binding site. The urease activity was further increased to 10.15 U/mL by adjusting the position of gene ureC. The optimal reaction condition of recombinant urease was 37 ℃ and pH 6.5~7.0. Under the optimal fermentation conditions, the enzyme activity reached 12.5 U/mL. The production of food-grade recombinant urease has potential application in traditional fermented food industry.