期刊
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
卷 66, 期 49, 页码 13011-13019出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.jafc.8b04566
关键词
urease; protein engineering; affinity; catalytic efficiency; ethyl carbamate; urea; rice wine
资金
- National Key Research and Development Program of China [2017YFC1600403, 2017YFC1600405]
- Fundamental Research Funds for the Central Universities [JUSRP51707A]
- National First-Class Discipline Program of Light Industry Technology and Engineering [LITE2018-16]
- Key Research and Development Program of Jiangsu Province [BE2016689]
Bacillus paralicheniformis urease (BpUrease) has been shown to be a promising biocatalyst for degrading the carcinogenic chemical ethyl carbamate (EC or urethane) in rice wine. However, low EC affinity and catalytic efficiency limit the practical application of BpUrease. In this study, we improved the EC degradation capability of BpUrease by site-saturation mutagenesis (SSM). The best variant L253P/L287N showed a 49% increase in EC affinity, 1027% increase in catalytic efficiency (k(cat)/K-m), and 583% increase in half-life (t(1/2)) at 70 degrees C. Homology modeling analysis suggest that mutation of Leu253 to Pro increased the BpUrease EC specificity by affecting the interaction between Arg339 with the catalytic residue His323, while Leu287Asn mutation benefits EC specificity and affinity by changing the interaction networks among the residues in the catalytic pocket. Our results show that the L253P/L287N variant efficiently degraded urea and EC in a model rice wine, making it a good candidate for practical application in the food industry.
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