Alteration of Chain-Length Selectivity and Thermostability of <i>Rhizopus oryzae</i> Lipase via Virtual Saturation Mutagenesis Coupled with Disulfide Bond Design
Jinsha Huang, Shuhan Dai, Xiying Chen, Li Xu, Jinyong Yan, Min Yang, Yunjun Yan
Abstract
Rhizopus oryzae lipase (ROL) is very attractive in biotechnology and industry as a safe and environmentally friendly biocatalyst. Functional expression of ROL in Escherichia coli facilitates effective high-throughput screening for positive variants. This work highlights a method to improve both selectivity and thermostability based on a combination of virtual saturation mutagenesis in the substrate pocket and disulfide bond prediction in the noncatalytic region. Using the method, ROL thermostability and activity to diverse 4-nitrophenyl esters could be substantially improved. The strategy of rational introduction of multiple mutations in different functional domains of the enzyme is a great prospect in the modification of biocatalysts.