title: Key residues responsible for enhancement of catalytic efficiency of Thermomyces lanuginosus lipase Lip revealed by complementary protein engineering strategy.
abstract: The variant Lip-T (S88T/A99N/V116D) of lipase Lip from Thermomyces lanuginosus has been proved to be a potential biocatalyst for kinetic resolution of 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester (CNDE) to produce valuable chiral intermediate of Pregabalin. In this study, random, site-directed and site-saturation mutagenesis were performed to further enhance the activity of Lip-T, and the key residues responsible for catalytic efficiency were revealed. A mutant S63L/D232A with improved activity toward CNDE was obtained after screening of approximately 2500 clones from random-mutant libraries. Site-directed mutagenesis at site 63 and 232 demonstrated that the single-point mutants S63L and D232A showed opposite effect on activity. S63L exhibited a significant improvement on activity, whereas D232A exerted a slight inhibitory effect. Then a mutant S63M with a 4.5-fold higher catalytic efficiency than Lip-T was obtained by site-saturation mutagenesis. Structural changes resulting from the mutations were analyzed and the mechanisms responsible for the enhanced activity were discussed. Moreover, the engineered lipase catalyzed enantioselective hydrolysis of CNDE at a very high substrate loading (765 g/l). As only 5% (w/v) resting cells were used, the bioprocess is much more cost-effective than Pfizer's process using 8% (w/v) commercially available lipase Lipolase(®). These results provide not only new insights into lipase structure-function relationships but also a novel robust biocatalyst for the production of Pregabalin.
authors.affiliation: Institute of Bioengineering, Zhejiang University of Technology, Hangzhou 310014, China; Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou 310014, China.
authors.forename: Xiao-Jun
authors.initials: XJ
authors.lastname: Li
authors.affiliation: Institute of Bioengineering, Zhejiang University of Technology, Hangzhou 310014, China; Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou 310014, China.
authors.forename: Ren-Chao
authors.initials: RC
authors.lastname: Zheng
authors.affiliation: Institute of Bioengineering, Zhejiang University of Technology, Hangzhou 310014, China; Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou 310014, China.
authors.forename: Hong-Ye
authors.initials: HY
authors.lastname: Ma
authors.affiliation: Institute of Bioengineering, Zhejiang University of Technology, Hangzhou 310014, China; Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou 310014, China.
authors.forename: Jian-Feng
authors.initials: JF
authors.lastname: Huang
authors.affiliation: Institute of Bioengineering, Zhejiang University of Technology, Hangzhou 310014, China; Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou 310014, China. Electronic address: zhengyg@zjut.edu.cn.
authors.forename: Yu-Guo
authors.initials: YG
authors.lastname: Zheng
mesh_terms: D001203:Ascomycota / Q000201:enzymology*; D002384:Catalysis; D007700:Kinetics; D008049:Lipase / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D015202:Protein Engineering; D013379:Substrate Specificity
chemical_list: D008049:Lipase
keywords: Catalytic efficiency; Error-prone polymerase chain reaction; Lipase; Pregabalin; Protein engineering