Format

Send to

Choose Destination
See comment in PubMed Commons below
J Biotechnol. 2014 Oct 20;188:29-35. doi: 10.1016/j.jbiotec.2014.08.004. Epub 2014 Aug 10.

Key residues responsible for enhancement of catalytic efficiency of Thermomyces lanuginosus lipase Lip revealed by complementary protein engineering strategy.

Author information

1
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.
2
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.

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.

KEYWORDS:

Catalytic efficiency; Error-prone polymerase chain reaction; Lipase; Pregabalin; Protein engineering

PMID:
25116359
DOI:
10.1016/j.jbiotec.2014.08.004
[Indexed for MEDLINE]
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for Elsevier Science
    Loading ...
    Support Center