کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
6491409 | 43411 | 2014 | 7 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Key residues responsible for enhancement of catalytic efficiency of Thermomyces lanuginosus lipase Lip revealed by complementary protein engineering strategy
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کلمات کلیدی
موضوعات مرتبط
مهندسی و علوم پایه
مهندسی شیمی
بیو مهندسی (مهندسی زیستی)
پیش نمایش صفحه اول مقاله
![عکس صفحه اول مقاله: Key residues responsible for enhancement of catalytic efficiency of Thermomyces lanuginosus lipase Lip revealed by complementary protein engineering strategy Key residues responsible for enhancement of catalytic efficiency of Thermomyces lanuginosus lipase Lip revealed by complementary protein engineering strategy](/preview/png/6491409.png)
چکیده انگلیسی
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 (765Â 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.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Biotechnology - Volume 188, 20 October 2014, Pages 29-35
Journal: Journal of Biotechnology - Volume 188, 20 October 2014, Pages 29-35
نویسندگان
Xiao-Jun Li, Ren-Chao Zheng, Hong-Ye Ma, Jian-Feng Huang, Yu-Guo Zheng,