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Directed Evolution Of2-CPA Dehalogenases

Posted on:2013-04-02Degree:MasterType:Thesis
Country:ChinaCandidate:P LiuFull Text:PDF
GTID:2250330401969876Subject:Biochemical Engineering
Abstract/Summary:PDF Full Text Request
2-chloropropionic acid (2-CPA) is a significant midbody in chemical industry, has a widely application in medicine, synthetic dyes, pesticides manufacturing and other industries. Using2-CPA dehalogenase enzyme split has important significance in preparation of optically pure2-CPA.Firstly, By means of the error-prone PCR of DehⅡ-S, and select a mutant DehⅡ-B2. Its optimum pH is9.5, the optimum temperature of60℃, Vmax and Km values were2.63×10-3mM·min-1and0.75mM. The specific activity of DehⅡ-B2is3.9times as large as that of DehⅡ-S. Through molecular modeling and molecular docking found that DehII-B2decreased the activation energy of1.4kcal/mol than DehⅡ-S, shorten the distance0.3216nm between the catalytic residues Asp10and the substrate a-carbon atom. The combined effect of both above factors increased the enzyme specific activity.Secondly, By means of site-directed mutagenesis to transform the site Asn203of DehDIV-R. Found that the mutation of Ser203, the catalytic selectivity is changed and enzyme specific activity dropped to0.101U/mg; When the mutation of Gln203, the selectivity remained unchanged, but the enzyme activity was reduced by17%. In addition, mutations of other amino acids are inactive. Analysis the results of homology modeling found that using steric effects and hydrogen bonding hypothesis may well explain the changing nature of mutant enzymes.Finally, By means of the error-prone PCR of DehDIV-R, screened mutants and created a mutant library. The mutant DehDIV-G2and DehDIV-E7of the enzyme specific activity increased by24.8%and39.6%. And through computer-aided design software SYBYL to molecular docking for the enzyme with substrate, docking scoring display that DehDIV-G2decreased the activation energy of0.23kcal/mol, DehDIV-E7decreased the activation energy of0.61kcal/mol. As the mutant enzymes decreased the activation energy, increased the affinity with R-2-CPA, thereby increasing enzyme specific activities.
Keywords/Search Tags:2-chloropropionic acid, dehalogenase, directed evolution, rational design, molecular docking
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