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Enhancing Thermostability Of Halohydrin Dehalogenase From Agrobacterium Radiobacter AD1(HheC) By Directed Evolution

Posted on:2016-05-06Degree:MasterType:Thesis
Country:ChinaCandidate:Z Y WuFull Text:PDF
GTID:2180330473455664Subject:Biochemistry and Molecular Biology
Abstract/Summary:PDF Full Text Request
Haloalcohol dehalogenase HheC from Agrobacterium radiobacter AD1 is a potentially useful enzyme for catalyzing enantioselective conversions under mild conditions. The enzyme catalyzes the dehalogenation of vicinal halohydrins to epoxides by an intramolecular nucleophilic displacement mechanism and their reverse ring-opening reactions with alternative nucleophiles as substrates. And these two reactions were proven to proceed with high enantioselectivity and regioselectivity,making these enzymes valuable tools for synthesis of pharmaceuticals and biologically active compounds. However, in some cases, the conditions of synthetic these pharmaceuticals are very harsh. Thus, the HheC used in this process need to be more thermostable. Protein thermostability arises from a combination of factors which are often difficult to rationalize. Therefore its improvement can be better addressed through directed evolution than by rational design approaches.In this work, a library with low mutagenesis frequency(2-3 a.a. mutation/gene)was constructed by using an error prone-PCR. After screening 2000 colonies, 6 mutants with total of 8 mutation sites were obtained. To further enhance the thermostability of HheC, an iterative site-directed mutagenesis(ISM) approach was adopted. ISM was started from B-2-3D(re-named to Var0), the most stable mutant among the 6 selected mutants. After several rounds iterative saturated mutagenesis, one mutant Var4 with a half life longer 3400 fold than WT was obtained after screen 2200 colonies. 75%residual activity was still retained for the mutant even after heat treatment at 70℃ for30 min. The catalytic activity of Var3 and Var4 were not reduced because of their enhanced thermostability. And they still retained most enantioselectivity to rac-2CPE.Our structural investigation highlight the importance of protein surface, loop and the hydrophobic group to protein thermostability, as well as the notion that introducing the protein-protein interactions to the multimer can yeild a large increase in stability.Overall, the results indicate the mutants we obtained would have great application prospect in industry and that directed evolution methodology could still be used as a powerful tool in modulating the thermostability of enzymes.
Keywords/Search Tags:directed evolution, Haloalcohol dehalogenase, thermostability, iterative site-directed mutagenesis
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