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| Directed Evolution and Application of S-2-CPA Dehalogenase |
| LIU Peng, YANG Chun-jiao, YANG Li-rong, XU Gang, WU Jian-ping |
| Zhejiang University, Hangzhou 310027, China |
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Abstract To enhance the functionality of dehalogenase from Pseudomonas sp. CGMCC 3267, Used error-prone PCR to create mutants with improved specific activity. Through error-prone PCR and high-throughput screening method, a desirable mutant DehII-B2 was obtained. Compare to initial strain, the specific activity of DehII-B2 was increased 3.9-fold. Three-dimensional structures of DehII-S and DehII-B2 were constructed by homology modeling, and molecular docking of dehalogenase and S-2-CPA. The results suggested that the mutant site is Ala7, the binding energy of DehII-B2 is reduced 1.4kcal/mol than DehII-S, and the distance of active site Asp10 and the α carbon atom of substrate is shorten 0.321 6nm, thus accelerate the enzyme reaction rate and increased enzyme specific activity. At present, the specific activity of DehII-B2 was higher than all enzyme in our lab. According to the results, the optimum pH and pH-stability were not changed, but the optimum temperature and thermo-stability of DehII-B2 were slightly increased than those of initial strain. The preliminary application of DehII-B2 was studied, and the best result was obtained at 40℃, 60mmol/L 2-CPA reacting for 10h.
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Received: 09 February 2012
Published: 25 May 2012
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