Directed evolution for increased chitinase activity |
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Authors: | Yanhua Fan Weiguo Fang Yuehua Xiao Xingyong Yang Yongjun Zhang Michael J Bidochka Yan Pei |
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Institution: | (1) Biotechnology Research Center, Southwest University, Beibei, Chongqing, People’s Republic of China;(2) Department of Biological Sciences, Brock University, 500 Glenridge Avenue St., Catharines, ON, L2S 3A1, Canada |
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Abstract: | Directed evolution through DNA shuffling and screening was used to enhance the catalytic ability of a fungal, Beauveria bassiana, chitinase, Bbchit1. The Bbchit gene was first linked to various prokaryotic signal sequences and expressed in Escherichia coli. The signal peptide, PelB, from Erwinia carotovora resulted in greatest chitinase secretion into broth. The nucleotide sequence expressing PelB signal peptide was then incorporated
into an E. coli vector to express Bbchit1 variants generated by three rounds of DNA shuffling. A Bbchit1 library with 150,000 variants was constructed with a nucleotide point mutation frequency of 0.6% and screened for chitinolytic
activity. Two Bbchit1 variants (SHU-1 and SHU-2) were selected that showed increased chitinolytic activity compared to the
wild type. Sequence analysis of these variants revealed mutations in amino acid residues that would not normally be considered
for rational design of improved chitinase activity. The amino acid substitutions occurred outside of the two putative substrate-binding
sites and the catalytic region. |
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Keywords: | Beauveria bassiana Chitinase Catalytic activity DNA shuffling |
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