title: Directed evolution for increased chitinase activity.
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.
authors.affiliation: Biotechnology Research Center, Southwest University, Beibei, Chongqing, People's Republic of China.
authors.forename: Yanhua
authors.initials: Y
authors.lastname: Fan
authors.forename: Weiguo
authors.initials: W
authors.lastname: Fang
authors.forename: Yuehua
authors.initials: Y
authors.lastname: Xiao
authors.forename: Xingyong
authors.initials: X
authors.lastname: Yang
authors.forename: Yongjun
authors.initials: Y
authors.lastname: Zhang
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Bidochka
authors.forename: Yan
authors.initials: Y
authors.lastname: Pei
mesh_terms: D052982:Beauveria / Q000201:enzymology / Q000235:genetics*; D002384:Catalysis; D002688:Chitinases / Q000235:genetics* / Q000378:metabolism; D032621:DNA Shuffling; D019020:Directed Molecular Evolution; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D015723:Gene Library; D016973:Pectobacterium carotovorum / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics
chemical_list: D005656:Fungal Proteins; D021382:Protein Sorting Signals; D002688:Chitinases