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Directed evolution of a Bacillus chitinase
Author(s) -
Songsiriritthigul Chomphunuch,
Pesatcha Putarika,
Eijsink Vincent G.H.,
Yamabhai Montarop
Publication year - 2009
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.200800258
Subject(s) - chitobiose , chitinase , bioconversion , chitin , bacillus licheniformis , directed evolution , mutant , chemistry , biochemistry , biology , enzyme , chitosan , bacteria , gene , genetics , bacillus subtilis , fermentation
Chitinases have potential in various industrial applications including bioconversion of chitin waste from crustacean shells into chito‐oligosaccharide‐based value‐added products. For industrial applications, obtaining suitable chitinases for efficient bioconversion processes will be beneficial. In this study, we established a straightforward directed evolution method for creating chitinase variants with improved properties. A library of mutant chitinases was constructed by error‐prone PCR and DNA shuffling of two highly similar (99% identical) chitinase genes from Bacillus licheniformis. Activity screening was done in two steps: first, activity towards colloidal chitin was screened for on culturing plates (halo formation). This was followed by screening activity towards the chitotriose analogue p ‐nitrophenyl‐β‐1,4‐ N, N' ‐diacetyl‐chitobiose at various pH in microtiter plates. From a medium‐throughput screening (517 colonies), we were able to isolate one mutant that demonstrated improved catalytic activity. When using p ‐nitrophenyl‐β‐1,4‐ N, N' ‐diacetyl‐chitobiose as substrate, the overall catalytic efficiency, k cat /K m of the improved chitinase was 2.7‐ and 2.3‐fold higher than the average k cat /K m of wild types at pH 3.0 and 6.0, respectively. The mutant contained four residues that did not occur in either of the wild types. The approach presented here can easily be adopted for directed evolution of suitable chitinases for various applications.