Display Settings:

Format

Send to:

Choose Destination
See comment in PubMed Commons below
Biotechnol J. 2009 Apr;4(4):501-9. doi: 10.1002/biot.200800258.

Directed evolution of a Bacillus chitinase.

Author information

  • 1National Synchrotron Research Center, Nakhon Ratchasima, Thailand.

Abstract

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-beta-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-beta-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.

Comment in

PMID:
19370717
[PubMed - indexed for MEDLINE]
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for John Wiley & Sons, Inc.
    Loading ...
    Write to the Help Desk