Directed evolution of a Bacillus chitinase.
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.
Songsiriritthigul
Chomphunuch
C
National Synchrotron Research Center, Nakhon Ratchasima, Thailand.
Pesatcha
Putarika
P
Eijsink
Vincent G H
VG
Yamabhai
Montarop
M
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D002021:Buffers; D020134:Catalytic Domain; D002686:Chitin / Q000235:genetics / Q000378:metabolism; D002688:Chitinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D032621:DNA Shuffling; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics; D005240:Feasibility Studies; D005798:Genes, Bacterial; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010710:Phosphates / Q000737:chemistry; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014169:Transformation, Bacterial
D002021:Buffers; D010710:Phosphates; D011994:Recombinant Proteins; D002686:Chitin; D002688:Chitinases