Disruption of the gene encoding the ChiB1 chitinase of Aspergillus fumigatus and characterization of a recombinant gene product.
The gene encoding a major, inducible 45 kDa chitinase of Aspergillus fumigatus was cloned and analysis of the deduced amino acid sequence identified a chitinase of the fungal/bacterial class which was designated ChiB1. Recombinant ChiB1, expressed in Pichia pastoris, was shown to function by a retaining mechanism of action. That is, the beta-conformation of the chitin substrate linkage was preserved in the product in a manner typical of family 18 chitinases. Cleavage patterns with the N-acetylglucosamine (GlcNAc) oligosaccharide substrates GlcNAc(4), GlcNAc(5) and GlcNAc(6) indicated that the predominant reaction involved hydrolysis of GlcNAc(2) from the non-reducing end of each substrate. Products of transglycosylation were also identified in each incubation. Following disruption of chiB1 by gene replacement, growth and morphology of disruptants and of the wild-type strain were essentially identical. However, during the autolytic phase of batch cultures the level of chitinase activity in culture filtrate from a disruptant was much lower than the activity from the wild-type. The search for chitinases with morphogenetic roles in filamentous fungi should perhaps focus on chitinases of the fungal/plant class although such an investigation will be complicated by the identification of at least 11 putative active site domains for family 18 chitinases in the A. fumigatus TIGR database (http://www.tigr.org/).
Jaques
Alex K
AK
Division of Microbiology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
Fukamizo
Tamo
T
Laboratory of Enzyme System Science, Department of Food and Nutrition, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan.
Hall
Diana
D
Division of Microbiology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
Barton
Richard C
RC
Division of Microbiology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
Escott
Gemma M
GM
Division of Microbiology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
Parkinson
Tanya
T
Department of Discovery Biology, Pfizer Global Research and Development, Sandwich Laboratories, Kent CT13 9NJ, UK.
Hitchcock
Christopher A
CA
Department of Discovery Biology, Pfizer Global Research and Development, Sandwich Laboratories, Kent CT13 9NJ, UK.
Adams
David J
DJ
Division of Microbiology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
D001232:Aspergillus fumigatus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D001483:Base Sequence; D015139:Blotting, Southern; D002688:Chitinases / Q000235:genetics* / Q000502:physiology*; D003001:Cloning, Molecular; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000378:metabolism
D011994:Recombinant Proteins; D002688:Chitinases