Overexpression and characterization of a novel chitinase from Trichoderma atroviride strain P1.
We describe the overexpression and characterization of a new 30 kDa family 18 chitinase (Ech30) from Trichoderma atroviride strain P1. Sequence alignments indicate that the active site architecture of Ech30 resembles that of endochitinases such as hevamine from the rubber tree (Hevea brasiliensis). The ech30 gene was overexpressed in Escherichia coli without its signal peptide and with an N-terminal His-tag. The enzyme was produced as inclusion bodies, from which active chitinase could be recovered using a simple refolding procedure. The enzyme displayed an acidic pH-optimum (pH 4.5-5.0), probably due to the presence of a conserved Asn residue near the catalytic glutamate, which is characteristic for acidic family 18 chitinases. Studies with oligomers of N-acetylglucosamine [(GlcNAc)(n)], 4-methylumbelliferyl (4-MU) labelled GlcNAc oligomers and beta-chitin reveal enzymatic properties typical of an endochitinase: 1) low activity towards short substrates (kinetic parameters for the hydrolysis of 4-MU-(GlcNAc)2 were K(m), 149+/-29 microM and k(cat), 0.0048+/-0.0005 s(-1)), and 2) production of relatively large amounts of trimers and tetramers during degradation of beta-chitin. Detailed studies with GlcNAc oligomers indicated that Ech30 has as many as seven subsites for sugar binding. As expected for a family 18 chitinase, catalysis proceeded with retention of the beta-anomeric configuration.
Hoell
Ingunn A
IA
Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, P.O. Box 5003, 1432 As, Norway.
Klemsdal
Sonja S
SS
Vaaje-Kolstad
Gustav
G
Horn
Svein J
SJ
Eijsink
Vincent G H
VG
D000117:Acetylglucosamine / Q000031:analogs & derivatives* / Q000737:chemistry; D000595:Amino Acid Sequence; D001216:Asparagine / Q000737:chemistry; D001665:Binding Sites; D020134:Catalytic Domain; D002686:Chitin / Q000737:chemistry; D002688:Chitinases / Q000096:biosynthesis* / Q000737:chemistry*; D004247:DNA / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D006923:Hymecromone / Q000031:analogs & derivatives* / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D011485:Protein Binding; D017510:Protein Folding; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014242:Trichoderma / Q000201:enzymology*
D017931:DNA Primers; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D002686:Chitin; C025158:4-methylumbelliferyl 2-acetamido-2-deoxy-beta-D-glucopyranoside; D018698:Glutamic Acid; D006923:Hymecromone; D001216:Asparagine; D004247:DNA; D002688:Chitinases; D000117:Acetylglucosamine