No abstract

Chitin is a homopolymer of b(1,4)-linked N-acetyl-dglucosamine (GlcNAc) residues and a major structural component of bacteria, fungi, and insects. In the ocean, chitin is produced in vast quantities by marine invertebrates, fungi, and algae [1]. This highly insoluble compound is utilized rapidly, as the sole source of carbon and nitrogen, by marine bacteria such as Vibrio spp. [2,3]. Two types of enzymes are required for the hydrolysis of chitin. The first, chitinases, are the major enzymes, which degrade the chitin polymer into chitooligosaccharides and subsequently into the disaccharide, (GlcNAc) 2 . (GlcNAc) 2 is then The enzymatic properties of chitinase A from Vibrio carchariae have been studied in detail by using combined HPLC and electrospray MS. This approach allowed the separation of a and b anomers and the simultaneous monitoring of chitooligosaccharide products down to picomole levels. Chitinase A primarily generated b-anomeric products, indicating that it catalyzed hydrolysis through a retaining mechanism. The enzyme exhibited endo characteristics, requiring a minimum of two glycosidic bonds for hydrolysis. The kinetics of hydrolysis revealed that chitinase A had greater affinity towards higher M r chitooligomers, in the order of (GlcNAc) 6 > (GlcNAc) 4 > (GlcNAc) 3 , and showed no activity towards (GlcNAc) 2 and pNP-GlcNAc. This suggested that the binding site of chitinase A was probably composed of an array of six binding subsites. Point mutations were introduced into two active site residues -Glu315 and Asp392 -by site-directed mutagenesis. The D392N mutant retained significant chitinase activity in the gel activity assay and showed 20% residual activity towards chitooligosaccharides and colloidal chitin in HPLC-MS measurements. The complete loss of substrate utilization with the E315M and E315Q mutants suggested that Glu315 is an essential residue in enzyme catalysis. The recombinant wild-type enzyme acted on chitooligosaccharides, releasing higher quantities of small oligomers, while the D392N mutant favored the formation of transient intermediates. Under standard hydrolytic conditions, all chitinases also exhibited transglycosylation activity towards chitooligosaccharides and pNP-glycosides, yielding picomole quantities of synthesized chitooligomers. The D392N mutant displayed strikingly greater efficiency in oligosaccharide synthesis than the wild-type enzyme.Abbreviations GlcNAc, N-acetyl-D-glucosamine; (GlcNAc) n , b1-4 linked oligomers of GlcNAc residues where n ¼ 2-6; pNP, p-nitrophenol; pNP-(GlcNAc) n , pNP-b-glycosides; SIM, single ion monitoring.

Chitinase A of Vibrio carchariae was expressed in Escherichia coli M15 host cells as a 575-amino-acid fragment with full enzymatic activity using the pQE60 expression vector. The yield of the highly purified recombinant protein was approximately 70 mg per litre of bacterial culture. The molecular mass of the expressed protein was determined by HPLC/ESI-MS to be 63 770, including the hexahistidine tag. Crystals of recombinant chitinase A were grown to a suitable size for X-ray structure analysis in a precipitant containing 10%(v/v) PEG 400, 0.1 M sodium acetate pH 4.6 and 0.125 M CaCl2. The crystals belonged to the tetragonal space group P422, with two molecules per asymmetric unit and unit-cell parameters a = b = 127.64, c = 171.42 A. A complete diffraction data set was collected to 2.14 A resolution using a Rigaku/MSC R-AXIS IV++ detector system mounted on an RU-H3R rotating-anode X-ray generator.