Enzymatic properties of wild‐type and active site mutants of chitinase A fromVibrio carchariae, as revealed by HPLC‐MS

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.

Role of Tyr-435 of Vibrio harveyi Chitinase A in Chitin Utilization

“…This enzyme degraded pNP-GlcNAc about 10-fold more efficiently than GlcNAc 2 . As already observed elsewhere [18], GlcNAc 6 is the best substrate and GlcNAc 3 is the poorest substrate. For long chain substrate, colloidal chitin is the best substrate among the long-chain chitin substrates (Fig.…”

“…We previously reported that a marine bacterium V. harveyi mainly secreted chitinase A (VhChiA), which is a member of family-18 chitinases, to degrade chitin-containing materials from marine biosphere for its energy source [7]. Further cloning and highly expressing recombinant VhChiA in an E. coli system allowed its enzymatic properties in chitin degradation to be investigated in detail [9,11,18]. Recently, crystal structures of VhChiA were solved in the absence or the presence of chitin oligomers [10].…”

“…S1). We reported previously using quantitative LC/MS technique [18] that VhChiA wild-type expressed greatest substrate specificity (k cat /K m ) towards chitohexaose. Similar results are also observed here with a more conventional (DNS) assay.…”

Synthesis of Long-Chain Chitooligosaccharides by a Hypertransglycosylating Processive Endochitinase of Serratia proteamaculans 568

“…Mutation of the middle Asp in the diagnostic DxDxE motif, which interacts with the catalytic Glu during the catalytic cycle, yielded strong TG mutants (50). Correlation between a lower K m and increased TG was observed for the ␤-mannanase Man5A from the blue mussel Mytilus edulis (19), endo-␤-N-acetylglucosaminidase from Mucor hiemalis (39), and chitinase A from Vibrio carchariae (33).…”

Microbial Chitinases for Chitin Waste Management

“…Methods that employ HPLC have also been described for the separation and quantifi cation of chito-oligosaccharides (Krokeide et al 2007 ) . Combination of HPLC and electrospray mass spectroscopy allows the simultaneous separation of a and b anomers, and monitoring of chitooligosaccharide products down to picomoles (Suginta et al 2005 ) . Mass spectroscopy and nuclear magnetic resonance (NMR) were utilized to determine the size of chito-oligosaccharides produced by the activity of chitinases or chemical depolymerisation of chitin (Howard et al 2003 ) .…”