An endochitinase A from Vibrio carchariae: cloning, expression, mass and sequence analyses, and chitin hydrolysis

Suginta, Wipa; Vongsuwan, Archara; Songsiriritthigul, Chomphunuch; Prinz, Heino; Estibeiro, Peter; Duncan, Rory R.; Svasti, Jisnuson; Fothergill‐Gilmore, Linda A.

doi:10.1016/j.abb.2004.01.017

Cited by 59 publications

(69 citation statements)

References 40 publications

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“…14) As expected, two motifs conserved in the catalytic regions of GH family 18 chitinases, S-x-G-G (amino acid no., 271-274) and x-D-x-x-D-x-D-x-E (amino acid no., 307-315) (E is a catalytic amino acid residue), 15) are present also in Pa-rChi. The carbohydrate binding domain classified in carbohydrate binding module (CBM) family 5 (http://afmb.cnrs-mrs.fr/CAZY/) was observed in the C-terminal region (amino acid no., 797-839) of Pa-rChi.…”

supporting

confidence: 61%

Production and Secretion of a RecombinantVibrio parahaemolyticusChitinase byEscherichia coliand Its Purification from the Culture Medium

Kadokura

Sakamoto

Saito

et al. 2007

Bioscience, Biotechnology, and Biochemistry

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supporting

confidence: 61%

Production and Secretion of a RecombinantVibrio parahaemolyticusChitinase byEscherichia coliand Its Purification from the Culture Medium

Kadokura

Sakamoto

Saito

et al. 2007

Bioscience, Biotechnology, and Biochemistry

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“…Chitinase A is highly expressed upon induction with chitin and is active as a monomer of M r 62 700. Analysis of chitin hydrolysis by using the viscosity assay and HPLC-ESI MS suggested that the newly isolated chitinase acts as an endochitinase [25]. We also reported isolation of the gene encoding chitinase A and functional expression of the recombinant enzyme in an Escherichia coli system.…”

mentioning

confidence: 80%

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

et al. 2005

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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.

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“…Vibrios are also able to break down chitin, a homopolymer of N-acetyl-D-glucosamine, which is one of the largest pools of amino sugars in the oceans (85)(86)(87)324). Vibrio harveyi, for instance, excretes at least ten different chitin-degrading enzymes (367,372). Accordingly, it was suggested that this ability may explain the ubiquitous occurrence of vibrios in aquatic settings (324).…”

Section: Nutrient Cyclingmentioning

confidence: 99%

Biodiversity of Vibrios

Thompson

Iida

Swings

2004

Microbiol Mol Biol Rev

1,062

888

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Vibrios are ubiquitous and abundant in the aquatic environment. A high abundance of vibrios is also detected in tissues and/or organs of various marine algae and animals, e.g., abalones, bivalves, corals, fish, shrimp, sponges, squid, and zooplankton. Vibrios harbour a wealth of diverse genomes as revealed by different genomic techniques including amplified fragment length polymorphism, multilocus sequence typing, repetetive extragenic palindrome PCR, ribotyping, and whole-genome sequencing. The 74 species of this group are distributed among four different families, i.e., Enterovibrionaceae, Photobacteriaceae, Salinivibrionaceae, and Vibrionaceae. Two new genera, i.e., Enterovibrio norvegicus and Grimontia hollisae, and 20 novel species, i.e., Enterovibrio coralii, Photobacterium eurosenbergii, V. brasiliensis, V. chagasii, V. coralliillyticus, V. crassostreae, V. fortis, V. gallicus, V. hepatarius, V. hispanicus, V. kanaloaei, V. neonatus, V. neptunius, V. pomeroyi, V. pacinii, V. rotiferianus, V. superstes, V. tasmaniensis, V. ezurae, and V. xuii, have been described in the last few years. Comparative genome analyses have already revealed a variety of genomic events, including mutations, chromosomal rearrangements, loss of genes by decay or deletion, and gene acquisitions through duplication or horizontal transfer (e.g., in the acquisition of bacteriophages, pathogenicity islands, and super-integrons), that are probably important driving forces in the evolution and speciation of vibrios. Whole-genome sequencing and comparative genomics through the application of, e.g., microarrays will facilitate the investigation of the gene repertoire at the species level. Based on such new genomic information, the taxonomy and the species concept for vibrios will be reviewed in the next years

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An endochitinase A from Vibrio carchariae: cloning, expression, mass and sequence analyses, and chitin hydrolysis

Cited by 59 publications

References 40 publications

Production and Secretion of a RecombinantVibrio parahaemolyticusChitinase byEscherichia coliand Its Purification from the Culture Medium

Production and Secretion of a RecombinantVibrio parahaemolyticusChitinase byEscherichia coliand Its Purification from the Culture Medium

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

Biodiversity of Vibrios

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