Abstract
We have previously reported the presence of three types of chitinase (acidic fish chitinase-1: AFCase-1, acidic fish chitinase-2: AFCase-2, fish chitinase-3: FCase-3) in Actinopterygii. In the present research, we report the identification of the novel chitinase genes HjChi (ORF: 1380 bp) and DkChi (ORF: 1440 bp) from the stomach of Chondrichthyes, Japanese bullhead shark (Heterodontus japonicas) and Kwangtung skate (Dipturus kwangtungensis), respectively. Organ-specific expression analysis identified the stomach-specific expression of HjChi, whereas DkChi was expressed widely in all organs. Chitinase activity was measured using pNP-(GlcNAc)n (n = 2, 3) as a substrate and β-N-acetylhexosaminidase (Hex) activity was measured using pNPGlcNAc. Relatively high values of chitinase activity were observed in the stomach, spleen, and gonads of the Japanese bullhead shark, H. japonicas , compared with that observed in the stomach of the Kwangtung skate D. kwangtungensis . However, Hex activity was detected throughout the body of both species. The optimal pH of chitinase in both the Japanese bullhead shark, H. japonicas, and the Kwangtung skate, D. kwangtungensis, were 3.5 - 5.5 and 3.5 - 4.0, respectively, and 4.0 for Hex in both species. Phylogenetic analysis revealed that Chondrichthyes chitinase forms a unique group (Chondrichthyes chitinase). These results suggested that the possibility of the formation of chitinase groups for each class in the phylogenetic analysis based on the observation of class-specific chitinase.
Highlights
Chitinase (EC 3.2.1.14) is an endo-chitinolytic enzyme that randomly hydrolyzes the β-1,4 glycosidic linkages of chitin to produce N-acetyl chitooligosaccharides ((GlcNAc)n) [1]. β-N-acetylhexosaminidase (Hex, EC 3.2.1.52) is an exo-type chitin-decomposing enzyme that sequentially hydrolyzes terminal residues, resulting in the formation of N-acetyl-D-glucosamine (GlcNAc) [2]
Using degenerate primers designed based on the deduced amino acid sequences of several vertebrate chitinases, the internal sequences of the cDNA of the stomach of H. japonicas and D. kwangtungensis were amplified by RT-PCR and fragments of 450 bp and 400 bp were obtained, respectively
Novel chitinase genes were obtained from the stomach of H. japonicas, HjChi (ORF: 1380 bp) and the stomach of D. kwangtungensis, D. Kwangtungensis chitinase cDNA (DkChi) (ORF: 1440 bp)
Summary
Chitinase (EC 3.2.1.14) is an endo-chitinolytic enzyme that randomly hydrolyzes the β-1,4 glycosidic linkages of chitin to produce N-acetyl chitooligosaccharides ((GlcNAc)n) [1]. β-N-acetylhexosaminidase (Hex, EC 3.2.1.52) is an exo-type chitin-decomposing enzyme that sequentially hydrolyzes terminal residues, resulting in the formation of N-acetyl-D-glucosamine (GlcNAc) [2]. Two or more kinds of chitinase isozymes have been discovered in the stomach of Actinopterygii, one of the most successful vertebrates on earth The activity of these chitinase isozymes is dependent on an acidic pH and the crystalline α-chitin decomposition activity of these enzymes has been demonstrated to be superior to that of other organisms [6]. Not even one chitinase gene of the AFCase-1, AFCase-2, and FCase-3 groups has been detected in the stomach of either Prionace glauca, classified as a Chondrichthyes, or the stomach of Latimeria chalmnae, classified as Sarcopterygii [12] These findings suggested the possibility that there may be a new chitinase group different from groups of AFCase-1, AFCase-2, and FCase-3, as there were large differences in the amino acid sequences of fish chitinases based on systematic positioning.
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