| Research Abstract |
1. Three chitinase isozymes were purified from the stomach of Marbled rockfish by ammonium sulfate fractionation, Chitopearl Basic BL-03 affinity column chromatography, and CM-Toyopearl 650S ion-exchange column chromatography. Substrate specificities of these chitinases were investigated by using insoluble long substrates, non-crystalline chitin, colloidal chitin, and two crystalline chitins,α-chitin from shrimp shell, crab shell, and silkworm cuticle, and β-chitin from squid pen. Hydrolyzing activity against soluble short substrates, N-acetylchitooligosaccharides ((GlcNAc)n, n=2 to 6) and p-nitrophenyl (GlcNAc)n (pNp-(GlcNAc)n, n=1 to 3), were also measured. The relative activities of HoChiA and SjChi toward various forms of chitin were as follows : shrimp shell or crab shell α-chitin > β-chitin >> silkworm cuticle α-chitin. On the other hand, the relative activities of HoChiB and HoChiC were β-chitin >> silkworm α-chitin > shrimp and crab α-chitin. The relative activities of HoChiA a
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nd SjChi toward soluble short substrates were also different to those of HoChiB and HoChiC.
2. β-N-Acetylhexosaminidase was purified from the liver of Japanese common squid Todarodes pacificus by ammonium sulfate fractionation (0-70%) and column chromatographies on Butyl-Toyopearl 650S and Toyopearl HW-55SS. The purified enzyme showed single protein band on PAGE. The molecular weight of the enzyme were estimated to be 125 kDa by gel filtration, 54 kDa by SDS-PAGE in non-reducing condition, and 33 kDa by SDS-PAGE in reducing condition. The optimum pH and temperature were 4.0 and 70℃, respectively. The enzyme was stable from pH 3.5 to 5.5, and below 60℃, respectively. The Km value of the β-N-acetylhexosaminidase for p-nitrophenyl N-acetylglucosaminide (pNp-GlcNAc) was 0.23 mM. As the GlcNAc-chain length of the substrate increases from pNp-GlcNAc to pNp Tri-N-acetylchitotorioside (pNp-GlcNAc_3), the release of pNp was delayed. The enzyme produced GlcNAc of β-anomer from GlcNAc_3 by enzymatic hydrolysis. These results indicate that β-N-acetylhexosaminidase from the liver of Japanese common squid releases GlcNAc from the non-reducing end side.
3. Three seaweed chitinase isozymes (Chi-A, B, and C) were purified from a red algae, Chondrus verrucosus. The molecular weights and isoelectric points were 24.5 kDa and 3.5 for Chi-A, 25.5 kDa and 4.6 for Chi-B, and 24.5 kDa and <3.5 for Chi-C. Optimum pH and temperature were observed at pH 2.0 and 80℃ for Chi-A and Chi-C, and pH 1.0 and 70℃ for Chi-B, respectively. Toward N-acetylchitooligosaccharide (GlcNAc_n) (n=2 to 6), Chi-A, B, and C hydrolyzed GlcNAc_5 and GlcNAc_6 and produced GlcNAc_n (n=2 to 4). GlcNAc_n (n=3, 4) with the reducing end-side of β anomer was detected from the hydrolysis products. These results indicated that the reactions of Chi-A, B, and C for GlcNAc_n were a retaining mechanism similar to that of family 18 chitinase. Toward crystalline chitins, Chi-A, B, and C degraded squid pen β-chitin more than crab shell and shrimp shell α-chitin.
4.Full length cDNA of chitinase was obtained from the stomach common mackerel. Less
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