Structure and Function of Useful Chitinolytic Enzymes for Utilization of Chitin from Marine Organisms

点击翻译按钮获取中文摘要

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 … More 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