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

1.采用硫酸铵分级沉淀、Chitopolymer Basic BL-03亲和层析和CM-Toyopolymer 650 S离子交换层析从大理石石斑鱼胃中分离纯化了3种几丁质酶同工酶。以不溶性长底物、非结晶甲壳素、胶体甲壳素和两种结晶甲壳素--虾壳、蟹壳、蚕壳的α-甲壳素和鱿鱼圈的β-甲壳素为底物，研究了这两种甲壳素酶的底物特异性。还测定了对可溶性短底物N-乙酰基壳寡糖（（GlcNAc）n，n=2至6）和对硝基苯基（GlcNAc）n（pNp-（GlcNAc）n，n=1至3）的水解活性。HoChiA和SjChi对各种形态甲壳素的相对活性为：虾壳或蟹壳α-甲壳素> β-甲壳素>>蚕壳α-甲壳素。而HoChiB和HoChiC的相对活性则为β-几丁质>>蚕α-几丁质>虾蟹α-几丁质。HoChiA a的相对活性 ...更多信息 和SjChi对可溶性短底物的反应也不同于HoChiB和HoChiC.从太平洋褶柔鱼肝中提取β-N-乙酰氨基己糖苷酶，经硫酸铵分级（0-70%）和Butyl-Toyoprotein 650 S和Toyoprotein HW-55 SS柱层析纯化。纯化后的酶在PAGE上呈单一蛋白带。凝胶过滤法测得酶的分子量为125 kDa，非还原条件下SDS-PAGE测得酶的分子量为54 kDa，还原条件下SDS-PAGE测得酶的分子量为33 kDa。最适pH为4.0，最适温度为70℃。该酶在pH3.5 ~ 5.5范围内稳定，在60℃以下稳定。β-N-乙酰氨基己糖苷酶对对硝基苯基N-乙酰氨基葡萄糖苷（pNp-GlcNAc）的Km值为0.23 mM，随着底物GlcNAc链长的增加，pNp的释放被延迟。该酶水解GlcNAc 3生成β-端基异构体GlcNAc。这些结果表明，日本鱿鱼肝脏中的β-N-乙酰氨基己糖苷酶从非还原端释放GlcNAc.从一种红藻角叉菜（Chondrus verrucosus）中分离纯化了3种海藻几丁质酶同工酶（Chi-A、B和C）。Chi-A、Chi-B和Chi-C的分子量分别为24.5kDa和3.5、25.5kDa和4.6、24.5kDa和<3.5。Chi-A和Chi-C的最适pH为2.0，最适温度为80℃; Chi-B的最适pH为1.0，最适温度为70℃。对于N-乙酰壳寡糖（GlcNAc_n）（n=2 ~ 6），Chi-A、B和C水解GlcNAc_5和GlcNAc_6，生成GlcNAc_n（n=2 ~ 4）。在水解产物中检测到GlcNAc_n（n=3，4），其末端为β端基异构体的还原端。这些结果表明，Chi-A、B和C对GlcNAc_n的反应是一种类似于家族18几丁质酶的保留机制。对于结晶几丁质，Chi-A、B和C对鱿鱼圈β-几丁质的降解作用大于对蟹壳和虾壳α-几丁质的降解作用。少

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