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

用于利用海洋生物甲壳素的有用几丁质分解酶的结构和功能

基本信息

批准号：
17580183
负责人：
MATSUMIYA Masahiro
金额：
$ 1.86万
依托单位：
Nihon University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17580183/
关键词：
CHITINASE BIOMASS CHITIN CHITOOLIGOSACCHARIDE SUBSTRATE SPECIFICITY N-ACECYLGLUCOSAMINE cDNA CLONING β-キチン N-末端アミノ酸配列

项目摘要

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

1.采用硫酸铵分级、Chitopearl Basic BL-03亲和柱层析、CM-Toyopearl 650S离子交换柱层析从大理石石斑鱼胃中纯化得到3种几丁质酶同工酶。通过使用不溶性长底物、非结晶甲壳素、胶体甲壳素和两种结晶甲壳素，虾壳、蟹壳和蚕角质层的α-甲壳素以及来自鱿鱼圈的β-甲壳素，研究了这些几丁质酶的底物特异性。还测量了针对可溶性短底物N-乙酰壳寡糖（(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对各种形式的几丁质的相对活性如下：虾壳或蟹壳α-几丁质>β-几丁质>>蚕角质层α-几丁质。另一方面，HoChiB和HoChiC的相对活性为β-几丁质>>蚕α-几丁质>虾和蟹α-几丁质。 HoChiA a ... More 和 SjChi 对可溶性短底物的相对活性也不同于 HoChiB 和 HoChiC.2。通过硫酸铵分级分离 (0-70%) 并在 Butyl-Toyopearl 650S 和 Toyopearl HW-55SS 上进行柱色谱，从日本普通鱿鱼的肝脏中纯化 β-N-乙酰己糖胺酶。纯化的酶在 PAGE 上显示单一蛋白带。通过凝胶过滤估计酶的分子量为125 kDa，在非还原条件下通过SDS-PAGE估计为54 kDa，在还原条件下通过SDS-PAGE估计为33 kDa。最适pH为4.0，最适温度为70℃。该酶在pH 3.5至5.5和60℃以下均稳定。 β-N-乙酰氨基己糖苷酶对对硝基苯基 N-乙酰氨基葡萄糖 (pNp-GlcNAc) 的 Km 值为 0.23 mM。随着底物的 GlcNAc 链长度从 pNp-GlcNAc 增加到 pNp Tri-N-乙酰壳托里奥苷 (pNp-GlcNAc_3)，pNp 的释放被延迟。该酶通过酶水解从 GlcNAc_3 产生 β-端基异构体的 GlcNAc。这些结果表明，来自日本鱿鱼肝脏的β-N-乙酰氨基己糖苷酶从非还原端释放GlcNAc。 3．从红藻（Chondrus verrucosus）中纯化了三种海藻几丁质酶同工酶（Chi-A、B 和 C）。 Chi-A 的分子量和等电点为 24.5 kDa 和 3.5，Chi-B 为 25.5 kDa 和 4.6，Chi-C 为 24.5 kDa 和<3.5。 Chi-A和Chi-C的最佳pH和温度分别为pH 2.0和80℃，Chi-B的最佳pH和温度分别为pH 1.0和70℃。对于N-乙酰壳寡糖(GlcNAc_n)(n=2至6)，Chi-A、B和C水解GlcNAc_5和GlcNAc_6并产生GlcNAc_n(n=2至4)。从水解产物中检测到具有β端基异构体还原端的GlcNAc_n (n=3, 4)。这些结果表明Chi-A、B和C对GlcNAc_n的反应是类似于家族18几丁质酶的保留机制。对于结晶几丁质，Chi-A、B、C对鱿鱼笔β-几丁质的降解程度高于对蟹壳和虾壳α-几丁质的降解能力。4.从鲭鱼胃中获得了几丁质酶的全长cDNA。较少的