Purification of a chitinase from the posterior salivary gland of common octopus Octopus vulgaris and its properties
Purification of a chitinase from the posterior salivary gland of common octopus Octopus vulgaris and its properties
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普通章鱼后唾液腺几丁质酶的纯化及其性质
DOI：
10.1166/jcc.2014.1049
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发表时间：
2014
期刊：
影响因子：
--
通讯作者：
and Masahiro Matsumiya
中科院分区：
文献类型：
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作者：
Tomohiro Ogino;Hirotaka Tabata;Mana Ikeda;Hiromi Kakizaki;and Masahiro Matsumiya
从普通章鱼Octopus vulgaris唾液腺中分离纯化了一种几丁质酶。使用以下柱色谱法处理：ChitinEX、TOYOPEARL Butyl-650 S和TOYOPEARL CM-650 S，随后进行表征。纯化的几丁质酶（OvChi）在SDS-PAGE上显示单一蛋白带，分子量为57 kDa。几丁质酶的N-末端氨基酸序列进行了分析，多达21个残基。它与糖苷水解酶（GH）家族18种几丁质酶具有很高的同源性。当pNp-（GlcNAc）3作为底物时，OvChi在pH4.5时表现出最佳活性，在pH4.0 -5.5范围内稳定。OvChi对几丁质酶的最适pH为5.0，在pH 6.0时酶活最高，达91%。OvChi的最适温度为40 °C，在pH4.5条件下预孵育10 min，其稳定性可达40 °C。降解难溶性长链底物的能力顺序为：甲壳素>鱿鱼圈α-甲壳素>胶体甲壳素>蚕蛹壳α-甲壳素>虾壳α-甲壳素>蟹壳α-甲壳素。以N-乙酰壳寡糖（GlcNAc）n（n = 2-6）为底物，OvChi降解（GlcNAc）4-6，生成（GlcNAc）2-4，并在水解产物的还原端观察到β-端基异构体的增加。它降解（GlcNAc）5产生（GlcNAc）2（56.5%）和（GlcNAc）3（43.5%）。观察到降解pNp-（GlcNAc）2-4的能力，并获得以下顺序：pNp-（GlcNAc）3 > pNp-（GlcNAc）2 > pNp-（GlcNAc）4。根据这些结果，我们得出结论，OvChi可能是一种几丁质分解酶，表现出强的内型，属于GH家族18几丁质酶。
A chitinase was purified from the salivary gland of the common octopus Octopus vulgaris. It was treated using the following column chromatographies: ChitinEX, TOYOPEARL Butyl-650S, and TOYOPEARL CM-650S, and subsequently characterized. The purified chitinase (OvChi) showed a single protein band on SDS-PAGE, and the molecular mass was estimated to be 57 kDa. The N-terminal amino acid sequence of the chitinase was analyzed up to 21 residues. It showed high homology to the glycoside hydrolase (GH) family 18 chitinases. When p-nitrophenyl N-acetyl-chitotrioside (pNp-(GlcNAc)3) was used as a substrate, OvChi exhibited optimum activity at pH 4.5 with stability from pH 4.0–5.5. The optimum pH of OvChi toward chitin nanofiber was pH 5.0 and 91% of the maximum activity was shown at pH 6.0. The optimum temperature of OvChi was 40 °C and it was stable up to 40 °C when it was preincubated for 10 min at pH 4.5. The ability to degrade insoluble long substrates was observed in the following order: chitin nanofiber squid pen α-chitin > colloidal chitin > silkworm cuticleα-chitin > shrimp shell α-chitin > crab shell α-chitin. When N-acetylchitooligosaccharides ((GlcNAc) n , n = 2–6) were used as substrates, OvChi degraded (GlcNAc)4–6 and produced (GlcNAc)2–4, and an increase of β-anomers was observed at the reducing end of the hydrolysis products. It degraded (GlcNAc)5 to produce (GlcNAc)2 (56.5%) and (GlcNAc)3 (43.5%). The ability to degrade pNp-(GlcNAc)2–4 was observed and the following order was obtained: pNp-(GlcNAc)3 > pNp-(GlcNAc)2 > pNp-(GlcNAc)4. On the basis of these results, we concluded that OvChi could be a chitinolytic enzyme that exhibits a strong endotype and belongs to the GH family 18 chitinases.