Laboratory of Marine Biotechnology and Microbiology, Graduate School of Fisheries Sciences, Hokkaido University, Minato-cho 3-1-1, Hakodate 041-8611, Japan.
Comp Biochem Physiol B Biochem Mol Biol. 2012 May;162(1-3):24-33. doi: 10.1016/j.cbpb.2012.02.003. Epub 2012 Feb 18.
A β-D-mannosidase (EC 3.2.1.25) with a molecular mass of approximately 100 kDa was purified from the digestive fluid of a marine gastropod Aplysia kurodai by ammonium sulfate fractionation followed by column chromatographies on TOYOPEARL Butyl-650 M, TOYOPEARL DEAE-650 M, and Superdex 200 10/300 GL. This enzyme, named AkMnsd in the present study, showed optimal activities at pH 4.5 and 40 °C and was stable at the acidic pH range from 2.0 to 6.7 and the temperature below 38 °C. The Km and Vmax values for AkMnsd determined at pH 6.0 and 30 °C with p-nitrophenyl β-d-mannopyranoside were 0.10 mM and 3.75 μmol/min/mg, respectively. AkMnsd degraded various polymer mannans as well as mannooligosaccharides liberating mannose as a major degradation product. Linear mannan from green alga Codium fragile was completely depolymerized by AkMnsd in the presence of AkMan, an endolytic β-mannanase, which we previously isolated from the same animal (Zahura et al., Comp. Biochem. Physiol. B 157, 137-148 (2010)). A cDNA encoding AkMnsd was amplified from the Aplysia hepatopancreas cDNA by the PCR using degenerated primers designed on the basis of N-terminal and internal amino-acid sequences of AkMnsd. The cloned AkMnsd cDNA consisted of 2985 bp and encoded an amino-acid sequence of 931 residues with the calculated molecular mass of 101,970 Da. The deduced sequence of AkMnsd showed 20-43% amino-acid identity to those of glycoside-hydrolase-family 2 (GHF2) β-mannosidases. The catalytically important amino-acid residues determined in GHF2 enzymes were completely conserved in AkMnsd. Thus, AkMnsd is regarded as a new member of GHF2 mannosidase from marine gastropod.
一种分子量约为 100 kDa 的β-D-甘露糖苷酶（EC 3.2.1.25）从海洋腹足纲动物 Aplysia kurodai 的消化液中通过硫酸铵分级和柱层析在 TOYOPEARL Butyl-650 M、TOYOPEARL DEAE-650 M 和 Superdex 200 10/300 GL 上纯化得到。该酶在本研究中被命名为 AkMnsd，其最适活性在 pH4.5 和 40°C 下，在 pH2.0 至 6.7 的酸性范围和低于 38°C 的温度下稳定。在 pH6.0 和 30°C 下，以对硝基苯-β-D-甘露吡喃糖苷为底物，AkMnsd 的 Km 和 Vmax 值分别为 0.10 mM 和 3.75 μmol/min/mg。AkMnsd 降解各种聚合物甘露聚糖以及甘露寡糖，释放甘露糖作为主要降解产物。绿藻 Codium fragile 的线性甘露聚糖在 AkMnsd 的存在下被完全解聚，AkMnsd 是我们之前从同一动物（Zahura 等人，《比较生物化学与生理学 B》157，137-148（2010 年））中分离出的一种内切β-甘露聚糖酶。通过使用基于 AkMnsd 的 N 末端和内部氨基酸序列设计的简并引物，从 Aplysia 肝胰腺 cDNA 通过 PCR 扩增出编码 AkMnsd 的 cDNA。克隆的 AkMnsd cDNA 由 2985 bp 组成，编码 931 个氨基酸的序列，计算分子量为 101970 Da。AkMnsd 的推导序列与糖苷水解酶家族 2（GHF2）β-甘露糖苷酶的 20-43%氨基酸同一性。在 GHF2 酶中确定的催化重要氨基酸残基在 AkMnsd 中完全保守。因此，AkMnsd 被认为是来自海洋腹足纲动物的 GHF2 甘露糖苷酶的新成员。
