College of Food Science and Light Industry, Nanjing Tech University, Nanjing, 211816, China.
College of Medicine and Life Science, Nanjing University of Chinese Medicine, Nanjing 210023, China.
Int J Biol Macromol. 2018 Mar;108:1331-1338. doi: 10.1016/j.ijbiomac.2017.11.040. Epub 2017 Nov 10.
Enzymatic preparation of carrageenan oligosaccharides has drawn increasing attention due to its advantages of mild reaction conditions and excellent product-specificity. A novel gene (CgkA) encoding a new κ-carrageenase was cloned, heterogeneously expressed and characterized from a newly isolated marine bacterium Pedobacter hainanensis NJ-02. It consisted of 1539bp and encoded 512 amino acid residues with a molecular weight of 57.12kDa. Multiple alignment analysis indicated that CgkA belongs to glycoside hydrolase (GH) family 16 and was most homologous to κ-carrageenase of Zobellia sp. M-2 with identity of 50%. The recombinant enzyme showed maximal activity of 3659.72U/mg at 40°C and pH 7.0. Additionally, it could retain more than 80% of its maximal activity after being incubated at pH of 5.0-9.0 below 40°C.K and Na with a wide range of concentration can activate the enzyme, while other divalent ions such as Cu, Zn showed inhibitory effect on the enzyme. The ESI-MS analysis of hydrolysates indicated that the enzyme can endolytically depolymerize the carrageenan into tetrasaccharides and hexasaccharides. The results suggest that it is an endo-type carrageenase and could be a valuable enzyme tool to produce carrageenan oligosaccharides with higher Dps.
由于其反应条件温和和产物特异性优异的特点，酶法制备卡拉胶寡糖引起了越来越多的关注。从新分离的海洋细菌 Pedobacter hainanensis NJ-02 中克隆、异源表达并表征了一种新型基因（CgkA），它编码一种新的 κ-卡拉胶酶。它由 1539bp 组成，编码 512 个氨基酸残基，分子量为 57.12kDa。多重比对分析表明，CgkA 属于糖苷水解酶（GH）家族 16，与 Zobellia sp. M-2 的 κ-卡拉胶酶的同源性最高，同源性为 50%。重组酶在 40°C 和 pH7.0 时表现出最大活性 3659.72U/mg。此外，它在 40°C 以下 pH5.0-9.0 孵育后仍能保持 80%以上的最大活性。K 和 Na 具有广泛的浓度可以激活酶，而其他二价离子如 Cu、Zn 对酶表现出抑制作用。水解产物的 ESI-MS 分析表明，该酶可以内切方式将卡拉胶降解为四糖和六糖。结果表明，它是一种内切型卡拉胶酶，可作为生产具有更高 Dps 的卡拉胶寡糖的有价值的酶工具。
