Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing, 100081, People's Republic of China.
Appl Biochem Biotechnol. 2012 Mar;166(6):1442-53. doi: 10.1007/s12010-011-9537-z. Epub 2012 Jan 19.
An endo-β-1,4-mannanase encoding gene, man5, was cloned from Bispora antennata CBS 126.38, which was isolated from a beech stump. The cDNA of man5 consists of 1,299 base pairs and encodes a 432-amino-acid protein with a theoretical molecular mass of 46.6 kDa. Deduced MAN5 exhibited the highest amino acid sequence identity of 58% to a β-mannanase of glycoside hydrolase family 5 from Aspergillus aculeatus. Recombinant MAN5 was expressed in Pichia pastoris and purified to electrophoretic homogeneity. The specific activity of the final preparation towards locust bean gum was 289 U mg(-1). MAN5 showed optimal activity at pH 6.0 and 70 °C and had good adaptation and stability over a broad range of pH values. The enzyme showed more than 60% of peak activity at pH 3.0-8.0 and retained more than 80% of activity after incubation at 37 °C for 1 h in both acid and alkaline conditions (pH 4.0-11.0). The K (m) and V (max) values were 1.33 mg ml(-1) and 444 μmol min(-1) mg(-1) and 1.17 mg ml(-1) and 196 μmol min(-1) mg(-1) for locust bean gum and konjac flour, respectively. Of all tested metal ions and chemical reagents, Co(2+), Ni(2+), and β-mercaptoethanol enhanced the enzyme activity at 1 mM, whereas other chemicals had no effect on or partially inhibited the enzyme activity. MAN5 was highly resistant to acidic and neutral proteases (trypsin, α-chymotrypsin, collagenase, subtilisin A, and proteinase K). By virtue of the favorable properties of MAN5, it is possible to apply this enzyme in the paper and food industries.
从一种山毛榉树桩中分离得到的双孢蘑菇 CBS 126.38 中克隆了一个内切-β-1,4-甘露聚糖酶编码基因 man5。man5 的 cDNA 由 1299 个碱基对组成，编码一个 432 个氨基酸的蛋白质，理论分子量为 46.6 kDa。推断的 MAN5 与从 Aspergillus aculeatus 糖苷水解酶家族 5 的 β-甘露聚糖酶具有最高的氨基酸序列同一性为 58%。重组 MAN5 在巴斯德毕赤酵母中表达并纯化至电泳均一性。最终制剂对罗望子胶的比活性为 289 U mg(-1)。MAN5 在 pH 6.0 和 70°C 时表现出最佳活性，在很宽的 pH 值范围内具有良好的适应性和稳定性。该酶在 pH 3.0-8.0 时表现出超过 60%的峰值活性，在酸性和碱性条件下（pH 4.0-11.0）在 37°C 孵育 1 小时后保留超过 80%的活性。Km 和 Vmax 值分别为 1.33 mg ml(-1)和 444 μmol min(-1) mg(-1)以及 1.17 mg ml(-1)和 196 μmol min(-1) mg(-1)，用于罗望子胶和魔芋粉。在所有测试的金属离子和化学试剂中，Co(2+)、Ni(2+)和β-巯基乙醇在 1 mM 时增强了酶的活性，而其他化学物质对酶的活性没有影响或部分抑制了酶的活性。MAN5 对酸性和中性蛋白酶（胰蛋白酶、α-糜蛋白酶、胶原酶、枯草杆菌蛋白酶 A 和蛋白酶 K）具有高度抗性。由于 MAN5 的优良特性，该酶有可能应用于造纸和食品工业。
