School of Medicine and Pharmaceutics, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, PR China.
Carbohydr Res. 2011 Oct 18;346(14):2149-55. doi: 10.1016/j.carres.2011.06.035. Epub 2011 Jul 8.
A parent strain Aspergillus niger LW-1 was mutated by the compound mutagenesis of vacuum microwave (VMW) and ethyl methane sulfonate (EMS). A mutant strain, designated as A. niger E-30, with high- and stable-yield β-mannanase was obtained through a series of screening. The β-mannanase activity of the mutant strain E-30, cultivated on the basic fermentation medium at 32°C for 96 h, reached 36,675 U/g dried koji, being 1.98-fold higher than that (18,50 1U/g dried koji) of the parent strain LW-1. The purified E-30 β-mannanase, a glycoprotein with a carbohydrate content of 19.6%, had an apparent molecular weight of about 42.0 kDa by SDS-PAGE. Its optimal pH and temperature were 3.5 and 65°C, respectively. It was highly stable at a pH range of 3.5-7.0 and at a temperature of 60°C and below. The kinetic parameters K(m) and V(max), toward locust bean gum and at pH 4.8 and 50°C, were 3.68 mg/mL and 1067.5 U/mg, respectively. The β-mannanase activity was not significantly affected by an array of metal ions and EDTA, but strongly inhibited by Ag(+) and Hg(2+). In addition, the hydrolytic conditions of konjak glucomannan using the purified E-30 β-mannanase were optimized as follows: konjak gum solution 240 g/L (dissolved in deionized water), hydrolytic temperature 50°C, β-mannanase dosage 120 U/g konjak gum, and hydrolytic time 8 h.
一株产内切-β-甘露聚糖酶的黑曲霉突变株的选育及酶学性质研究
采用真空微波（VMW）和硫酸二甲酯（EMS）复合诱变处理出发菌株黑曲霉 LW-1，经一系列筛选获得了一株产酶活力高且稳定的突变株，命名为黑曲霉 E-30。在基础发酵培养基中于 32°C 培养 96 h，突变株 E-30 的β-甘露聚糖酶酶活达到 36675 U/g 干曲，比出发菌株 LW-1（18501 U/g 干曲）提高了 1.98 倍。E-30 所产β-甘露聚糖酶经纯化后为糖蛋白，含糖量为 19.6%，SDS-PAGE 电泳显示其表观分子量约为 42.0 kDa。该酶的最适 pH 和温度分别为 3.5 和 65°C，在 pH3.5-7.0 和 60°C 以下均具有较高的稳定性。在 pH4.8、50°C 条件下，该酶对罗望子豆胶的动力学参数 K_(m)和 V_(max)分别为 3.68 mg/mL 和 1067.5 U/mg。该酶对多种金属离子和 EDTA 不敏感，但 Ag(+)和 Hg(2+)对其有强烈的抑制作用。此外，用纯化的 E-30 β-甘露聚糖酶对魔芋葡甘聚糖的水解条件进行了优化，结果表明：魔芋胶溶液 240 g/L（溶解于去离子水中），水解温度 50°C，β-甘露聚糖酶用量 120 U/g 魔芋胶，水解时间 8 h。
