耐高温β-甘露聚糖酶基因的高效表达与水解魔芋精粉条件的优化

High-level expression of a thermal stable β-mannanase and optimization on the hydrolysis conditions for konjac flour

β-1,4-D-甘露聚糖酶(EC3.2.1.78)能够随机催化水解甘露聚糖和葡甘露聚糖等中的β-1,4-甘露聚糖苷键。魔芋(Amorphophallus konjac)精粉是葡甘露聚糖类物质,以之为原粉在甘露聚糖酶的作用下可制作益生元类低聚甘露糖。采用串联多拷贝表达盒的方式提高耐高温甘露聚糖酶的表达量,获得具有高产特性的菌株;探究甘露聚糖酶的酶学性质以及水解魔芋精粉制作低聚甘露糖的最适条件,为甘露聚糖酶的产业化及低聚甘露糖的酶法制备奠定基础。结果表明:该酶的最适温度为70℃,最适p H5.0;所获得的多拷贝菌株在14 L发酵罐下酶活可达10810 U/m L;在最适反应条件下,该酶可高效地将魔芋精粉水解为低聚甘露糖。

β-1,4-D-mannanase （EC 3.2.1.78） was able to catalyze the hydrolysis of mannan and glucomannan. Konjac （Amorphophallus konjac） flour Js a glucomanic substance which can be used as the materials to produce probiotic oligomeric mannose under the hydrolysis of mannanase. In this study, the high level expression of mannanase was achieved by constructing multiple-copy expression cassettes in genome. The enzymatic characteristics and the conditions for hydrolysis of konjac flour were checked and optimized to faciliate the industrial application of mannanase. The results have shown that the optimal temperatrue and pH of mannanase were 70 ℃ and pH5.0, respectively; The enzyme activity of mannanase could reach 10810 U/mL in 14 L bench top bioreactor; Under the optimal conditions, the konjac flour could be efficiently hydrolvzed into olioomannose.