耐热β-甘露聚糖酶的异源表达及低聚寡糖制备研究

Heterologous expression of thermotolerant β-mannanase and preparation of oligosaccharides

研究从魔芋基地筛选产甘露聚糖酶菌株,并克隆甘露聚糖酶基因。设计兼并引物获得目的基因,通过电转化法至毕赤酵母GS115中实现异源表达,研究该重组酶的酶学特性。结果显示,重组甘露聚糖酶DL4man基因为1 104 bp,蛋白分子量为41.5 kDa,糖苷水解酶26家族。最适反应pH值为5.5,最适反应温度为55℃,70℃下半衰期为70 min,80℃下耐受100 min后剩余46%的酶活,属于耐高温甘露聚糖酶,具有良好的胃-胰蛋白酶耐受性。酶解角豆胶的产物主要是聚合度为2、3、4、5的甘露寡糖。酶解魔芋粉的主要产物是聚合度为2、3的甘露寡糖。研究实现耐高温甘露聚糖酶在毕赤酵母GS115中的异源表达。研究表明,该酶酶解魔芋粉、角豆胶均能够生成甘露低聚糖,可为甘露低糖的制备提供参考。

In the study, the mannanase-producing strain was screened from the konjac base and the mannanase gene was cloned for further study. A merger primer was designed to obtain the target gene, which was heterologous expressed in Pichia pastoris GS115 by electro-transformation, and the enzymatic characteristics of the recombinant enzyme were studied. The results showed that the size of recombinant mannanase DL4 man gene was 1 104 bp, the molecular weight of the protein was 41.5 kDa, glycosidic hydrolase 26 family. The optimal pH value and temperature was 5.5 and 55 ℃, the half-life was 70 min under 70 ℃, and 46% enzyme activity remained after 100 min tolerance under 80 ℃, which belonged to high temperature resistant mannanase and had good tolerance to pepsin-trypsin. The product of enzymatic hydrolysis of carrageenan was mainly mannan-oligosaccharide with polymerization degree of 2, 3, 4, 5. The main product of enzymatic hydrolysis of konjac powder was mannan-oligosaccharide with polymerization degree of 2 and 3. The heterologous expression of high temperature resistant mannanase in pichia pastoris GS115 was achieved in the study. The The study indicates that mannanase can be used to produce mannan-oligosaccharide by enzymolysis of konjac powder and carrageenan, which provides a reference for preparation of mannan-oligosaccharide.