一种鉴定多糖水解酶类及其产生菌的新方法

A new plate method for screening of polysaccharide-degrading enzymes and their producing microorganisms

摘要:

利用曲里苯蓝能与多糖形成复合物的原理,建立起一种简便、快捷、灵敏的筛选多糖水解酶类及其产生菌的平板鉴定方法。曲里苯蓝对供试微生物无毒害作用,不影响酶的活性,并可高温灭菌。其最适浓度在0.005%~0.01%(W/V)之间。可用于纤维素水解酶类、淀粉水解酶类、甘露聚糖酶、普鲁兰酶等多糖水解酶,与传统方法相比,该方法不仅有着同样高的灵敏度,而且能够提高筛选效率,避免污染问题,同时适合于多种多糖底物。但不能用于木聚糖酶和菊粉酶的检测。

Abstract:

A plate assay based on the formation of haloes on Petri dishes, containing the trypan blue dye and polysaccharides as substrates, provides a specific, reliable and rapid detection of corresponding polysaccharide degrading enzymes and their producing microorganisms. A blue complex was formed by mixing trypan blue and polysaccharides as substrates. It has been proved by testing three strains that the trypan blue was neither harmful to microorganisms nor enzymes and could stand the normal sterilization. It's optimum concentration was from 0. 005 % to 0. 01 % (W/V). It do not need to prepare dye-labelled polysaccharides, so is a money and time-consuming method. The sensitivity of trypan blue method was the same as traditional method and it has potential for increasing the efficacy of screening of microorganisms, utilizing different polysaccharides, especially for large-scale searching programs, such as screening of large numbers of natural samples and engineering bacteria. Using this method, polysaccharide-degrading enzyme genes also has potential of as a new kind of marker gene in gene engineering techniques. According to the result, this method is suitable for detecting cellulase, amylase, pullulanase and mannase, but not suitable for detecting xylanase and inulinase.