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

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.