Abstract:

A high-throughput screening method based on membrane for selecting cholesterol oxidase mutants with improved thermal activity was proposed. This method takes advantage of the fact that the process of enzyme treatment and the reaction between enzyme and substrate was separated effectively，thus preventing the enzyme from reaction with substrate before heat treatment. High temperature treatment only involved the membrane not the agar plate，thereby the problem of softening or melting of the agar plate was resolved. This method is applicable to the screening of mutant colonies with high activity which produce obvious reddish brown transparent circle on the agar plate. The nitrocellulose filter membrane used for transfer printing is cheap and the major advantages of this method are reliability and simple，rapid screening.

摘要：

建立了一种以滤膜为基础的高通量筛选耐高温胆固醇氧化酶产生菌的方法。利用酶偶联及胆固醇不溶于水的特性产生显色透明圈。酶的热处理过程和酶与底物的反应过程分开，有效地防止酶在热处理前与底物反应。高温处理是在滤膜上进行，从而克服了高温琼脂平板变软或熔化的缺点。用该方法筛选时，产生的红褐色透明圈较明显，易于挑出性能改善的突变菌株。转印所用NC膜的价格便宜，操作比较简便。