【摘要】 为提高木霉几丁质酶检测方法的准确性和灵敏度,建立一种快速检测几丁质酶同工酶的方法。采用活性凝胶电泳、变性凝胶电泳、原位显色凝胶电泳结合荧光增白剂(Calcofluor white M2R)显色从绿色木霉LTR-2发酵产物中检测几丁质酶同工酶。活性凝胶电泳在粗酶液浓缩5倍时显示两条活性谱带,变性凝胶电泳在浓缩10倍时显示一条活性谱带,原位显色凝胶电泳在浓缩20倍时显示两条不清晰的活性谱带,SDS-PAGE显示这两条活性谱带的分子量分别为65kDa和42kDa。结果表明活性聚丙烯酰胺凝胶电泳和Calcofluor white M2R显色相结合的方法在几丁质酶上样量为0.47U时具有较好的分辨能力,是检测木霉几丁质酶同工酶的有效的方法。更多还原

【Abstract】 In order to improve the accuracy and sensitivity in detecting of chitinase isozymes from Trichoderma spp, a rapid sensitive method is established. It was employed to detect chitinase isozymes from Trichoderma viride LTR-2 through native gel electrophoresis, denatured gel electrophoresis and in situ gel electrophoresis with Calcofluor white M2R. Two chitinase isozymes native bands CHI65 and CHI42 were revealed in solid plate containing chitin and Calcofluor white M2R with native gel electrophoresis after chitin grown culture was concentrated 5 times. The two bands showed a smear instead of well-defined band with in situ gel electrophoresis after 20 times concentration. While only CHI65 chitinase was found with denatured gel electrophoresis after 10 times concentration. The molecular weight of CHI65 and CHI42 was separately about 65kDa and 42kDa. As a result, it was suitable for detecting chitinase isozymes through active gel electrophoresis with Calcofluor white M2R. The result showed that it was effective for differentiation among chitinase isozymes when total chitinase loading amount was 0.47U.更多还原