一种源于蜗牛的壳聚糖水解酶的纯化和定性

目的研究属于蜗牛的壳聚糖水解酶的纯化方法,得到壳聚糖水解酶的纯品,从而为氨基酸序列分析、基因克隆及工业菌制备奠定前期基础。方法建立检测蜗牛壳聚糖水解酶活性的手段并考察影响酶活性的各种因素,比较现有层析方法纯化蜗牛壳聚糖水解酶的实际效果,确定纯化的最佳条件,从而设计出最合理的纯化方案。结果经苯基琼脂糖柱层析,DEAE-Sepharose离子交换层析和Sephacryl S-300凝胶过滤分离,得到高纯高活性蛋白质,在SDS-PAGE上用银染的方法呈单一蛋白质条带,比活性提高33.333倍,纯化倍数为18.272,得率为0.15。结论实验建立了1种从蜗牛中分离高效高纯度壳聚糖水解酶的方法,为壳寡糖的酶解工业生产提供了新思路、新方法。

Purification and characterization of chitosanase from snail

Objectives To investigate the purification method of chitosanase.The ultimate goal of this research was to prepare purified onzyme to provide genetic information for cloning the gene coding for the enzyme by molecular biology tochniques.Methods The p-hydroxybenzonic acide hydrazidde(PAHBAH) assay was tested and confirmed for measurement of reducing sugar as the detection of enzyme activity.Different types of purification columns were frun for screening suitable chromatographhy methods.Results To complete purify the enzyme protein,a series of columns was determined as hydrophobic interaction followed by DEAE-Sepharose ion exchange,and Sephacry1 S-300 gel filtration.The purity of purified enzyme was checked by SDS-PAGE,and the enzyme showed a single band.The specific activity was increased by 24 folds during purification.The purification fold of the chitosanase was 18.272 and the its yield was 15%,specific activity is raised 33.333.Conclusions The text approach diversity purify methods of helicase,setting up a high performance method of separate chitosanase from helicase,to provid new approach for enzymolysis industry of oligosaccharide.