木霉几丁质酶和几丁寡糖的制备及提纯  

Preparation and Purification of Trichodema Chitinase and Chitooligosaccharide

作　　者：沈奕[1] 伍万荣[1] 高智谋[1] 王革 

机构地区：[1]安徽农业大学植物保护学院,安徽合肥230036 [2]云南玉溪红塔集团生化研究中心,云南玉溪653100

出　　处：《安徽农业科学》2009年第16期7332-7333,7369,共3页Journal of Anhui Agricultural Sciences

基　　金：安徽省教育厅自然科学重点项目(2006KJ057A)

摘　　要：[目的]为木霉几丁质酶和几丁寡糖的开发应用提供必要的技术支持。[方法]由高产几丁质酶的木霉菌株YHS-1液体振荡培养得到木霉几丁质酶粗酶液,分别测定了木霉菌株YHS-1在不同时间、不同温度下的几丁质酶活值,优化木霉产酶条件。并研究了几丁寡糖的制备及提纯方法。[结果]供试木霉最佳产酶温度为35℃,最佳产酶时间为4 d,此时酶活值为55 U。几丁质酶粗酶液通过(NH4)2SO4分级沉淀、阴离子交换柱层析、SDS-PAGE提纯后可以达到层析纯,出现单一并且峰值很高的层析峰,电泳确认2条带,分别为几丁质外切酶和几丁质内切酶。几丁寡糖用初提纯的几丁质酶与过量的胶态几丁质反应得到粗溶液,再经过蛋白质沉淀和冷冻离心后得到纯化。[结论]建立了木霉几丁质酶和几丁寡糖的制备及提纯方法,为木霉几丁质酶和几丁寡糖的开发应用提供了必要的技术支持。[Objective] The technical support for the preparation and purification of Trichodema chifinase and chitooligosaccharide was provided. [ Method]Tbe crude solution of Trichodema chitinase was obtained from the liquid of Trichodema strain-YHS-I cultured and the chitinase activity of Trichodema strain-YHS-I at the different times（ 1,2, 3,4 and 5 days） and temperatures （20, 30, 35 and 40℃ ） were determined, respectively, for the research on the its preparation and purification method. [ Results ] The results showed that the optimum temperature for the production of ehitinase was 35 ℃ and the optimum time, the forth day and at the condition, enzyme activity was 55 U. Chromatographically pure chitinase could be obtained by （ NH4 ） 2 SO4 fractional precipitation, anion-exchange chromatography, SDS-PAGE. A singular and high chromatography peak could be found and two electrophoretic bands could be validated from gel electrophoresis. The two bands are exochitinase and endochitinase. The crude extract of chitlnooligosaccharide was produced by enzymolysis of ehitinase and colloid chitin. The pure chitinooligosaccharide could be obtained through the purification of the crude extract with protein sedimentation and refrigerated eentrifugation. [ Con- clusion ] The preparation and purification method of Trichodema chitinase and chitooligosaccharide was established.

关 键 词：木霉 几丁质酶 几丁寡糖 制备 提纯 

分 类 号：S188[农业科学—农业基础科学]