木霉几丁质酶和几丁寡糖的制备及提纯

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。

Preparation and Purification of Trichodema Chitinase and Chitooligosaccharide

Objective] The technical support for the preparation and purification of Trichodema chitinase and chitooligosaccharide was provided.Method]The crude solution of Trichodema chitinase was obtained from the liquid of Trichodema strain-YHS-1 cultured and the chitinase activity of Trichodema strain-YHS-1at 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 chitinase 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)2SO4 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 chitinooligosaccharide was produced by enzymolysis of chitinase and colloid chitin.The pure chitinooligosaccharide could be obtained through the purification of the crude extract with protein sedimentation and refrigerated centrifugation.Conclusion] The preparation and purification method of Trichodema chitinase and chitooligosaccharide was established.