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目的:研究重组内切几丁质酶的分离纯化及其催化壳聚糖制备壳寡糖的反应条件优化。方法:重组菌发酵液经PEG20000浓缩和DEAE-Sepharose FF离子交换层析后,测定总蛋白含量和内切几丁质酶活力。用纯化的内切几丁质酶催化壳聚糖制备壳寡糖,探讨其最佳的反应条件。结果:纯化的内切几丁质酶比活力41.34U/mg,纯化倍数2.49,酶总回收率89.63%。内切几丁质酶催化壳聚糖降解制备壳寡糖的最优化反应条件是:壳聚糖质量分数4%,p H7.0,温度30℃,时间12 h。结论:本研究结果为内切几丁质酶和壳寡糖的产业化应用奠定了良好基础。
OBJECTIVE: To study the isolation and purification of recombinant endochitinase and the optimization of the reaction conditions for preparing chitosan oligosaccharide from chitosan. Methods: The total protein content and endochitinase activity of the recombinant bacteria fermentation broth were determined by PEG20000 concentration and DEAE-Sepharose FF ion exchange chromatography. Chitosan oligosaccharide was prepared by chitosan catalyzed by purified endochitinase, and the optimal reaction conditions were discussed. Results: The specific activity of purified endochitinase was 41.34U / mg, the purification fold was 2.49 and the total recovery of the enzyme was 89.63%. The optimum reaction conditions for the chitosan oligosaccharide degradation catalyzed by chitosan were: chitosan mass fraction 4%, p H7.0, temperature 30 ℃, time 12 h. Conclusion: The results of this study laid a good foundation for the industrial application of endochitinase and chitooligosaccharides.