【摘要】 目的:克隆壳聚糖酶基因于大肠杆菌中实现高表达,制备壳寡糖。方法:以枯草芽孢杆菌总DNA为模板扩增壳聚糖酶基因(CSN),克隆至载体pET23a(+)上,转化菌株BL21(DE3)。重组子经0.5 mmol/L IPTG诱导后,SDS-PAGE和质谱检测与鉴定重组酶。酶纯化后水解壳聚糖,薄层色谱分析其水解产物。结果:质谱证明壳聚糖酶(31.5kDa)成功表达,表达量占菌体总蛋白的45%左右。纯化后重组酶浓度为900 mg/L,纯度95%、回收率85%,酶活力为10 000 U/mg。壳聚糖降解产物为壳二糖至壳四糖。结论:原核表达载体pET23a(+)-CSN构建正确,壳聚糖酶表达量与活性高,适用于水解壳聚糖制备壳寡糖。更多还原

【Abstract】 Objective:High-expression of chitosanase through the cloning in E.coli and preparation of chitooligosaccharides.Method:A chitosanase(CSN) DNA fragment was amplified from the total DNA of Bacillus subtilis by PCR,and cloned to plasmid pET23a(+).The recombinant plasmid pET23a(+)-CSN was transformed into E.coli BL21(DE3),and the expression was induced by 0.5 mmol/L IPTG.The expressed product was identified by SDS-PAGE and mass spectrometry,and the recombinant protein was purified by His-Tag Purification Kit.Chitosan was hydrolyzed by purified CSN and the product was analyzed by thin layer chromatography.Result:Mass spectrometry proved that the target protein,with a relative molecular weight of 31.5 kDa,contained about 45% of total protein.The recombinant protein could specifically bind to His-Tag kit,about 95% of recombinant protein was purified and the recovery rate was 85%.The protein concentration was 900 mg/L and the specific activity was 10 000 U/mg.The major hydrolysis products of chitosan were chitodimer,chitotrimer and chitotetramer.Conclusion:The cloned DNA sequence of pET23a(+)-CSN was identical to that of reported CSN cDNA,and the chitosanase was successfully expressed in E.coli.The purified recombinant protein could be used for preparation of chitooligosaccharides.更多还原