摘要
用LB培养基培养转化重组嗜碱耐盐芽孢杆菌(XJU-1)乙醛脱氢酶aldA基因的工程菌BL21(DE3),提取粗酶液,然后利用SDS-PAGE电泳考察重组ALDH的表达量和分子量;测定其活性;并对表达产物的最适pH、最适温度、金属离子的影响和Km值进行研究。结果表明,工程菌BL21(DE3)表达的ALDH量明显高于对照菌,亚基分子量约为56 kDa;乙醛脱氢酶活性比对照菌增加了24倍;最适反应温度为20～40℃,最适pH为9.0～9.5,K+和Na+对酶有激活作用,而Mn2+,Mg2+对酶有抑制作用;Km值为1.73 mmol/L。说明重组嗜碱耐盐芽孢杆菌(XJU-1)乙醛脱氢酶aldA基因在工程菌BL21(DE3)中能够高效表达。
In this experiment, the expression and the enzymological characters of acetaldehyde dehydrogenae （ALDH） of Bacillus halodurans XJU-1 in E.coli BL21（DE3） were studied. E.coli BL21（DE3） containing recombinant acetaldehyde dehydrogenae （ALDH） of Bacillus halodurans XJU-1 was grown in LB medium, induced by IPTG, Sonication was carried out on ice in an ultrasonic processor to obtain the crude enzyme solution, and then SDS-PAGE electrophoresis was used to study the expression of recombinant ALDH and molecular weight and to determine the en- zymatic activity of ALDH. And the optimum pH, the optimum temperature, the impact of metal ions and values of Km of the expression product were investigated. The results showed that engineering strain BL21 （DE3） expression of ALDH was significantly higher than the control strains, the molecular weight of the enzyme as estimated by SDS-PAGE was approximately 56,000. In this experiment, the optimum pH and temperature of the enzyme was found to be pH 9.0-9.5 and at 20℃-40℃ .The enzymatic activity of ALDH was activated by K＋ and Na＋. In contrast, it was more or less inhibited by Mn2＋and Mg2＋. TheKm values of the enzyme were 1.73 mmol/L for acetaldehyde. In conclusion, acetaldehyde dehydrogenae gene aldA of Bacillus halodurans XJU -1 is highly expressed in E. coli BL21（DE3）.
出处
《酿酒科技》
2010年第7期21-23,共3页
Liquor-Making Science & Technology
基金
四川大学"214"振兴计划科研启动基金资助(No.0082204127092)
