源自重组菌BL21-pET28a-cdE降氰酶的基本性质研究  

Study on basic property of cyanide-degrading enzyme from a recombinant strain BL21-pET28a-cdE

作　　者：和晓贤 李青云[1,2] 唐爱星 刘幽燕[1,2] HE Xiao-xian;LI Qing-yun;TANG Ai-xing;LIU You-yan(School of Chemistry and Chemical Engineering,Guangxi University,Nanning,530004,China;Guangxi Key Laboratory of Biorefinery,Nanning 530004,China)

机构地区：[1]广西大学化学化工学院,广西南宁530004 [2]广西生物炼制重点实验室,广西南宁530004

出　　处：《广西大学学报（自然科学版）》2018年第3期1258-1266,共9页Journal of Guangxi University（Natural Science Edition）

基　　金：国家自然科学基金资助项目(51108098);广西大学科研基金资助项(XJZ130360)

摘　　要：在前期研究工作的基础上,对含降氰酶基因重组菌BL21-pET28a-cdE开展遗传稳定性以及降氰酶基本特性的研究,比较分析原代菌与传代重组菌的菌落形态特点、质粒稳定性、蛋白表达、酶活水平,并对镍柱纯化获得的95%纯度的降氰酶进行了最适温度、最适pH、保藏稳定性的实验考察。结果显示,重组菌在无抗生素压力下传至100代时的质粒稳定性可保持在73%,菌落形态、蛋白表达、酶活水平等指标与原代的相一致。降氰酶的最适氰降解反应条件为30℃、pH 7.0。添加2%甘油可在4℃条件下稳定保藏降氰酶50 h,其相对酶活力为102.2%,而添加2%乙醇可使降氰酶在-20℃条件下稳定保藏1个月,其相对酶活力仍可达到80.9%。研究结果表明,重组菌BL21-pET28a-cdE具有良好的遗传稳定性,为未来大规模发酵制备降氰酶处理氰污染提供了可能。Based on the previous research,the genetic stability of the recombinant strain BL21-pET28 a-cdE and enzymatic basic property of cyanide-degrading enzyme were studied. The colonial morphology,plasmid stability, protein expression and enzyme activity of primary strains and subcultured strains were compared and analysed. The optimum reaction temperature and p H,as well as the preservation stability of the cyanide-degrading enzyme with 95% purity obtained by Nichelating affinity chromatography were experimented. The results showed that the heredity character of BL21-pET28 a-cdE was stable. The plasmid stability was 73% after 100 generations under nokanamycin selection pressure. The growth state and the expression of protein were consistent with those of the original generation. The cyanide-degrading enzyme had an optimal activity at a temperature of 30 ℃ and a pH of 7. 0. The enzyme was preserved for 50 h at 4 ℃ by adding 2% glycerin as the stabilizer,and the relative enzyme activity was 102. 2%. Furthermore,the relative enzyme activity was 80. 9% by adding 2% ethanol alcohol for one month preservation at-20 ℃. The results indicate that the recombinant strain BL21-pET28 a-cdE with a good genetic stability offers the possibility for treating cyanide-containing waste by producing the cyanide-degrading enzyme in mass industrial production.

关 键 词：降氰酶 重组菌 氰化物 酶学性质 

分 类 号：Q89[生物学]