CAJ | 학술논문

目的构建表达重组粗糙脉孢菌乙醇脱氢酶的工程菌,并对乙醇脱氢酶酶活测定条件进行优化,为氧化还原酶酶法催化在手性药物中间体合成中的应用提供指导.方法采用 PCR技术,以粗糙脉孢菌基因组 DNA为模板,克隆得到粗糙脉孢菌乙醇脱氢酶基因,连接到表达载体 pET-28a上,得到重组质粒 pET-ADH.将该重组质粒导入大肠杆菌 BL21中,并经培养、筛选、酶切、测序鉴定.用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达并测酶活力,同时对影响酶活的条件进行研究.结果粗糙脉孢菌乙醇脱氢酶活力较高,最适反应介质为 Tris-HCl缓冲液,在 30 ~ 35℃、pH 7 ~ 8时酶活力较稳定,较适底物为 2-氯苯乙酮.结论通过采用 PCR技术,成功克隆表达粗糙脉孢菌乙醇脱氢酶,并优化得到最优酶活条件.
목적 구건표체중조조조맥포균을순탈경매적공정균,병대을순탈경매매활측정조건진행우화,위양화환원매매법최화재수성약물중간체합성중적응용제공지도.방법 채용 PCR기술,이조조맥포균기인조 DNA위모판,극륭득도조조맥포균을순탈경매기인,련접도표체재체 pET-28a상,득도중조질립 pET-ADH.장해중조질립도입대장간균 BL21중,병경배양、사선、매절、측서감정.용이병기-β-D-류대반유당감(IPTG)유도표체병측매활력,동시대영향매활적조건진행연구.결과 조조맥포균을순탈경매활력교고,최괄반응개질위 Tris-HCl완충액,재 30 ~ 35℃、pH 7 ~ 8시매활력교은정,교괄저물위 2-록분을동.결론 통과채용 PCR기술,성공극륭표체조조맥포균을순탈경매,병우화득도최우매활조건.
Objective To study the most optimized biotransformation conditions of alcohol dehydrogenase (ADH) cloned fromNeurospora crassa, so as to provide help for exploring the synthesis of chiral intermediates by enzymatic biocatalysis with oxidoreductases. Methods Theadh gene was cloned from the genomic DNA ofNeurospora crassa by PCR, linked to expression vector pET-28a to construct pET-ADH, and then pET-ADH was transformed into BL21(DE3)pLSs. Culturing, screening, enzyme digesting and sequencing were performed for identification. The recombinant strains were induced by IPTG to express ADH. The biotransformation conditions of ADH were optimized by single factor and orthogonal experiments. Results The results showed that optimized biotransformation conditions were Tris HCl buffer, pH 7.0 - 8.0, 30 - 35℃, and 2-chloro-1-phenylethanone as the most suitable substrate. By comparison, no ADH appeared in hosted E.coli BL21(DE3)pLSs. Conclusion The expressed alcohol dehydrogenase showes potent oxidoreductase activities influenced by pH, temperature and substrate.