摘要
目的：筛选获得高活力的微生物脂肪酶，实现其异源高效表达。方法：通过富集培养、平板筛选、单菌落发酵验证以获得产脂肪酶菌株，克隆脂肪酶基因，构建重组质粒在大肠杆菌中表达，纯化后进行酶学性质研究。结果：获得一株高产脂肪酶的菌株，经16SrDNA鉴定属于Proteussp。根据已报道的来源于Proteus vulgaris的脂肪酶基因设计引物，克隆其全长基因，大小为864bp，编码287个氨基酸。构建重组表达质粒pET32a—tipK19，在大肠杆菌中表达酶活达1500U／mL。该脂肪酶的最适温度为40℃，最适pH为9，在pH8～10之间稳定性较好，Ca^2＋对酶有激活作用，表面活性剂等对酶抑制作用明显，对有机溶剂有一定的耐受性。结论：克隆表达了-Proteussp脂肪酶，并对其性质进行了研究，为其应用奠定基础。
Objective：Screening for high active microbial lipase, and achieving its effectively heterologous expression. Method：Acquiring lipase - producing strains by enrichment culture, plate screening and single colony fermentation validation. Further, cloning the full - length sequence of lipase, and constructing the recombinant pET plasmid and expressing in E. coli. Then, purifying the recombinant lipase for characterization. Result：A lipase -producing strain identified as Proteus sp by 16S rDNA was screened. According to the reported lipase gene from Proteus vulgaris K80, a pair of primers was designed to clone the full length lipase gene of K19, which was named as lipK19 with the size of 864 bp, and encoding 287 amino acids. Recombinant expression plasmid pET32a - lipK19 was constructed, which was trans- ferred in E. coli and induced with IPTG,the expressed LipK19 activity attained to 1 500 U/mL. The optimum temperature of LipK19 was 40 ℃ ,and the optimum pH was 9 with high stability between pH 8 - 10. The Ca^2＋ could activate the activity of LipK19 ,but the surfactants inhibited the enzyme activity significantly. This lipase showed a certain degree of tolerance to organic solvent. Conclusion： Cloning and ex- pressing the lipase from Proteus sp. ,and investigating its properties ,founding the basis for its application.
出处
《生物技术》
CAS
CSCD
北大核心
2014年第1期66-72,共7页
Biotechnology
基金
国家高技术研究发展计划(863计划)项目("果蔬洗涤用酶与有机物降解酶研制"
2012AA022206B)
生物反应器工程国家重点实验室开放课题("深共溶剂中脂肪酶催化机制研究")
南京林业大学引进高层次人才科研基金
南京林业大学"青年拔尖人才培养计划"专项经费
江苏高校优势学科建设工程项目资助
