摘要
从南极普利兹湾深海900米深的沉积物中提取获得宏基因组DNA,并通过设计引物,从中克隆到全长为948bp的低温脂肪酶(lip3)开放阅读框完整序列,该基因编码一个由315个氨基酸残基组成、预计分子质量为34.557ku酶蛋白(Lip3);氨基酸序列上的GFGNS(GXGXS)和G-N-S-M-G(GXSXG)在许多脂肪酶中有很高的保守性,它们是水解机制所必需的序列,也是丝氨酸水解酶中最保守的序列.构建了lip3基因重组表达载体,并在大肠杆菌中获得表达,采用镍离子亲和层析柱对表达的酶蛋白Lip3进行纯化,得到约35ku蛋白条带,酶学性质的分析表明,该酶的最适作用温度为25℃,在0℃时表现为最高活力的22%,最适pH值为8.0,对热敏感,35℃热处理60min剩余酶活为10%,以硝基苯棕榈酸酯为底物,Lip3的酶促反应常数Km值随着反应温度的升高而升高,是典型的低温酶.
The metagenomic DNA was extracted from the deep sea sediment with the depth of 900m of Prydz Bay, Antarctic. A lipase gene （lip3） with the size of 948bp was cloned from the metagenomic DNA by PCR with the primers designed. The deduced Lip3 protein was composed of 315 amino acids （AA） with a molecular mass of 34.577 ku. The motifs GFGNS（GXGXS）and G-N-S-M-G（GXSXG）in the AA sequences of Lip3 were found to be conserved in other lipase. They were most conserved sequence among the serine hydrolase and were necessary for the activity. A 35 ku of Lip3 was purified by Ni-NTA chelating sepharose column from the extract of recombinant E.coli Top 10F＇ cell harboring a pLLP-OmpA plasmid inserted with lip3. The purified Lip3 was most active at 25℃ and kept 22% of activity at 0℃. Only 10% of activity was retained after it was incubated at 35℃ for 60 min. The optimal pH value for the Lip3 activity was 8.0. The Km value of the enzyme towards p-nitrophenyl palmitate increased with the increasing of assayed temperature. These results indicated that Lip3 was a typical alkaline cold active enzyme.
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
2006年第12期1207-1214,共8页
Progress In Biochemistry and Biophysics
基金
国家自然科学青年基金资助项目(40406029)~~
关键词
南极深海沉积物
宏基因组
低温脂肪酶
克隆与表达
Antarctic deep sea sediment, metagenome, cold active lipase, cloning and expression
