破囊壶菌脂肪酶基因的克隆、双温控自诱导表达及酶学性质研究  

Cloning, auto-induced expression by dual temperature controlling and characterization of lipase from Aurantiochytrium sp. PKU#SW7

作　　者：梁园梅 刘瑛[1] Maurycy Daroch 成家杨[1] 

机构地区：[1]北京大学环境与能源学院,广东深圳518055

出　　处：《微生物学通报》2016年第6期1190-1198,共9页Microbiology China

基　　金：海洋公益性行业科研专项经费项目(No.201305022);国家外国青年专家基金项目(No.31450110424);中国博士后科学基金项目(No.2014M560855);深圳市海外高层次人才创新创业专项基金项目(No.KQCX201405211502553)~~

摘　　要：【目的】克隆破囊壶菌Aurantiochytrium sp.PKU#SW7的脂肪酶基因,实现其在大肠杆菌细胞中异源高效表达,并进行初步酶学性质研究。【方法】基于转录组数据注释,获得脂肪酶基因lip,构建重组基因工程菌Rosetta(DE3)/p ET30-lip,利用双温控自诱导方法高效表达蛋白,表达产物(LIP)经Ni-Agarose His亲和层析柱纯化后进行酶学性质研究。【结果】从Aurantiochytrium sp.PKU#SW7中克隆得到一个大小为873 bp的脂肪酶基因(Gen Bank登录号为KT305964),该酶对p-NPB最适反应温度和pH分别为40°C和8.0。以不同浓度的金属离子Ca^(2+)和Co^(2+)溶液分别保温处理酶液30 min可使酶活提高1.3倍左右;甲醇对脂肪酶的抑制作用不明显。在最适反应条件下对p-NPP与p-NPB的酶活力分别为70.0±3.1 U/mg和102.5±2.6 U/mg。【结论】Aurantiochytrium sp.PKU#SW7脂肪酶具有良好的特性,符合生物柴油生物催化剂基本要求。[Objective] The lipase gene of Aurantiochytrium sp. PKU#SW7 was cloned and expressed in Escherichia coli, and the lipase enzymatic properties were characterized. [Methods] Sequence of lipase gene was identified with transcriptome data（Liu, et al; unpublished data）. The recombinant plasmid p ET30-lip was over expressed in Rosetta（DE3） by auto-induced and dual temperature control strategy. The recombinant lipase（LIP） was purified with Ni-Agarose His affinity chromatography and the enzymatic properties were further determined. [Results] A length of 873 bp lipase gene was obtained（Gen Bank accession number： KT305964）. The optimal temperature and p H for lipase hydrolytic activity on p-NPB were 40 °C and p H 8.0, respectively. The LIP activity was increased to about 130% after treated by Ca^（2＋） and Co^（2＋） for 30 min. No significant inhibition of LIP activity was observed by methanol treatment. Under the optimal conditions, the LIP specific activities for p-NPP and p-NPB were 70.0±3.1 U/mg and 102.5±2.6 U/mg. [Conclusion] The LIP of Aurantiochytrium sp. PKU#SW7 possess favorable characteristics and meets the basic requirements of biodiesel catalyst.

关 键 词：破囊壶菌 脂肪酶 双温控自诱导表达 

分 类 号：Q78[生物学—分子生物学] Q936