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

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)/pET30-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(GenBank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Ca2+和Co2+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。

Abstract:

[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 [Methods] Sequence of lipase gene was identified with transcriptome data (Liu, et al; unpublished data).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 pET30-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 (GenBank accession number: KT305964). The optimal temperature and pH for lipase hydrolytic activity on p-NPB were 40 °C and pH 8.0, respectively. The LIP activity was increased to about 130% after treated by Ca2+ and Co2+ 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.