腐生葡萄球菌M36耐有机溶剂脂肪酶基因的克隆与原核表达

Cloning and expression of organic solvent tolerant lipase gene from Staphylococcus saprophyticus M36

摘要:

脂肪酶是重要的工业用酶, 在食品加工、生物柴油的合成等领域具有广泛的应用。但是在应用中有机溶剂对脂肪酶具有一定的毒性, 因此获得耐有机溶剂的脂肪酶基因并实现高效表达是脂肪酶规模化应用的前提。本研究应用PCR技术首次从耐有机溶剂脂肪酶产生菌腐生葡萄球菌M36基因组DNA中扩增得到脂肪酶Ⅲ基因lip3(GenBank Accession No. FJ979867), 其编码区长度为741 bp, 编码247个氨基酸, 推测蛋白分子量大小为31.6 kD。它与腐生葡萄球菌lip3推测的基因(GenBank Accession No. AP008934)只有83%的同源性。将该基因与大肠杆菌表达载体pET-DsbA连接, 转化大肠杆菌Escherichia coli BL21(DE3)获得重组菌株BL21(DE3)/pET-DsbA-lip3, 在pH 8、25 oC条件下, OD600为1.0时用0.4 mmol/L IPTG诱导12 h酶活达到25.8 U/mL。重组酶在甲醇、正己烷、异辛烷、正庚烷等有机溶剂中具有较好的耐性。lip3基因的克隆及在大肠杆菌中有效表达的研究为进一步进行基因工程改造和脂肪酶应用奠定了基础。

Abstract:

Lipases are important biocatalysts that are widely used in food processing and bio-diesel production. However, organic solvents could inactivate some lipases during applications. Therefore, the efficient cloning and expression of the organic solvent-tolerant lipase is important to its application. In this work, we first found out an organic solvent-tolerant lipase from Staphylococcus saprophyticus M36 and amplified the 741 bp Lipase gene lip3 (GenBank Accession No. FJ979867), by PCR, which encoded a 31.6 kD polypeptide of 247 amino acid residues. But the lipase shared 83% identity with tentative lip3 gene of Staphylococcus saprophyticus (GenBank Accession No. AP008934). We connected the gene with expression vector pET-DsbA, transformed it into Escherichia coli BL21 (DE3), and obtained the recombinant pET-DsbA-lip3. With the induction by 0.4 mmol/L of isopropyl beta-D-thiogalactopyranoside at pH 8.0, OD600 1.0, 25oC for 12 h, the lipase activity reached up to 25.8 U/mL. The lipase expressed was stable in the presence of methanol, n-hexane, and isooctane, n-heptane.