Tang Yanchong, Lu Yaping, Lü Fengxia, Bie Xiaomei, Guo Yao, Lu Zhaoxin
Enzyme Engineering Laboratory, College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China.
Sheng Wu Gong Cheng Xue Bao. 2009 Dec;25(12):1989-95.
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, 25 degrees C 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.
脂肪酶是重要的生物催化剂，广泛应用于食品加工和生物柴油生产。然而，在应用过程中有机溶剂可能会使某些脂肪酶失活。因此，高效克隆和表达耐有机溶剂脂肪酶对其应用至关重要。在本研究中，我们首先从腐生葡萄球菌M36中发现了一种耐有机溶剂脂肪酶，并通过PCR扩增出741 bp的脂肪酶基因lip3（GenBank登录号：FJ979867），该基因编码一个由247个氨基酸残基组成的31.6 kD多肽。但该脂肪酶与腐生葡萄球菌的推测lip3基因（GenBank登录号：AP008934）有83%的同源性。我们将该基因与表达载体pET-DsbA连接，转化到大肠杆菌BL21（DE3）中，得到重组质粒pET-DsbA-lip3。在pH 8.0、OD600为1.0、25℃条件下，用0.4 mmol/L异丙基-β-D-硫代半乳糖苷诱导12 h，脂肪酶活性可达25.8 U/mL。所表达的脂肪酶在甲醇、正己烷、异辛烷和正庚烷存在下具有稳定性。
