本文克隆表达了来源于嗜热细菌Thermoanaerobacter sp. X514的嗜热脂肪酶LipTX基因(Teth514_0029)，利用亲和层析纯化了脂肪酶LipTX，并详细研究了该脂肪酶的酶学性质。采用PCR技术扩增脂肪酶LipTX基因，克隆到载体pET15b中，构建重组载体pET15b-LipTX；将重组载体转入大肠杆菌BL21（DE3）中并利用IPTG诱导表达目的蛋白，经70 ℃热处理和Ni-NTA亲和层析两步纯化，得到纯化的重组酶。SDS-PAGE结果表明，脂肪酶LipTX分子量为28 ku。酶学性质研究表明，该酶最适反应温度和pH分别为70 ℃和7.5；通过水解底物特异性和动力学实验，发现LipTX可以水解不同长链酰基（C8-C12）的对硝基苯酚酯底物，对硝基苯酚癸酸酯是该酶最适合底物；同时该酶能够水解链长在C4到C16范围的三甘油酯底物，其中以甘油三丁酸酯为底物的酶活力最高。该酶对非极性有机溶剂（甲醇、乙醇、丙酮、DMF、DMSO和正己烷和氯仿）和变性剂（SDS、Tween-20和TritonX-100）具有较强的抗性，因此在生物催化和有机化学合成中具有广阔的开发应用前景。

The gene coding for a thermostable lipase LipTX (Teth514_0029) from thermophilic bacterium Thermoanaerobacter sp. strain X514 was cloned and the lipase LipTX was purified using affinity chromatography. The gene coding for lipase LipTX was amplified using polymerase chain reaction (PCR) method, and cloned into a pET15b vector, which was used to construct the recombinant plasmid pET15b-LipTX. The constructed pET15b-LipTX was transformed into host Escherichia coli BL21 (DE3) to express the recombinant enzyme via isopropyl β-D-1-thiogalactopyranoside (IPTG) induction. The purified recombinant enzyme was obtained after heat treatment and nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) result showed that the relative molecular mass of lipase LipTX was approximately 28 ku. Study of the enzymatic properties indicated that LipTX displayed optimal activity at 70 ℃ and pH 7.5. Substrate specificity and kinetics experiments showed that LipTX could hydrolyze p-nitrophenyl ester (pNP-ester) substrates with different acyl chain lengths (from C8 to C12), and the most suitable substrate was pNP-decanoate. Additionally, this enzyme could hydrolyze triacylglycerols with long acyl chains (from C4 to C16) and showed the strongest enzymatic activity with tributyrin as the substrate. Furthermore, the recombinant lipase LipTX was found to have strong resistance to non-polar organic solvents (methanol, ethanol, acetone, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), hexane, and chloroform) and denaturants (sodium dodecyl sulfate (SDS), Tween-20, and Triton X-100). These results demonstrate that the lipase LipTX has a broad application prospect for biocatalysis and organic synthesis.

国家自然科学基金资助项目（21406210）；河南省科技创新杰出人才项目（144200510011）