Abstract: Cyrophilic lipase has important application values in feed, laundry, and organic synthesis. However, the discovered cyrophilic lipases are limited in number and the knowledge about their biodiversity is also restricted. Therefore, it is necessary to mine the natural environment for new cyrophilic lipase genes. To obtain cyrophilic lipases, a lipase gene, namely Pseudomonas guguanensis lipase 1 (PgLip1) was identified and cloned from P. guguanensis by homology search against its genomic sequence. The gene sequence was analyzed  and  the protein was predicted. After expression, PgLip1 was investigated for its optimal substrate, pH and temperature. In addition, the effects of surfactant, denaturing agents, EDTA metal ions and organic solvents on the activity of PgLip1 were also studied. As a result, with a length of 840 bp, PgLip1 was predicted to encode a protein of 279 amino acids, which had 92.6% sequence similarity to a lipase from Pseudomonas mendocina. With an optimal temperature at 10 ℃, PgLip1 had 60.2% relative activity at 0 ℃ and was therefore a cryophilic lipase. With an optimal pH of 8.5, it had more than 62% relative activity at  pH 3.0~12.0. After being treated at 40 ℃ for 45 min, PgLip1 still had 726% residual activity. The best substrate of PgLip1 was p-nitrophenyl butyrate (pNPB). PgLip1 retained higher activity in higher concentrations of certain organics such as isopropanol, isoamyl alcohol and ethyl acetate than in lower concentrations of these reagents. Meanwhile, the surfactants Tween-20, Tween-80, and Triton X-100 could largely stimulate the activity of PgLip1 by 3.2~5.9 folds. Therefore, PgLip1 was a cyrophilic lipase that was stimulated by surfactants and tolerant to high concentrations of certain organics, suggestive of good application potentials in the future.

Key words: lipase, Pseudomonas guguanensis, cryophilic enzyme, organic solvent, surfactant

摘要： 低温脂肪酶在饲料、洗涤、有机合成等行业中具有重要的应用价值，然而目前已发现的低温脂肪酶数量不多，对其基因多样性了解不够深入，因此有必要从自然环境中挖掘新的低温脂肪酶基因。为了获得低温脂肪酶基因资源，通过基因组序列比对分析，从谷关假单胞菌（Pseudomonas guguanensis）中发现并克隆获得一个脂肪酶基因P. guguanensis lipase 1 (PgLip1),并对该基因进行序列分析和蛋白质预测，表达后检测PgLip1的最适底物、最适pH、最适温度，同时研究了表面活性剂、变性剂、EDTA、金属离子及有机溶剂对该酶活性的影响。结果显示：该基因片段大小为840 bp，预测编码含279个氨基酸残基的蛋白质，氨基酸序列与来源于门多萨假单胞菌（Pseudomonas mendocina）的脂肪酶相似性92.6%。该酶的最适温度为10 ℃，在0 ℃相对酶活为60.2%，是一个低温脂肪酶；其最适pH为8.5，在pH 3.0~12.0时，仍然具有62%以上的相对酶活。40 ℃处理45 min后，PgLip1还能保持72.6%的残余酶活。该酶的最适反应底物为对硝基苯基丁酸酯(pNPB)；在高浓度异丙醇、异戊醇和乙酸乙酯中的酶活反而高于相应较低浓度中，吐温-20、吐温-80和Triton X-100表面活性剂能较大程度激活PgLip1（3.2~5.9倍）。研究丰富了对低温脂肪酶生物多样性的科学认识，所获得的PgLip1是一个受表面活性剂特异激活且对部分高浓度有机溶剂耐受的低温脂肪酶，具有较好的应用前景。

关键词: 脂肪酶, 谷关假单胞菌, 低温酶, 有机溶剂, 表面活性剂