关键词: 脂肪酶, 谷关假单胞菌, 低温酶, 有机溶剂, 表面活性剂

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