Abstract:

A thermophilic fungus that have high lipase activity was screened from the acid waste water of a tine mine in Yunnan Province. It was identified as Neosartorya fischeri through morphological observation and internal transcribed spacer (ITS) sequence analysis，and named Neosartorya fischeri P1. The lipase gene Lip024 was cloned from N. fischeri P1 genome and expressed in Escherichia coli BL21 (DE3) and Pichia pastoris GS115， with the activity of recombinant lipase 0.09U/mL and 0.26U/mL，respectively. High density fermentation was performed in 3.7 L fermenter for the recombinant strain，and the recombinant enzyme activity reached 5.22U/mL. Lip024 was purified to homogeneity by ultrafiltration and anion exchange chromatography. The optimal pH of recombinant Lip024 was pH 7.0， and the enzyme showed high stability at pH3.0~7.0. The Lip024 was highly thermostable, and the optimal temperature was found to be 50℃. In addition，Ca2+ and Mg2+ can significantly improve the activity of Lip024.

Key words: lipase；Escherichia coli；Pichia pastoris；thermophilic fungus

摘要：

从云南酸矿废水中获得了一株脂肪酶活性较高的嗜热真菌，经显微形态及转录间隔区（ITS）序列分析鉴定为新萨托菌，命名为Neosartorya fischeri P1。通过同源克隆，从该真菌中获得了脂肪酶基因Lip024，并在Escherichia coli BL21 (DE3) 和Pichia pastoris GS115 中进行表达，表达量分别为0.09 U/mL、0.26 U/mL。重组酵母菌株在3.7L发酵罐进行高密度发酵后，重组酶酶活达5.22 U/mL。重组蛋白经超滤浓缩和阴离子交换柱纯化后达到电泳纯。重组酶酶学性质分析表明：该酶的最适pH为7.0，在pH 3.0~7.0的范围内非常稳定，酶活力均保持在95%以上；最适温度为50℃，并具有一定的热稳定性。此外，Ca2+和Mg2+能够显著提高Lip024重组脂肪酶的酶活。

关键词: 脂肪酶；大肠杆菌；毕赤酵母；嗜热真菌