| 嗜热真菌Neosartorya fischeri P1脂肪酶基因的克隆、表达及酶学性质分析 |
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| 引用本文： | 孙乔乔,谭笑,吕依,黄火清,张会图,路福平.嗜热真菌Neosartorya fischeri P1脂肪酶基因的克隆、表达及酶学性质分析[J].中国农业科技导报,2014,16(5):53-59. |
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| 作者姓名： | 孙乔乔 谭笑 吕依 黄火清 张会图 路福平 |
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| 作者单位： | (1.天津科技大学生物工程学院, 天津 300457,2.中国农业科学院饲料研究所, 北京 100081) |
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| 基金项目： | 国家863计划项目（2013AA102803）；国家自然科学基金项目（2011BADB02）资助。 |
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| 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。
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| 关 键 词： | 脂肪酶 大肠杆菌 毕赤酵母 嗜热真菌 |
Cloning|Expression of Thermophilic Fungus Neosartorya fischeri P1 Lipase Gene and Analysis of its Enzymatic Property |
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| SUN Qiao\|qiao,TAN Xiao,LV Yi,HUANG Huo\|qing,ZHANG Hui\|tu,LU Fu\|pi.Cloning|Expression of Thermophilic Fungus Neosartorya fischeri P1 Lipase Gene and Analysis of its Enzymatic Property[J].Journal of Agricultural Science and Technology,2014,16(5):53-59. |
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| Authors: | SUN Qiao\|qiao TAN Xiao LV Yi HUANG Huo\|qing ZHANG Hui\|tu LU Fu\|pi |
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| Institution: | (1.College of Biotechnology, Tianjin University of Science and Technology, Tianjing 300457|;
2.Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China) |
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| 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. |
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| Keywords: | lipase Escherichia coli Pichia pastoris thermophilic fungus |
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