嗜热栖热放线菌（BT107）脂肪酶I在毕赤酵母中的高效表达及特性研究
High-level expression and characterization of Galactomyces geotrichum (BT107) lipase I in Pichia pastoris.
作者信息
Fernández Layla, Pérez-Victoria Ignacio, Zafra Alberto, Benítez Pedro L, Morales Juan C, Velasco Javier, Adrio José L
机构信息
Department of Biotechnology, Puleva Biotech, Camino de Purchil, 66, 18004-Granada, Spain.
出版信息
Protein Expr Purif. 2006 Oct;49(2):256-64. doi: 10.1016/j.pep.2006.06.015. Epub 2006 Jun 27.
The mature lipI gene, encoding the lipase I from Galactomyces geotrichum BT107, was obtained by PCR from genomic DNA, sequenced and cloned into a Pichia pastoris expression vector. Clones containing multiple copies of lipI integrated in their genome were analyzed to achieve high-level expression of the recombinant lipase I. One strain with four or more copies of the expression cassette was able to produce more than 200mg/L of extracellular heterologous protein. The lipase I was partially purified using anion exchange chromatography and its activity on monounsaturated (triolein) and polyunsaturated (triEPA) triglycerides was analyzed by a novel HPLC-MS assay.
通过PCR从基因组DNA中获得了编码来自地丝酵母BT107脂肪酶I的成熟lipI基因，进行测序后克隆到毕赤酵母表达载体中。对基因组中整合有多个lipI拷贝的克隆进行分析，以实现重组脂肪酶I的高水平表达。一个含有四个或更多表达盒拷贝的菌株能够产生超过200mg/L的细胞外异源蛋白。使用阴离子交换色谱法对脂肪酶I进行部分纯化，并通过一种新型的HPLC-MS测定法分析其对单不饱和（三油酸甘油酯）和多不饱和（三二十碳五烯酸甘油酯）甘油三酯的活性。
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