Isolation, Characterization, and Molecular Cloning of the cDNA Encoding a Novel Phytase from Aspergillus niger 113 and High Expression in Pichia pastoris

文献类型： 外文期刊

第一作者： Xiong, AS

作者： Xiong, AS;Yao, QH;Peng, RH;Li, M;Fan, HQ;Guo, MJ;Zhang, SL

作者机构：

关键词： Synthetic phyI1 gene;Fermentation;Phytase production;Pichia pastoris;Recombinant DNA

期刊名称：JOURNAL OF BIOCHEMISTRY AND MOLECULAR BIOLOGY （ 影响因子：2.021； 五年影响因子：2.1 ）

ISSN：

年卷期：

页码：

收录情况： SCI

摘要： Phytases catalyze the release of phosphate from phytic acid. Phytase-producing microorganisms were selected by culturing the soil extracts on agar plates containing phytic acid. Two hundred colonies that exhibited potential phytase activity were selected for further study. The colony showing the highest phytase activity was identified as Aspergillus niger and designated strain 113. The phytase gene from A. niger 113 (phyI1) was isolated, cloned, and characterized. The nucleotide and deduced amino acid sequence identity between phyI1 and phyA from NRRL3135 were 90% and 98%, respectively. The identity between phyI1 and phyA from SK-57 was 89% and 96%. A synthetic phytase gene, phyI1s, was synthesized by successive PCR and transformed into the yeast expression vector carrying a signal peptide that was designed and synthesized using P. pastoris biased codon. For the phytase expression and secretion, the construct was integrated into the genome of P. pastoris by homologous recombination. Over-expressing strains were selected and fermented. It was discovered that ～4.2 g phytase could be purified from one liter of culture fluid. The activity of the resulting phytase was 9.5 U/mg. Due to the heavy glycosylation, the expressed phytase varied in size (120, 95, 85, and 64 kDa), but could be deglycosylated to a homogeneous 64 kDa species. An enzymatic kinetics analysis showed that the phytase had two pH optima (pH 2.0 and pH 5.0) and an optimum temperature of 60 ℃.

分类号： Q81

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