熊爱生 姚泉洪 彭日荷 李 贤 范惠琴 郭美锦 张嗣良
《Journal of Biochemistry and Molecular Biology
摘 要 植酸酶能将植酸水解成肌醇和磷酸，在饲料中添加植酸酶不仅可以提高植物性饲料中磷的利用率、减少粪便中排出的磷对环境污染，而且能够通过降低植酸盐的抗营养作用，增加动物对蛋白质和一些金属离子的吸收能力。从土壤中分离得到一株表达植酸酶的菌株，命名为黑曲霉-113，从该菌株中克隆一编码新的植酸酶基因，命名为phyI1，phyI1基因与来源于NRRL3135的phyA和来源于SK-57的phyA基因分别有90% 和89%同源性。为了在甲醇酵母中高表达，对基因进行改造，表达量达到4.2g/L，该蛋白比活是9.5 U/mg，酶动力学分析表面该酶有两个最适pH，最适温度为60℃.
ISOLATION, CHARACTERIZATION, MOLECULAR CLONING OF THE CDNA ENCODING A NOVEL PHYTASE FROM ASPERGILLUS NIGER 113 AND HIGH EXPRESSION IN PICHIA PASTORIS
Aisheng Xionga) Quanhong Yaoa) Rihe Penga) Xian Lia) Huiqin Fana) Meijin Guoa)
(a) Agro-Biotechnology Research Center of Shanghai Academy of Agricultural Sciences, Shanghai Key Laboratory of Agricultural Genetics and Breeding, Beidi RD 2901, 201106, Shanghai, China;
b) East China University of Science and Technology, Shanghai 200237, China)
Abstract Phytases catalyze the release of phosphate from phytic acid. Phytase-producing microorganisms were selected by culturing soil extracts on agar plates containing phytic acid. Two hundred colonies exhibiting potential phytase activity were selected for further study. The colony showing 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. Nucleotide and deduced amino acid sequence identity between phyI1 and phyA from NRRL3135 were 90% and 98% respectively; Identity existed between phyI1 and phyA from SK-57 were 89% and 96%. A synthetic phytase gene, phyI1s, was synthesized by successive PCR and transformed into the yeast expression vector carrying a signal peptide designed and synthesized using P.pastoris biased codon. For phytase expression and secretion, the construct was integrated into the genome of P.pastoris by homologous recombination. Over-expressing strains were selected and fermented, and it was found that ~4.2 g phytase could be purified from a single 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. Enzymatic kinetics analysis showed that the phytase had two pH optima (pH 2.0 and pH 5.0), and an optimum temperature of 60 ℃.
Key words Isolation and synthetic phyI1 gene; Recombinant DNA; Phytase production; Fermentation; Pichia pastoris