Bei J L, Chen Z, Yang L, Liao L, Wang X Z, Jiang Z Y
Biopharmaceutical Center, Zhongshan University, GuangZhou 510275, China.
Sheng Wu Gong Cheng Xue Bao. 2001 May;17(3):254-8.
The phytase gene of Aspergillus niger NRRL3135 was modified with a deletion of intron and signal coding sequence. Then, according to the codon preference of Pichia pastoris, modified phyA gene was artificially synthesized and cloned into expression vector of pPICZ alpha A. The recombinant plasmid was transformed into chromosome of Pichia pastoris X-33 strain by electroporation. The results of SDS-PAGE and enzymatic kinetic analysis proved that the recombinant phytase was secreted into culture medium with nearly same character of natural phytase. After screening for high level productive yeast strains, a strain named SPAN-III produced recombinant phytase with 165,000 u/mL under the condition of shake cultivation. It will satisfy the demand for industrialized production in some degree.
对黑曲霉NRRL3135的植酸酶基因进行修饰，删除了内含子和信号编码序列。然后，根据巴斯德毕赤酵母的密码子偏好性，人工合成修饰后的phyA基因，并将其克隆到pPICZ alpha A表达载体中。通过电穿孔将重组质粒转化到巴斯德毕赤酵母X-33菌株的染色体中。SDS-PAGE和酶动力学分析结果证明，重组植酸酶分泌到培养基中，其性质与天然植酸酶几乎相同。筛选出高产酵母菌株后，一株名为SPAN-III的菌株在摇瓶培养条件下产生了165,000 u/mL的重组植酸酶。这在一定程度上满足了工业化生产的需求。
