植酸酶基因的多点突变及在毕赤酵母中的高效表达

根据毕赤酵母基因的密码子选择偏爱性,不改变其编码氨基酸序列，对来源于黑曲霉N25植酸酶phyA基因，进行了突变，构建了含有正确突变的酵母表达载体pPIC9k-phyAm-4,电击转化毕赤酵母，获得优化了密码子的重组酵母转化子。经PCR鉴定表明，植酸酶基因已整合到酵母基因组中； 表达产物的SDS-PAGE分析表明，酶蛋白分子大小为70.15KD。Southern blotting结果表明,phyA基因整合到酵母染色体DNA中；转化子酶活测定结果表明，经密码子优化的重组酵母PP-NPm-4-2酶活可达136900U/ml，比Arg没有优化的PP-NPm-8 (47600 Uoml-1)酶活高约2.8倍。

Multipoint mutation and Over-expression in Pichia pastoris

According to bias in codon choice of Pichia pastoris, The phytase phyA gene from Aspergillus niger N25 was mutated without changing its amino acid sequence. The expression plasmid pPIC9k-phyAm was constructed and transformed into GS115 strain. Positive clones,of which the chromosomes were integrated with phyA gene,were identified by the phenotype and PCR. SDS-PAGE analysis suggust that the size of enzyme protein of the expression product was about 70.15kDa.Southern blotting analysis to the yeast transformants showed that phyA gene was intergrated into the chromosome genome. The phytase activity of PP-NP m-4-4 with codons optimized reached 136 000U/ml in malt wort culture medium after being induced with 36h, which was the 2.8 times of the original strain PP-NPm-8.