来源于柠檬酸杆菌的高比活植酸酶基因在毕赤酵母中的高效表达

柠檬酸杆菌(Citrobacterbraakii)来源的植酸酶是目前报道的比活最高的植酸酶。按照毕赤酵母(Pichiapastoris)对密码子的选择偏向性,对来源于柠檬酸杆菌的高比活植酸酶基因AppA进行了密码子优化改造。改造后的基因AppA(m)按正确的阅读框架融合到毕赤酵母表达载体pPIC9的α-因子信号肽编码序列3′端,通过电击转化得到重组转化子。通过PCR验证,AppA(m)已整合在酵母染色体上。SDS-PAGE分析和表达产物的研究表明,植酸酶得到了高效分泌表达,在5L发酵罐中植酸酶蛋白表达量达到3·2mg/mL发酵液,发酵效价达到每毫升发酵液1·4×107IU以上,高于目前报道的各种植酸酶基因工程菌株的发酵效价。

Overexpression of Citrobacter braakii phytase with high specific activity in Pichia pastoris

Utilization of the phytase with high specific activity is an effective way to improve the fermentation potency of phytase in recombinant host and decrease the production cost. Up to now, the phytase APPA from Citrobacter braakii exhibits the highest specific activity in the all phytases recorded previously. The gene AppA encoding phytase was modified according to the bias in codon choice of the high expression gene in Pichia pastoris without changing the amino acid sequence and artificially synthesized. The modified gene, AppA ( m) , was inserted into the Pichia pastoris expression vector pPIC9 under the control of AOX1 promoter, and the resulted expression vector pPIC9-AppA ( m) was introduced into the host Pichia pastoris by electroporation. PCR analysis of the recombinant yeast indicated that AppA (m) gene was integrated into the chromosome of Pichia pastoris. The Pichia pastoris recombinants for phytase overexpression were screened by enzyme activity analysis and SDS-PAGE. The recombinant phytase APPA was purified by simple methods, such as dialysis, ultrafiltration and chromatography. After the simple purification, the purity of the recombinant phytase reached to electrophoresis purity, and the recombinant phytase was shown to be glycosylated by Endo-H treatment. The specific activity of the purified recombinant APPA was 3.5 x 10(6) IU/mg of protein. Recombinant phytase APPA showed activity at pH values from 2.0 through 7.0 with the optimum at 4.5. The temperature optimum was 55 degrees C at pH 4.5.The Km value for sodium phytate was 0.165mmol/L with a Vmax of 3.3 x 10(6)IU/mg min. In 5-liter fermentor in fed-batch fermentation, the expression level of phytase in recombinant Pichia pastoris was 3.2mg/mL and the fermentation potency exceeded 1.4 x 10(7) IU/mL, which is the highest level among all of the reported phytase recombinant strains at present.