Huang Huo-qing, Luo Hui-ying, Bai Ying-guo, Wang Ya-ru, Yao Bin, Meng Kun, Yuan Tie-zheng, Yang Pei-long
Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
Wei Sheng Wu Xue Bao. 2006 Dec;46(6):945-50.
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.
利用高比活性的植酸酶是提高重组宿主中植酸酶发酵效价并降低生产成本的有效途径。到目前为止，来自布氏柠檬酸杆菌的植酸酶APPA在先前记录的所有植酸酶中表现出最高的比活性。编码植酸酶的基因AppA根据毕赤酵母中高表达基因的密码子选择偏好进行修饰，且不改变氨基酸序列，然后人工合成。将修饰后的基因AppA（m）插入到AOX1启动子控制下的毕赤酵母表达载体pPIC9中，并通过电穿孔将所得的表达载体pPIC9-AppA（m）导入宿主毕赤酵母。对重组酵母的PCR分析表明AppA（m）基因已整合到毕赤酵母的染色体中。通过酶活性分析和SDS-PAGE筛选出用于植酸酶过表达的毕赤酵母重组体。重组植酸酶APPA通过透析、超滤和色谱等简单方法进行纯化。经过简单纯化后，重组植酸酶达到电泳纯，并且经内切糖苷酶H处理显示该重组植酸酶被糖基化。纯化后的重组APPA的比活性为3.5×10⁶ IU/mg蛋白质。重组植酸酶APPA在pH值2.0至7.0范围内具有活性，最适pH为4.5。在pH 4.5时最适温度为55℃。植酸钠的Km值为0.165mmol/L，Vmax为3.3×10⁶ IU/mg·min。在5升发酵罐中进行分批补料发酵时，重组毕赤酵母中植酸酶的表达水平为3.2mg/mL，发酵效价超过1.4×10⁷ IU/mL，这是目前所有报道的植酸酶重组菌株中的最高水平。
