Cao Shi-shu, Hu Zhi-qiu
Department of Plant Pathology and Microbiology, University of California, Riverside, CA 92521, USA.
J Microbiol Methods. 2009 Oct;79(1):106-10. doi: 10.1016/j.mimet.2009.08.018. Epub 2009 Sep 4.
An optimized Citrobacter braakii phytase gene, appA-c, was chemically synthesized by oligonucleotides synthesis and over-lap PCR method. The appA-c gene encoding 423 amino acids was cloned into expression vector pPIC9 and transformed into methylotropic yeast Pichia pastoris. From about 2000 transformants, 400 transformants exhibiting phytase activity were obtained. One transformant showing the strongest phytase activity was selected for detailed analyses in 5 L bioreactor. Under control of the highly-inducible alcohol oxidase gene (AOX1) promoter, the transformant was able to secrete 3.85 mg/ml protein to the culture supernatant in about 110 h methanol induction, which comprises of 12,116 U ml(-1) phytase activity. Further characterization of the recombinant phytase was conducted. The optimal pH and temperature for this recombinant phytase was about 4.0 and 50 degrees C, respectively. Fe3+, Zn2+ and Cu2+ could significantly inhibit the recombinant phytase enzyme activity. The specific activity of this recombinant enzyme was 3147 U mg(-1). The K(m) and V(max) values for sodium phytate were determined to be 0.5 mM and 3085 U/mg, respectively. To our knowledge, this is the first report of a chemically synthesized C. braakii appA gene heterologous expression with the highest expression level and highest phytase activity achieved. The novel gene optimization and synthesis method can be applied to other related researches.
通过寡核苷酸合成和重叠PCR方法化学合成了优化的布氏柠檬酸杆菌植酸酶基因appA-c。将编码423个氨基酸的appA-c基因克隆到表达载体pPIC9中，并转化到甲基营养型酵母毕赤酵母中。从大约2000个转化子中获得了400个具有植酸酶活性的转化子。选择一个显示最强植酸酶活性的转化子在5 L生物反应器中进行详细分析。在高诱导性醇氧化酶基因（AOX1）启动子的控制下，该转化子在约110 h的甲醇诱导下能够向培养上清液中分泌3.85 mg/ml的蛋白质，其中包含12,116 U/ml的植酸酶活性。对重组植酸酶进行了进一步表征。该重组植酸酶的最佳pH和温度分别约为4.0和50℃。Fe3+、Zn2+和Cu2+可显著抑制重组植酸酶的酶活性。该重组酶的比活性为3147 U/mg。植酸钠的K(m)和V(max)值分别测定为0.5 mM和3085 U/mg。据我们所知，这是首次报道化学合成的布氏柠檬酸杆菌appA基因异源表达获得最高表达水平和最高植酸酶活性。该新的基因优化和合成方法可应用于其他相关研究。
