Rodriguez E, Mullaney E J, Lei X G
Department of Animal Science, Cornell University, Ithaca, New York 14853, USA.
Biochem Biophys Res Commun. 2000 Feb 16;268(2):373-8. doi: 10.1006/bbrc.2000.2121.
Aspergillus fumigatus phytase is a heat-stable enzyme of great potential. Our objective was to determine if a high level of functional expression of the A. fumigatus phytase gene could be produced in Pichia pastoris and how the recombinant phytase reacted to different substrates, heating conditions, and proteases. A 1.4-kb DNA fragment containing the coding region of the gene was inserted into the expression vector pPICZalphaA and expressed in P. pastoris as an active, extracellular phytase (r-Afp). The yield was 729 mg of purified protein per liter of culture, with a specific activity of 43 units/mg of protein. The enzyme r-Afp shared similar pH and temperature optima, molecular size, glycosylation extent, and specificity for p-nitrophenyl phosphate and sodium phytate to those of the same enzyme expressed in A. niger. Given 20 min of exposure to 65 to 90 degrees C, the enzyme retained 20 to 39% higher residual activity in 10 and 200 mM sodium acetate than that in sodium citrate. The enzyme seemed to be resistant to pepsin digestion, but was degraded by high levels of trypsin. In conclusion, P. pastoris is a potential host to express high levels of A. fumigatus phytase and the thermostability of the recombinant enzyme is modulated by the specificity of buffer used in the heat treatment.
烟曲霉植酸酶是一种极具潜力的热稳定酶。我们的目标是确定烟曲霉植酸酶基因在毕赤酵母中能否实现高水平的功能性表达，以及重组植酸酶对不同底物、加热条件和蛋白酶的反应情况。将包含该基因编码区的1.4 kb DNA片段插入表达载体pPICZalphaA，并在毕赤酵母中表达为一种活性胞外植酸酶（r - Afp）。每升培养物的纯化蛋白产量为729 mg，比活性为43单位/mg蛋白。r - Afp酶在pH和温度最佳值、分子大小、糖基化程度以及对磷酸对硝基苯酯和植酸钠的特异性方面，与在黑曲霉中表达的同一种酶相似。在65至90摄氏度下暴露20分钟，该酶在10 mM和200 mM醋酸钠中的残留活性比在柠檬酸钠中高20%至39%。该酶似乎对胃蛋白酶消化有抗性，但会被高水平的胰蛋白酶降解。总之，毕赤酵母是表达高水平烟曲霉植酸酶的潜在宿主，重组酶的热稳定性受热处理所用缓冲液特异性的调节。
