Han Y, Lei X G
Department of Animal Science, Cornell University, Ithaca, New York, 14853, USA.
Arch Biochem Biophys. 1999 Apr 1;364(1):83-90. doi: 10.1006/abbi.1999.1115.
Economical and thermostable phytase enzymes are needed to release phytate-phosphorus in plant foods for human and animal nutrition and to reduce phosphorus pollution of animal waste. Our objectives were to determine if a methylotrophic yeast, Pichia pastoris, was able to express a phytase gene (phyA) from Aspergillus niger efficiently and if suppression of glycosylation by tunicamycin affected its functional expression. The gene (1.4 kb) was inserted into an expression vector pPICZalphaA with a signal peptide alpha-factor, under the control of AOX1 promoter. The resulting plasmid was transformed into two P. pastoris strains: KM71 (methanol utilization slow) and X33 (wild-type). Both host strains produced high levels of active phytase (25-65 units/ml of medium) that were largely secreted into the medium. The expressed enzyme was cross-reacted with the polyclonal antibody raised against the wild-type enzyme and showed two pH optima, 2.5 and 5.5, and an optimal temperature at 60 degrees C. Compared with the phyA phytase overexpressed by A. niger, this phytase had identical capacity in hydrolyzing phytate-phosphorus from soybean meal and slightly better thermostability. Deglycosylation of the secreted phytase resulted in reduction in the size from 95 to 55 kDa and in thermostability by 34%. Tunicamycin (20 microg/ml of medium) resulted in significant reductions of both intracellular and extracellular phytase activity expression. Because there was no accumulation of intracellular phytase protein, the impairment did not seem to occur at the level of translocation of phytase. In conclusion, glycosylation was vital to the biosynthesis of the phyA phytase in P. pastoris and the thermostability of the expressed enzyme.
为了释放植物性食物中的植酸磷用于人类和动物营养，并减少动物粪便中的磷污染，需要经济且热稳定的植酸酶。我们的目标是确定甲基营养型酵母毕赤酵母是否能够高效表达黑曲霉的植酸酶基因（phyA），以及衣霉素对糖基化的抑制是否会影响其功能表达。将该基因（1.4 kb）插入带有信号肽α-因子的表达载体pPICZalphaA中，在AOX1启动子的控制下。将所得质粒转化到两种毕赤酵母菌株中：KM71（甲醇利用缓慢型）和X33（野生型）。两种宿主菌株都产生了高水平的活性植酸酶（25 - 65单位/毫升培养基），且大部分分泌到培养基中。表达的酶与针对野生型酶产生的多克隆抗体发生交叉反应，显示出两个最适pH值，分别为2.5和5.5，最适温度为60℃。与黑曲霉过表达的phyA植酸酶相比，这种植酸酶在水解豆粕中植酸磷方面具有相同的能力，并且热稳定性略好。分泌的植酸酶去糖基化导致其大小从95 kDa减小到55 kDa，热稳定性降低34%。衣霉素（20微克/毫升培养基）导致细胞内和细胞外植酸酶活性表达显著降低。由于细胞内植酸酶蛋白没有积累，这种损害似乎不是在植酸酶转运水平发生的。总之，糖基化对于毕赤酵母中phyA植酸酶的生物合成以及表达酶的热稳定性至关重要。
