Wyss M, Pasamontes L, Friedlein A, Rémy R, Tessier M, Kronenberger A, Middendorf A, Lehmann M, Schnoebelen L, Röthlisberger U, Kusznir E, Wahl G, Müller F, Lahm H W, Vogel K, van Loon A P
VFB Department, F. Hoffmann-La Roche Ltd., CH-4070 Basel, Switzerland.
Appl Environ Microbiol. 1999 Feb;65(2):359-66. doi: 10.1128/AEM.65.2.359-366.1999.
Phytases (myo-inositol hexakisphosphate phosphohydrolases) are found naturally in plants and microorganisms, particularly fungi. Interest in these enzymes has been stimulated by the fact that phytase supplements increase the availability of phosphorus in pig and poultry feed and thereby reduce environmental pollution due to excess phosphate excretion in areas where there is intensive livestock production. The wild-type phytases from six different fungi, Aspergillus niger, Aspergillus terreus, Aspergillus fumigatus, Emericella nidulans, Myceliophthora thermophila, and Talaromyces thermophilus, were overexpressed in either filamentous fungi or yeasts and purified, and their biophysical properties were compared with those of a phytase from Escherichia coli. All of the phytases examined are monomeric proteins. While E. coli phytase is a nonglycosylated enzyme, the glycosylation patterns of the fungal phytases proved to be highly variable, differing for individual phytases, for a given phytase produced in different expression systems, and for individual batches of a given phytase produced in a particular expression system. Whereas the extents of glycosylation were moderate when the fungal phytases were expressed in filamentous fungi, they were excessive when the phytases were expressed in yeasts. However, the different extents of glycosylation had no effect on the specific activity, the thermostability, or the refolding properties of individual phytases. When expressed in A. niger, several fungal phytases were susceptible to limited proteolysis by proteases present in the culture supernatant. N-terminal sequencing of the fragments revealed that cleavage invariably occurred at exposed loops on the surface of the molecule. Site-directed mutagenesis of A. fumigatus and E. nidulans phytases at the cleavage sites yielded mutants that were considerably more resistant to proteolytic attack. Therefore, engineering of exposed surface loops may be a strategy for improving phytase stability during feed processing and in the digestive tract.
植酸酶（肌醇六磷酸磷酸水解酶）天然存在于植物和微生物中，尤其是真菌。植酸酶补充剂能提高猪和家禽饲料中磷的利用率，从而减少集约化畜牧生产地区因过量磷酸盐排泄造成的环境污染，这一事实激发了人们对这些酶的兴趣。来自六种不同真菌（黑曲霉、土曲霉、烟曲霉、构巢曲霉、嗜热毁丝霉和嗜热栖热菌）的野生型植酸酶在丝状真菌或酵母中过表达并纯化，然后将它们的生物物理特性与大肠杆菌植酸酶的特性进行比较。所有检测的植酸酶都是单体蛋白。大肠杆菌植酸酶是一种非糖基化酶，而真菌植酸酶的糖基化模式差异很大，不同的植酸酶之间、在不同表达系统中产生的给定植酸酶以及在特定表达系统中产生的给定植酸酶的不同批次之间均有所不同。当真菌植酸酶在丝状真菌中表达时，糖基化程度适中；而在酵母中表达时，糖基化程度过高。然而，不同程度的糖基化对单个植酸酶的比活性、热稳定性或重折叠特性没有影响。当在黑曲霉中表达时，几种真菌植酸酶易受培养上清液中存在的蛋白酶的有限蛋白水解作用。对片段进行N端测序表明，切割总是发生在分子表面暴露的环上。对烟曲霉和构巢曲霉植酸酶在切割位点进行定点诱变，得到的突变体对蛋白水解攻击的抗性明显增强。因此，改造暴露的表面环可能是提高植酸酶在饲料加工和消化道中稳定性的一种策略。
