Wyss M, Brugger R, Kronenberger A, Rémy R, Fimbel R, Oesterhelt G, Lehmann M, van Loon A P
VFB Department, F. Hoffmann-La Roche Ltd., 4070 Basel, Switzerland.
Appl Environ Microbiol. 1999 Feb;65(2):367-73. doi: 10.1128/AEM.65.2.367-373.1999.
Supplementation with phytase is an effective way to increase the availability of phosphorus in seed-based animal feed. The biochemical characteristics of an ideal phytase for this application are still largely unknown. To extend the biochemical characterization of wild-type phytases, the catalytic properties of a series of fungal phytases, as well as Escherichia coli phytase, were determined. The specific activities of the fungal phytases at 37 degreesC ranged from 23 to 196 U. (mg of protein)-1, and the pH optima ranged from 2.5 to 7.0. When excess phytase was used, all of the phytases were able to release five phosphate groups of phytic acid (myo-inositol hexakisphosphate), which left myo-inositol 2-monophosphate as the end product. A combination consisting of a phytase and Aspergillus niger pH 2.5 acid phosphatase was able to liberate all six phosphate groups. When substrate specificity was examined, the A. niger, Aspergillus terreus, and E. coli phytases were rather specific for phytic acid. On the other hand, the Aspergillus fumigatus, Emericella nidulans, and Myceliophthora thermophila phytases exhibited considerable activity with a broad range of phosphate compounds, including phenyl phosphate, p-nitrophenyl phosphate, sugar phosphates, alpha- and beta-glycerophosphates, phosphoenolpyruvate, 3-phosphoglycerate, ADP, and ATP. Both phosphate liberation kinetics and a time course experiment in which high-performance liquid chromatography separation of the degradation intermediates was used showed that all of the myo-inositol phosphates from the hexakisphosphate to the bisphosphate were efficiently cleaved by A. fumigatus phytase. In contrast, phosphate liberation by A. niger or A. terreus phytase decreased with incubation time, and the myo-inositol tris- and bisphosphates accumulated, suggesting that these compounds are worse substrates than phytic acid is. To test whether broad substrate specificity may be advantageous for feed application, phosphate liberation kinetics were studied in vitro by using feed suspensions supplemented with 250 or 500 U of either A. fumigatus phytase or A. niger phytase (Natuphos) per kg of feed. Initially, phosphate liberation was linear and identical for the two phytases, but considerably more phosphate was liberated by the A. fumigatus phytase than by the A. niger phytase at later stages of incubation.
添加植酸酶是提高籽实类动物饲料中磷利用率的有效方法。适用于此应用的理想植酸酶的生化特性仍 largely 未知。为了扩展野生型植酸酶的生化特性研究，测定了一系列真菌植酸酶以及大肠杆菌植酸酶的催化特性。真菌植酸酶在 37℃时的比活性范围为 23 至 196 U·(mg 蛋白质)-1，最适 pH 范围为 2.5 至 7.0。当使用过量植酸酶时，所有植酸酶都能够释放植酸（肌醇六磷酸）的五个磷酸基团，最终产物为肌醇 2-单磷酸。由植酸酶和黑曲霉 pH 2.5 酸性磷酸酶组成的组合能够释放所有六个磷酸基团。在检测底物特异性时，黑曲霉、土曲霉和大肠杆菌植酸酶对植酸具有较高的特异性。另一方面，烟曲霉、构巢曲霉和嗜热栖热菌植酸酶对包括苯磷酸、对硝基苯磷酸、糖磷酸、α 和β甘油磷酸、磷酸烯醇丙酮酸、3-磷酸甘油酸、ADP 和 ATP 在内的多种磷酸化合物表现出相当高的活性。通过使用高效液相色谱法分离降解中间体的磷酸释放动力学和时间进程实验均表明，烟曲霉植酸酶能够有效裂解从六磷酸肌醇到二磷酸肌醇的所有肌醇磷酸。相比之下，黑曲霉或土曲霉植酸酶的磷酸释放量随孵育时间减少，肌醇三磷酸和二磷酸肌醇积累，这表明这些化合物作为底物不如植酸。为了测试广泛的底物特异性是否对饲料应用有利，通过在每千克饲料中添加 250 或 500 U 的烟曲霉植酸酶或黑曲霉植酸酶（Natuphos）的饲料悬浮液进行体外磷酸释放动力学研究。最初，两种植酸酶的磷酸释放呈线性且相同，但在孵育后期，烟曲霉植酸酶释放的磷酸量比黑曲霉植酸酶多得多。
