Department of Biotechnology, Lund University, SE-221 00 Lund, Sweden.
Anal Biochem. 2011 Mar 15;410(2):177-84. doi: 10.1016/j.ab.2010.10.034. Epub 2010 Nov 2.
Phytase (EC 3.1.3.-) hydrolyzes phytate (IP(6)) present in cereals and grains to release inorganic phosphate (P(i)), thereby making it bioavailable. The most commonly used method to assay phytase, developed nearly a century ago, measures the P(i) liberated from IP(6). This traditional endpoint assay is time-consuming and well known for its cumbersomeness in addition to requiring extra caution for handling the toxic regents used. This article reports a simple, fast, and nontoxic kinetic method adaptable for high throughput for assaying phytase using IP(6)-lysozyme as a substrate. The assay is based on the principle that IP(6) forms stable turbid complexes with positively charged lysozyme in a wide pH range, and hydrolysis of the IP(6) in the complex is accompanied by a decrease in turbidity monitored at 600 nm. The turbidity decrease correlates well to the released P(i) from IP(6). This kinetic method was found to be useful in assaying histidine acid phytases, including 3- and 6-phytases, a class representing all commercial phytases, and alkaline β-propeller phytase from Bacillus sp. The influences of temperature, pH, phosphate, and other salts on the kinetic assay were examined. All salts, including NaCl, CaCl(2), and phosphate, showed a concentration-dependent interference.
植酸酶（EC 3.1.3.-）能够水解谷物和谷物中存在的植酸（IP(6)），从而释放出无机磷（P(i)），使其具有生物利用度。近一个世纪前开发的最常用的植酸酶测定方法是测量从 IP(6)中释放的 P(i)。这种传统的终点测定方法耗时且繁琐，除了需要格外小心处理使用的有毒试剂外，还很麻烦。本文报道了一种简单、快速且无毒的动力学方法，可用于使用 IP(6)-溶菌酶作为底物测定植酸酶，适用于高通量。该测定基于以下原理：在宽 pH 范围内，IP(6)与带正电荷的溶菌酶形成稳定的混浊复合物，复合物中的 IP(6)水解伴随着浊度降低，在 600nm 处监测。浊度降低与从 IP(6)释放的 P(i)相关。该动力学方法已被证明可用于测定组氨酸酸性植酸酶，包括 3-植酸酶和 6-植酸酶，它们代表所有商业植酸酶，以及来自芽孢杆菌的碱性β-螺旋桨植酸酶。还研究了温度、pH、磷酸盐和其他盐对动力学测定的影响。所有盐，包括 NaCl、CaCl(2)和磷酸盐，均显示出浓度依赖性干扰。
