Enzymatic Characterization of Aminoacylase from Thermophilic Bacteria and Its Application to Amino Acid Production

本研究旨在建立一种新的氨基酰化酶速率测定方法，阐明嗜热菌耐热性氨基酰化酶的酶学特性，并与其他耐热性酶进行比较，同时开发一种用该耐热性氨基酰化酶由外消旋体合成氨基酸的新酶学方法。研究结果总结如下：1。根据猪肾、真菌和细菌的氨基酰化酶可将合成的n -乙酰基不饱和氨基酸乙酰氨基丙烯酸酯水解为乙酸、氨和丙酮酸酯，建立了一种简便的氨基酰化酶光度测定方法。该方法与时间和酶浓度呈线性关系，可用于氨基酰化酶的动力学研究。从热葡萄芽孢杆菌的细胞提取物中纯化出耐热性氨基酰化酶，并对其进行了表征。这种酶的分子量约为17.5万，由4个相同的亚基组成，每摩尔酶蛋白含有4克锌原子。这种酶的一个特点是在高温下和与有机溶剂和蛋白质变性剂有很大的稳定性。研究了嗜热硬脂芽孢杆菌耐热二肽酶与氨基酰化酶的酶学性质。建立了酶法生产l -苯丙氨酸的新方法。

This study was undertaken to establish a new rate assay method of aminoacylase, to elucidate enzymological properties of thermostable aminoacylase from thermophilic bacteria to compare with other thermolabile enzymes, and to develop a new enzymatic procedure for the synthesis of amino acids from racemates with the thermostable aminoacylase. The results are summarized as follows:1. A convenient spectrophotometric rate assay for aminoacylase was developped based on the observation that acetamidoacrylate, a synthetic N-acetyl unsaturated amino acid, was hydrolyzed to acetate, ammonia and pyruvate by hog kidney, fungal and bacterial aminoacylases. This assay is linear with time and enzyme concentration, and is useful for kinetic studies of aminoacylase.2. The thermostable aminoacylase was purified to homogeneity from cell extracts of Bacillus thermoglucosidius and characterized. The enzyme has a molecular weight of about 175,000, and is composed of four identical subunits, containing 4 g atoms of zinc per mole of enzyme protein. Great stability at high temperatures and with organic solvents and protein denaturants is a characteristic of the enzyme.3. Enzymological properties of the thermostable dipeptidase from B. stearothermophilus were compared with those of aminoacylase.4. A new enzymatic method for L-phenylalanine production was established.