Tramper J, Angelino S A, Müller F, van der Plas H C
Biotechnol Bioeng. 1979 Oct;21(10):1767-86. doi: 10.1002/bit.260211006.
Xanthine dehydrogenase (EC 1.2.1.37) was isolated from chicken livers and immobilized by adsorption to a Sepharose derivative, prepared by reaction of n-octylamine with CNBr-activated Sepharose 4B. Using a crude preparation of enzyme for immobilization it was observed that relatively more activity was adsorbed than protein, but the yield of immobilized activity increased as a purer enzyme preparation was used. As more activity and protein were bound, relatively less immobilized activity was recovered. This effect was probably due to blocking of active xanthine dehydrogenase by protein impurities. The kinetics of free and immobilized xanthine dehydrogenase were studied in the pH range 7.5-9.1. The Km and V values estimated for free xanthine dehydrogenase increase as the pH increase; the K'm and V values for the immobilized enzyme go through a minimum at pH 8.1. By varying the amount of enzyme activity bound per unit volume of gel, it was shown that K'm is larger than Km are result of substrate diffusion limitation in the pores of the support material. Both free and immobilized xanthine dehydrogenase showed substrate activation at low concentrations (up to 2 microM xanthine). Immobilized xanthine dehydrogenase was more stable than the free enzyme during storage in the temperature range of 4-50 degrees C. The operational stability of immobilized xanthine dehydrogenase at 30 degrees C was two orders of magnitude smaller than the storage stability, t 1/2 was 9 and 800 hr, respectively. The operational stability was, however, better than than of immobilized milk xanthine oxidase (t 1/2 = 1 hr). In addition, the amount of product formed per unit initial activity in one half-life, was higher for immobilized xanthine dehydrogenase than for immobilized xanthine oxidase. Unless immobilized milk xanthine oxidase can be considerable stabilized, immobilized chicken liver xanthine dehydrogenase is more promising for application in organic synthesis.
黄嘌呤脱氢酶（EC 1.2.1.37）从鸡肝中分离出来，并通过吸附到一种琼脂糖衍生物上进行固定化，该琼脂糖衍生物由正辛胺与溴化氰活化的琼脂糖4B反应制备而成。使用粗酶制剂进行固定化时，观察到吸附的活性相对比蛋白质更多，但随着使用更纯的酶制剂，固定化活性的产率有所增加。随着更多的活性和蛋白质被结合，回收的固定化活性相对较少。这种效应可能是由于蛋白质杂质对活性黄嘌呤脱氢酶的阻断所致。在pH值7.5 - 9.1范围内研究了游离和固定化黄嘌呤脱氢酶的动力学。游离黄嘌呤脱氢酶的Km和V值随pH值升高而增加；固定化酶的K'm和V值在pH 8.1时出现最小值。通过改变每单位体积凝胶结合的酶活性量，结果表明K'm大于Km是由于底物在载体材料孔中的扩散限制所致。游离和固定化黄嘌呤脱氢酶在低浓度（高达2 microM黄嘌呤）时均表现出底物活化作用。在4 - 50摄氏度温度范围内储存期间，固定化黄嘌呤脱氢酶比游离酶更稳定。固定化黄嘌呤脱氢酶在30摄氏度下的操作稳定性比储存稳定性小两个数量级，t 1/2分别为9小时和800小时。然而，其操作稳定性优于固定化牛奶黄嘌呤氧化酶（t 1/2 = 1小时）。此外，在一个半衰期内，每单位初始活性形成的产物量，固定化黄嘌呤脱氢酶比固定化黄嘌呤氧化酶更高。除非固定化牛奶黄嘌呤氧化酶能够得到显著稳定，否则固定化鸡肝黄嘌呤脱氢酶在有机合成中的应用更具前景。
