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Development of a soluble quinoproteins and applications

可溶性醌蛋白的研制及应用

基本信息

批准号：
05556016
负责人：
ADACHI Osao
金额：
$ 10.94万
依托单位：
Yamaguchi University, Faculty of Agriculture
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1995
项目状态：
已结题

项目摘要

Quinoproteins in which a novel prosthetic group pyrroloquinoline quinone (PQQ) catalyze an irreversible reaction of substrate oxidations. Thus, the characteristics of quinoproteins have an attractive properties suitable for biosensors or diagnostic enzymes for clinical control. Enzymes used in such field so far have been restricted to NAD (P) -dependent ones which generally catalyze a reversible reaction. Therefore, they are usually accompanied by basic troubles, for examples, it is theoretically impossible to measure a trace amount of substrate by the end point measurement. Alternatively, a trick which can overcome the reaction equilibrium to drive the enzymatic estimation forwards by adding a huge amounts of ingredient of the reactants.Different from such reversible enzymes, quinoproteins catalyze one directional oxidative reaction and make the end point measurement possible. A trace amount of substrate can be assayd with high accuracy with most quinoproteins. The quinoproteins so far developed for the purpose have come from membrane-bound and shown some hydrophobicity which makes the enzyme difficult to handle like a soluble enzyme.In this research, one microbial strain, Pseudomonas putida HK5, was isolated from soil by an enrichment technique. Many kinds of quinoproteins are induced in the cells when it is grown on different substrates. Alcohol dehydrogenase is formed when it is grown on n-butanol, whereas glycerol specific alcohol dehydrogenase does on 1,2-butanediol. The former has been evaluated as a diagnostic enzyme for alcohol. On the other hand, the latter for glycerol which comes from neutral fat. Such enzymes are soluble in nature and show a great number of priority as diagnostic enzymes. It is quite promising that the enzymes developed in this research should open a new aereas in biosensor.

以新型假体基吡咯喹啉醌（PQQ）催化底物氧化的不可逆反应的醌蛋白。因此，藜蛋白的特性具有吸引人的特性，适合用于临床控制的生物传感器或诊断酶。迄今为止，用于该领域的酶仅限于NAD (P)依赖性酶，这些酶通常催化可逆反应。因此，它们通常伴随着基本的麻烦，例如，理论上不可能通过终点测量来测量痕量的衬底。另一种方法是，通过添加大量的反应物成分来克服反应平衡来推动酶的估计。与这些可逆酶不同，藜蛋白催化单向氧化反应，使终点测量成为可能。痕量底物可以用大多数藜蛋白进行高精度测定。迄今为止开发的藜蛋白都是膜结合的，并且表现出一定的疏水性，这使得酶很难像可溶性酶那样处理。本研究采用富集技术从土壤中分离出一株恶臭假单胞菌HK5。在不同的基质上生长时，细胞内可诱导产生多种藜蛋白。醇脱氢酶是在正丁醇上生长时形成的，而甘油特异性醇脱氢酶是在1,2-丁二醇上形成的。前者已被评价为酒精的诊断酶。另一方面，后者是来自中性脂肪的甘油。这类酶在自然界中是可溶的，作为诊断酶具有很大的优先性。本研究开发的酶可望在生物传感器领域开辟新的领域。