Development of a soluble quinoproteins and applications

一种新型的基团吡咯喹啉醌(PQQ)催化底物氧化的不可逆反应的藜麦蛋白。因此，藜麦蛋白的特性具有吸引人的特性，适合作为生物传感器或诊断酶用于临床控制。到目前为止，用于这一领域的酶仅限于NAD(P)依赖的酶，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微量的衬底。与这种可逆酶不同的是，藜麦蛋白催化单向氧化反应，使终点测量成为可能。用大多数藜麦蛋白可以高准确度地测定微量底物。目前开发的藜麦蛋白来源于膜结合蛋白，具有一定的疏水性，这使得该酶很难像可溶性酶一样处理。当它生长在不同的底物上时，细胞中会诱导出多种藜麦蛋白。乙醇脱氢酶在正丁醇上生长时形成，而甘油专一性酒精脱氢酶在1，2-丁二醇上生长时形成。前者已被评估为酒精的诊断酶。另一方面，后者用于甘油，它来自中性脂肪。这类酶在自然界中是可溶的，并显示出作为诊断酶的大量优先地位。本研究开发的酶在生物传感器领域具有广阔的应用前景。

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.