Development of a soluble quinoproteins and applications

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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.