Development of Thermotolerant Acetic Acid Bacteria Useful for Vinegar Fermentation at Higher Temperatures

Abstract

A Gram negative bacterial strain that can use cholate or deoxycholate as the sole carbon and energy sources was isolated from soil and tentatively identified as Pseudomonas putida. The strain formed 3α-hydroxysteroid dehydrogenase (EC 1.1.1.50) in the cytosolic fraction. The enzyme was purified and crystallized from the cell-free extract by a procedure involving chromatographic separation and ammonium sulfate fractionation. The crystallized enzyme showed a single protein band on both polyacrylamide gel electrophoresis and SDS-PAGE, giving a molecular weight of 25, 000. An alternative molecular weight measurement by gel filtration also indicated that the enzyme has such a low molecular weight. This is the lowest molecular weight among the related enzymes so far reported. In addition to high enzyme content in the cell-free extract, since the enzyme was stable in solution for a long period and tended to be crystallized readily, a simple purification method for the enzyme with high recovery was proposed that is convenient for a large scale rapid enzyme preparation. The enzyme required NAD specifically as the primary electron acceptor but not NADP or other compounds. The apparent Michaelis constant, K_m, for cholate was 15.0μM. The enzyme also showed high substrate specificity to other 3α-hydroxysteroids. Besides cholate, deoxycholate, androsterone, glycocholate, taurocholate, and ursodeoxycholate were readily oxidized by the enzyme and showed a similar order of kinetic parameters. Judging from the enzymatic properties obtained, it is reasonable to propose that the enzyme can be available for enzymatic diagnosis of bile acids, though the enzyme reaction is virtually reversible.