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Membrane-bound aldehyde dehydrogenase (ALDH) was purified from the membrane fraction of an industrial-vinegar-producing strain, Acetobacterpolyoxogenes sp. nov. NBI1028 by solubilization with Triton X-100 and sodium N-lauroyl sarcosinate and subsequent column chromatography on DEAE-Sepharose CL-6B and hydroxyapatite. The purified enzyme was homogeneous on polyacrylamide disc gel electrophoresis. Upon sodium dodecyl sulphate-polyacrylamide gelelectrophoresis, the enzyme showed the presence of two subunits with a molecular mass of 75000 daltons and 19000 daltons, respectively. From the absorption and fluorescence spectra, the absence of cytochrome c and the presence of pyrroloquinoline quinone in the purified enzyme were demonstrated. The ALDH preferentially oxidized aliphatic aldehyde with a straight carbon chain except for formaldehyde. The apparent Km for acetaldehyde was 12 mM. The optimum pH and temperature were 7.0 and 500-60 ° C, respectively. The enzyme remained active after storage at 4 ° C for 20 days. p-Chloromercuribenzoic acid and heavy metal salts such as CuSO4 were inhibitory to the enzyme. Ferricyanide was effective as an electron acceptor.

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Spheroplasts of auxotrophic mutants derived from Acetobacter aceti subsp. aceti No. 1023 were efficiently prepared by treatment with lysozyme, using sucrose as an osmotic stabilizer, and regenerated on an agar plate containing sorbitol and gelatin. In addition, spheroplast fusion between the several auxotrophic mutants was achieved in the presence of polyethylene glycol and CaCl2. The frequency of fusion was found to be about 5 x 10~5. Spheroplast fusion between A. aceti subsp. aceti No. 2 with the ability to grow at high temperature and A. aceti subsp. xylinum NBI1002 with high resistance to acetic acid was also achieved by the same method, with a frequency of 6.0 x 10~6. The fusants showed various degrees of resistance to acetic acid and ability to grow at high temperature. One of the fusants, named No. 116, could produce acetic acid from ethanol continuously under conditions under which both parent strains were unable to grow. This suggests that spheroplast fusion is applicable to the breeding of strains for vinegar production. The genus Acetobacter is industrially important for vinegar production. To improve strains of Acetobacter genetically, recombinant DNAtechniques are considered to be useful, and host-vector systems and an efficient transformation method for Acetobacter have been developed.1~4) However, cell fusion may also be applicable for such a purpose, especially

A new enzymatic method for microdetermination of ethanol has been established with particulate alcohol dehydrogenase from acetic acid bacteria and applied to the practical purposes. The enzyme had an optimum pH for ethanol oxidation at a fairly acidic region. Trace amounts of ethanol could be assayed by measuring the initial reaction rate as successful as by reading the end point of the reaction.Some advantages in using this enzyme for ethanol determination were pointed out comparing with NAD-linked alcohol dehydrogenase from yeast or horse liver. Impurity in the enzyme preparations, stability of reagents and coexistence of other substances in the assay mixture were not as critical as in NAD-linked enzyme. Acidic samples could also be directly determined for ethanol without preadjustment of sample pH.