Polyvinyl alcohol (PVA)-utilizing cultures were obtained from various sources. They were mixed cultures even after cyclical transfer to liquid and plate media with PVA as a sole source of carbon. Component bacteria were isolated from the several mixed cultures, and it was shown that PVA was utilized symbiotically by two bacterial members which could not utilize PVA in each respective pure culture. From a mixed culture, strains VM15, VM15A (Pseudomonasputida) and VM15C (Pseudomonas sp.) were isolated as members essential for PVA utilization. VM15C was the predominant strain in the mixed-culture population and produced PVA-degrading enzyme. The culture supernatant of VM15A enabled VM15C to grow on PVA. VM15A was presumed to supply VM15C with a unique growth stimulant which was distinct from usual growth factors.

A novel enzyme, formaldehyde dismutase, was purified and crystallized from the cell extract of an isolated bacterium, Pseudomonas putida F 61. The enzyme catalyzes the dismutation of aldehydes and alcohol: aldehyde oxidoreduction in the absence of an exogenous electron acceptor. The enzyme is composed of four identical subunits with a M, of 44000. Each subunit contains 1 mol NAD(H) and 2 mol zinc/mol. The ratio of NAD+ and NADH in a crystalline preparation of the enzyme was about 7: 3. The enzyme-bound coenzyme was completely reduced and oxidized on the addition of a large amount of an alcohol and an aldehyde respectively. Both the oxidized and reduced enzymes catalyzed the dismutation reaction to the same extent. Steady-state kinetics of the enzyme were investigated using an oxidoreduction reaction between an alcohol and p-nitroso-N,N-dimethylaniline. The enzyme obeys a ping-pong mechanism and is competitively inhibited by an alcoholic substrate analogue, pyrazole, but not coenzyme analogues, such as AMP, N-methylnicotinamide. These results indicate that NAD(H) binds firmly (but not covalently) at each active site. The enzyme-bound NAD(H) was reduced and oxidized only by the added second substrates, alcohol and aldehyde respectively, and not by exogenous electron acceptors [including NAD(H)].In the preceding studies [I -31 we found a novel enzyme, which was given the trivial name of formaldehyde dismutase, in an isolated bacterium, Pseudomonas putida F61. This enzyme catalyzed the dismutation of aldehydes (including formaldehyde), leading to the formation of equimolar amounts of the corresponding alcohols and acids. Preferable substrates,for the reaction were aldehydes that are hydrated to a great extent, such as formaldehyde, acetaldehyde and methylglyoxal. A variety of non-hydrated aldehydes was also reduced through the cross-dismutation with the hydrated aldehydes. Thus, the dismutation and cross-dismutation reactions can be seen as coupled oxidoreduction of an aldehyde (RCHO) and an 'alcohol' [RCH(OH),] formed through the hydration. Furthermore, this enzyme catalyzes the oxidoreduction of a natural alcohol and an aldehyde, leading to the exchange of the alcohol and aldehyde species. Analogous reactions with limited substrates have been reported to be catalyzed by native horse liver alcohol dehydrogenase in the presence of NAD [4-61, and by the dehydrogenase covalently bound an NAD analogue in the absence of an exogenous coenzyme [7, 81. However, formaldehyde dismutase, which is a naturally occurring bacterial enzyme, is distinct from these native and modified alcohol dehydrogenases in that it shows no requirement of an electron acceptor and there is no enhancement of its activity on the addition of an excess amount of NAD'. The oxidoreduction in the dismutation reaction is mediated by a non-dissociable coenzyme of this enzyme. In this work we report that the crystalline enzyme contains nonCorrespondence to N. Kato,

Under UV irradiation in the presence of an inorganic base, aqueous formaldehyde was found to give pentaerythritol and 2-hydroxymethylglycerol as the main products, accompanied by the concomitant formation of a mixture of sugars and sugar alcohols. The results indicate that this photochemical formose reaction is considerably different in product distribution from the thermal formose reaction using the Ca(OH)2 catalyst. The detailed examination of the photochemical formose reaction was carried out in the presence of Na2CO3, and a possible scheme for the formation of pentaerythritol and 2-hydroxymethylglycerol is proposed.

A newly found methanol-using bacterium, Mycobacterium gastri MB19, is a facultative methylotroph which assimilates methanol via the ribulose monophosphate pathway. 3-Hexulose phosphate synthase was purified from the organism and characterized. This enzyme was found to use glycolaldehyde {Km=A3mu) and methylglyoxal {Km=5.1mM) as well as formaldehyde {Km= 1.4 him) in the presence ofD-ribulose 5-phosphate as an acceptor. The product of the condensation of glycolaldehyde with D-ribulose 5-phosphate was isolated by ionexchange chromatography. The dephosphorylated product was tentatively identified as a heptulose with the molecular formula C7H14O7from its spectrophotometric properties and GC-MSresults.

A novel enzyme, pyrroloquinoline quinone (PQQ)-dependent polyvinyl alcohol (PVA) dehydrogenase, was found in and partially purified from the membrane fraction of a PVA-degrading symbiont, Pseudomonas sp. strain VM15C. The enzyme required PQQ for PVA dehydrogenation with phenazine methosulfate, phenazine ethosulfate, and 2,6-dichlorophenolindophenol as electron acceptors and did not show PVA oxidase activity leading to H202 formation. The enzyme was active toward low-molecular-weight secondary alcohols rather than primary alcohols. A membrane-bound PVA oxidase was also present in cells of VM15C. Although the purified oxidase showed a substrate specificity similar to that of PQQ-dependent PVA dehydrogenase and about threefold-higher PVA-dehydrogenating activity with phenazine methosulfate or phenazine ethosulfate than PVA oxidase activity with H202 formation, it was shown that the enzyme does not contain PQQ as the coenzyme, and PQQ did not affect its activity. Incubation of the membrane fraction of cells with PVA caused a reduction in the cytochrome(s) of the fraction.