Masayuki Shimao

Biodegradation of plastics.

Widespread studies on the biodegradation of plastics have been carried out in order to overcome the environmental problems associated with synthetic plastic waste. Recent work has included studies of the distribution of synthetic polymer-degrading microorganisms in the environment, the isolation of new microorganisms for biodegradation, the discovery of new degradation enzymes, and the cloning of genes for synthetic polymer-degrading enzymes.

L-serine production by a methylotroph and its related enzymes.

The production process of l-serine from methanol and glycine has been developed using a methylotroph with the serine pathway. Consecutive reactions of two enzymes, methanol dehydrogenase (MDH) and serine hydroxymethyltransferase (SHMT) are involved in the production. We screened a high producer, Hyphomicrobium methylovorum, which is an obligate methylotroph. With resting cells of the bacterium, 24 mg/ml of l-serine was produced from 100 mg/ml of glycine and 48 mg/ml of methanol in 3 days under optimal conditions. Next, a glycine-resistant mutant GM2 showed improved serine production (32–34 mg/ml). The mutant GM2 was found to have elevated activities of MDH and SHMT. Since there has so far been little report on the systematic characterization of enzymes of the serine pathway in methylotrophs, not only the above two enzymes but also the other three enzymes in H. methylovorum were purified and characterized: MDH, SHMT and hydroxypyruvate reductase (HPR) were crystallized; serine-glyoxylate aminotransferase (SGAT) and glycerate kinase (GK) were purified to homogeneity. As a result, all these enzymes were found to be stable against preservation and to exist abundantly in the bacterium. The gene of SHMT was cloned and its deduced amino acid sequence had homology to those of Escherichia coli (55%) and rabbit liver (44%), whereas the enzyme of the bacterium was immunochemically distinguishable from those of microorganisms other than Hyphomicrobium strains and mammalian livers.

Dihydroxyacetone production from methanol by a dihydroxyacetone kinase deficient mutant of Hansenula polymorpha

SummaryA mutant, No. 65, of Hansenula polymorpha CBS 4732 was isolated which was impaired in its ability to grow on methanol and dihydroxyacetone. Mutant No. 65 produced dihydroxyacetone and glycerol from methanol with a 18.8% yield in a resting-cells reaction. The absence of dihydroxyacetone kinase activity in the mutant is believed to be the reason for its inability to grow on methanol and for the accumulation of trioses. This mutant, however, was able to grow on glycerol, and dihydroxyacetone kinase was found in the cells. The growth on glycerol was almost completely inhibited by the addition of methanol (0.1% v/v). As far as tested with partially purified enzymes, no property was found that could be used to distinguish between the kinases from methanol- and glycerol-grown cells. The evidence suggests that the phenotype of No. 65 is a lesion not in the structural gene but in its regulatory gene.

Production of 3-hydroxybutyric acid trimer by Bacillus megaterium B-124

Abstract Newly isolated Bacillus megaterium B-124, which accumulated poly(3-hydroxybutyric acid) (PHB) amounting to about 20% of its dry cell weight, was found to secrete 3-hydroxybutyric acid and its trimer into the culture broth. The culture conditions were investigated and it was found that optimum conditions gave 7.6 g of the trimer per liter in 3 d of culture under the optimum conditions. The changes in the amounts of PHB, 3-hydroxybutyric acid, and its trimer during culture did not simply show that the trimer was the direct product of intracellular degradation of PHB.

Dismutation and cross-dismutation of aldehydes, and alcohol: aldehyde oxidoreduction by resting-cells of Pseudomonas putida F61-a

Abstract Pseudomonas putida F61-a defective in formaldehyde dehydrogenase was derived from the parent strain (F61). The bacterial strain grown on a nutrient broth supplemented with 1% glucose exhibited high formaldehyde dismutase activity. The dismutation and cross-dismutation of aldehydes occurred stoichiometrically in the resting-cells reaction. Many kinds of aliphatic and aromatic aldehydes that are scarcely soluble in water were utilized in these reactions as well as soluble ones. Formaldehyde at an extremely high concentration (0.5 M) was almost completely converted to equimolar amounts of methanol and formic acid by the resting-cells, which could be used three times without a loss of activity. The cross-dismutation between acrolein and formaldehyde occurred efficiently in the resting-cells reaction with 0.1 M each substrate. The alcohol: aldehyde oxidoreduction of the short-chain substrates was also shown by the resting-cells of a mutant (M6) unable to grow on n -propanol.

Dihydroxyacetone kinase from a methylotrophic yeast, Hansenula polymorpha CBS 4732

Dihydroxyacetone (DHA) kinase was purified to electrophoretic homogeneity from methanol-grown Hansenula polymorpha CBS 4732. The enzyme was a dimer with a molecular weight of 150,000, and had an isoelectric point of 4.9. The enzyme was active toward DHA, and D- and L-glyceraldehydes as phosphorylation acceptors, and only ATP served as a donor. ADP inhibited the enzyme at a physiological concentration. Magnesium ion was essential for the activity and stability. Some other divalent cations can substitute in part the magnesium ion. The DHA kinases found in cells grown on methanol and glycerol were immunologically identical, but were different from those of other methylotrophic yeasts as shown by immunotitration. A mutant (204D) derived from the yeast, which could not grow on methanol or DHA but could so on glycerol, was deficient in DHA kinase. Glycerol kinase activity was found in glycerol-grown 204D cells as well as the parent strain.

Formate production from methanol by formaldehyde dismutase coupled with a methanol oxidation system

SummaryFormaldehyde dismutase was greatly stabilized by immobilization in a urethane prepolymer (PU-6). The immobilized enzyme exhibited stochiometrical dismutation of formaldehyde to methanol and formate in several repeated reactions. Conversion of methanol to formate occurred in a reaction with an immobilized enzyme system consisting of alcohol oxidase, catalase and formaldehyde dismutase, and with an intact cell-mixture of Hansenula polymorpha and Pseudomonas putida. Furthermore, the stability of the cell-mixture during repeated reactions was greatly improved by the immobilization, the 600 mM methanol added periodically being converted to formate in a 75% yield in 12 h. The immobilized cellsystem was also effective for the conversion of several aliphatic alcohols, C1 to C4, to the corresponding acids.

Pyruvate production by Enterococcus casseliflavus A-12 from gluconate in an alkaline medium

Abstract A newly isolated lactic acid bacterium, Enterococcus casseliflavus A-12, produced pyruvic acid (16 g/l) during aerobic culture in an alkaline medium containing sodium gluconate (50 g/l) as the carbon source. The production was dependent on the pH of the culture, the optimum initial pH being 10.0. With static culture, the organism produced lactic acid (2.7 g/l) from both gluconate and glucose. Pyruvate did not accumulate in growing cultures on glucose, but resting cells obtained from a culture on gluconate produced pyruvate from glucose as well as gluconate. The enzyme profiles of the organism, which grew on gluconate and glucose, suggested that gluconate was metabolized via the Entner-Doudoroff and Embdem-Meyerhof-Parnas pathways in aerobic culture, and that glucose was oxidized mainly via the latter pathway under both aerobic and anaerobic conditions. Gluconokinase, a key enzyme in the aerobic metabolism of gluconate, was partially purified from this strain and characterized.

Purification and properties of aldehyde reductases from yeasts that convert ethyl 2-acetamido-3-oxobutyrate to optically active ethyl 2-acetamido-3-hydroxybutyrate

Yeasts were screened for strains that converted ethyl 2-acetamido-3-oxobutyrate (AAOB) to optically active ethyl 2-acetamido-3-hydroxybutyrate (AAHB). Sporobolomyces sp. AKU4430 was found to accumulate the D-threo isomer of AAHB by a whole-cell reaction. Candida albicans AKU4596 accumulated mainly the D-threo and L-threo isomers. The enzymes that reduced AAOB were purified from these two yeasts, and characterized. To judge from their substrate specificity, inhibition pattern, molecular structure, and reaction mechanisms, the enzymes were of the NADPH-dependent aldo-keto reductase family (probably aldehyde reductase, EC 1.1.1.2). The enzyme of Sporobolomyces sp. reduced AAOB more strongly than that of C. albicans. The stereoselectivity of the enzymes was low; three isomers of AAHB (L-threo, L-allo, and D-threo) were produced by each purified enzyme. The selective accumulation of an isomer(s) of AAHB by reaction in yeast cells probably occurred because of differences in isomer degradation.

Crystallization and preliminary diffraction studies of hydroxypyruvate reductase (D-glycerate dehydrogenase) from Hyphomicrobium methylovorum

Two crystal forms of hydroxypyruvate reductase (D-glycerate dehydrogenase) from the methylotrophic bacterium Hyphomicrobium methylovorum have been grown from ammonium sulphate solutions. One crystal form is triclinic, with unit cell parameters a = 60.4 A, b = 60.5 A, c = 66.3 A, alpha = 102.3 degrees, beta = 113.7 degrees and gamma = 102.7 degrees, suggesting that a dimer (monomer M(r) 38,000) occupies the unit cell. This crystal form diffracts to beyond 2.4 A resolution and is suitable for crystallographic structure analysis.