Crystallization and Characterization of Agmatine Oxidase from Penicillium chrysogenum

Agmatine oxidase was purified and crystallized with an overall yield of about 30% from a mycelial extract of Penicillium chrysogenum by a procedure involving ammonium sulfate fractionation, and DEAE-cellulose, hydroxyapatite, 1,8-diaminooctane-Sepharose 4B and Sephadex G-200 column chromatographies. The purified enzyme was homogeneous on disc gel electrophoresis and the pink crystals appeared as a hexagonal board on addition of solid ammonium sulfate. The molecular weight of the native monomer form was determined to be 160,000 by gel filtration, and it was composed of two identical subunits. The prosthetic group was identified as copper and its content was determined to be 2 mol per mol of the enzyme. The enzyme was inhibited by hydroxylamine, hydrazine, phenylhydrazine, semicarbazide, KCN, PCMB, Ag⁺, Hg²⁺ and Cu²⁺. The apparent Km values for agmatine, histamine and putrescine were calculated to be 2.51 × 10^?4m, 4.25 × 10^?4m and 1.64 × 10^?2m, respectively.     

Purification and Characterization of Formaldehyde Dehydrogenase in a Methanol-utilizing Yeast,Kloeckera sp. No. 2201

An NAD-linked formaldehyde dehydrogenating enzyme was found in the cell-extract of Kloeckera sp. No. 2201, which utilized methanol as a sole source of carbon. The enzyme was inducibly formed in methanol-grown cells. This fact suggests that the enzyme may play a significant role in the methanol metabolism of this yeast. The enzyme was purified from a cell-extract by ammonium sulfate fractionation, column chromatographies on DEAE-cellulose and on hydroxylapatite, and Sephadex G-200 gel filtration. From an experiment with the purified enzyme, it was found that the enzyme specifically required reduced glutathione for activity, and was reactive toward methylglyoxal as well as formaldehyde. The enzyme catalyzed the following reaction:

the enzyme was concluded to be a kind of formaldehyde dehydrogenase (formaldehyde: NAD oxidoreductase, EC 1.2.1.1). Other properties of the enzyme were also investigated.     

Derepression of FAD Pyrophosphorylase and Flavin Changes during Growth of Kloeckera sp. No. 2201 on Methanol

A methanol-utilizing yeast Kloeckera sp. No. 2201, when grown with methanol as a sole carbon and energy source, accumulated about three times much flavin as those grown with glucose, ethanol, or glycerol. A high proportion of the total flavin was FAD in methanol-grown cells. A remarkable derepression of FAD pyrophosphorylase accompanied by an inducible formation of an FAD-dependent alcohol oxidase which catalyzes oxidation of methanol, the first step in the oxidation sequence, was observed during growth of the yeast on methanol. Significant elevations of riboflavin synthetase and flavokinase were also found. Formate, as well as methanol, effectively induced both FAD pyrophosphorylase and methanol-oxidizing enzymes (alcohol oxidase, formaldehyde dehydrogenase, formate dehydrogenase, and catalase). Observations with other methanol-utilizing yeasts also gave essentially same results. These results led to the conclusion that cellular flavin level might be under control with level of flavoprotein physiologically required.     

Biochemical Studies on Fatty Liver

The effects on the fat content in livers of amino acid supplements to various cereal diets at 1.12% dietary nitrogen level, which causes fatty livers in young rats, have been investigated. When rats are fed on diets supplemented with amino acid to retain the requirement pattern of essential amino acids, they grow very well, but the accumulation of liver fat fails to decrease. However, when a part of the requirement pattern diets is replaced with 0.30% of l-threonine, the deposition of liver fat shows a clear decrease. The results obtained suggest that this excess part of threonine has a specific action for the prevention of fat deposition in livers regardless of protein utilization, together with the so-called “supplementary effects” of amino acids.     

A New Preparation Method of Coenzyme A with Microorganisms

The structure of aggregates formed by heating dilute BSA solution was analyzed with the fractal concept using light scattering methods. BSA was dissolved in HEPES buffer of pH 7.0 and acetate buffer of pH 5.1 to 0.1% and 0.001% solutions, respectively, and heated at 95°C, varying the heating time t_a. The fractal dimension D_f of the aggregate in the solution was evaluated from static light scattering experiments. The polydispersity exponent τ and the average hydrodynamic radius <R_h> of the aggregates were calculated from dynamic light scattering experiments using master curves obtained by Klein et al. The values of D_f and τ of heat-induced aggregates of BSA at pH 7.0 were about 2.1 and 1.5, respectively, the values of which agreed with those predicted by the reaction-limited cluster–cluster aggregation (RLCCA) model. On the other hand, D_f of heat-induced aggregates at pH 5.1 was about 1.8, which agreed with that predicted by the diffusion-limited cluster–cluster aggregation (DLCCA) model. The dependence of <R_h> for the sample of pH 7.0 on t_a was similar to that of the polystyrene colloids reported previously.     

Some Aspects of the Enzyme Activities Involved in Coenzyme A Biosynthesis in Various Microorganisms

The distribution of the enzyme activities relating to CoA biosynthesis from pantothenic acid in various microorganisms and the effect of CoA on these activities are described.

High activities of partial reactions involved in CoA biosynthesis were surveyed in various type culture strains involving bacteria, actinomycetes, lactic acid bacteria, molds, and yeasts. Generally, higher activities were found in bacteria. CoA inhibited the phosphorylation of pantothenic acid, and resulted in a decrease of CoA production in all the CoA producing strains, while only a little inhibition by CoA was observed in the other reactions, and CoA production from pantothenic acid 4′-phosphate by Brevibacterium ammoniagenes IFO 12071 was not repressed even in the presence of 4mm of CoA. Extracellular excretion of the enzymes of CoA biosynthesis was observed when cells were in contact with sodium lauryl sulfate. Degrading activity against CoA and that against AMP were relatively lower in CoA producing strains when compared with those in other strains. It was confirmed that Brown’s route of CoA biosynthesis operates in Brevibacterium ammoniagenes IFO 12071.     

Studies on Vitamin B6 Metabolism in Microorganisms

A new phosphotransferring reaction which phosphorylated pyridoxine through the phosphoryl group transfer from p-nitrophenylphosphate was found, and the distribution of the reaction in several microorganisms was investigated. The transferring activity was widely distributed in various kinds of microorganisms, especially in fungi belonging to genus such as Aspergillus. The phosphorylated product was isolated from the reaction mixture with the dried cells of Aspergillus flavus and identified as pyridoxine 5′-phosphate.     

Effects of Organic Solvents and Chemical Modification on the Activity of Glycerol Dehydrogenase

The addition of an organic solvent to the reaction mixture led to a change in the activity of glycerol dehydrogenase. The activity on glycerol decreased to lower than that on 1,2-propanediol. This was due to the apparent increase in the Km value for each substrate. The system was applied to determination of 1,2-propanediol. The standard curve for 1,2-propanediol with a rate assay method was not affected by a 10-fold amount of glycerol in the presence of «-butanol. Chemical modification, except for succinylation of the NH₂-residue, did not cause any change in substrate specificity. The participation of a His-residue in the active site was suggested by the results of chemical modification.