Mitsunori Hayashi

Production of 5-Aminolevulinic Acid by Photosynthetic Bacteria

Abstract Extracellular formation of 5-aminolevulinic acid (ALA) by adding levulinic acid (LA), an inhibitor of ALA dehydratase, was examined in the anaerobic-light culture of Rhodobacter sphaeroides . The addition of LA (10–25 mmol/ l ) during the middle log phase retarded the growth and accelerated the extracellular formation of ALA, while over 50 mmol/ l completely suppressed both growth and formation. The formation of ALA was closely related to intracellular ALA synthetase activity. Light intensity was also an important factor for enhancing ALA formation. The optimal condition, addition of 15 mmol/ l of LA during the middle log phase with 3 klx illumination, resulted in ALA formation of 0.26 mol/ l . In addition, supplementation with glycine (30 mmol/ l ) and succinate (30 mmol/ l ), precursors of ALA biosynthesis, enhanced ALA formation up to ca. 2 mmol/ l .

Production of a herbicide, 5-aminolevulinic acid, by Rhodobacter sphaeroides using the effluent of swine waste from an anaerobic digestor

SummaryFor the production of a herbicide, 5-amino-levulinic acid (ALA), from anaerobic digestion liquor, the utilization of the photosynthetic bacterium, Rhodobacter sphaeroides was examined. This bacterium could produce ALA extracelularly from this liquor with the addition of levulinic acid (LA), an inhibitor of ALA dehydratase (ALAD), and glycine, a precursor of ALA biosynthesis in the Shemin pathway. Succinate (another precursor) addition was unnecessary for ALA production. When repeated additions of LA were made together with glycine ALA production was significantly enhanced. However, above three additions of LA, ALA production was not further enhanced. The maximum value of ALA production attained was 4.2 mM (0.63 g/ 1), which was over double that of other ALA producers such as Chlorella vulgaris. Propionic acid was predominantly utilized compared with other lower fatty acids, suggesting that this might be converted to ALA via succinyl-coenzyme A (CoA) in the methylmalonyl-CoA pathway.

Enhanced production of 5-aminolevulinic acid by repeated addition of levulinic acid and supplement of precursors in photoheterotrophic culture of Rhodobacter sphaeroides

Abstract When levulinic acid (LA) was added repeatedly in the photosynthetic culture of Rhodobacter sphaeroides to maintain its concentration at a constant (low) level, the activity of 5-aminolevulinic acid (ALA) dehydratase could be kept low enough to reduce the synthesis of porphobilinogen from ALA formed. This brought about the extracellular accumulation of ALA, while cellular growth was partially retarded. In this condition, the intermittent or continuous supplement of a fixed amount of the pre-cursors (glycine and succinate) to reduce the inhibitory effects of glycine on growth could enhance the ALA accumulation twice as much as when the same amount of the precursors was supplied all at once.

Conversion of D-xylose into xylitol by xylose reductase from Candida pelliculosa coupled with the oxidoreductase system of methanogen strain HU

SummaryA preliminary test for the enzymatic conversion of D-xylose into xylitol by the intact cells of Candida pelliculosa (xylose reductase) coupled with the intact cells of a formate-utilizing methanogen strain HU (hydrogenase and F420-NADP oxidoreductase) was conducted by using H2 as an electron donor of NADP+. In the system, NADP(H) was well regenerated via the methanogen cells and about 90% conversion of xylose to xylitol (ca. 8 g/l) could be achieved at 35°C and pH 7.5 after 24 h incubation.

Production of corrinoids including vitamin B-12 byMethanosarcina barkeri growing on methanol

SummaryA preliminary attempt was made for producing vitamin B-12 byMethanosarcina barkeri strain Fusaro in a fed-batch culture with a methanol minimum medium. After 11 days, total methanol consumption, cell density and corrinoid concentration were 145 g/l, 8.5 g(dry cell weight)/l, and 135 mg/l (73% in supernatant) respectively. Electrophoretic separation revealed that 33% of the total corrinoids was B-12 Factor III (5-hydroxybenzimidazolylcobamide) and the remaining corrinoids were cobinamide (Factor B) and its derivatives.

Regeneration and retention of NADP (H) for xylitol production in an ionized membrane reactor

SummaryA sulfonated polysulfone membrane reactor was used forin situ regeneration and retention of coenzymes NADP (H) using the xylose reductase ofCandida pelliculosa coupled with oxidoreductase system ofMethanobacterium sp. in the reduction of xylose to xylitol with hydrogen gas. The membrane could almost completely reject the permeation of NADP (H) (92 and 97%), F420 (97%) and the required enzymes (100%), but not reject for the permeation of xylitol (product). After 4-h reaction for the production of xylitol from xylose (93% yield), although 25% NADP (H) initially added was lost its activity due to unavoidable degradation, the membrane could reject the permeation of the remaining NADP (H) and F420 at the level of 90 and 95%, respectively.

Influence of iron on the excretion of 5-aminolevulinic acid by a photosynthetic bacterium, Rhodobacter sphaeroides

Abstract Fe 2+ and/or Fe 3+ supplemented to the culture of Rhodobacter sphaeroides enhanced intracellular 5-aminolevulinic acid (ALA) synthetase, but ALA excretion could not be observed, even though the ALA dehydratase inhibitor (levulinic acid) was added. The reason for this was investigated, and it was found that Fe 2+ directly inhibits ALA synthetase activity. The supplemented Fe 2+ was accumulated in the cells.

Isolation of 5,6-dimethylbenzimidazolyl cobamide coenzyme from Rhodopseudomonas spheroides.

A photosynthetic bacterium, Rhodopseudomonas spheroides, has often been used for studies of the biosynthesis of porphyrins and bacteriochlorophyll. It is also known that the bacterium can be grown nonphotosynthetically, e.g. aerobically in the dark on a medium containing organic acids and natural nutrients such as yeast extract, peptone, etc. This is why the organism has been used for the studies of organic acid metabolism under aerobic as well as anaerobic conditions, photosynthetic reactions and regulation mechanism of porphyrin biosynthesis [1 ]. Recently, Lascelles et al. [2] reported that vitamin B12 activity was found microbiologically in ceils ofR. spheroides grown on medium containing cobalt chloride. However, the forms of vitamin B12 produced and the quantitative relationship among the vitamin B12 analogues have not been elucidated. The pathway of vitamin B12 biosynthesis in R. spheroides seems to be the same as that of porphyrins, at least from glycine to porphobilinogen, but this has not been confirmed. It is of interest to elucidate the relationship between porphyrin and vitamin B12-factor formation with special reference to the mechanism of tetrapyrrole biosynthesis. We have tried to identify the forms of vitamin B12 produced aerobically in shake culture.

Biosynthesis and properties of 2-thioadenyl cobamide and its coenzyme form

Barker et al. [l-2] first isolated adenyl cobamide coenzyme and subsequently cobalamin coenzyme from cultures of Clostridium tetanomorphum and later from Propionibacterium sp. Thereafter, many reports have appeared on the roles of the coenzyme forms of cobalamin in a number of important biochemical reactions. Kamikubo et al. [3] reported that biosynthetic 2-chloroadenyl cobamide coenzyme from growing Propionibacterium arabinosum cells was active as a coenzyme for propanediol dehydrase. However, no further reports have appeared on the biochemical functions of cobamide coenzymes with purines in the nucleotide moiety. In this paper, the biosyhthesis of 2-thioadenyl cobamide and its coenzyme form using resting Pr. arabinosum cells, their physico-chemical properties and biological as well as biochemical activities are dealt with.

Isolation of 5,6-dimethylbenzimidazolyl cobamine coenzyme as a cofactor for glutamate formation from Acetobacter suboxydans

Abstract In order to know the kinds and forms of vitamin B 12 coenzymes which participate in the formation of glutamic acid through the pathway via citramalate, mesaconate and β-methyla spartate by Acetobacter suboxydans , isolation and identification of the compounds from the bacteria were attempted. From 500 l fermentation of A. suboxydans , 1.7 kg of the freeze-dried cells were obtained. Corrinoids were extracted from the cells with 80% ethanol with or without cyanide, purified with p -chlorophenol-chloroform, fractionated by aluminacolumn chromatography and crystallized in acetone. The paper-ionophoretic, paper-chromatographic, spectrophotometric and microbiological investigations of the cyanocorrinoid showed that the compound obtained might be 5,6-dimethylbenzimidazolyl cobamide. The investigation on the nucleoside, a Ce(III)-decomposition product of the corrinoid proved to be α-ribazole and therefore gave an additional evidence for 5,6-dimethylbenzimidazolyl cobamide. For coenzyme forms, cautions were taken to operate in every process without cyanide and in the dark. Similar physicochemical tests indicated that the coenzyme form of the compound is cobalamin coenzyme.