Conversion ofL-sorbose to 2-keto-L-gulonic acid by mixtures of immobilized cells ofGluconobacter melanogenus IFO 3293 andPseudomonas species

Summary L-Sorbose was converted to 2-keto-L-gulonic acid by mixtures ofGluconobacter melanogenus IFO 3293 andPseudomonas syringae NRRL B-865 entrapped simultaneously in polyacrylamide gel. Since the temperature optima of both enzymatic reactions involved differed, a two-stage process with cells immobilized separately seems to offer a more efficient method to prepare 2-keto-L-gulonic acid.For preceeding paper in this series see Martin & Perlman (1976).     

Conversion of L-sorbose to L-sorbosone by immobilized cells of Gluconobacter melanogenus IFO 3293.

Gluconobacter melanogenus IFO 3293 cells capable of converting L-sorbose to L-sorbosone were immobilized in polyacrylamide gel. The preferred polymer composition for high activity and stability was determined to contain a total monomer concentration of 7.2% and 16.6% crosslinking agent. No significant differences in optimal conditions for conversion, e.g., pH and temperature, were found in comparison with free cell suspensions. However, in the absence of L-sorbose, the thermal stability of immobilized cells was lower. After the initial loss, the conversion activity of immobilized cells increased, possibly due to lysis, and this increase was related to the polymerization conditions and the incubation temperature for the L-sorbose conversion. The enzymatic activity and stability of the immobilized cells also depended on the physical form of the gel and the aeration levels. Addition of electron acceptors or addition of L-sorbosone to the medium reduced, while addition of neomycin, ampicillin, chloramphenicol, and tetracycline increased the stability of the enzymatic activity.     

Application of Oxygen-Enriched Aeration in the Conversion of Glycerol to Dihydroxyacetone by Gluconobacter melanogenus IFO 3293

Gluconobacter melanogenus 3293 converts glycerol to dihydroxyacetone(DHA) during exponential growth on a yeast extract-phosphate medium at pH 7. The efficiency of this conversion in 25-liter batch fermentations has been found to increase over threefold, when oxygen tension is controlled by increasing the partial pressure of oxygen in the aeration. Conversion of glycerol to DHA does not occur under oxygen-limited fermentation conditions. When the dissolved oxygen tension was maintained at 0.05 atmospheres (using oxygen-enriched air), quantitative conversion of up to 100 g of glycerol/liter to DHA was obtained in 33 h. The amount of glycerol converted can be increased without increasing impeller speed or aeration rate. This increase is not the result of increased production of cell mass. The specific conversion of glycerol to DHA increased from 12.2 g of DHA/g of cell mass at the point of maximum conversion to 35.8 with oxygen enrichment. This increased specific production occurred even though the specific growth rate during the period of oxygen enrichment decreased from 0.23 to 0.06/h.     

Stimulation by organic solvents and detergents of conversion of L-sorbose to L-sorbosone by Gluconobacter melanogenus IFO 3293.

Treatment of Gluconobacter melanogenus IFO 3293 cells with benzene, carbon tetrachloride, cyclohexane, deoxycholate, toluene, or xylene stimulated their conversion of L-sorbose to L-sorbosone two- to threefold. The degree of stimulation depended upon the length of exposure time to the agent and the age of the G. melanogenus cells. A rapid decrease in viability of the cells and degradation of cell RNA was noted after treatment with the effective agents. The G. melanogenus cells were unable to absorb L-sorbose actively after toluene treatment.     

Fermentation of glucose by Acetobacter melanogenus.

Growing cultures of Acetobacter melanogenus ATCC 9937 concerted D-glucose to 2,5-diketo-D-gluconic acid with D-gluconic acid and 5-keto-D-gluconic acid as intermediates. The 2,5-diketo-D-gluconic acid was isolated from the fermented medium by treatment with an anion exchange resin.     

Conversion of L-sorbose to L-sorbosone by Gluconobacter melanogenus

Growing cultures, washed cells, and cell-free extracts of Gluconobacter melanogenus IFO 3293 were found to convert L -sorbose to L -sorbosone. The product was identified by thin layer chromatography of the 2, 4-dinitrophenylhydrazone, and by paper partition chromatography using chemically prepared materials as standards. Factors influencing the conversion included incubation temperature and composition of the growth medium. Addition of betaine or choline to the growing cultures stimulated conversion of L -sorbose to L -sorbosone.     

生黑醋酸杆菌的太空诱变选育

目的:通过太空育种的方法选育获得高活性的生黑醋酸杆菌,实现维生素C的高效发酵.方法:采用太空育种的方法,对VC一步发酵菌生黑醋酸杆菌进行神州七号搭载诱变.结果:经过多轮试管初筛、摇瓶复筛,获得1株高效菌,编号为G454.23%醇浓摇瓶发酵,发酵周期缩短约2h;在适度提高通风下,可完成高醇浓度(33%)发酵,发酵周期约24h.该菌经发酵条件优化,连续5批常规生产.结果:新菌株G454的发酵稳定性好,周期均在20h以内,平均周期18.4h;与对照相比,发酵周期缩短2h,山梨糖产率12.34mg/ml/h,较对照提高7.68%.结论:筛选所得菌株454可缩短发酵周期10%.,提高山梨糖产率7.68%.     

The fermentation of L-sorbose by Gluconobacter melanogenus. I. General characteristics of the fermentation

Growing cultures, washed cells, and cell-free preparations of Gluconobacter melanogecnus IFO 3293 converted L -sorbose to 2-keto-L -gulonic acid, to D -sorbitol (which was metabolized further) and to 5-keto-D -fructose.     

Osmotically Regulated Transport of Proline by Lactobacillus acidophilus IFO 3532

[This corrects the article on p. 2831 in vol. 57.].     

生黑葡萄糖酸杆菌转化高浓度山梨醇的条件优化

目前,国内维生素C主要采用二步发酵法生产,其中第一步为生黑葡萄酸杆菌(Gluconobacter melano-genus)将D-山梨醇转化为L-山梨糖。考察了该菌株在提高培养基中山梨醇浓度时的发酵特性和发酵条件。实验室摇瓶实验结果显示,通风量、发酵前期及后期pH值控制、接种种液类型都影响高浓度山梨醇摇瓶发酵的转化率。以35%山梨醇浓度发酵液做种子液明显优于生产上采用的三级种子液(12%~17%山梨醇浓度),培养基前期pH值5.0~6.0,后期pH值4.2~3.9,装液量180 mL,发酵周期在30 h之内,山梨醇转化率在98%以上。培养基山梨醇浓度由23%提高到35%,发酵周期延长8 h。上述实验结果对指导生产工艺优化具有重要意义。     

Metabolism of xenobiotic compounds by enzymes in cell extracts of the fungus Cunninghamella elegans.

Cell extracts of the filamentous fungus Cunninghamella elegans contain epoxide hydrolase (EC 3.3.2.3), glutathione S-transferase (EC 2.5.1.18) and UDP-glucuronosyltransferase (EC 2.4.1.17) activities. Epoxide hydrolase activity was determined with p-nitrostyrene oxide as substrate and was shown to be associated with the 100 000 g pellet obtained from disrupted mycelia. Glutathione S-transferase activity was demonstrated with 1-chloro-2,4-dinitrobenzene and p-nitrobenzyl chloride as substrates. The presence of two or more glutathione S-transferase activities was indicated by different activity ratios for the two substrates in different extracts, and by distinct thermal denaturation curves. UDP-glucuronosyltransferase activity with 3-hydroxybenzo[a]pyrene as substrate was found only with the non-sedimentable fraction prepared from ruptured mycelia.     

Osmotically regulated transport of proline by Lactobacillus acidophilus IFO 3532.

We reported previously that, when exposed to high osmotic pressure, Lactobacillus acidophilus IFO 3532 cells accumulated N,N,N-trimethylglycine (glycine betaine), which serves as a compatible intracellular solute. When grown in medium with high osmotic pressure, these cells also accumulated one amino acid, proline. The uptake of [3H]proline by resting, glucose-energized cells was stimulated by increasing the osmotic pressure of the assay medium with 0.5 to 1.0 M KCl, 1.0 M NaCl, or 0.5 M sucrose. The accumulated [3H]proline was not metabolized further. In contrast, there was no osmotic stimulation of [3H]leucine uptake. The uptake of proline was activated rather than induced by exposure of the cells to high osmotic pressure. Only one proline transport system could be discerned from kinetics plots. The affinity of the carrier for proline remained constant over a range of osmotic pressures from 650 to 1,910 mosM (Kt, 7.8 to 15.5 mM). The Vmax, however, increased from 15 nmol/min/mg of dry weight in 0.5 M sucrose to 27 and 40 nmol/min/mg of dry weight in 0.5 M KCl and in 1.0 M KCl or NaCl, respectively. The efflux of proline from preloaded cells occurred rapidly when the osmotic pressure of the suspending buffer was lowered.     

Osmotically regulated transport of proline by Lactobacillus acidophilus IFO 3532.

We reported previously that, when exposed to high osmotic pressure, Lactobacillus acidophilus IFO 3532 cells accumulated N,N,N-trimethylglycine (glycine betaine), which serves as a compatible intracellular solute. When grown in medium with high osmotic pressure, these cells also accumulated one amino acid, proline. The uptake of [3H]proline by resting, glucose-energized cells was stimulated by increasing the osmotic pressure of the assay medium with 0.5 to 1.0 M KCl, 1.0 M NaCl, or 0.5 M sucrose. The accumulated [3H]proline was not metabolized further. In contrast, there was no osmotic stimulation of [3H]leucine uptake. The uptake of proline was activated rather than induced by exposure of the cells to high osmotic pressure. Only one proline transport system could be discerned from kinetics plots. The affinity of the carrier for proline remained constant over a range of osmotic pressures from 650 to 1,910 mosM (Kt, 7.8 to 15.5 mM). The Vmax, however, increased from 15 nmol/min/mg of dry weight in 0.5 M sucrose to 27 and 40 nmol/min/mg of dry weight in 0.5 M KCl and in 1.0 M KCl or NaCl, respectively. The efflux of proline from preloaded cells occurred rapidly when the osmotic pressure of the suspending buffer was lowered.     

Fermentative Production of Succinic Acid from n-Paraffin by Candida brumptii IFO 0731

An investigation of succinic acid production from n-paraffin under various culture conditions was carried out with Candida brumptii IFO 0731. Ammonium nitrogen was required for both cell growth and succinic acid production. Favorable culture conditions for succinic acid production were ascertained. The productivity was markedly increased by the additions of CaCO₃ and organic nutrients. Under the best condition, the largest quantity of succinic acid production, 23.6 mg/ml, was obtained in a 67% yield from super heavy n-paraffin after 8 days cultivation.     

Two different conformations of the antitumor beta-D-glucan produced by Sclerotinia sclerotiorum IFO 9395

The antitumor beta-D-glucan [SSG, (1----3)-linked with a D-glucosyl group attached to position 6 of alternate units] produced by Sclerotinia sclerotiorum IFO 9395 afforded a gel in neutral aqueous solution which gave no 13C-n.m.r. signals. Signals appeared on the addition to the gel of sodium hydroxide to 0.15M and the line-width was narrowest near 0.25M base (gel-to-sol transition). SSG bound to Congo Red and shifted the lambda max in a manner similar to that of curdlan. Neutralisation of the sol regenerated the gel. SSG and regenerated SSG showed different c.p.-m.a.s. 13C-n.m.r. spectra, especially the signals for C-3 which appeared at 86 and 89 p.p.m., respectively. The c.p.-m.a.s. spectrum of regenerated SSG was similar to that of curdlan, suggesting a helical structure. Thus, SSG and regenerated SSG appear to have different conformations.