Journal of Fermentation Technology最新文献

From Chinese hamster ovary (CHO) cells which were producing Erythroid Differentiation Factor (EDF) in a culture medium, anchorage-independent cells, named as CHO-SPN, were produced by repeated cultivating in a suspension system. The growth time and maximum cell density of the CHO-SPN cells were 48 h and 7.8×10⁵ viable cells/ml. CHO-SPN cells accumulated 8,000 units/ml (corresponding to 4 mg/ml) of EDF in 4d. After 20 cycles of culture, CHO-SPN cells still possessed the same EDF productivity and the same growth kinetics. Furthermore, in an appropriate dissolved oxygen concentration and pH controlled culture system, the growth time and cell density became 24 h and 1×10⁶ viable cells/ml. The critical level of dissolved oxygen for cell growth was 0.015 atm. The maximum oxygen demand was 3.3×10⁻⁹ mole of O₂/ml/min.

Fetal bovine serum (FBS) was indispensable for cell growth. However, a FBS-free medium (ASF201) was available for maintenance of the CHO-SPN cells, and EDF production occurred in the same medium.

Screening of a strain which contained a large amount of ubiquinone Q-10 and a variety of isoprenoid compounds using different culture conditions and mutations was carried out.

Protomonas extorquens TK 0045, which was found to contain carotenoid pigments, Hop-22(29)-ene, and Hopan-22-ol, was selected on the basis of cell yield and the content of ubiquinone Q-10. The contents of ubiquinone and sterols increased as the age of the culture increased, and reached a maximum level during the stationary phase.

The contents of ubiquinone, sterols and carotenoid pigments, and ubiquinone homologs produced by P. extorquens TK 0045 were varied using mutagenesis. Mutants that had increased or decreased contents of carotenoid pigments were obtained with a high frequency. Most mutants had varying contents of other isoprenoid compounds. The ubiquinone homologs obtained by mutagenesis varied with a high frequency, and mutants which possessed increased levels of ubiquinone Q-9, Q-11 or Q-12 were isolated. However, the major ubiquinone component in these mutants was Q-10 the same as that in the wild strain. The production of ubiquinone was increased considerablyby repeated mutagenesis, with the content of ubiquinone produced by the third generation mutant (strains HB-5) being approximately 3.3 mg·g dry cell⁻¹ (2.5 times that of the wild strain). The acquisition of mutants exhibiting altered synthesis of carotenoid pigments would be useful for increasing the content of ubiquinone Q-10 in bacterial cells.

The new enzyme, propioin synthase, concerned with the formation of propioin from propionaldehyde was purified 270-fold from the crude enzyme in a yield of 28% by protamine sulfate precipitation, ammonium sulfate fractionation and G-200 gel chromatography using citrate-phosphate buffer (0.1 M Na₂HPO₄–0.02 M citric acid, pH 6.8, containing 0.33 mM MgSO₄, 0.1 mM thiamine pyrophosphate, 2.5 mM MnSO₄ and 30 mM β-mercaptoethanol). The purified enzyme was homogeneous on disc gel electrophoresis. It was most active at pH 6.8–7.0 and 37°C, and stable at pH 7–8 and below 45°C. Its activity was enhanced by FeSO₄·7H₂O, MnSO₄, thiamine pyrophosphate, β-mercaptoethanol, MgSO₄, CaCO₃, and NaCl, and inhibited by AgNO₃, HgCl₂, CuSO₄, ZnSO₄, SnCl₂, NH₄Cl, (CH₃COO)₂Pb·3H₂O, iodoacetic acid, FeCl₃·6H₂O, and (NH₄)₂SO₄. Its molecular weight was 96,000 by sedimentation equilibrium, and 100,000 by Sephadex G-200 column chromatography.

Six strains of methylotrophic yeast were examined for production of l-methionine-enriched cells. Candida biodinii (Kloeckera sp.) No. 2201, which accumulated 0.54 mg/g-dry cell weight (DCW) of free l-methionine (pool methionine), was selected as the parental strain for breeding l-methionine-rich mutants. Ethionine-resistant mutants were derived from the strain by UV irradation. A mutant strain, E500-78, which was resistant to 500 μg/ml of dl-ethionine, accumated 6.02 mg/g-DCW of pool methionine. The culture conditions for mutant strain E500-78 to increase pool methionine accumulation were o-ptimized. As a result, the mutant strain accumulated 8.80 mg/g-DCW of pool methionine and contained 16.02 mg/g-DCW total methionine.

The economic significance of increasing cell mass to high concentrations for metabolite production has been discussed theoretically. First, the direct cost of the metabolite production has been formulated as the sum of the raw material cost, the running cost, and the direct labor cost. By definition, the raw material cost is related to the metabolite yield from the nutrient consumed and the running cost is related to the productivity (or space-time yield) of the metabolite formed. Two characteristics of fed-batch culture at high cell mass concentration, i.e. the increase in the total volume of the culture broth and a definite volumetric occupation of wet cells in the total culture broth, have been taken into mathematical consideration.

• 1.

  (1) When the metabolite accumulates intracellularly, the direct production cost is minimized by obtaining the greatest amount of cells from a fixed amount of the nutrient consumed in the shortest operating time at which the cells have the highest content of the metabolite under consideration. In the two stage cultivation method (the early cell growth phase followed by the metabolite accumulation phase), an increase in cell mass concentration reduces the raw material cost only a little, but it does reduce the running cost.

• 2.

  (2) When the metabolite accumulates extracellularly in the culture supernatant, the raw material cost rises with increasing yield of the cell mass which is formed from a given amount of the raw material. The raw material cost cannot be lowered unless the metabolite yield is raised. The running cost tends to be reduced by increases in the cell mass concentration. In the two-stage cultivation method of extracellular metabolite production, the raw material cost increases with increasing cell mass concentration unless the metabolite yield at the second phase is predominant, while there is usually a particular cell mass concentration at which the running cost is minimized.

The syntheses of fatty acyl residues in a salt-tolerant yeast, Zygosaccharomyces rouxii, were compared under 4 growth conditions as follows, transfer from; (A) NaCl-free medium to the same medium, (B) NaCl-free medium to 2 M NaCl medium, (C) 2 M NaCl medium to the same medium, and (D) 2 M NaCl medium to NaCl-free medium. The synthesis of linoleyl residue 18:2) was depressed by the presence of 2 M NaCl while that of oleyl residue (18:1) was not. Under condition D, however, the synthesis of 18:2 recovered in the NaCl-free medium. These phenomena were confirmed through pulse-labeling and chase experiments. It is suggested that desaturation from 18:1 to 18:2 may be depressed by the presence of 2 M NaCl.

The biotransformation of commodity aromatic chemicals into dihydroxy derivatives was studied. A strain isolated from the invironment, Pseudomonas JI104, used benzene, toluene, and other hydrocarbons as sole carbon and energy sources. We selected mutants unable to grow with benzene, and among these, screened for strains with deficient cis-benzenglycol dehydrogenase able to stably produce cis-benzeneglycol when another carbon source was co-metabolized.

We exained the possibility of cis-benzeneglycol production by growing the mutant strain in the presence of benzene vapor. Ethanol was the carbon and energy source most adapted to the cis-benzeneglycol production phase, and lactate or propanol could also be used. Glucose inhibited the production of the metabolite.

The growth rates were barely affected by the presence of benzene at a reduced partial pressure (less than 20% of saturation), showing that continuous culture is possible. In a batch process, 0.54g·1⁻¹ of a cell suspension produced 5.1 mmol·1⁻¹cis-benzeneglycol in 27 h, using ethanol as the energy source.

Naringinases from both Penicillium sp. and Aspergillus niger were compared for their enzyme kinetics and the effects of sugars on the enzyme activities. Lineweaver-Burk plots showed that glucose, fructose and rhamnose were all competitive inhibitors for α-rhamnosidase of naringinase from Penicillium sp. and non-competitive inhibitors for the same enzyme from A. niger, When naringinase from Penicillium sp. was immobilized on chitin and used successively for the hydrolysis of p-nitrophenyl-α-rhamnoside or naringin in a simulated fruit juice system or grapefruit juice, it was observed that the enzyme column was very stable. Such results are in contrast to what has bee observed for naringinase fron A. niger. Therefore, it is quite possible that the sugars in the fruit juice which play a role as competitive or non-competitive inhibitors on naringinase may account for the stability of the enzyme column during successive debittering of grapefruits juice.