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  • 1
    ISSN: 1432-072X
    Keywords: Yeast flocculation ; Chemical modification of cell surface components ; Floc-forming ability ; Brewer's yeast ; Saccharomyces cerevisiae ; Deflocculation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Effects of treatments with proteolytic enzymes and protein-modifying reagents on flocculation of brewer's yeast IFO 2018 were investigated. The floc-forming ability of the yeast cells was irreversibly eliminated by treatment with papain, trypsin, chymotrypsin or pepsin, indicating that certain proteins on the cell surface participate in the yeast flocculation. Chemical modification with reagents, known to act on disulfide bridges, carboxyl and/or phosphate groups, phenolic groups, amino groups, and imidazole groups, also destroyed the ability to flocculate, although in some cases a high concentration (8 M) of urea was necessary in addition to protein-modifying reagents. Thus, it is suggested strongly that these functional groups of amino acid residues of the proteins are essential for the floc-forming ability of brewer's yeast cells.
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  • 2
    ISSN: 1432-072X
    Keywords: Pseudomonas denitrificans ; Denitrification ; Nitrite accumulation ; Formate ; Nitrite reductase ; Nitrate reductase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The growth of Pseudomonas denitrificans ATCC 13867 under denitrifying conditions was significantly stimulated by adding an appropriate amount of formate (2.5 mM or above) to the growth medium. The accumulation of nitrite in the culture was markedly depressed so long as formate remained in the culture above a certain level. Cellular activities of enzymes participating in denitrification also changed. The cells grown in the presence of formate exhibited a lower nitrate reductase activity and, in contrast, a higher nitrite reductase activity than the cells grown without added formate.
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  • 3
    ISSN: 1432-072X
    Keywords: Flocculation of brewer's yeast ; Flocculation of cell walls ; Metal ions ; Protein denaturants ; Enzyme treatment ; Deflocculation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Effects of metal ions, protein-denaturants and enzyme treatments on flocculation of cell walls of Beer Yeast IFO 2018 were investigated. Cell walls from flocculent cells grown in a complete medium were able to form flocs as were whole cells, but cell walls from non-flocculent cells, such as “Mg2+-deficient” cells, “early-phase” cells and “low-pH” cells, were not. The cell walls dispersed in distilled water reflocculated in solutions containing Ca2+ or other metal ions. Of the alkali metal ions tested, only Na+ inhibited flocculation of flocculent cell walls at a concentration more than 0.1 M. Ca2+ or Sn4+ was absolutely required for flocculation of cell walls in the physiological saline (NaCl, 150 mM), but the effect of Sn4+ seems rather non-specific, because it promoted flocculation of non-flocculent cell walls as well. Sr2+ and Ba2+ were antagonistic to Ca2+ and inhibited flocculation. Flocculation of cell walls was also depressed by high concentrations of protein-denaturants, e.g. urea and guanidine·HCl. Treatment with proteolytic enzymes deprived cell walls of floc-forming ability. Effect of metal ions, protein-denaturants and treatment with enzymes on the flocculation of intact cells was investigated as control. Since flocculating properties of cell walls were very similar to those of intact cells, flocculation must be an inherent property of cell walls.
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  • 4
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary Whole cells of Corynebacterium sp. having steroid 9α-hydroxylation system were immobilized by entrapment with photo-crosslinkable resin prepolymers, urethane prepolymers or several kinds of polysaccharides. Of various entrapment methods tested, cells entrapped in photo-crosslinked gels showed the highest activity to hydroxylate 4-androstene-3,17-dione at 9α-position. The properties of the photo-crosslinkable resin prepolymers, such as the hydrophobicity and the chain length of the prepolymers, affected markedly the activity of the entrapped cells. Addition of dimethyl sulfoxide to a buffer system at 15 vol. % was effective to solubilize the product, 9α-hydroxy-4-androstene-3,17-dione, and gave the highest yield. In an aqueous system, the activity of hydrophilic gel-entrapped cells was higher than that of hydrophobic gel-entrapped cells. In 15% of dimethyl sulfoxide, the hydrophobic gel-entrapped cells showed almost the same activity as the hydrophilic gel-entrapped cells probably due to extraction of the product from gels to the external solvent before its metabolic degradation. Entrapment significantly enhanced the conversion ratio at high substrate concentrations and the operational stability of the 9α-hydroxylation system in the cells. In the presence of nutrients in the reaction mixture, entrapped growing cells maintained fully the original activity at least during 10 times of repeated batch reactions for 5 days.
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  • 5
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary Two-step bioconversion of cortexolone (Reichstein's Compound S) to its Δ 1-dehaydro-11β-hydroxy derivative, prednisolone, was successfully performed by the combined use of immobilized Curvularia lunata mycelia and immobilized Arthrobacter simplex cells. Immobilized living mycelia of C. lunata having a high 11β-hydroxylation activity were prepared by in situ germination of spores entrapped in photo-crosslinked resin gels of a suitable net-work structure. Acetone-dried cells of A. simplex having an induced steroid Δ 1-dehydrogenase activity were also entrapped with photo-crosslinkable resin prepolymers and used for Δ- dehydrogenation of hydrocortisone to prednisolone. For the production of prednisolone from cortexolone, the combination of sequential steps, 11β-hydroxylation and subsequent Δ 1-dehydrogenation, was found to be suitable. Each immobilized microbial cell system was stable and could be used for the sequential reactions repeatedly (operational period, 25 days).
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  • 6
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary The effects of cerulenin, an anti-lipogenic antibiotic, on the growth and cellular fatty acid composition ofCandida lipolytica were investigated by changing the chain length of n-alkane, the growth substrate. The antibiotic inhibited almost completely the growth of the yeast on glucose, n-undecane and n-dodecane, but partly that on n-tridecane. The yeast growth on longer alkanes, e.g., from n-tetradecane to n-octadecane, was not affected by this antibiotic, indicating that a chain elongation system and/or intact incorporation system predominantly operate in the formation of cellular fatty acids from such longer chain n-alkanes. Comparison of the fatty acid profiles between the cells grown on n-alkanes of different chain lengths, especially on n-pentadecane, in the presence and absence of cerulenin, supported the supposition that only the de novo synthesis system of the yeast would be affected by the antibiotic, whereas the chain elongation system would not.
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  • 7
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary Attempts were made to entrap enzymes or microbial cells with water-insoluble photo-crosslinkable resin prepolymers of different types in organic solvent systems in the presence or absence of water. Acetone-dried cells of Artbrobacter simplex immobilized in a maleic polybutadiene gel (PBM-2000) converted hydrocortisone to prednisolone in a phosphate buffer. 4-Androstene-3,17-dione was converted to androst-1,4-diene-3,17-dione in benzene-n-heptane solution by Nocardia rhodocrous which was immobilized by a hydrophobic prepolymer, ENTP-2000. The ENTP-2000 had been synthesized from poly(propylene glycol)-2000, hydroxyethylacrylate and isophorone diisocyanate. Even enzymes catalyzing aqueous phase reactions, such as catalase and invertase, were immobilized in a polybutadiene resin (PB-200k) to give active gel-entrapped preparations. The cells and enzymes immobilized in these hydrophobic resins exhibited moderate activities compared with those of the free cells and enzymes.
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  • 8
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary Microbial cells and cellular organelles were immobilized by mixing aqueous suspensions of the biocatalysts with water-miscible urethane prepolymers. Thus immobilized preparations of acetone-dried cells of Arthrobacter simplex and thawed cells of Nocardia rhodocrous showed appreciable {ie351-1} activities in the transformation of hydrocortisone into prednisolone and 4-androstene-3,17-dione to androst-1,4-diene-3,17-dione, respectively. The activities of catalase and alcohol oxidase were observed in the immobilized peroxisomes (microbodies) of a methanol-grown yeast Kloeckera sp. No. 2201. Yeast mitochondria entrapped with the prepolymer showed adenylate kinase activity. These results indicate the usefulness of the urethane prepolymers as convenient materials for entrapment of not only enzymes, but also organelles and microbial cells.
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  • 9
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary Production of L-tryptophan, L-tyrosine, or their analogues was attempted using immobilized tryptophanase or β-tyrosinase. The immobilized tryptophanase used in this study was first prepared by the present authors by coupling of free apoenzyme fromEscherichia coli B/1t-7A to pyridoxal 5′-phosphate (PLP) previously bound on Sepharose. This immobilization method involves the formation of Schiff base linkage between 4-formyl group of Sepharose-bound PLP and the α-amino group of the lysine residue of the catalytic center of one subunit of tetrameric apotryptophanase, followed by reductive fixation of the Schiff base linkage with NaBH4. In the case of β-tyrosinase fromEscherichia intermedia having two catalytic centers, however, immobilization by direct coupling to CNBr-activated Sepharose or a bromoacetyl derivative of Sepharose was more suitable than by the coupling to Sepharose-bound PLP. In each case, the affinity for substrate or coenzyme was scarcely affected by the immobilization. The immobilized enzymes thus obtained were shown to possess higher thermal stability and higher resistance to denaturing agents than the free counterparts. The optimal temperature for a short time reaction (10 min) was ca. 70°C for immobilized tryptophanase or 55°C for immobilized β-tyrosinase. In each case the optimal reaction temperature mediated by the immobilized enzyme was fairly higher than that catalyzed by the respective free enzyme. Addition of ethanol (5%, V/V) to the reaction mixtures favored the tryptophanase and β-tyrosinase reactions. The equilibrium of α, β-elimination reactions of L-tryptophan and β-tyrosine lied so far to the synthetic side (70% in tryptophanase and 80% in β-tyrosinase reactions, respectively). By continuous flow methods using these immobilized enzyme columns, L-tryptophan, L-tyrosine, and their analogues, such as L-DOPA and L-5-hydroxytryptophan, were conveniently synthesized in good yields.
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  • 10
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary The effects of vitamin B12 on the growth and mitomycin biosynthesis ofStreptomyces caespitosus were studied. In a medium lacking cobalt ion and vitamin B12, both the growth and the antibiotic formation were very low. Addition of vitamin B12 to the medium markedly enhanced both yields. Methionine added to the medium also showed the same effect as the vitamin. Studies on the effects of vitamin B12 and methionine indicated that vitamin B12 does not participate directly in the methyl incorporation into the mitomycin molecule but might contribute to the antibiotic biosynthesis as a result of its involvement in methionine formation. An enzymatic system was found in cell-free extracts ofS. caespitosus which catalyzed the transfer of the methyl group from methionine into Compound II-f, a demethylated derivative of mitomycin obtained by Webb et al. (1962). The methylated product was characterized as mitomycin A or its closely related substance from its antibacterial and chromatographic properties.
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