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Biomedical subjects

D D Ryu

Publications and source records attributed to D D Ryu.

At least 37 records · Page 2Linked to original sources
PubMed1986

Construction of lactose-assimilating and high-ethanol-producing yeasts by protoplast fusion.

The availability of a yeast strain which is capable of fermenting lactose and at the same time is tolerant to high concentrations of ethanol would be useful for the production of ethanol from lactose. Kluyveromyces fragilis is capable of fermenting lactose, but it is not as tolerant as Saccharomyces cerevisiae to high concentrations of ethanol. In this study, we have used the protoplast fusion technique to construct hybrids between auxotrophic strains of S. cerevisiae having high ethanol tolerance and an auxotrophic strain of lactose-fermenting K. fragilis isolated by ethyl methanesulfonate mutagenesis. The fusants obtained were prototrophic and capable of assimilating lactose and producing ethanol in excess of 13% (vol/vol). The complementation frequency of fusion was about 0.7%. Formation of fusants was confirmed by the increased amount of chromosomal DNA per cell. Fusants contained 8 x 10 to 16 x 10 mug of DNA per cell as compared with about 4 x 10 mug of DNA per cell for the parental strains, suggesting that multiple fusions had taken place.

Journal Article↗
PubMed1985

Relationship between butirosin biosynthesis and sporulation in Bacillus circulans.

The relationship between butirosin biosynthesis and certain biochemical characteristics related to sporulation in a strain of Bacillus circulans NRRL B-3313 was examined. The cellular content of dipicolinic acid increased while the amount of poly-beta-hydroxybutyrate decreased with changes in antibiotic productivity. Oligosporogenous mutants failed to synthesize the antibiotic and to degrade poly-beta-hydroxybutyrate. These observations suggest that spore formation may be related to antibiotic production in this strain of B. circulans.

Anti-Bacterial Agents↗
PubMed1984

An improved synthesis of captopril.

An improved synthesis of captopril using methacrylic acid as the starting material is described. Treatment of methacrylic acid (I) with a hydrogen halide gave the 3-halogeno-2-methylpropanoic acids II and III, which were treated with thionyl chloride to yield the corresponding 3-halogeno-2-methylpropanoyl chlorides IV and V. Treatment of IV or V with L-proline yielded the N-(R,S-3-halogeno-2-methylpropanoyl)-L-prolines VI and VII, which were separated into optically pure R- and S-diastereoisomers using dicyclohexylamine. Treatment of halides of VI or VII with methanolic ammonium hydrosulfide gave captopril in 28% yield.

Captopril↗
PubMed1984

Enzymatic synthesis of phenoxymethylpenicillin using Erwinia aroideae enzyme.

Enzymatic synthesis of phenoxymethylpenicillin from 6-aminopenicillanic acid and phenoxyacetic acid methyl ester was attempted by using partially purified alpha-acylamino-beta-lactam acylhydrolase I (ALAHase I) enzyme from Erwinia aroideae NRRL B-138. The reaction rates were carefully followed by determination of 6-aminopenicillanic acid (6-APA), phenoxymethylpenicillin (PNV), phenoxyacetic acid (POA), phenoxyacetic acid methyl ester (POM), and phenoxyacetylglycine (POG) using high performance liquid chromatography. Among the acyl donors tested, POM gave the highest yield (12.2% based on 6-APA). The overall conversion increased almost linearly with an increase in molar ratio of POM to 6-APA up to 4:1. The effects of organic solvents on the overall yield were also evaluated. Some improvement of PNV yield was observed when ethanol, 2-propanol, and acetone were used. ALAHase I was found to carry out three reactions simultaneously: transfer of acyl group to acyl acceptor to form semisynthetic beta-lactam antibiotic; hydrolysis of acyl donor in amide or ester bond, and hydrolysis of semisynthetic beta-lactam antibiotic which was produced by the enzyme. It was also observed that the hydrolysis reactions of POM and PNV were irreversible in this reaction system. The optimal pH for the three reactions was different. They were: pH 9.0 for POM hydrolysis, 6.8 for the transfer of phenoxyacetyl group to 6-APA, and 6.0 for the PNV hydrolysis. The apparent Km values for POM, 6-APA and PNV were estimated as 33, 25 and 31 mM, respectively.

Erwinia↗
PubMed1983

Production of rosamicin: improvement of synthetic medium.

Rosamicin is one of the important macrolide antibiotics that has clinical efficacy and broad-spectrum antibacterial activity. Using a mutant strain of Micromonospora rosaria (NRRL 3718), a chemically defined medium was developed, and some fermentation conditions that are important to rosamicin biosynthesis were optimized to achieve rosamicin productivity of 230 mug/ml. Soluble starch and l-asparagine were found to be the best carbon and nitrogen sources, and a stimulative effect of magnesium and zinc ions was also found. The medium developed contains: soluble starch, 4%; l-asparagine, 0.15%; K(2)HPO(4), 0.075%; CaCO(3), 0.6%; MgSO(4) . 7H(2)O, 0.05%; FeSO(4) . 7H(2)O, 10 M; CuSO(4) . 5H(2)O, 10 M; ZnSO(4) . 7H(2)O, 10 M; and MnSO(4) . (4-6)H(2)O, 10 M. The required air supply was about 40 mmol of O(2) liter . h . atm, and the favorable culture temperature was 28 to 29 degrees C.

Journal Article↗
PubMed1983

Genetic recombination in Micromonospora rosaria by protoplast fusion.

Auxotrophic strains of Micromonospora rosaria were isolated by N-methyl-N'-nitro-N'-nitrosoguanidine mutagenesis and used in intraspecific recombination by protoplast fusion. High-frequency fusion of protoplasts of M. rosaria strains was induced by polyethylene glycol (molecular weight, 1,000) (PEG 1,000). The optimum concentration of PEG 1,000 for fusion of M. rosaria was 50% (wt/vol). PEG 4,000 was slightly better than PEG 1,000 at concentrations lower than 50% (wt/vol). The recombinant frequency did not increase after treatment with PEG 1,000 (50% [wt/vol]) for longer than 20 min. Under these conditions, fusion with many auxotrophic strains of M. rosaria resulted in a high frequency of formation of true recombinants (sometimes more than 10%). Additionally, when ros (rosamicin nonproducing) strains were crossed by protoplast fusion; about 5% of the resultant prototrophic recombinants were shown to have the ros+ (rosamicin producing) characteristic restored. Rosamicin production by M. rosaria colonies was clearly distinguished by the broth overlay method. The results of fusion experiments between ros and ros+ strains indicated that either the chromosomal mutation or pleiotrophic effect of some auxotrophic markers is involved.

Genes, Bacterial↗
PubMed1983

Mutagenesis of Micromonospora rosaria by using protoplasts and mycelial fragments.

Both mycelial fragments and protoplasts were successfully employed for mutagenesis of Micromonospora rosaria NRRL 3718, and the results were compared. The optimal conditions and effective procedures for mutagenesis of M. rosaria by a chemical mutagen, N-methyl-N'-nitro-N-nitrosoguanidine, have been determined. Mutation was efficiently induced when mycelial fragments were treated with N-methyl-N'-nitro-N-nitrosoguanidine at a concentration of 0.3 to 0.5 mg/ml in the reaction buffer of pH 7.0. Optimal treatment time was 20 to 40 min. Ampicillin treatment was very effective for enrichment of auxotrophs. Protoplasts showed much higher sensitivity to the lethal effect of N-methyl-N'-nitro-N-nitrosoguanidine. Although protoplasts have some advantage of single cell characteristics, the frequency of auxotrophs obtained was somewhat lower. Up to 4% of the colonies were shown to be auxotrophs under the well-defined conditions. This mutagenesis method with protoplasts or fragmented mycelia (or both) should be applicable to other actinomycetes that have limited or no sporulation.

Cell Fractionation↗
PubMed1980

Enzymatic biosynthesis of cephalexin.

The reaction kinetics of the enzymatic synthesis of cephalexin from 7-aminodeacetoxy cephalosporanic acid and phenylglycine methylester was studied using the synthesizing enzyme obtained from Xanthomonas citri. The activation energy, Km values for 7-aminodeacetoxy cephalosporanic acid and phenylglycine methylester, and Ki value for phenylglycine methylester were determined as 8.63 kcal/mol, 3.7mM, 14.5mM, and 70mM, respectively. The enzyme was found to be constitutive and susceptible to deactivation.

Amidohydrolases↗
PubMed1979

Biochemical properties of penicillin amidohydrolase from Micrococcus luteus.

Some biochemical properties of whole-cell penicillin amidohydrolase from Micrococcus luteus have been studied. This whole-cell enzyme showed its maximal activity at 36 degrees C at pH 7.5. It was found that the activation energy of this enzyme was 8.03 kcal (ca. 33.6 kJ) per mol, and this amidohydrolase showed first-order decay at 36 degrees C. The penicillin amidohydrolase was deactivated rapidly at temperatures above 50 degrees C during storage or preincubation for 24 h. The Michaelis constant, Km, for penicillin G was determined as 2.26 mM, and the substrate inhibition constant, Kis, was 155 mM. The whole-cell penicillin amidohydrolase from M. luteus was capable of hydrolyzing penicillin G, penicillin V, ampicillin, and cephalexin, but not cephalosporin C and cloxacillin. This whole-cell enzyme also had synthetic activity for semisynthetic penicillins or cephalosporins from D-(--)-alpha-phenylglycine methyl ester and 6-alpha-aminopenicillanic acid or 7-amino-3-deacetoxycephalosporanic acid.

Amidohydrolases↗
PubMedDate not supplied

High cell density culture of Yarrowia lipolytica using a one-step feeding process.

Yarrowia lipolytica is a potentially useful host for heterologous protein production. To develop an efficient culture method for high cell density cultivation and heterologous gene expression of Y. lipolytica, the effects of medium components and their concentrations on the growth of Y. lipolytica have been investigated. Addition of yeast extract to the culture media was found to significantly reduce the long lag phase encountered when Y. lipolytica was cultivated in synthetic culture media containing high concentrations of glycerol. Therefore, by enriching with 0.3% yeast extract the synthetic culture medium containing 15% glycerol, we could cultivate Y. lipolytica up to 83 g/L dry cell weight in a batch culture. Furthermore, over 100 g/L and 88 units/mL of rice alpha-amylase activity were obtained in less than 50 h with a one-step feeding process in which a recombinant Y. lipolytica expressing rice alpha-amylase was cultivated in the 10% glycerol medium enriched with 0.3% yeast extract and fed only once with the concentrated feeding medium (60% glycerol). The easy cultivation of recombinant Y. lipolytica to a high cell density may strengthen its position as a host for heterologous protein production.

Culture Media↗
PubMedDate not supplied

Optimal temperature control policy for a two-stage recombinant fermentation process.

The optimal temperature control policy to be followed in the operation of a two-stage fermentation system in which gene expression is induced by a temperature-sensitive gene switching system was studied. A genetically structured model was used to describe product formation, and kinetic equations based on experimental data were used to quantify the specific gene expression rate and parameters that affect plasmid instability. A constant temperature control policy and temperature profiling control policy including temperature cycling were studied and compared. Maximum average production rate was obtained from a temperature control policy in which the second stage was operated initially at about 40.5 degrees C and the temperature decreased slightly to a constant value at 40.0 degrees C. The maximum average production rate, which corresponds to the optimal temperature control policy, for an operation of 180 h was 29.7 units of protein (mg of cells)-1 h-1.

DNA, Recombinant↗
PubMedDate not supplied

Propagation of recombinant vaccinia virus in HeLa cells: adsorption kinetics and replication in batch cultures.

The influence of various culture parameters on infection and replication of recombinant vaccinia virus in HeLa cells was examined during various phases of viral replication. A modified form of the model of Valentine and Allison (Biochim. Biophys. Acta 1960, 40, 393-399) model was used to predict successfully the viral adsorption rates in cell suspensions. An experimentally determined aggregation factor, epsilon, was included in the model to account for deviations of the observed adsorption rates from those predicted by the earlier model. It was also shown that the ionic strength, ionic species, and serum proteins present in the medium significantly altered the adsorption kinetics of the virus. The lysosomotropic base chloroquine was found to enhance viral infection more than 2-fold during the penetration step of viral infection. It was also demonstrated that cells infected during the exponential growth phase gave higher viral yields than those infected during the lag or stationary growth phases and the initial viral MOI did not significantly alter viral yields. Finally, it was demonstrated that viral infection of HeLa cells grown in 4-L bioreactor batch cultures resulted in increased death and glucose uptake rates and significantly lower growth rates.

Cells, Cultured↗
PubMedDate not supplied

Studies on scale-up parameters of an immunoglobulin separation system using protein A affinity chromatography.

Effects of operational and system parameters on process scale-up of murine immunoglobulin (IgG2a kappa) purification using Protein A affinity chromatography are investigated. Parameters studied are those related to sample application, elution, ligand concentration on support, and column size change. Between sample application velocities of 0.004 and 0.030 cm/s (16-108 cm/h), the product concentration profiles in eluate did not show significant differences. With given system parameters, the retention time and bandwidth of the peak could be predicted by moment theory. The mean equilibrium constant during elution showed a strong effect of pH between 2 and 4. Within the range of protein A concentrations studied, 0.15-1.22 mg/mL of gel, the column capacity shows a linear relationship with the concentration of protein A immobilized. Dimensional scale-up of the column in the radial direction increased the total purification capacity linearly as the cross-sectional area increased without increasing pressure drop; however, the product concentration was diluted. Scale-up of the column dimension in the axial direction enables higher concentrations of product in the eluate, although the retention time increases linearly as the gel height increases.

Adsorption↗
PubMedDate not supplied

Effects of the par locus on the growth rate and structural stability of recombinant cells.

A decreased growth rate of recombinant cells is observed when a cloned gene protein encoded in a multicopy plasmid is induced from a strong promoter. This negative effect on the cell growth rate may be the consequence of alternate use or reallocation of energy, precursor metabolites, and protein synthesizing machinery. In order to analyze the causes for this adverse effect, we have studied how genetic elements present in multicopy plasmids affect the growth of recombinant Escherichia coli (K12 delta H1 delta trpEA). Turning on of the PL promoter, whether or not protein-coding sequences were present downstream of the promoter, decreased the growth of the recombinant cells by 15-50%. This result suggests that the essential factor(s) for cell growth may be sequestered by the genetic elements present in the plasmids and thus may cause the decreased growth. The negative effect of the de-repressed PL promoters on cell growth was partially reversed when the par sequence from pSC101 was inserted into the same plasmid. In each case when the plasmid carried only the PL promoter, the PL promoter followed by the N-terminal part of the protein-coding region, or the PL promoter followed by complete sequences encoding a protein, the par sequence exhibited a positive effect on the growth rate of the recombinant cells, which usually grow at a slower rate than the host cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗