• Korean Journal of Microbiology
• /
• 제37권2호
• /
• pp.164-169
• /
• 2001

The conversion of cephalosporin C to 7$\alpha$-hydroxycephalosporin C was examined with the cell-free extract of several cephamycin producing strains. Streptomyces clavuligerus ATCC 27064 was the most potent strain for the activity of cephalosporin 7$\alpha$-hydroxylase. Partially purified and immobilized cephalosporin 7$\alpha$-hydroxylase with resins were used to synthesize 7$\alpha$-hydroxycephalosporin C from the substrate, cephalosporin C. The molecular weight of the product isolated from the reaction mixture were determined to be 431 by ESI-Mass. $^1H$ NMR also support the conversion of cephalosporin C to 7$\alpha$-hydroxycephalosporin C by immobilized enzyme.

• Microbiology and Biotechnology Letters
• /
• 제20권2호
• /
• pp.178-182
• /
• 1992

For an industrial-scale purification and production of cephalosporin C from a culture broth of Ceplzalos#mium nmemonium CSA-2.8A3 mutant, ultrafiltration, column chromatography, reverse osmosis, and spray drying were empolyed. Above 90% of yield and high purity of cephalosporin C were obtained through WA-30, HP-20, XAD-2000 and SK-1B column chromatographies. Especially, in the tendom operation of the columns, the recovery yield was increased up to 96%. The purified cephalosporin C was stable at $4^{\circ}C$ and in acidic condition, while it was unstable at room temperature and in alkaline condition at pH above 8.0. Cephalosporin C powder or a final product prepared by spray drying contained 85.554 of sodium cephalosporin C, 6.3%' of water, 4.63% of free $Na^+$ ions. and traces of metal ions.

• Microbiology and Biotechnology Letters
• /
• 제23권5호
• /
• pp.559-564
• /
• 1995

Twenty microbial strains producing the acylase were isolated from soil by using Micrococcus luteus ATCC 9341 as an indicator strain, using either D-($\alpha $)-phenylglycine methylester and 7-aminocephalosporanic acid (7-ACA) or glutaric acid dimethylester and 7-ACA as substrates. Among the isolates, only one strain was turned out to be the 7-ACA producer from either cephalosporin C or glutaryl 7-ACA as the substrates by using the overlay of 7-ACA sensitive strain (SS5). 7-ACA produced from cephalosporin C by an isolate (APS20) was detected by high performance liquid chromatography. The isolated strain (APS20) was identified to Bacillus macerans on the basis of cellular fatty acid profile by gas chromatography. Bacillus macerans APS20 had no $\beta $-lacta-mase activity on cephalosporin C, and that is very important for the enzymatic production process of 7-ACA. However, this strain was resistant up to 100 $\mu $g/ml of cephalosporin C.

• Microbiology and Biotechnology Letters
• /
• 제19권5호
• /
• pp.521-535
• /
• 1991

A kinetic model incorporating cell morphology in cephalosporin C biosynthesis by Cephalosporium amemoniurn was developed. The double-substrate Double-substrate kinetic model was used to describe cell growth. Methionine controlled the rate of growth while glucose ultimately controlled the extent of growth. The changes in specific product formation rate were associated with morphologenesis, especially cell differentiation. To increase the productivity of cephalosporin C, the proposed model equations were applied to a fed-batch culture. The algorithm to optimize the fed-batch culture consists of two steps; cell growth was maximized in the growth phase and then cephalosporin C production was maximized in the production phase. The increase of about 33% in the cephalosporin C titre was obtained by the optimal feeding scheduling in comparison with that of batch culture.

• Microbiology and Biotechnology Letters
• /
• 제23권5호
• /
• pp.556-558
• /
• 1995

A strain which showed cephalosporin C resistance and 7-aminocephalosporanic acid sensitivity was isolated from nature. Among the isolates, SS5 was sensitive to cephalosporin C, penicillin G, ampicillin, 7-aminocephalosporanic acid, 6-aminopenicillanic acid, and 7-aminodeacetoxy cephatosporanic acid at concentrations of 1,000 $\mu $g/ml, 2,000 $\mu $g/ml, 3,000 $\mu $g/ml, 30 $\mu $g/ml 100 $\mu $g/ml and 100 $\mu $g/ml, respectively. But SS5 was sensitive at very low concentration of chloramphenicol, kanamycin, neomycin, streptomycin and tetracycline. Since SS5 was sensitive to 7-ACA (30 $\mu $g/ml) and didn't have $\beta $-lactamase activity on the cephalosporin C, SS5 could be useful as an indicator strain for the production of 7-ACA, which is an important precursor for the synthesis of many semisynthetic cephalosporins.

• Applied Chemistry for Engineering
• /
• 제10권4호
• /
• pp.563-567
• /
• 1999

Reverse osmosis concentration for cephalosproin C was studied using a polyamide composite membrane, FT-30 in spiral wound type with high solute rejection. The experiments were carried out in the aqueous solution of cephalosporin C for water flux, solute rejection and mass transfer coefficient under applied pressure of $4{\sim}20kg/cm^2$, feed concentration of 100~1000 mg/L and feed velocity of 2.8 and 5.6 L/min at room temperature. The effect of operating pressure on the separation of cephalosporin C showed that permeate flux increased with increasing operation pressure. These results are consistent with those predicted by Kedem-Katchalsky model. Solute rejection was nearly 1. The increase of feed concentration caused the reduction of cephalosporin C rejection, which was higher at low concentration than at high concentration, but degree of reduction was small.

• KSBB Journal
• /
• 제14권2호
• /
• pp.235-240
• /
• 1999

Simple and rapid screening methods were developed to screen mutant strains of Trigonopsis variabilis ATCC10679 (TW). D-amino acid oxidase (D-AAO) from a mutant strain, T26, showed about 30% higher specific activity against cephalosporin C than from its wild type, TW. D-AAO genes from both TW and T26 strains were cloned and sequenced. There was one nucleotide changed from T to C at 811 position, resulting in an amino acid codon changed from Phe-258 to Ser-258.

• BMB Reports
• /
• 제34권3호
• /
• pp.274-277
• /
• 2001

Lipase of Staphylococcus sp. was purified from the culture supernatant, and its molecular mass estimated to be 44 kDa by SDS-PAGE. Its optimum temperature and pH for the hydrolysis of p-nitrophenyl substrates was $28^{\circ}C$ and pH 8.5, respectively The gene encoding the lipase was cloned in Escherichia coli in the $NH_2$-teminally truncated form by using the shotgun method, and sequenced. The mature enzyme had a 49-93% amino acid sequence homology with other staphylococcal lipases. This lipase was used for the hydrolysis of the 3-O-acetate of cephalosporin-C to give an intermediate, deacetylated cephalosporin-C that is useful for further chemical elaborations.

• Microbiology and Biotechnology Letters
• /
• 제32권3호
• /
• pp.224-229
• /
• 2004

Several microorganisms (esterase-producing group) were isolated by the solid selective media containing-naphtylacetate. Among them, strain 366M-9 having a high activity of cephalosporin-C deacetylase (CAH; EC 3.1.1.41) was selected. The strain 366M-9 was identified as Bacillus sphaericus on the basis of morphological, physiological, and biochemical characteristics. The production of CAH reached at maximum value after 32 hrs, when cultivated in the optimal medium containing dextrin 2.5%, peptone 2.5%, sodium chloride 0.5%, dipotassium phosphate 0.25%, ferrous sulfate 0.02%, and 7-ACA 0.1% at $30^{\circ}C$ with initial pH 6.0. The CAH was purified by 3 steps with ammonium sulfate precipitation, adsorption chromatography on hydroxyapatite column, and Sephadex G-200 gel chromatography. The final enzyme preparation was homogeneous as judged by the analysis of SDS-PAGE and HPLC. Optimum temperature and pH for CAH activity were $50{\circ}C$ and around 7.0, respectively. And the enzyme was stable at pH 6.0～8.0, up to $50^{\circ}C$. The Michaelis-Menten constants ($K_{m}$ ), $V_{max}$ were 0.87 mM and 1.22 unit/ml, respectively.

• Microbiology and Biotechnology Letters
• /
• 제27권2호
• /
• pp.96-101
• /
• 1999

Various side-chains are introduced to the 7-amino position of 7-aminocepha-losporanic acid (7-ACA) to make semi-synthetic cephalosporin antibiotics. In order to convert cephalosporin C (CPC) to 7-ACA, two enzymatic reactions are generally imployed. Glutary1-7-aminocephalosporanic acid (Gl-7-ACA) acylase is involved in the second step where the reaction intermediate, Gl-7-ACa is converted into 7-ACA. It was recently reported that CPC amidase can convert CPC directly into 7-ACA in a single enzymatic reaction. A study was undertaken to screen microorganisms conferring enzyme activity to convert Gl-7-ACA or CPC into 7-ACA by one or two enzymatic reactions. In order to screen the microorganisms rapidly, a non-$\beta$-lactam model compund, glutaryl-$\rho$-nitroanilide, was utilized in an early stage, thereafter the selected microorganisms were examined with real substrates. One microorganism exhibiting both Gl-7-ACA acylase and CPC amidase activities was obtained by the colorimetry method and HPLC assay, and was identified as a strain of Serratia species, designated as Serratia sp. N14.4. The optimal fermentation conditions for Serratia sp. N14.4 was pH9.0 and 3$0^{\circ}C$.