• Journal of Microbiology and Biotechnology
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• 제6권5호
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• pp.336-339
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• 1996

Glutaryl 7-aminocephalosporanic acid acylase of Pseudomonas sp. SY-77-1 was immobilized with oxiran acrylic beads for the production of 7-aminocephalosporanic acid (7-ACA) from glutaryl 7-aminocephalosporanic acid (GL 7-ACA). The immobilized enzyme maintained its activity at a constant level for 7 days, but lost 30$%$ of its activity after 20 days. Optimal reaction conditions for the synthesis of 7-ACA were found to be $30^{\circ}C$ and pH 8.0 using the immobilized enzyme. For the economic production of 7-ACA, substrate and enzyme concentrations were optimized to 60 mM and 0.5 g wet weight per 10 $m\ell$ of reaction volume, respectively. Under optimized conditions, 50 mM 7-ACA was produced from 60mM GL 7-ACA within 8 h, resulting in a conversion yield of 83$%$.

• Journal of Microbiology
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• 제37권4호
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• pp.200-205
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• 1999

7-Aminocephalosporanic acid (7-ACA) is the initial compound in preparation of cephalosporin antibiotics widely used in clinical treatment. Bacteria producing glutaryl 7-ACA acylase, which convert cephalosporin C to 7-ACA, has been screened in soil samples. A bacterial strain exhibiting high glutaryl 7-ACA acylase activity, designated KAC-1, was isolated and identified as a strain of Pseudomonas diminuta by characterizing its morphological and physiological properties. The screening procedures include culturing on enrichment media containing glutaric acid, glutamate, and glutaryl 7-aminocephalosporanic acid as selective carbon sources. To enhance enzyme production, optimal cultivation conditions were investigated. This strain grew optimally at pH 7 to 9 and in temperatures of 20 to 40 C, but acylase production was higher when the strain was grown at 25 C. Glutaric acid, glutamate and glucos also acted as inducers for acylase production. In a jar fermenter culture, P. diminuta KAC-1 produce acylase in a growth-associated manner. The substrate specificity of KAC-1 acylase by cell extract showed that this enzyme had specificity toward glutaryl 7-ACA, glutaryl 7-ADCA, but not cephalosporin C.

• 한국미생물·생명공학회지
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• 제23권5호
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• pp.556-558
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• 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.

• Biotechnology and Bioprocess Engineering:BBE
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• 제2권2호
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• pp.105-108
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• 1997

A search was undertaken to screen microorganisms that produce an enzyme capable of deacylating glutary1-7-amincephalosporanic acid to 7-aminocephalosporanic acid in soil samples. The screening was carried out by preparing enrichment cultures containing glutary-7ACA and cephalosporin C as selective carbon sources. A non-${\beta}$-lactam model compound,, glutary-p-nitroanilide, was synthesized as a substrate suitable for the rapid screening of microorganisms isolated from the enrichment cultures. Two isolates exhibiting acylase activity, designated BY7.4 and BY8.1, were identified as strains of Pseudomonas species. Pseudomonas BY8.1 showed higher acylase activity toward G1-7ACA than Pseudomonas BY7.4. Environmental conditions for the optimal acylase activity of Pseudomonas BY8.1 were shown to be pH9 and 30$^{\circ}C$.

• 공업화학
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• 제8권4호
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• pp.700-703
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• 1997

Phthalic anhydride를 출발물질로 halogenation, phosphorylation하여 diethyl phthalimidoalkylphosphonate를 합성하였다. 이 화합물을 chlorination하여 O-ethyl phthalimidoalkylphosphonate을 만든후 diphenylmethyl 7-$\beta$-amino-3-acetoxymethyl-3-cephem-4-carboxylate와 coupling하여 지금까지 알려져 있지 않은 화합물 diphenylmethyl-7-$\beta$-(O-ethylphthalimidomethylphosphony1)-3-acetoxymethyl-3-cephem-4-carboxylate와 diphenylmethyl-7-$\beta$-[O-ethylphthalimidoethylphosphonyl]-3-acetoxymethyl-3-cephem-4-carboxylate를 각각 19%, 43%의 수율로 합성하였다.

• Journal of Microbiology and Biotechnology
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• 제11권3호
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• pp.452-457
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• 2001

The glutaryl 7-aminocephalosporanic acid (glutaryl 7-ACA) acylase was purified from Pseudomonas diminuta KAC-1 cells isolated from soil, and characterized. The acylase was purified by procedures including ammonium sulfate fractionation and column chromatographies on DEAE-Sepharose, Phenyl-Sepharose, Q-Sepharose, and Superose 12H/R. The negative acylase was found to be composed of two subunits with molecular masses of approximately 55 kDa and 17 kDa, respectively. The isoelectric point of the enzyme was 4.0. The specific activities of the purified acylase were 8.0 and 7.0 U/mg on glutaryl 7-ACA and glutaryl 7-aminodesacetoxy cephalosporanic acid (glutaryl 7-ADCA), respectively, and $K_m$ values were 0.45 mM for glutaryl 7-ADCA and 0.67 mM for glutaryl 7-ADCA. The enzyme had a pH optimum at 8.0 and a tmperature optimum at $40^{\circ}C$. The acylase catalyzed the synthesis of glutaryl 7-ACA from glutaric acid and 7-ACA as well as the hydrolysis of glutaryl 7-ADCA, although the reaction rate of the synthesis was slower than that of the hydrolysis. In addition, it was found that the enzyme had a glutaryl transferase activity, thereby transferring the glutaryl group from one cephalosporin nucleus to another.

• 한국미생물·생명공학회지
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• 제23권5호
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• pp.559-564
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• 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.

• 한국미생물·생명공학회지
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• 제27권2호
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• pp.96-101
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• 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$0^{\circ}C$.

• Biotechnology and Bioprocess Engineering:BBE
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• 제10권6호
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• pp.510-515
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• 2005

Pseudomonas cepacia BY21 was found to produce glutaryl acylase that is capable of deacylating glutaryl-7-aminocephalosporanic acid (glutaryl-7-ACA) to 7-aminocephalosporanic acid (7-ACA), which is a starting material for semi-synthetic cephalosporin antibiotics. Amino acids of the reported glutaryl acylases from various Pseudomonas sp. strains show a high similarity (>93% identity). Thus, with the known nucleotide sequences of Pseudomonas glutaryl acylases in GenBank, PCR primers were designed to clone a glutaryl acylase gene from P. cepacia BY21. The unknown -subunit gene of glutaryl acylase from chromosomal DNA of P. cepacia BY21 was cloned successfully by PCR. The -subunit amino acids of P. cepacia BY21 acylase (GenBank accession number AY948547) were similar to those of Pseudomonas diminuta KAC-1 acylase except that Asn408 of P. diuminuta KAC-1 acylase was changed to Leu408.

• KSBB Journal
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• 제10권3호
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• pp.264-270
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• 1995

Trigonopsis variabilis는 버l타락탐 항생제인 cephalosporin C (ceph C)를 ${\alpha}$-keto-adipyl-7 a aminocephalosporanic acid(AKA-7 ACA)로 생변 환하는 강력한 D-amino acid oxidase 효소를 갖고 있다. 본 연구는 이 D-AAO 효소의 유전자를 추출하기 위하여 polymerase chain reaction (PCR)을 사용하였다. PCR 단편을 콜로닝하기 위하여 Taq D DNA polymerase, Klenow, T4 DNA polymerase I, Alkaline phosphatase Calf Intestinal와 T4 kinase 의 효소반응을 이용하여 4가지의 방법을 샤용한 결 과, blunt - end 의 PCR fragment 를 phosphory­l lation하고 blunt -end의 pUC18 plasmid를 dephos phorylation 한 후 ligation 한 것 이 가장 좋은 클로 닝 효율을 보였다. Ceph C에 대한 D-AAO 효소의 활성은 재조합 E. coli의 세포추출액과 permea bilized cells에서 모두 확인할 수 있었다.