• Microbiology and Biotechnology Letters
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• v.27 no.2
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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$0^{\circ}C$.

• Microbiology and Biotechnology Letters
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• v.22 no.3
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• pp.296-303
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• 1994

An immobilized Trigonopsis variabilis cells having an high activity of D-amino acid oxidase(DAO) was used to convert CPC into GL-7-ACA. The optimal pH of the reaction system was 8.0-8.5, and the optimal temperature was 40$\circ$C. When immobilized cell was used repeatedly in semi-batchwise reaction, the system retained 80% of the initial activity after used of 12 times for over 12 hours. The storage stability of the immobilized cell was maintained for 30 days at 4$\circ$C. The CPC concentration for the maximal reaction rate was about 30 mM and 40 mM for free and immobilized cells, respectively. Substrate inhibition of CPC concentration more than 50 mM was overcomed by 20~25% by immobilization. Pure oxygen supply into reaction system was most efficient in D-amino acid oxidase reaction. Continuous conversion to GL-7-ACA from CPC has been developed with an bioreactor system containing immobilized T variabilis cells. By opera- tion of the reactor for 5 hours, the average conversion yield of >80% and GL-7-ACA production of 40~45 mM per hour could be obtained.

• Journal of Microbiology and Biotechnology
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• v.6 no.5
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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 and Biotechnology
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• v.6 no.6
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• pp.375-380
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• 1996

The gene coding for glutaryl-7-aminocephalosporanic acid (GL-7-ACA) acylase was cloned from Pseudomonas sp. GK16 and some of its characteristics were analyzed. The complete nucleotide sequence revealed that the putative open reading frame is 2160 bases long and encodes 720 amino acids. By SDS-PAGE three proteins, approximately corresponding to 70, 54 and 16 kDa of molecular weight, were detected in E. coli cells carrying pGAP18. The largest protein should be a precursor which is not processed yet, while the other two proteins must be derived from the precursor by the proteolytic processing.

• 한국생물공학회:학술대회논문집
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• 2000.04a
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• pp.463-465
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• 2000

부분 정제한 VHb-DAO와 CPC를 30분 이상 반응시키면 CPC가 GL-7ACA로 80%이상 변환되었다. VHb-DAO와 D-form의 아미노산이 반응한 결과 $K_m$은 D-Methionine, D-Valine이 낮으며, $V_{max}$는 D-${\alpha}$-aminophenylacetic acid, D-Leucine, D-Phenyalanine가 높은 수치를 나타내었다.

• Journal of Microbiology and Biotechnology
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• v.11 no.2
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• pp.199-203
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• 2001

A simple quantitative method to measure the degree of silanization was developed, based on the reaction of ninhydrin with the silanization reagent (3-aminopropyltriethoxysilane, 3-APTES). At low concentrations (0.001-0.005%, v/v) of 3-APTES, a good linearity was obtained when 3-APTES reacted with undiluted ninhydrin for 30 min. On the other hand, at high levels of 3-APTES, a linearity was obtained when 3-APTES reacted with 3-fold diluted ninhydrin for 20 min. The reliability of regression curves mentioned above was expressed as a regression coefficient ($R^2$) of more than 0.99. Immobilization of different enzymes was introduced via silanization by using the 3-APTES in order to confirm the validity of the ninhydrin method. When yield for each step in the immobilizatio process were compared, yields of both glutaraldehyde and protein were founc to have the same tendency to silanization. These results shw that the ninhydrin method was suitable for quatitative analysis of silanization and that yields of immobilization could be pre-estimated by measuring silanization levels using the ninhydrin method.