• Journal of Microbiology and Biotechnology
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• 제20권2호
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• pp.325-331
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• 2010

Rhodococcus erythropolis amidase was expressed in Escherichia coli cells. The crude amidase in the cell-free extract was immobilized using the cross-linked enzyme aggregate (CLEA) method. The crude amidase was mixed with bovine serum albumin and then precipitated with ammonium sulfate. The resultant precipitant was subsequently cross-linked with glutaraldehyde. Scanning electron microscopy revealed that this co-CLEA had a ball-like shape with a diameter of approximately $1\;{\mu}m$. This co-CLEA evidenced hydrolytic activity toward a variety of amide substrates. The amidase co-CLEA evidenced an optimum temperature of $60^{\circ}C$ and an optimum pH of 8.0, results that were similar to those of the soluble amidase. The reaction stability of the co-CLEA was increased. That is, it was stable up to $50^{\circ}C$ and in a pH range of 5.0-12.0. Additionally, the co-CLEA could be recovered by centrifugation, and retained 96% activity after 3 repeated cycles. This amidase co-CLEA may prove useful as a substitute for soluble amidase as a biocatalyst in the pharmaceutical and chemical industries.

• Journal of Microbiology and Biotechnology
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• 제24권7호
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• pp.936-942
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• 2014

Using racemic (R,S)-2-(4-chlorophenyl)-3-methylbutyramide, an intermediate for the chiral pyrethroid insecticide Esfenvalerate, as a sole nitrogen source in a minimal medium, several strains with high enatioselectivity (${\geq}98%$) were isolated by enrichment techniques. One of the strains, LG 31-3, was identified as Burkholderia multivorans, based on physiological and morphological tests by a standardized Biolog station for carbon source utilization. A novel amidase was purified from B. mutivorans LG 31-3 and characterized. The enzyme exhibited (S)-selective amidase activity on racemic (R,S)-2-(4-chlorophenyl)-3-methylbutyramide. Addition of the racemic amide induced the production of the enantioselective amidase. The molecular mass of the amidase on SDS-PAGE analysis was shown to be 50 kDa. The purified amidase was subjected to proteolytic digestion with a modified trypsin. The N-terminal and internal amino acid sequences of the purified amidase showed a high sequence homology with those deduced from a gene named YP_366732.1 encoding indole acetimide hydrolase from Burkholderia sp. 383.

• Journal of Microbiology and Biotechnology
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• 제18권3호
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• pp.552-559
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• 2008

Ethyl (S)-4-chloro-3-hydroxybutyrate is an intermediate for the synthesis of Atorvastatin, a chiral drug used for hypercholesterolemia. A Rhodococcus erythropolisstrain (No.7) able to convert 4-chloro-3-hydroxybutyronitrile into 4-chloro-3-hydroxybutyric acid has recently been isolated from soil. This activity has been regarded as having been caused by the successive actions of the nitrile hydratase and amidase. In this instance, the corresponding amidase gene was cloned from the R. erythropolis strain and expressed in Escherichia coli cells. A soluble active form of amidase enzyme was obtained at $18^{\circ}C$. The Ni column-purified recombinant amidase was found to have a specific activity of 3.89 U/mg toward the substrate isobutyramide. The amidase was found to exhibit a higher degree of activity when used with mid-chain substrates than with short-chain ones. Put differently, amongst the various amides tested, isobutyramide and butyramide were found to be hydrolyzed the most rapidly. In addition to amidase activity, the enzyme was found to exhibit acyltransferase activity when hydroxyl amine was present. This dual activity has also been observed in other enzymes belonging to the same amidase group (E.C. 3.5.1.4). Moreover, the purified enzyme was proven to be able to enantioselectively hydrolyze 4-chloro-3-hydroxybutyramide into the corresponding acid. The e.e. value was measured to be 52% when the conversion yield was 57%. Although this e.e. value is low for direct commercial use, molecular evolution could eventually result in this amidase being used as a biocatalyst for the production of ethyl (S)-4-chloro-3-hydroxybutyrate.

• Journal of Microbiology and Biotechnology
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• 제18권12호
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• pp.1932-1937
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• 2008

The R-amidase production by a newly isolated strain of Delftia tsuruhatensis ZJB-05174 was optimized in this paper. Effects of factors such as carbon sources, nitrogen sources, and inducers on amidase production were investigated. The medium composition was optimized using central composite designs and response surface analysis. The optimal medium components for enhanced amidase production were found to be as follows: glucose, 8.23 g/l; yeast extract, 11.59 g/l; 2,2-(R,S)-dimethylcyclopropane carboxamide, 1.76 g/l; NaCl, 1 g/l; ${KH_2}{PO_4}$ 1 g/l; and ${K_2}{HPO_4}$ 1 g/l. A maximum enzyme production of 528.21 U/l was obtained under the optimized conditions, which was 4.7 times higher than that obtained under initial conditions.

• 자연과학논문집
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• 제17권1호
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• pp.55-64
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• 2006

이 연구에서는 6개의 히스티딘이 첨부된 파지 K11 라이소자임의 제조 분리 및 특성을 알아보고자 하였다. 첨부된 히스티딘에 의한 이 효소의 활성도의 변화는 없었으며, 효소 활성의 최적 pH는 7.2-7.4 이었다. 여러 다른 종류의 양이온 존재하의 활성도를 측정한 결과 칼슘과 마그네슘 이온에 의해 효소 활성이 완전히 억제되었으나, 아연이나 나트륨 이온은 효소의 활성도를 이 이온들이 없을 때와 같은 정도로 유지하였다. 단지 100 mM 이상의 아연 이온 농도에서 K11 라이소자임 효소 활성도를 완전히 억제하였다.

• 농약과학회지
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• 제6권1호
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• pp.31-35
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• 2002

본 논문의 목적은 benfuracarb 원제(90.2%)에 함유된 불순물의 glutathione-S-transferase와 amidase에 대한 저해 특성과 해당 불순물의 구조를 밝히는데 있다. Benfuracarb 원제, 유효성분 및 불순물은 glutathione-S-transferase(GST)를 효과적으로 저해하였으나, 그 저해력은 GST 효소 저해제인 ethacrynic acid의 저해력보다는 낮았다. 즉, GST에 대한 benfuracarb 원제, 유효성분 및 불순물의 $I_{50}$은 각각 $9.7{\times}10^{-4}M,\;>1.0{\times}10^{-3}M,\;1.8{\times}10^{-4}M$이었으나, ethacrynic acid의 $I_{50}$은 $1.7{\times}10^{-5}M$이었다. Benfuracarb 원제, 유효성분 및 불순물은 amidase를 저해하였는데, 이들의 효소저해력은 iprobenfos의 저해력($I_{50},\;8.2{\times}10^{-7}M$)보다는 낮은 $6.0{\times}10^{-5}M,\;4.3{\times}10^{-4}M,\;7.6{\times}10^{-5}M$이었다. Benfuracarb 원제에는 4종의 불순물(IM $1{\sim}4$)이 검출되었는데, 이들 중 IM 2와 3은 GST와 amidase의 활성을 저해하였던 반면, IM 4는 효소활성을 저해하지 않았다. 이들 불순물 중 효소활성 저해특성을 갖는 IM 2와 3을 IR, $^1H$-NMR, $^{13}C$-NMR, LC-MS를 이용하여 구조를 분석한 결과, IM 2는 ethyl-N-isopropylamino propionate로, 그리고 IM 3은 ethyl-N-isopropyl-N-(chlorosulfenyl) aminopropionate로 확인되었다.

• Bulletin of the Korean Chemical Society
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• 제21권10호
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• pp.1020-1024
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• 2000

• 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$0^{\circ}C$.

• 한국미생물·생명공학회지
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• 제36권4호
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• pp.314-319
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• 2008

Penicillin G amidase(PGA, benzylpenicillinamidohydrolase, EC 3.5.1.11)는 penicillin G를 phenylacetic acid(PAA)와 6-aminopenicillanic acid(6-APA)로 분해하는 효소이다. Escherichia coli(E. coli) ATCC 11105의 PGA는 24 kDa의 small subunit과 65 kDa의 large subunit으로 구성되어 있고, precursor polypeptide에서 signal peptide와 spacer peptide가 절단되어 활성을 가진 heterodimer가 형성된다. 본 연구에서는 E. coli ATCC 11105에서 PCR(polymerase chain reaction)을 통해 증폭한 pga gene을 expression vector에 넣어 pET-pga plasmid를 제작하였고, 이것을 E. coli BL21 (DE3) 균주에 형질 전환하여 PGA를 발현하고 그 활성을 분석하였다. E. coli BL21(DE3)/pET-pga 균주의 고밀도 배양액을 SDS-PAGE로 분석 했을 때, PGA의 precursor, large subunit, 그리고 small subunit으로 보이는 protein band가 나타났으며, PGA가 soluble form의 precursor로 발현되어 processing을 거쳐서 large subunit과 small subunit으로 절단되기도 하고, 일부는 insoluble form의 precursor로 발현되기도 하는 것으로 생각된다. 유가배양시 온도변화 전략을 사용하여 고농도 배양에서 발현을 유도하였다. 온도변화 전략은 $37^{\circ}C$에서 $28^{\circ}C$를 거쳐 $22^{\circ}C$로 3단계로 변화시켰다. 이러한 전략으로 PGA활성은 19.6 U/mL이며 균체량은 600 nm에서 흡광도가 62까지 도달하였다.

• Journal of Microbiology and Biotechnology
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• 제8권4호
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• pp.347-352
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• 1998

A new strain of Pseudomonas aeruginosa growing in a rice field contaminated with herbicide and effluents of a factory manufacturing explosives was isolated. This isolate showed excellent growth in unusually high concentration of acrylamide (60 mM). It utilized acrylamide as the sole source of carbon and nitrogen for growth. Other amides such as acetamide, butyramide, isobutyramide, and methacrylamide were also utilized for the growth by this isolate. Acrylamide was degraded into acrylic acid and ammonia by the enzyme amidase. More than $65\%$ of added acrylamide (40 mM) was converted into acrylic acid after 40 h of growth of the culture. Amidase activity was inducible, the highest activity being observed with isobutyramide ($12.5{\mu}M$ ammonia/mg protein/min). These results demonstrate that this bacterium can degrade a variety of amides.