• Journal of Microbiology and Biotechnology
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• v.20 no.2
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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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• v.24 no.7
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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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• v.18 no.3
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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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• v.18 no.12
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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.

• The Journal of Natural Sciences
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• v.17 no.1
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• pp.55-64
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• 2006

The construction, purification, and characterization of hexahistidine-tagged phage K11 lysozyme are carried out in this study. The results showed that the enzymatic activities of K11 lysozyme are not affected by the purification tag. The optimum pH of K11 lysozyme is 7.2-7.4. The amidase activity of K11 lysozyme was also measured in the presence of different cations. The addition of $Ca^2+$ and $Mg^2+$ almost completely shut down the amidase activity but $Zn^2+$ and $Na^+$ maintained the amidase activity. In the presence of 100 mM $Zn^2+$ the amidase activity was nearly abolished.

• The Korean Journal of Pesticide Science
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• v.6 no.1
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• pp.31-35
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• 2002

Objectives of this study were to determine if impurities in technical grade benfuracarb inhibit glutathione-S-transferase and amidase and to identify structures of impurities in technical grade benfuracarb. Technical grade benfuracarb, active ingredient, and impurity inhibited glutathione-S-transferase, and their $I_{50}$ were $9.7{\times}10^{-4}M,\;>1.0{\times}10^{-3}M,\;1.8{\times}10^{-4}M$, respectively. Such inhibition, however, was not higher than that by ethacrynic acid, a selective inhibitor to GST. Technical grade benfuracarb, active ingredient, and impurity also inhibited amidase, and their $I_{50}$ were $6.0{\times}10^{-5}M,\;4.3{\times}10^{-4}M,\;7.6{\times}10^{-5}M$, respectively. Our results show that the inhibition of both detoxifying enzymes by impurities in benfuracarb was 10-fold lower than that by active ingredient, suggesting that both active ingredient and impurities are involved in the inhibition of both detoxifying enzymes. Of four impurities (IM $1{\sim}4$) that were separated from technical grade benfuracarb, IM 2 and IM 3 inhibited GST and amidase. Based on data from IR, $^1H$-NMR, $^{13}C$-NMR and MS, it was determined that IM 2 is ethyl-N-isopropylamino propionate and IM 3 is ethyl-N-isopropyl-N(chlorosulfenyl)aminopropionate.

• Bulletin of the Korean Chemical Society
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• v.21 no.10
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• pp.1020-1024
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• 2000

• 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.36 no.4
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• pp.314-319
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• 2008

Penicillin G amidase (PGA, benzylpenicillinaminohydrolase, EC 3.5.1.11) is industrially important enzyme which converts penicillin G to 6-aminopenicillanic acid (6-APA) and phenylacetic acid (PAA). The PGA in E. coli ATCC 11105 is secreted into the periplasm after removing signal sequences and becomes heterodimer which composed of two subunits, small subunit (24 kDa) and large subunit (65 kDa). In this study, the PGA gene was obtained from E. coli ATCC 11105 using PCR (polymerase chain reaction) technique. The active PGA was successfully secreated into periplasm in E. coli BL2 1(DE3) harboring pET-pga plasmid. The optimized fed-batch fermentation, consisting of a three-step shift of culture temperature from $37^{\circ}C$ to $22^{\circ}C$, gave a productivity of 19.6 U/mL with a cell growth of 62 O.D. at 600 nm.

• Journal of Microbiology and Biotechnology
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• v.8 no.4
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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.