• Journal of Microbiology and Biotechnology
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• v.10 no.5
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• pp.573-579
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• 2000

Knowledge on the enzymes acting on p-amino-acid-containing peptides appears to be somewhat limited when compared with those acting on peptides composed on L-amino acids. Less than ten D-stereospecific enzymes are hitherto known. This review describes about several novel D-stereospecific peptidases and amidases of microbial origin, including D-aminopeptidase (E.C. 3.4.11.19), alkaline D-peptidase, and D-amino aicd amidase, which are applied to the synthesis of D-amino acid/or D-amino acid derivatives.

• Korean Journal of Microbiology
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• v.15 no.4
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• pp.145-153
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• 1977

Penicillin amidase (EC 3.5.1.11) was produced by a mutant strain of Bacillius megaterium ATCC 14945. Hydroxylamine assay method for the determination of 6-APT was modified by using "HCl addition techniques" in order to simplify the time consuming orginal assay method without sacrifice of accuracy. Using the new mutant strain, the effects of fermentation conditions on enzyme production were studied.e studied.

• Journal of Life Science
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• v.27 no.10
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• pp.1145-1151
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• 2017

In this study, we report on a simple, efficient method for obtaining penicillin G amidase (PGA) from recombinant Escherichia coli using a formulation mixed with detergent and lysozyme. Research was conducted on the extraction efficiency of PGA from the periplasmic space in cells in terms of the type of detergent, detergent concentration, pH, reaction time, and temperature of permeabilization. The extraction yield of PGA in the formulated surfactant/lysozyme treatment was increased by approximately (55-65 U/ml) in comparison with that in the single surfactant treatment. The released PGA solution was concentrated and exchanged with buffer using an ultrafiltration (U/F) system. The yields of diatomite filtration, membrane filtration (M/F), and U/F were 69.7%, 93.8%, and 77.3%, respectively. A total of 212 KU of PGA was recovered. At the 25-L culture scale, the overall yield of extraction using the mixed surfactant/lysozyme method was 49.2%. The specific activity of extracted PGA was 11 U/mg in protein. The concentrated PGA solution was immobilized on microporous silica beads without further purification of PGA. The total immobilization yield of PGA on the resin was 48.7%, while the enzyme activity was 101 U/g. The immobilized PGA was successfully used to produce 6-APA from penicillin G. Our results indicated that a simple extraction method from periplasmic space in E. coli may be used for the commercial scale production of ${\beta}-lactam$ antibiotics using immobilized PGA.

• Biomedical Science Letters
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• v.18 no.4
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• pp.438-444
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• 2012

N-acylethanolamines (NAEs) including endocannabinoids, anadamide, are long chain fatty acid ethanolamines and express ubiquitously in animal and plant tissues. NAEs have several pharmacological effects including anti-inflammatory, analgesic and anorexic effects. The levels of NAEs in tissues are strictly regulated by synthesizing and hydrolyzing enzymes because NAEs are not stored in the cell but rather made on demand. NAEs are hydrolyzed to free fatty acids and ethanolamines by fatty acid amide hydrolase and N-acylethanolamine-hydrolyzing acid amidase (NAAA). Here, we suggest the fluorescence-based assay system for NAAA. We developed N-(4-methy-2-oxo-2H-chromen-7-yl)palmitamide (PAAC) as a fluorogenic substrate for NAAA and we also generated NAAA stably expressing COSM6 cell line. When extracts of cells expressing NAAA were incubated with PAAC, NAAA specifically hydrolyzed PAAC to palmitic acids and fluorogenic dye, coumarin. Release of coumarin was monitored by using fluorometer. NAAA hydrolyzed PAAC with an apparent Km of $20.05{\mu}M$ and Vmax of 32.18 pmol/mg protein/min. This assay system can be used to develop inhibitors or activators of NAAA.

• Journal of Microbiology and Biotechnology
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• v.16 no.9
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• pp.1416-1421
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• 2006

We identified a gene encoding the N-acetylmuramoyl L-alanine amidase (amiB) of Vibrio anguillarum, which catalyzes the degradation of peptidoglycan in bacteria. The entire open reading frame (ORF) of the amiB gene was composed of 1,722 nucleotides and 573 amino acids. The deduced amino acid sequence of AmiB showed a modular structure with two main domains; an N-terminal region exhibiting an Ami domain and three highly conserved, continuously repeating LysM domains in the C-terminal portion. An amiB mutant was constructed by homologous recombination to study the biochemical function of the AmiB protein in V. anguillarum. Transmission electron microscopy (TEM) revealed morphological differences, and that the mutant strain formed trimeric and tetrameric unseparated cells, suggesting that this enzyme is involved in the separation of daughter cells after cell division. Furthermore, inactivation of the amiB gene resulted in a marked increase of sensitivity to oxidative stress and organic acids.

• Microbiology and Biotechnology Letters
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• v.23 no.6
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• pp.671-677
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• 1995

An extracellular enzyme showing lytic activity on E. coli peptidoglycan had been isolated from Bacillus sp. BL-29. The lytic enzyme was purified to homogeneity by ion-exchange chromatography and gel filtration, with a recovery of 5%. The enzyme was monomeric and had an estimated molecular weight of 31,000 Da. The mode of action of the purified enzyme was also investigated. When the purified lytic enzyme was incubated with cell wall peptidoglycan, N-terminal amino groups were released without the release of reducing groups. The N-terminal amino acid released was identified as dinitrophenylalanine (DNP-alanine) by analysis of terminal amino acid by dinitrophenylation method. This result suggests that the lytic enzyme should be a kind of N-acetylmura-myl-L-alanine amidase.

• Microbiology and Biotechnology Letters
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• v.9 no.1
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• pp.35-44
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• 1981

Whole cell penicillin amidase of Escherichia coli was immobilized by entrapment in gelatin followed by extrusion and crosslinking with glutaraldehyde. The immobilized engyme preparation demonstrated the recovery yield of activity up to 70% and good stability during storage and operation. The half life of activity decay during the operation was estimated to be about 50 days. The optimum pH and temperature for both of immobilized and soluble enzyme are 8.5 and 5$0^{\circ}C$, respectively. No significant change was demonstrated in the effect of pH and temperature, but the increase in heat stability at high temperature was observed in the case of the immobilized enzyme. It was found that the plug flow reactor could be operated favorably since the pH drop along the column path due to tile reaction product was minimized by employing substrate solution with moderate buffer strength. The optimal condition of reactor operation was discussed with regard to the effect of substrate concentration and the residence time on the conversion efficiency and productivity.

• Microbiology and Biotechnology Letters
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• v.9 no.3
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• pp.165-171
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• 1981

The penicillin amidase from Escherichia coli (ATCC 9637) was immobilized by entrappment in gelatin and DEAE-cellulose mixture cross-linked with glutaraldehyde, and the kinetics in a differential column reactor was studied. The optimal operating condition of a differential reactor was reasonably met when the enzyme loading was 1g, and 30 mM substrate solution in 0.1 M phosphate buffer (pH 8.0) was fed at flow rate 4$m\ell$/min and 4$0^{\circ}C$. The optimal pH and temperature were found to be 8.0 and 55$^{\circ}C$, respectively. The Michaelis-Menten constant was 4.8 mM while the maximum velocity was 308 units/g of the immobilized enzyme under the condition of the differential reactor. The effect of substrate inhibition disappeared in the immobilized enzyme preparation. The differential reactor was proved to be good for studying the true kinetics since the pH drop and the external diffusional resistance could be eliminated.

• Microbiology and Biotechnology Letters
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• v.34 no.3
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• pp.204-210
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• 2006

Ethyl (S)-4-chloro-3-hydroxybutyrate is a useful intermediate for the synthesis of Atorvastatin, a chiral drug to hypercholesterolemia. In this research, two 4-chloro-3-hydroxybutyro-nitrile-degrading strains were isolated from soil sample. They were identified as Rhodococcus erythropolis strains by 16S rRNA analysis. The nitrile-degrading enzyme(s) were suggested to be nitrile hydratase and amidase rather than nitrilase from the result of thin layer chromatography analysis. The corresponding genes were obtained by PCR cloning method. The predicted protein sequences had identities more than 96% with nitrile hydratase ${\alpha}-subunit$, nitrile hydratase ${\beta}-subunit$, and amidase of R. erythropolis. The 4-chloro-3-hydroxybutyronitrile-hydrolyzing activities in both strains were increased dramatically by ${\varepsilon}-caprolactam$ which was known as good inducer for nitrile hydratase. Both intact cells and cell-free extract could hydrolyze the nitrile compound. So, the intact cell and the enzymes could be used as potential biocatalyst for the production of 4-chloro-3-hydroxybutyric acid.

• Microbiology and Biotechnology Letters
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• v.9 no.3
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• pp.159-164
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• 1981

Reinforced Calcium-alginate gel entrappment method for enzyme immobilization is described with an example of penicillin amidase from Bacillus megaterium KFCC 10029, a partially constitutive mutant of B. megaterium ATCC 14945. Penicillin amidase recovered from the fermentation broth by adsorption on celite is mixed with alginate and gelatin solution, and cast into a pellet or noodle form by coagulation in calcium salt solution followed by crosslinking with glutaraldehyde. Optimum pH and temperature of the immobilized enzyme preparation were 8.0 and 6$0^{\circ}C$, respectively. Kinetic constants such as Km value and the inhibition constant of 6-APA and phenylacetic acid were 2.6 mM, 7.4 mM and 21.2 mM, respectively. The enzyme leakage from the adsorbent during operation was successfully prevented owing to the increase of physical strength of gel coat. The half lives in a column reactor were 6 and 30 days at the respective temperature of 4$0^{\circ}C$ and 3$0^{\circ}C$, which were the 6-8 fold increased values as compared with those of without entrappment. The results highly recommended the use of reinforced Calcium-alginate gel entrappment method for the enhancement of physical strength and the operational stability of alginate gel entrapped enzyme.