• Biotechnology and Bioprocess Engineering:BBE
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• v.6 no.1
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• pp.11-17
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• 2001

To enhance the productivity and activity of nitrile hydratase in Rhodococcus rhodochrous M33, a glucose-limited fed-batch culture was performed. In a fed-batch culture where the glucose was controlled at a limited level and cobalt was supplemented during the fermentation period, the cell mass and total activity of nitrile hydratase both increased 3.3-fold compared to that in the batch fermentation. The productivity of nitrile hydratase also increased 1.9-fold compared to that in the batch fermentation. The specific activity of nitrile hydratase in the whole cell preparation when using a fed-batch culture was 120 units/mg-DCW, which was similar to that in the batch culture.

• Microbiology and Biotechnology Letters
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• v.17 no.5
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• pp.429-435
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• 1989

A bacterial strain of Brevibaterium sp. CH1 was isolated and used to produce an enzyme (nitrile hydratase) necessary for earring out the bioconversion of acrylonitrile to acrylamide. The culture and reaction conditions, and medium optimization were studied for the strain. The conversion yield was nearly 100％ with a trace amount of acrylic acid produced. The strain showed strong activity of nitrile hydratase toward acrylonitrile and extremely low activity of the amidase toward acrylamide. We sought optimum culture conditions for the formation of nitrile hydratase by Brevibacterium sp. CH1. The effects of temperature and pH on the activity of free and immobilized tells were investigated. The nitrite hydratase of Brevibacterium sp. CH1 acted not only on various aliphatic nitrites such as acrylonitrile, propionitrile and acetonitrile, but also on aromatic nitrile as nicotinonitrile.

• Microbiology and Biotechnology Letters
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• v.18 no.1
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• pp.56-60
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• 1990

A bacterial strain of Brevibacterium sp. CHI was isolated from soil and used to produce an enzyme (nitrile hydratase) necessary for carrying out the bioconversion of acrylonitrile to acrylamide. Various immobilization methods and enzyme stability were investigated. The nitrile hydratase showed the maximum stability at pH 7 for the free cells. EDTA and phenyl methyl sulfonyl fluoride were selected as the protease inhibitor and the enzyme stability was evaluated by changing inhibitor concentration. Acrylamide beads were the best carriers among four carriers we tested in terms of stability and physicoehemical strength. The storage stability of the immobilized cells decreased with increasing acrylamide concentration of the gel phase at 4$^{\circ}C$, and was very low at acrylarnide concentration above 25%.

• Microbiology and Biotechnology Letters
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• v.20 no.5
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• pp.614-618
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• 1992

The batch and fed-batch cultivations of Brevibacteriurn CHI were carried out for the production of nitrile hydratase. In batch culture, with pH control the cell mass and the specific activity increased more 20% and 30%. respectively. The maximum growth rate was obtained at a glucose concentration of $20g/{\ell}$ because of substrate inhibition. The fed-batch culture of Brevibacteriurn CHI with constant substrate feeding gave a cell density of up to $68g/{\ell}$ and nitrile hydratase activity was maintained at above 6.1units/mg. The cell growth yield on carbon .source was ca. 0,68 g/g glucose consumed. The total nitrile hydratase activity in this fed-batch mode increased up to 414.8 units/m${\ell}$, which amounted to 4.4 times that of the batch culture.

• Biotechnology and Bioprocess Engineering:BBE
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• v.7 no.4
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• pp.194-200
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• 2002

The cells of Rhodococcus rhodochrous M33, which produce a nitrile hydratase enzyme, were immobilized in acrylamide-based polymer gels. The optimum pH and temperature for the activity of nitrile hydratase in both the free and Immobilized cells were 7.4 and 45$\^{C}$, respectively, yet the optimum temperature for acrylamide production by the immobilized cells was 20$\^{C}$. The nitrile hydratase of the immobilized cells was more stable with acrylamide than that of the free cells. Under optimal conditions, the final acrylamide concentration reached about 400 g/L with a conversion yield of almost 100% after 8 h of reaction when using 150 g/L of immobilized cells corresponding to a 1.91 g-dry cell weight/L. The enzyme activity of the immobilized cells rapidly de-creased with repeated use. However, the quality of the acrylamide produced by the immobilized cells was much better than that produced by the free cells in terms of color, salt content, turbidity, and foam formation. The quality of the aqueous acrylamide solution obtained was found to be of commercial use without further purification.

• Journal of Microbiology and Biotechnology
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• v.1 no.2
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• pp.136-141
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• 1991

Optimum culture conditions for the formation of nitrile hydratase by Brevibacterium sp. CH2 were investigated. Addition of ferric and ferrous ions greatly increased the nitrile hydratase formation. The effects of nitriles, amides, and acids as an inducer on the formation of nitrile hydratase were investigated. Isobutyramide was the best inducer among the tested compounds. When Brevibacterium sp. CH2 was cultivated for 23 h at $30^{\circ}C$ in a optimized medium containing 15 g of glucose, 5 g of bacto peptone, 3 g of yeast extract, 3 g of malt extract, 1 g of $KH_2$$PO_4$, 1 g of $K_2$$HPO_4$, 1 g of NaCl, 0.5 g of isobutyramide, 0.2 g of MgSO$_4$ㆍ7$H_2O$, and 0.02g of $FeSO_4$ㆍ$7H_2$O per liter of distilled water with pH controlled at 7.1, the maximum total activity was 665 units/ml of the culture broth and the specific activity was 70 units/mg of the dry cells. The medium optimization increased the specific activity of Brevibacterium sp. CH2 2.2 times.

• 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.

• Journal of Applied Biological Chemistry
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• v.43 no.4
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• pp.207-210
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• 2000

As a typical example of the screening for a microbial biocatalyst from nature, isolation of nitrilesynthesizing microorganisms, characterization of a new enzyme aldoxime dehydratase, and its function in the aldoxime-nitrile pathway are introduced. Catalytic properties of some of our enzymes were improved through a direct evolutionary approach.

• Journal of Microbiology and Biotechnology
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• v.1 no.3
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• pp.182-187
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• 1991

The effects of acrylonitrile and acrylamide on the enzyme action of nitrile hydratase of Brevibacterium sp. CH1 and CH2 strains used for the biotransformations of nitriles were studied. The excessive substrate (acrylonitrile) and product (acrylamide) inhibited the enzyme activity competitively. In comparison with 0.2 mol/l of CH1 strain, the substrate inhibition of CH2 strain began to appear only at a high acrylonitrile concentration of 0.91 mol/l. In a packed bed reactor, dispersed plug flow model was proposed and this model was proved to be valid by the experiment. Also acrylamide productivity decreased sharply when acrylamide concentration in the substrate solution exceeded 20％ (wt/v).

• Journal of Microbiology and Biotechnology
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• v.26 no.2
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• pp.337-346
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• 2016

Three combinations of molecular chaperones from Escherichia coli (i.e., DnaK-DnaJ-GrpE-GroEL-GroES, GroEL-GroES, and DnaK-DnaJ-GrpE) were overproduced in E. coli BL21, and their in vitro stabilizing effects on a nitrile hydratase (NHase) were assessed. The optimal gene combination, E. coli groEL-groES (ecgroEL-ES), was introduced into Rhodococcus ruber TH3. A novel engineered strain, R. ruber TH3G was constructed with the native NHase gene on its chromosome and the heterologous ecgroEL-ES genes in a shuttle plasmid. In R. ruber TH3G, NHase activity was enhanced 37.3% compared with the control, TH3. The in vivo stabilizing effect of ecGroEL-ES on the NHase was assessed using both acrylamide immersion and heat shock experiments. The inactivation behavior of the in vivo NHase after immersion in a solution of dynamically increased concentrations of acrylamide was particularly evident. When the acrylamide concentration was increased to 500 g/l (50%), the remaining NHase activity in TH3G was 38%, but in TH3, activity was reduced to 10%. Reactivation of the in vivo NHases after varying degrees of inactivation was further assessed. The activity of the reactivated NHase was more than 2-fold greater in TH3G than in TH3. The hydration synthesis of acrylamide catalyzed by the in vivo NHase was performed with continuous acrylonitrile feeding. The final concentration of acrylamide was 640 g/l when catalyzed by TH3G, compared with 490 g/l acrylamide by TH3. This study is the first to show that the chaperones ecGroEL-ES work well in Rhodococcus and simultaneously possess protein-folding assistance functions and the ability to stabilize and reactivate the native NHases.