• Biotechnology and Bioprocess Engineering:BBE
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• 제6권1호
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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.

• 한국미생물·생명공학회지
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• 제17권5호
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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.

• 한국미생물·생명공학회지
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• 제18권1호
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• pp.56-60
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• 1990

Brevibacterium sp. CH1 균주가 흙으로부터 분리하여 아크릴로니트릴을 아크릴아마이드로 생변화를 수행하는데 필요한 효소를 생산하기 위하여 사용하였다. 여러가지 고정화 방법과 효소 안정성이 조사 되었다. Nitrile hydratase는 free cell에 대하여 pH7에서 최대한 안정성을 보여주었다. EDTA와 phenyl menthl fluoride을 protease inhibitor로 선정하여 inhibitor 농도를 변화시키면서 효소의 저장안정성을 평가하였다. 아크릴아마이드가 안정성 및 물리화학적 강도를 고려 할 때 가장 좋은 carrier였다. 고정화 세포의 저장안정성은 4$^{\circ}C$에서 gel상의 아크릴아마이드 농도가 증가함에 따라 감소하였고, 25% 이상의 아크릴아마이드 농도에서 안정성이 매우 낮았다.

• 한국미생물·생명공학회지
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• 제20권5호
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• pp.614-618
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• 1992

Brevibacterium CH1의 회분배양 결과 최적의 초기 포도당 농도는 $20g/{\ell}$이었고, 균체의 최대 성장속도는 $0.21h^{-1}$이었다. pH 를 조절하여 줌으로써 균체농도와 효소의 비활성이 각각 20, 30 정도 증가하였다. 따라서 pH 조절은 균체농도 및 효소생성에 상당한 영향을 주는 것을 알 수 있었다. M3 배지를 연속적으로 공급하면서 유가식 회분배양 한 결과 88시간 배양 후 균체농도는 $68g/{\ell}$로 고농도 배양이 되었으며, 비활성은 6.1units/mg으로 유지되었다. 효소의 총활성은 배양시간에 따라 증가하여 80시간 배양 후 최대 414.8 units/$m{\ell}$ 이었으며, 회분배양에 비해 4.4배 증가하였다. 이 경우에 Yx/s(cell growth yield)는 0.68g cells/g glucose consumed이었다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제7권4호
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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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• 제1권2호
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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.

• 한국미생물·생명공학회지
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• 제34권3호
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• pp.204-210
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• 2006

각종 니트릴 화합물은 키랄 의약품의 합성에 사용되는 유용한 중간체이다. 본 연구에서는 토양 분리균 중에서 4-chloro-3-hydroxy butyronitrile(CHBN)기질로부터 고지혈증 치료제인 Atorvastatin을 합성하는 데에 필요한 4-chloro-3-hydroxy butyric acid(CHBAc)를 생성하는 균주 2종류를 선발하였다. 16S rRNA 분석을 통해서 균 동정을 수행한 결과, 모두 Rhodococcus erythropolis에 속하는 것으로 밝혀졌으며, TLC 분석 결과로부터 CHBN 기질을 분해하는 효소는 니트릴 히드라타아제(NHase)와 아미다아제(amidase)인 것으로 추정되었다. 분리균의 CHBN 분해효소는 ${\varepsilon}$-카프로락탐에 의해서 발현이 유도되었으며, 균체와 세포 추출액에서 모두 기질 분해활성을 나타났다. 기존에 보고된 효소의 유전자 염기서열로부터 프리머를 제조하고 PCR을 수행함으로써 분리균으로부터 니트릴 히드라타아제와 아미다아제 유전자를 확보하게 되었다. 발굴된 유전자의 염기서열을 분석한 결과, 이미 보고된 Rhodococcus erythropolis의 니트릴 히드라타아제 ${\alpha}$-서브유니트과 ${\beta}$-서브유니트 및 아미다아제와 96% 이상의 상동성을 보였다. 따라서 CHBN기질은 분리균의 니트릴 히드라타아제와 아미다아제 효소에 의해서 아미(CHBAm)를 거쳐 산(CHBAc)으로 전환되는 것을 알게 되었다.

• Journal of Applied Biological Chemistry
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• 제43권4호
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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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• 제1권3호
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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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• 제26권2호
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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.