• Journal of Microbiology and Biotechnology
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• 제27권2호
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• pp.289-296
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• 2017

Lysine decarboxylase (CadA) converts ${\small{L}}-lysine$ into cadaverine (1,5-pentanediamine), which is an important platform chemical with many industrial applications. Although there have been many efforts to produce cadaverine through the soluble CadA enzyme or Escherichia coli whole cells overexpressing the CadA enzyme, there have been few reports concerning the immobilization of the CadA enzyme. Here, we have prepared a cross-linked enzyme aggregate (CLEA) of E. coli CadA and performed bioconversion using $CadA^{CLEA}$. $CadA^{free}$ and $CadA^{CLEA}$ were characterized for their enzymatic properties. The optimum temperatures of $CadA^{free}$ and $CadA^{CLEA}$ were $60^{\circ}C$ and $55^{\circ}C$, respectively. The thermostability of $CadA^{CLEA}$ was significantly higher than that of $CadA^{free}$. The optimum pH of both enzymes was 6.0. $CadA^{free}$ could not be recovered after use, whereas $CadA^{CLEA}$ was rapidly recovered and the residual activity was 53% after the $10^{th}$ recycle. These results demonstrate that $CadA^{CLEA}$ can be used as a potential catalyst for efficient production of cadaverine.

• 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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• 제21권11호
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• pp.1159-1165
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• 2011

Biodiesel is methyl and ethyl esters of long-chain fatty acids produced from vegetable oils or animal fats. Lipase enzymes have occasionally been used for the production of this biofuel. Recently, biodiesel production using immobilized lipase has received increased attention. Through enhanced stability and reusability, immobilized lipase can contribute to the reduction of the costs inherent to biodiesel production. In this study, methanol-tolerant lipase M37 from Photobacterium lipolyticum was immobilized using the cross-linked enzyme aggregate (CLEA) method. Lipase M37 has a high lysine content (9.7%) in its protein sequence. Most lysine residues are located evenly over the surface of the protein, except for the lid structure region, which makes the CLEA preparation yield quite high (~93%). CLEA M37 evidences an optimal temperature of $30^{\circ}C$, and an optimal pH of 9-10. It was stable up to $50^{\circ}C$ and in a pH range of 4.0-11.0. Both soluble M37 and CLEA M37 were stable in the presence of high concentrations of methanol, ethanol, 1-propanol, and n-butanol. That is, their activities were maintained at solvent concentrations above 10% (v/v). CLEA M37 could produce biodiesel from olive oil and alcohols such as methanol and ethanol. Additionally, CLEA M37 generated biodiesel via both 2-step methanol feeding procedures. Considering its physical stability and reusability, CLEA M37 may potentially be used as a catalyst in organic synthesis, including the biodiesel production reaction.

• 한국미생물·생명공학회지
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• 제46권3호
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• pp.234-243
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• 2018

남극해에는 산업적으로 유용한 신규 효소촉매를 생산하는 미생물들이 들어 있다. 우리는 로스해(Ross Sea)로부터 분리한 여러 저온성 박테리아를 조사하였으며, 그 중에서 지방분해 능력이 뛰어난 Croceibacter atlanticus (No. 40-F12)를 찾았다. Shotgun 클로닝 방법으로 리파아제 유전자(lipCA)를 찾았으며 Escherichia coli 균에서 LipCA 효소를 발현하였다. Spain Arreo metagenome alpha/beta hydrolase를 기준으로 LipCA 상동구조모델을 만들어서 분석한 결과, ${\alpha}/{\beta}$ hydrolase fold, Gly-X-Ser-X-Gly motif, 그리고 lid 구조를 갖고 있기 때문에 전형적인 리파아제 효소임이 밝혀졌다. Ammonium sulfate 침전법과 겔여과 크로마토그래피를 통해서 세포추출액으로부터 LipCA 효소를 순수하게 분리한 후, 최적 온도, pH, 안정성, 기질특이성, 유기용매 안정성 등의 효소특성을 규명하였다. LipCA를 cross-linked enzyme aggregate (CLEA) 방법으로 고정화하고 효소특성을 조사, 비교하였다. 고정화를 통해 온도, pH, 유기용매에 대한 안정성이 증가하였고 기질특이성의 변화는 관찰되지 않았다. $LipCA^{CLEA}$는 원심분리 방법으로 쉽게 회수되었고 4번의 재사용 후에 40% 이상의 활성이 잔재하였다. 이 논문은 C. atlanticus 리파아제의 발현, 특성규명, Cross-linked Enzyme Aggregated 고정화를 바탕으로 안정성을 높여 산업적 활용 가능성을 제시한 최초의 보고이다.

• 분석과학
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• 제29권3호
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• pp.105-113
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• 2016

고정화 지지체로 사용된 실리카 나노입자와 실리카 코팅된 자성 나노입자에 작용기를 부착시켜 기능성을 부가한 후 효소인 리파아제를 고정화하여 리파아제의 안정성을 향상시키고자 연구를 수행하였다. 지지체에 부착하는 작용기가 고정화된 효소의 활성과 안정성에 미치는 영향도 살펴보았다. 실리카 나노입자와 실리카 코팅된 자성 나노입자에 부착한 작용기인 epoxy group과 amine group은 glycidyl methacrylate과 aminopropyl triethoxysilane을 통해 실리카 나노입자와 실리카 코팅된 자성 나노입자 표면에 각각 부착하였다. 작용기가 부착된 실리카 나노입자와 실리카 코팅된 자성 나노입자에 고정화한 Candida rugosa lipase는 자유효소에 비해 초기반응속도는 다소 낮았지만, 3 회 재사용한 후 측정한 활성이 최초 활성 대비 92 % 이상의 활성을 유지하였다. 또한, 실리카 코팅된 자성 나노입자에 glutaraldehyde를 이용한 cross-linked enzyme aggregate (CLEA) 방법과 공유결합법을 통해 라파아제를 각각 고정화한 연구를 수행한 결과, 실리카 나노입자와 실리카 코팅된 자성 나노입자에 CLEA 방법과 공유결합법으로 각각 고정화한 Candida rugosa lipase는 자유효소에 비해 초기반응속도 뿐만 아니라 최종 활성도 높았고, 5 회 재사용한 후 측정한 활성이 최초 활성 대비 73 % 이상의 활성을 유지하였다.