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유영제,박경문,박재연,Myung Jin Shin,Seung Hoon Song 한국생물공학회 2007 Biotechnology and Bioprocess Engineering Vol.12 No.6
A novel sol-gel immobilization method employing a detergentless micro-emulsion system that consisted of n-hexane/iso-propanol/water was developed and used to immobilize a horseradish peroxidase (HRP). Micro-sized gel powder containing enzymes was generated in the ternary solution without drying and grinding steps or the addition of detergent, therefore, the method described in this study is a simple and straightforward process for the manufacture of gel powder. The gel powder made in this study was able to retain 84% of its initial enzyme activity, which is higher than gel powders produced through other immobilization methods. Furthermore, the HRP immobilized using this method, was able to maintain its activity at or above 95% of its initial activity for 48 h, whereas the enzyme activities of free HRP and HRP that was immobilized using the other sol-gel method decreased dramatically. In addition, even when in the presence of excess hydrogen peroxide, the enzyme immobilized using the novel sol-gel method described here was more stable than enzymes immobilized using the other method.
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유영제 한국미생물학회 1990 微生物과 産業 Vol.16 No.2
1970년대의 유전자재조합 및 세포융합 기술개발에 의하여 insulin, interferon 등의 많은 의약품이 개발되고 있으며, 최근에는 아미노산, 효소 등 정밀 화학 제품의 개발, 석유화학 대체공정의 개발, 환경 보존에의 응용, 대체에너지의 개발등을 목표로 많은 연구가 수행되고 있어 생물공학의 분야가 계속 영역을 넓혀가고 있다. 실험실에서 개발한 생물공학의 연구결과를 실용화하기 위하여는 경제성있는 생물 공정기술이 뒷받침되어야 하는데 이러한 생물공정 기술 중에서 핵심이 되는 부분의 하나가 생물반응기 기술이라고 하겠다. 우수한 생물반응기를 설계하고 경제적으로 운전하기 위해서는 공정연구및 개발단계에서 최적화 및 제어에 대한 개념을 포함시켜야하며, 실제 조업을 하는 경우에도 제품 품질의 균일성, 에너지 및 자원의 절약, 운전실수의 방지및 안전을 목적으로 조업조건을 최적화하고 제어하여야한다. 또한 접종상태 및 생물반응 조건의 사소한 차이에도 원하는 제품의 생합성이 다륵 변화하므로 생물공정을 monitoring하여 사소한 변화라도 공정의 최적화및 제어전략에 반영되도록 하여야 한다. 본고에서는 생물반응기의 최적화 및 제어기술에 대하여 몇가지 예를 들어 그 필요성과 최근의 연구동향을 간단히 소개하고자 한다.
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고정화 효소반응기에서의 민감도 분석 및 연속 변수 추정
유영제,이중헌 한국화학공학회 1989 Korean Chemical Engineering Research(HWAHAK KONGHA Vol.27 No.4
생촉매 반응기를 on-line으로 최적화하여 제어하기 위해서는 반응 속도상수를 정확히 추정하여야 한다. 고정화 효소에 의하여 포도당이 과당으로 전환되는 반응을 예로들어 생성물의 농도를 측정하여 반응속도식에서 민감도가 제일 큰 비활성화 속도상수를 추정하였다. 온도가 일정하게 유지되는 운전조건 및 시간에 따라 온도가 변하는 최적제어 조건하에서 시간에 따른 민감도가 변수 추정에 미치는 영향을 분석하였다. 민감도를 Kalman filtering에 고려함으로서 민감도가 낮은 운전초기에 추정값의 진동이 감소하며 민감도가 증가함에 따라 참값에 잘 수렴함을 알 수 있었다. Accurate estimation of the kinetic parameters is essential for on-line optimization and control in biocatalytic reactor system. Fructose production from glucose using immobilized enzyme was chosen as a model system and deactivation constant which is the most sensitive among the kinetic parameters was estimated from the measurements of product concentration. The effects of sensitivity on the sequential parameter estimation were analyzed under the operating conditions of both constant temperature and varying temperature. By considering the parameter sensitivity on Kalman filtering, estimated values showed reduced fluctuations in the initial period of operations where the sensitivity was low and better convergence to the true value as the sensitivity increased.
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Selection of Mediators for Bioelectrochemical Nitrate Reduction
유영제,Seung Hwan Kim,Seung Hoon Song 한국생물공학회 2005 Biotechnology and Bioprocess Engineering Vol.10 No.1
The bioelectrochemical reduction of nitrate in the presence of various mediators including methyl viologen and azure A was studied using a 3-electrode voltammetric system. The catalytic potential for the reduction of the mediators was observed in the reactor, which for methyl viologen and azure A were -0.74 V and -0.32 V, respectively, with respect to the potential of Ag/AgCl reference electrode. This potential was then applied to a working electrode to reduce each mediator for enzymatic nitrate reduction. Nitrite, the product of the reaction, was measured to observe the enzymatic nitrate reduction in the reaction media. Methyl viologen was observed as the most efficient mediator among those tested, while azure A showed the highest electron efficiency at the intrinsic reduction potential when the mediated enzyme reactions were carried out with the freely solubilized mediator. The electron transfer of azure A with respect to time was due to the adhesion of azure A to the hydrophilic surface during the reduction. In addition, the use of the adsorbed mediator on conductive activated carbon was proposed to inhibit the change in the electron transfer rate during the reaction by maintaining a constant mediator concentration and active surface area of the electrode. Azure A showed better than nitrite formation than methyl viologen when used with activated carbon.
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유영제,송승훈,Seung Hwan Kim 한국생물공학회 2006 Biotechnology and Bioprocess Engineering Vol.11 No.1
In this study, we have purified and characterized the membrane bound nitrate reductase obtained from the denitrifying bacteria, Ochrobactrum anthropi SY509, which was isolated from soil samples. O. anthropi SY509 can grow in minimal medium using nitrate as a nitrogen source. We achieved an overall purification rate of 15-fold from the protein extracted from the membrane fraction, with a recovery of approximately 12% of activity. The enzyme exhibited its highest level of activity at pH 5.5, and the activity was increased up to 70℃. Periplasmic and cytochromic proteins, including nitrite and nitrous oxide reductase, were excluded during centrifugation and were verified using enzyme essay. Reduced methyl viologen was determined to be the most efficient electron donor among a variety of anionic and cationic dyestuffs, which could be also used as an electron donor with dimethyl dithionite. The effects of purification and storage conditions on the stability of enzyme were also investigated. The activity of the membrane-bound nitrate reductase was stably maintained for over 2 weeks in solution. To maintain the stability of enzyme, the cell was disrupted using sonication at low temperatures, and enzyme was extracted by hot water without any surfactant. The purified enzyme was stored in solution with no salt to prevent any significant losses in activity levels.
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