조효소를 함유하지 않는 효모의 Homocysteine 분해효소, Cystathionine β-Synthase의 생화학적 특성

지광환 ( Kwang Hwan Jhee ),조현남 ( Hyun Nam Cho ),양선아 ( Seun Ah Yang ),이인선 ( In Seun Lee ) 한국미생물생명공학회 2007 한국미생물·생명공학회지 Vol.35 No.3

Spectroscopic Characterization of the Active Site Mutation of Cystathionine β-Synthase which causing Homocystinuria

Kwang-Hwan Jhee(지광환) 한국생물공학회 2005 한국생물공학회 학술대회 Vol.2005 No.10

HPLC를 이용한 신기능성 고분자, 폴리감마 글루탐산의 입체구조 조성비의 정량

유동찬,지광환 금오공과대학교 2005 論文集 Vol.26 No.-

Poly-v-glutamic acid (γ-PGA) is an unusual anionic polypeptide in which D-and/or L-glutamate is polymerized via γ-amide linkages. Reversed-phase high-performance liquid chromatography and pre-column derivation with 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (Marley's reagent) were used for assaying the stereotype composition of L and D-glutamic acid followed by the total hydrolysis of γ-PGA The determination of the stereotype ratio for the γ-PGA is important for the application for its industrial use. Our rrethod is more convenient and reproducible compared with the previous method that is using chiral colurrm for the separation of the racemates of amino acids. Our data obtained with HPLC method exhibited good quantitative correlation with that from enzymatic analysis of γ-PGA Our data would be a useful and convenient method for quality control for the mass production of γ-PGA.

γ-PGA에 포함된 D-glutamate의 효소화학적 정량분석

조현정,지광환 금오공과대학교 2005 論文集 Vol.26 No.-

Bacillus subtilis chungkookjang produces a new biofunctional polymer, poly y-glutamic acid (γ-PGA). γ-PGA consists of D and/or L-glutamate and its ratio is different depending microorganisms and culturing environrrents, Here we are reporting the new enzymatic method to determine the stereochemistry of D-glutamic acid composition ratio in γ-PGA D-amino acid aminotransferase (D-AAT) can catalyzes transamination between D-amino acids and a-keto acids. Thus the amount of D-glutamate in γ-PGA can be determined by the assaying the amount of produced D-alanine followed by the reaction of D-AAT in the presence of pyruvic acid. D-glutamate and D-alanine can be separated by thin layer chromatography (TLC) in NH₄0H : ethanol (2:8, v/v) solvent system D-alanine can be visualized by ninhydrin (0.5% in 75% ethanol) spraying and quantitated by the measurement the absorbance of TLC spot followed by the elution with 0.005% copper sulfate in 75% ethanol at EOOnm. Partially purified D-AAT is good enough to carry out this new and very convenient method to determine the amount of D-glutamic acid in γ-PGA.

나노 은을 광증착시킨 폴리(에틸렌 테레프탈레이트)/나노-TiO₂ 복합체의 항균특성 연구

조성민,지광환,김정현,권오경,임상규,민병길,Jo, Seong-Min,Jhee, Kwang Hwan,Kim, Jung Hyun,Kwon, Oh Kyung,Lim, Sang Kyoo,Min, Byung Gil 한국섬유공학회 2012 한국섬유공학회지 Vol.49 No.6

Nanocomposite films of poly(ethylene terephthalate)(PET) and nano titania($TiO_2$) were prepared by melt compounding with a twin-screw extruder followed by hot pressing for the purpose of investigating antibacterial activity. FE-SEM, EDS and XRD measurements confirmed that nano $TiO_2$ was successfully dispersed at the level of individual particles in the PET matrix. Silver was introduced through photocatalytic reduction by nano $TiO_2$ under UV (254 nm) irradiation to the nanocomposite films after being immersed in aqueous solution $AgNO_3$ (100 ppm Ag ion). Even at 1 wt% of $TiO_2$ content, the nanocomposite with silver photodeposition exhibited strong antimicrobial activity against Klebsiella pneumoniae and Staphylococcus aureus in measurement by shaking flask method.

인간 Cystathionine ${\gamma}$-Lyase에 의한 Selenomethionine의 Methylselenol로의 직접분해

조현남,지광환,Cho, Hyun-Nam,Jhee, Kwang-Hwan 한국미생물·생명공학회 2014 한국미생물·생명공학회지 Vol.42 No.1

Selenium(Se)은 사람에게 필수성분이지만 독성이 강한 물질이다. 따라서 생체에서 Se(셀레늄)의 농도조절은 정확하고 효율적으로 이루어져야 한다. 사람은 Se을 음식의 형태로 섭취해야 하고 주로 seleno-L-methionine (L-SeMet)의 형태로 섭취한다. 섭취된 L-SeMet은 methionine 대사와 동일한 효소를 이용하여 Se-adenosyl-L-SeMet으로 대사된다고 알려져 있었다. 그러나 최근 쥐의 간 추출물의 실험에서, L-SeMet이 cystathionine ${\gamma}$-lyase (mouse CGL)의 작용으로 methylselenol ($CH_3SeH$)로 직접적으로 대사된다는 보고가 있었다. CGL은 원래 cystathionine을 L-cysteine과 ${\alpha}$-ketobutyrate, 그리고 $NH_3$로 분해하는 효소로 알려져 있었다. 따라서 본 연구에서는 쥐의 간 추출물 대신에 인간의 CGL을 분리 정제하여 L-SeMet에서 methylselenol의 형성을 확인하고자 하였다. Methylselenol의 표준시료는 dimethyldiselenide를 sodium tetrahydroborate로 환원시켜 준비하였다. 그리고 L-SeMet을 기질로 사용한 효소 반응액 중에서 가스상의 생성물은 GC/MS 스펙트럼으로 분석하였다. 분석 결과 methylselenol의 유도체인 dinitrophenyl selenoether와 일치하였다. 또한 인간 CGL이 L-SeMet에서 methylselenol을 형성하는 반응의 kinetic parameter를 mouse CGL과 비교 분석하였다. 결과적으로 human CGL은 섭취된 L-SeMet의 대사를 책임지고 있는 중요한 효소이다. Selenium is an essential trace element for mammals, but it is very toxic. Therefore, the control of selenium concentrations should be precisely and effectively monitored. Selenium is naturally obtained through foods and seleno-L-methionine (LSeMet) is a major form of selenium. It has been reported that L-SeMet is only converted into Se-adenosyl-L-SeMet. However, a recent study suggested that L-SeMet was directly metabolized into methylselenol ($CH_3SeH$) in mouse liver extract by the reaction of cystathionine ${\gamma}$-lyase (CGL). The canonical reaction of CGL was known to catalyze the cleavage of L-cystathionine to L-cysteine, ${\alpha}$-ketobutyrate and $NH_3$. In the present study, we found that L-SeMet could be directly converted to $CH_3SeH$ using purified homogenous human CGL instead of mouse liver cytosol. Authentic $CH_3SeH$ was prepared by reduction of dimethyldiselenide with sodium tetrahydroborate. The gaseous product of the enzymatic reaction with L-SeMet was analyzed by GC/MS spectrometry. The GC/MS data was identical to that of authentic dinitrophenyl selenoether. We also analyzed the kinetic parameters for the formation of $CH_3SeH$ from L-SeMet by human and mouse CGL. These results suggest that human CGL is a critical enzyme which is responsible for L-SeMet metabolism.

산성 pH 조건에서 차아염소산나트륨의 항균 활성 향상

손현빈,배원빈,지광환,Son, Hyeon-Bin,Bae, Won-Bin,Jhee, Kwang-Hwan 한국미생물·생명공학회 2022 한국미생물·생명공학회지 Vol.50 No.2

차아염소산나트륨(NaClO)은 병원 및 식품산업 분야에서 널리 사용되는 소독제로 세균, 곰팡이, 바이러스에 대해서도 항균 활성이 있다. 차아염소산나트륨의 항균 활성은 용액의 pH에 의해 조절되는 안정적인 HClO 농도의 유지에 있다. 차아염소산(HClO)은 화학적으로 중성이므로 세균의 막에 쉽게 침투할 수 있으며 차아염소산나트륨의 항균 활성은 차아염소산염 이온(ClO^-)보다는 용액 내 HClO 농도에 의존하리라 사료된다. 본 연구에서 pH 조절에 따른 차아염소산나트륨의 항균 활성을 time kill test와 차아염소산나트륨 처리 전후의 활성산소종(ROS) 및 ATP 농도 변화로 조사하였다. 또한 전계방출형 주사 전자 현미경(FE-SEM)을 통하여 세포벽의 파괴정도를 확인하였다. pH 5 조건에서 5 ppm 차아염소산나트륨은 Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) 균에 대하여 99.9%의 항균 활성을 나타내었고, ROS 생성량은 pH 7 조건보다 48% 증가하였다. 또한, pH 5 조건의 차아염소산나트륨에 노출된 E. coli와 S. aureus의 ATP 농도가 각각 94%와 91% 감소하였다. FE-SEM 결과, pH 5 조건에 노출된 균의 세포벽이 파괴된 것을 확인하였다. 본 연구결과를 종합해보면, pH를 조절하는 것 만으로 5 ppm 농도의 차아염소산나트륨의 항균 활성을 향상시킬 수 있음을 시사한다. Sodium hypochlorite (NaClO) is a disinfectant widely used in hospitals and food industries because of its antimicrobial activity against not only bacteria but also fungi and virus. The antibacterial activity of NaClO lies in the maintenance of a stable hypochlorous acid (HClO) concentration, which is regulated by pH of the solution. HClO can easily penetrate bacterial cell membrane due to its chemical neutrality and the antibacterial activity of NaClO is thought to depend on the concentration of HClO in solution rather than hypochlorite ions (ClO^-). In this study, we investigated the antibacterial activity of NaClO according to pH adjustment by means of time kill test and assays of Reactive Oxygen Species (ROS) and adenosine triphosphate (ATP) concentration changes before and after NaClO treatment. We also investigated that the degree of cell wall destruction through field emission scanning electron microscopy (FE-SEM). Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) exposed to 5 ppm NaClO at pH 5 exhibited 99.9% mortality. ROS production at pH 5 was 48% higher than that produced at pH 7. In addition, the ATP concentration in E. coli and S. aureus exposed to pH 5 decreased by 94% and 91%, respectively. As a result of FE-SEM, it was confirmed that the cell wall was destroyed in the bacteria by exposing to pH 5 NaClO. Taken together, our results indicate that the antibacterial activity of 5 ppm NaClO can be improved simply by adjusting the pH.

목분-폴리프로필렌 복합체의 기계적, 열적 및 난연특성

박기훈,신백우,지광환,방대석 한국방재학회 2014 한국방재학회논문집 Vol.14 No.2

We manufactured wood flour-polypropylene(PP) composites by a modular intermeshing co-rotating twin screw extruder. The limitingoxygen index(LOI) and cone calorimeter experiments were carried out in order to analyze flame retardant characteristics. Andmechanical properties were measured by universal testing machine(UTM). The specimens for these tests were prepared by addingfour types of flame retardants such as DBDPO(decabromodiphenyl oxide), APP(ammonium polyphosphate), ATH(aluminium trihydroxide)and MP(melamine phosphate). It was found that mechanical properties of the composites with flame retardants showedhigher tensile strength and flexural strength than those of control wood flour-PP composites. Also the results of cone calorimeter testindicated that adding flame retardants produced lower heat release rate and mean heat release rate compared to the wood flour-PPcomposites. Among the four flame retardants, melamine phosphate had the most effective flame retarding characteristics. 모듈라 치합형 동방향회전 이축스크류식 압출기를 이용하여 목분-PP 복합체를 제조하였다. 난연 특성을 분석하기 위하여 한계산소지수 실험 및 콘칼로리미터 실험을 실시하였으며, UTM 시험을 통하여 목분-폴리프로필렌 복합체의 기계적 특성을 분석하였다. 할로겐계(DBDPO), 인계(APP), 무기계(ATH), 멜라민계(MP) 난연제 네 종류를 각각 첨가한 복합체 시편을 준비하였고 이들 복합체의 난연및 기계적 특성을 측정하여 순수 목분-PP 복합체보다 난연제가 첨가된 복합체의 인장강도 및 굴곡강도가 향상되었음을 확인하였다. 또한 콘칼로리미터 실험결과 난연제를 첨가한 복합체가 열방출률 및 평균열방출량 모두 순수 목분-PP 복합체 보다 낮았다. 네 가지 난연제 중 멜라민 포스페이트의 난연특성이 가장 효과적임을 확인하였다.

목련 꽃 에센셜 오일의 항균 및 항염증 활성

이재열(Jae-Yeul Lee),지광환(Kwang-Hwan Jhee),양선아(Seun-Ah Yang) 한국생명과학회 2020 생명과학회지 Vol.30 No.3

목련은 항산화효과와 진정작용과 같은 다양한 효능을 가진 것으로 알려져 있다. 본 연구에서는 목련 에센셜오일의 항균 활성 및 항염증 활성을 검증하고자 한다. 목련 에센셜 오일은 증류법으로 추출한 것을 사용하였으며, 항균 활성은 S. aureus와 E. coli, P. aeruginosa에 대하여 paper disk diffusion법과 최소저해농도(minimum inhibitory concentration, MIC) 값을 측정하여 확인하였다. 목련 에센셜 오일은 S. aureus 균주에 대하여 18 mm의 clear zone형성과 0.25 mg/ml의 MIC 값을 나타내는 우수한 항균효과를 보였다. P. aeruginosa 균주와 E. coli 균주는 clear zone 형성이 각각 14 mm와 17 mm로 나타났다. 또한, MIC값은 각각 1 mg/ml와 0.5 mg/ml로 측정되었다. 항염증 활성은 LPS 유도된 RAW264.7 세포에서 nitric oxide (NO)와 PGE₂의 생성억제율을 조사하였다. 목련 에센셜 오일, 500 μg/ml의 농도에서 RAW264.7세포에 대한 세포 독성은 나타내지 않았지만, NO (37.7%)와 PGE₂ (24.0%)억제를 보였다. GC-MS를 이용하여 목련 에센셜 오일을 분석하였다. 목련 에센셜 오일의 3 가지 주요 방향제 성분은 3-carene (77.07%), β-elemene (6.92%) 및 caryphyllene (2.86%)로 분석되었다. 이러한 결과는 목련 에센셜 오일이 항균 및 항염증 효과를 갖는 화장품 기능성 물질로서 잠재력을 가지고 있음을 시사한다. Magnolia kobus is known to exert various biological effects, such as antioxidant and hypnotic activity. In this study, we investigated the antimicrobial and anti-inflammatory activity of M. kobus essential oil extracted using steam distillation. Its antimicrobial activity was tested against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa by the paper disk diffusion and minimum inhibitory concentration (MIC) methods. Its anti-inflammatory activity was evaluated by measuring its inhibition ratio on the production of nitric oxide (NO) and PGE₂ in lipopolysaccharide (LPS)-induced RAW264.7 cells. Its composition was analyzed by gas chromatography–mass spectrometry (GC-MS). The results showed that M. kobus essential oil exhibited excellent antibacterial activity against S. aureus, with a clear zone of 18 mm and an MIC value of 0.25 mg/ml. Its clear zones against P. aeruginosa and E. coli were 14 mm and 17 mm, respectively, while its MIC values were 1 mg/ml and 0.5 mg/ml, respectively. The essential oil exhibited no cytotoxicity to the RAW264.7 cells at a concentration of 500 μg/ml while showing NO (37.7%) and PGE₂ inhibition (24.0%). Its three main fragrance ingredients identified were 3-carene (77.07%), β-elemene (6.92%), and caryphyllene (2.86%). The results suggest that M. kobus essential oil has potential as a cosmetic functional material with antimicrobial and anti-inflammatory effects.

인간 Cystathionine γ-Lyase에 의한 Selenomethionine의 Methylselenol로의 직접분해

조현남 ( Hyun Nam Cho ),지광환 ( Kwang Hwan Jhee ) 한국미생물생명공학회(구 한국산업미생물학회) 2014 한국미생물·생명공학회지 Vol.42 No.1

Selenium(Se)은 사람에게 필수성분이지만 독성이 강한 물질이다. 따라서 생체에서 Se(셀레늄)의 농도조절은 정확하고 효율적으로 이루어져야 한다. 사람은 Se을 음식의 형태로 섭취해야 하고 주로 seleno-L-methionine (L-SeMet)의 형태로 섭취한다. 섭취된 L-SeMet은 methionine 대사와 동일한 효소를 이용하여 Se-adenosyl-L-SeMet으로 대사된다고 알려져 있었다. 그러나 최근 쥐의 간 추출물의 실험에서, L-SeMet이 cystathionine γ-lyase (mouse CGL)의 작용으로 methylselenol(CH3SeH)로 직접적으로 대사된다는 보고가 있었다. CGL은 원래 cystathionine을 L-cysteine과 α-ketobutyrate, 그리고 NH3로 분해하는 효소로 알려져 있었다. 따라서 본 연구에서는 쥐의 간 추출물 대신에 인간의 CGL을 분리 정제하여 LSeMet에서 methylselenol의 형성을 확인하고자 하였다. Methylselenol의 표준시료는 dimethyldiselenide를 sodium tetrahydroborate로 환원시켜 준비하였다. 그리고 L-SeMet을 기질로 사용한 효소 반응액 중에서 가스상의 생성물은 GC/MS 스펙트럼으로 분석하였다. 분석 결과 methylselenol의 유도체인 dinitrophenyl selenoether와 일치하였다. 또한 인간 CGL이 L-SeMet에서 methylselenol을 형성하는 반응의 kinetic parameter를 mouse CGL과 비교 분석하였다. 결과적으로 human CGL은 섭취된 L-SeMet의 대사를 책임지고 있는 중요한 효소이다. Selenium is an essential trace element for mammals, but it is very toxic. Therefore, the control of selenium concentrations should be precisely and effectively monitored. Selenium is naturally obtained through foods and seleno-L-methionine (LSeMet) is a major form of selenium. It has been reported that L-SeMet is only converted into Se-adenosyl-L-SeMet. However, a recent study suggested that L-SeMet was directly metabolized into methylselenol (CH3SeH) in mouse liver extract by the reaction of cystathionine γ-lyase (CGL). The canonical reaction of CGL was known to catalyze the cleavage of L-cystathionine to L-cysteine, α-ketobutyrate and NH3. In the present study, we found that L-SeMet could be directly converted to CH3SeH using purified homogenous human CGL instead of mouse liver cytosol. Authentic CH3SeH was prepared by reduction of dimethyldiselenide with sodium tetrahydroborate. The gaseous product of the enzymatic reaction with L-SeMet was analyzed by GC/MS spectrometry. The GC/MS data was identical to that of authentic dinitrophenyl selenoether. We also analyzed the kinetic parameters for the formation of CH3SeH from L-SeMet by human and mouse CGL. These results suggest that human CGL is a critical enzyme which is responsible for L-SeMet metabolism.