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토양에서 분리된 Aeromonas hydrophila AM-28이 생산하는 단백질 가수분해효소의 특성
김인숙,김형권,이정기,배경숙,오태광,Kim, In-Sook,Kim, Hyung-Kwoun,Lee, Jung-Kee,Bae, Kyung-Sook,Oh, Tae-Kwang 한국미생물학회 1994 미생물학회지 Vol.32 No.4
토양으로부터 탈지대두박을 잘 분해하는 단백질 가수분해효소를 생산하는 미생물을 분리하여 형태적, 생화학적 특성과 세포벽 지방산조성을 조사한 결과, 분리된 세균은 Aeromonas hydrophila로 동정되었다. 분리된 세균은 glycerol을 탄소원으로, tryptone 및 $(NH_4)_2HPO_4$을 진소원으로, $CaCl_2$를 무기원으로 사용한 배지에서 효소를 가장 많이 생산하였으며, 생산된 효소의 반응최적 pH는 8.0이고, 반응최적온도는 $65^{\circ}C$였다. 이 효소는 $55^{\circ}C$ 이하의 온도에서, pH 7.0에서 13.0의 범위에서 비교적 안정한 것으로 나타났다. 또한 이 효소는 금속이온 $Cu^{2+},\;Hg^{2+}.\;Mn^{2+}$ 및 $Zn^{2+}$에 의해서 효소활성이 저해를 받고, 효소저해제 phenylmethylsulfonyl fluoride (PMSF)와 ethylenediaminetetraacetic acid (EDTA)에 의해서 효소활성이 강력히 저해되는 성질로부터 효소활성부위에 serine기와 금속이온을 갖고 있는 것으로 밝혀졌다. A bacterial strain NO. AM-28, showing proteolytic activity against defatted soybean was isolated from domestic soil. The isolated strain was identified as Aeromonas hydrophila by both the biochemical tests using API kit and the analysis of cellular fatty acid profile with MIDI system. The protease production from A. hydrophila AM-28 was highly enhanced when it was cultivated in the medium containing glycerol as a carbon source, tryptone or $(NH_4)_2HPO_4$ as a nitrogen source, and $CaCl_2$ as a mineral source. The optimal pH and temperature for the enzyme was 8.0 and $65^{\circ}C$, respectively. The enzyme was stable up to $55^{\circ}C$ and at pH values ranging from 7.0 to 13.0. The enzyme activity was inhibited by phenylmethylsulfonyl fluoride and EDTA, indicating that serine residue and metal ions be involved in enzyme activity.
남극 로스해에서 분리한 Croceibacter atlanticus 균 유래 리파아제의 생산, 고정화, 효소특성 연구
박채경 ( Chae Gyeong Park ),김형권 ( Hyung Kwoun Kim ) 한국미생물생명공학회(구 한국산업미생물학회) 2018 한국미생물·생명공학회지 Vol.46 No.3
The Antarctic Ocean contains numerous microorganisms that produce novel biocatalysts that can have applications in various industries. We screened various psychrophilic bacterial strains isolated from the Ross Sea and found that a Croceibacter atlanticus strain (Stock No. 40-F12) showed high lipolytic activity on a tributyrin plate. We isolated the corresponding lipase gene (lipCA) by shotgun cloning and expressed the LipCA enzyme in Escherichia coli cells. Homology modeling of LipCA was carried out using the Spain Arreo lake metagenome alpha/beta hydrolase as a template. According to the model, LipCA has an α/β hydrolase fold, Gly-X-Ser-X-Glymotif, and lid sequence, indicating that LipCA is a typical lipase enzyme. Active LipCA enzyme was purified fromthe cell-free extract by ammonium sulfate precipitation and gel filtration chromatography. We determined its enzymatic properties including optimum temperature and pH, stability, substrate specificity, and organic solvent stability. LipCA was immobilized by the cross-linked enzyme aggregate (CLEA) method and its enzymatic properties were compared to those of free LipCA. After cross-linking, temperature, pH, and organic solvent stability increased considerably, whereas substrate specificities did not changed. The LipCA CLEA was recovered by centrifugation and showed approximately 40% activity after 4th recovery. This is the first report of the expression, characterization, and immobilization of a C. atlanticus lipase, and this lipase could have potential industrial application.
Proteus vulgaris에서 유래한 리파아제의 고정화 및 바이오디젤 생산
윤신아 ( Shin Ah Yoon ),한진이 ( Jin Yee Han ),김형권 ( Hyung Kwoun Kim ) 한국미생물생명공학회(구 한국산업미생물학회) 2011 한국미생물·생명공학회지 Vol.39 No.3
바이오디젤은 긴 사슬 지방산의 알킬 에스테르로서 동물성 지방 또는 식물성 오일과 알코올이 반응하여 에스테르 교환반응에 의해 생성되는 대체연료이다. 지난 십여 년 동안, 다양한 리파아제를 이용한 바이오디젤 생산에 대해 연구되었다. 하지만 효소 촉매 공정을 통한 바이오디젤 생산의 경우, 높은 효소 단가로 산업적 공정에 쉽게 적용할 수 없었다. 이러한 문제점을 극복하기 위해, 저렴한 오일 원료를 선택하거나, 바이오디젤 생산에 적합한 리파아제를 스크리닝하는 과정 또는 리파아제 고정화 방법이 활발히 연구되었다. 이번 연구에서는 P. vulgaris에서 유래한 리파아제 K80을 E. coli균에서 발현하여 얻은 효소액으로 바이오디젤을 생산하였다. 재조합리파아제 K80은 높은 발현량을 보였으며, 높은 가수분해 반응의 비활성도(specific activity)와 유기용매에서 높은 안정성을 확인했다. 리파아제 K80은 올리브 오일과 메탄올을 3-stepwise 방법을 이용하여 바이오디젤을 생산할 수 있었다. 리파아제 K80을 소수성 결합을 이용하여 담체 표면에 흡착시켜 얻은 고정화 K80을 이용하여 수용성 리파아제 K80과 동일한 방법으로 바이오디젤을 생산한 결과, 효율적으로 바이오디젤 생산을 확인했다. 고정화 K80은 다양한 식물성 오일과 메탄올을 사용하여 효과적으로 바이오디젤을 생산하였다. 고정화 K80을 이용하여 바이오디젤 생산뿐만 아니라 다른 산업적 공정에서도 활용할 수 있을 것으로 기대한다. Biodiesel, mono-alkyl esters of long chain fatty acids, is one of the alternative fuels derived from renewable lipid feedstock, such as vegetable oils or animal fats. For decade, various lipases have been used for the production of biodiesel. However, the production of biodiesel by enzymatic catalyst has profound restriction in industry application due to high cost. To overcome these problems, many research groups have studied extensively on the selection of cheap oil sources, the screening of suitable lipases, and development of lipase immobilization methods. In this study, we produced biodiesel from plant oil using Proteus vulgaris lipase K80 expressed in Escherichia coli cells. The recombinant lipase K80 was not only expressed in high level but also had high specific lipase activity and high stability in various organic solvents. Lipase K80 could produce biodiesel from olive oil by 3-stepwise methanol feeding method. The immobilized lipase K80 also produced biodiesel using the same 3-stepwise method. The immobilized lipase could produce biodiesel efficiently from various plant oils and waste oils.
메타게놈유래의 저온성 에스터라제 EM2L8의 효소적 특성과 이를 활용한 고지혈증 치료제 키랄소재의 생산
정지혜 ( Ji Hye Jung ),최윤희 ( Yun Hee Choi ),이정현 ( Jung Hyun Lee ),김형권 ( Hyung Kwoun Kim ) 한국미생물생명공학회 2009 한국미생물·생명공학회지 Vol.37 No.2
Esterase EM2L8 gene isolated from deep sea sediment was expressed in Escherichia coli BL21 (DE3) and the esterase activity of the cell-free extract was assayed using p-nitrophenyl butyrate-spectrophotometric method. Its optimum temperature was 40-45℃ and 45% activity of the maximum activity was retained at 15℃. The activation energy at 15-45℃ was calculated to be 4.9 kcal/mol showing that esterase EM2L8 was a typical cold-adapted enzyme. Enzyme activity was maintained for 6 h and 4 weeks at 30℃ and 4℃, respectively. When each ethanol, methanol, and acetone was added to the reaction mixture to 15% concentration, enzyme activity was maintained. In the case of DMSO, enzyme activity was kept up to 40% concentration. (S)-4-Chloro-3-hydroxy butyric acid is a chiral intermediate for the synthesis of Atorvastatin, a hyperlipemia drug. When esterase EM2L8 (40 U) was added to buffer solution (1.2 mL, pH 9.0) containing ethyl-(R,S)-4-chloro-3-hydroxybutyrate (38 mM), it was hydrolyzed into 4-chloro-3-hydroxy butyric acid with a rate of 6.8 μmole/h. The enzyme hydrolyzed (S)-substrate more rapidly than (R)-substrate. When conversion yield was 80%, e.e.s value was 40%. When DMSO was added, hydrolysis rate increased to 10.4 μmole/h. The plots of conversion yield vs e.e.s in the presence or absence of DMSO were almost same, implying that the reaction enantioselectivity was not changed by the addition of DMSO. Taken together, esterase EM2L8 had high activity and stability at low temperatures as well as in various organic solvents/aqueous solutions. These properties suggested that it could be used as a bioctalyst in the synthesis of useful pharmaceuticals.
고온성 Bacillus amyloliquefaciens NS 15-4가 생산하는 내열성 Protease의 특성
김형권,김기현,이정기,김영옥,남희섭,오태광 한국산업미생물학회 1995 한국미생물·생명공학회지 Vol.23 No.3
탈지대두박의 분해능이 뛰어난 단백질가수분해 효소를 생산하는 고온성 세균을 토양으로부터 분리하였다. 이 균은 형태적, 생리적 특성으로부터 Bacillus amyloliquefaciens로 동정되었다. 이 균을 탈지대두박이 포함된 배지에 접종하고 50℃에서 진탕배양한 후, 균이 생산한 단백질가수분해효소를 ammonium sulfate 침전, DEAE-, CM-sepharose, phenyl-sepharose column을 통해 분리하였다. SDS-PAGE로부터 이 효소의 분자량이 약 30,000 임이 밝혀졌으며 N 말단의 아미노산 서열이 AQSVPYGISQIKAPA인 것으로 분석되었다 .이 효소의 반응최적온도는 60℃, 반응최적 pH는 11이었고, Ca^(++)에 의해 효소의 열안정성이 증가하였다. 또한 이 효소는 PMSF에 의해서 효소활성이 저해되는 serine protease로 판명되었다. 특히 기존의 다른 단백질가수분해효소와 비교하였을 때 물에 대한 용해도가 낮은 탈지대두박에 대해서 가수분해 역가가 큰 것으로 밝혀졌다. A thermophilic bacteria showing proteolytic activity against defatted soybean was isolated from soil. It was identified as Bacillus amyloliquefaciens based on its morphological and physiological characteristics. The Bacillus amyloliquefaciens NS 15-4 was cultivated at 50℃ by rotary shaking in a medium containing defatted soybean. An extracellular protease from this strain was purified to homogeneity by ammonium sulfate precipitation, ion exchange, and hydrophobic interaction chromatographies. The molecular weight of the enzyme was estimated to be approximately 30,000 by SDS-PAGE and the N-terminal amino acid sequence of the enzyme was turned out to be AQSVPYGISQIKAPA. The optimum temperature and pH for the enzyme reaction were 60℃ and 11, respectively, and its thermostability was increased by the addition of calcium ion. The enzyme was inactivated by phenylmethylsulfonylfluoride, suggesting it be a serine protease. Comparing with other commercial proteases, the enzyme showed relatively high proteolytic activity against defatted soybean, a water-insoluble protein substrate.