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Purification and Characterization of a Thermostable Protease from Pseudomonas aeruginosa NS-83
Kim, Hyung Kwoun,Kim, Kee Hyun,Lee, Jung Kee,Bae, Kyung Sook,Sung, Chang,Oh, Tae Kwang 한국미생물 · 생명공학회 1994 Journal of microbiology and biotechnology Vol.4 No.2
A bacterial strain NS-83 isolated from soil was able to produce an extracellular thermostable protease. The strain was identified as Pseudomonas aeruginosa based on its morphological and physiological characteristics. A thermostable protease from this strain has been purified to homogeneity as judged by SDS-PAGE and isoelectric focusing. The purification procedures included hydrophobic interaction, ion exchange, and gel filtration chromatography. The M_r and the pl of the enzyme were 32,000 and 5.9, respectively. The optimal pH at 55℃ and the optimal temperature at pH 7.0 were 8.0 and 60℃, respectively. The D-values of the enzyme at 60, 65, and 70℃ were 22, 2.1, and 0.75 hrs, respectively. The enzyme activity was significantly inhibited in the presence of 1 mM o-phenanthroline or EDTA, suggesting that the enzyme is metalloprotease. The K_m and V_max for Hammarsten casein were found to be 3.2 ㎎/㎖ and 0.918 unit/㎖, respectively. These enzymatic properties were similar to those of elastase produced from P. aeruginosa IFO 3455, but the enzyme was clearly different from the reported elastase, in respect to Ca^++ effects on enzyme-thermostability. This property, together with amino acid composition analysis, confirmed that the enzyme differs from the known P. aeruginosa elastase.
Kim, Young-Ok,Park, In-Suk,Kim, Hyung-Kwoun,Nam, Bo-Hye,Kong, Hee Jeong,Kim, Woo-Jin,Kim, Dong-Gyun,Kim, Bong-Seok,Jee, Young-Ju,Song, Jung-Hun,Lee, Sang-Jun The Korean Society for Applied Biological Chemistr 2013 Applied Biological Chemistry (Appl Biol Chem) Vol.56 No.1
A bacterial strain that produces a cold-adapted esterase was isolated from tidal flats and identified as Shewanella sp. Ke75. In the present study, the corresponding gene was cloned using the shotgun method. The amino acid sequence deduced from the nucleotide sequence (957 bp) corresponded to a protein of 318 amino acid residues with a calculated molecular weight of 34875 Da. The esterase showed 68 and 57% identities with the putative esterases of Shewanella amazonensis SB2B and Colwellia psychrerythraea 34H, respectively. The esterase contained a putative leader sequence, as well as the conserved catalytic triad (Ser, His, Asp), consensus pentapeptide GXSXG, and oxyanion hole sequence (HG). The protein Ke75 was produced in both soluble and insoluble forms when Escherichia coli cells harboring the gene were cultured at $30^{\circ}C$. The enzyme showed specificity for C4 (butyrate) as a substrate, with little activity toward the other p-nitrophenyl esters tested. The optimum pH and temperature for enzyme activity were pH 9.0 and $30^{\circ}C$, respectively. Relative activity remained up to 60% even at $5^{\circ}C$ with an activation energy of 6.29 kcal/mol, which indicated that it was a cold-adapted enzyme. Enzyme activity was enhanced in the presence of $Mn^{2+}$ ions, but inhibited by $Cd^{2+}$, $Cu^{2+}$, $Hg^{2+}$, and $Zn^{2+}$ ions.
Gene Cloning and Characterization of a Cold-Adapted Esterase from Acinetobacter venetianus V28
( Kim Young Ok ),( Yu Li Heo ),( Hyung Kwoun Kim ),( Bo Hye Nam ),( Hee Jeong Kong ),( Dong Gyun Kim ),( Woo Jin Kim ),( Bong Seok Kim ),( Young Ju Jee ),( Sang Jun Lee ) 한국미생물 · 생명공학회 2012 Journal of microbiology and biotechnology Vol.22 No.9
Acinetobacter venetians V28 was isolated from the intestine of righteye flounder, Poecilopsetta plinthus caught in Vietnam seawater, and the esterase gene was cloned using a shotgun method, The amino acid sequence deduced from the nucleotide sequence (1,017bp) corresponded to a protein of 338 amino acid residues with a molecular weight of 37,186. The esterase had 87% and 72% identities with the lipases of A. junii SH205 and A. calcoaceticus RUH2202, respectively. The esterase contained a putative leader sequence, as well as the conserved catalytic triad (Ser, His, Asp), consensus pentapeptide GXSXG, and oxyanion hole sequence (HG). The protein from the strain V28 was produced in both a soluble and an insoluble form when the Escherichia coli cells harboring the gene were cultured at 18oC. The maximal activity of the purified enzyme was observed at a temperature of 40oC and pH 9.0 using p-NP-caprylate as substrate; however, relative activity still reached to 70% even at 5oC with an activation energy of 3.36 kcal/mol, which indicated that it was a cold-adapted enzyme. The enzyme was a nonmetalloprotein and was active against p-nitrophenyl esters of C4, C8, and C14. Remarkably, this enzyme retained much of its activity in the presence of commercial detergents and organic solvents. This cold-adapted esterase will be applicable as catalysts for reaction in the presence of organic solvents and detergents.
Kim, Hyung Kwoun,Lee, Seung A 가톨릭대학교 자연과학연구소 2003 자연과학논문집 Vol.24 No.-
효소의 열안정에 미치는 칼슘이온의 영향이 세가지 리파제에 대해 실험적으로 상이하게 나타났다. BaciL1us pumilus 리파제(B26)는 칼슘의 영향을 전혀 받지 않았으나, BaciL1us stearother-mophilus 리파제(L1)는 칼슘의 영향이 관찰되었고, Proteus vulgaris 리파제(K80)는 칼슘에 대해 절대적으로 큰 영향을 받았다. 이 세가지 리파제 단백질의 3차원 구조를 분석하여 비교하였다. BaciL1us subilis 리파제(PDB-id, 1ISP)와 Pseudomonas aeruginosa 리파제(1EX9)를 주형구조로 이용하고 Geno3D 프로그램(http://pbil.ibcp.fr/)을 활용함으로써 리파제 B26과 리파제K80의 구조를 각각 구하였다. 리파제 L1의 구조는 이미 밝혀진 3차원 구조를 사용하였다. 비교분석결과에 의하면, 리파제 B26의 구조에는 칼슘결합부위가 없으나, 리파제 L1의 경우, 4개의 아미노산(Glu360, Asp365, Pro366, Gly286)이 한 개의 칼슘결합부위를 형성하는 것으로 밝혀졌으며, 리파제 K80의 경우에도 4개의 아미노산(Asp213, Asp 256, Gln260, Leu264)이 1개의 칼슘결합부위을 형성함으로써, 효소의 열안정성에 높이는데 기여하는 것으로 밝혀졌다. The effects of calcium ion on enzyme thermostability were quite different experimentaL1y among BaciL1us pumilus lipase (B26), BaciL1us stearothermophilus lipase (L1), and Proteus vulgaris lipase (K80). As reported previously, lipase B26 was calcium-independent and lipase L1 was moderately calcium-dependent. In this research, lipase K80 was turned out to be dramaticaL1y calcium-dependent. The three dimensional structure models of lipase B26 and lipase K80 were made by using Geno3D tool based on the structures of BaciL1us subtilis lipase (PDB-id, LISP) and Pseudomonas aeruginosa lipase (PDB-id, 1EX9), respectively. The structure of lipase L1 has been already elucidated. Investigation of the three protein structures showed that lipase 1326 had no calcium-binding site and that lipase L1 and lipase K80 had one calcium-binding site, respectively. In case of lipase L1, two carboxyl oxygen atoms of G1u360 and Asp365 and twc main chain carbonyl oxygen atoms of Pro366 and Gly286 were coordinated with one calcium icn. In the model of lipase K80, on the other hands, two carboxyl oxygen atoms of Asp213 and Asp256 and two main chain carbonyl oxygen atoms of Gln260 and Leu264 could be properly coordinated with one calcium ion. Calcium ions seemed to bind to these calcium-binding sites and increase their structural stability at higher temperatures.
Cloning and characterization of a new cold-active lipase from a deep-sea sediment metagenome
Jeon, Jeong Ho,Kim, Jun-Tae,Kim, Yun Jae,Kim, Hyung-Kwoun,Lee, Hyun Sook,Kang, Sung Gyun,Kim, Sang-Jin,Lee, Jung-Hyun Springer-Verlag 2009 Applied microbiology and biotechnology Vol.81 No.5
<P>To search for new cold-active lipases, a metagenomic library was constructed using cold-sea sediment samples at Edison Seamount and was screened for lipolytic activities by plating on a tricaprylin medium. Subsequently, a fosmid clone was selected, and the whole sequence of 36 kb insert of the fosmid clone was determined by shotgun sequencing. The sequence analysis revealed the presence of 25 open reading frames (ORF), and ORF20 (EML1) showed similarities to lipases. Phylogenetic analysis of EML1 suggested that the protein belonged to a new family of esterase/lipase together with LipG. The EML1 gene was expressed in Escherichia coli, and purified by metal-chelating chromatography. The optimum activity of the purified EML1 (rEML1) occurred at pH 8.0 and 25 degrees C, respectively, and rEML1 displayed more than 50% activity at 5 degrees C. The activation energy for the hydrolysis of olive oil was determined to be 3.28 kcal/mol, indicating that EML1 is a cold-active lipase. rEML1 preferentially hydrolyzed triacylglycerols acyl-group chains with long chain lengths of > or = 8 carbon atoms and displayed hydrolyzing activities toward various natural oil substrates. rEML1 was resistant to various detergents such as Triton X-100 and Tween 80. This study represents an example which developed a new cold-active lipase from a deep-sea sediment metagenome.</P>
KIM, MYUNG HEE,KIM, HYUNG-KWOUN,OH, BYUNG-CHUL,OH, TAE-KWANG 한국미생물 · 생명공학회 2000 Journal of microbiology and biotechnology Vol.10 No.6
The lipase gene (lip) from Bacillus stearothermophilus was recombined in vitro by utilizing the DNA shuffling technique. After four rounds of shuffling, transformation, and screening based on the initial rate of clear zone formation on a tricaprylin plate, a clone (M10) was isolated, the cell extract of which showed about 2.8-fold increased lipase activity. The DNA sequence of the mutant lipase gene (m10) showed 3base changes, resulting in two cryptic mutations and one amino acid substitution; S113 (AGC→AGT), L252 (TTG→TTA), and G275E (GGA→GAA). SDS-PAGE analysis revealed that the increased enzyme activity observed in M10 was partly caused by high expression of the m10 lipase gene. The amount of the expressed G275E lipase was estimated to comprise as much as 41% of the total soluble proteins of the cell. The maximum velocity (V_max) of the purified mutant enzyme for the hydrolysis of olive oil was measured to be 3,200U/㎎, which was 10% higher than that of the parental (WT) lipase (2,900U/㎎). Its optimum temperature for the hydrolysis of olive oil was 68℃ and it showed a typical Ca^2+-dependent thermostability, properties of which were the same as those of the WT lipase. However, the mutant enzyme exhibited a high enantiospecificity towards (S)-naproxen compared with the WT lipase. In addition, it showed increased hydrolytic activity towards triolein, tricaprin, tricaprylin, and tricaproin.
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.