• 대한의생명과학회지
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• 제12권3호
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• pp.225-231
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• 2006

Fibrinolytic enzyme was purified from the fruiting bodies of Lepista nuda, using DEAE-Cellulose chromatography, Phenyl Sepharose chromatography, and Mono-S column chromatography. The substance has a molecular weight of 30006.62 Da as measured by MALD-TOF mass spectrometry. The N-terminal amino acid sequence of the enzyme was Tyr-Pro-Ser-Pro-Ser-His-Gln-Thr-Ala-Val-Asn-Ala-Ile-Ile-X. The activity of the enzyme was inhibited by PMSF, indicating that the enzyme is a serine protease. No inhibition was found with E-64, pepstatin, and EDTA. It has broad substrate specificity for synthetic peptides. The enzyme was stable up to $30^{\circ}C$. The enzyme hydrolyzes both Aa and y chains of human fibrinogen but did not show any reactivity for $B{\beta}$ chain of human fibrinogen.

• Journal of Microbiology and Biotechnology
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• 제23권12호
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• pp.1699-1707
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• 2013

During the fermentative production of 1,3-propanediol under high substrate concentrations, accumulation of intracellular 3-hydroxypropionaldehyde will cause premature cessation of cell growth and glycerol consumption. Discovery of oxidoreductases that can convert 3-hydroxypropionaldehyde to 1,3-propanediol using NADPH as cofactor could serve as a solution to this problem. In this paper, the yqhD gene from Klebsiella pneumoniae DSM2026, which was found encoding an aldehyde reductase (KpAR), was cloned and characterized. KpAR showed broad substrate specificity under physiological direction, whereas no catalytic activity was detected in the oxidation direction, and both NADPH and NADH can be utilized as cofactors. The cofactor binding mechanism was then investigated employing homology modeling and molecular dynamics simulations. Hydrogen-bond analysis showed that the hydrogen-bond interactions between KpAR and NADPH are much stronger than that for NADH. Free-energy decomposition dedicated that residues Gly37 to Val41 contribute most to the cofactor preference through polar interactions. In conclusion, this work provides a novel aldehyde reductase that has potential applications in the development of novel genetically engineered strains in the 1,3-propanediol industry, and gives a better understanding of the mechanisms involved in cofactor binding.

• Journal of Microbiology and Biotechnology
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• 제22권7호
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• pp.917-922
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• 2012

Homology modeling of Streptomyces peucetius CYP147F1 was constructed using three cytochrome P450 structures, CYP107L1, CYPVdh, and CYPeryF, as templates. The lowest energy SPCYP147F1 model was then assessed for stereochemical quality and side-chain environment by Accelrys Discovery Studio 3.1 software. Further activesite optimization of the SPCYP147F1 was performed by molecular dynamics to generate the final SPCYP147F1 model. The substrate limonene was then docked into the model. The model-limonene complex was used to validate the active-site architecture, and functionally important residues within the substrate recognition site were identified by subsequent characterization of the secondary structure. The docking of limonene suggested that SPCYP147F1 would have broad specificity with the ligand based on the two different orientations of limonene within the active site facing to the heme. Limonene with C7 facing the heme with distance of $3.4{\AA}$ from the Fe was predominant.

• BMB Reports
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• 제39권1호
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• pp.46-54
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• 2006

The coenzyme A-acylating 2-oxoacid:ferredoxin oxidoreductase and ferredoxin (an effective electron acceptor) were purified from the hyperthermophilic archaeon, Sulfolobus solfataricus P1 (DSM1616). The purified ferredoxin is a monomeric protein with an apparent molecular mass of approximately 11 kDa by SDS-PAGE and of $11,180{\pm}50$ Da by MALDI-TOF mass spectrometry. Ferredoxin was identified to be a dicluster, [3Fe-4S][4Fe-4S], type ferredoxin by spectrophotometric and EPR studies, and appeared to be zinc-containing based on the shared homology of its N-terminal sequence with those of known zinc-containing ferredoxins. On the other hand, the purified 2-oxoacid: ferredoxin oxidoreductase was found to be a heterodimeric enzyme consisting of 69 kDa $\alpha$ and 34 kDa $\beta$ subunits by SDS-PAGE and MALDI-TOF mass spectrometry. The purified enzyme showed a specific activity of 52.6 units/mg for the reduction of cytochrome c with 2-oxoglutarate as substrate at $55^{\circ}C$, pH 7.0. Maximum activity was observed at $70^{\circ}C$ and the optimum pH for enzymatic activity was 7.0 -8.0. The enzyme displays broad substrate specificity toward 2-oxoacids, such as pyruvate, 2-oxobutyrate, and 2-oxoglutarate. Among the 2-oxoacids tested (pyruvate, 2-oxobutyrate, and 2-oxoglutarate), 2-oxoglutarate was found to be the best substrate with $K_m$ and $k_{cat}$ values of $163\;{\mu}M$ and $452\;min^{-1}$, respectively. These results provide useful information for structural studies on these two proteins and for studies on the mechanism of electron transfer between the two.

• BMB Reports
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• 제31권1호
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• pp.53-57
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• 1998

The CMCax gene from Acetobacter xylinum ATCC 23769 was cloned and expressed in E. coli. With this gene, three gene products - mature CMCax, CMCax containing signal peptide(pre-CMCax), and a glutathione-S-transferase(GST)-CMCax fusion enzyme - were expressed. CMCax and pre-CMCax are aggregated to multimeric forms which showed high CMC hydrolysis activity, whereas GST-CMCax was less aggregated and showed lower activity, indicating that oligomerization of CMCax controbutes to the cellulose hydrolysis activity to achieve greater efficiency. The enzyme was identified to be an $\beta$-1,4-endoglucanase, which catalyzes the cleavage of internal $\beta$-1,4-glycosidic bonds of cellulose. The reaction products, cellobiose and cellotriose, from cellopentaose as a substrate, were identified by HPLC. Substrate specificity of cellotetraose by this enzyme was poor, and the reaction products consisted of glucose, cellobiose, and cellotriose in a very low yield. Theses results suggested that cellopentaose might be the oligosaccharide substrate consisting of the lowest number of glucose. The optimum pH of CMCax and pre CMCax was about 4.5, whereas that of GST-CMCas was rather broad at pH 4.5-8. The physiological significance of cellulose-hydrolyzing enzyme, CMCax, having such low $\beta$-1,4-endoglucanase activity and low optimum pH in cellulose-producing A. xylinum is not clearly known yet, but it seems to be closely related to the production of cellulose.

• 미생물학회지
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• 제50권2호
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• pp.128-136
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• 2014

Acyl homoserine lactone (AHL) 분해효소인 lactonase는 높은 기질 특이성을 지니기 때문에 경제적이고 효율적인 쿼럼 저해 기술로 이용될 가능성을 지니고 있다. 본 연구에서는 Chromobacterium violaceum CV026과 Agrobacterium tumefaciens NTL4를 바이오센서로 이용하여 biofouling이 일어난 역삼투막 시료로부터 쿼럼 센싱과 관련된 생물막 형성을 억제하는 6종의 균주를 분리 연구하였다. 분리된 균주는 모두 Bacillus 속으로 동정되었으며, AHL 분자의 acyl 사슬 길이나 치환 종류에 상관 없이 쿼럼 저해활성을 보여주었다. 균주들은 Pseudomonas aeruginosa PAO1에 의한 생물막 형성을 46.7-58.3% 정도 감소시켰으며 이 때 저해물질은 열처리에 민감한 특성을 보여주었다. 분리 균주 중 RO1S-5를 이용하여 N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12 AHL)과 반응시킨 결과, 상응하는 acyl homoserine (3-oxo-C12-HS)이 생성되는 것을 LC-MS로 확인하여 쿼럼 저해가 lactonase 활성에 의한 것임을 규명하였다. AHL 물질에 대한 높은 특이성 등을 감안할 때 분리 균주 RO1S-5는 생물막 형성과 관련된 질병이나 산업공정 중 발생하는 biofouling을 해결하는데 유용하게 쓰일 수 있을 것으로 기대된다.

• Journal of Microbiology and Biotechnology
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• 제29권3호
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• pp.373-381
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• 2019

Site-directed mutagenesis was employed to generate five different triple point mutations in the double mutant (C295A/I86A) of Thermoanaerobacter ethanolicus alcohol dehydrogenase (TeSADH) by computer-aided modeling with the aim of widening the small alkyl-binding pocket. TeSADH engineering enables the enzyme to accept sterically hindered substrates that could not be accepted by the wild-type enzyme. The underline in the mutations highlights the additional point mutation on the double mutant TeSADH introduced in this work. The catalytic efficiency ($k_{cat}/K_M$) of the ${\underline{M151A}}$/C295A/I86A triple TeSADH mutant for acetophenone increased about 4.8-fold higher than that of the double mutant. A 2.4-fold increase in conversion of 3'-methylacetophenone to (R)-1-(3-methylphenyl)-ethanol with a yield of 87% was obtained by using ${\underline{V115A}}$/C295A/I86A mutant in asymmetric reduction. The ${\underline{A85G}}$/C295A/I86A mutant also produced (R)-1-(3-methylphenyl)-ethanol (1.7-fold) from 3'-methylacetophenone and (R)-1-(3-methoxyphenyl)-ethanol (1.2-fold) from 3'-methoxyacetophenone, with improved yield. In terms of thermal stability, the ${\underline{M151A}}$/C295A/I86A and ${\underline{V115A}}$/C295A/I86A mutants significantly increased ${\Delta}T_{1/2}$ by $+6.8^{\circ}C$ and $+2.4^{\circ}C$, respectively, with thermal deactivation constant ($k_d$) close to the wild-type enzyme. The ${\underline{M151A}}$/C295A/I86A mutant reacts optimally at $70^{\circ}C$ with almost 4 times more residual activity than the wild type. Considering broad substrate tolerance and thermal stability together, it would be promising to produce (R)-1-(3-methylphenyl)-ethanol from 3'-methylacetophenone by ${\underline{V115A}}$/C295A/I86A, and (R)-1-phenylethanol from acetophenone by ${\underline{M151A}}$/C295A/I86A mutant, in large-scale bioreduction processes.

• The Korean Journal of Physiology and Pharmacology
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• 제1권2호
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• pp.185-193
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• 1997

The characteristics of $Na^+$-dependent cycloleucine uptake was investigated in OK cells with regard to substrate specificity and regulation by protein kinase C (PKC). Inhibition studies with different synthetic and natural amino acids showed a broad spectrum affinity to neutral amino acids regardless of their different side chains including branched or aromatic, indicating that the $Na^+$-dependent cycloleucine uptake in OK cells is mediated by System $B^o$ or System $B^o$-like transporter rather than the classical System A or ASC. Phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate, but not $4{\alpha}-PMA$ elicited a time-dependent biphasic stimulation of $Na^+$-dependent cycloleucine uptake, which produced early transient peak at 30 min and late sustained peak at 180min. Both the early and late stimulations by PMA were due to an increase in Vmax and not due to a change in Km. PKC inhibitors blocked both the early and late stimulation by PMA, while protein synthesis inhibitors blocked the late stimulation only. These results suggest the existence and regulation by PKC of System $B^o$ or System $B^o$-like broad spectrum transport system for neutral amino acids in OK cells.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2002년도 생물공학의 동향 (X)
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• pp.457-460
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• 2002

In an effort to isolate novel strains expressing a thermostable esterase that hydrolyzed the rac-ketoprofen ethyl ester to ketoprofen in the stereospecific manner, we screened various soils and composts from broad ecological niches in which the activity was expected to be found. Three hundreds of microbial strains were tested to determine their ester-hydrolyzing activity by using an agar plate containing insoluble tributyrin as an indicative substrate, and then further screened by activity on the (R,S)-ketoprofen ethyl ester. Twenty-six strains were screened primarily at high growth and incubation temperature and further compared the ability to ethyl ester-hydrolyzing activity in terms of conversion yield and chiral specificity. Consequently, a strain JYl44 was isolated as a novel strain that produced a (R)-stereospecific esterase with high stability and systematically identified as a Bacillus stearothermophilus JY144. The enzyme indeed stables at a broad range of temperature, upto 65 $^{\circ}C$, and pH ranging from 6.0 to 10.0. The optimal temperature and pH for enzymatic conversion were 50 $^{\circ}C$ and 9.0, respectively. Based on the observations that resulted a poor cell growth, and enzyme expression in wild type strain, we further attempted the gene cloning into a general host Escherichia coli and determined its primary structure, concomitantly resulting a high level expression of the enzyme. The cloned gene had an open reading frame (250 amino acids) with a calculated molecular mass of 27.4 kDa, and its primary structure showed a relative high homology (45-52 %) to the esterases from Streptomyces and Bacillus strains. The recombinant whole cell enzyme could efficiently convert the rac-ketoprofen ethyl ester to (R)-ketoprofen, with optical purity of 99 % and yield of 49 %.

• 생명과학회지
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• 제16권3호
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• pp.375-381
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• 2006

Saccharomyces cerevisiae 로부터 알데히드 환원효소를 정제하였다. 정제된 알데히드 환원효소를 biocatalyst로 사용하여 치환기가 있는 카르보닐 화합물의 선택적 환원을 시도하였다. 효소를 이용한 환원반응의 생성물의 구조를 TLC, GC, Mass, NMR, FT-IR을 이용하여 확인하였으며 효소를 이용한 환원반응이 높은 선택성을 가지고 진행됨을 확인하였다. 또한 이 반응은 알데히드 환원효소의 억제제인 벤조산에 의해 크게 억제되었다. 치환기가 있는 카르보닐 화합물의 선택적 환원반응은 의약품 제조 분야에서 매우 중요한 반응이며 미생물에서 정제한 알데히드 환원효소가 biocatalyst 로서 선택적 환원반응에 이용될 수 있으리라 사료된다.