• 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.

• Journal of Microbiology and Biotechnology
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• 제18권9호
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• pp.1544-1549
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• 2008

MhMTS and MhMTH are trehalose ($\alpha$-D-glucopyranosyl-[1,1]-$\alpha$-D-glucopyranose) biosynthesis genes of the thermophilic microorganism Metallosphaera hakonensis, and encode a maltooligosyltrehalose synthase (MhMTS) and a maltooligosyltrehalose trehalohydrolase (MhMTH), respectively. In this study, the two genes were fused in-frame in a recombinant DNA, and expressed in Escherichia coli to produce a bifunctional fusion enzyme, MhMTSH. Similar to the two-step reactions with MhMTS and MhMTH, the fusion enzyme catalyzed the sequential reactions on maltopentaose, maltotriosyltrehalose formation, and following hydrolysis, producing trehalose and maltotriose. Optimum conditions for the fusion enzyme-catalyzed trehalose synthesis were around $70^{\circ}C$ and pH 5.0-6.0. The MhMTSH fusion enzyme exhibited a high degree of thermostability, retaining 80% of the activity when pre-incubated at $70^{\circ}C$ for 48 h. The stability was gradually abolished by incubating the fusion enzyme at above $80^{\circ}C$. The MhMTSH fusion enzyme was active on various sizes of maltooligosaccharides, extending its substrate specificity to soluble starch, the most abundant natural source of trehalose production.

• BMB Reports
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• 제38권6호
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• pp.676-682
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• 2005

To date, no 8-oxoguanine-specific endonuclease-coding gene has been identified in Thermotoga maritima of the order Thermotogales, although its entire genome has been deciphered. However, the hypothetical protein Tm1821 from T. maritima, has a helix-hairpin-helix motif that is considered to be important for DNA binding and catalytic activity. Here, Tm1821 was overexpressed in Escherichia coli and purified using Ni-NTA affinity chromatography, protease digestion, and gel filtration. Tm1821 protein was found to efficiently cleave an oligonucleotide duplex containing 8-oxoguanine, but Tm1821 had little effect on other substrates containing modified bases. Moreover, Tm1821 strongly preferred DNA duplexes containing an 8-oxoguanine:C pair among oligonucleotide duplexes containing 8-oxoguanine paired with four different bases (A, C, G, or T). Furthermore, Tm1821 showed AP lyase activity and Schiff base formation with 8-oxoguanine in the presence of $NaBH_4$, which suggests that it is a bifunctional DNA glycosylase. Tm1821 protein shares unique conserved amino acids and substrate specificity with an 8-oxoguanine DNA glycosylase from the hyperthermophilic archaeon. Thus, the DNA recognition and catalytic mechanisms of Tm1821 protein are likely to be similar to archaeal repair protein, although T. maritima is an eubacterium.

• BMB Reports
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• 제34권3호
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• pp.244-249
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• 2001

The anabolic acetolactate synthase III was purified to homogeneity from Serratia marcescens using DEAE-Sepharose, Phenyl-Sepharose, and hydroxylapatite column chromatography The native molecular weight of the enzyme was approximately 165 kDa. The enzyme is composed of two large and two small subunits with molecular weights of 64 and 15 kDa, respectively. The N-terminal sequence of the large and small subunit of the enzyme was Ser-Ala-Thr-Pro-Gln-Pro-Ser-Thr-Arg-Phe-Thr-Cys-Ala-Gln-Leu-Ile-Ala-His-Leu and Met-Leu-Gln-Pro-Gln-Asp-Lys-Pro-Gln-Val-Ile-Leu-Glu-Leu-Ala-Val-Arg-Asn-His-Pro-Gly-Val-Met-Ser-His-Val, respectively. The optimum pH and pI value were 7.5 and 5.5, respectively The $IC_{50}$ values were $20\;{\mu}M$ and $14\;{\mu}M$ for valine and herbicide SU7, respectively. The substrate specificity ratio, R value, was determined to be approximately 40, which suggests that this enzyme prefers the formation of $\alpha$-aceto-$\alpha$-hydroxybutyrate leading to the synthesis of isoleucine.

• Journal of Life Science
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• 제9권1호
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• pp.40-44
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• 1999

Glutathione S-transferase (GST) is a multifunctional protein that catalyzes the catalyzes the conjugation of glutathione with electrophilic compounds. It exists in a variety of isoenzy-matic froms with a wide range of substrate specificity and plays a pivotal role in detoxification of various drugs. In order to elucidate the GST-${\pi}$'s involvement of multidrug resistance (MDR) in drug-resistant tumor cell lines, we determined GST-${\pi}$ content by "1 step sandwich method". Consequently, adriamycin resistant cells of MCF-7 (MCF-7/ADM) have 7-fold increase of GST-${\pi}$ content than that of MCF-7 cells, while its {TEX}$IC_{50}${/TEX} was 116-fold greater than parent cell line. By northrn blotting, we compared whether MCF-7/ADM cells express GST-${\pi}$ mRNA. The GST-${\pi}$ mRNA expression in these cells was not inducible, but constitutive when treated for 24 h with a concentration of 0, 20, 200, and 2000 nM of adriamycin, respectively. Taken together, these results suggest that GST-${\pi}$ may not be directly associated with multidrug resistance in these human cancer cell lines.ell lines.

• Journal of Microbiology and Biotechnology
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• 제15권4호
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• pp.745-748
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• 2005

A phytase from Aeromonas sp. LIK 1-5 was partially purified by ammonium sulfate precipitation and DEAE-Sephacel column chromatography. Its molecular weight was 44 kDa according to SDS-PAGE gel. Enzyme activity was optimal at pH 7 and at $50^{\circ}C$. The purified enzyme was strongly inhibited by 2 mM EDTA, $Zn^{2+},\;Co^{2+},\;or\;Mn^{2+}$, and activated by 2 mM $Ca^{2+}$. The K_m value for sodium phytate was 0.23 mM, and the enzyme was resistant to trypsin. The N-terminal amino acid sequence of the phytase was similar to that of other known alkaline phytases. The phytase was specific for ATP and sodium phytate, which is different from other known alkaline phytases. Based on the substrate specificity, the phytase may therefore be a novel alkaline phytase.

• Journal of Microbiology and Biotechnology
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• 제17권1호
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• pp.74-80
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• 2007

An enantioselective lipase gene (esf) for the kinetic resolution of optically active (S)-flurbiprofen was cloned from the new strain Serratia marcescens ES-2. The esf gene was composed of a 1,845-bp open reading frame encoding 614 amino acid residues with a calculated molecular mass of 64,978 Da. The lipase expressed in E. coli was purified by a three-step procedure, and it showed preferential substrate specificity toward the medium-chain-length fatty acids. The esf gene encoding the enantioselective lipase was reintroduced into the parent strain S. marcescens ES-2 for secretory overexpression. The transformant S. marcescens BESF secreted up to 217kU/ml of the enantioselective lipase, about 54-fold more than the parent strain, after supplementing 3.0% Triton X-207. The kinetic resolution of (S)-flurbiprofen was carried out even at an extremely high (R,S)-flurbiprofen ethyl ester [(R,S)-FEE] concentration of 500 mM, 130 kU of the S. marcescens ES-2 lipase per mmol of (R,S)-FEE, and 1,000 mM of succinyl ${\beta}-cyclodextrin$ as the dispenser at $37^{\circ}C$ for 12h, achieving the high enantiomeric excess and conversion yield of 98% and 48%, respectively.

• Journal of Microbiology and Biotechnology
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• 제19권10호
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• pp.1184-1190
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• 2009

A thermostable extracellular $\beta$-mannanase from the culture supernatant of a fungus Aspergillus niger gr was purified to homogeneity. SDS-PAGE of the purified enzyme showed a single protein band of molecular mass 66 kDa. The $\beta$-mannanase exhibited optimum catalytic activity at pH 5.5 and $55^{\circ}C$. It was thermostable at $55^{\circ}C$, and retained 50% activity after 6 h at $55^{\circ}C$. The enzyme was stable at a pH range of 3.0 to 7.0. The metal ions $Hg^{2+}$, $Cu^{2+}$, and $Ag^{2+}$ inhibited complete enzyme activity. The inhibitors tested, EDTA, PMSF, and 1,10-phenanthroline, did not inhibit the enzyme activity. N-Bromosuccinimide completely inhibited enzyme activity. The relative substrate specificity of enzyme towards the various mannans is in the order of locust bean gum>guar gum>copra mannan, with $K_m$ of 0.11, 0.28, and 0.33 mg/ml, respectively. Since the enzyme is active over a wide range of pH and temperature, it could find potential use in the food-processing industry.

• Journal of Microbiology and Biotechnology
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• 제18권6호
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• pp.1044-1049
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• 2008

In order to study functional nucleotides in prototype foamy virus (PFV) DNA on specific recognition by PFV integrase (IN), we designed chimeric U5 long terminal repeat (LTR) DNA substrates by exchanging comparative sequences between human immunodeficiency virus type-1 (HIV-1) and PFV U5 LTRs, and investigated the 3'-end processing reactivity using HIV-1 and PFV INs, respectively. HIV-1 IN recognized the nucleotides present in the fifth and sixth positions at the 3'-end of the substrates more specifically than any other nucleotides in the viral DNA. However, PFV IN recognized the eighth and ninth nucleotides as distinctively as the fifth and sixth nucleotides in the reactions. In addition, none of the nucleotides present in the twelfth, sixteenth, seventeenth, eighteenth, nineteenth, and twentieth positions were not differentially recognized by HIV-1 and PFV INs, respectively. Therefore, our results suggest that the functional nucleotides that are specifically recognized by its own IN in the PFV U5 LTR are different from those in the HIV-1 U5 LTR in aspects of the positions and nucleotide sequences. Furthermore, it is proposed that the functional nucleotides related to the specific recognition by retroviral INs are present inside ten nucleotides from the 3'-end of the U5 LTR.

• Journal of Microbiology and Biotechnology
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• 제16권7호
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• pp.1047-1052
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• 2006

A bile salt hydrolase (BSH) was purified from Lactobacillus plantarum CK 102 and its enzymatic properties were characterized. This enzyme was successfully purified using ion-exchange chromatography with Q-Excellose and hydrophobic interaction chromatography with Butyl-Excellose. The purified enzyme showed a single protein band of 37 kDa by SDS-polyacrylamide gel electrophoresis, which was similar to the molecular weight of known BSHs. The amino acid sequence of GLGLPGDLSSMSR, determined by MALDI-TOF, was identical to that of BSH of L. plantarum WCFS1. Although this BSH hydrolyzed all of the six major human bile salts, glycine-conjugated bile acid was the best substrate, based on its specificity and $K_{m}$ value. Among the various substrates, the purified enzyme maximally hydrolyzed glycocholate with apparent $K_{m}$ and $V_{max}$ values of 0.5 mM and 94 nmol/min/mg, respectively. The optimal pH of the enzyme ranged from 5.8 to 6.3. This enzyme was strongly inhibited by thiol enzyme inhibitors such as iodoacetate and periodic acid.