• Journal of Microbiology and Biotechnology
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• v.23 no.2
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• pp.144-148
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• 2013

Lycopene cyclase converts lycopene to ${\beta}$-carotene by catalyzing the formation of two beta-rings at each end of the linear carotene structure. This reaction takes place as a two-step reaction in which both sides of of the lycopene molecule are cyclized into ${\beta}$-carotene rings via the monocyclic ${\gamma}$-carotene as an intermediate. The crtY gene coding for lycopene cyclase from Paracoccus haeundaensis consists of 1,158 base pairs encoding 386 amino acids residues. An expression plasmid containing the crtY gene (pET44a-CrtY) was constructed and expressed in Escherichia coli, and produced a recombinant protein of approximately 43 kDa, corresponding to the molecular mass of lycopene cyclase. The expressed protein was purified to homogeneity by His-tag affinity chromatography and showed enzymatic activity corresponding to lycopene cyclase. We also determined the lycopene substrate specificity and NADPH cofactor requirements of the purified protein. The $K_m$ values for lycopene and NADPH were 3.5 ${\mu}M$ and 2 mM, respectively. The results obtained from this study will provide a wider base of knowledge on the enzyme characterization of lycopene cyclase at the molecular level.

• Molecules and Cells
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• v.21 no.2
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• pp.302-307
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• 2006

Two Epstein-Barr virus (EBV) types, type 1 and type 2, maintain the same allelic specificity at four genomic loci encoding the EBNA2, -3A, -3B, and -3C proteins. We have previously described 16 EBV-transformed B-lymphoblastoid cell lines derived from Korean cancer patients, and the EBNA2 types of the EBV isolates therein. In this study, the allelic types of the EBNA2, -3A, -3B, and -3C genes of these EBV isolates were determined. We report the identification of two distinct types of naturally occurring intertypic recombinants, one with genotype EBNA2 type1/EBN3A, -3B, -3C type 2 and the other with genotype EBNA2, -3A type 1/EBNA3B, -3C type 2. The existence of these intertypic recombinants indicates that various intertypic EBV strains may be circulating in the human population, in addition to typical EBV-1 and EBV-2 strains.

• Journal of environmental and Sanitary engineering
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• v.23 no.2
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• pp.1-18
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• 2008

The tetrachloroethylene (PCE) dehalogenase of Clostridium bifermentans DPH-1 (a halorespiring organism) was purified, cloned, and sequenced. This enzyme is a homodimer with a molecular mass of ca. 70 kDa and exhibits dehalogenation of dichloroethylene isomers along with PCE and trichloroethylene (TCE). Broad range of substrate specificity for chlorinated aliphatic compounds (PCE, TCE, cis-1,2-dichloroethylene, trans-1,2-dichloroethylene, 1,1-dichloroethylene, 1,2-dichloropropene, and 1,1,2-trichloroethane) for this enzyme was also observed. A mixture of propyl iodide and titanium citrate caused a light-reversible inhibition of enzymatic activity suggesting the involvement of a corrinoid cofactor. A partial sequence (81 bp) of the encoding gene for PCE dehalogenase was amplified and sequenced with degenerateprimers designed from the N-terminal sequence (27 amino acid residues). Southern analysis of C. bifermentans genomic DNA using the polymerase chain reaction product as a probe revealed restriction fragment bands. A 5.0 kb ClaI fragment, harboring the relevant gene (designated pceC) was cloned (pDEHAL5) and the complete nucleotide sequence of pceC was determined. The gene showed homology mainly with microbial membrane proteins and no homology with any known dehalogenase, suggesting a distinct PCE dehalogenase. So, C. bifermentans could play some important role in the initial breakdown of PCE and other chlorinated aliphatic compounds in sites contaminated with mixtures of halogenated substances.

• Biomolecules & Therapeutics
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• v.2 no.2
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• pp.155-160
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• 1994

Human immunodeficiency virus type 1 (HIV-1) protease is essential for the replication of the virus and it is therefore an attractive target for antiviral drugs of HIV-1. Several dipeptide isosteres containing 2-isoxazoline or $\alpha$-hydroxy ketomethylene have been synthesized and their inhibitory effects on the HIV-1 protease examined. The enzymatically active HIV-1 protease was purified to homogeniety from E. coli transformed with a recombinant plasmid (pMAL-pro) containing the entire gene encoding the protease. The purified protease had the substrate specificity with Km value of 9.8$\mu$M when an undecapeptide His-Lys-Ala-Arg-Val-Leu-(p-nitro)Phe-Glu-Ala-Nle-Ser-amide was used as a substrate, and the products from the substrate after specific cleavage by HIV-1 protease were analyzed by HPLC. The synthetic compounds containing dipeptide isosteres showed specific inhibitory effects while a dipeptide isostere containing an isoxazoline ring inhibited the HIV-1 protease competitively with Ki value of 500 $\mu$M. Even if the inhibition effects of HIV-1 protease were not very high, these novel dipeptide isosteres can be used as key structural moieties for developing specific inhibitors of HIV-1 protease.

• Parasites, Hosts and Diseases
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• v.36 no.3
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• pp.183-190
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• 1998

A cDNA expression library of Clonorchis sinensis adult worm was constructed, and screened out immunologically. One clone, pBCs31, was selected ill view of its predominant reactivity with an experimentally infected rabbit serum. Recombinant C. slnensis antigen iIi 28 kDa as a if-falactosidase fusion protein produced in EscherichiG coli was identified by immunoblot analysis. The cloned gene was composed of 16 copies or a 30 base pair repeat and an additional 320 bases. The deduced amino acid seqiLence of the tandem repeat was AQPPKSGDGG. On RNA slot blot analysis, C. sinensis adult worm RNA showed a positive reaction with the cloned gene Enzyme-linked immunosorbent assay using a purified recombinant antigen of pBCs31 showed higtl specificity for diagnosis of clonorchiasis.

• Journal of Microbiology and Biotechnology
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• v.17 no.8
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• pp.1271-1283
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• 2007

A fibrinolytic protease (PoFE) was purified from the cultured mycelia of the edible oyster mushroom Pleurotus ostreatus, using a combination of various chromatographies. The purification protocol resulted in an 876-fold purification of the enzyme, with a final yield of 6.5%. The apparent molecular mass of the purified enzyme was estimated to be 32 kDa by SDS-PAGE, fibrin-zymography, and size exclusion using FPLC. The optimal reaction pH value and temperature were pH 6.5 and $35^{\circ}C$, respectively. PoFE effectively hydrolyzed fibrinogen, preferentially digesting the $A{\alpha}$-chain and the $B{\beta}$-chain over the ${\gamma}$-chain. Enzyme activity was enhanced by the addition of $Ca^{2+},\;Zn^{2+},\;and\;Mg^{2+}$ ions. Furthermore, PoFE activity was potently inhibited by EDTA, and it was found to exhibit a higher specificity for the chromogenic substrate S-2586 for chymotrypsin, indicating that the enzyme is a chymotrypsin-like metalloprotease. The first 19 amino acid residues of the N-terminal sequence were ALRKGGAAALNIYSVGFTS, which is extremely similar to the metalloprotease purified from the fruiting body of P. ostreatus. In addition, we cloned the PoFE protein, encoding gene, and its nucleotide sequence was determined. The cDNA of cloned PoFE is 867 nucleotides long and consists of an open reading frame encoding 288 amino acid residues. Its cDNA showed a high degree of homology with PoMEP from P. ostreatus fruiting body. The mycelia of P. ostreatus may thus represent a potential source of new therapeutic agents to treat thrombosis.

• Journal of Microbiology
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• v.45 no.2
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• pp.158-167
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• 2007

In this study, we have cloned a novel cDNA encoding for a papain-family cysteine protease from the Uni-ZAP XR cDNA library of the polychaete, Periserrula leucophryna. This gene was expressed in Escherichia coli using the T7 promoter system, and the protease was characterized after partial purification. First, the partial DNA fragment (498 bp) was amplified from the total RNA via RT-PCR using degenerated primers derived from the conserved region of cysteine protease. The full-length cDNA of cysteine protease (PLCP) was prepared via the screening of the Uni-ZAP XR cDNA library using the $^{32}P-labeled$ partial DNA fragment. As a result, the PLCP gene was determined to consist of a 2591 bp nucleotide sequence (CDS: 173-1024 bp) which encodes for a 283-amino acid polypeptide, which is itself composed of an 59-residue signal sequence, a 6-residue propeptide, a 218-residue mature protein, and a long 3'-noncoding region encompassing 1564 bp. The predicted molecular weights of the preproprotein and the mature protein were calculated as 31.8 kDa and 25 kDa, respectively. The results of sequence analysis and alignment revealed a significant degree of sequence similarity with other eukaryotic cysteine proteases, including the conserved catalytic triad of the $Cys^{90},\;His^{226},\;and\;Asn^{250}$ residues which characterize the C1 family of papain-like cysteine protease. The nucleotide and amino acid sequences of the novel gene were deposited into the GenBank database under the accession numbers, AY390282 and AAR27011, respectively. The results of Northern blot analysis revealed the 2.5 kb size of the transcript and ubiquitous expression throughout the entirety of the body, head, gut, and skin, which suggested that the PLCP may be grouped within the cathepsin F-like proteases. The region encoding for the mature form of the protease was then subcloned into the pT7-7 expression vector following PCR amplification using the designed primers, including the initiation and termination codons. The recombinant cysteine proteases were generated in a range of 6.3 % to 12.5 % of the total cell proteins in the E. coli BL21(DE3) strain for 8 transformants. The results of SDS-PAGE and Western blot analysis indicated that a cysteine protease of approximately 25 kDa (mature form) was generated. The optimal pH and temperature of the enzyme were determined to be approximately 9.5 and $35^{\circ}C$, respectively, thereby indicating that the cysteine protease is a member of the alkaline protease group. The evaluation of substrate specificity indicated that the purified protease was more active towards Arg-X or Lys-X and did not efficiently cleave the substrates with non-polar amino acids at the P1 site. The PLCP evidenced fibrinolytic activity on the plasminogen-free fibrin plate test.

• Korean Journal of Microbiology
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• v.51 no.1
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• pp.68-74
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• 2015

Based on the genomic analysis of Thermococcus pacificus P-4, we identified a putative GH1 ${\beta}$-glucosidase-encoding gene (Tpa-glu). The gene revealed a 1,464 bp encoding 487 amino acid residues, and the deduced amino acid residues exhibited 77% identity with Pyrococcus furiosus ${\beta}$-glucosidase (accession no. NP_577802). The gene was cloned and expressed in Escherichia coli system. The recombinant protein was purified by metal affinity chromatography and characterized. Tpa-Glu showed optimum activity at pH 7.5 and $75^{\circ}C$, and thermostability with a half life of 6 h at $90^{\circ}C$. Tpa-Glu exhibited hydrolyzing activity against various pNP-glycopyranosides, with kcat/Km values in the order of pNP-${\beta}$-glucopyranoside, pNP-${\beta}$-galactopyranoside, pNP-${\beta}$-mannopyranoside, and pNP-${\beta}$-xylopyranoside. In addition, the enzyme exhibited exo-hydrolyzing activity toward ${\beta}$-1,3-linked polysaccharide (laminarin) and ${\beta}$-1,3- and ${\beta}$-1,4-linked oligosaccharides. This is the first description of an enzyme from hyperthermophilic archaea that displays exo-hydrolyzing activity toward ${\beta}$-1,3-linked polysaccharides and could be applied in combination with ${\beta}$-1,3-endoglucanase for saccharification of laminarin.

• Journal of the Korean Chemical Society
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• v.57 no.1
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• pp.81-87
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• 2013

Staphylococcus aureus is the most important pathogen in nosocomial infections, including bloodstream infections. Prompt identification of S. aureus from blood cultures and detection of methicillin resistance are essential in cases of suspected sepsis. We have studied a new method for the sequence-specific visual detection of minute amounts of nucleic acids using intercalating reaction by addition of SYBR Green to amplicons of LAMP, and it's a unique gene amplification method in which DNA can be isothermally amplified using only one enzyme. Staphylococcus-LAMP, which targets the spa gene, encoding S.aureus-specific protein A, and the mecA gene, encoding penicillin-binding protein-2' for methicillin resistance, detected MRSA and MRSE. In this study, by using LAMP assay, I detected for Staphylococcus aureus and Staphylococcus epidermidis concentration in the clinical sample. The detection of Staphylococcus aureus and Staphylococcus epidermidis was tested by using serial 10-fold dilutions standard solution. I have accurate detected the limit of detection, sensitity, specificity and reproducibility of the assay. The Bio-chip based LAMP assay allowed easy, rapid, accurate and sensitive detection of infection with Staphylococcus and especially applicable in a resource-limited situation.

• Korean Journal of Culture and Social Issue
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• v.20 no.4
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• pp.347-364
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• 2014

The present study was conducted to investigate how emotional expression change, test delay, and background influence on face recognition. In experiment 1, participants were presented with negative faces at study phase and administered for standard old-new recognition test including targets of negative and neutral expression for the same faces. In experiment 2, participants were studied negative faces and tested by old-new face recognition test with targets of negative and positive faces. In experiment 3, participants were presented with neutral faces at study phase and had to identify the same faces with no regard for negative and neutral expression at face recognition test. In all three experiments, participants were assigned into either immediate test or delay test, and target faces were presented in both white and black background. Results of experiments 1 and 2 indicated higher rates for negative faces than neutral or positive faces. Facial expression consistency enhanced face recognition memory. In experiment 3, the superiority of facial expression consistency were demonstrated by higher rates for neutral faces at recognition test. If facial expressions were consistent across encoding and retrieval, memory performance on face recognition were enhanced in all three experiments. And the effect of facial expression change have different effects on background conditions. The findings suggest that facial expression change make face identification hard, and time and background also affect on face recognition.