• 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.

• Korean Journal of Microbiology
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• v.42 no.1
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• pp.77-81
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• 2006

Degenerate primers corresponding to the sequences of the N-terminal regions of Mn-peroxidase isozymes were used to isolate the genomic fragments encoding the isozymes of Mn-peroxidase, CVMP1, CVMP2, CVMP3 and CVMP5 from the white-rot fungus Trametes versicolor KN9522. Three isozymes except one gave the expected PCR products (cmp1, cmp2 and cmp5) of about 900 base pairs, respectively. DNA sequence data obtained from each PCR products were used to analyze the BLAST program search on the National Center for Biotechnology Information. cmp1, cmp2 and cmp5 were similar to MPG-I (GenBank accession number Z30668) and PGV-II (GenBank accession number, Z54279) gene T. versicolor PRL572. PCR products of cmp1 and cmp2 showed 77%, 95% base sequence similarities to MPG-I gene and cmp5 showed about 88% similarity to PGV-II gene from T. versicolor PRL572. From this experiment, we could isolate genomic DNA fragments with degenerate primers designed from the N-terminal amino acid sequences of Mn-peroxidase isozyme family.

• Journal of Applied Biological Chemistry
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• v.65 no.4
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• pp.383-386
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• 2022

A gene encoding thermostable cellulase B (TmCelB) was isolated from Thermotoga maritima. The open reading frame (ORF) of TmCelB gene was 825bp long which predicted to encode 274 amino acid residues with a molecular weight of 31,732 Da. The 17 amino acid residues from N-terminal of the TmCelB was known as signal peptides. To analyze the enzymatic activity and biochemical properties, the ORF of TmCelB gene excluding a putative signal sequence encoding 17 amino acids were introduced into the E. coli expression vector, pRSET-B, and overexpressed in E. coli BL21. The optimum temperature of recombinant TmCelB was around 95 ℃, and the optimum pH of recombinant TmCelB was around pH 4.5. The recombinant TmCelB was stable at temperature below 100 ℃.

• Journal of Microbiology and Biotechnology
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• v.6 no.6
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• pp.402-406
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• 1996

Inulin fructotransferase (depolymerizing) (EC 2.4.1.93) was purified 34-fold from the culture broth of Arthrobacter sp. A-6 by using a combination of ammonium sulfate fractionation, DEAE-Sepharose CL-6B chromatography and Sephacryl S-200 gel filtration. The purified enzyme converts inulin into di-D-fructofuranose dianhydride III(DFA III) and small quantities of fructo-oligosaccharides. The temperature and pH optima of the enzyme were $70^{\circ}C$ and 6.0, respectively. Molecular weight of the enzyme was determined to be 49 kDa by 12$%$ SDS-polyacrylamide gel electrophoresis, and 145 kDa by Sephacryl S-200gel filtration. This indicates that the functional inulin fructotransferase of Arthrobacter sp. A-6 has a homomeric trimer structure. The enzyme had an isoelectric point of pH 4.6. The N-terminal amino acid sequence of the purified enzyme subunit was Ala-Asp-Asn-Pro-Asp-Gly(\ulcorner)-Ser-Asn-Met(or Glu)-Tyr-Asp-Val.

• Journal of Microbiology and Biotechnology
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• v.5 no.2
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• pp.92-95
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• 1995

Halophilic Pseudomonas sp.W7 isolated from laver in the southem sea of Korea showed alginate lyase activity. Gene (aly) encoding alginate lyase was cloned in E.coli JM83 and the N-terminal amino acid sequence of the enzyme was determined after purificaion. The recombinant enzyme has been shown to have a molecular weight of about 40kDa after 12% SDS-polyacrylamide gel electrophoresis.

• Korean Journal of Microbiology
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• v.30 no.6
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• pp.421-424
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• 1992

Escherichia coli JM83 harboring penicilin G acylase gene of Bacillus megaterium ATCC14945 produced a protein in large amount (>20% of the total protein). The protein was identified as GroEL, one of the E. coli heat shock protein, by N-terminal amino acid sequence analysis. It was found that GroEL was induced by the expressed foreign penicilin G acylase at both 27 and $37^{\circ}C$.

• Biomedical Science Letters
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• v.12 no.3
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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.

• Biomedical Science Letters
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• v.8 no.4
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• pp.245-250
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• 2002

Fibrinolytic enzyme has been purified from the edible mushroom, Tricholoma sejunctum using DEAE-cellulose chromatography, Phenyl-Sepharose chromatography and Mono-S column chromatography. The apparent molecular mass of purified enzyme was estimated to be 17100 Da by SDS-polyacrylamide gel electrophoresis and 19000 Da by gel filtration, Indicating that it was a monomer. The N-terminal amino acid sequence of the enzyme was Ala-Thr-Tyr-Lys-Ile-X-Ser-Ala-Thr-His-Gln-X-X-Leu-Val. It has a pH optimum at pH 9.5, suggested that purified enzyme was a alkaline protease. The activity of purified enzyme was inhibited by EDTA and 1,10-phenanthroline, indicating that purified enzyme is a metalloprotease. The activity of purified enzyme was increased by Zn$^{2+}$ and Co$^{2+}$, however, the enzyme activity was totally inhibited by Hg$^{2+}$.

• 한국생물공학회:학술대회논문집
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• 2002.04a
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• pp.62-65
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• 2002

We have previously shown that the addition of silkworm hemolymph to a culture medium increases the longevity of insect and mammalian cells by inhibiting apoptosis. This indicates that the component which inhibits apoptosis is contained in the silkworm hemolymph, The apoptosis-inhibiting component was isolated from silkwonn hemolymph and characterized in our previous study. A database search using the N-terminal amino acid sequence of this component as a template resulted in a 95% homology with a low molecular weight lipoprotein, the so called ’30K protein' of unknown function. In this study, the 30K protein gene was expressed in mammalian and insect cells to confirm the apoptosis-inhibiting effect. The overexpression of 30K protein in mammalian cell inhibited the staurosporin-induced apoptosis by the prevention of the activation of caspase 3. Using an Autographa californicanuclear polyhedrosis virus (AcNPV) system, the 30K protein was overexpressed also in insect cells. The expression of the 30K protein increased the longevity of baculovirus-infected insect cells by inhibiting apoptosis. These results suggest that the 30K protein is a novel anti-apoptotic protein.

• Animal cells and systems
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• v.2 no.3
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• pp.367-370
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• 1998

Two molecular species of apolipophorin-III (spoLp-III) were purified from the last instar larval hemolymph of Galleria mellonella by gel permeation chromatography (Sephadex G-100), ion exchange chromatography (DE-52), heat treatment (90C for 30 min) and Mono S FPLC, and were named apoLp-III-a and apoLp-lll-b, respectively. They were indistinguishable by SDS-PAGE but could be separated by native PAGE. The molecular mass of apoLp-III determined by SDS-PAGE was approximately 18 kDa. The N-terminal amino acid sequence of apoLp-III-b revealed high similarities with the apoLp-III from Manduca sexta.