• Journal of Applied Biological Chemistry
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• v.42 no.4
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• pp.197-201
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• 1999

Antagonistic bacteria active against phytopathogenic fungi, Phytophthora capsici, Pythium ultimum, Rhizoctonia solani, Botrytis cinerea, and Fusarium oxysporum were isolated from greenhouse soils. An antifungal compound was extracted by ethyl acetate from acidified culture filtrate and purified through column chromatography and thin layer chromatography. Activity-guided bioassay was followed throughout the purification steps using Pythium ultimum as a test organism. The purified antifungal compound was identified as phenylacetic acid (PAA) based on the data obtained from IR, EI/MS, $^1H-NMR$, and $^{13}C-NMR$. Two different isolates, which had vast differences in differential characteristics except 16S rDNA sequence homology, produced the same compound, phenylacetic acid. $ED_{50}$ values of the phenylacetic acid against P. ultimum, P. capsici, R. solani, B. cinerea, and F. oxysporum were 45, 21, 318, 360, and 226 ppm, respectively.

• Journal of Microbiology and Biotechnology
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• v.14 no.2
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• pp.284-289
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• 2004

A PCR assay for the rapid detection of Vibrio vulnificus strains was developed using a virulence gene for elastase found in various Vibrio species. The DNA sequences in the elastase gene facilitated the identification of a species-specific probe for pathogenic V. vulnificus strains from both clinical and environmental sources. Using an elastase gene-based PCR reaction, a species-specific 507-bp PCR product was visualized by agarose gel electrophoresis. Three different DNA extraction methods were then compared to improve the simplicity and rapidity of detection. A PCR assay using the conventional DNA extraction or boiling method was able to detect as few as 25 V. vulnificus cells, making the detection limits at least 1-log-scale lower than that for the EDT A-treated DNA extraction method. In particular, the boiling method, which does not require purification of the chromosomal DNA, was very effective in terms of simple and rapid detection. Meanwhile, the detection limit in a mixed bacterial culture that included other bacteria, such as Escherichia coli or Bacillus subtilis, was two V. vulnificus cells, which was 1-log-scale lower than that for the control. Accordingly, when coupled with a new DNA extraction method, the elastase gene-based PCR can provide a rapid, specific, and sensitive method for identifying V. vulnificus in clinical and environmental samples.

• Microbiology and Biotechnology Letters
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• v.38 no.2
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• pp.158-162
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• 2010

The gene encoding N-terminally truncated Tod polymerase ($\Delta$Tod polymerase) from Thermus thermophilus HJ6 was expressed in Escherichia coli under the control of the lambda pR and pL tandem promoters on the expression vector pJLA503. The N-terminal domain (250 amino acids) of Tod polymerase was removed without significant effect on enzyme activity and stability, while no 5'$\rightarrow$3' exonuclease activity was detected. The $\Delta$Tod polymerase was verified to possess very efficient reverse transcriptase (RT) activity in the presence of $MgCl_2$. The cDNA can also be amplified in the polymerase chain reaction (PCR) with this mutant enzyme. The $\Delta$Tod polymerase was exhibited higher activity than the Taq polymerase in a one-step RT-PCR.

• Journal of Applied Biological Chemistry
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• v.44 no.4
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• pp.167-172
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• 2001

A cDNA fragment encoding the chloroplastic fructose-1, 6-bisphosphatase (FBPase) was cloned via PCR from the cDNA library of pea leaves. The cloned cDNA, about 1.05 kbp without signal sequence, was introduced into a pET-28a vector for expression in E. coli strain BL21(DE3)pLysS. The recombinant FBPase was purified through $Ni^+-NTA$ affinity chromatography and characterized. Molecular mass of the monomer was about 42,000. Enzymatic activity of the purified enzyme as the native pea chloroplastic FBPase was the highest at alkaline pH (pH 9.0). The recombinant enzyme was activated by a reducing agent DTT and was insensitive to AMP. The activation energy (Ea) and Arrehenius frequency factor were 42.67 kcal/mol and $2.65{\times}10^{14}/s$, respectively, slightly higher than those of the native enzyme. $K_M$ and $V_{max}$ were $99.98{\mu}M$ and $52.9{\mu}M/min$, respectively, which were comparable with the native enzyme.

• BMB Reports
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• v.39 no.5
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• pp.578-585
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• 2006

The cDNA of a firefly luciferase from lantern mRNA of Lampyroidea maculata has been cloned, sequenced and functionally expressed. The cDNA has an open reading frame of 1647 bp and codes for a 548-residue-long polypeptide. Noteworthy, sequence comparison as well as homology modeling showed the highest degree of similarity with H. unmunsana and L. mingrelica luciferases, suggesting a close phylogenetic relationship despite the geographical distance separation. The deduced amino acid sequence of the luciferase gene of firefly L. maculata showed 93% identity to H. unmunsana. Superposition of the three-dimensional model of L. maculata luciferase (generated by homology modeling) and three dimensional structure of Photinus pyralis luciferase revealed that the spatial arrangements of Luciferin and ATP-binding residues are very similar. Putative signature of AMP-binding domain among the various firefly species and Lampyroidea maculata was compared and a striking similarity was found. Different motifs and sites have been identified in Lampyroidea maculata by sequence analysis. Expression and purification of luciferase from Lampyroidea maculata was carried out using Ni-NTA Sepharose. Bioluminescence emission spectrum was similar to Photinus pyralis luciferase.

• Archives of Pharmacal Research
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• v.23 no.2
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• pp.139-146
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• 2000

7- Bromomethylbenz[a]anthracene is a known mutagen and carcinogen. The two major DNA adducts produced by this carcinogen, i.e., $N^2$-(benz[a]anthracen-7-yl methyl)-2'-deoxyguanosine (2, b[a]$a^2$G) and $N^6$-(benz[a]anthracen-7-ylmethyl)-2'-deoxyadenosine (4, b[a]$a^6$/A), as wel 1 as the simpler benzylated analogs,$N^2$-benzyl-2'deoxyguanosine (1, $bn^2$G) and $N^6$-benzyl-2'-deoxyadenosine (3, $bn^6$/A), were prepared by direct aralkylation of 2'-deoxyguanosine and 2'-deoxyadenosine. To determine the site-specific mutagenicity of these bulky exocyclic amino-substituted adducts, the suitably protected nucleosides were incorporated into 16-base oligodeoxyribonucleotides in place of a normal guanine or adenine residues which respectively are part of the ATG initiation codon for the lac Z' \alpha-complementation gene by using an in situ activation approach and automated phosphite triester synthetic methods. The base composition and the incorporation of the bulky adducts into synthetic oligonucleotides were characterized after purification of the modified oligonucleotides by enzymatic digestion and HPLC analysis.

• Mycobiology
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• v.31 no.4
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• pp.196-199
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• 2003

A very reliable and specific method for the identification of fungi in ectotrophic mycorrhizal symbiosis was developed using a specific PCR assay based on the amplification of the ITS1 region. To obtain specific data, an ITS-diagnostic assay was carried out that reveals genera and species specific sequences. Here, an application of one method is presented, which covers the identification of pure mycelia, basidiocarps as well as mixed samples such as ectomycorrhizal roots that were mingled with remains of the host plant. For this purpose a protocol was established that allowed the extraction of DNA from single mycorrhizal roots. In order to perform a specific ITS analysis we generated a new ITS-primer(ITS8) by a multiple alignment of five different genera and species of mycorrhizal fungi. The utilization of ITS1 and ITS8 resulted in specific PCR amplicons, which were characterized by sequencing without purification steps, even when the template DNA was associated with roots.

• Journal of Marine Bioscience and Biotechnology
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• v.2 no.3
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• pp.142-148
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• 2007

An agar-degrading marine bacterium, strain AS-3 was isolated from the seawater. The strain AS-3 was identified as Algibacter lectus AS-3 by 16S rDNA sequence. The optimum medium for agarase activity of the isolated strain was determined to be marine medium, marine broth 2216 containing 0.1% agar as carbon source. An extracellular agarase was purified 6.9-fold from the culture supernatant by ammonium sulfate precipitation, ion exchange chromatography and gel filtration methods. The optimum pH and temperature for this enzyme were 7.0 and $40-50^{\circ}C$, respectively. Antioxidative activity of the strain AS-3 was 62.4% in the supernatant cultured for 12 h.

• Microbiology and Biotechnology Letters
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• v.39 no.3
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• pp.218-223
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• 2011

To obtain eicosapentaenoic acid (EPA)-producing bacteria, some 600 strains of bacteria were isolated from Antarctic sediment and marine organisms during the summer expedition of 1999-2000 and 7 EPA-producing bacteria were obtained through screening with TLC and GC. A strain designated as L93 showed the highest EPA production, which was gram-negative, rod-shaped bacterium. L93 strain was identified as Shewanella sp., from the sequence analysis of 16S rDNA. Optimal conditions temperature and pH for the growth and EPA production were about $4^{\circ}C$ and pH 7. In addition, its production was optimized by 50%(w/v) sea salt. We establish the optimal production system to produce about 320 mg per liter by using this optimal EPA production conditions. EPA-methyl ester was purified from cultured L93 strain to a purity of higher than 97% and typical purification yield is greater than 72% of the input amount via urea complexation and HPLC.

• Journal of Microbiology and Biotechnology
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• v.32 no.5
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• pp.551-563
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• 2022

L-asparaginase (E.C. 3.5.1.1) purified from bacterial cells is widely used in the food industry, as well as in the treatment of childhood acute lymphoblastic leukemia. In the present study, the Burkholderia pseudomallei L-asparaginase gene was cloned into the pGEX-2T DNA plasmid, expressed in E. coli BL21 (DE3) pLysS, and purified to homogeneity using Glutathione Sepharose chromatography with 7.26 purification fold and 16.01% recovery. The purified enzyme exhibited a molecular weight of ~33.6 kDa with SDS-PAGE and showed maximal activity at 50℃ and pH 8.0. It retained 95.1, 89.6%, and 70.2% initial activity after 60 min at 30℃, 40℃, and 50℃, respectively. The enzyme reserved its activity at 30℃ and 37℃ up to 24 h. The enzyme had optimum pH of 8 and reserved 50% activity up to 24 h. The recombinant enzyme showed the highest substrate specificity towards L-asparaginase substrate, while no detectable specificity was observed for L-glutamine, urea, and acrylamide at 10 mM concentration. THP-1, a human leukemia cell line, displayed significant morphological alterations after being treated with recombinant L-asparaginase and the IC₅₀ of the purified enzyme was recorded as 0.8 IU. Furthermore, the purified recombinant Lasparaginase improved cytotoxicity in liver cancer HepG2 and breast cancer MCF-7 cell lines, with IC₅₀ values of 1.53 and 18 IU, respectively.