• KSBB Journal
• /
• v.11 no.4
• /
• pp.431-437
• /
• 1996

The nar promoter of Escherichta coli, which is maximally induced under anaerobic conditions in the presence of nitrate, was characterized to see whether the nar promoter cloned onto pBR322 can be used as an expression promoter. The modified nar promoter, in which several bases in the -10 region was mutated to the consensus sequence by site-directed mutagenesis, was characterized in E. coli, on which chromosome the fnr gene affecting expression of the nar promoter according to dissolved oxygen level was mutated. The E. coli lacZ gene was used as a reporter gene. The following effects were investigated to find optimal conditions to induce the modified nar promoter: induction methods, optimal nitrate concentrations, the amount of ${\beta}$-galactosidase expressed at the different growth conditions, and induction characteristics. The following results were obtained from the experiments : expression of ${\beta}$-galactosidase from the modified nar promoter was not affected much by nitrate concentrations. The maximal specific ${\beta}$-galactosidase activity was obtained when E. coli was grown under aerobic conditions, and then the modified nar promoter was induced at OD600=2.2 under microaerobic conditions (DO=1∼2%), under which conditions the maximal specific ${\beta}$-galactosidase activity was 13,000 Miller units. However, the specific ${\beta}$-galactosidase activity was approximately 6,000 Miller units even before the modified nar promoter was induced. Therefore, the modified nar promoter seemed to be useful when the cloned gene wants to be expressed in E. coli constitutively.

• Korean Journal of Microbiology
• /
• v.44 no.1
• /
• pp.74-79
• /
• 2008

The purpose of this study is to clone cgt gene from Paenibacillus sp. JB-13 and to overexpress the protein in E. coli. For this purpose, the cgt gene was amplified from Paenibacillus sp. JB-13 genomic DNA by PCR using degenerate oligonucleotide primers. The sequence analysis results showed that the cgt gene from Paenibacillus sp. JB-13 has 98% homology with the cgt gene of Bacillus sp. To overexpress the protein, the cgt gene was cloned into pEXP7 expression vector and transformed into E. coli. The production of CGTase by recombinant E. coli was optimized under following conditions: 0.5% glucose, 3.0% polypeptone, 0.3% $K_2HPO_4$, 0.5% NaCl, and 7.0 of initial pH, 2.0% of inoculum, $37^{\circ}C$ of culture temperature for 14 hr. And the optimal agitation was found at 0.1 vvm. The synthesis of 2-O-${\alpha}$-D-Glucopyranosyl L-Ascorbic acid (AA-2G) using the CGTase expressed in E. coli was identified as AA-2G by HPLC and HPLC confirmed that treating AA-2G made by cloned CGTase with ${\alpha}$-glucosidase substantially produced AA and glucose.

• Microbiology and Biotechnology Letters
• /
• v.34 no.3
• /
• pp.211-215
• /
• 2006

AfsR2 is a global regulatory protein involved in the stimulation of secondary metabolite biosynthesis in various Streptomyces species including avermectin-producing S. avermitilis. Among several AfsR2-dependent genes identified from the comparative proteomics, the polyribonucleotide nucleotidyltransferase (PNP) and the glyceraldehyde-3-phosphate dehydrogenase (GPD) genes were previously proposed to regulate the actinorhodin production in S. lividans upon afsR2 over-expression positively and negatively, respectively. To show the biological significance of the PNP and GPD genes in the S. avermitilis strains, these two genes were functionally expressed in both the wild-type and the avermectin-overproducing mutant strains. The PNP gene expression stimulated secondary metabolite production in the wild-type S. avermitilis ATCC31267, but not in the avermectin-overproducing S. avermitilis ATCC31780. Interestingly, the GDP gene expression stimulated secondary metabolite production by 4-fold in the wild-type S. avermitilis ATCC31267 and by 2.5-fold in the avermectin-overproducing S. avermitilis ATCC31780, respectively. These results suggest that the biological significance of the afsR2-dependent PNP and GPD gene expressions on antibiotic biosynthetic regulation could be significantly different depending on Streptomyces species.

• Journal of the korean academy of Pediatric Dentistry
• /
• v.32 no.2
• /
• pp.357-369
• /
• 2005

Streptococci are Gram-positive, facultative anaerobes and have no catalase activities. Among mutans streptococci containing ${\alpha}-type$ hemolytic activity, S. mutans is a causative agent for dental caries. As well as acid production yielding the demineralization of tooth enamel, adherence and colonization of S. mutans to the teeth are also important for its virulence. These early colonization are accomplished by the bacterial fibrillar protein, Antigen I/II (Ag I/II) and glucosyltransferase (GTF). Therefore, Ag I/II and GTF are reasonable targets for the development of vaccine against S. mutans GS-5. The ag I/II and gtfD genes from S. mutans GS-5 were cloned and sequenced. Sequence analyses showed the nucleotides sequence of cloned genes had high homology to the sequences previously reported. The sequence alignment of 280 nucleotides between the cloned Ag I/II and the available sequence of the corresponding S. mutans GS-5 showed the perfect match. Comparing with the sequence of gtfD from S. mutans UA159, the corresponding nucleotide sequence of S. mutans GS-5 showed some mismatches and the mismatches introduced changes in four residues out of 105 amino acids, yielding four missense mutations.

• Korean Journal of Microbiology
• /
• v.46 no.2
• /
• pp.213-218
• /
• 2010

Lactate dehydrogenases (LDHs) have been highly focused for long time, due to their important roles in biochemical and metabolic pathways of cells. On the basis of genome-wide searching results, three putative LDH genes from Bacillus cereus ATCC 14579 genome have been PCR-amplified, cloned, and well-expressed in E. coli. All three BcLDH isozymes are supposed to share highly conserved catalytic amino acid residues in common $NAD^+$-dependent LDHs. Meanwhile, BcLDH1 consisting of 314 amino acids shares 86 and 49% of identities with BcLDH2 and 3, respectively. Interestingly, only BcLDH1 showed the converting activities between L-lactate and pyruvate in the presence of $NAD^+$ coenzyme, while the other isozymes are likely to have almost no activity. As a result, it was revealed that BcLDH1 can be a typical $NAD^+$-dependent L-lactate-specific dehydrogenase.

• Journal of Life Science
• /
• v.22 no.11
• /
• pp.1545-1551
• /
• 2012

Authors report the glycoside hydrolase (GH) family 16 ${\beta}$-agarase from the strain of Agarivorans sp. JA-1, which authors previously stated as recombinant expression and characterization of GH-50 and GH-118 ${\beta}$-agarase. It comprised an open reading frame of 1,362 base pairs, which encodes a protein of 49,830 daltons consisting of 453 amino acid residues. Valuation of the total sequence showed that the enzyme has 98% nucleotide and 99% amino acid sequence similarities to those of GH-16 ${\beta}$-agarase from Pseudoalteromonas sp. CY24. The gene corresponding to a mature protein of 429 amino acids was recombinantly expressed in Escherichia coli, and the enzyme was purified to homogeneity by affinity chromatography. It showed maximal activity at $40^{\circ}C$ and pH 5.0, representing 67.6 units/mg. Thin layer chromatography revealed that mainly neoagarohexaose and neoagarotetraose were produced from agarose. The enzyme would be valuable for the industrial production of functional neoagarooligosaccharides.

• Journal of Life Science
• /
• v.26 no.10
• /
• pp.1113-1120
• /
• 2016

Two bacterial strains producing extracellular man nanase were isolated from doenjang, a traditionally fermented soybean paste in Korea. The isolates, WL-6 and WL-11, were identified as Bacillus subtiis on the basis of their 16S rRNA gene sequences, morphological, and biochemical properties. Two genes encoding the mannanase of both B. subtilis WL-6 and B. subtilis WL-11 were each cloned into Escherichia coli, and their nucleotide sequences were determined. Both mannanase genes consisted of 1,086 nucleotides, encoding polypeptides of 362 amino acid residues. The deduced amino acid sequences of the two WL-6 and WL-11 mannanases, designated Man6 and Man11, respectively, differed from each other by eight amino acid residues, and they were highly homologous to those of mannanases belonging to the glycosyl hydrolase family 26. The 26 amino acid stretch in the N-terminus of Man6 and Man11 was a predicted signal peptide. Both Man6 and Man11 were localized at the level of 94–95% in an intracellular fraction of recombinant E. coli cells. The enzymes hydrolyzed both locust bean gum and mannooligosaccharides, including mannotriose, mannotetraose, mannopentaose, and mannohexaose, forming mannobiose and mannotriose as predominant products. The optimal reaction conditions were 55°C and pH 6.0 for Man6, and 60°C and pH 5.5 for Man11. Man11 was more stable than Man6 at high temperatures.

• Microbiology and Biotechnology Letters
• /
• v.35 no.2
• /
• pp.104-111
• /
• 2007

In this study, aniline dioxygenase genes responsible for initial catabolism of aniline in Burkholderia sp. HY1 and Delftia sp. HY99 were cloned and the amino acid sequences were comparatively analyzed, which already have been reported as bacteria utilizing aniline as a sole source of carbon and nitrogen, B. sp. HY1 was found to have at least a plasmid, and the plasmld-cured strain, B. sp. HY1-PC obtained using mitomycin C was tested with wild type strain to investigate whether the former maintained the degradability for aniline. This proved that the aniline oxygenase gene from B. sp. HY1 was located in chromosomal DNA, not in plasmid DNA. Aniline dioxygenase small subunits from B. sp. HY1 and D. sp. HY99 were found, based on 146 amino acids, to share 79% similarity. Notably, ado2 genes from B. sp. HY1 and D. sp. HY99 which were found to be terminal dioxygenase of aniline dioxygenase small subunit showed 99% similarity in the deduced amino acid sequences with tdnA2 of Frateuria sp. ANA-18 and danA2 of D. sp. AN3, respectively. Besides, enzyme assay and amino acid sequence analysis of catechol dioxygenase supported the previous report that B. sp. HY1 might occupy ortho-cleavage pathway using catechol 1,2-dioxygenase, while D. sp. HY99 might occupy catechol 2,3-dioxygenase for meta-cleavage pathway.

• Journal of Life Science
• /
• v.24 no.9
• /
• pp.995-1000
• /
• 2014

The studies of marine viruses in terms of viral isolation and detection have been limited due to the high mutation rate and genetic diversity of marine viruses. Of the modern methods currently used to detect marine viruses, serological methods based on enzyme-linked immunosorbent assay (ELISA) are the most common. They depend largely on the quality of the antibodies and on highly purified suitable antigens. Recently, a new experimental system for using viral capsid protein as an antigen has been developed using the yeast surface display (YSD) technique. In the present study, the capsid protein gene of the red-spotted grouper nervous necrosis virus (RGNNV) was expressed and purified via YSD and HA-tagging systems, respectively. Two regions of the RGNNV capsid protein gene, RGNNV1 and RGNNV2, were individually synthesized and subcloned into a yeast expression vector, pCTCON. The expressions of each RGNNV capsid protein in the Saccharomyces cerevisiae strain EBY100 were indirectly detected by flow cytometry with fluorescently labeled antibodies, while recognizing the C-terminal c-myc tags encoded by the display vector. The expressed RGNNV capsid proteins were isolated from the yeast surface through the cleavage of the disulfide bond between the Aga1 and Aga2 proteins after ${\beta}$-mercaptoethanol treatment, and they were directly detected by Western blot using anti-HA antibody. These results indicated that YSD and HA-tagging systems could be applicable to the expressions and purification of recombinant RGNNV capsid proteins.

• Journal of Sericultural and Entomological Science
• /
• v.52 no.2
• /
• pp.102-109
• /
• 2014

We constructed the fibroin H-chain expression system to produce enhanced yellow fluorescent proteins (EYFP) in the silk of transgenic silkworm. Fluorescent silk could be made by fusing EYFP cDNA to the heavy chain gene and injecting it into a silkworm. The EYFP fusion protein, each with N- and C-terminal sequences of the fibroin H-chain, was designed to be secreted into the lumen of the posterior silk glands. The expression of the EYFP/H-chain fusion gene was regulated by the fibroin H-chain promoter. The yellow fluorescence proving that the fusion protein was present in the silk. Accordingly, we suggest that the EYFP fluorescence silk will enable the production of novel biomaterial based on the transgenic silk.