• Food Science of Animal Resources
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• v.39 no.4
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• pp.601-609
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• 2019

Bifidobacterium longum KACC 91563 secretes family 5 extracellular solute-binding protein via extracellular vesicle. In our previous work, it was demonstrated that the protein effectively alleviated food allergy symptoms via mast cell specific apoptosis, and it has revealed a therapeutic potential of this protein in allergy treatment. In the present study, we cloned the gene encoding extracellular solute-binding protein of the strain into the histidine-tagged pET-28a(+) vector and transformed the resulting plasmid into the Escherichia coli strain BL21 (DE3). The histidine-tagged extracellular solute-binding protein expressed in the transformed cells was purified using Ni-NTA affinity column. To enhance the efficiency of the protein purification, three parameters were optimized; the host bacterial strain, the culturing and induction temperature, and the purification protocol. After the process, two liters of transformed culture produced 7.15 mg of the recombinant proteins. This is the first study describing the production of extracellular solute-binding protein of probiotic bacteria. Establishment of large-scale production strategy for the protein will further contribute to the development of functional foods and potential alternative treatments for allergies.

• Journal of Microbiology
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• v.44 no.3
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• pp.301-310
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• 2006

Two pathways of ammonium assimilation and glutamate biosynthesis have been identified in microorganisms. One pathway involves the NADP-linked glutamate dehydrogenase, which catalyzes the amination of 2-oxoglutarate to form glutamate. An alternative pathway involves the combined activities of glutamine synthetase, which aminates glutamate to form glutamine, and glutamate synthase, which transfers the amide group of glutamine to 2-oxoglutarate to yield two molecules of glutamate. We have cloned the large subunit of the glutamate synthase (GOGAT) from Salmonella typhimurium by screening the expression of GOGAT and complementing the gene in E. coli GOGAT large subunit-deficient mutants. Three positive clones (named pUC19C12, pUC19C13 and pUC19C15) contained identical Sau3AI fragments, as determined by restriction mapping and Southern hybridization, and expressed GOGAT efficiently and constitutively using its own promoter in the heterologous host. The coding region expressed in Escherichia coli was about 170 kDa on SDS-PAGE. This gene spans 4,732 bases, contains an open reading frame of 4,458 nucleotides, and encodes a mature protein of 1,486 amino acid residues (Mr =166,208). The EMN-binding domain of GOGAT contains 12 glycine residues, and the 3Fe-4S cluster has 3 cysteine residues. The comparison of the translated amino acid sequence of the Salmonella GOGAT with sequences from other bacteria such as Escherichia coli, Salmonella enterica, Shigella flexneri, Yersinia pestis, Vibrio vulnificus and Pseudomonas aeruginosa shows sequence identity between 87 and 95%.

• Journal of Microbiology and Biotechnology
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• v.15 no.4
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• pp.749-755
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• 2005

The ccpA gene encoding catabolite control protein A (CcpA) of Leuconostoc mesenteroides SYl, a strain isolated from kimchi, was cloned, sequenced, analyzed for transcript, and overexpressed in Escherichia coli. The ccpA ORF (open reading frame) is 1,011 bp in size, which can encode a protein of 336 amino acid residues with a molecular mass of 36,739 Da. The transcription start site was mapped at a position 49 nucleotides upstream of the start codon, and promoter sequences were also identified. The putative cre site overlapped with the -35 promoter sequence. The deduced amino acid sequence of the CcpA contained the helix-turn-helix motif found in many DNA-binding regulatory proteins. CcpA from 1. mesenteroides SY1 had $54.6\%$ identity with CcpA from Lactobacillus casei. The Northern blot experiment showed that ccpA was transcribed as a single 1.1 kb transcript, and transcription was repressed when grown on media containing glucose. CcpA was overproduced in E. coli BL21(DE3) cells using the pET expression vector, and purified to an apparent homogeneity. Gel Mobility Shift Assay with purified CcpA and a DNA fragment containing the ere sequence of the $\alpha$-galactosidase gene (aga) from L. mesenteroides SY1 revealed that CcpA bound specifically to the cre site of aga.

• Journal of Microbiology and Biotechnology
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• v.24 no.9
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• pp.1260-1268
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• 2014

Screening of a gene library from Paenibacillus sp. PBS-2 generated in Escherichia coli led to the identification of a clone with lipolytic activity. Sequence analysis showed an open reading frame encoding a polypeptide of 378 amino acid residues with a predicted molecular mass of 42 kDa. The esterase displayed 69% and 42% identity with the putative ${\beta}$-lactamases from Paenibacillus sp. JDR-2 and Clostridium sp. BNL1100, respectively. The esterase contained a Ser-x-x-Lys motif that is conserved among all ${\beta}$-lactamases found to date. The protein PBS-2 was produced in both soluble and insoluble forms when E. coli cells harboring the gene were cultured at $18^{\circ}C$. The enzyme is a serine protein and was active against p-nitrophenyl esters of $C_2$, $C_4$, $C_8$, and $C_{10}$. The optimum pH and temperature for enzyme activity were pH 9.0 and $30^{\circ}C$, respectively. Relative activity of 55% remained at up to $5^{\circ}C$ with an activation energy of 5.84 kcal/mol, which indicates that the enzyme is cold-adapted. Enzyme activity was inhibited by $Cd^{2+}$, $Cu^{2+}$, and $Hg^{2+}$ ions. As expected for a serine esterase, activity was inhibited by phenylmethylsulfonyl fluoride. The enzyme was remarkably active and stable in the presence of commercial detergents and organic solvents. This cold-adapted esterase has potential as a biocatalyst and detergent additive for use at low temperatures.

• Journal of Microbiology and Biotechnology
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• v.15 no.2
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• pp.254-258
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• 2005

We have conducted in vitro reconstitution study of ferritin from its subunits FerH and FerL. For the reconstitution, FerH was produced from an expression vector construct in Escherichia coli and was purified from a heat treated cell extract by using one-step column chromatography. FerL was expressed as inclusion bodies. The denatured form of FerL was obtained by a simple washing step of the inclusion bodies with 3 M urea. The reconstitution experiment was conducted with various molar ratios of urea-denatured FerH and FerL to make the ferritin nanoparticle with a controlled composition of FerH and FerL. SDS-PAGE analysis of the reconstituted ferritins revealed that the reconstitution required the presence of more than 40 molar$\%$ of FerH in the reconstitution mixture. The assembly of the subunits into the ferritin nanoparticle was confmned by the presence of spherical particles with diameter of 10 nm by the atomic force microscopic image. Further analysis of the particles by using a transmission electron microscope revealed that the reconstituted particles exhibited different percentages of population with dense iron core. The reconstituted ferritin nanoparticles made with molar ratios of [FerH]/[FerL]=l00/0 and 60/40 showed that 80 to $90\%$ of the particles were apoferritin, devoid of iron core. On the contrary, all the particles formed with [FerH]/[FerL]=85/ 15 were found to contain the iron core. This suggests that although FerH can uptake iron, a minor portion of FerL, not exceeding $40\%$ at most, is required to deposit iron inside the particle.

• Korean Journal of Veterinary Research
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• v.41 no.2
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• pp.189-195
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• 2001

Single base substitution and deletion mutation have been introducted into the verotoxin 2 (VT2)A subunit gene from O157:H7 isolates to reduce cytotoxicity of VT2 and the cytotoxicity between wild type toxin and mutant toxoid were compared. A M13-derived recombinant plasmid pEP19RF containing a 940bp EcoRI-PstI fragment of VT2A gene was constructed for oligonucleotide-directed mutagenesis. The duoble mutant pDOEX was constructed by point and deletion mutation of two different highly conserved regions of VT2A encoding active site cleft of enzymatic domain. The key residue, Glu 167(GAA) and the pentamer(WGRIS) consisting of the enzymatic domain were replaced by ASP(GAC) and completely deleted in nucleotide sequence analysis of mutant, respectively. In the comparision of vero cell cytotoxicity between wide type toxin and toxoid from mutant, the wild type toxin expressed cytotoxicity in dilution of $10^{-6}$, but the toxid from mutant did not show cytotoxicity to vero cells.

• Journal of Microbiology and Biotechnology
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• v.12 no.4
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• pp.603-609
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• 2002

DFA IV is di-D-fructose-2,6':6,2'-dianhydride, consisting of two fructose residues. It can be enzymatically synthesized from levan by levan fructotransferase, and can be used for mineral absorption. Understanding of the structure and composition of levan is important to obtain high-level production of DFA IV. A bacterial strain, Pseudomonas aurantiaca 5-4380, was identified to produce low-branched levan, and the levansucrase gene (lsch) from this bacterium was found to be composed of 1,275 Up coding for a protein of 424 amino acids, with an estimated molecular weight of 47 kDa. The bacterial levansucrase gene was expressed in Escherichia coli DH5${\alpha}$ by its own promoter and lac promoter. The recombinant levansucrase was produced in soluble form with 170U of levansucrase activity from 1-ml E. coii culture broth. The expressed enzyme from the clone showed similar biochemical properties, such as size of active levansucrase, degree of branching, and optimum temperature, with P.aurantiaca 5-4380 levansucrase.

• Journal of Microbiology and Biotechnology
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• v.20 no.5
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• pp.917-924
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• 2010

The practicability of transgenic tobacco engineered to express bacterial native mercuric reductase (MerA), responsible for the transport of $Hg^{2+}$ ions into the cell and their reduction to elemental mercury ($Hg^0$), without any codon modification, for phytoremediation of mercury pollution was evaluated. Transgenic tobacco plants reduce mercury ions to the metallic form; take up metallic mercury through their roots; and evolve the less toxic elemental mercury. Transformed tobacco produced a large amount of merA protein in leaves and showed a relatively higher resistance phenotype to $HgCl_2$ than wild type. Results suggest that the integrated merA gene, encoding mercuric reductase, a key enzyme of the bacterial mer operon, was stably integrated into the tobacco genome and translated to active MerA, which catalyzes the bioconversion of toxic $Hg^{2+}$ to the least toxic elemental $Hg^0$, and suggest that MerA is capable of reducing the $Hg^{2+}$, probably via NADPH as an electron donor. The transgenic tobacco expressing merA volatilized significantly more mercury than wild-type plants. This is first time we are reporting the expression of a bacterial native merA gene via the nuclear genome of Nicotiana tabacum, and enhanced mercury volatilization from tobacco transgenics. The study clearly indicates that transgenic tobacco plants are reasonable candidates for the remediation of mercurycontaminated areas.

• Applied Biological Chemistry
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• v.39 no.6
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• pp.430-436
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• 1996

The induction by xylose and repression by glucose of xylose isomerase(XI) were investigated to elucidate the regulation for production of XI in Escherichia coli. Regulation for expression of xyIA gene which codes XI is under control of xylR which is a regulatory gene for xylose catabolism. When xyIR gene was resided in chromosome, the inductions of XI by the addition of 0.4% xylose were increased to 1.9 and 1.7-fold in case of locating on multicopy(pEX202/DH77) and low copy Plasmid(pEX102/DH77), respectively, as compared with that of xylA gene which was resided in chromosome(JM109). xyIR gene product derived from xyIR gene on chromosome might react to xylA gene on the plasmid as same as xylA gene on chromosome. In JM109 and xylA transformant; pEX202/DH77 and pEX102/DH77, the inductions of XI were completely repressed by the addition of 0.2% glucose and these catabolite repressions were derepressed by the addition of 1 mM cAMP In comparison with the addition of 0.4% xylose only for the induction XI was inductively produced 1.7 to 2-fold with the addition of xylose plus 1 mM cAMP in DM minimal media. pEX13/TP2010, xylA transformant of the deficient mutant($xyl^-,\;cya^-$; TP2010) of XI and cAMP production, did not induce XI by the addition of xylose only but induced in case of simultaneous addition of xylose and cAMP. These results show that cAMP and xylose are the indispensable effectors for the induction and derepression of Xl in E. coli.

• Toxicological Research
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• v.26 no.3
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• pp.171-175
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• 2010

The human genome contains approximately 13 orphan cytochrome P450 (P450, CYP) genes, of which the apparent function or substrate has not been identified. However, they seem to possess their own biological relevance in some tissues or developmental stages. Here, we characterized the heterologously expressed CYP2W1, an orphan P450 enzyme. The recombinant CYP2W1 protein containing a $6{\times}$(His)-tag at Nterminus has been expressed in Escherichia coli and purified. Expression level of CYP2W1 holoenzyme was around 500 nmol P450 holoenzyme per liter culture medium. The reduced CO difference spectrum of CYP2W1 showed a maximum absorption at 449 nm. CYP2W1 indicated the significant induction to bioactivate Trp-P-1, MeIQ, and IQ in E. coli DJ701 tester strain. However, the bioactivation of B[$\alpha$]P, and NNK by CYP2W1 was relatively low. The model structure of CYP2W1 suggested the characteristic P450 folds with the lengths and orientations of the individual secondary elements. The F-G loop is situated on the distal side of heme to accommodate the flexibility of active site of CYP2W1. These studies can provide useful information for the finding of its biological roles and structure-function relationships of an orphan CYP2W1 enzyme.