• Journal of Microbiology and Biotechnology
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• v.10 no.5
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• pp.612-619
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• 2000

To investigate the biochemical properties of V. mimicus metalloprotease, whose gene was isolated previously from Vibrio mimicus ATCC33653, overexpression and purification were attempted. The 1.9 kb of open reading frame was amplified by PCR from pVMC193 plasmid which ligated the VMC gene with pUC19 and introduced into Escherichia coli BL21 (DE3) using the overexpression vector, pET22b (+). The overexpressed metalloprotease (VMC) was purified with Ni-NTA column chromatography and characterized with various protease inhibitors, pHs, temperatures, and substrates. The purified VMC showed the proteolytic activity against gelatin, soluble and insoluble collagens, and synthetic peptides. Unlike the observations made with all metalloproteases originated from other Vibrio sp., the VMC did not hydrolyze the casein. The proteolytic activity was critically decreased when the VMC was treated with metal chelating reagents, such as EDTA, 2,2-bipyridine, and 1, 10-phenanthroline. In particular, the 71 kDa VMC exhibited the hemagglutinating activity against human erythrocyte. As the purified VMC was treated with $CuCl_2$ and $NiCl_2$ for the chemical modification of metal binding, the proteolytic activity and hemagglutinating activity were profoundly influenced. The multialignment analysis made on the reported Vibrio metalloproteases showed the difference of amino acid sequence similarity between the two distinctive classes of Vibrio metalloproteases.

• Journal of Applied Biological Chemistry
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• v.52 no.3
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• pp.109-115
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• 2009

The gene encoding a putative aspartate aminotransferase in Xanthomonas oryzae pv. oryzae (Xoo) was cloned using PCR technique. The gene was ligated with pET-21(a) vector containing His6 tag and expressed in E. coli BL21(DE3). Affinity purification of the recombinant aspartate aminotransferase with Ni-NTA resin resulted in one band by SDS-PAGE analysis. The purified enzyme showed a molecular weight of 43 kDa, as expected. The enzyme was the most active toward L-aspartate as an amino donor, indicating that the purified enzyme is one of aspartate aminotrans-ferases exist in Xoo. Optimal activity of the enzyme was observed at around pH 7.5 and stability was much higher at alkaline pH rather than acidic pH values. The enzyme was considerably activated by the presence of manganese ion, showing about 157% of control activity at 1.0 mM.

• Journal of the Korean Institute of Electrical and Electronic Material Engineers
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• v.20 no.1
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• pp.80-85
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• 2007

We report the electro-optical properties of the sensory rhodopsin II using a near-field scanning microwave microscope(NSMM). Rhodopsin was known as a photoreceptor pigment with a retinal as a chromophore via a protonated Schiff base and consists of seven ${\alpha}-helical$ transmembrane segments. The sensory rhodopsin II, expressing E. coli UT5600 with endogenous retinal biosynthesis system and purified with $Ni^{-2}-NTA$ affinity chromatography in the presence of 0.02 % DM (Dodecyl Maltoside) from Natronomonas pharaonis. We measured the absorption spectra and the transients difference of sensory rhodopsin II from Natronomonas pharaonis using a UV/VIS spectrophotometer with Nd-Yag Laser (532 nm). The absorption spectra of NpSR II showed a typical rhodopsin spectrum with a left shoulder region and the photointermediates spectra of NpSR II-ground state (${\lambda}max=498\;nm$), NpSR II-M state (${\lambda}max=390\;nm$), and NpSR II-O state (${\lambda}max=550\;nm$) during the photocycle. The observed photocycle reaction was confirmed by measuring the microwave reflection coefficient $S_{11}$ at an operating frequency of f=3.93-3.95 GHz and compared with the results of a photocycle of NpSR II.

• Research in Plant Disease
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• v.18 no.3
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• pp.231-235
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• 2012

Indian citrus ring spot virus (ICRSV) is known to cause a serious disease to citrus, especially to Kinnow mandarin, the popular cultivated citrus species in India. In this study, we developed diagnostic systems based on enzyme-linked immunosorbent assay (ELISA). In order to generate antibodies against ICRSV coat protein, we overexpressed the coat protein in Escherichia coli using the pET15b expression vector containing an optimized ICRSV coat protein gene. The recombinant ICRSV coat protein was overexpressed as soluble form at $37^{\circ}C$ upon IPTG induction. The protein was purified to 95% in purity by Ni-NTA column chromatography. The purified protein was immunized to rabbit for the generation of polyclonal antibody (PAb). The PAb showed a specific immunoreaction to recombinant ICRSV coat protein in western blot analysis and ELISA. Diluted rabbit antisera (10,000 fold) could detect less than 10 ng and 5 ng of the target protein in western blot and ELISA analysis, respectively.

• Journal of Microbiology and Biotechnology
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• v.16 no.1
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• pp.145-148
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• 2006

A new functional lipolytic enzyme (AT4) has recently been found from Agrobacterium tumefaciens C58 Cereon using a genome-wide approach. The enzyme has some sequence similarity to E. coli acetyl hydrolase, Emericella nidulans lipase, Moraxella sp. lipase, Acinetobacter lwoffii esterase, and Streptomyces hygroscopicus acetyl hydrolase. However, the sequence similarities are very low (less than $25\%$), suggesting that it is a new lipase/esterase enzyme. ill the present study, intact cell of the A. tumefaciens strain was shown to have lipolytic activity on a tributyrin-LB plate. The AT4 gene was then expressed at a high level in E. coli BL21 (DE3) cells and the enzyme was purified simply by Ni-NTA column chromatography. The purified enzyme showed hydrolytic activity toward p-nitrophenyl caproate, but not toward olive oil, suggesting that the AT4 enzyme was a typical esterase rather than lipase. AT4 esterase had a maximum hydrolytic activity at $45^{\circ}C$ and pH 8.0, when p-nitrophenyl caproate was used as a substrate. It was relatively stable up to $40^{\circ}C$ and at pH 5.0-9.0. Calcium ion and EDT A did not affect the activity and thermal stability of the enzyme. As for substrate specificity, AT4 enzyme could rapidly hydrolyze acetyl and butyl groups from p-nitrophenyl esters and 1-naphthyl esters. In addition, it also released acetyl residues from acetylated glucose and xylose substrates. Therefore, this new esterase enzyme might be used as a biocatalyst in acetylation and deacetylation reactions performed in the fine chemical industry.

• Journal of Microbiology and Biotechnology
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• v.20 no.6
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• pp.995-1000
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• 2010

A novel deoC gene was identified from Paenibacillus sp. EA001 isolated from soil. The gene had an open reading frame (ORF) of 663 base pairs encoding a protein of 220 amino acids with a molecular mass of 24.5 kDa. The amino acid sequence was 79% identical to that of deoxyribose 5-phosphate aldolase (DERA) from Geobacillus sp. Y412MC10. The deoC gene encoding DERA was cloned into an expression vector and the protein was expressed in Escherichia coli. The recombinant DERA was purified using Ni-NTA affinity chromatography and then characterized. The optimum temperature and pH of the enzyme were $50^{\circ}C$ and 6.0, respectively. The specific activity for the substrate deoxyribose 5-phosphate (DR5P) was $62\;{\mu}mol/min/mg$. The $K_m$ value for DR5P was determined to be 145 mM with the $k_{cat}$ value of $3.2{\times}10^2/s$ from Lineweaver-Burk plots. The EA001 DERA showed stability toward a high concentration of acetaldehyde (100 mM).

• Microbiology and Biotechnology Letters
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• v.46 no.1
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• pp.51-58
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• 2018

The expression, purification, and characterization of cold-adapted lipase from the psychrophile, Janthinobacterium sp. were investigated. The gene encoding lipase from Janthinobacterium sp. PAMC 25641 was cloned into a pET28a(+) vector and heterologously expressed in Escherichia coli BL21 (DE3). The amino acid sequence deduced from the nucleotide sequence (930 bp) corresponded to a protein having 309 amino acid residues with a molecular weight of 32.7 kDa and a pI of 5.55. Recombinant E. coli harboring the Janthinobacterium lipase gene were induced by addition of isopropyl-${\beta}$-D-thiogalactopyranoside. $Ni^{2+}$-NTA affinity chromatography was used to purify the lipase, which had a specific activity of 107.9 U/mg protein. The effect of temperature and pH on the activity of lipase was measured using p-nitrophenyl octanoate as a substrate. The stability of the lipase at low temperatures indicated it is a cold-adapted enzyme. The lipase activity was increased by $Na^{2+}$, $Mg^{2+}$, and $Mn^{2+}$, and decreased by $Zn^{2+}$ and $Co^{2+}$. Analysis of the lipase activity using various p-nitrophenyl esters showed a strong preference toward short acyl chains of the esters, indicating the ability of the cold-adapted lipase to hydrolyze short-chain esters.

• Journal of Microbiology and Biotechnology
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• v.16 no.11
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• pp.1809-1813
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• 2006

A gene corresponding to 4-${\alpha}$-glucanotransferase (${\alpha}GTase$) was cloned from the thermophilic bacterium Thermus brockianus. The nucleotide sequence analysis showed that the ${\alpha}GTase$ gene is composed of 1,503 nucleotides and encodes a polypeptide that is 500 amino acids long with a calculated molecular mass of 57,221 Da. The deduced amino acid sequences of Thermus brockianus ${\alpha}GTase$ (TBGT) exhibited a high level of similarity to the amino acid sequence of ${\alpha}GTase$ of Thermus thermophilus (86%), but low level of homology to that of E. coli (26%). The TBGT gene was overexpressed in E. coli BL21, and the corresponding recombinant enzyme was efficiently purified by Ni-NTA affinity chromatography. The enzymatic characteristics revealed that optimal pH and temperature were pH 6 and $70^{\circ}C$, respectively. Most interestingly, TBGT reacted with small oligosaccharides, especially maltotriose, to form various maltooligosaccharides by using its disproportionation activity.

• Journal of Microbiology and Biotechnology
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• v.25 no.1
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• pp.18-25
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• 2015

To understand the metabolism of flavonoid rhamnoglycosides by human intestinal microbiota, we measured the metabolic activity of rutin and poncirin (distributed in many functional foods and herbal medicine) by 100 human stool specimens. The average α-L-rhamnosidase activities on the p-nitrophenyl-α-L-rhamnopyranoside, rutin, and poncirin subtrates were 0.10 ± 0.07, 0.25 ± 0.08, and 0.15 ± 0.09 pmol/min/mg, respectively. To investigate the enzymatic properties, α-L-rhamnosidase-producing bacteria were isolated from the specimens, and the α-L-rhamnosidase gene was cloned from a selected organism, Bifidobacterium dentium, and expressed in E. coli. The cloned α-L-rhamnosidase gene contained a 2,673 bp sequcence encoding 890 amino acid residues. The cloned gene was expressed using the pET 26b(+) vector in E. coli BL21, and the expressed enzyme was purified using Ni²⁺-NTA and Q-HP column chromatography. The specific activity of the purified α-L-rhamnosidase was 23.3 µmol/min/mg. Of the tested natural product constituents, the cloned α-L-rhamnosidase hydrolyzed rutin most potently, followed by poncirin, naringin, and ginsenoside Re. However, it was unable to hydrolyze quercitrin. This is the first report describing the cloning, expression, and characterization of α-L-rhamnosidase, a flavonoid rhamnoglycosidemetabolizing enzyme, from bifidobacteria. Based on these findings, the α-L-rhamnosidase of intestinal bacteria such as B. dentium seem to be more effective in hydrolyzing (1 →6) bonds than (1 →2) bonds of rhamnoglycosides, and may play an important role in the metabolism and pharmacological effect of rhamnoglycosides.

• The Journal of Korean Society of Virology
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• v.28 no.3
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• pp.215-224
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• 1998

Cloning, sequencing and expressing in E. coli of the thymidine kinase (TK) gene of Herpes simplex virus type-1 (HSV-1) strain F was investigated. The TK gene, located in the BamHI 3.74 kb DNA fragment of the plasmid pHLA-12, was amplified by polymerase chain reaction (PCR). The 1,131 kb PCR product was cloned into the BamHI and EcoRI sites of pBacPAK9 plasmid and then named pBac-TK recombinant. The TK gene was subcloned into the BamHI and BglII sites of pQE-30, and named pQE-TK recombinant. The nucleotide sequence of the 1,131 kb TK gene was determined, and the GC content was 65.13%. There were deduced 367 amino acid residues with a total molecular weight of 43 kDa. The weight was confirmed by the protein produced by E. coli M15/pQE-TK on the SDS-PAGE and Western blot. The production of the TK protein in the IPTG induced cells was measured over 4 h. At the end of 1, 2 and 3 h the level increased by 146, 204 and 242%, respectively. The amount of the protein at the highest fraction purified with Ni-NTA resin chromatography was $0.68\;{\mu}g$ per ml. The soluble state TK protein was present in the cytoplasm. In these results the F strain was different in base sequence and amino acid sequence from that of the CL101 strain, which caused difference in their strains.