• Journal of Microbiology and Biotechnology
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• v.10 no.6
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• pp.829-835
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• 2000

The ptsH and ptsI genes of Lactococus lactis subsp. lactis ATCC 7962 (L. lactis 7962), encoding the general proteins of phosphotransferase system (PTS) components, HPr and enzyme I, respectively, were cloned and characterized. A 1.3 kb PCR product was obtained using a primer set that was hybridized to the internal region of the L. lactis 7962 pts HI genes and then subcloned into a low-copy number vector, pACYC184. The 5' upstream and 3' downstream region from the 1.3 kb fragment were subsequently clone using the chromosome walking method. The complete ptsHI operon was constructed and the nucleotide sequences determined. Two ORFs corresponding to HPr (88 amino acids) and enzyme I (575 amino acids) were located. The ptsHI genes of L. lactis 7962 showed a very high homology (84-90%) with those genes from other Gram-positive bacteria. A primer extension analysis showed that the transcription started at either one of two adjacent bases upstream of the start codon. Using a Northern analysis, two transcripts were detected; the first, a 0.3 kb transcript corresponding to ptsH and the second, a 2 kb transcript corresponding to ptsH and ptsI. The transcription level of ptsH was higher than that of ptsI. The concentration of the ptsH transcript in cells grown on glucose was similar to that in cells grown on lactose, yet higher than that in cells grown on galactose. The ptsI transcript was scarcely detected in cell grown on lactose or galactose. The ptsI transcript was scarcely detected in cells grown on lactose or galactose. The results of a sequence analysis and Northern blot confirmed that the ptsH and ptsI genes of L. lactis 7962 were arranged in an operon like other known ptsHI genes and the expression of the ptsHI genes was regulated at the transcriptional level in response to the carbon source.

• Journal of Microbiology and Biotechnology
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• v.22 no.9
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• pp.1245-1252
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• 2012

Acinetobacter venetians V28 was isolated from the intestine of righteye flounder, Poecilopsetta plinthus caught in Vietnam seawater, and the esterase gene was cloned using a shotgun method. The amino acid sequence deduced from the nucleotide sequence (1,017 bp) corresponded to a protein of 338 amino acid residues with a molecular weight of 37,186. The esterase had 87% and 72% identities with the lipases of A. junii SH205 and A. calcoaceticus RUH2202, respectively. The esterase contained a putative leader sequence, as well as the conserved catalytic triad (Ser, His, Asp), consensus pentapeptide GXSXG, and oxyanion hole sequence (HG). The protein from the strain V28 was produced in both a soluble and an insoluble form when the Escherichia coli cells harboring the gene were cultured at $18^{\circ}C$. The maximal activity of the purified enzyme was observed at a temperature of $40^{\circ}C$ and pH 9.0 using p-NP-caprylate as substrate; however, relative activity still reached to 70% even at $5^{\circ}C$ with an activation energy of 3.36 kcal/mol, which indicated that it was a cold-adapted enzyme. The enzyme was a nonmetallo-protein and was active against p-nitrophenyl esters of $C_4$, $C_8$, and $C_{14}$. Remarkably, this enzyme retained much of its activity in the presence of commercial detergents and organic solvents. This cold-adapted esterase will be applicable as catalysts for reaction in the presence of organic solvents and detergents.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1994.04a
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• pp.186-186
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• 1994

Many agonists have been known to activate the hydrolysis of membrane phospholipids through the bindings with corresponding receptors on the various cells. Diacylglycerol and inositol 1,4,5-trisphosphate(IP3) generated by the action of phosphoinositide-specific phospholipase C (PI-PLC) are well known second messengers for the activation of protein kinase C and the mobilization of Ca2+ in many cells. Three types of PI-PLC isozyme (${\alpha}$,${\gamma}$, and $\delta$) and several subtrpes for each type have been identified from mammalian sources by purification of enzymes and cloning of their cDNAs. Each type PI-PLC isozyme is coupled to different receptors and mediators, for example, ${\beta}$-types are coupled to the seven-transmembrane-receptors via Gq family of G-proteins and ${\beta}$-types directly to the receptor tyrosine kinases. Specific modulators for the signaling pathway through each type of PI-PLC should be very useful as potential potential candidates for lend substances in developing novel drugs. To establish the sensitive and convenient screening systems for searching modulators on PI-PLC mediated signaling, two kinds of approaches have been tried. (1) Establishment of in vitro assay condition for each type of PI-PLC isozyme: Overexpression by using vaccinia virus and purification of each isozyme was carried out for the preparation of large amounts of enaymes. Optimum and sensitive assay condition for the measurements of PI-ELC activities were established. (2) Development of the cell lines in which each type of PI-PLC is permanently overexpressed: A fibroblast cell line (3T3${\gamma}$1-7) in which PI-PLC-${\gamma}$1 was overexpressed by using pZip-neo expression vector was developed and used for the measurement of PDGF-induced IP3 formation. The responses for IP3 formed in 3T3${\gamma}$1-7 cells by the treatment of PDGF is 8 times more sensitive than those in control cells. 3T3${\gamma}$l-7 cell is useful for the screening of the inhibitors on the PDGF-induced cellular responses from large number of samples in a small volume(50 ${\mu}$l) and short time(5-15 min). Using these systems, we screened hundreds of herb-extracts for the inhibition of PDGF-induced IP3 formation and selected several extracts that showed the inhibition as the candidates for isolation and characterization of active substances. The determination of the acting point of selected extracts or fractions in the PDGF signaling pathway has been analyzing.

• Korean Journal of Veterinary Research
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• v.48 no.4
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• pp.441-449
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• 2008

The genus Lactobacillus is the largest of the genera included in lactic acid bacteria and is associated with mucosal membranes of human and animal. Only a few Lactobacillus plasmid-encoded functions have been discovered and used. In this study, a novel plasmid (pILR091) was isolated from a wild L. reuteri isolated from pig and described the characteristics of its replicons, genetic organization, and relationship with other plasmids. After digestion of the plasmid, pILR091, with SalI, plasmid DNA was cloned into the pQE-30Xa vector and sequenced. The complete sequence was confirmed by the sequencing of PCR products and analyzed with the Genbank database. The isolate copy number and stability were determined by quantitative-PCR. The complete sequence of L. reuteri contained 7,185 nucleotides with 39% G-C content and one cut site by two enzymes, SalI and HindIII. The similar ori sequence of the pC194- rolling circle replication family (TTTATATTGAT) was located 63 bp upstream of the protein replication sequence, ORF 1. Total of five ORFs was identified and the coding sequence represented 4,966 nucleotides (70.4%). ORF1 of pILR091 had a low similarity with the sequence of pTE44. Other ORFs also showed low homology and E-values. The average G-C content of pILR091 was 39%, similar with that of genomic DNA. The copy number of pILR091 was determined at approximately 24 to 25 molecules per genomic DNA. These results suggested that pILR091 might be a good candidate to construct a new vector, which could be used for cloning and expression of foreign genes in lactobacilli.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.4
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• pp.607-615
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• 2018

Objective: This study was conducted to isolate the cellulolytic microorganism from the rumen of Holstein steers and characterize endoglucanase gene (Cel5A) from the isolated microorganism. Methods: To isolate anaerobic microbes having endoglucanase, rumen fluid was obtained from Holstein steers fed roughage diet. The isolated anaerobic bacteria had 98% similarity with Eubacterium cellulosolvens (E. cellulosolvens) Ce2 (Accession number: AB163733). The Cel5A from isolated E. cellulolsovens sp. was cloned using the published genome sequence and expressed through the Escherichia coli BL21. Results: The maximum activity of recombinant Cel5A (rCel5A) was observed at $50^{\circ}C$ and pH 4.0. The enzyme was constant at the temperature range of $20^{\circ}C$ to $40^{\circ}C$ but also, at the pH range of 3 to 9. The metal ions including $Ca^{2+}$, $K^+$, $Ni^{2+}$,$Mg^{2+}$, and $Fe^{2+}$ increased the endoglucanase activity but the addition of $Mn^{2+}$, $Cu^{2+}$, and $Zn^{2+}$ decreased. The Km and Vmax value of rCel5A were 14.05 mg/mL and $45.66{\mu}mol/min/mg$. Turnover number, Kcat and catalytic efficiency, Kcat/Km values of rCel5A was $96.69(s^{-1})$ and 6.88 (mL/mg/s), respectively. Conclusion: Our results indicated that rCel5A of E. cellulosolvens isolated from Holstein steers had a broad pH range with high stability under various conditions, which might be one of the beneficial characteristics of this enzyme for possible industrial application.

• Journal of Marine Bioscience and Biotechnology
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• v.2 no.4
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• pp.234-237
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• 2007

Calmodulin (CaM) is a $Ca^{2+}$-binding protein essential for biological functions mediated through $Ca^{2+}$-dependent mechanism. A cDNA clone for CaM was isolated from a cDNA library of olive flounder Paralichthys olivaceus. The CaM cDNA concists of 782 bp and encodes a polypeptide of 149 amino acids with four $Ca^{2+}$-binding motifs EF-hands (EF-I, EF-II, EF-III, and EF-IV). The deduced amino acid sequence of CaM shows 97-100% amino acid sequence identity to other CaM sequences. Semi-quantitative PCR analysis revealed that the CaM transcription was began during early development and the CaM mRNA is expressed highly in brain and intestine, and moderately in kidney, gill, and eye of healthy olive flounder. Taken together, CaM may be necessary for early olive flounder development and that it may have a part in homeostasis.

• Microbiology and Biotechnology Letters
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• v.36 no.3
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• pp.182-188
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• 2008

A hyperthermophilic bacteria (strain HJ6) was isolated from a hot springs located in the Arima-cho, Hyogo, Japan. The cells were long-rod type ($2-4{\mu}m$), about $0.4{\mu}m$ in diameter. The pH and temperature for optimal growth were 6.5 and $80^{\circ}C$, respectively. Phylogenetic analysis based on the 16S rDNA sequence and biochemical studies indicated that HJ6 belonged to the genus Thermus thermophilus (Tt). The gene encoding the Trehalose synthase (TS) was cloned and sequenced. The open reading frame (ORF) of the TtTS gene was composed of 2,898 nucleotides and encoded a protein (975 amino acids) with a predicted molecular weight of 110.56 kDa. The deduced amino acid sequence of TtTS showed 99% and 83% identities to the Thermus caldophilus TS and Meiothermus ruber TS, respectively. TtTS gene was expressed in Escherichia coli cells, and the recombinant protein was purified to homogeneity. The optimal temperature and pH for Trehalose synthase activity were found to be $80^{\circ}C$ and 7.5, respectively. The half-life of heat inactivation was about 40 min at $90^{\circ}C$. The maximum trehalose conversion rate of maltose into trehalose by the enzyme increased as the substrate concentration increased, and reached 55.7% at the maltose concentration of 500 mM, implying that the enzyme conversion was dependent of the substrate concentration.

• Journal of Microbiology and Biotechnology
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• v.21 no.8
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• pp.861-868
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• 2011

A xylanase gene, xyn7c, was cloned from Paenibacillus sp. 12-11, an alkalophilic strain isolated from the alkaline wastewater sludge of a paper mill, and expressed in Escherichia coli. The full-length gene consists of 1,296 bp and encodes a mature protein of 400 residues (excluding the putative signal peptide) that belongs to the glycoside hydrolase family 10. The optimal pH of the purified recombinant XYN7C was found to be 8.0, and the enzyme had good pH adaptability at 6.5-8.5 and stability over a broad pH range of 5.0-11.0. XYN7C exhibited maximum activity at $55^{\circ}C$ and was thermostable at $50^{\circ}C$ and below. Using wheat arabinoxylan as the substrate, XYN7C had a high specific activity of 1,886 U/mg, and the apparent $K_m$ and $V_{max}$ values were 1.18 mg/ml and 1,961 ${\mu}mol$/mg/min, respectively. XYN7C also had substrate specificity towards various xylans, and was highly resistant to neutral proteases. The main hydrolysis products of xylans were xylose and xylobiose. These properties make XYN7C a promising candidate to be used in biobleaching, baking, and cotton scouring processes.

• The Plant Pathology Journal
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• v.35 no.4
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• pp.351-361
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• 2019

In our previous study, pyrrolnitrin produced in Pseudomonas chlororaphis G05 plays more critical role in suppression of mycelial growth of some fungal pathogens that cause plant diseases in agriculture. Although some regulators for pyrrolnitrin biosynthesis were identified, the pyrrolnitrin regulation pathway was not fully constructed. During our screening novel regulator candidates, we obtained a white conjugant G05W02 while transposon mutagenesis was carried out between a fusion mutant $G05{\Delta}phz{\Delta}prn::lacZ$ and E. coli S17-1 (pUT/mini-Tn5Kan). By cloning and sequencing of the transposon-flanking DNA fragment, we found that a vfr gene in the conjugant G05W02 was disrupted with mini-Tn5Kan. In one other previous study on P. fluorescens, however, it was reported that the deletion of the vfr caused increased production of pyrrolnitrin and other antifungal metabolites. To confirm its regulatory function, we constructed the vfr-knockout mutant $G05{\Delta}vfr$ and $G05{\Delta}phz{\Delta}prn::lacZ{\Delta}vfr$. By quantifying ${\beta}-galactosidase$ activities, we found that deletion of the vfr decreased the prn operon expression dramatically. Meanwhile, by quantifying pyrrolnitrin production in the mutant $G05{\Delta}vfr$, we found that deficiency of the Vfr caused decreased pyrrolnitrin production. However, production of phenazine-1-carboxylic acid was same to that in the wild-type strain G05. Taken together, Vfr is required for pyrrolnitrin but not for phenazine-1-carboxylic acid biosynthesis in P. chlororaphis G05.

• Journal of Bio-Environment Control
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• v.11 no.3
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• pp.139-143
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• 2002

Cotton Glutathione S-Transferase (GST: EC 2.5.1.18) was cloned and overexpressed in tobacco (Nicotiana tabacum) plants. Northern blot analysis confirmed the successful transformation of cotton gst gene in tobacco plant. Type I and Type ll transcript patterns were identified in transgenic tobacco plants and only Type I transcripts were discussed in this paper, The activity of GST in the type II transgenic plants was about 1.5-fold higher than those of the wild type and non-expresser by using 1-chloro-2,4-dinitrobenzene (CDNB) and reduced glutathione as the substrate. The expression of cotton GST in tobacco plants proved that Gh-5 could be translated into functional protein. Type II transgenic plants produced functional GST in the cells. The effects of cotton GST in the seedlings was evaluated by growing the control and transgenic seedlings at $15^{\circ}C$ in the growth chamber in the light. Overexpressors were grown well compared to the control plants (non-expressors). lo test far tolerance to salinity, seeds of Gh-5 overexpressors and the wild type Xanthi seedlings were grown at 0, 50, 100, 150, and 200 mM NaCl solution. Gh-5 transgenic seedlings showed higher growth rate over control seedlings on 50 and 100 mM NaCl solution. There was no difference in growth rate at 150 and 200mM NaCl concentration.