• Journal of Microbiology
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• 제43권1호
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• pp.34-37
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• 2005

In the present study, the xylA gene encoding a thermostable xylose (glucose) isomerase was cloned from Streptomyces chibaensis J-59. The open reading frame of xylA (1167 bp) encoded a protein of 388 amino acids with a calculated molecular mass of about 43 kDa. The XylA showed high sequence homology (92% identity) with that of S. olivochromogenes. The xylose (glucose) isomerase was expressed in Escherichia coli and purified. The purified recombinant XylA had an apparent molecular mass of 45 kDa, which corresponds to the molecular mass calculated from the deduced amino acid and that of the purified wild-type enzyme. The N-terminal sequences (14 amino acid residues) of the purified protein revealed that the sequences were identical to that deduced from the DNA sequence of the xylA gene. The optimum temperature of the purified enzyme was $85^{\circ}C$ and the enzyme exhibited a high level of heat stability.

• Preventive Nutrition and Food Science
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• 제9권4호
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• pp.324-329
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• 2004

In order to investigate the molecular mechanism of $\gamma$-aminobutyric acid (GABA) production in lactic acid bacteria, we cloned a glutamate decarboxylase (GAD) gene from Lactobacillus plantarum using polymerase chain reaction (PCR). One PCR product DNA was obtained and inserted into a TA cloning vector with a T7 promoter. The recombinant plasmid was used to transform E. coli. The insertion of the product was con­firmed by EcoRI digestion of the plasmid purified from the transformed E. coli. Nucleotide sequence analysis showed that the insert is a full-length Lactobacillus plantarum GAD and that the sequence is $100\%$ and $72\%$ identical to the regions of Lactobacillus plantarum GAD and Lactococcus lactis GAD sequences deposited in GenBank, accession nos: NP786643 and NP267446, respectively. The amino acid sequence deduced from the cloned Lactobacillus plantarum GAD gene showed $100\%$ and $68\%$ identities to the GAD sequences deduced from the genes of the NP786643 and NP267446, respectively. To express the GAD protein in E. coli, an expression vector with the GAD gene (pkk/GAD) was constructed and used to transform the UT481 E. coli strain and the expression was confirmed by analyzing the enzyme activity. The Lactobacillus plantarum GAD gene obtained may facilitate the study of the molecular mechanisms regulating GABA metabolism in lactic acid bacteria.

• Journal of Microbiology and Biotechnology
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• 제7권1호
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• pp.8-12
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• 1997

In order to overproduce D-xylose isomerase, the Escherichia coli D-xylose isomerase (D-xylose ketol-isomerase, EC 5.3.1.5) gene (xylA) was fused to ${\lambda}P_{L}$ promoter. The promoterless xylA gene containing the ribosome binding site and coding region for D-xylose isomerase was cloned into a site 0.3 kb downstream from the ${\lambda}P_{L}$ promoter on a high copy number plasmid. An octameric XbaI linker containing TAG amber codon was inserted between 33rd codon of ${\lambda}N$ and the promoterless xylA gene. The resulting recombinant plasmid (designated as pPX152) was transformed into E. coli M5248 carrying a single copy of the temperature sensitive ${\lambda}cI857$ gene on its chromosomal DNA. When temperature-induced, the transformants produced 15 times as much D-xylose isomerase as that of D-xylose-induced parent strain. The amount of overproduced D-xylose isomerase was found to be about 60% of total protein in cell-free extracts.

• Journal of Microbiology
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• 제33권3호
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• pp.201-207
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• 1995

We have previously reported that SPP2 gene product of yeast Saccharomyces cerevisiae is involved in the pre-mRNA splicing. To investigate the rol ein the splicing pathway of the Spp2p protein, the SPP2 gene was overexpressed in Escherichia coli and polyclonal antibodies to Spp2p were generated from rabbits. First, a DNA fragment containing the SPP2 GENE without its promoter was subcloned into an E. coli expression vector, pKK233-3. The resulting recombinant plasmid pBQ14 contained an IPTG inducible tac promoter and the SPP2 structural gene. Overexpression of the SPP2 gene was achieved by additionof 0.1 to 1.0 mM IPTG to a logarithmic culture of E. coli JM103(pBQ14) for 90 min at 37.deg.C. Sequence of N-terminal 15 amino acids of the overproduced protein was well matched to the deduced one from the SPP2 reading frame. Then, polyclonal antibodies were generated from rabbits immunized with gel-purified SppSp protein. These antibodies reacted specifically with the Spp2p protein extracted from yeast cells expressing the SPP2 gene to a great extent. The antibodies could also block the activity of yeast splicing extracts.

• Journal of Plant Biotechnology
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• 제5권2호
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• pp.69-77
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• 2003

The ornamental industry encompasses cut flower, pot plant, turfgrass and nursery stock production and is an important part of the agricultural sector. As internationally traded commodities, cut flowers and plants are an integral part of the economy of a number of developing countries in South America, the Caribbean and Africa. Genetic modification (GM) is a tool with great potential to the ornamental horticulture industry. The rapid progress in our knowledge of plant molecular biology can accelerate the breeding ornamental plants using recombinant DNA technology techniques. Not only is there the possibility of creating new, novel products the driver of the industry but also the potential to develop varieties requiring less chemical and energy inputs. As an important non-food agricultural sector the use of genetically modified (GM) ornamental crops may also be ideal for the intensive farming necessary to generate pharmaceuticals and other useful products in GM plants. To date, there are only a few ornamental GM products in development and only one, a carnation genetically modified for flower colour, in the marketplace. International Flower Developments, a joint venture between Florigene Ltd. in Australia and Suntory Ltd. of Japan, developed the GM carnations. These flowers are currently on sale in USA, Japan and Australia. The research, development and commercialization of these products are summarized. The long term prospects for ornamental GM products, like food crops, will be determined by the regulatory environment, and the acceptance of GM products in the marketplace. These critical factors will be analysed in the context of the current legislative environment, and likely public and industry opinion towards ornamental genetically modified organisms (GMO's).

• Journal of Plant Biotechnology
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• 제29권3호
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• pp.199-207
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• 2002

The tropane alkaloids hyoscyamine (its racemic form being atropine) and scopolamine are used medicinally as anticholinergic agents that act on the parasympathetic nerve system. Because they differ in their actions on the central nervous system, currently there is a 10-fold higher commercial demand for scopolamine, in the N-butylbromide form, than there is for hyoscyamine and atropine combined. Several solanaceous species have been used as the commercial sources of these alkaloids, but the scopolamine contents in these plants often are much lower than those of hyoscyamine. For this reason there has been long-standing interest in increasing the scopolamine contents of cultivated medicinal plants. Naturally occurring and artificial interspecific hybrids of Duboisia have high scopolamine contents and are cultivated as a commercial source of scopolamine in Australia and other countries. Anther culture combined with conventional interspecific hybridization also has been used to breed high scopolamine-containing plants in the genera Datura and Hyoscyamus, but without much success. The use of recombinant DNA technology for the manipulation of metabolic processes in cells promises to provide important contributions to basic science, agriculture, and medicine. In this review, I introduce on the enzymes and genes involved in tropane alkaloid biosynthesis and current progress in metabolic engineering approaches for tropane alkaloid, especially scopolamine, production.

• Plant Breeding and Biotechnology
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• 제6권4호
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• pp.454-462
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• 2018

Yield and grade are the key factors that affect production value of peanut. The objective of this study was to identify QTLs for pod yield, hundred-seed weight, and total sound mature kernel (TSMK). A total of 90 recombinant inbred lines, derived from Tamrun OL07 and a breeding line Tx964117, were used as a mapping population and planted in Brownfield and Stephenville, Texas. A genetic map was developed using 1,211 SNP markers based on double digest restriction-site associated DNA sequencing (ddRAD-seq). A total of 10 QTLs were identified above the permutation threshold, three for yield, three for hundred-seed weight and four for TSMK, with LOD score values of 3.7 - 6.9 and phenotypic variance explained of 12.2% - 35.9%. Among those, there were several QTLs that were detected in more than one field experiment. The commonly detected QTLs in this study may be used as potential targets for future breeding program to incorporate yield and grade related traits through molecular breeding.

• Journal of Microbiology and Biotechnology
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• 제1권3호
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• pp.157-162
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• 1991

Conditions for glutathione production in E. coli cells which possess pGH501 (2 gshI+gshII) were studied. In terms of ATP supply for the glutathione synthesis, two different systems have been constructed and compared. When the acetate kinase reaction of E. coli was used for ATP generation, 20 mM of L-cysteine was completely converted to glutathione by toluene-treated E. coli cells (100 mg/ml) harboring pGH501 within 2 h at $37^{\circ}C$. However, considering the economical aspects, the glycolytic pathway of yeast was chosen as a better system for ATP generation. The optimal concentrations of reactants for glutathione production were determined to be as follows; 80 mM L-glutamate, 20 mM L-cysteine, 20 mM glycine, 20 mM $MgCl_2$, 50 mM potassium phosphate buffer (pH 7.5), 400 mM glucose, polyoxyethylene stearylamine ($5\;\mul/ml$), toluene-treated E. coli HB101/pGH501 (100 mg/ml), and dried yeast cells (400 mg/ml). The conversion ratio of L-cysteine to glutathione was 80% (about 5 mg/ml) under optimal condition within 6 h at $37^{\circ}C$.

• Proceedings of the Korean Society of Plant Biotechnology Conference
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• 한국식물생명공학회 2003년도 식물바이오벤처 페스티발
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• pp.39-48
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• 2003

The ornamental industry encompasses cut flower, pot plant, turfgrass and nursery stock production and is an important part of the agricultural sector. As internationally traded commodities, cut flowers and plants are an integral part of the economy of a number of developing countries in South America, the Caribbean and Africa. Genetic modification (GM) is a tool with great potential to the ornamental horticulture industry. The rapid progress in our knowledge of plant molecular biology can accelerate the breeding ornamental plants using recombinant DNA technology techniques. Not only is there the possibility of creating new, novel products the driver of the industry but also the potential to develop varieties requiring less chemical and energy inputs. As an important non-food agricultural sector the use of genetically modified (GM) ornamental crops may also be ideal for the intensive farming necessary to generate pharmaceuticals and other useful products in GM plants. To date, there are only a few ornamental GM products in development and only one, a carnation genetically modified for flower colour, in the marketplace. International Flower Developments, a joint venture between Florigene Ltd. in Australia and Suntory Ltd.of Japan, developed the GM carnations. These flowers are currently on sale in USA, Japan and Australia. The research, development and commercialisation of these products are summarised. The long term prospects for ornamental GM products, like food crops, will be determined by the regulatory environment, and the acceptance of GM products in the marketplace. These critical factors will be analysed in the context of the current legislative environment, and likely public and industry opinion towards ornamental genetically modified organisms (GMO's).

• Parasites, Hosts and Diseases
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• 제61권4호
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• pp.428-438
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• 2023

Clonorchis sinensis is commonly found in East Asian countries. Clonorchiasis is prevalent in these countries and can lead to various clinical symptoms. In this study, we used overlap extension polymerase chain reaction (PCR) and the Xenopus laevis oocyte expression system to isolate a cDNA encoding the choline transporter of C. sinensis (CsChT). We subsequently characterized recombinant CsChT. Expression of CsChT in X. laevis oocytes enabled efficient transport of radiolabeled choline, with no detectable uptake of arginine, α-ketoglutarate, p-aminohippurate, taurocholate, and estrone sulfate. Influx and efflux experiments showed that CsChT-mediated choline uptake was time- and sodium-dependent, with no exchange properties. Concentration-dependent analyses of revealed saturable kinetics consistent with the Michaelis-Menten equation, while nonlinear regression analyses revealed a Km value of 8.3 µM and a Vmax of 61.0 pmol/oocyte/h. These findings contribute to widen our understanding of CsChT transport properties and the cascade of choline metabolisms within C. sinensis.