• KSBB Journal
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• 제26권6호
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• pp.557-560
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• 2011

It has been known that Δ-desaturase (TAD5) in the biosynthetic pathway of long chain polyunsaturated fatty acids of Thraustochytrium aureumis responsible for the conversion of di-homo-${\gamma}$-linolenic acid (C20：4) into arachidonic acid (C20：4). The genetic sequence analysis on TAD5 of Thraustochytrium aureum ATCC34304 used in this study showed that it has two amino acid changes when compared to that of Thraustochytrium aureum TAD5 first reported in 2003. Accordingly, Thraustochytrium aureum ATCC34304 TAD5 was named TAD5_1. TAD5_1-inserted methylotropic Pichia pastoris was prepared and then cultured with a precursor fatty acid, di-homo-${\gamma}$-linolenic acid. GC analysis confirmed that a certain amount of the precursor fatty acid was converted into arachidonic acid. In this study, not only a recombinant Pichia pastoris with the typical activity of ${\Delta}5$-desaturase which plays an essential role in the biosynthesis of LCPUFAs was successfully made but also the preparationpotential of a recombinant Pichia pastoris strain which may synthesize eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) that are important in maintaining and improving human's brain function was proposed.

• Asian-Australasian Journal of Animal Sciences
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• 제15권12호
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• pp.1776-1783
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• 2002

A population segregating for the naked neck (Na) gene was used to evaluate its effect on fast growing broilers at heat stress. An experimental stock comparable to those of modern broilers was established by backcrossing to colour synthetic male and female lines. Matings between heterozygous (Na/na) males and females produced normally feathered (na/na), heterozygous (Na/na) and homozygous (Na/Na) chicks for the present study. Day old to seven week old coloured broilers of three genotypes viz. normally feathered (na/na), heterozygous naked neck (Na/na) and homozygous naked neck (Na/Na) were compared for heat dissipation, growth performance, body conformation traits, blood biochemical parameters and carcass traits in tropical climate. In hot climate, naked neck broilers had significantly less body temperature and better heat dissipation capabilities as compared to normal broilers. The naked neck broilers had significantly higher body weight and better feed conversion ratio than na/na broilers. The Na/Na or Na/na broilers exhibited higher giblet yield, blood loss and lower feather mass compared to na/na broilers. The results indicated that the reduction in feather coverage in Na/Na and Na/na broilers facilitates better heat dissipation with lower body temperature, more body weight gain, better FCR and carcass traits compared to normal broilers.

• BMB Reports
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• 제29권4호
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• pp.308-313
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• 1996

An 1.4 kb of the fad3 cDNA encoding microsomal linoleic acid desaturase catalyzing the conversion of linoleic acid (18:2, ${\omega}-6$) to linolenic acid (18:2, ${\omega}-3$) was introduced into tobacco plants by the Agrobacterium-mediated plant transformation, Among the transgenic tobacco plants conferring kanamycin resistance, five transformants showing increment in unsaturated fatty acid contents were selected and further analyzed for the transgenecity, In genomic Southern blot analyses, copy numbers of the integrated fad3 DNA in chromosomal DNA of the five transgenic tobacco plants were varied among the transgenic lines. By Northern blot analyses, the abundancy of the fad3 mRNA transcript directed by Cauliflower Mosaic Virus 35S promoter was consistent with the relative copy number of the fad3 DNA integrated in the chromosome of transgenic tobacco plants. When compared with the wild type, accumulation of linolenic acid in transgenic tobacco roots was elevated 3.7- to 4.7-fold showing a corresponding decrease in the linoleic acid contents; however, slight increments for linolenic acid were noticed in transgenic leaf tissues. These results indicated that the elevated level of fad3 expression is achieved in transgenic tobacco plants.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2002년도 생물공학의 동향 (X)
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• pp.445-448
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• 2002

The Pseudomonas fluorescens KCTC 1767, a selected and identified as potential candidate for stereo-specific resolution of rac-ketoprofen ethyl ester, was systematically investigated in order to induce the high level expression and detailed characterization of the expressing enzyme esterase. We cloned the esterase gene from chromosomal DNA of Pseudomonas fluorescens KCTC 1767 by PCR with two synthetic primers that desinged for simple purification. The recombinant esterase from Pseudomonas fluorescens KCTC 1767 exibited a high conversion rate and enantioselectivity to the (S)-ketoprofen ethyl ester as expected. The enzyme was easily purified to homogeniety by using a metal chelating affinity chromatography as a protein with poly histidine taq, and thus obtained 0.6 mg of protein from a 100 mL culture broth in a single step. The purified enzyme was steadily stable at the pH range from 7.0 to 10. The activity was also retained to be about 70% after the preincubation at $40^{\circ}C$ but over $50^{\circ}C$ lost the activity completely. The molecular mass of the esterase was estimated to be about 43 kDa on SDS-PAGE, and an identical result was also shown in gel filteration chromatography. The specific activity was calculated 27 mM/mg-protein/min by using the rac-ketoprofen ethly ester as a substrate.

• KSBB Journal
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• 제27권6호
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• pp.341-346
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• 2012

Indican (indoxyl-${\beta}$-D-glucoside) is a colorless natural compound and can be used as a precursor for the production of indigo. This production step only require an enzyme, ${\beta}$-glucosidase, that readily screened from microbial resource by using selective media supplemented with indican as a sole carbon source. Agrobacterium tumefaciens was well grown in this media and thus presumed to produce a related enzyme. The corresponding gene, encoding a protein with a calculated molecular mass of 51 kDa, was cloned and overexpressed as MBP fusion proteins. The purified enzyme was determined to be a dimer and showed the maximum activity for indican at pH 7.0 and $40^{\circ}C$. The kinetic parameters for indican, Km and Vmax, were determined to be 1.4 mM and 373.8 ${\mu}M/min/mg$, respectively. The conversion yield of indican into indigo using this enzyme was about 1.7-1.8 folds higher than that of previously isolated enzyme from Sinorhizobium meliloti. Additionally, this enzyme was able to hydrolyze various ${\beta}$-1,4 glycoside substrates.

• Journal of Microbiology and Biotechnology
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• 제20권3호
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• pp.579-586
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• 2010

A genomic DNA fragment encoding a putative maltooligosyltrehalose synthase (NfMTS) for trehalose biosynthesis was cloned by the degenerate primer-PCR from cyanobacterium Nostoc flagelliforme. The ORF of NfMTS was 2,799 bp in length and encoded 933 amino acid residues constituting a 106.6 kDa protein. The deduced amino acid sequence of NfMTS contained 4 regions highly conserved for MTSs. By expression of NfMTS in E. coli, it was demonstrated that the recombinant protein catalyzed the conversion of maltohexaose to maltooligosyl trehalose. The $K_m$ of the recombinant enzyme for maltohexaose was 1.87 mM and the optimal temperature and pH of the recombinant enzyme was at $50^{\circ}C$ and 7.0, respectively. The expression of MTS of N. flagelliforme was upregulated, and both trehalose and sucrose contents increased significantly in N. flagelliforme during drought stress. However, trehalose accumulated in small quantities (about 0.36 mg/g DW), whereas sucrose accumulated in high quantities (about 0.90 mg/g DW), indicating both trehalose and sucrose were involved in dehydration stress response in N. flagelliforme and sucrose might act as a chemical chaperone rather than trehalose did during dehydration stress.

• The Plant Pathology Journal
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• 제30권2호
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• pp.220-227
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• 2014

The GacS/GacA system in the root colonizer Pseudomonas chlororaphis O6 is a key regulator of many traits relevant to the biocontrol function of this bacterium. Proteomic analysis revealed 12 proteins were down-regulated in a gacS mutant of P. chlororaphis O6. These GacS-regulated proteins functioned in combating oxidative stress, cell signaling, biosynthesis of secondary metabolism, and secretion. The extent of regulation was shown by real-time RT-PCR to vary between the genes. Mutants of P. chlororaphis O6 were generated in two GacS-regulated genes, trpE, encoding a protein involved in tryptophan synthesis, and prnA, required for conversion of tryptophan to the antimicrobial compound, pyrrolitrin. Failure of the trpE mutant to induce systemic resistance in tobacco against a foliar pathogen causing soft rot, Pectobacterium carotovorum SCCI, correlated with reduced colonization of root surfaces implying an inadequate supply of tryptophan to support growth. Although colonization was not affected by mutation in the prnA gene, induction of systemic resistance was reduced, suggesting that pyrrolnitrin was an activator of plant resistance as well as an antifungal agent. Study of mutants in the other GacS-regulated proteins will indicate further the features required for biocontrol-activity in this rhizobacterium.

• Journal of Microbiology and Biotechnology
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• 제20권5호
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• pp.853-861
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• 2010

High substrate costs decrease the profitability of polyhydroxyalkanoates (PHAs) production, and thus low-cost carbon substrates coming from agricultural and industrial residuals are tested for the production of these biopolymers. Among them, crude glycerol, formed as a by-product during biodiesel production, seems to be the most promising source of carbon. The object of this study was to characterize the mixed population responsible for the conversion of crude glycerol into PHAs by cultivation-dependent and -independent methods. Enrichment of the microbial community was monitored by applying the Ribosomal Intergenic Spacer Analysis (RISA), and the identification of community members was based on 16S rRNA gene sequencing of cultivable species. Molecular analysis revealed that mixed populations consisted of microorganisms affiliated with four bacterial lineages: ${\alpha}$, ${\gamma}$-Proteobacteria, Actinobacteria, and Bacteroides. Among these, three Pseudomonas strains and Rhodobacter sp. possessed genes coding for polyhydroxyalkanoates synthase. Comparative analysis revealed that most of the microorganisms detected by direct molecular analysis were obtained by the traditional culturing method.

• Journal of Microbiology and Biotechnology
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• 제28권8호
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• pp.1346-1351
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• 2018

Oxidoreductases are effective biocatalysts, but their practical use is limited by the need for large quantities of NAD(P)H. In this study, a whole-cell biocatalyst for NAD(P)H cofactor regeneration was developed using the economical substrate glycerol. This cofactor regeneration system employs permeabilized Escherichia coli cells in which the glpD and gldA genes were deleted and the gpsA gene, which encodes $NAD(P)^+-dependent$ glycerol-3-phosphate dehydrogenase, was overexpressed. These manipulations were applied to block a side reaction (i.e., the conversion of glycerol to dihydroxyacetone) and to switch the glpD-encoding enzyme reaction to a gpsA-encoding enzyme reaction that generates both NADH and NADPH. We demonstrated the performance of the cofactor regeneration system using a lactate dehydrogenase reaction as a coupling reaction model. The developed biocatalyst involves an economical substrate, bifunctional regeneration of NAD(P)H, and simple reaction conditions as well as a stable environment for enzymes, and is thus applicable to a variety of oxidoreductase reactions requiring NAD(P)H regeneration.

• Journal of Microbiology and Biotechnology
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• 제31권5호
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• pp.740-746
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• 2021

Efficient cellulolytic enzyme production is important for the development of lignocellulose-degrading enzyme mixtures. However, purification of cellulases from their native hosts is time- and labor-consuming. In this study, a constitutive expression system was developed in Penicillium oxalicum for the secreted production of proteins. Using a constitutive polyubiquitin gene promoter and cultivating with glucose as the sole carbon source, nine cellulolytic enzymes of different origins with relatively high purity were produced within 48 h. When supplemented to a commercial cellulase preparation, cellobiohydrolase I from P. funiculosum and cellobiohydrolase II from Talaromyces verruculosus showed remarkable enhancing effects on the hydrolysis of steam-exploded corn stover. Additionally, a synergistic effect was observed for these two cellobiohydrolases during the hydrolysis. Taken together, the constitutive expression system provides a convenient tool for the production of cellulolytic enzymes, which is expected to be useful in the development of highly efficient lignocellulose-degrading enzyme mixtures.