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Won-Kyung Hong Sun-Yeon Heo Baek-Rock Oh Chul Ho Kim Jung-Hoon Sohn Ji-Won Yang Akihiko Kondo Jeong-Woo Seo 《Bioprocess and biosystems engineering》2013,36(9):1191-1197
In the present study, we established a genetic system for manipulating the oleaginous heterotrophic microalgae Aurantiochytrium sp. KRS101, using cycloheximide resistance as the selectable marker. The gene encoding ribosomal protein L44 (RPL44) of Aurantiochytrium sp. KRS101 was first identified and characterized. Proline 56 was replaced with glutamine, affording cycloheximide resistance to strains encoding the mutant protein. This resistance served as a novel selection marker. The gene encoding the Δ12-fatty acid desaturase of Mortierella alpina, used as a reporter, was successfully introduced into chromosomal DNA of Aurantiochytrium sp. KRS101 via 18S rDNA-targeted homologous recombination. Enzymatic conversion of oleic acid (C18:1) to linoleic acid (C18:2) was detected in transformants but not in the wild-type strain. 相似文献
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Park DS Oh HW Heo SY Jeong WJ Shin DH Bae KS Park HY 《Journal of microbiology (Seoul, Korea)》2007,45(5):409-417
Burkholderia sp. HY-10 isolated from the digestive tracts of the longicorn beetle, Prionus insularis, produced an extracellular lipase with a molecular weight of 33.5 kDa estimated by SDS-PAGE. The lipase was purified from the culture supernatant to near electrophoretic homogenity by a one-step adsorption-desorption procedure using a polypropylene matrix followed by a concentration step. The purified lipase exhibited highest activities at pH 8.5 and 60 degrees . A broad range of lipase substrates, from C4 to C18 rho-nitrophenyl esters, were hydrolyzed efficiently by the lipase. The most efficient substrate was rho-nitrophenyl caproate (C6). A 2485 bp DNA fragment was isolated by PCR amplification and chromosomal walking which encoded two polypeptides of 364 and 346 amino acids, identified as a lipase and a lipase foldase, respectively. The N-terminal amino acid sequence of the purified lipase and nucleotide sequence analysis predicted that the precursor lipase was proteolytically modified through the secretion step and produced a catalytically active 33.5 kDa protein. The deduced amino acid sequence for the lipase shared extensive similarity with those of the lipase family I.2 of lipases from other bacteria. The deduced amino acid sequence contained two Cystein residues forming a disulfide bond in the molecule and three, well-conserved amino acid residues, Ser131, His330, and Asp308, which composed the catalytic triad of the enzyme. 相似文献
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Won-Kyung Hong Chul-Ho Kim Sun-Yeon Heo Lian Hua Luo Baek-Rock Oh Dina Rairakhwada Jeong-Woo Seo 《Bioprocess and biosystems engineering》2011,34(2):231-236
Currently, 1,3-propanediol (1,3-PD) is an important chemical widely used in polymer production, but its availability is being
restricted owing to its expensive chemical synthesis. A methylotrophic yeast Hansenula polymorpha was engineered by expression of dhaB1, dhaB2, dhaB3, dhaB
RA1
and dhaB
RA2
encoding glycerol dehydratase complex and dhaT encoding 1,3-PD oxidoreductase from Klebsiella pneumoniae under direction of promoter of glyceraldehyde-3 phosphate dehydrogenase (GAPDH). The engineered recombinant yeast strain
can produce 1,3-PD from glucose (2.4 g L−1) as well as glycerol (0.8 g L−1), which might lead to a safe and cost-effective method for industrial production of 1,3-PD from various biomass resources. 相似文献
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Pil-Soo Seo Sun-Yeon Heo Eun Jong Han Jeong-Woo Seo Shin-Je Ghim Chul Ho Kim 《Biotechnology and Bioprocess Engineering》2009,14(2):168-174
The human papillomavirus (HPV) 18 L1 gene, which encodes the L1 major capsid protein, was isolated from a female patient in
Pusan, Korea Republic and was cloned into pGEX-4T-1 vector. The HPV-18L1 gene was expressed in Escherichia coli as a fusion protein with a glutathione-S-transferase (GST) tag. The soluble recombinant fusion protein, GST-18 L1 fusion,
was isolated to high purity. HPV-18 L1 was purified from the GST-18 L1 fusant after biotinylated thrombin cleavage, and then
the treated thrombin was removed serially using streptavidin conjugated resin. The purified HPV-18 L1 was confirmed by western
blotting using a rabbit anti-denatured papillomavirus polyclonal antibody. The virus-like particles (VLP) from the purified
full-length 18 L1 protein without any extra amino acid sequences was observed through the analysis of the electron microscope.
This is the first study to report the expression and purification of HPV-18 L1 in E. coli. This expression and purification system offers a simple method of expressing and purifying HPV L1 protein, and could potentially
be an effective route for the development and manufacturing of highly purified HPV-18 L1-based cervical cancer vaccines. 相似文献
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Jang Min Park Baek-Rock Oh In Yeong Kang Sun-Yeon Heo Jeong-Woo Seo Seung-Moon Park Won-Kyung Hong Chul Ho Kim 《Journal of industrial microbiology & biotechnology》2017,44(7):1107-1113
A Bacillus sp. strain named BRC1 is capable of producing 2,3-butanediol (2,3-BD) using hydrolysates of the Jerusalem artichoke tuber (JAT), a rich source of the fructose polymer inulin. To enhance 2,3-BD production, we undertook an extensive analysis of the Bacillus sp. BRC1 genome, identifying a putative gene (sacC) encoding a fructan hydrolysis enzyme and characterizing the activity of the resulting recombinant protein expressed in and purified from Escherichia coli. Introduction of the sacC gene into Bacillus sp. BRC1 using an expression vector increased enzymatic activity more than twofold. Consistent with this increased enzyme expression, 2,3-BD production from JAT was also increased from 3.98 to 8.10 g L?1. Fed-batch fermentation of the recombinant strain produced a maximal level of 2,3-BD production of 28.6 g L?1, showing a high theoretical yield of 92.3%. 相似文献
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Lian Hua Luo Jeong-Woo Seo Sun-Yeon Heo Baek-Rock Oh Dae-Hyuk Kim Chul Ho Kim 《Bioprocess and biosystems engineering》2013,36(9):1319-1326
Klebsiella pneumoniae produces 3-hydroxypropionic acid (3-HP) from glycerol with oxidation of 3-hydroxypropionaldehyde (3-HPA) to 3-HP in a reaction catalyzed by aldehyde dehydrogenase (ALDH). In the present study, two putative ALDHs of K. pneumoniae, YneI and YdcW were identified and characterized. Recombinant YneI was specifically active on 3-HPA and preferred NAD+ as a cofactor, whereas YdcW exhibited broad substrate specificity and preferred NADP+ as a cofactor. Overexpression of ALDHs in the glycerol oxidative pathway-deficient mutant K. pneumoniae AK resulted in a significant increase in 3-HP production upon shake-flask culture. The final titers of 3-HP were 2.4 and 1.8 g L?1 by recombinants overexpressing YneI and YdcW, respectively. Deletion of the ALDH gene from K. pneumoniae did not affect the extent of 3-HP synthesis, implying non-specific activity of ALDHs on 3-HPA. The ALDHs might play major roles in detoxifying the aldehyde generated in glycerol metabolism. 相似文献
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Jang Min Park Won-Kyung Hong Sung-Mok Lee Sun-Yeon Heo You Ree Jung In Yeong Kang Baek-Rock Oh Jeong-Woo Seo Chul Ho Kim 《Journal of industrial microbiology & biotechnology》2014,41(9):1425-1433
Klebsiella pneumoniae synthesize large amounts of l-2,3-butanediol (l-2,3-BD), but the underlying mechanism has been unknown. In this study, we provide the first identification and characterization of an l-2,3-BD dehydrogenase from K. pneumoniae, demonstrating its reductive activities toward diacetyl and acetoin, and oxidative activity toward l-2,3-BD. Optimum pH, temperature, and kinetics determined for reductive and oxidative reactions support the preferential production of 2,3-BD during cell growth. Synthesis of l-2,3-BD was remarkably enhanced by increasing gene dosage, reaching levels that, to the best of our knowledge, are the highest achieved to date. 相似文献