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Kim G.-E.,Chonnam National University | Lee J.-H.,Chonnam National University | Jung S.-H.,Chonnam National University | Seo E.-S.,Chonnam National University | And 6 more authors.
Journal of Agricultural and Food Chemistry | Year: 2010

Hydroquinone galactoside (HQ-Gal) as a potential skin whitening agent was synthesized by the reaction of lactase (β-galactosidase) from Kluyveromyces lactis, Aspergillus oryzae, Bacillus circulans, and Thermus sp. with lactose as a donor and HQ as an acceptor. Among these lactases, the acceptor reaction involving HQ and lactose with K. lactis lactase showed a higher conversion ratio to HQ-Gal (60.27%). HQ-Gal was purified using butanol partitioning and silica gel column chromatography. The structure of the purified HQ-Gal was determined by nuclear magnetic resonance, and the ionic product was observed at m/z 295 (C12H16O7Na)+ using matrix assisted laser desorption ionization time-of-flight mass spectrometry. HQ-Gal was identified as 4-hydroxyphenyl-β-d-galactopyranoside. The optimum conditions for HQ-Gal synthesis by K. lactis determined using response surface methodology were 50 mM HQ, 60 mM lactose, and 20 U mL-1 lactase. These conditions produced a yield of 2.01 g L-1 HQ-Gal. The half maximal inhibitory concentration (IC50) of diphenylpicrylhydrazyl scavenging activity was 3.31 mM, indicating a similar antioxidant activity compared to β-arbutin (IC50 = 3.95 mM). The Ki value of HQ-Gal (0.75 mM) against tyrosinase was smaller than that of β-arbutin (K i = 1.97 mM), indicating its superiority as an inhibitor. HQ-Gal inhibited (23%) melanin synthesis without being significantly toxic to the cells, while β-arbutin exhibited only 8% reduction of melanin synthesis in B16 melanoma cells compared with the control. These results indicate that HQ-Gal may be a suitable functional component in the cosmetics industry. © 2010 American Chemical Society.

Jeong D.-E.,Korea Research Institute of Bioscience and Biotechnology | Park S.-H.,Korea Research Institute of Bioscience and Biotechnology | Park S.-H.,Korean University of Science and Technology | Pan J.-G.,Korea Research Institute of Bioscience and Biotechnology | And 3 more authors.
Nucleic Acids Research | Year: 2015

Genome engineering without leaving foreign DNA behind requires an efficient counter-selectable marker system. Here, we developed a genome engineering method in Bacillus subtilis using a synthetic gene circuit as a counter-selectable marker system. The system contained two repressible promoters (B. subtilis xylA (Pxyl) and spac (Pspac)) and two repressor genes (lacI and xylR). Pxyl-lacI was integrated into the B. subtilis genome with a target gene containing a desired mutation. The xylR and Pspac-chloramphenicol resistant genes (cat) were located on a helper plasmid. In the presence of xylose, repression of XylR by xylose induced LacI expression, the LacIs repressed the Pspac promoter and the cells become chloramphenicol sensitive. Thus, to survive in the presence of chloramphenicol, the cell must delete Pxyl-lacI by recombination between the wild-type and mutated target genes. The recombination leads to mutation of the target gene. The remaining helper plasmid was removed easily under the chloramphenicol absent condition. In this study, we showed base insertion, deletion and point mutation of the B. subtilis genome without leaving any foreign DNA behind. Additionally, we successfully deleted a 2-kb gene (amyE) and a 38-kb operon (ppsABCDE). This method will be useful to construct designer Bacillus strains for various industrial applications. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Jeong Y.-S.,Korea Advanced Institute of Science and Technology | Choi S.-L.,Korea Research Institute of Biosciences and Biotechnology | Kyeong H.-H.,Korea Advanced Institute of Science and Technology | Kim J.-H.,Korea Advanced Institute of Science and Technology | And 6 more authors.
Protein Engineering, Design and Selection | Year: 2012

Synthetic organophosphates (OPs) have been used as nerve agents and pesticides due to their extreme toxicity and have caused serious environmental and human health problems. Hence, effective methods for detoxification and decontamination of OPs are of great significance. Here we constructed and used a high-throughput screening (HTS) system that was based on phenolics-responsive transcription activator for directed evolution of OP-degrading enzymes. In the screening system, phenolic compounds produced from substrates by OP-degrading enzymes bind a constitutively expressed transcription factor DmpR, initiating the expression of enhanced green fluorescent protein located at the downstream of the DmpR promoter. Fluorescence intensities of host cells are proportional to the levels of phenolic compounds, enabling the screening of OP-degrading enzymes with high catalytic activities by fluorescence-activated cell sorting. Methyl parathion hydrolase from Pseudomonas sp. WBC-3 and p-nitrophenyl diphenylphosphate were used as a model enzyme and an analogue of G-type nerve agents, respectively. The utility of the screening system was demonstrated by generating a triple mutant with a 100-fold higher kcat/Km than the wild-type enzyme after three rounds of directed evolution. The contributions of individual mutations to the catalytic efficiency were elucidated by mutational and structural analyses. The DmpR-based screening system is expected to be widely used for developing OP-degrading enzymes with greater potential. © 2012 The Author.

Yang T.H.,Genofocus Inc. | Kwon M.-A.,Korea Research Institute of Chemical Technology | Lee J.Y.,Korea Research Institute of Chemical Technology | Choi J.-E.,Korea Research Institute of Chemical Technology | And 2 more authors.
Applied Biochemistry and Biotechnology | Year: 2015

Photobacterium lipolyticum M37 lipase (LipM37) was immobilized on the surface of intracellular polyhydroxybutyrate (PHB) granules in Escherichia coli. LipM37 was genetically fused to Cupriavidus necator PHA synthase (PhaCCn), and the engineered PHB operon containing the lipM37-phaCCn successfully mediated the accumulation of PHB granules (85 wt.%) inside E. coli cells. The PHB granules were isolated from the crude cell extract, and the immobilized LipM37 was comparable with the free form of LipM37 except for a favorable increase in thermostability. The immobilized LipM37 was used to synthesize oleic acid methyl ester (biodiesel) and oleic acid dodecyl ester (wax ester), and yielded 98.0 % conversion in esterification of oleic acid and dodecanol. It was suggested that the LipM37–PhaCCn fusion protein successfully exhibited bifunctional activities in E. coli and that in situ immobilization of lipase to the intracellular PHB could be a promising approach for expanding the biocatalytic toolbox for industrial chemical synthesis. © 2015 Springer Science+Business Media New York

Ryu H.-J.,Chonnam National University | Jin X.,Genofocus Inc. | Lee J.-H.,Chonnam National University | Woo H.-J.,Chonnam National University | And 8 more authors.
Enzyme and Microbial Technology | Year: 2010

Long isomalto-oligosaccharides (IMOs) were generated via an engineered fusion enzyme of dextransucrase and dextranase (DSXR). To increase the expression level, response surface methodology (RSM) was utilized for optimization of protein expression conditions for enhancement of protein production by the effects of three-level-three-factors and their mutual interaction in Escherichia coli. Seventeen experiments were designed and conducted for investigation of cell density to start induction, induction temperature, and induction time. Optimal induction conditions included a cell density to start induction (A600) of 0.76 at 12.16°C for 18h for dextransucrase activity and a cell density to start induction (A600) of 0.75 at 10.5°C for 20.9h for dextranase activity. The produced dextransucrase or dextranase activity was obtained at 120.1±7.2 or 871±58U, respectively, from 1L cultures. © 2010 Elsevier Inc.

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