Genofocus Inc.

Daejeon, South Korea

Genofocus Inc.

Daejeon, South Korea

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Patent
Genofocus Inc. | Date: 2017-03-29

The present invention relates to novel beta-galactosidase and, more specifically, to novel beta-galactosidase derived from Bacillus circulans, a gene encoding the same, a recombinant vector and a recombinant microorganism containing the gene, a method for preparing beta-galactosidase using the recombinant microorganism, and a method for preparing galactooligosaccharide using the beta-galactosidase. The use of novel beta-galactosidase according to the present invention can achieve efficient mass-production of galactooligosaccharide.


Patent
Genofocus Inc. | Date: 2015-05-19

The present invention relates to a novel beta-galactosidase, and more particularly to a novel beta-galactosidase derived from Bacillus circulans, a gene encoding the beta-galactosidase, a recombinant vector and a recombinant microorganism, which contain the gene, a method for producing a beta-galactosidase using the recombinant microorganism, and a method for producing galactooligosaccharide using the beta-galactosidase. The use of the novel beta-galactosidase according to the present invention makes it possible to efficiently produce a large amount of galactooligosaccharide.


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 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


PubMed | Genofocus Inc. and Korea Research Institute of Chemical Technology
Type: Journal Article | Journal: 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 (PhaC Cn ), and the engineered PHB operon containing the lip M37 -phaC Cn 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.


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.


Cho E.-A.,Korea Research Institute of Bioscience and Biotechnology | Cho E.-A.,Soongeui Womens College and 8 3 | Kim E.-J.,Genofocus Inc. | Pan J.-G.,Korea Research Institute of Bioscience and Biotechnology
Enzyme and Microbial Technology | Year: 2011

The immobilization of enzymes on edible matrix supports is of great importance for developing stabilized feed enzymes. In this study, probiotic Bacillus spores were explored as a matrix for immobilizing Escherichia coli phytase, a feed enzyme releasing phosphate from phytate. Because Bacillus spore is inherently resistant to heat, solvents and drying, they were expected to be a unique matrix for enzyme immobilization. When mixed with food-grade Bacillus polyfermenticus spores, phytases were adsorbed to their surface and became immobilized. The amount of phytase attached was 28.2±0.7mg/g spores, corresponding to a calculated activity of 63,960U/g spores; however, the measured activity was 41,120±990.1U/g spores, reflecting a loss of activity upon adsorption. Immobilization increased the half life (t 1/2) of the enzyme three- to ten-fold at different temperatures ranging from 60 to 90°C. Phytase was bound to the spore surface to the extent that ultrasonication treatment was not able to detach phytases from spores. Desorption of spore-immobilized phytase was only achieved by treatment with 1M NaCl, 10% formic acid in 45% acetonitrile, SDS, or urea, suggesting that adsorption of phytase to the spore might be via hydrophobic and electrostatic interactions. We propose here that Bacillus spore is a novel immobilization matrix for enzymes that displays high binding capacity and provides food-grade safety. © 2011 Elsevier Inc.


Jeong Y.-S.,Korea Advanced Institute of Science and Technology | Choi J.M.,Korea Advanced Institute of Science and Technology | Kyeong H.-H.,Korea Advanced Institute of Science and Technology | Choi J.-Y.,Genofocus Inc. | And 2 more authors.
Biochemical and Biophysical Research Communications | Year: 2014

V-type nerve agents, known as VX, are organophosphate (OP) compounds, and show extremely toxic effects on human and animals by causing cholinergic overstimulation of synapses. The bacterial organophosphorus hydrolase (OPH) has attracted much attention for detoxifying V-type agents through hydrolysis of the P-S bond. However, low catalytic efficiency of OPH has limited the practical use of the enzyme. Here we present rational design of OPH with high catalytic efficiency for a V-type nerve agent. Based on the model structure of the enzyme and substrate docking simulation, we predicted the key residues that appear to enhance the access of the substrate to the active site of the enzyme, and constructed numerous OPH mutants. Of them, double mutant, L271/Y309A, was shown to exhibit a 150-fold higher catalytic efficiency for VX than the wild-type. © 2014 Elsevier Inc. All rights reserved.


Pan J.-G.,Korea Research Institute of Bioscience and Biotechnology | Choi S.-K.,Korea Research Institute of Bioscience and Biotechnology | Jung H.-C.,Korea Research Institute of Bioscience and Biotechnology | Kim E.-J.,Genofocus Inc.
FEMS Microbiology Letters | Year: 2014

In principle, protein display is enabled by fusing target proteins to naturally secreted, surface-anchored protein motifs. In this work, we developed a method of native protein display on the Bacillus spore surface that obviates the need to construct fusion proteins to display a motif. Spore coat proteins are expressed in the mother cell compartment and are subsequently assembled and deposited on the surface of spores. Therefore, target proteins overexpressed in the mother cell compartment during the late sporulation phase were expected to be targeted and displayed on the spore surface. As a proof of principle, we demonstrated the display of carboxymethylcellulase (CMCase) in its native form on the spore surface. The target protein, CMCase, was expressed under the control of the cry1Aa promoter, which is controlled by σE and σK and is expressed in the mother cell compartment. The correct display was confirmed using enzyme activity assays, flow cytometry, and immunogold electron microscopy. In addition, we demonstrated the display of a β-galactosidase tetramer and confirmed its correct display using enzyme activity assays and protein characterization. This native protein display system, combined with the robust nature of Bacillus spores, will broaden the range of displayable target proteins. Consequently, the applications of display technology will be expanded, including high-throughput screening, vaccines, biosensors, biocatalysis, bioremediation, and other innovative bioprocesses. © 2014 Federation of European Microbiological Societies.

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