Hyungdon Yun

Konkuk University, Sŏul, Seoul, South Korea

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Publications (58)179.32 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Optically pure β-amino acids are of high pharmacological significance since they are used as key ingredients in many physiologically active compounds. Despite a number of enzymatic routes to these compounds, an efficient synthesis of β-amino acids continues to pose a major challenge for researchers. ω-transaminase has emerged as an important class of enzymes for generating amine compounds. However, only a few ω-transaminases have been reported so far which show activity towards aromatic β-amino acids. In this study, (S)-ω-transaminase from Burkholderia graminis C4D1M has been functionally characterized and used for the production of chiral aromatic β-amino acids via kinetic resolution. The enzyme showed a specific activity of 3.1 U/mg towards rac-β-phenylalanine at 37°C. The Km and Kcat values of this enzyme towards rac-β-phenylalanine with pyruvate as the amino acceptor were 2.88mM and 91.57min(-1) respectively. Using this enzyme, racemic β-amino acids were kinetically resolved to produce (R)-β-amino acids with an excellent enantiomeric excess (>99%) and ∼50% conversion. Additionally, kinetic resolution of aromatic β-amino acids was performed using benzaldehyde as a cheap amino acceptor. Copyright © 2015. Published by Elsevier B.V.
    Journal of biotechnology. 01/2015; 196.
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    ABSTRACT: FoCYP53A19, a novel cytochrome P450 capable of performing benzoate hydroxylation, was identified and characterized from the ascomycete Fusarium oxysporum f.sp. lycopersici. Comparative functional analysis of FoCYP53A19 with the heterologous and homologous cytochrome P450 reductases (CPR) such as Saccharomyces cerevisiae (ScCPR), Candida albicans (CaCPR) and F. oxysporum (FoCPR) revealed novel catalytic properties. The catalytic efficiency and substrate specificity of FoCYP53A19 was significantly influenced and altered by the source of the reductase employed. The yeast reconstitution system of FoCYP53A19 with ScCPR performed the hydroxylation of benzoic acid (BA) and demethylation of 3-methoxybenzoic acid (3-MBA); but when reconstituted with CaCPR, FoCYP53A19 performed only the essential hydroxylation of fungal benzoate catabolism. Remarkably, FoCYP53A19 with its homologous reductase FoCPR, not only demonstrated the improved conversion rates of BA and 3-MBA, but also exhibited activity towards the hydroxylation of 3-hydroxybenzoic acid. The electron transfer compatibility and the coupling efficiency between the homologous FoCYP-FoCPR system is significant and it favored enhanced monooxygenase activity with broader substrate specificity.
    Enzyme and Microbial Technology 12/2014; 70. · 2.97 Impact Factor
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    ABSTRACT: In general, conventional enzyme engineering utilizes 20 canonical amino acids to alter and improve the functional properties of proteins such as stability, and activity. In this study, we utilized the noncanonical amino acid incorporation technique to enhance the functional properties of ω-transaminase (ω-TA). Herein, we enhanced the stability of ω-TA by residue-specific incorporation of (4R)-fluoroproline [(4R)-FP] and successfully immobilized onto chitosan or polystyrene (PS) beads with site-specifically incorporated L-3,4-dihydroxyphenylalanine (DOPA) moiety. The immobilization of ω-TAdopa and ω-TAdp[(4R)-FP] onto PS beads showed excellent reusability for 10 cycles in the kinetic resolution of chiral amines. Compared to the ω-TAdopa, the ω-TAdp[(4R)-FP] immobilized onto PS beads exerted more stability that can serve as suitable biocatalyst for the asymmetric synthesis of chiral amines.
    ChemCatChem 12/2014; 7(3). · 5.18 Impact Factor
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    ABSTRACT: Lead is one of the most hazardous metals ubiquitous in the environment, causing serious health hazards to organisms. Recently, fluorescent proteins such as GFP and Dsred were utilized for the development of reagent-less rapid metal sensors. Here, we demonstrate the development of a lead-sensing GFP that is highly sensitive to lead in micro molar concentrations
    Chemical Communications 11/2014; · 6.38 Impact Factor
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    ABSTRACT: The iLOV protein belongs to a family of blue-light photoreceptor proteins containing a light-oxygen-voltage sensing domain with a non-covalently bound flavin mononucleotide (FMN) as its chromophore. Due to advantages such as small size, oxygen-independent nature, and pH stability, iLOV is an ideal candidate over other reporter fluorescent proteins such as GFP and DsRed. Here, for the first time, we describe the feasibility of applying LOV domain-based fluorescent iLOV as a metal sensor by measuring the fluorescence quenching of a protein with respect to the concentration of metal ions. In the present study, we demonstrate the inherent copper sensing property of the iLOV protein and identify the possible amino acids responsible for metal binding. The fluorescence quenching upon exposure to Cu(2+) was highly sensitive and exhibited reversibility upon the addition of the metal chelator EDTA. The copper binding constant was found to be 4.72 ± 0.84 µM. In addition, Cu(2+)-bound iLOV showed high fluorescence quenching at near physiological pH. Further computational analysis yielded a better insight into understanding the possible amino acids responsible for Cu(2+) binding with the iLOV protein.
    Journal of microbiology and biotechnology. 10/2014;
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    ABSTRACT: Misaminoacylation of 3,4-dihydroxyphenylalanine (Dopa) molecules to tRNATyr by endogenous tyrosyl-tRNA synthetase allowed the quantitative replacement of tyrosine residues with a yield of over 90 % by an in vivo residue-specific incorporation strategy, to create, for the first time, engineered mussel adhesive proteins (MAPs) in Escherichia coli with a very high Dopa content, close to that of natural MAPs. The Dopa-incorporated MAPs exhibited a superior surface adhesion and water resistance ability by assistance of Dopa-mediated interactions including the oxidative Dopa cross-linking, and furthermore, showed underwater adhesive properties comparable to those of natural MAPs. These results propose promising use of Dopa-incorporated engineered MAPs as bioglues or adhesive hydrogels for practical underwater applications.
    Angewandte Chemie 09/2014;
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    ABSTRACT: In this study, we successfully synthesized enantiomerically pure (R)- and (S)-γ-amino acids (>99% ee) using ω- transaminase (ω-TA) through kinetic resolution and asymmetric synthesis respectively. The present study demonstrates the high potentiality of ω-TA reaction for the production of chiral γ-amino acids.
    Chemical Communications 08/2014; · 6.38 Impact Factor
  • Giyoung Shin, Sam Mathew, Hyungdon Yun
    Journal of Industrial and Engineering Chemistry 08/2014; · 2.06 Impact Factor
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    ABSTRACT: The research work was aimed for isolation and identification of Aspergillus flavus from poultry feed samples using conventional and molecular techniques. Out of 23 samples, 8 (34.78%) were found positive for A. flavus. The molecular techniques comprise comparison of ITS1-5.8S-ITS2 region. In addition, the ability of the isolates to express aflatoxin synthase (CYP64A1) gene in synthetic liquid medium was also analyzed. Based on cultural characteristics, unique nature of the ITS 1 and 2 genes and expression of CYP64A1 at mRNA level, A. flavus was accurately identified from the feed samples. The isolated A. flavus would be a potential candidate for further studies and application.
    Pakistan Journal of Agricultural Sciences 06/2014; 51(2):287-291. · 1.05 Impact Factor
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    ABSTRACT: Daidzein C6 hydroxylase (6-DH, nfa12130), which is a class I type of cytochrome P450 enzyme, catalyzes a hydroxylation reaction at the C6-position of the daidzein A-ring and requires auxiliary electron transfer proteins. Current utilization of cytochrome P450 (CYP) enzymes is limited by low coupling efficiency, which necessitates extramolecular electron transfers, and low driving forces, which derive electron flows from tightly regulated NADPH redox balances into the heterogeneous CYP catalytic cycle. To overcome such limitations, the heme domain of the 6-DH enzyme was genetically fused with the NADPH-reductase domain of self-sufficient CYP102D1 to enhance electron transfer efficiencies through intramolecular electron transfer and switching cofactor preference from NADH into NADPH. 6-DH-reductase fusion enzyme displayed distinct spectral properties of both flavoprotein and heme proteins and catalyzed daidzein hydroxylation more efficiently with a k cat/K m value of 120.3 ± 11.5 [10(3) M(-1) s(-1)], which was about three times higher than that of the 6-DH-FdxC-FdrA reconstituted system. Moreover, to obtain a higher redox driving force, a Streptomyces avermitilis host system was developed for heterologous expression of fusion 6-DH enzyme and whole cell biotransformation of daidzein. The whole cell reaction using the final recombinant strain, S. avermitilisΔcyp105D7::fusion 6-DH (nfa12130), resulted in 8.3 ± 1.4 % of 6-OHD yield from 25.4 mg/L of daidzein.
    Applied Microbiology and Biotechnology 04/2014; 98(19). · 3.81 Impact Factor
  • Advanced Synthesis & Catalysis 03/2014; · 5.54 Impact Factor
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    ABSTRACT: Here we enhanced the stability and biophysical properties of mRFP1 through a combination of canonical and non-canonical amino acid mutagenesis. The global replacement of proline residue with (2S, 4R)-4-fluoroproline [(4R)-FP] into mRFP1 led to soluble protein but lost its fluorescence, whereas (2S, 4S)-4-fluoroproline [(4S)-FP] incorporation resulted in insoluble protein. The bioinformatics analysis revealed that (4R)-FP incorporation at Pro63 caused fluorescence loss due to the steric hindrance of fluorine atom of (4R)-FP with the chromophore. Therefore, Pro63 residue was mutated with the smallest amino acid Ala to maintain non coplanar conformation of the chromophore and helps to retain its fluorescence with (4R)-FP incorporation. The incorporation of (4R)-FP into mRFP1-P63A showed about 2∼3 fold enhancement in thermal and chemical stability. The rate of maturation is also greatly accelerated over the presence of (4R)-FP into mRFP1-P63A. Our study showed that a successful enhancement in the biophysical property of mRFP1-P63A[(4R)-FP] using non-canonical amino acid mutagenesis after mutating non-permissive site Pro63 into Ala.
    Biochemical and Biophysical Research Communications 09/2013; · 2.28 Impact Factor
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    ABSTRACT: In this study, we developed a one-pot one-step deracemization method for the production of various enantiomerically pure amines using two opposite enantioselective ω-TAs. Using this method, various aromatic amines were successfully converted to their (R)-forms (>99%) with good conversion.
    Chemical Communications 06/2013; · 6.38 Impact Factor
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    ABSTRACT: A current challenge in high-throughput screening (HTS) of hydroxylation reactions by P450 is a fast and sensitive assay for regioselective hydroxylation against millions of mutants. We have developed a solid-agar plate-based HTS assay for screening ortho-specific hydroxylation of daidzein by sensing formaldehyde generated from the O-dealkylation reaction. This method adopts a colorimetric dye, pararosaniline, which has previously been used as an aldehyde-specific probe within cells. The rationale for this method lies in the fact that the hydroxylation activity at ortho-carbon position to COH correlates with a linear relationship to O-dealkylation activity on chemically introduced methoxy group at the corresponding COH. As a model system, a 4',7-dihydroxyisoflavone (daidzein) hydroxylase (CYP102D1 F96V/M246I), which catalyzes hydroxylation at ortho positions of the daidzein A/B-ring, was examined for O-dealklyation activity, by using permethylated daidzein as a surrogate substrate. By using the developed indirect bishydroxylation screening assay, the correlation coefficient between O-dealkylation and bishydroxylation activity for the template enzyme was 0.72. For further application of this assay, saturation mutants at A273/G274/T277 were examined by mutant screening with a permethylated daidzein analogue substrate (A-ring inactivated in order to find enhanced 3'-regioselectiviy). The whole-cell biotransformation of daidzein by final screened mutant G1 (A273H/G274E/T277G) showed fourfold increased conversion yield, with 14.3 mg L(-1) production titer and greatly increased 3'-regioselectiviy (3'/6=11.8). These results show that there is a remarkably high correlation (both in vitro and in vivo), thus suggesting that this assay would be ideal for a primary HTS assay for P450 reactions.
    ChemBioChem 06/2013; 14(10). · 3.06 Impact Factor
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    ABSTRACT: Among over 180 Aspergilli, Aspergillus fumigatus is the most common etiological agent to cause invasive mold infection in immunocompromised human and animal. Besides, the fungus is used for various useful purposes. Accurate identification of A. fumigatus is crucial. Here, the research work was aimed for isolation and identification of A. fumigatus from feed samples using a combined traditional-molecular approach. Out of 23 feed samples, 2 (8.7%) were found positive for A. fumigatus. The internal transcribed spacer (ITS) 1 and ITS 2 regions and the 5.8S ribosomal DNA (rDNA) region of the fungus were amplified by polymerase chain reaction (PCR). The ITS regions are located between the 18S and 28S rRNA genes, and rRNA gene for 5.8S RNA separates the two ITS regions. The isolated gene has been sequenced and deposited in GenBank (accession no. KC142152). The gene was 100% similar to other reference species of A. fumigatus, whereas in phylogenetic analysis, a clear distance was found in the cases of other Aspergilli. Based on morphological characteristics, unique nature of the ITS1 and ITS2 regions and phylogenetic analysis of the genes, A. fumigatus was correctly identified. The isolate has been named as Aspergillus fumigatus BAU-1. The isolate could be a good candidate for further studies especially for the utilization in the field of biotechnology.
    BSVER ASCon XIX; 02/2013
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    ABSTRACT: Recently, ω-transaminases have been increasingly used to synthesize amine compounds by reductive amination of prochiral ketones which are of high pharmacological significance. However, the conventional methods for evaluating these enzymes are time consuming and have often been regarded as a bottle neck in developing these enzymes as industrial biocatalysts. In the past few years, several high throughput screening methods have been developed for fast evaluation and identification of ω-transaminase. This review summarizes the various methodologies developed for rapidly screening ω-transaminases.
    Biotechnology and Bioprocess Engineering 02/2013; 18:1-7. · 1.28 Impact Factor
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    ABSTRACT: Diversification of protein sequence-structure space is a major concern in protein engineering. Deletion mutagenesis can generate a protein sequence-structure space different from substitution mutagenesis mediated space, but it has not been widely used in protein engineering compared to substitution mutagenesis, because it causes a relatively huge range of structural perturbations of target proteins which often inactivates the proteins. In this study, we demonstrate that, using green fluorescent protein (GFP) as a model system, the drawback of the deletional protein engineering can be overcome by employing the protein structure with high stability. The systematic dissection of N-terminal, C-terminal and internal sequences of GFPs with two different stabilities showed that GFP with high stability (s-GFP), was more tolerant to the elimination of amino acids compared to a GFP with normal stability (n-GFP). The deletion studies of s-GFP enabled us to achieve three interesting variants viz. s-DL4, s-N14, and s-C225, which could not been obtained from n-GFP. The deletion of 191-196 loop sequences led to the variant s-DL4 that was expressed predominantly as insoluble form but mostly active. The s-N14 and s-C225 are the variants without the amino acid residues involving secondary structures around N- and C-terminals of GFP fold respectively, exhibiting comparable biophysical properties of the n-GFP. Structural analysis of the variants through computational modeling study gave a few structural insights that can explain the spectral properties of the variants. Our study suggests that the protein sequence-structure space of deletion mutants can be more efficiently explored by employing the protein structure with higher stability.
    PLoS ONE 12/2012; 7(12):e51510. · 3.53 Impact Factor
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    ABSTRACT: Residue-specific incorporation is a technique used to replace natural amino acids with their close structural analogs, unnatural amino acids (UAAs), during protein synthesis. This is achieved by exploiting the substrate promiscuity of the wild type amino acyl tRNA synthetase (AARS) towards the close structural analogs of their cognate amino acids. In the past few decades, seleno-methionine was incorporated into proteins, using the substrate promiscuity of wild type AARSs, to resolve their crystal structures. Later, the incorporation of many UAAs showed that the AARSs are polyspecific to the close structural analogs of their cognate amino acids and that they maintain fidelity for the 19 natural amino acids. This polyspecificity helps to expand the use of this powerful tool to incorporate various UAA residues specifically through in vivo and in vitro approaches. Incorporation of UAAs is expensive, tedious and time-consuming. For the efficient incorporation of UAAs, it is important to screen substrate selectivity prior to their incorporation. As an initial study, using a docking tool, we analyzed the polyspecificity of the methionyl-tRNA synthetases (MetRSs) towards multiple reported and virtually generated methionine analogs. Based on the interaction result of these docking simulations, we predicted the substrate selectivity of the MetRS and the key residues responsible for the recognition of methionine analogs. Similarly, we compared the active site residues of the MetRSs of different species and identified the conserved amino acids in their active sites. Given the close similarity in the active site residues of these systems, we evaluated the polyspecificity of MetRSs.
    Journal of molecular graphics & modelling 11/2012; 39C:79-86. · 2.17 Impact Factor
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    ABSTRACT: Phenolic acid decarboxylase (PAD) catalyzes the non-oxidative decarboxylation of p-coumaric acid (pCA) to p-hydroxystyrene (pHS). PAD from Bacillus amyloliquefaciens (BAPAD), which showed k (cat)/K (m) value for pCA (9.3 × 10(3) mM(-1) s(-1)), was found as the most active one using the "Subgrouping Automata" program and by comparing enzyme activity. However, the production of pHS of recombinant Escherichia coli harboring BAPAD showed only a 22.7 % conversion yield due to product inhibition. Based on the partition coefficient of pHS and biocompatibility of the cell, 1-octanol was selected for the biphasic reaction. The conversion yield increased up to 98.0 % and 0.83 g/h/g DCW productivity was achieved at 100 mM pCA using equal volume of 1-octanol as an organic solvent. In the optimized biphasic reactor, using a three volume ratio of 1-octanol to phosphate buffer phase (50 mM, pH 7.0), the recombinant E. coli produced pHS with a 88.7 % conversion yield and 1.34 g/h/g DCW productivity at 300 mM pCA.
    Applied Microbiology and Biotechnology 10/2012; · 3.81 Impact Factor
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    ABSTRACT: A cytochrome P450 (CYP) enzyme, 3'-daidzein hydroxylase, CYP105D7 (3'-DH), responsible for daidzein hydroxylation at the 3'-position, was recently reported. CYP105D7 (3'-DH) is a class I type of CYP that requires electrons provided through electron transfer proteins such as ferredoxin and ferredoxin reductase. Presently, we constructed an artificial CYP in order to develop a reaction host for the production of a hydroxylated product. Fusion-mediated construction with the reductase domain from self-sufficient CYP102D1 was done to increase electron transfer efficiency and coupling with the oxidative process. An artificial self-sufficient daidzein hydroxylase (3'-ASDH) displayed distinct spectral properties of both flavoprotein and CYP. The fusion enzyme catalyzed hydroxylation of daidzein more efficiently, with a kcat/Km value of 16.8 μM-1 min-1, which was about 24-fold higher than that of the 3'-DH-camA/B reconstituted enzyme. Finally, a recombinant Streptomyces avermitilis host for the expression of 3'-ASDH and production of the hydroxylated product was developed. The conversion that was attained (34.6%) was 5.2-fold higher than that of the wild-type.
    Microbial Cell Factories 06/2012; 11:81. · 4.25 Impact Factor
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Publication Stats

549 Citations
179.32 Total Impact Points

Institutions

  • 2014
    • Konkuk University
      • Department of Bioscience and Technology
      Sŏul, Seoul, South Korea
  • 2008–2013
    • Yeungnam University
      • School of Biotechnology
      Onyang, South Chungcheong, South Korea
  • 2003–2013
    • Seoul National University
      • • Department of Chemical and Biological Engineering
      • • School of Chemical and Biological Engineering
      • • Institute of Molecular Biology and Genetics
      Seoul, Seoul, South Korea