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ABSTRACT: Although transplantation of microencapsulated islets has been proposed as a therapy for the treatment of diabetes mellitus, limited retrievability of the cells has impeded its medical usage. To achieve retrieval of microencapsulated islets, capsules were attached to polydimethylsiloxane(PDMS) with a biocompatible adhesive. Because the hydrophobic nature of the PDMS surface prevents attachment, surface modification is essential. Alginate microcapsules were attached to modified PDMS sheets, and the mechanical stability of the resulting constructs was determined. Acrylic acid (AA) and acrylamide (AM) mixtures were grafted on the surfaces of PDMS sheets using a two-step oxygen plasma treatment (TSPT). TSPT-PDMS was characterized according to water contact angle and zeta-potential measurements. The contact angle was altered by changing the ratio of AM to AA to generate hydrophilic surface. Evaluation of the surface charge at pH 2, 7, and 12 confirmed the presence of polar groups on the modified surface. Microcapsules were attached to TSPT-PDMS using Histoacryl® and shown to be in a mono-layered and half-exposed state. The shear stress resistance of alginate capsules attached to the PDMS sheet indicates the possibility of transplantation of encapsulated cells without scattering in vivo. This method is applicable to retrieve microencapsulated porcine islets when required. This article is protected by copyright. All rights reserved.
Biotechnology and Applied Biochemistry 05/2013; · 1.53 Impact Factor
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ABSTRACT: The activity of Candida antarctica lipase B was improved by mutation of the area surrounding the active site. We changed the edges of four helices surrounding the active site to flexible amino acids. Two mutants, V139E and I255E, obtained as a result of Pichia pastoris expression, showed enhanced specific activity of 9.9 and 8.1 U/mg while that of wild type was 2.3 U/mg for p-nitrophenyl caprylate hydrolysis. It was nearly 5.4-fold and 3.5-fold, respectively. The stability of both mutants on organic solvent was slightly decreased but almost similar with that of wild type. In the kinetic assay, k cat values were shown as dominant factor for the enhancement of catalytic efficiency, k cat/K m, since it was 4.1-fold and 3.8-fold, respectively.
Applied biochemistry and biotechnology 04/2013; · 1.94 Impact Factor
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ABSTRACT: The relative occurrences of amino acids, residual properties, and secondary structure type found in the residual structure states were compared between thermophilic and mesophilic proteins to find out the protein-thermostabilizing factors. The thermostabilizing patterns in each residual structure state are as follows: (1) in fully exposed state, higher relative occurrences of GLN, ILE, and PHE; (2) in exposed state, higher relative occurrences of ARG, GLU, salt bridges, the residue with low solvation energy, and the residues in 3/10 helix, and lower relative occurrences of ALA, SER, and VAL; (3) in partially exposed state, higher relative occurrence of flexible residue and lower relative occurrence of SER; (4) in buried state, higher relative occurrences of ARG and GLU, and lower relative occurrence of MET; and (5) in well-buried state, higher relative occurrences of ALA, cation-pi interaction, the residues in 3/10 helix, and lower relative occurrences of ASP, GLY, and the residues in the extended beta strand. These findings could be useful for developing protein thermostabilization strategies according to each residual structure state.
Applied biochemistry and biotechnology 04/2013; · 1.94 Impact Factor
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ABSTRACT: Electrostatic interactions are important in protein folding, binding, flexibility, stability and function. The pH at which the enzyme is maximally active is determined by the pKas of the active site residues, which are modulated by several factors including the change in electrostatics in its vicinity. As the acidic xylanases are important in food and animal feed industries, electrostatic interactions are introduced in Bacillus circulans xylanase to shift their pH optima towards the acidic side. Arg substitutions are made to modulate the pKas of the active site residues. Neutral residues are substituted by Arg in such a way that the substituted residue can make direct interaction with the catalytic residues. However, the mutations with other titratable residues (Asp, Arg, Lys, His, Tyr, and Ser) present in between the catalytic sites and the substituted sites are avoided. Site directed mutagenesis was conducted to confirm the strategy. The results show the shift in pH optima of the mutants towards the acidic side by 0.5 - 1.5 unit. Molecular dynamics simulation of the mutant V37R reveals that the decrease in activity is due to the increase in distance between the substrate oxygen atoms and catalytic glutamates.
Biotechnology and Bioprocess Engineering 02/2013; 18(1):35-42. · 1.28 Impact Factor
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ABSTRACT: Enzyme reactions in organic solvent such as for organic synthesis have great industrial potential. However, enzymes lose their stability in hydrophilic organic solvents due to the deformation of the enzyme by the solvent. It is thus important to enhance the stability of enzymes in hydrophilic organic solvents. Previous approaches have not considered on the interaction between enzymes and solvents due to the lack of information. In this study, the structural motions of the enzyme in methanol cosolvent and the interaction between the enzyme surface and the solvent molecule were investigated using molecular dynamics simulation (MD). By analyzing the MD simulation results, the surface residues of Candida antarctica lipase B (CalB) with higher root mean square deviation (RMSD) in a methanol solvent were considered as methanol affecting site and selected for site-directed mutagenesis. The methanol affecting site was computationally redesigned by lowering the RMSD. Among the candidate mutants, the A8T, A92E, N97Q and T245S mutants showed higher organic solvent stability at various methanol concentrations. The rational approach developed in this study could be applied to the stabilization of other industrial enzymes used in organic solvents.
Journal of biotechnology 11/2012; · 2.88 Impact Factor
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ABSTRACT: Levodopa or L-3,4-dihydroxyphenylalanine (L-DOPA) is the precursor of the neurotransmitter dopamine. L-DOPA is a famous treatment for Parkinson's disease symptoms. In this study, electroenzymatic synthesis of L-DOPA was performed in a three-electrode cell, comprising a Ag/AgCl reference electrode, a platinum wire auxiliary electrode, and a glassy carbon working electrode. L-DOPA had an oxidation peak at 376 mV and a reduction peak at -550 mV. The optimum conditions of pH, temperature, and amount of free tyrosinase enzyme were pH 7, 30 degrees C, and 250 IU, respectively. The kinetic constant of the free tyrosinase enzyme was found for both cresolase and catacholase activity to be 0.25 and 0.4 mM, respectively. A cyclic voltammogram was used to investigate the electron transfer rate constant. The mean heterogeneous electron transfer rate (ke) was 5.8 × 10(-4) cm/s. The results suggest that the electroenzymatic method could be an alternative way to produce L-DOPA without the use of a reducing agent such as ascorbic acid.
Journal of Microbiology and Biotechnology 10/2012; 22(10):1446-51. · 1.38 Impact Factor
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ABSTRACT: One widely known drawback of enzymes is their instability in diverse conditions. The thermostability of enzymes is particularly relevant for industrial applications because operation at high temperatures has the advantage of a faster reaction rate. Protein stability is mainly determined in this study by intra-molecular hydrophobic interactions that have a collective and 3-dimensional clustering effect. To interpret the thermostability of enzymes, network analysis was introduced into the protein structure, and a network parameter of structural hierarchy, k of k-clique, was used to discern more developed hydrophobic interaction clusters in the protein structure. The favorable clustering conformations of hydrophobic residues, which seemed to be important for protein thermostability, were discovered by the application of a network analysis to hydrophobic interactions of GH11 xylanases. Coordinating higher k-clique hydrophobic interaction clusters through the site-directed mutagenesis of the model enzyme, Bacillus circulans xylanase, stabilized the local structure and thus improved thermostability, such that the enzyme half-life and melting temperature increased by 78 fold and 8.8 °C, respectively. This study highlights the advantages of interpreting collective hydrophobic interaction patterns and their structural hierarchy and the possibility of applying network analysis to the thermostabilization of enzymes.
Journal of biotechnology 05/2012; 161(1):49-59. · 2.88 Impact Factor
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ABSTRACT: The enzymatic production of biodiesel has been considered as an eco-friendly process. Candida antarctica lipase B (CalB) has been studied for its application for biodiesel production because of its high activity and stability.
Enzyme deactivation caused by alcohol and effect of glycerol has to be resolved for the industrial application of this process.
In traditional kinetic studies of biodiesel production, the effects of alcohol and oil were only considered in the kinetic
equation, while the effect by glycerol was neglected. A new kinetic model incorporating glycerol effect is proposed in this
paper. The proposed kinetic equation is applied by predicting the supplying rate of methanol in a fed-batch addition of methanol.
The conversion rate was improved from 59.7% to 94.6% in a fed-batch by considering glycerol effect.
Key wordsBiodiesel–Enzyme–Kinetic Model–Oil–Glycerol–Lipase
Korean Journal of Chemical Engineering 04/2012; 28(9):1908-1912. · 0.99 Impact Factor
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ABSTRACT: For effective toluene degradation, the effects of a nitrogen source were studied with Pseudomonas putida BZ912, which was isolated from crude oil contaminated soil and is capable of degrading VOC. Two nitrogen sources, ammonia
and nitrate, showed different effects on specific growth rates (0.25 hr−1 and 0.12 hr−1, respectively), biomass yields (0.56 vs. 0.39) and specific toluene degradation rates (0.51 hr−1 vs. 0.26 hr−1). Under the resting cell conditions, the cells pre-cultured in the ammonia-containing medium showed higher specific toluene
degradation rate than that in nitrate-containing medium (0.045 hr−1 vs. 0.038 hr−1). Ammonia as a nitrogen source was effective for degradation in high toluene concentration because high cellular biomass
was accomplished. Nitrate showed slow growth rate compared to ammonia. The resting cell conditions demonstrated that it was
able to degrade toluene efficiently without increasing biomass. These conditions could be a solution for degrading VOC after
high cellular biomass was obtained in a biofilter. By changing the nitrogen source and the growth conditions according to
the toluene concentration, the control of cell biomass and the desired removal capacity were accomplished.
Korean Journal of Chemical Engineering 04/2012; 25(1):139-143. · 0.99 Impact Factor
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ABSTRACT: The bioelectrochemical reduction of nitrate in the presence of various mediators including methyl viologen and azure A was
studied using a 3-electrode voltammetric system. The catalytic potential for the reduction of the mediators was observed in
the reactor, which for methyl viologen and azure A were −0.74 V and −0.32 V, respectively, with respect to the potential of
Ag/AgCl reference electrode. This potential was then applied to a working electrode to reduce each mediator for enzymatic
nitrate reduction. Nitrite, the product of the reaction, was measured to observe the enzymatic nitrate reduction in the reaction
media. Methyl viologen was observed as the most efficient mediator among those tested, while azure A showed the highest electron
efficiency at the intrinsic reduction potential when the mediated enzyme reactions were carried out with the freely solubilized
mediator. The electron transfer of azure A with respect to time was due to the adhesion of azure A to the hydrophilic surface
during the reduction. In addition, the use of the adsorbed mediator on conductive activated carbon was proposed to inhibit
the change in the electron transfer rate during the reaction by maintaining a constant mediator concentration and active surface
area of the electrode. Azure A showed better than nitrite formation than methyl viologen when used with activated carbon.
Biotechnology and Bioprocess Engineering 04/2012; 10(1):47-51. · 1.28 Impact Factor
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ABSTRACT: Permeabilization is known to overcome cell membrane barriers of whole cell biocatalysts. The use of organic solvents is advantageous
in terms of cost, simplicity, and efficiency. In this study,Ochrobactrum anthropi SY509 was permeabilized with various organic solvents. Treatment with organic solvents resulted in lower permeability barriers
due to falling out lipids of the cell membrane. Therefore, permeabilized cells showed higher enzyme activity with no cell
viability. Among various organic solvents, 0.5% (v/v) chloroform was selected as the most efficient permeabilizing reagent.
Changes in the cell membrane structure were observed and the residual amounts of phospholipids of the cell membrane were measured
to investigate the mechanism of the improved permeability.
Biotechnology and Bioprocess Engineering 04/2012; 9(3):147-150. · 1.28 Impact Factor
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ABSTRACT: In this study, we report the first functional cloning and heterogeneous expression of 3-ketovalidoxylamine C-N lyase (E.C.
4.3.3.1) from Flavobacterium saccharophilum IFO 13984. This gene is 1,098 bp in length and encodes a peptide of 366 amino acids. The recombinant C-N lyase was successfully
overexpressed in E. coli, and its functional activity, degradation of 3-ketovalidoxylamine A, was confirmed by HPLC analysis. The sequence and phylogenetic
analysis showed that the C-N lyase has no similarity with other amine lyases (E.C. 4.3.3) but has similarity with the conserved
domain present in SusD and RagB. Thus, the C-N lyase may have a similar binding domain for sugar moieties with SusD/RagB.
This genetic information may lead to improvements in C-N lyase function for industrial applications.
Keywords3-ketovalidoxylamine C-N lyase–
Flavobacterium saccharophilum
–gene cloning–expression
Biotechnology and Bioprocess Engineering 04/2012; 16(2):366-373. · 1.28 Impact Factor
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ABSTRACT: Biodiesel has been greatly interested as an alternative fuel and is produced by a transesterification reaction of oil with
alcohol. Recently, microbial lipases have been used for biodiesel production. Among the microbial lipase, immobilized Candida antartica lipase B (CALB) is the most widely used. However, CALB is unstable and shows low catalytic efficiency in the reaction media
because the reaction media contains a high concentration of methanol and the lipase is also inhibited by the by-product glycerol.
In this study, to overcome these limitations, we developed an amphiphilic matrix to immobilize CALB. The immobilized lipase
in an amphiphilic matrix with 80% ethyltrimethoxysilane (ETMS) in tetramethoxysilane (TMOS) and pretreated with oil showed
the highest specific activity and biodiesel conversion ratio; about 90% biodiesel conversion in 24 h at an initial molar ratio
of 1: 1 (oil: methanol) with stepwise methanol feeding in order to adjust the net molar ratio to be 1: 3.
Keywordsamphiphilic matrix-
Candida antartica lipase B (CALB)-immobilization-biodiesel production
Biotechnology and Bioprocess Engineering 04/2012; 15(4):603-607. · 1.28 Impact Factor
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ABSTRACT: In this study, a mediator-free glucose/O2 bio-fuel cell was developed based on a 3-dimensional carbon nanomaterial/polypyrrole composite with glucose oxidase and tyrosinase
as the anodic and cathodic catalysts, respectively. This mediator-free biofuel cell has the following merits: (1) the biocatalyst
was unaffected by toxic mediators and (2) current generation is independent, because there is no problem associated with mediator
leakage from the electrode. The carbon nanomaterial in this 3-dimensional composite was used not only as immobilization support
for the biocatalyst, but also as an electron carrier. This would be advantageous for glucose oxidation on the bioanode and
O2 reduction on the biocathode in the glucose/O2 biofuel cell. This biofuel cell showed enhanced power density and half-life compared to other glucose/O2 biofuel cells previously reported, producing 157.4 μW/cm3 with 1 mM glucose as fuel and 0.5 M NaCl as the electrolyte, at a cell voltage of +85 mV over 29 h with continuous 1 mM glucose
feeding.
Keywordsbiofuel cell-3-dimensional enzyme/carbon nanomaterial/polypyrrole composite-glucose oxidase-tyrosinase-mediator-free-stability
Biotechnology and Bioprocess Engineering 04/2012; 15(3):371-375. · 1.28 Impact Factor
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ABSTRACT: Gluconate and glucose were selected as the carbon substrates in the production of poly-3-hydroxybutyrate (PHB). Gluconate
was utilized to maximize the specific growth rate during the first stage of cell growth, whereas glucose was used to maximize
PHB biosynthesis during the second stage of PHB accumulation. The sequential feeding of gluconate and glucose resulted in
a 50% enhancement of PHB productivity as compared to the cultures cultivated on glucose alone. In conjunction with secondary
glucose uptake, the presence of a trace amount of ammonium increased the rate of PHB biosynthesis during the stage of PHB
accumulation. Via the feeding of 0.03 mmol/h of NH4Cl solution prior to the exhaustion of the initial amount of NH4Cl, PHB productivity was significantly enhanced as compared to the cultures raised on glucose alone. The glucose-grown culture
evidenced a higher level of NADPH during the NH4Cl-exausted PHB accumulation stage than was observed in the gluconate-grown culture, which reflects that the reason of higher
PHB production observed when glucose was used as a carbon source. NH4Cl feeding following the depletion of initial NH4Cl resulted in elevated levels of both ATP and NADPH, which increased the PHB biosynthesis rate, and also in a decrease in
the level of NADH, which reflected the alleviation of the inhibitory effects on the cells caused by nitrogen depletion.
Biotechnology and Bioprocess Engineering 04/2012; 13(1):14-24. · 1.28 Impact Factor
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ABSTRACT: Effects of artificial electron donors to deliver reducing power on enzymic denitrification were investigated using nitrate
reductase and nitrite reductase obtained fromOchrobactrum antropi. The activity of nitrite reductase in the soluble portion was almost the same as that in the precipitated portion of the
cell extract. Nitrate removal efficiency was higher with benzyl viologen than with methyl viologen or NADH as an artificial
electron donor. The turn-over numbers of nitrate and nitrite reductase were 14.1 and 1.9 μmol of nitrogen reduced/min·mg cell
extracts, respectively when benzyl viologen was used as an electron donor.
Biotechnology and Bioprocess Engineering 04/2012; 7(2):112-116. · 1.28 Impact Factor
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ABSTRACT: A novel sol-gel immobilization method employing a detergentless micro-emulsion system that consisted ofn-hexane/iso-propanol/water was developed and used to immobilize a horseradish peroxidase (HRP). Micro-sized gel powder containing
enzymes was generated in the ternary solution without drying and grinding steps or the addition of detergent, therefore, the
method described in this study is a simple and straightforward process for the manufacture of gel powder. The gel powder made
in this study was able to retain 84% of its initial enzyme activity, which is higher than gel powders produced through other
immobilization methods. Furthermore, the HRP immobilized using this method, was able to maintain its activity at or above
95% of its initial activity for 48h, whereas the enzyme activities of free HRP and HRP that was immobilized using the other
sol-gel method decreased dramatically. In addition, even when in the presence of excess hydrogen peroxide, the enzyme immobilized
using the novel sol-gel method described here was more stable than enzymes immobilized using the other method.
Biotechnology and Bioprocess Engineering 04/2012; 12(6):640-645. · 1.28 Impact Factor
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ABSTRACT: Although much research has been conducted on the separation of single species of heavy metal, the selective adsorption of
two or more heavy metals in mixture is relatively little known. In this study, polysaccharide beads were prepared to selectively
remove the targeted heavy metal ion from mixture. Among the biomasses, polysaccharide was examined due to its low cost and
easy accessibility. In a single metal ion system, chitosan, λ-carrageenan, and alginic acid showed high affinity to mercury,
copper, and lead, respectively. In the ion mixture, the same trend shown in the single metal ion solution was observed. The
optimum electrolyte concentraion was investigated to adsorb the metal ion selectively, and it was possible to remove the targeted
metal ion selectively with chitosan, alginic acid and λ-carrageenan at 1 mmol concentration of electrolyte. In order to demonstrate
the feasibility of selective biosorption, two packed-bed reactors in series containing chitosan and alginic acid beads in
each were studied and selective adsorption to Hg2+ and Pb2+, respectively, was observed.
Korean Journal of Chemical Engineering 04/2012; 21(6):1168-1172. · 0.99 Impact Factor
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ABSTRACT: Aeration was found to affect the biological denitrification byOchrobactrum anthropi SY509. Although cell growth was vigorous under 1 vvm of aeration and an agitation speed of 400 rpm in a 3-L jar fermentor,
almost no nitrate was removed. Yet under low agitation speeds (100, 200, and 300 rpm), denitrification occurred when the dissolved
oxygen was exhausted shortly after the inoculation of the microorganism.Ochrobactrum anthropi SY509 was found to express highly active denitrifying enzymes under anaerobic conditions. The microorganism also synthesized
denitrifying enzymes under aerobic conditions (1 vvm and 400 rpm), yet their activity was only 60% of the maximum level under
anaerobic conditions and the nitrate removal efficiency was merely 15%. However, although the activities of the denitrifying
enzymes were inhibited in the presence of oxygen, they were fully recovered when the conditions were switched to anaerobic
conditions.
Biotechnology and Bioprocess Engineering 04/2012; 7(6):352-356. · 1.28 Impact Factor
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ABSTRACT: Binary mixtures of hydrophilic and hydrophobic solvents were assessed for their ability to balance enzyme activity with the conservation of enzyme stability in organic media. Acetone, dioxane and dodecane were chosen as model organic solvents, and subtilisin Carlsberg and horseradish peroxidase (HRP) were chosen as model enzymes. Residual enzyme activities were measured to monitor enzyme stability, and the fluorescence intensity of HRP was monitored to investigate structural changes due to the presence of an organic solvent. Enzyme stability increased with the increasing hydrophobicity of the solvent mixture used, and a solvent mixture with a high log P value (~ >4) was capable of conserving enzyme stability. Enzyme stability in organic media can be conserved therefore with a mixture of hydrophilic and hydrophobic solvents: this approach might be used as a general and practical strategy for optimizing enzyme activity and stability for industrial applications.
Biotechnology Letters 02/2012; 34(6):1131-5. · 1.68 Impact Factor
