• KSBB Journal
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• v.10 no.1
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• pp.105-112
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• 1995

The fiber-optic biosensor using enzyme-immobilized Langmuir-Blodgett film is developed fort the measurement of ethanol. The enzyme, alcohol dehydrogenase, is immobilized at the molecular level on the arachidic acid monolayer using Langmuir-Blodgett film technique. Based on the ordered multisubstrate mechanism, the immobilized enzyme kinetics is investigated. The optical sensing system is proposed, and sensor signal is proportional to ethanol concentration and is related wish the number of enzyme layers. As the number of deposited LB film layer increases up to 20 1ayers, the high ethanol concentration of 45mM can be measured without the saturation of signal. Surface pressure-area isotherm is measured for the three-different charged-lipids. Arachidic acid is the most suitable for the adsorption of alcohol dehydrogenase based on electrostatic force.

• Journal of Environmental Health Sciences
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• v.29 no.2
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• pp.80-86
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• 2003

To evaluate the effect of cyclohexane(CH) treatment on the serum levels of glutathion S-transferase(GST) activity in liver damaged animals, damaged liver was induced with pretreatment of 50% $CCl_4$ dissolved in olive oil (0.1 m1/100g body weight) intraperitoneally 17 times every other day. To $CCl_4$-treated rats, CH (1.56 g/kg body weight, i.p) was injected once and then the animals were sacrificed at 4 hours after injection of CH. The $CCl_4$-treated animals were identified as severe liver damage on the basis of liver functional findings, 1,e, increased serum levels of alanine aminotransferase(ALT), alkaline phosphate(ALP) and xanthine oxidase(XO) activities. On the other hand, $CCl_4$-treated animals injected with CH once($CCl_4$-pretreated animals) showed more decreased serum levels of ALT and XO, and more increased those of ALP rather than $CCl_4$-treated animals. In case of comparing the GST with ALT activity in liver, both $CCl_4$-treated and pretreated animals showed similar changing pattern of enzyme actvity. Especially $CCl_4$-pretreated animals showed significantly increased serum level of GST actvity compared with the $CCl_4$-treated those, whereas those of ALT showed reversed tendency. In aspects of GST enzyme kinetics, $CCl_4$-pretreated animals showed higher Vmax of liver GST enzyme than $CCl_4$-treated animals. In conclusion, injection of CH to the liver damaged rats led to enhanced liver damage and more increased activity of serum GST which may be chiefly caused by the enzyme induction.

• Journal of Life Science
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• v.21 no.1
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• pp.127-133
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• 2011

${\beta}$-Galactosidase was immobilized on chitosan bead by covalent bonding using glutaraldehyde. The characteristics of the immobilized enzyme were investigated. Maximum immobilization yield of 75% was obtained on chitosan bead. Optimum pH and temperature for the immobilized enzyme was 7.0 and $50^{\circ}C$, respectively. The immobilized enzyme showed a broader range of pH and temperature compared to a free one. A mathematical model for the operation of the immobilized enzyme in a packed-bed reactor was established and solved numerically. Under different inlet lactose concentrations and feed flow rate conditions, lactose conversion was measured in a packed-bed reactor. The experimental results of continuous operation in a packed-bed reactor were compared to theoretic results using Michaelis-Menten kinetics with competitive product inhibition and external mass transfer resistance. The model predicted the experimental data with errors less than 5%. Process optimization of continuous operation in a packed-bed reactor was also conducted. In a recirculation packed-bed operation, conversion of lactose was 97% in 3 hours. In a continuous packed-bed operation, the effect of flow rate and initial lactose concentration was investigated. Increasing flow rates and initial lactose concentration decreased the conversion of substrate.

• Journal of Microbiology and Biotechnology
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• v.29 no.4
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• pp.577-586
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• 2019

The engineered Aspergillus oryzae has a high NADPH demand for xylose utilization and overproduction of target metabolites. Glucose-6-phosphate dehydrogenase (G6PDH, E.C. 1.1.1.49) is one of two key enzymes in the oxidative part of the pentose phosphate pathway, and is also the main enzyme involved in NADPH regeneration. The open reading frame and cDNA of the putative A. oryzae G6PDH (AoG6PDH) were obtained, followed by heterogeneous expression in Escherichia coli and purification as a his6-tagged protein. The purified protein was characterized to be in possession of G6PDH activity with a molecular mass of 118.0 kDa. The enzyme displayed maximal activity at pH 7.5 and the optimal temperature was $50^{\circ}C$. This enzyme also had a half-life of 33.3 min at $40^{\circ}C$. Kinetics assay showed that AoG6PDH was strictly dependent on $NADP^+$ ($K_m=6.3{\mu}M$, $k_{cat}=1000.0s^{-1}$, $k_{cat}/K_m=158.7s^{-1}{\cdot}{\mu}M^{-1}$) as cofactor. The $K_m$ and $k_{cat}/K_m$ values of glucose-6-phosphate were $109.7s^{-1}{\cdot}{\mu}M^{-1}$ and $9.1s^{-1}{\cdot}{\mu}M^{-1}$ respectively. Initial velocity and product inhibition analyses indicated the catalytic reaction followed a two-substrate, steady-state, ordered BiBi mechanism, where $NADP^+$ was the first substrate bound to the enzyme and NADPH was the second product released from the catalytic complex. The established kinetic model could be applied in further regulation of the pentose phosphate pathway and NADPH regeneration of A. oryzae to improve its xylose utilization and yields of valued metabolites.

• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.1
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• pp.52-59
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• 2005

The study was targeted to saccharify foodwastes with the cellulolytic and amylolytic enzymes obtained from culture supernatant of Trichoderma harzianum FJ1 and analyze the kinetics of the saccharification in order to enlarge the utilization in industrial application. T. harzianum FJ1 highly produced various cellulolytic (filter paperase 0.9, carboxymethyl cellulase 22.0, ${\beta}$-glucosidase 1.2, Avicelase 0.4, xylanase 30.8, as U/mL-supernatant) and amylolytic (${alpha}$-amylase 5.6, ${\beta}$-amylase 3.1, glucoamylase 2.6, as U/mL-supernatant) enzymes. The $23{\sim}98\;g/L$ of reducing sugars were obtained under various experimental conditions by changing FPase to between $0.2{\sim}0.6\;U/mL$ and foodwastes between $5{\sim}20\%$ (w/v), with fixed conditions at $50^{\circ}C$, pH 5.0, and 100 rpm for 24 h. As the enzymatic hydrolysis of foodwastes were performed in a heterogeneous solid-liquid reaction system, it was significantly influenced by enzyme and substrate concentrations used, where the pH and temperature were fixed at their experimental optima of 5.0 and $50^{\circ}C$, respectively. An empirical model was employed to simplify the kinetics of the saccharification reaction. The reducing sugars concentration (X, g/L) in the saccharification reaction was expressed by a power curve ($X=K{\cdot}t^n$) for the reaction time (t), where the coefficient, K and n. were related to functions of the enzymes concentrations (E) and foodwastes concentrations (S), as follow: $K=10.894{\cdot}Ln(E{\cdot}S^2)-56.768,\;n=0.0608{\cdot}(E/S)^{-0.2130}$. The kinetic developed to analyze the effective saccharification of foodwastes composed of complex organic compounds could adequately explain the cases under various saccharification conditions. The kinetics results would be available for reducing sugars production processes, with the reducing sugars obtained at a lower cost can be used as carbon and energy sources in various fermentation industries.

• Applied Biological Chemistry
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• v.38 no.2
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• pp.118-122
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• 1995

Escherichia Coli ornithine transcarbamylase is the enzyme which catalyzes the L-citrulline biosynthesis from L-ornithine and carbamyl phosphate. To facilitate the purification of enzyme which will be used for many biochemical studies such as structure and function relationships and catalytic mechanisms, the cloning and expression of E. coli argI gene for ornithine transcarbamylase was conducted. argI was amplified from genomic DNA of E. coli strain of $DH5{\alpha}$, by polymerization chain reaction (PCR) method. The amplified argI gene was ligated to the prokaryotic expression vector pKK223-3 and used for transformation of E. coli TB2 which was deficient of ornithine transcarbamylase. The over-produced enzyme by the tnansformant was purified by ammonium sulfate fractionation, heat denaturation and affinity chromatography. The result of SDS denaturation gel electrophoresis for the purified enzyme showed a single band of about 38 kDa of ornithine transcarbamylase. Kinetic data for the expressed enzyme gave almost the s?????? values as those of the wild type enzyme. The $k_{cat}$, of the enzyme was $1.0{\times}10^5min^{-1}$, and $K_ms$ for ornithine and carbamyl phosphate were 0.35 mM and 0.06 mM, respectively.

• Biotechnology and Bioprocess Engineering:BBE
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• v.1 no.1
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• pp.46-50
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• 1996

In order to design industrial scale reactors and proceises for multi-phase biocatalytic reactions, it is essential to understand the mechanisms by which such systems operate. To il-lustrate how such mechanisms can be modeled, the hydrolysis of the primary ester groups of triglycerides to produce fatty acids and monoglycerides by lipased (glycerol-ester hydrolase) catalysis has been selected as an example of multiphase biocatalysis. Lipase is specific in its behavior such that it can act only on the hydrolyzed (or emulsified) part of the substrate. This follows because the active center of the enzyme is catalytically active only when the substrate contacts it in its hydrolyzed form. In other words, lipase acts only when it can shuttleback and forth between the emulsion phase and the water phase, presumably within an interphase or boundary layer between these two phases. In industrial applications lipase is employed as a fat splitting enzyme to remove fat stains from fabrics, in making cheese, to flavor milk products, and to degrade fats in waste products. Effective use of lipase in these processes requires a fundamental understanding of its kinetic behavior and interactions with substrates under various environmental conditions. Therefore, this study focuses on modeling and simulating the enzymatic activity of the lipase as a step towards the basic understanding of multi-phase biocatalysis processes.

• Journal of Environmental Science International
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• v.18 no.7
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• pp.797-802
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• 2009

To control degradation rate of biodegradable poly(lactide)s (PLA), the stereochemical PLAs with different ratios of d-lactide and l-lactide units were synthesized by the ring open polymerization and the their degradation kinetics were measured by a Langmuir film balance. The alkaline (pH=11) degradation of poly(l-lactide) (l-PLA) monolayer showed the faster rate at a surface pressure of 4 mN/m in the ranges from to 0 to 7 mN/m. However, the enzymatic degradation of l-PLA with Proteinase K did not occur until 4 mN/m. Above a constant surface pressure of 4 mN/m, the degradation rate was increased with a constant surface pressure. These behaviors might be attributed to the difference in the contacted area with degradation medium: alkaline ions need small contact area with l-PLA while enzymes require much bigger one to be activated due to different medium sizes. The stereochmical PLA monolayers showed that the alkaline degradation was increased with their optical impurities while the enzymatic one was inversed. These results could be explained by the decrease of crystallinity with the optical impurity and the inactivity of enzyme to d-LA unit.

• Journal of Pharmaceutical Investigation
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• v.24 no.1
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• pp.1-9
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• 1994

In order to study systemic peptide delivery through the ocular route, the stabilities of methionine enkephalin (Met-Enk) and $[D-ala^2]-methionine$ enkephalinamide (YAGFM) in the corneal extracts of rabbits were investigated using reversed phase HPLC. Met-Enk was found to be hydrolyzed most rapidly in the corneal epithelium, but YAGFM was relatively stable. Aminopeptidases appeared to contribute over 60% to the degradation of Met-Enk and the degradation rate of Met-Enk followed the first order kinetics. The half-lives of Met-Enk in the extracts of the corneal epithelium and endothelium were 36 and 673 min, respectively. From the effects of enzyme inhibitors, it was found that the application of the mixture of amastatin, thimerosal and EDTA was very useful for the inhibition of peptide degradation.

• Journal of Environmental Science International
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• v.17 no.11
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• pp.1221-1226
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• 2008

A variety of fungal species are known to degrade cyanide through the action of cyanide hydratase, a specialized nitrilases which hydrolyze cyanide to formamide. This work is a report on two unknown and un-characterized members from Neurospora crassa and Aspergillus nidulans. Recombinant forms of three cyanide hydratases (CHT) originated from N. crassa, Gibberella zeae, and A. nidulans were prepared after their genes were cloned with N-terminal hexahistidine purification tags, expressed in E. coli and purified using immobilized metal affinity chromatography. These enzymes were compared according to their pH activity profiles, and kinetic parameters. Although all three were similar, the N. crassa CHT has the widest pH range of activity above 50% and highest turnover rate ($6.6{\times}10^8min^{-1}$) among them. The CHT of A. nidulans has the highest Km value of the three nitrilases evaluated in here. Expression of CHT in both N. crassa and A. nidulans were induced by the presence of KCN, regardless of any presence of nitrogen sources. These data can be used to determine optimal procedures for the enzyme uses in the remediation of cyanide-containing wastes.