• Food Science and Biotechnology
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• v.27 no.6
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• pp.1761-1769
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• 2018

Enzyme-assisted extraction is a cost-effective, safe, and efficient method to obtain bioactives from plant materials. During this study, 10 different marine algae from Sri Lanka were individually extracted by using five commercial food-grade carbohydrases. The enzymatic and water extracts of the seaweeds were analyzed for their antioxidant and anti-inflammatory activities. The highest DPPH, hydrogen peroxide ($H_2O_2$) and intracellular $H_2O_2$ scavenging abilities were observed from the Celluclast extract of Sargassum polycystum (CSp). CSp exerted protective effects against oxidative stress-induced cell death in hydrogen peroxide-induced Chang cells and in model zebrafish. The Celluclast extract of Chnoospora minima (CCm) showed the strongest anti-inflammatory activity against lipopolysaccharide (LPS)-induced NO production in RAW 264.7 macrophages ($IC_{50}=44.47{\mu}g/mL$) and in model zebrafish. CCm inhibited the levels of iNOS, COX-2, $PGE_2$, and TNF-${\alpha}$ in LPS stimulated RAW 264.7 macrophages. Hence, CSp and CCm could be utilized in developing functional ingredients for foods, and cosmeceuticals.

• Genomics & Informatics
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• v.20 no.4
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• pp.41.1-41.9
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• 2022

The pathogen Gallibacterium anatis has caused heavy economic losses for commercial poultry farms around the world. However, despite its importance, the functions of its hypothetical proteins (HPs) have been poorly characterized. The present study analyzed the functions and structures of HPs obtained from Gallibacterium anatis (NCTC11413) using various bioinformatics tools. Initially, all the functions of HPs were predicted using the VICMpred tool, and the physicochemical properties of the identified virulence proteins were then analyzed using Expasy's ProtParam server. A virulence protein (WP_013745346.1) that can act as a potential drug target was further analyzed for its secondary structure, followed by homology modeling and three-dimensional (3D) structure determination using the Swiss-Model and Phyre2 servers. The quality assessment and validation of the 3D model were conducted using ERRAT, Verify3D, and PROCHECK programs. The functional and phylogenetic analysis was conducted using ProFunc, STRING, KEGG servers, and MEGA software. The bioinformatics analysis revealed 201 HPs related to cellular processes (n = 119), metabolism (n = 61), virulence (n = 11), and information/storage molecules (n = 10). Among the virulence proteins, three were detected as drug targets and six as vaccine targets. The characterized virulence protein WP_013745346.1 is proven to be stable, a drug target, and an enzyme related to the citrate cycle in the present pathogen. This enzyme was also found to facilitate other metabolic pathways, the biosynthesis of secondary metabolites, and the biosynthesis of amino acids.

• Biomedical Science Letters
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• v.12 no.3
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• pp.281-287
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• 2006

Cigarette smoke causes atypical structure of pulmonary and oxidative damage. Therefore, we carried out to determine if exposure to cigarette smoke alters pulmonary structure and anti-oxidant related enzyme in a animal model, when natural product extracts using by Nebulizer. The rat were divided into four groups: $H_2O-treated$ (Control), natural product (Camellia sinensis) extracts-treated (CS), natural product extracts-treated with cigarette smoke-exposed (CS+SM) and cigarette smoke-expose (SM). All groups are similar to Control group in weight, but SM group is lower than the other groups. Microscopic image of the pulmonary structure in SM group showed deleterious alterations in the morphology, but the other groups are maintained in normal structure. In anti-oxidant related enzymes, SOD (superoxide dismutase) and catalase, SM group represents the lowest enzyme activity among all groups. But G6PD (glucose-6-phosphate dehydrogenase) and LPO (lipid peroxidation) is SM group represents the highest enzyme activity among all groups. These result indicate that the natural product extracts is an efficient tissue protective substance against smoke-induced lung injury.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1994.11a
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• pp.69-80
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• 1994

Although ureases play important roles in microbial nitrogen metabolism and in the pathogenesis of several human diseases, little is known of the mechanism of metallocenter biosynthesis in this Ni-Containing enzyme. Klebsiella aerogenes urease apo-protein was purified from cells grown in the absence of Ni. The purified apo-enzyme showed the same native molecular weight, charge, and subunit stoichiometry as the holo-enzyme. Chemical modification studies were consistent with histidinyl ligation of Ni. Apo-enzyme could not be activated by simple addition of Ni ions suggesting a requirement for a cellular factor. Deletion analysis showed that four accessory genes (ureD, ureE, ureF, and ureG) are necessary for the functional incorporation of the urease metallocenter. Whereas the $\Delta$ureD, $\Delta$ureF, and $\Delta$ureG mutants are inactive and their ureases lack Ni, the $\Delta$ureE mutants retain partial activity and their ureases possess corresponding lower levels of Ni. UreE and UreG peptides were identified by SDS-polyacrylamide gel comparisons of mutant and wild type cells and by N-terminal sequencing. UreD and UreF peptides, which are synthesized at ve교 low levels, were identified by using in vitro transcription/translation methods. Cotransformation of E. coli cells with the complementing plasmids confirmed that ureD and ureF gene products act in trans. UreE was purified and characterized. immunogold electron microscopic studies were used to localize UreE to the cytoplasm. Equilibrium dialysis studies of purified UreE with $^{63}$ NiC1$_2$ showed that it binds ～6 Ni in a specific manner with a $K_{d}$ of 9.6 $\pm$1.3 $\mu$M. Results from spectroscopic studies demonstrated that Ni ions are ligated by 5 histidinyl residues and a sixth N or O atom, consistent with participation of the polyhistidine tail at the carboxyl termini of the dimeric UreE in Ni binding. With these results and other known features of the urease-related gene products, a model for urease metallocenter biosynthesis is proposed in which UreE binds Ni and acts as a Ni donor to the urease apo-protein while UreG binds ATP and couples its Hydrolysis to the Ni incorporation process.ouples its Hydrolysis to the Ni incorporation process.s.

• BMB Reports
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• v.34 no.6
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• pp.559-565
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• 2001

Protein carboxyl O-methyltransferase (E.C.2.1.1.24) may play a role in the repair of aged protein that is spontaneously incorporated with isoaspartyl residues. The porcine brain carboxyl O-methyltransferase was cloned in the pET32 vector, and overexpressed in E.coh (BL21) that harbors pETPCMT, which encodes 227 amino acids, including tagging proteins at the N-terminus. The protein sequence of the cloned porcine brain PCMT (r-pbPCMT) shares a 98% identity with that of human erythrocyte PCMT and rat brain PCMT. It is 100% identical with that of bovine brain. The r-pbPCMT was purified using Ni-NTA affinity chromatography and digested by enterokinase in order to remove the protein tags. Then Superdex 75HR gel filtration chromatography was performed. The r-pbPCMT exhibited similar in vitro substrate specificities with the PCMT that was purified from porcine brain. The molecular weight of the enzyme was estimated to be 24.5 kDa on the SDS polyacrylamide gel electrophoresis. The $K_m$ value was $1.1{\times}10^{-7}\;M$ for S-adenosyl-L-methionine. S-adnosyl-L-homocysteine was a competitive type of inhibitor with the $K_i$ value of $1.38{\times}10^{-4}\;M$. The enzyme has optimal activity at pH 6.0 and $37^{\circ}C$. These results indicate that the expressed enzyme is functionally similar to the natural protein. It also suggests that it may be a suitable model to further understand the function of the mammalian enzyme.

• Korean Journal of Pharmacognosy
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• v.36 no.2 s.141
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• pp.81-87
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• 2005

The effects of the DWP-04 [DDB:selenium yeast:glutathione (31.1 : 6.8 : 62.1 (w/w%)] on acetaminophen detoxification enzyme system were studied in rats. Treatment with DWP-04 was prevented againt acetaminophen-induiced hepatotoxicity in rat as evidenced by the decreased formation of lipid peroxide. Effect of DWP-04 on the activities of free radical-generating enzymes, free radical scavenging enzymes and glutathione-related enzymes as well as detoxification mechanism of DWP-04 against acetaminophen-treated was investigated in rat. Activities of cytochrome p450, cytochrome b5, aminopyrine demethylase and aniline hydroxylase as free radical-generating enzymes activities were decreased by the treatment with DWP-04 against acetaminophen treated. Although acetaminophen-induced hepatotoxicity results in the significantly decrease in the level of hepatic glutathione and activities of glutathine S-transferase, quinone reductase, glutathione reductase and ${\gamma}-glutamyl-$cysteine synthetase, these decreasing effects were markedly lowered in the DWP-04-treated rat. Therefore, it was concluded that the mechanism for the observed preventive effect of DWP-04 against the acetaminophen-induced hepatotoxicity was associated with the decreased activities in the free radical-generating enzyme system.

• Animal cells and systems
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• v.8 no.4
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• pp.307-312
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• 2004

Fast kinetics of transient pH changes and difference spectrum formation have been investigated following mixing of ADP/ATP with partially purified plasma membrane PM-ATPase of the pathogenic yeast Candida albicans in the presence of five nutrients: glucose, glutamic acid, proline, lysine, and arginine and two analogs of glucose: 2-deoxy D-glucose and xylose. Average $H^+$- absorption to release ratio, indicative of population of ATPase undergoing complete hydrolytic cycle, was found to be 0.27 for control. This ratio varied between 0.25 (proline) to 0.36 (arginine) for all other compounds tested, except for glucose. In the presence of glucose, $H^+$- absorption to release ratio was exceptionally high (0.92). While no UV difference spectrum was observed with ADP, mixing of ATP with ATPase led to a large conformational change. Exposure to different nutrients restricted the magnitude of the conformational change; the analogs of glucose were found to be ineffective. This suppression was maximal in the case of glucose (80%); with other nutrients, the magnitude of suppression ranged from 40-50%. Rate of $H^+$- absorption, which is indicative of E~P complex dissociation, showed positive correlation with suppression of conformational change only in the case of glucose and no other nutrient/analog. Mode of interaction of glucose with plasma membrane $H^+$-ATPase thus appears to be strikingly distinct compared to that of other nutrients/analogs tested. The results obtained lead us to propose a model for explaining glucose stimulation of plasma membrane $H^+$-ATPase activity.

• Journal of the Korean Chemical Society
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• v.53 no.5
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• pp.499-504
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• 2009

Metals have often played important roles to some enzymatic reactions that are essential to biological processes. Therefore many scientists have studied the reaction mechanisms of catalytic reactions in metaloenzymes for many years. Methane MonoOxygenase (MMO) is an enzyme that oxidize methane to methyl alcohol. Recently Tolman et al. studied a model reaction for MMO, which is a hydroxide transfer reaction in Bis-($\mu$-oxo)-dicopper complex, and suggested several possible mechanisms. Later a two-step mechanism, which is hydrogen transfer followed by hydroxide rebound, was proposed from theoretical studies. In this study we calculated the reactant, product, and the transition state structures, and energetics of the first hydrogen transfer reaction using various DFT methods including recently developed the MO6 family of DFT, namely, MO6, MO6L, and MO6-2X. We found that the M06/6-31G(d,p)/LANL2DZ method reproduce the experimental XRD structure of reactants very well. The TS structures, barrier heights, and reaction energies depend very much on the size of the basis sets.

• 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.

• Food Science of Animal Resources
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• v.43 no.1
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• pp.46-60
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• 2023

Slaughterhouse blood is a by-product of animal slaughter that can be a good source of animal protein. This research purposed to examine the functional qualities of the blood plasma from Hanwoo cattle, black goat, and their hydrolysates. Part of the plasma was hydrolyzed with proteolytic enzymes (Bacillus protease, papain, thermolysin, elastase, and α-chymotrypsin) to yield bioactive peptides under optimum conditions. The levels of hydrolysates were evaluated by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis. The antioxidant, metal-chelating, and angiotensin I-converting enzyme (ACE) inhibitory properties of intact blood plasma and selected hydrolysates were investigated. Accordingly, two plasma hydrolysates by protease (pH 6.5/55℃/3 h) and thermolysin (pH 7.5/37℃/3-6 h) were selected for analysis of their functional properties. In the oil model system, only goat blood plasma had lower levels of thiobarbituric acid reactive substances than the control. The diphenyl picrylhydrazyl radical scavenging activity was higher in cattle and goat plasma than in proteolytic hydrolysates. Ironchelating activities increased after proteolytic degradation except for protease-treated cattle blood. Copper-chelating activity was excellent in all test samples except for the original bovine plasma. As for ACE inhibition, only non-hydrolyzed goat plasma and its hydrolysates by thermolysin showed ACE inhibitory activity (9.86±5.03% and 21.77±3.74%). In conclusion, goat plasma without hydrolyzation and its hydrolysates can be a good source of bioactive compounds with functional characteristics, whereas cattle plasma has a relatively low value. Further studies on the molecular structure of these compounds are needed with more suitable enzyme combinations.