• Journal of Microbiology and Biotechnology
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• v.28 no.7
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• pp.1078-1085
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• 2018

A salt-tolerant cellulase secreted by a marine Bacillus sp. SR22 strain with wide resistance to temperature and pH was purified and characterized. Its approximate mass was 37 kDa. The endoglucanase, named as Bc22Cel, was purified by ammonium sulfate precipitation, gel filtration chromatography, and extraction from the gel after non-reducing sodium dodecyl sufate-polyacrylamide gel electrophoresis. The optimal pH value and temperature of Bc22Cel were 6.5 and $60^{\circ}C$, respectively. The purified Bc22Cel showed a considerable halophilic property, being able to maintain more than 70% of residual activity even when pre-incubated with 1.5 M NaCl for 1 h. Kinetic analysis of the purified enzyme showed the $K_m$ and $V_{max}$ to be 0.704 mg/ml and $29.85{\mu}mol{\cdot}ml^{-1}{\cdot}min^{-1}$, respectively. Taken together, the present data indicate Bc22Cel as a potential and useful candidate for industrial applications, such as the bioconversion of sugarcane bagasse to its derivatives.

• Bulletin of the Korean Chemical Society
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• v.23 no.6
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• pp.837-845
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• 2002

Based on ab initio calculations at the MP2(FULL)/6-31+G**//RHF/6-31G** level, we compare the energetic and mechanistic features of a model reaction for catalytic action of Δ?-3-ketosteroid isomerase (KSL,E.C.5.3,3.1) with those of a corresponding nonenzymatic reaction in aqueous solution. The results show that the two catalytic acid residues,Tyr14 and Asp99, can lower the free energy of activation by 8.6kcal/mol, which is in good agreement with the experimentally predicted~9 kcal/mol,contribution of electrophilic catalyses to the whole enzymatic rate enhancement. The dienolate intermediate formed by proton transfer from the substrate carbon acid to the catalytic base residue (Asp38) ins predicted to be stabilized by 12.0 kcal/mol in the enzymatic reaction, making its formation thermodynamically favorable. It has been argued that enzymes catalyzing the reactions of carbon acids should resolve the thermodynamic problem of stabilizing the enolate intermediate as well as the kinetic porblem of lowering the free energy of activation for porton abstraction. We find that KSI can successfully overcome the thermodynamic difficulty ingerent in the nonenzymatic reaction through the electrophilic catalyses of the two acid residues. Owing to the stabilization of dienolate intermediate, the reketonization step could influence the overall reaction rate more significantly in the KSI- catalyzed reaction than in the nonenzymatic reaction, further supporting the previous experimental findings. However, the electrophilic catalyses alone cannot account for the whole catalygic capability (12-13 kcal/mol), confiming the earlier experimental implications for the invement of additional catalytic components. The present computational study indicates clearly how catalytic residues of KSI resolve the fundamental problems associated with the entropic penalty for forming the rate-limiting transition state and its destabilization in the bulk solvation environment.

• Journal of Microbiology and Biotechnology
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• v.24 no.11
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• pp.1551-1558
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• 2014

The esterase gene Est8 from the thermophilic bacterium Bacillus sp. K91 was cloned and expressed in Escherichia coli. The monomeric enzyme exhibited a theoretical molecular mass of 24.5 kDa and an optimal activity around $50^{\circ}C$ at pH 9.0. A model of Est8 was constructed using a hypothetical YxiM precursor structure (2O14_A) from Bacillus subtilis as template. The structure showed an ${\alpha}/{\beta}$-hydrolase fold and indicated the presence of a typical catalytic triad consisting of Ser-11, Asp-182, and His-185, which were investigated by site-directed replacements coupled with kinetic characterization. Asp-182 and His-185 residues were more critical than the Ser-11 residue in the catalytic activity of Est8. A comparison of the amino acid sequence showed that Est8 could be grouped into the GDSL family and further classified as an SGNH hydrolase. Est8 is a new member of the SGNH hydrolase subfamily and may employ a different catalytic mechanism.

• BMB Reports
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• v.41 no.11
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• pp.784-789
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• 2008

Mac-2BP is a ligand of the galectin family that has been suggested to affect tumor proliferation and metastasis formation. We assessed Mac-2BP expression at the transcriptional and translational levels to evaluate nerve growth factor (NGF)-induced Mac-2BP expression. A time kinetic analysis using reverse transcription-polymerase chain reaction showed that NGF-induced Mac-2BP transcript levels were 4-5 times higher than in controls. Mac-2BP enzyme-linked immunosorbent assay and immuno-fluorescence staining showed a 2-3-fold increase in intracellular and secreted Mac-2BP as a result of NGF stimulation. This increase was regulated by Akt activation and NF-${\kappa}B$ binding. p65 and p50-NF-${\kappa}B$ are major transcriptional factors in the Mac-2BP promoter region, and were shown to be regulated in accordance with the Akt activation states. Collectively, these results suggest that NGF induces Mac-2BP expression via the PI3K/Akt/NF-${\kappa}B$ pathway.

• Journal of Society of Preventive Korean Medicine
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• v.12 no.2
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• pp.37-49
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• 2008

In this study, we experimented the influence of three herbal medicines, which are Saussurea lappa Clarke, Poncirus trifoliata Rafin, Citrus aurantium Linne, which are called 'Yigiyak(理氣藥)' on drug metabolizing enzyme cytochrome P450 3A4 in Human Liver Microsome. Above all, the reason for this study is that herbal medicines can be assumed that herbs might have interactions with drugs, other herbs, alcohol and chemicals whether those are much better synergy effects than expected effects when the medicine was treated alone or not. As a result, we showed that all of five traditional herbal medicines had no CYP 3A4 inhibition effect on 10, 20, 30, 40, $50{\mu}g/m{\ell}$ doses in Human Liver Microsome even Saussurea lappa Clarke showed a little inhibition as about 93% and 79% inhibition rate of control. However, this result are mostly not enough to prove that SLC has a CYP 3A4 inhibition effect. Moreover, it is not that those rates showed that those herbal medicines have CYP 3A4 induction effect. In conclusion, the result could support that those herbal medicines are more safe than chemical drugs even if this is the basic step to prove that result. Therefore, more specific studies to support this result, which are Kinetic study, cell and animal study then finally until clinical research, are required.

• Journal of Microbiology and Biotechnology
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• v.18 no.5
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• pp.933-941
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• 2008

An extracellular $\beta$-glucosidase produced by Monascus purpureus NRRL1992 in submerged cultivation was purified by acetone precipitation, gel filtration, and hydrophobic interaction chromatography, resulting in a purification factor of 92-fold. A $2^2$ central-composite design (CCD) was performed to find the best temperature and pH conditions for enzyme activity. Maximum activity was observed in a wide range of temperature and pH values, with optimal conditions set at $50^{\circ}C$ and pH 5.5. The $\beta$-glucosidase showed moderate thermostability, was inhibited by $HgCl_2$, $K_2Cr_O_4$, and $K_2Cr_2O_7$, whereas other reagents including $\beta$-mercaptoethanol, SDS, and EDTA showed no effect. Activity was slightly stimulated by low concentrations of ethanol and methanol. Hydrolysis of p-nitrophenyl-$\beta$-D-glucopyranoside (pNPG), cellobiose, salicin, n-octyl-$\beta$-D-glucopyranoside, and maltose indicates that the $\beta$-glucosidase has broad substrate specificity. Apparently, glucosyl residues were removed from the nonreducing end of p-nitrophenyl-$\beta$-D-cellobiose. $\beta$-Glucosidase affinity and hydrolytic efficiency were higher for pNPG, followed by maltose and cellobiose. Glucose and cellobiose competitively inhibited pNPG hydrolysis.

• Journal of Microbiology
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• v.35 no.3
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• pp.200-205
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• 1997

Bothl $Ca^{2+}$ and Sr$^{2+}$-containing methanol dehydrogenases (MDH) were purified to homogeneity with yields of 48% and 42%, respectively, from Methylabacillus methanolovorus sp. strain SK5. Most of the biochemical and structural properties were similar to each other. However, some differences were found: (1) although the overall shape of the absorption spectrum of Sr$^{2+}$-MDH was very similar to that of $Ca^{2+}$-MDH, the absorption intensity originating from the cofactor in Sr$^{2+}$. MDH was higher than that in $Ca^{2+}$-MDH. Small blue shift of the maximum was also observed. These are probably due to a difference in redox state of the cofactors in $Ca^{2+}$ and Sr$^{2+}$-MDH; (2) Sr$^{2+}$-MDH was more heat-stable than $Ca^{2+}$-MDH above 56$^{\circ}C$; (3) the V$_{max}$ values for the methanol-dependent activities of Sr$^{2+}$- and $Ca^{2+}$-MDH in the presence of 3 mM KCN were 2.038 and 808 nmol/mg protein/min, respectively. In addition, the $K_{m}$ values of Sr$^{2+}$ and $Ca^{2+}$ MDH for methanol were 12 and 21 $\mu$M, respectively; (4) the endogenous activity of $Ca^{2+}$-MDH was more sensitive than that of Sr$^{2+}$-MDH in the presence of cyanide; (5) Diethyl pyrocarbonate treatment increased the enzyme activities of $Ca^{2+}$- and Sr$^{2+}$-MDH 4.2- and 1.4-folds, respectively. These results indicate that Sr$^{2+}$ stabilizes the structural conformation and enhances the activity of MDH more than $Ca^{2+}$.

• Toxicological Research
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• v.12 no.2
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• pp.155-161
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• 1996

The enzymatic properties for NADPH-P450 reductase domain of a fusion protein between human cytochrome P450 1A1 and rat NADPH-P450 reductase expressed in Escherichia coli were investigated. The fusion plasmid pCW/1A1OR-expressed E. coli membrane showed high NADPH-cytochrome c reductase activity ($830.1\pm 85.8 nmol\cdot min^{-1}\cdot mg protein^{-1}$), while pCW control vector and P 450 1A1 expression vector pCW/1A1 showed relatively quite low activity ($4.35\pm 0.49, 3.27\pm 0.50 nmol\cdot min^{-1}\cdot mg protein^{-1}$, respectively). The kinetic curves for NADPH-cytochrome c reductase followed typical Michaelis-Menten kinetics. The $K_{max}$ and $V_{max}$ for NADPH-dependent reductase activity were $8.24\pm 2.61\mu $and $817.9\pm 60.8 nmol\cdot min^{-1}\cdot mg protein^{-1}$, respectively, whereas those for cytochrome c-dependent reductase activity were $19.97\pm 2.86\mu M$ and $1303.5\pm 67.1 nmol\cdot min^{-1}\cdot mg protein^{-1}$. The reductase activities were also compared with those of rat, porcine and human liver microsomes. The activity of pCW/ 1A1OR-expressed E. coli membrane was 15.2-fold higher than that of rat liver microsome. Treatment with benzo(a)pyrene, 7-ethoxyresorufin and $\alpha$-naphthofiavone which are known as specific substrates or inhibitor for human P450 1A1 increased NADPH-cytochrome c reductase activity of fusion protein in E. coli membrane dose-dependently. These results demonstrate that the membrane topology of fused enzyme may be important for activity of its NADPH-P450 reductase domain.

• Natural Product Sciences
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• v.28 no.3
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• pp.143-152
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• 2022

In our study, sixteen known phenolic compounds, including quercetin (1), methyl gallate (2), caesalpiniaphenol C (3), 8S,8'S,7'R-(-)-lyoniresinol (4), 7,3',5'-trihydroxyflavanone (5), sappanchalcone (6), sappanone A (7), taxifolin (8), fisetin (9), fustin (10), (+)-catechin (11), brazilin (12), 3,4,5-trimethoxyphenyl β-ᴅ-glucopyranoside (13), 1-(2-methylbutyryl)phloroglucinol-glucopyranoside (14), (+)-epi-catechin (15), and astragalin (16) and one mixture of two conformers of protosappanin B (17/18) were isolated from the stems of Caesalpinia decapetala var. japonica. Their structures were elucidated based on a comparison of their physicochemical and spectral data with those of literature. To the best of our knowledge, this represents the first isolation of compounds 3, 4, 8, 9, and 10 from C. decapetala and compounds 13 and 14 from the Caesalpinia genus. All the isolated compounds were evaluated for their inhibitory effect against the α-glucosidase enzyme. Among them, two flavonols (1 and 9), one chalcone (6), and one homoisoflavanone (7) exhibited an inhibitory effect on α-glucosidase action with an IC₅₀ range value of 5.08-15.01 μM, stronger than that of the positive control (acarbose, IC₅₀ = 152.22 μM). Kinetic analysis revealed that compounds 1 and 9 showed non-competitive α-glucosidase inhibition, while the inhibition type was mixed for compounds 6 and 7.

• Animal Bioscience
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• v.35 no.10
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• pp.1592-1605
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• 2022

Objective: The objective of this study was to select an effective in vitro digestion-fermentation model to estimate the effect of decreasing dietary crude protein (CP) on odor emission during pig production and to suggest potential prediction markers through in vitro and in vivo experiments. Methods: In the in vitro experiment, three diet formulations with different CP contents (170 g/kg, 150 g/kg, and 130 g/kg) but containing the same standardized ileal digestible essential amino acids (SID-EAA) were assessed. Each diet was evaluated by two different in vitro gastric-intestinal phase digestion methods (flask and dialysis), combined with fresh pig feces-ferment inoculation. Eighteen growing barrows (31.9±1.6 kg) were divided into three groups: control diet (180 g CP/kg, without SID-EAA adjustment), 170 g CP/kg diet, and 150 g CP/kg diet for 4 weeks. Results: The in vitro digestion results indicated that in vitro digestibility was affected by the gastric-intestinal phase digestion method and dietary CP level. According to the gas kinetic and digestibility results, the dialysis method showed greater distinguishability for dietary CP level adjustment. Nitrogen-related odor compounds (NH₃-N, indole, p-cresol, and skatole) were highly correlated with urease and protease activity. The feeding study indicated that both EAA-adjusted diets resulted in a lower odor emission especially in p-cresol and skatole. Both protease and urease activity in feces were also closely related to odor emissions from nitrogen metabolism compounds. Conclusion: Dialysis digestion in the gastric-intestinal phase followed by fresh fecal inoculation fermentation is suitable for in vitro diet evaluation. The enzyme activity in the fermentation and the fecal samples might provide a simple and effective estimation tool for nitrogen-related odor emission prediction in both in vitro and in vivo experiments.