• 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.

• Journal of Applied Biological Chemistry
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• v.48 no.1
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• pp.7-10
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• 2005

Trichoderma sp. SY most effectively produces an extracellular ${\gamma}$-L-arabinofuranosidase (AF) using arabinose as a carbon source. AF grown on cellulose as a carbon source was purified 28-fold with 4.4% yield by DEAE exchange and HQ/20 cation exchange chromatographies The purified enzyme was found to be homogeneous on SDS-PAGE with molecular weight of 89 kDa. It exhibited a high level of activity with p-nitrophenyl ${\alpha}$-L-arabinofuranoside, showing $K_m$ and $V_{max}$ values of $0.15\;{\mu}M$ and $239.85U{\cdot}mg^{-1}$, respectively and did not require any metal ion for activity. It also released p-nitrophenol from p-nitrophenol conjugated ${\beta}$-D-xylopyranoside, and ${\beta}$-D-galactopyranoside not from ${\beta}$-D-glucopyranoside.

• Journal of Microbiology and Biotechnology
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• v.8 no.3
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• pp.270-276
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• 1998

Extracellular ${\alpha}$-glucosidase was purified to homogeneity from moderately thermophilic Bacillus sp. DG0303. The thermostable ${\alpha}$-glucosidase was purified by ammonium sulfate fractionation, ion-exchange chromatography, preparative polyacrylamide gel electrophoresis (PAGE), and electroelution. The molecular weight of the enzyme was estimated to be 60 kDa by SDS-PAGE. The optimum temperature for the action of the enzyme was at $60^{\circ}C$. It had a half-life of 35 min at $60^{\circ}C$. The enzyme was stable at the pH range of 4.5~7.0 and had an optimum pH at 5.0. The enzyme preparation did not require any metal ion for activity. The thermostable ${\alpha}$-glucosidase hydrolyzed the ${\alpha}$-1,6-linkages in isomaltose, isomaltotriose, and panose, and had little or no activity with maltooligosaccharides and other polysaccharides. The $K_m$ (mM) for p-nitrophenyl-${\alpha}$-D-glucopyranoside (pNPG), panose, isomaltose, and isomaltotriose were 4.6, 4.7, 40.8, and 3.7 and the $V_{max}$(${\mu}mol{\cdot}min^-1$$mg^-1$) for those substrates were 5629, 1669, 3410, and 1827, respectively. The N-terminal amino acid sequence of the enzyme was MERVWWKKAV. Based on its substrate specificity and catalytic properties, the enzyme has been assigned to be an oligo-1,6-glucosidase.

• Food Science and Biotechnology
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• v.18 no.6
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• pp.1342-1350
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• 2009

This paper investigates the production and optimization of $\beta$-galactosidase enzyme using synthetic medium by Kluyveromyces lactis NRRL Y-8279 in stirred tank bioreactor. Response surface methodology was used to investigate the effects of fermentation parameters on $\beta$-galactosidase enzyme production. Maximum specific enzyme activity of 4,622.7 U/g was obtained at the optimum levels of process variables (aeration rate 2.21 vvm, agitation speed 173.4 rpm, initial sugar concentration 33.8 g/L, incubation time 24.0 hr). The optimum temperature and pH of the $\beta$-galactosidase enzyme produced under optimized conditions were $37^{\circ}C$ and pH 7.0, respectively. The enzyme was stable over a pH range of 6.0-7.5 and a temperature range of $25-37^{\circ}C$. The $K_m$ and $V_{max}$ values for O-nitrophenol-$\beta$-D-galactopyranoside (ONPG) were 1.20 mM and $1,000\;{\mu}mol/min{\cdot}mg$ protein, respectively. The response surface methodology was found to be useful in optimizing and determining the interactions among process variables in $\beta$-galactosidase enzyme production. Hence, this study fulfills the lack of using mathematical and statistical techniques in optimizing the $\beta$-galactosidase enzyme production in stirred tank bioreactor.

• Korean Journal of Microbiology
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• v.16 no.2
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• pp.47-61
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• 1978

Strptomyces albus T-12 which ahd been isolated and identified in the laboratory, was selected for the studies on the cultural conditions on the production of D-xylose iosmerase and the enzymological characteristics using the partially purified enzyme. The best results in the enzyme production came from D-xylose medium than wheat bran. The divalent metla ions as $Co^{2+},\;Fe^{2+},\;Zn^{2+}\;and\;Cu^{2+}$ retard or inhibit the cell-growth at the early stages of mycelia propagations, and T-12 strain is especially sensitive to $Co^{2+}$. After 60 hours of shaking cultivation at $30^{\circ}C$ and 200 rpm, a maximum enzyme activitz, 0.49 enzyme units, was obtained. Cell-free enzyme obtained from mycelia heat-treated in the prescence of 0.5mM $Co^{2+}$, showed a 2.4-fold increase in specific than the enzyme from untreated mycelia. The specific activity of the purified enzyme through Sephadex G-150 columm showed 180 fold to the crude enzyme. The effective activators of the enzyme appeared to be $Mg^{2+}\;and\;Co^{2+}$ ions, and it exhibited the maximal enzyme activity showed at pH 7.0 and at tempersture around $80^{\circ}C$ when $Mg^{2+}\;and\;Co^{2+}$ ions were added. The enzyme isomerized D-glucose, D-xylose, D-ribose, L-arabinose, D-mannose, and L-rhamnose in the present of $Mg^{2+}\;and\;Co^{2+}$ ions as an activatiors. $Mg^{2+}\;and\;Co^{2+}$ ions were non-competitively bound at different allosterix sites of enzyme molecule. $Mg^{2+}(5mM)\;or\;Co^{2+}(1.0mM)$ protected against the thermal denaturations of the enzyme activities. The michelis constant(Km) and $V_{max}$ values of the emzyme for D-glucose and D-xylose were 0.52M, $2.12{\mu}moles/ml{\cdot}min.\;and\;0.28M,\;0.65moles/ml{\cdot}min.$, respectively.

• Korean Journal of Food Science and Technology
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• v.49 no.3
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• pp.274-278
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• 2017

In this study, predictive mathematical models were developed to estimate the kinetics of Staphylococcus aureus growth in processed meat product galbitang. Processed meat product galbitang was inoculated with 0.1 mL of S. aureus culture and stored at 4, 10, 20, $37^{\circ}C$. The ${\mu}_{max}$ (maximum specific growth rate) and LPD (lag phase duration) values were calculated. The primary model was used to develop a response surface secondary model. The growth parameters were analyzed using the square root model as a function of storage temperature. The developed model was confirmed by calculating RMSE (Root Mean Square Error) values as statistic parameters. The LPD decreased, but ${\mu}_{max}$ increased with an increase in the storage temperature. At 4, 10, 20 and $37^{\circ}C$, $R^2$ was 0.99, 0.98, 0.99 and 0.99, respectively; RMSE was 0.39. The developed predictive growth model can be used to predict the risk of S. aureus contamination in processed meat product galbitang; hence, it has potential as an input model for the risk assessment.

• Journal of Ginseng Research
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• v.36 no.4
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• pp.418-424
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• 2012

This study focused on the enzymatic biotransformation of the major ginsenoside Rb1 into Rd for the mass production of minor ginsenosides using a novel recombinant ${\beta}$-glucosidase from Flavobacterium johnsoniae. The gene (bglF3) consisting of 2,235 bp (744 amino acid residues) was cloned and the recombinant enzyme overexpressed in Escherichia coli BL21(DE3) was characterized. This enzyme could transform ginsenoside Rb1 and gypenoside XVII to the ginsenosides Rd and F2, respectively. The glutathione S-transferase (GST) fused BglF3 was purified with GST-bind agarose resin and characterized. The kinetic parameters for ${\beta}$-glucosidase had apparent $K_m$ values of $0.91{\pm}0.02$ and $2.84{\pm}0.05$ mM and $V_{max}$ values of $5.75{\pm}0.12$ and $0.71{\pm}0.01{\mu}mol{\cdot}min^{-1}{\cdot}mg$ of $protein^{-1}$ against p-nitrophenyl-${\beta}$-D-glucopyranoside and Rb1, respectively. At optimal conditions of pH 6.0 and $37^{\circ}C$, BglF3 could only hydrolyze the outer glucose moiety of ginsenoside Rb1 and gypenoside XVII at the C-20 position of aglycon into ginsenosides Rd and F2, respectively. These results indicate that the recombinant BglF3 could be useful for the mass production of ginsenosides Rd and F2 in the pharmaceutical or cosmetic industry.

• Journal of Microbiology and Biotechnology
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• v.17 no.1
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• pp.58-66
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• 2007

Isolation, expression, and characterization of a novel $endo-{\beta}-1,3(4)-D-glucanase$ with high specific activity and homology to Bacillus lichenases is described. One clone was screened from a genomic library of Paenibacillus sp. F-40, using lichenan-containing plates. The nucleotide sequence of the clone contains an ORF consisting of 717 nucleotides, encoding a ${\beta}-glucanase$ protein of 238 amino acids and 26 residues of a putative signal peptide at its N-terminus. The amino acid sequence showed the highest similarity of 87% to other ${\beta}-1,3-1,4-glucanases$ of Bacillus. The gene fragment Bg1 containing the mature glucanase protein was expressed in Pichia pastoris at high expression level in a 3-1 high-cell-density fermenter. The purified recombinant enzyme Bg1 showed activity against barley ${\beta}-glucan$, lichenan, and laminarin. The gene encodes an $endo-{\beta}-1,3(4)-D-glucanase$ (E. C. 3.2.1.6). When lichenan was used as substrate, the optimal pH was 6.5, and the optimal temperature was $60^{\circ}C$. The $K_m,\;V_{max},\;and\;k_{cat}$ values for lichenan are 2.96mg/ml, $6,951{\mu}mol/min{\cdot}mg,\;and\;3,131s^{-1}$, respectively. For barley ${\beta}-glucan$ the values are 3.73mg/ml, $8,939{\mu}mol/min{\cdot}mg,\;and\;4,026s^{-1}$, respectively. The recombinant Bg1 had resistance to pepsin and trypsin. Other features of recombinant Bg1 including temperature and pH stability, and sensitivity to some metal ions and chemical reagents were also characterized.

• Journal of Microbiology and Biotechnology
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• v.19 no.9
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• pp.873-880
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• 2009

A novel xylanase gene, Kxyn, was cloned from Kocuria sp. Mn22, a bacteria isolated from the deep sea of the east Pacific. Kxyn consists of 1,170 bp and encodes a protein of 390 amino acids that shows the highest identity (63%) with a xylanase from Thermohifida fusca YX. The mature protein with a molecular mass of approximately 40 kDa was expressed in Escherichia coli BL21 (DE3). The recombinant Kxyn displayed its maximum activity at $55^{\circ}C$ and at pH 8.5. The $K_m,\;V_{max}$, and $k_{cat}$ values of Kxyn for birchwood xylan were 5.4 mg/ml, $272{\mu}mol/min{\cdot}mg$, and 185.1/s, respectively. Kxyn hydrolyzed birchwood xylan to produce xylobiose and xylotriose as the predominant products. The activity of Kxyn was not affected by $Ca^{2+},\;Mg^{2+},\;Na^+,\;K^+$, ${\beta}$-mercaptoethanol, DTT, or SDS, but was strongly inhibited by $Hg^{2+},\;Cu^{2+},Zn^{2+}$, and $Pb^{2+}$. It was stable over a wide pH range, retaining more than 80% activity after overnight incubation at pH 7.5-12. Kxyn is a cellulase-free xylanase. Therefore, these properties make it a candidate for various industrial applications.

• Journal of Pharmaceutical Investigation
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• v.35 no.1
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• pp.51-55
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• 2005

A high-performance liquid chromatographic method was employed for the determination of bumetanide in human plasma. After addition of internal standard (IS, naproxen) and acidification of the plasma with 1 M hydrochloric acid, the drug and IS were extracted into dichloromethane. The organic phase was back-extracted into 1 M sodium bicarbonate solution and 50 ${\mu}l$ of the aqueous phase was injected onto a reversed-phase C18 column with a mobile phase consisting of methanol: water: glacial acetic acid = 65 : 35 : 1. The samples were detected utilizing a fluorescence detector (excitation wavelength 235 nm, emission wavelength 405 nm). The method was specific and validated with a lower limit of 5 ng/mL. Intra- and inter-day precision and accuracy were acceptable for all quality control samples including the lower limit of quantification. The applicability of the method was demonstrated by analysis of plasma after oral administration of a single 2 mg dose to 24 healthy subjects. From the plasma bumetanide concentration vs. time curves, the mean AUC was $246.5{\pm}73.8\;ng{\cdot}hr/mL$ and $C_{max}$ of $132.1{\pm}40.9$ ng/mL reached 1.2 hr after administration. The mean biological half-life of burnet ani de was $1.1{\pm}0.2$hr. Based on the results, this simple and validated assay method could readily be used in any pharmacokinetic or bioequivalence studies using humans.