• Journal of Microbiology and Biotechnology
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• v.18 no.3
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• pp.443-448
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• 2008

Multidomain proteins for the biochemical analysis of the scouring efficiency of cotton fabrics were constructed by the fusion of a reporter moiety in the N-terminal and the cellulose binding domain (CBD) in the C-terminal. Based on the specific binding of the CBD of Cellulomonas fimi exoglucanase (Cex) to crystalline cellulose (Avicel), the reporter protein is guided to the cellulose fibers that are increasingly exposed as the scouring process proceeds. Among the tested reporter proteins, a thermostable ${\beta}$-glycosidase (BglA) from Thermus caldophilus was found to be most appropriate, showing a higher applicability and stability than GFP, DsRed2, or a tetrameric ${\beta}$-glycosidase (GUS) from Escherichia coli, which were precipitated more seriously during the expression and purification steps. When cotton fabrics with different scouring levels were treated with the BglA-CBD and incubated with X-Gal as the chromogenic substrate, an indigo color became visible within 2 h, and the color depth changed according to the conditions and extent of the scouring.

• Journal of Microbiology and Biotechnology
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• v.20 no.6
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• pp.1011-1017
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• 2010

A novel dioscin-glycosidase that specifically hydrolyzes multi-glycosides, such as 3-O-${\alpha}$-L-($1{\to}4$)-rhamnoside, 3-O-${\alpha}$-L-($1{\to}2$)-rhamnoside, 3-O-${\alpha}$-L-($1{\to}4$)-arabinoside, and ${\beta}$-D-glucoside, on diosgenin was isolated from the Absidia sp.d38 strain, purified, and characterized. The molecular mass of the new dioscin-glycosidase is about 55 kDa based on SDS-PAGE. The dioscin-glycosidase gradually hydrolyzes either 3-O-${\alpha}$-L-($1{\to}4$)-Rha or 3-O-${\alpha}$-L-($1{\to}2$)-Rha from dioscin into 3-O-${\alpha}$-L-Rha-${\beta}$-D-Glc-diosgenin, further rapidly hydrolyzes the other ${\alpha}$-L-Rha from 3-O-${\alpha}$-L-Rha-${\beta}$-D-Glc-diosgenin into the main intermediate products of 3-O-${\beta}$-D-Glc-diosgenin, and subsequently hydrolyzes these intermediate products into aglycone as the final product. The enzyme also gradually hydrolyzes 3-O-${\alpha}$-L-($1{\to}4$)-arabinoside, 3-O-${\alpha}$-L-($1{\to}2$)-rhamnoside, and ${\beta}$-D-glucoside from [3-O-${\alpha}$-L-($1{\to}4$)-Ara, 3-O-${\alpha}$-L-($1{\to}4$)-Rha]-${\beta}$-D-Glc-diosgenin into diosgenin as the final product, exhibiting significant differences from previously reported glycosidases. The optimal temperature and pH for the new dioscin-glycosidase is $40^{\circ}C$ and 5.0, respectively. Whereas the activity of the new dioscin-glycosidase was not affected by $Na^+$, $K^+$, and $Mg^{2+}$ ions, it was significantly inhibited by $Cu^{2+}$ and $Hg^{2+}$ ions, and slightly affected by $Ca^{2+}$ ions.

• Journal of Microbiology and Biotechnology
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• v.18 no.2
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• pp.287-294
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• 2008

Three amino acid residues (His119, Glu164, and Glu338) in the active site of Thermus caldophilus GK24 ${\beta}$-glycosidase (Tca ${\beta}$-glycosidase), a family 1 glycosyl hydrolase, were mutated by site-directed mutagenesis. To verify the key catalytic residues, Glu164 and Glu338 were changed to Gly and Gln, respectively. The E164G mutation resulted in drastic reductions of both ${\beta}$-galactosidase and ${\beta}$-glucosidase activities, and the E338Q mutation caused complete loss of activity, confirming that the two residues are essential for the reaction process of glycosidic linkage hydrolysis. To investigate the role of His119 in substrate binding and enzyme activity, the residue was substituted with Gly. The H119G mutant showed 53-fold reduced activity on 5mM p-nitrophenyl ${\beta}$-D-galactopyranoside, when compared with the wild type; however, both the wild-type and mutant enzymes showed similar activity on 5mM p-nitrophenyl ${\beta}$-D-glucopyranoside at $75^{\circ}C$. Kinetic analysis with p-nitrophenyl ${\beta}$-D-galactopyranoside revealed that the $k_{cat}$ value of the H119G mutant was 76.3-fold lower than that of the wild type, but the $K_m$ of the mutant was 15.3-fold higher than that of the wild type owing to the much lower affinity of the mutant. Thus, the catalytic efficiency $(k_{cat}/K_m)$ of the mutant decreased to 0.08% to that of the wild type. The $k_{cat}$ value of the H119G mutant for p-nitrophenyl ${\beta}$-D-glucopyranoside was 5.l-fold higher than that of the wild type, but the catalytic efficiency of the mutant was 2.5% of that of the wild type. The H119G mutation gave rise to changes in optima pH (from 5.5-6.5 to 5.5) and temperature (from $90^{\circ}C\;to\;80-85^{\circ}C$). This difference of temperature optima originated in the decrease of H119G's thermostability. These results indicate that His119 is a crucial residue in ${\beta}$-galactosidase and ${\beta}$-glucosidase activities and also influences the enzyme's substrate binding affinity and thermostability.

• Journal of Microbiology and Biotechnology
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• v.29 no.4
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• pp.562-570
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• 2019

${\beta}$-Glucosylglycerol (${\beta}-GG$) and their derivatives have potential applications in food, cosmetics and the healthcare industry, including antitumor medications. In this study, ${\beta}-GG$ and its unnatural glycosides were synthesized through the transglycosylation of two enzymes, Sulfolobus shibatae ${\beta}$-glycosidase (SSG) and Deinococcus geothermalis amylosucrase (DGAS). SSG catalyzed a transglycosylation reaction with glycerol as an acceptor and cellobiose as a donor to produce 56% of ${\beta}-GGs$ [${\beta}$-$\text\tiny{D}$-glucopyranosyl-($1{\rightarrow}1/3$)-$\text\tiny{D}$-glycerol and ${\beta}$-$\text\tiny{D}$-glucopyranosyl-($1{\rightarrow}2$)-$\text\tiny{D}$-glycerol]. In the second transglycosylation reaction, ${\beta}$-$\text\tiny{D}$-glucopyranosyl-($1{\rightarrow}1/3$)-$\text\tiny{D}$-glycerol was used as acceptor molecules of the DGAS reaction. As a result, 61% of ${\alpha}$-$\text\tiny{D}$-glucopyranosyl-($1{\rightarrow}4$)-${\beta}$-$\text\tiny{D}$-glucopyranosyl-($1{\rightarrow}1/3$)-$\text\tiny{D}$-glycerol and 28% of ${\alpha}$-$\text\tiny{D}$-maltopyranosyl-($1{\rightarrow}4$)-${\beta}$-$\text\tiny{D}$-glucopyranosyl-($1{\rightarrow}1/3$)-$\text\tiny{D}$-glycerol were synthesized as unnatural glucosylglycerols. In conclusion, the combined enzymatic synthesis of the unnatural glycosides of ${\beta}-GG$ was established. The synthesis of these unnatural glycosides may provide an opportunity to discover new applications in the biotechnological industry.

• Food Science and Preservation
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• v.8 no.1
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• pp.66-73
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• 2001

This study was carried out to know whether $\beta$-galactosidase is directly important or not on fruit softening during the development of peach fruits compared to those in the stage stage. It was investigated that the flesh firmness, cell wall components, and the glycosidase activities of the peach fruits with a fast softening cultivar, 'Mibeakdo', a slow softening cultivar,'Yumyung'and a middle softening cultivar, 'Okubo$\beta$, at different developmental stages, on 13 May, 16 June, 16 July, and 5 August and on 28 August which harvested only 'Yumyung' fruits. In order to investigate the amounts of total sugar and non-cellulosic neutral sugar, the cell wall materials of each fruit were solubilized in distilled water, 0.05M CDTA, 0.05M Na$_2$CO$_3$, 4% KOH, and 24% KOH sequentially. During the fruit development, the fruit firmness of three cultivars decreased and the fruit firmness of 'Yumyung' was higher than that fo 'Mibeakdo' and 'Okubo' in the overall period. During the fruit development, the changes of total sugar amounts of each measured fractions were similar among peach cultivars. Arabinose and galactose were the predominant non-cellulosic neutral sugars in all the fractions including cell wall material of the three cultivars. There was an active relationship between the changes of flesh firmness in three cultivars and the mol % changes of rhamnose on 5 August which was the harvest date of 'Mibeakdo' and 'Okubo' fruits. The activity of soluble $\beta$-galactosidase was high at the early developmental stage and then dropped to a very low activity level in all cultivars. The activity of cell wall-bound $\beta$-galactosidase was high at the early developmental stage and then decreased continuously through the harvest date. In addition the changes of other glycosidase activities were similar among cultivars.

• Biomolecules & Therapeutics
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• v.6 no.3
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• pp.242-246
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• 1998

The inhibitory activities of Amberlite active fraction, which was obtained from methanol soluble fraction of freeze dried slikworm urine, on the rat intestinal glycosidase-catalyzed enzymatic reaction were examined in in viro and in vivo experiments. Amberlite active fraction showed significant inhibitory effects on the hydrolysis of o-glycosidic bond, especially $\alpha$-1,4 bond. On the other hand, the inhibition on the hydrolysis of $\beta$-glycosidic bond was very weak. Oral administration of Amberlite active fraction resulted in a dose-dependent decrease in the blood glucose after an oral maltose load, and postprandial hyperglycemia in carbohydrate-loaded mice was suppressed by Amberlite active fraction at 60 mgHg in decreasing order of maltose, starch, sucrose and lactose. 60 mg/kg of Amberlite active fraction lowered the blood glucose level markedly after 18, 35, and 60 min after an oral maltose load and the antihyperglycemic activity was maintained upto 90 min. In alloxan-induced hyperglycemic mice, Amberlite active fraction at a dose of 100 mg/kg also significantly lowered blood glucose after an oral maltose load, and its efficacy was almost equivalent to that of acarbowe.

• Archives of Pharmacal Research
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• v.21 no.1
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• pp.17-23
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• 1998

Flavonoid glycosides were metabolized to phenolic acids via aglycones by human intestinal microflora producing ${\alpha}$-rhamnosidase, exo-${\beta}$-glucosidase, endo- ${\beta}$-glucosidase and/or ${\beta}$-glucuronidase. Rutin, hesperidin, naringin and poncirin were transformed to their aglycones by the bacteria producing ${\alpha}$-rhamnosidase and ${\beta}$-glucosidase or endo- ${\beta}$-glucosidase, and baicatin, puerarin and daidzin were transformed to their aglycones by the bacteria producing ${\beta}$glucuronidase, C-glycosidase and ${\beta}$-glycosidase, respectively. Anti-platelet activity and cytotoxicity of the metabolites of flavonoid glycosides by human intestinal bacteria were more effective than those of the parental compounds. 3,4-Dihydroxyphenylacetic acid and 4-hydroxyl-phenylacetic acid were more effective than rutin and quercetin on anti-platelet aggregation activity. 2,4,6-Trihydroxybenzaidehyde, quercetin and ponciretin were more effective than rutin and ponciretin on the cytotoxicity for tumor cell lines. We insist that these flavonoid glycosides should be natural prodrugs.

• Journal of Microbiology and Biotechnology
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• v.28 no.2
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• pp.255-261
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• 2018

Aglycon protopanaxatriol (APPT) has valuable pharmacological effects such as memory enhancement and tumor inhibition. ${\beta}$-Glycosidase from the hyperthermophilic bacterium Dictyoglomus turgidum (DT-bgl) hydrolyzes the glucose residues linked to APPT, but not other glycoside residues. ${\beta}$-Glycosidase from the hyperthermophilic bacterium Pyrococcus furiosus (PF-bgl) hydrolyzes the outer sugar at C-6 but not the inner glucose at C-6 or the glucose at C-20. Thus, the combined use of DT-bgl and PF-bgl is expected to increase the biotransformation of PPT-type ginsenosides to APPT. We optimized the ratio of PF-bgl to DT-bgl, the concentrations of substrate and enzyme, and the reaction time to increase the biotransformation of ginsenoside Re and PPT-type ginsenosides in Panax ginseng leaf extract to APPT. DT-bgl combined with PF-bgl converted 1.0 mg/ml PPT-type ginsenosides in ginseng leaf extract to 0.58 mg/ml APPT without other ginsenosides, with a molar conversion of 100%. We achieved the complete biotransformation of ginsenoside Re and PPT-type ginsenosides in ginseng leaf extract to APPT by the combined use of two ${\beta}$-glycosidases, suggesting that discarded ginseng leaves can be used as a source of the valuable ginsenoside APPT. To the best of our knowledge, this is the first quantitative production of APPT using ginsenoside Re, and we report the highest concentration and productivity of APPT from ginseng extract to date.

• Journal of Ginseng Research
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• v.42 no.4
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• pp.504-511
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• 2018

Background: The biological activities of ginseng saponins (ginsenosides) are associated with type, number, and position of sugar moieties linked to aglycone skeletons. Deglycosylated minor ginsenosides are known to be more biologically active than major ginsenosides. Accordingly, the deglycosylation of major ginsenosides can provide the multibioactive effects of ginsenosides. The purpose of this study was to transform ginsenoside Rb2, one of the protopanaxadiol-type major ginsenosides, into minor ginsenosides using ${\beta}$-glycosidase (BG-1) purified from Armillaria mellea mycelium. Methods: Ginsenoside Rb2 was hydrolyzed by using BG-1; the hydrolytic properties of Rb2 by BG-1 were also characterized. In addition, the influence of reaction conditions such as reaction time, pH, and temperature, and transformation pathways of Rb2, Rd, F2, compound O (C-O), and C-Y by treatment with BG-1 were investigated. Results: BG-1 first hydrolyzes 3-O-outer ${\beta}$-$\text\tiny{D}$-glucoside of Rb2, then 3-O-${\beta}$-$\text\tiny{D}$-glucoside of C-O into C-Y. C-Y was gradually converted into C-K with a prolonged reaction time, but the pathway of Rb2 ${\rightarrow}$ Rd ${\rightarrow}$ F2 ${\rightarrow}$ C-K was not observed. The optimum reaction conditions for C-Y and C-K formation from Rb2 by BG-1 were pH 4.0-4.5, temperature $45-60^{\circ}C$, and reaction time 72-96 h. Conclusion: ${\beta}$-Glycosidase purified from A. mellea mycelium can be efficiently used to transform Rb2 into C-Y and C-K. To our best knowledge, this is the first result of transformation from Rb2 into C-Y and C-K by basidiomycete mushroom enzyme.

• Journal of Microbiology and Biotechnology
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• v.17 no.3
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• pp.454-460
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• 2007

Enzymatic properties and substrate specificity of recombinant $\beta-glycosidases$ from a hyperthermophilic archaeon, Sulfolobus shibatae (rSSG), were analyzed. rSSG showed its optimum temperature and pH at $95^{\circ}C$ and pH 5.0, respectively. Thermal inactivation of rSSG showed that its half-life of enzymatic activity at $75^{\circ}C$ was 15 h whereas it drastically decreased to 3.9 min at $95^{\circ}C$. The addition of 10 mM of $MnCl_2$ enhanced the hydrolysis activity of rSSG up to 23% whereas most metal ions did not show any considerable effect. Dithiothreitol (DTT) and 2-mercaptoethanol exhibited significant influence on the increase of the hydrolysis activity of rSSG rSSG apparently preferred laminaribiose $(\beta1\rightarrow3Glc)$, followed by sophorose $(\beta1\rightarrow2Glc)$, gentiobiose $(\beta1\rightarrow6Glc)$, and cellobiose $(\beta1\rightarrow4Glc)$. Various. intermolecular transfer products were formed by rSSG in the lactose reaction, indicating that rSSG prefers lactose as a good acceptor as well as a donor. The strong intermolecular transglycosylation activity of rSSG can be applied in making functional oligosaccharides.