• Journal of Microbiology and Biotechnology
• /
• v.20 no.6
• /
• pp.1011-1017
• /
• 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 Sericultural and Entomological Science
• /
• v.48 no.1
• /
• pp.25-27
• /
• 2006

[ ${\alpha}-Glycosidase$ ] inhibitors slows the velocity of the uptake of monosaccharides in the small intestine by retarding the speed of degradation of disaccharides to monosaccharides, which made it possible to develop ${\alpha}-glycosidase$ inhibitors as the antihyperglycemic ('antihyperglycemic' means 'blood-glucose-level-lowering') reagent for the diabetic patients such as acarbose and miglitol. Twenty kinds of ${\alpha}-glycosidase$ inhibitors have been reported to exist in mulberry, Morns alba, and some of them are also found in the silkworm, Bombyx mari, as the result of its daily feeding of mulberry leaves as the sole diet. 1-Deoxynojirimycin (DNJ), one of the most potent ${\alpha}-glycosidase$ inhibitor, is the most abundant among polyhydroxylated alkaloids with ${\alpha}-glycosidase$ inhibiting activity in both M alba and B. mari, therefore considered the antihyperglycemic criterion of the mulberry- or silkworm-based neutroceutical products. DNJ is thought to be accumulated in the body of silkworm because the its concentration in the silkworm body is two to three times as much as that in the mulberry leaves. Eighteen silkworm F1 varieties have been recommended for industrial rearing in Korea by some standards such as pathological strength. DNJ concentration in 18 recommendedwere measured at 3rd day in the 5th instar after lyophilization to determine the varieties that accumulates DNJ in its body most. GeumOk-Jam was the highest in the DNJ concentration of 5.45 mg/gDW among the recommended F1 varieties.

• Journal of Microbiology and Biotechnology
• /
• v.14 no.3
• /
• pp.584-592
• /
• 2004

A thermostable $\beta$-glycosidase gene, tfi $\beta$-gly, was cloned from the genomic library of Thermus filiformis Wai33 A1. ifi $\beta$-gly consists of 1,296 bp nucleotide sequence and encodes a polypeptide of 431 amino acids. It shares a strong amino acid sequence similarity with the $\beta$-glycosidases from other Thermus spp. belonging to the glycosyl hydrolase family 1. In the present study, the enzyme was overexpressed in Escherichia coli BL21 (DE3) using the pET21b(+) vector system. The recombinant enzyme was purified to homogeneity by heat treatment and a $Ni^{2+}$-affinity chromatography. Polyacrylamide gel electrophoresis (PAGE) showed that the recombinant Tfi $\beta$-glycosidase was a monomeric form with molecular mass of 49 kDa. The temperature and pH range for optimal activity of the purified enzyme were 80- $90^{\circ}C$ and 5.0-6.0, respectively. Ninety-three percent of the enzyme activity was remained at $70^{\circ}C$ after 12 h, and its half-life at $80^{\circ}C$ was 6 h, indicating that Tfi $\beta$-glycosidase is highly thermostable. Based on its K_m$, or $K_{cat}K_m$, ratio, Tfi $\beta$-glycosidase appeared to have higher affinity for $\beta$-D-glucoside than for $\beta$-D-galactoside, however, $K_{cat} for \beta$-D-galactoside was much higher than that for $\beta$-D-glucoside. The activity for lactose hydrolysis was proportionally increased at $70^{\circ}C$ and pH 7.0 without substrate inhibition until reaching 250 mM lactose concentration. The specific activity of Tfi TEX>$\beta$-glycosidase on 138 mM lactose at $70{^\circ}C$ and pH 7.0 was 134.9 U/mg. Consequently, this newly cloned enzyme appears to have a valuable advantage of conducting biotechnological processes at elevated temperature during milk pasteurization in the production of low-lactose milk.

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

• BMB Reports
• /
• v.31 no.3
• /
• pp.269-273
• /
• 1998

The objective of this study was to evaluate the reversal of multi drug resistance of human cell lines by specific inhibitors of ${\alpha}-glycosidase$ and mannosidases that had been reported to be involved in N-linked oligosaccharide processing of glycoproteins. N-methyldeoxynojirimycin, I-deoxynojirimycin, and castanospermine, which were known to be potent inhibitors of both ${\alpha}-glycosidase$ I and II, showed no activity against the multidrug resistant phenotype of the cell lines of SNU1DOX, KB-V1, and MCF-7/ADR. In contrast, I-deoxymannojirimycin, an inhibitor of mannosidase I, resulted in a slight reversal for the vinblastine resistance of the KB-V1 cell line, but did not show any activity toward the other cell lines. Parallel experiments with tunicamycin, an inhibitor of N-linked glycosylation, also resulted in no significant changes in multidrug resistant (MDR) phenotype of the cell lines tested in this work. These observations suggest that the unglycosylation of P-glycoprotein associated with the inhibitor treatments might not be correlated with the reversal of multidrug resistance of the cell lines tested in this study.

• Reproductive and Developmental Biology
• /
• v.35 no.2
• /
• pp.185-190
• /
• 2011

To evaluate the effect of spermatozoa culture on glycosidase activity of frozen-thawed spermatozoa in human, the spermatozoa were treated experimentally and assayed for activities of ${\alpha}$-L-fucosidase, ${\alpha}$-D-mannosidase, ${\beta}$-D-galactosidase and N-acetyl-${\beta}$-D-glucosaminidase (${\beta}$-GlcNAc'ase). The ${\beta}$-GlcNAc'ase activity was at least two-folds higher than other glycosidases regardless of spermatozoa incubation (p<0.05). The spermatozoa motility was decreased with incubation periods, but no effects by different glycosidases on the changes of spermatozoa motility during the various periods of incubation. In all glycosidases, the spermatozoa-zona binding rates in spermatozoa without incubation were higher than in spermatozoa incubated for 2 h (p<0.05). ${\beta}$-GlcNAc'ase is present mainly in the plasma membrane of spermatozoa frozen-thawed in human. It was also shown that the glycosidase activity was increased in all glycosidases in spite of lower sperm-zona binding by spermatozoa incubation.

• BMB Reports
• /
• v.32 no.3
• /
• pp.317-319
• /
• 1999

A leucine aminopeptidase has been isolated from the culture medium of the soil fungus, Aspergillus flavus. The enzyme was found to be a glycoprotein, as judged by electrophoresis analysis and the subsequent staining by the periodic acid-Schiff's reagent. Carbohydrate moieties could be cleaved by N-glycosidase, but not by O-glycosidase, indicating that the glucans are linked to the asparagine residue in the protein. Removal of N-glucans was observed without prior denaturation of the protein, implying that the N-glycosidic linkage is exposed and accessible to glycosidase. When the activity of native or deglycosylated enzyme was measured in the presence of various metal ions, removal of carbohydrates increased the aminopeptidase activity of the enzyme.

• Korean Journal of Food Science and Technology
• /
• v.25 no.2
• /
• pp.191-195
• /
• 1993

The intestinal microflora of humans is an extraordinarily complex mixture of microorganisms, the majority of which are anaerobic bacteria. Amongst them, most prevalent bacteria are Bacteroides, Eubacterium, Peptococcus, Bifidobacteria. We isolated a Bacteroides fragilis strain from a Korean adult and examined various glycosidase activities of this strain. The activities of $N-acetyl-{\beta}-glucosaminidase,\;{\alpha}-fucosidase$, ${\beta}-glucuronidase$, chitobiase and PNPCase were stronger in Bacteroides fragilis Roid 8 than in other intestinal anaerobic bacteria. $N-acetyl-{\beta}-glucosaminidase$ was strongest, followed by ${\alpha}-fucosidase$, ${\beta}-glucuronidase$ and PNPCase. The activities of ${\beta}-galactosidase$, ${\beta}-xylosidase,\;{\alpha}-arabinofuranosidase$ were not present or very low. The activities of ${\alpha}-glucosidase$, ${\beta}-glucosidase$ and ${\alpha}-galactosidase$ were present but at a lower level than in Bifidobacterium. The effect of the carbon sources on the production of $N-acetyl-{\beta}-glucosaminidase$, ${\alpha}-fucosidase$, ${\beta}-glucuronidase$ and PNPCase of Bacteroides fragilis Roid 8 was investigated. :.actose and glucose lowered the production of the varous glycosidase enzymes studied in this work. In addition, we investigated the optimum temperature and pH of each glycosidase from Bacteroides fragilis Roid-8 using crude enzyme preparations.

• 한국생물공학회:학술대회논문집
• /
• 2000.11a
• /
• pp.721-724
• /
• 2000

The possibility of the enzymatic in vitro glycosylation using peptide-N-glycosidase F was examined. Oligosaccharide chains in the glycoproteins are important for the biological activity, solubility, immunogenecity, recognition, and prevention of degradation. After 4 h incubation of deglycosylated glycoprotein with excess glucose oligomer and ammonia in acetone at $50^{\circ}C$, upper shift of protein band was observed on SDS-PAGE. And the different deglycosylation characteristics of glucose oxidase and fetuin were investigated.

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