• Journal of Microbiology and Biotechnology
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• 제20권6호
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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.

• 한국잠사곤충학회지
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• 제48권1호
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• pp.25-27
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• 2006

누에장려품종 중 혈당강하제 개발에 적합한 품종 결정을 위하여 DNJ 함량을 정량한 결과, 봄에 사육한 금옥잠이 5.45 mg/gDW으로 DNJ 함량이 가장 높았다. 또한 누에 유전자원 원종 66품종의 DNJ 함량을 결정한 결과, 품종간 DNJ 함량 차이가 매우 큰 것으로 밝혀져, 혈당 강하제용 누에 육종시 기초자료가 될 수 있으며, 또한 DNJ 함량이 낮은 품종은 DNJ 축적 기작 연구에 많은 도움이 될 것으로 사료된다.

• Journal of Microbiology and Biotechnology
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• 제14권3호
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• pp.584-592
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• 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
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• 제18권3호
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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.

• BMB Reports
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• 제31권3호
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• pp.269-273
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• 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
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• 제35권2호
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• pp.185-190
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• 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
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• 제32권3호
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• pp.317-319
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• 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.

• 한국식품과학회지
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• 제25권2호
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• pp.191-195
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• 1993

인체의 대장은 여러 종류의 균들에 의하여 상재균총이 이루어져 있는데 이들중 혐기성 균들이 주종을 이루고 있다. 이들 혐기성 균들 중 가장 많은 수가 Bacteroides이다. 본 연구에서 한국인으로부터 분리된 Bacteroides fragilis Roid 8은 장내의 다른 혐기성 균주들에 비하여 $N-acetyl-{\beta}-glucosaminidase$, ${\alpha}-fucosidase$, ${\beta}glucuronidase$ chitobiase, PNPCase 등의 활성이 높았다. ${\beta}-galactosidase$, ${\beta}-xylosidase$, ${\alpha}-arabinofuranosidae$활성은 없었고 ${\alpha}-glucosidase$, ${\beta}-glucosidase$, ${\alpha}-galactosidae$ 등의 생산은 Bifidobacteria 에 비하여 낮았다. BHI 기본배지에 여러 종류의 탄수화물을 첨가하여 배양한 뒤 생산된 $N-acetyl-{\beta}-glucosaminidae$, ${\alpha}-fucosidase$, ${\beta}-glucuronidase$ chitobiase, PNPCase, ${\beta}-glucosidase$, ${\beta}-glucosidase$ ${\alpha}-galactosidase$ 활성을 조사한 결과 모두 glucose와 lactose 첨가배지에서 이들 효소들의 활성이 낮았다. 조사된 모든 효소들에 대하여 특이적으로 현저히 생산을 증가시키는 당은 없었다. 이들 8개의 효소에 대하여 최적 pH와 최적온도가 조사되었다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2000년도 추계학술발표대회 및 bio-venture fair
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• pp.721-724
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• 2000

재조합 단백질 생산에서 문제가 되고 있는 번역 후 과정인 glycosylation 을 in vitro 상에서 수행하였다. 원핵생물 시스템에서 재조합 단백질을 생산하고, 이후 효소를 이용하여 올리고당을 붙여 원래의 당단백질과 유사한 단백질을 생산하는 것이 산업적으로 경쟁력을 가질 수 있으므로 이를 위하여 glucose oxidase와 fetuin을 모델 당단백질로, 가수분해 효소인 peptide-N-glycosidase F 의 역반응 활성을 이용하여 glycosylation 을 시도하였다. 역가수분해로의 평형 이동을 위하여 그 기질인 올리고당과 암모니아를 과량 첨가하고, 반응 온도를 높였다. Glucose oxidase의 경우에는 denaturation 했을 때 완전한 deglycosylation 이 일어났지만, fetuin의 경우에는 그렇지 못했다. Glucose oxidase 의 glycosylation 은 수용액상에서는 불가능 했지만 acetone 을 media로 사용하여 $50^{\circ}C$에서 4 시간동안 반응시켰을 때 SDS-PAGE 분석 결과 reglycosylation이 일어나 단백질 밴드가 위로 올라감을 관찰할 수 있었다.

• Journal of Microbiology and Biotechnology
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• 제18권2호
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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.