• Title, Summary, Keyword: ${\alpha}$-galactosidase

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Enzymatic Studies on the α-Galactosidases from Soybean and Aspergillus niger (대두(大豆) 및 Aspergillus niger α-galactosidase의 효소학적(酵素學的) 연구(硏究))

  • Keum, Jong-Hwa;Oh, Man-Jin
    • Korean Journal of Agricultural Science
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    • v.18 no.1
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    • pp.49-73
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    • 1991
  • To elucidate enzymatic properties of $\alpha$-galactosidases (EC3, 2, 1, 22) from germinated soybean and Aspergillus niger changes in the enzyme activities and oligosaccharide contents during germination of soybean were determined and $\alpha$-galactosidases from germinated soybean and wheat bran culture of Aspergillus niger were purified by ammonium sulfate fractionation, ion exchange chromatography and gel filtration. Their chemical and enzymatic properties were investigated and the results obtained were summarized as follows : 1. $\alpha$-Galactosidase activity of soybean was maximized when it was germinated at $25^{\circ}C$ for 120 hours. And raffinose and stachyose in soybean were decomposed completely after 96 hours and 120 hours of germination, respectively. 2. The highest level of $\alpha$-Galactosidase activity was obtained when Aspergillus niger was grown on wheat bran medium at $30^{\circ}C$ for 96 hours. 3. Soybean $\alpha$-galactosidase was purified by 6.6 fold by ammonium slufate fractionation, ion exchange chromatography on DEAE-Cellulose and Sephadex A-50., and gel filtration on Sephadex G-150. Its specific activity was 825 units/mg protein and the yield was 2.5% of the total activity of crude extracts. 4. Aspergillus niger $\alpha$-galactosidase was purified by 23.7 fold. Its specific activity was 1,229 units/mg protein and the yield was 14% of the total activity of wheat bran culture. 5. The purified $\alpha$-galactosidases of soybean and Aspergillus niger were found to be homogeneous by polyacrylamide gel electrophoresis and by HPLC. 6. Chemical properties of the purified $\alpha$-galactosidases were : 1) The soybean $\alpha$-galactosidase was monomeric and its molecular weight was estimated to be 30,000 by SDS-PAGE whereas the Aspergillus niger $\alpha$-galactosidase was a tetrameric glycoprotein which consisted of identical subunits with molecular weight of 28,000 each.

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Kinetic Properties of $\alpha$-Galactosidase from Aspergillus niger ATCC 16513 and Soybean(Glycine max. L) (Aspergillus niger ATCC 16513과 대두(Glycine max. L) $\alpha$-galactosidase의 kinetic 성질)

  • Geum, Jong-Hwa;Lee, Jong-Su;Sin, Cheol-Seung
    • The Journal of Natural Sciences
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    • v.5 no.1
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    • pp.53-57
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    • 1992
  • This experiment was carried out to elucidate some kinetic properties of the $\alpha$-galactosidase which produced and purified from Aspergillus niger ATCC 16513 and soybean(Glycine max. L). The Km value of Asp. niger and soybean $\alpha$-galactosidase were 37.0mM and 50.0mM for raffinose and55.5mM and 55.5mM for stachyose, respectively. The activity of Asp. niger and soybean $\alpha$-galactosidase were inhibited by galactose. Among the amino acids in active sites of both Asp. niger and soybean $\alpha$-galactosidase, histidine was identified by chemical modification of diethyl pyrocarbonate. Number of amino acids residues per mole of Asp. niger and soybean $\alpha$-galactosidase were 902 and 286, respectively.

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Purification and Properties of $\alpha$-Galactosidase from Aspergillus niger (Aspergillus niger $\alpha$-Galactosidase의 정제 및 성질)

  • 금종화;오만진;김찬조
    • Microbiology and Biotechnology Letters
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    • v.19 no.5
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    • pp.477-486
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    • 1991
  • To elucidate enzymatic properties of a-glactosidase (EC 3.2.1.22) from Asp. niger, a-galactosidase from wheat bran culture was purified by ammonium sulfate fractionation, ion exchange chromatography and gel filtration. And then its enzymatic propeties were investigated. The highest level of $\alpha$-galactosidase activity was obtained when Asp. niger was grown on wheat bran medium at $30^{\circ}C$ for 96 hours. The $\alpha$-galactosidase was purified by 23.7 fold by ammonium sulfate fractionation, ion exchange chromatography on DEAE-Celluose and Sephadex A-50, and gel filtration on Sephadex G-150 and its specific activity was 1,229 Unitslmg protein and the yield was 14% of the total activity of wheat bran culture. The purified $\alpha$-galactosidase was found to be homogeneous by polyacrylamide gel electrophoresis and HPLC. The $\alpha$-galactosidase was a tetrameric glycoprotein which consisted of identical subunits with molelcular weight of 28,000 each by SDS-PAGE and isoelectric point was determined analytical isoelectric focusing to be pH 4.6. The optimal temperature and pH for the $\alpha$-galactosidase activity were $40^{\circ}C$ and pH 6.5, respectively, and 54% of its activity was lost by heating at $60^{\circ}C$ for 10 mins, It was appeared to have higher affinty to raffinose than to stachyose. The K, value and activation energy of $\alpha$-galactosidase were 5.0 mM and 8.515 Kcal per mole for p-nitrophenyl- $\alpha$--D-galactopyranoside, respectively.

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Detection of Bifidobacteria by ${\alpha}-Galactosidase$ activity (${\alpha}-Galactosidase$의 활력차이에 의한 Bifidobacteria의 선별)

  • Min, Hae-Ki;Lee, See-Kyung;Kang, Kook-Hee
    • Applied Biological Chemistry
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    • v.36 no.3
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    • pp.191-196
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    • 1993
  • This method using the synthesis substrate of $5-bromo-4-chloro-3-indolyl-{\alpha}-galactoside\;(X-{\alpha}-Gal)$ was examined for the differential enumeration of Bifidobacteria and lactic acid-producing bacteria. Bifidobacteria possess a high level of ${\alpha}-galactosidase$ activity. Bifidobacterium longum KCTC 3215 exhibited the highest ${\alpha}-galactosidase$ specific activity (8.57 units/mg protein). Determination of ${\alpha}-galactosidase$ activity using the PNPG procedure showed that Lactobacillus, Streptococcus, Pediococcus, and Leuconostoc strain had lower ${\alpha}-galactosidase$ activity as compared to Bifidobacteria. The $X-{\alpha}-Gal$ based medium is useful to identify Bifidobacteria among lactic acid-producing bacteria since the enzyme action of ${\alpha}-galactosidase$ spills $X-{\alpha}-Gal$ substrate and releases indol which impacts a blue color to Bifidobacterial colonies on agar plates. All strains of Bifidobacteria appeared as blue colonies on MRS agar medium supplemented with $100\;{\mu}M\;X-{\alpha}-Gal$ while colonies of other lactic acid-producing bacteria appeared white or light blue.

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Characterization of Extracellular $\alpha$-Galactosidase Produced by Bacillus licheniformis YB-42. ($\alpha$-Galactosidase를 생산하는 Bacillus lichennformis YB-42의 분리와 효소 특성)

  • 김현숙;이경섭;소재호;이미성;최준호;윤기홍
    • Microbiology and Biotechnology Letters
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    • v.32 no.2
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    • pp.128-134
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    • 2004
  • A bacterium producing the $\alpha$-galactosidase was isolated from Korean soybean paste. The isolate YB-42 has been identified as Bacillus licheniformis on the basis on its 16S rRNA sequence, morphology and biochemical properties. The $\alpha$-galactosidase activity was detected in both the culture supernatant and the cell extract of B. licheniformis YB-42. The partially purified extracellular $\alpha$-galactosidase was obtained from the culture supernatant by DEAE-Sepharose column and Q-Sepharose column chromatography. The enzyme showed the maximum activity for hydrolysis of para-nitrophenyl-$\alpha$-D-galactopyranoside (pNP-$\alpha$Gal) at pH 6.5 and $45^{\circ}C$. It was able to hydrolyze oligomeric substrates such as melibiose, raffmose and stachyose to liberate galactose residue, indicating that the a-galactosidase of B. licheniformis YB-42 hydrolyzed $\alpha$-1,6 linkage. The hydrolyzing activity of $\alpha$-galactosidase for both pNP-$\alpha$Gal and melibiose was dramatically decreased by galactose. Both glucose and mannose inhibited the activity for pNP-$\alpha$Gal less than galactose.

Characterization of Extracellular \alpha-galactosidase Produced by Streptomyces sp. YB-4. (균체외 \alpha-galactosidase를 생산하는 Streptomyces sp. YB-4의 분리 및 효소 특성)

  • 김소영;조기행;김창진;박동진;윤기홍
    • Microbiology and Biotechnology Letters
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    • v.30 no.4
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    • pp.332-338
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    • 2002
  • A strain YB-4 producing the extracellular $\alpha$-galactosidase was isolated from soil, and has been identified as Streptomyces sp. on the basis of its cultural, morphological and physiological properties. The partially purified $\alpha$-galactosidase was most active on paranitrophenyl-$\alpha$-D-galactopyranoside at pH 6.0 and 6$0^{\circ}C$. The enzyme retained 90% of its maximum activity between pH 4.0 and pH 10.0 after pre-incubation for 1 h. The enzyme was able to hydrolyze oligomeric substrates such as melibiose, raffinose and stachyose to liberate galactose residue, indicating that the $\alpha$-galactosidase of Steptomyces sp. YB-4 hydrolyzed $\alpha$-1,6 linkage.

Purification and Characterization of an α-D-Galactosidase from Grape Berry

  • Kang, Han-Chul;Kim, Tae-Su
    • Journal of Applied Biological Chemistry
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    • v.43 no.3
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    • pp.141-146
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    • 2000
  • Glycosidase activities were tested from the grape berries, Vitis labruscana B. Takasumi. Among various glycosidases, $\alpha$-D-galactosidase was found to be the most active in the flesh and other glycosidases were considerably active in the order of the following: $\alpha$-D-mannosidase>$\alpha$-D-glucosidase>$\beta$-D-glucosidase>$\beta$-D-galactosidase. In the seeds, $\alpha$-D-glucosidase activity was the highest and other glycosidases such as $\alpha$-D-galactosidase, $\beta$-D-glucosidase, and $\beta$-D-galactosidase were still significantly active. The $\alpha$-D-galactosidase in the grape flesh was purified over 83-folds through salting-out with $(NH_4)_2SO_4$ and a series of chromatographies employing Sephadex G-50, Octyl-Sepharose, Q-Sepha- rose, and Biogel P-100. The enzyme was a monomer of 45 kDs as determined through SDS-PAGE and Sephacryl S-200 chromatography. The purified enzyme showed a preference of $\alpha$-D-galactose to $\beta$-D-galactose as a substrate about 5.4 times. Sulfhydryl specific reagents such as N-ethylmaleimide and iodoacetamide significantly inhibited the enzyme activity to the extents of 48 and 52% of its initial activity, respectively. The optimumpH range of $\alpha$-D-galactosidase was around 6.5-7.0. The enzyme activity increased by 46% in the presence of 1mM $Fe^{2+}$.

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Purification and properties of soybean ${\alpha}-galactosidase$ (대두 ${\alpha}-galactosidase$의 정제 및 성질)

  • Keum, Jong-Hwa;Oh, Man-Jin;Kim, Seong-Yeol
    • Applied Biological Chemistry
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    • v.34 no.3
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    • pp.249-257
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    • 1991
  • To elucidate enzymatic properties of ${\alpha}-galactosidase$ (EC 3, 2, 1, 22) from germinated soybean, changes in the enzyme activities and oligosaccharide contents during germination of soybean were determined. ${\alpha}-Galactosidase$ from germinated soybean was purified by ammonium sulfate fractionation, ion exchange chromatography and gel filtration. Their chemical and enzymatic properties was investigated. ${\alpha}-galactosidase$ activity of sobeam was maximized when it was germinated at $25^{\circ}C$ for 120 hour. Raffinose and stachyose in soybean were decomposed completely after 96 hours and 120 hours of germination, respectively. Soybean ${\alpha}-galactosidase$ was purified by 6.6 fold by ammonium sulfate fractionation, ion exchange chromatography on DEAE-Cellulose and Sephadex A-50, and gel filtration on Sephadex G-150. Its specific activity was 825 Units/mg protein and the yield was 2.5% of the total activity of crude extracts. The purified ${\alpha}-galactosidase$ of soybean was found to be homogeneous by polyacrylamide gel electrophoresis and by HPLC. Isoelectric point of soybean ${\alpha}-galactosidase$ was determined analytical isoelectric focusing to be pH 4.8. The soybean ${\alpha}-galactosidase$ was monomeric and its molecular weight was estimated to be 30,000 by SDS-PAGE. The optimal temperature and pH for the soybeam ${\alpha}-galactosidase$ activity were $40^{\circ}C$ and pH 6.0 and 75% of its activity was lost by heating at $60^{\circ}C$ for 10 min. The enzyme was appeared to have higher affinity to raffinose than to stachyose. The Km value of soybean enzyme was 5.3 mM for ${\rho}-nitrophenyl-{\alpha}-D-galactopyranoside$ and the activation energy on PNPG was calculated to be 13.02 Kcal per mole.

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Comparison of Cultured Soymilk by Bifidobacterium and Various Human Intestinal Bacteria (Bifidobacterium과 기타 장내 세균에 의한 두유 배양 비교)

  • Lee, Se-Kyung;Son, Heon-Soo;Ji, Geun-Eog
    • Korean Journal of Food Science and Technology
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    • v.25 no.6
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    • pp.694-697
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    • 1993
  • Soymilk was cultured by various human large intestinal bacteria and lactic acid bacteria; Bifidobacterium longum, Lactobacillus acidophilus, Baeteroides fragilis, Eubacterium limosum, Clostridium perfringens and Escherichia coli. Among them, only B. longum utilized raffinose and stachyose actively which are major oligosaccharides present in soymilk by producing active ${\alpha}-galactosidase$ and produced greatest acid. Number of colony forming unit of B. longum reached $1.5{\times}10^{8}$ after 16 hr culture in soymilk. Also Bifidobacterium longum produced the highest level of ${\alpha}-galactosidase,\;{\beta}-galactosidase\;and\;{\alpha}-galactosidase$, in soymilk during culture.

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The Changes of $\alpha$-galactosidase Activities and Stachyose and Raffinose Contents During Fermentation of Soybeans (대두의 발효에 따른 $\alpha$-Galactosidase활성 및 Stachyose, Raffinose 함량 변화)

  • Kim, Sung-Soo;Yoon, Sun
    • Korean journal of food and cookery science
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    • v.14 no.5
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    • pp.509-512
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    • 1998
  • Changes in the contents of stachyose and raffinose were determined during soybean fermentation. ${\alpha}$-Galactosidase activities were also monitored in soybean and its fermented products. The stachyose contents were 31.8239 mg/g of soybean, 4.2217 mg/g of Meju, and 2.1184 mg/g of Doenjang. The raffinose contents were 2.6914 mg/g of soybean, 1.7413 mg/g of Meju, and negligible of Doenjang. ${\alpha}$-Galactosidase activities was distinct in soybean and Meju. They were 14.5954 units/mg protein of soybean, 13.1489 units/mg protein of Meju, and 1.9157 units/mg protein of Doenjang. The results suggested that the decrease of stachyose and raffinose contents in fermented soy products were due to the ${\alpha}$-galactosidase activity.

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