• The Korean Journal of Food And Nutrition
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• v.12 no.1
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• pp.63-68
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• 1999

Production of galactooligosaccharides by an immobilized $\beta$-galactosidase from Aspergillus niger CAD 1 in sodium alginate was investigated. The ranges of temperature and pH for the maximum stability of im-mobilized $\beta$-galactosidase were 20~45$^{\circ}C$ and 4.0~5.5, respectively. The activation energy for the immob-illized $\beta$-galactosidase was 13,400 cal/mole At the concentration of the immobilized $\beta$-galactosidase 0.12 unit/g in sodium alginate the yield of galactooligosaccharides in cheese whey containing 20% lactose was 18% after incubation for 72 hr at 45$^{\circ}C$. The remaining activity for the immobilized $\beta$-galactosidase 10 times repeated use 87%.

• 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.

• Journal of Microbiology and Biotechnology
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• v.11 no.1
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• pp.106-111
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• 2001

A ${\beta}-galactosidase$ gene of Bifidobacterium adolescentis Int57 (INT57) was cloned using the shotgun method. The sequence of the ${\beta}-galactosidase$ gene existing in the sequenced 3,260-bp fragment showed higher than 40% homology with other bacterial ${\beta}-galactosidase$ genes. The expression in Escherichia coli suggested that the ${\beta}-galactosidase$ might have a monomeric, dimeric, or tetrameric protein structure. This is probably the first peer-reviewed sequence analysis of the ${\beta}-galactosidase$ gene of the genus Bifidobacterium.

• 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 $Fe^{2+}$.

• Microbiology and Biotechnology Letters
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• v.18 no.6
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• pp.566-570
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• 1990

Regulation of $\beta$ -galactosidase formation was studied with Lactobacillus sporogenes. Synthesis of the enzyme was effectively induced by isopropyl- $\beta$-D-thiogalactopyranoside (IPTG) or galactose, and to a much lower level by lactose. When 15 mM glucose was added at the different intervals to the cultures that had been in contact with IPTG, the same levels of inhibition of the enzyme synthesis were observed (approximately one-third the differential rate of a control culture without glucose). This suggests that glucose did not interfere with the entry of the inducer into the cells, but interfere with the formation of $\beta$ -galactosidase through catabolite repression. The glucose inhibitory effect was not overcome by adding CAMP or cGMP to the culture media.

• 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.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.12
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• pp.1751-1757
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• 2004

Recombinant heterologous proteins can be produced as insoluble aggregates partially or perfectly inactive in Escherichia coli. One of the strateges to improve the solubility of recombinant proteins is fusion with a partner that is excellent in producing soluble fusion proteins. To improve the production of soluble $\beta$-galactosidase, the gene of Thermus thermophilus KNOUC112 $\beta$-galactosidase (KNOUC112 $\beta$-gal) was fused with thioredoxin gene, and optimization of its expression in E. coli TOP10 was performed. KNOUC112 $\beta$-gal in pET-5b was isolated out, fused with thioredoxin gene in pThioHis C, and transformed to E. coli TOP10. The $\beta$-galactosidase fused with thioredoxin was produced in E. coli TOP10 as dimer and trimer. The productivity of fusion $\beta$ -galactosidase expressed via pThioHis C at 37$^{\circ}C$ was about 5 times higher than that of unfused $\beta$-galactosidase expressed via pET-5b at 37$^{\circ}C$. Inclusion body of $\beta$-galactosidase was formed highly, regardless of the induction by IPTG when KNOUC112 $\beta$ -gal was expressed via pET-5b at 37$^{\circ}C$. Fusion $\beta$ -galactosidase expressed at 37$^{\circ}C$ via pThioHis C without the induction by IPTG was soluble, but the induction by IPTG promoted the formation of inclusion body. Lowering the incubation temperature for the expression of fusion gene under 25$^{\circ}C$ prevented the formation of inclusion body, optimally at 25$^{\circ}C$. 0.07 mM of IPTG was sufficient for the soluble expression of fusion gene at 25$^{\circ}C$. The soluble production of Thermus thermophilus KNOUC112 $\beta$-galactosidase could be increased about 10 times by fusion with thioredoxin, and optimization of incubation temperature and IPTG concentration for induction.

• 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.

• Korean Journal of Microbiology
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• v.27 no.3
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• pp.181-187
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• 1989

Plasmid DNA was isolated from Lactobacillus casei SW-M1($Lac^{+}$strain). The curing frequencies of pPLac plasmid from L. casei SW-M1 showed 43% for acriflavin treatment and 53% for ethidium bromide treatment after 3 times transfer. On the charaterization of pPLac plasmid, it was found that the plasmid contained gene encoding phospho-$\beta$-galactosidase for lactose utilization. Lactose-PTS(phosphotransferase system)was involved in membrane transport system in $Lac^{+}$ strain. Induction of phospho-$\beta$-galactosidase was specially effective by galactose, lower effect with lactose and glucose but not by IPTG(isopropyl-$\beta$-D-thiogalactoside). This result showed that induction of phospho-$\beta$-galactosidase by IPTG did not appeared. The catabolite repression of phospho-$\beta$-galactosidase synthesis by glucose was not found in L. casei.

• Microbiology and Biotechnology Letters
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• v.31 no.2
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• pp.151-156
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• 2003

A strain YB-10 was isolated from soil as a producer of the extracellular $\beta$-D-galactosidase, which catalyzes the hydrolysis of lactose. The strain YB-10 was identified as Streptomyces sp. on the basis of its cultural, morphological and physiological properties. After treating culture supernatant of the isolate with ammonium sulfate, the precipitated protein was used as a crude $\beta$-galactosidase for analyzing its reaction properties with para-nitrophenyl-$\beta$-D-galactosidase(pNP-$\beta$Gal) as a substrate. The $\beta$-galactosidase showed its maximal activity at pH 6.0 and 6$0^{\circ}C$. The enzyme was also active on lactose. The hydrolyzing activity of $\beta$-galactosldase for pNP-$\beta$Gal and lactose was decreased by galactose. Its hydrolyzing activity far lactose was also decreased by glucose, but the activity for pNP-$\beta$Gal was increased to 1.8-folds by glucose.