• Korean Journal of Food Science and Technology
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• v.12 no.1
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• pp.45-52
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• 1980

The nylon (poly 6, 10) microcapsules containing ${\beta}-galactosidase$ were obtained by the interfacial polymerization of 1, 6-diaminohexane and sebacoyl chloride with ${\beta}-galactosidase$ from Escherichia coli. They were generally spherical and had a mean diameter of $80{\mu}$ with 45 % of the activity recovery. In particular, there was no transport hamper of lactose through the membrane of microcapsules. The characteristics of the microencapsulated enzyme were similar to those of soluble enzyme optimal pHs, $7.0{\sim}7.2$ for the soluble and $7.3{\sim}7.5$ for the microencapsulated ; optimal temperatures, $50^{\circ}C$ for both ; apparent $K_m,\;3.33{\times}10^{-4}(on ONPG),$ $2.86{\times}10^{-3}$ M(on lactose) for the soluble and $5.28{\times}10^{-4}$ (on ONPG), $4.25{\times}10^{-3}$ M (on lactose) for the microencapsulated ; activation energies, 8.94 for the soluble and 9.78 Kcal/mole for the microencapsulated enzyme. Using this microencapsulated ${\beta}-galactosidase$, hydrolyses of lactose and milk lactose were carried out and 80 % of 5 % lactose solution and 70 % of lactose in skim milk were hydrolyzed in 40 hr at $27^{\circ}C$. The reusability and operational stability showed that the remaining activity was 50 % of the original activity after 5 runs and 120 hr of total operating time at $27^{\circ}C$.

• Food Science and Preservation
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• v.11 no.3
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• pp.417-423
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• 2004

Botanical antimicrobial agent-grapefruit seed extract mixture (BAAG) have an unknown compounds which exhibit the antibiotic activities aganist microorganisms including bacteria and fungi. We have examined the effects of BAAG on the physiological function of Botrytis cinerea which was isolated from necrotic lesions of decayed fruits and vegetables such as cucumbers, grapes, tomatoes, and red peppers during storage. In the results of enzymatic activities related to the energetic metabolism there was no inhibitory effect of BAAG on the activities of several enzymes in vitro including glucose 6-phosphate dehydrogenase and malate dehydrogenase, while there was inhibitory effect of BAAG on the activities of hexokinase and succinate dehydrogenase. O-nitrophenyl-$\beta$-D-galactopyranoside(ONPG), the artificial substrate of $\beta$-galactosidase was hydrolyzed in the presence of BAAG, indicating that the membrane was pertubated by the BAAG. From the results we suggested that the antibiotic activity of BAAG is due to the change of membrane permeability of the cell. BAAG was fractionated and purified by silica gel and sephadex column chromatography. Among active fractions two peaks were identified as naringin and limonin when they were analyzed by by NMR and Fast atomic bombardment.

• Journal of Life Science
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• v.26 no.5
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• pp.503-508
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• 2016

Biosensors have been used as first-step monitoring tools to detect on-site samples in a simple and cost-effective manner. Numerous recombinant microbial biosensors have been exploited for monitoring on-site toxic chemicals and biological signals. Herein, a recombinant microbial biosensor was constructed for monitoring cadmium. The cadmium responding cadC regulatory gene and it’s promoter from Staphylococcus aureus was amplified through PCR, fused with the lacZ gene, and transformed into Escherichia coli BL21 (DE3) cells. In the presence of cadmium, the biosensor cells express β-galactosidase showing red color development with chlorophenol red β-galactopyranoside (CPRG) as the enzymatic substrate. The biosensor cells showed the best β-galactosidase activity after 3 hr induction with cadmium at pH 5 and a detection range from 0.01 μM to 10 mM cadmium with a linearity from 0.01 to 0.1 μM cadmium (y = 0.98 x + 0.142, R² = 0.98). Among the heavy metals, cadmium and lead showed good responses, tin and cobalt showed medium responses, and mercury and copper showed no responses. The biosensor cells showed good responses to several waste waters similar to buffer solution, all spiked with cadmium. The biosensor described herein could be applied for on-site cadmium monitoring in a simple and cost-effective manner without sample pretreatments.

• Applied Biological Chemistry
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• v.46 no.4
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• pp.299-304
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• 2003

A strain YB-9 was isolated from soil as a producer of the extracellular ${\beta}-D-galactosidase$, which catalyzes the hydrolysis of lactose. The strain YB-9 was identified as Streptomyces sp. on the basis of its cultural, morphological and physiological properties. After treating culture supematant of the isolate with ammonium sulfate $(15{\sim}70%)$, the precipitated protein was used as a crude ${\beta}-galactosidase$ for analyzing its reaction properties with $para-nitrophenyl-{\beta}-D-galactoside$ $(pNP-{\beta}Gal)$ and lactose as substrates. The {\beta}-galactosidase showed its maximal activity at pH $6.0{\sim}6.5$ and $60^{\circ}C$. The hydrolyzing activity of ${\beta}-galactosidase$ for both $pNP-{\beta}Gal$ and lactose was decreased by galactose. Its hydrolyzing activity for lactose was slightly decreased by glucose, but the activity for $pNP-{\beta}Gal$ was increased to 1.3-folds by glucose. Especially, its hydrolyzing activity was not affected for lactose and was increased to 1.6-folds for $pNP-{\beta}Gal$ by xylose.

• Applied Biological Chemistry
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• v.26 no.4
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• pp.217-221
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• 1983

$\beta$-Galactosidase($\beta$-D-galactoside galactohydrolase, E.C. 3. 2. 1. 23) from E. coli K-12 CSH 36 was immobilized on porous cellulose beads which were previously activated with tannin and p-benzoquinone. Their general properties and applicational possibities were investigated. The most effective, enzyme immobilization was obtained when tannin and p-benzoquinone, pH 11.0, were used together as activation reagents and a period of 6 hours of activation. The optimum pH of $\beta$-galactosidase was 5.5 for free enzyme and 6. 0 for the immobilized enzyme, the optimum temperatures for native and immobilized enzyme were both $50^{\circ}C$. Kms of native $\beta$-galactosidase and immobilized enzyme for ONPG(o-nitrophenyl galactopyranoside) were about $4.0{\times}10^(-4)M$ and $7.5{\times}10^(-4)M$, respectively. In the case of tannin : p-benzoquinone activated cellulose beads, the immobilized enzyme retained over 80% of the initial enzyme activity after 20 runs, which is very promising result far a possible industrial application.

• Journal of Microbiology and Biotechnology
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• v.28 no.4
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• pp.606-612
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• 2018

The enzyme xylose isomerase (E.C. 5.3.1.5, XI) is responsible for the conversion of an aldose to ketose, especially xylose to xylulose. Owing to the ability of XI to isomerize glucose to fructose, this enzyme is used in the food industry to prepare high-fructose corn syrup. Therefore, we studied the characteristics of XI from Anoxybacillus kamchatkensis G10, a thermophilic bacterium. First, the gene coding for XI (xylA) was inserted into the pET-21a(+) expression vector and the construct was transformed into the Escherichia coli competent cell BL21 (DE3). The expression of recombinant XI was induced in the absence of isopropyl-thio-${\beta}$-galactopyranoside and purified using Ni-NTA affinity chromatography. The optimum temperature of recombinant XI was $80^{\circ}C$ and measurement of the heat stability indicated that 55% of residual activity was maintained after 2 h incubation at $60^{\circ}C$. The optimum pH was found to be 7.5 in sodium phosphate buffer. Magnesium, manganese, and cobalt ions were found to increase the enzyme activity; manganese was the most effective. Additionally, recombinant XI was resistant to the presence of $Ca^{2+}$ and $Zn^{2+}$ ions. The kinetic properties, $K_m$ and $V_{max}$, were calculated as 81.44 mM and $2.237{\mu}mol/min/mg$, respectively. Through redundancy analysis, XI of A. kamchatkensis G10 was classified into a family containing type II XIs produced by the genera Geobacillus, Bacillus, and Thermotoga. These results suggested that the thermostable nature of XI of A. kamchatkensis G10 may be advantageous in industrial applications and food processing.

• Journal of Microbiology and Biotechnology
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• v.22 no.11
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• pp.1532-1539
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• 2012

The ${\alpha}$-galactosidase-coding gene agaAJB13 was cloned from Sphingomonas sp. JB13 showing 16S rDNA (1,343 bp) identities of ${\leq}97.2%$ with other identified Sphingomonas strains. agaAJB13 (2,217 bp; 64.9% GC content) encodes a 738-residue polypeptide (AgaAJB13) with a calculated mass of 82.3 kDa. AgaAJB13 showed the highest identity of 61.4% with the putative glycosyl hydrolase family 36 ${\alpha}$-galactosidase from Granulicella mallensis MP5ACTX8 (EFI56085). AgaAJB13 also showed <37% identities with reported protease-resistant or Sphingomonas ${\alpha}$-galactosidases. A sequence analysis revealed different catalytic motifs between reported Sphingomonas ${\alpha}$-galactosidases (KXD and RXXXD) and AgaAJB13 (KWD and SDXXDXXXR). Recombinant AgaAJB13 (rAgaAJB13) was expressed in Escherichia coli BL21 (DE3). The purified rAgaAJB13 was characterized using p-nitrophenyl-${\alpha}$-D-galactopyranoside as the substrate and showed an apparent optimum at pH 5.0 and $60^{\circ}C$ and strong resistance to trypsin and proteinase K digestion. Compared with reported proteaseresistant ${\alpha}$-galactosidases showing thermolability at $50^{\circ}C$ or $60^{\circ}C$ and specific activities of <71 U/mg with or without protease treatments, rAgaAJB13 exhibited a better thermal stability (half-life of >60 min at $60^{\circ}C$) and higher specific activities (225.0-256.5 U/mg). These sequence and enzymatic properties suggest AgaAJB13 is the first identified and characterized Sphingomonas ${\alpha}$-galactosidase, and shows novel protease resistance with a potential value for basic research and industrial applications.

• Microbiology and Biotechnology Letters
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• v.11 no.3
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• pp.205-210
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• 1983

Extracellular $\beta$-galactosidase was prepared from a culture of Lactobacillus sporogenes, a spore-forming lactic acid bacterium. The enzyme functioned optimally at pH 6.8 and at 6$0^{\circ}C$ o-nitrophenyl-$\beta$-D-galactopyranoside (ONPG) in 0.05M sodium phosphate buffer. The activation energy of the enzymatic hydrolysis of ONPG was about 16,000 cal/mole below $50^{\circ}C$ and 11,300 cal/mole above the temperature. It was fairly stable over a pH range from 4.0 to 8.0 losing only less than 30% of its activity after hearting at 6$0^{\circ}C$ and pH 6.8 for 3 hours. Metal ions showed no significant effect on the enzyme activity, whereas L-cysteine exerted a slight stimulatory effect at the concentration of 10mM. The km values were 1.48mM for ONPG and 64.5mM for lactose. Hydrolysis of ONPG by the enzyme was product-inhibited by galactose (Ki=13.3mM, competitive inhibition) and by glucose(Ki= 11.4mM, uncompetitive type). The enzyme activity was also noncompetitively inhibited in the presence of lactose (Ki= 17.8mM).

• Korean Journal of Food Science and Technology
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• v.27 no.6
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• pp.985-990
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• 1995

Grapefruit seed extracts(GFSE) have some unknown compounds which exhibit the antibiotic activities aganist microorganisms including bacteria and fungi. We have examined the effects of GFSE on the growth of Enterobacter pyrinus which was isolated from necrotic lesions of pear trees. During the cultivation, the growth of the bacteria was strongly inhibited at the low concentration(0.01%, w/w) of GFSE. Hydrophobic fraction extracted from GFSE by mixed solvents (chloroform : methanol : water, 1 : 2 : 0.8, v/v/v) had components which inhibited the growth of bacteria. There was, however, no inhibitory effect of GFSE on the activities of several enzymes including hexokinase, glucose 6-phosphate dehydrogenase, malate dehydrogenase and succinate dehydrogenase. $O-nitrophenyl-{\beta}-D-galactopyranoside(ONPG)$, the artificial substrate of ${\beta}-galactosidase$ was hydrolyzed in the presence of GFSE, indicating that the membrane was pertubated by the GFSE. From the results it was suggested that the antibiotic activity of GFSE is due to the change of membrane permeability of cell. GFSE was fractionated by high performance liquid chromatography equipped with $C_{18}$ reverse phase column. Among active fractions, three peaks were identified as 1-chloro-2-methyl-benzene (o-toluene), N,N-dimethyl-benzenemethaneamine, 1-[2-(2-ethylethoxy)ethoxy]-4- (1,1,3,3-tetramethyl)-bezene, respectively, while the other three remained unidentified.

• 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.