• Journal of Microbiology and Biotechnology
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• v.10 no.5
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• pp.663-669
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• 2000

The fur (ferric uptake regulation) expression vector pMON2064 was modified to produce a Fur-fusion expression vector. A kinker site, factor Xa cleavage site, and several restriction endonuclease sites were introduced to facilitate easy cloning and isolating of the fusion protein. The resulting fusion expression vector, pMONSTER, was then used to make fusion expression vector, pMONSTER, was then used to make fusion proteins with $\beta$-galactosidase and the protease of the human immunodeficiency virus type 1 (HIV-1 PR). Strain SW4020 harboring the Fur $\beta$-galactosidase fusion vector produced blue colonies on a 5-bromo-4-chloro-3-indolyl-$\beta$-D-galactoside plate and the resulting 133 kDa fusion protein reacted with an anti-Fur antibody. The strain harboring the Fur-HIV-1 PR fusion vector produced a 29 kDa fusion protein, which also reacted with an anti-Fur antibody. The Fur-HIV-1 PR fusion protein was purified by a single column application that was designed to isolate the Fur protein. The purified Fur-HIV-1 PR fusion protein digested with factor Xa cleaved a recombinant Gag protein to release smaller fragments, including a p24 capsid protein. The Fur-HIV-1 PR fusion protein itself did not exhibit any proteolytic activity.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.6
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• pp.989-993
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• 2007

A lactose-hydrolyzed milk with low sweetness was developed using nanofiltration. Raw milk was treated with 0.03% ${\beta}$-galactosidase at $4^{\circ}C$ for 24 h to hydrolyze lactose partially. The resultant lactose-hydrolyzed milk containing 0.43% lactose was then concentrated using a nanofiltration membrane to reach concentration factor of 2.13. The concentration factors and coefficients of retention of milk components in nanofiltration were determined. The concentration factor of milk fat was 2.20 which was the highest of the milk components. The coefficient of retention of calcium and riboflavin was 0.96 and 0.76, respectively. However, the coefficient of retention of glucose, galactose, and sodium was 0.21, 0.15, and 0.22, respectively. Raw milk was treated with 0.1% ${\beta}$-galactosidase at $4^{\circ}C$ for 40 h to hydrolyze lactose fully and then concentrated to reach a concentration factor of 1.6 by using nanofiltration. The concentrated milk was reconstituted with water. The lactose-hydrolyzed milk had sweetness similar to milk. The compositional ratios of crude protein, calcium, sodium, and riboflavin of lactose-hydrolyzed nanofiltrated milk to those of raw milk were 99%, 97%, 77%, and 80%, respectively. This study showed that nanofiltration of lactose-hydrolyzed milk to remove galactose and glucose did not cause significant loss of calcium. The lactose-hydrolyzed nanofiltrated milk contained 0.06% lactose and had sweetness similar to milk.

• Journal of Microbiology and Biotechnology
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• v.8 no.4
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• pp.318-324
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• 1998

A ${\beta}$-galactosidase with high transgalactosylic activity was purified from a Bacillus species, registered as KFCC10855. The enzyme preparation showed a single protein band corresponding to a molecular mass of 150 kDa on SDS-PAGE and gave a single peak with the estimated molecular mass of 250 kDa on Sephacryl S-300 gel filtration, suggesting that the enzyme is a homodimeric protein. The amino acid and sugar analyses revealed that the enzyme is a glycoprotein, containing 19.2 weight percent of sugar moieties, and is much more abundant in hydrophilic amino acid residues than in hydrophobic residues, the mole ratio being about 2:1. The pI and optimum pH were determined to be 5.0 and 6.0, respectively. Having a temperature optimum at $70^{\circ}C$ for the hydrolysis of lactose, the enzyme showed good thermal stability. The activity of the enzyme preparation was markedly increased by the presence of exogenous Mg (II) and was decreased by the addition of EDTA. Among the metal ions examined, the most severely inhibitory effect was seen with Ag (I) and Hg (II). Further, results of protein modification by various chemical reagents implied that 1 cysteine, 1 histidine, and 2 methionine residues occur in certain critical sites of the enzyme, most likely including the active site. Enzyme kinetic parameters, measured for both hydrolysis and transgalactosylation of lactose, indicated that the enzyme has an excellent catalytic efficiency for formation of the transgalactosylic products in reaction mixtures containing high concentrations of the substrate.

• Journal of Microbiology and Biotechnology
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• v.1 no.4
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• pp.274-280
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• 1991

Effects of IPTG induction on cell growth and production of ${\beta}$-galactosidase-preS2 fusion protein (${\beta}$gal-preS2) were studied in a defined medium using a recombinant Escherichia coli JM109/pCMHB30. IPTG was added (0.2 mM) to induce the cloned-gene expression in the early-, mid-, and late-log growth phases. The most serious decreases in growth rate and plasmid stability were observed for the induction in the early-log growth phase. The expression level of ${\beta}$gal-preS2 attained by the induction in the mid-log phase was about 0.51 mg fusion protein/mg total cellular protein, which was 2- and 5-fold improvement over the levels obtained with the inductions in the early- and late-log phases. Formation of acidic byproducts including acetate and pyruvate showed different profiles during the fermentation period for each cases of induction; pyruvate was the major byproduct for the induction in the early-log phase while acetate production became more significant for the cases of inductions in the mid- and late-log phases.

• Journal of Microbiology and Biotechnology
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• v.26 no.7
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• pp.1267-1277
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• 2016

Currently, enzymatic synthesis of lactulose, a synthetic prebiotic disaccharide, is commonly performed with glycosyl hydrolases. In this work, a new type of lactulose-producing biocatalyst was developed by displaying β-galactosidase from Bacillus stearothermophilus IAM11001 (Bs-β-Gal) on the surface of Bacillus subtilis 168 spores. Localization of β-Gal on the spore surface as a fusion to CotX was verified by western blot analysis, immunofluorescence microscopy, and flow cytometry. The optimum pH and temperature for the resulting spore-displayed β-Gal was 6.0 and 75℃, respectively. Under optimal conditions, it showed maximum activity of 0.42 U/mg spores (dry weight). Moreover, the spore-displayed CotX-β-Gal was employed as a whole cell biocatalyst to produce lactulose, yielding 8.8 g/l from 200 g/l lactose and 100 g/l fructose. Reusability tests showed that the spore-displayed CotX-β-Gal retained around 30.3% of its initial activity after eight successive conversion cycles. These results suggest that the CotX-mediated spore-displayed β-Gal may provide a promising strategy for lactulose production.

• Korean Journal of Microbiology
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• v.32 no.1
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• pp.12-19
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• 1994

KEM1 is known to control the spindle pole body or microtubule function, probably in response to the cellular nutritional conditions in Saccharomyces cerevisiae. Transposon insertions were performed in the cloned KEM1 gene using mini-Tn10-LUK element carrying E. coli ${\beta}$-galactosidase structural gene. A collection of ranfom Tn10-LUK insertions defined an approximately 3.5 kb region required for the KEM1 function. From this collection functional KEM1::lacZ protein fusions were identified. Indirect immunofluorescence using anti-${\beta}$-galacatosidase antibodies localized the KEM1::lacZ fusion protein to the periphery of the nucleus.

• Genomics & Informatics
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• v.1 no.2
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• pp.113-118
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• 2003

Among a number of antigens characterized in M leprae, an etiological agent of Leprosy, the 18 kDa antigen, is unique to M leprae. We have previously determined a sequence specific element in the 18 kDa gene of M leprae, which confers transcriptional repression. In this report, we have examined if the element could be applied to genes other than the 18 kDa gene of M leprae. To identify the roles of the regulatory sequence in heterologous promoter, we have constructed pB3 vector series, which contains BCG hsp65 promoter and the M leprae 18 kDa transcription repression responsive element in tandem using LacZ gene as a reporter gene. Cloning of hsp65 promoters of M bovis BCG or M smegmatis in front of LacZ gene resulted in normal $\beta$­galactosidase activity as expected. However, when the sequence element was placed between the promoter and the LacZ gene, $\beta$-galactosidase activity was reduced 10-fold less. Also we have examined with pB3(-) vector, that harbors the transcription repression responsive element in a reversed orientation, the $\beta$-galactosidase activity was found to be similar to pB3(+) vector. Thus, these results further confirm that M leprae 18 kDa transcription repression responsive element could regulate BCG hsp65 heterologous promoter and that the element could act as an operator for the transcription of mycobacteria.

• Food Science and Biotechnology
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• v.15 no.6
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• pp.908-913
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• 2006

Recombinant ${\beta}$-galactosidase from Bifidobacterium longum LL04 was expressed in Escherichia coli and partially purified by ammonium sulphate precipitation and anion-exchange chromatography (Mono-Q). The optimum temperature and pH of the partially purified enzyme were $50^{\circ}C$ and pH 7.0-8.0, respectively, when o-nitrophenyl-${\beta}$-D-galactopyranoside was used as a substrate. The enzyme was stable over the pH range of 5.0-9.0, and was active at $40^{\circ}C$ for more than 60 min at pH 7.0. The enzyme was significantly activated by $Na^+$ and $K^+$. Maximal activity was observed at the concentration of 10 mM for both $Na^+$ and $K^+$. The enzyme activity was strongly inhibited by most bivalent metal ions. The Km and Vmax on ONPG at 37 and $50^{\circ}C$ were 0.72, 167.9, and 0.507 mM, 310.9 U/mL, respectively.

• Asian-Australasian Journal of Animal Sciences
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• v.25 no.6
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• pp.852-860
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• 2012

An Antarctic bacterial isolate displaying extracellular ${\alpha}$-galactosidic activity was named Paenibacillus sp. LX-20 based on 16S rRNA gene sequence analysis. Optimal activity for the LX-20 ${\alpha}$-galactosidase occurred at pH 6.0-6.5 and $45^{\circ}C$. The enzyme immobilized on the smart polymer Eudragit L-100 retained 70% of its original activity after incubation for 30 min at $50^{\circ}C$, while the free enzyme retained 58% of activity. The enzyme had relatively high specificity for ${\alpha}$-D-galactosides such as p-nitrophenyl-${\alpha}$-galactopyranoside, melibiose, raffinose and stachyose, and was resistant to some proteases such as trypsin, pancreatin and pronase. Enzyme activity was almost completely inhibited by $Ag^+$, $Hg^{2+}$, $Cu^{2+}$, and sodium dodecyl sulfate, but activity was not affected by ${\beta}$-mercaptoethanol or EDTA. LX-20 ${\alpha}$-galactosidase may be potentially useful as an additive for soybean processing in the feed industry.

• KSBB Journal
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• v.21 no.4
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• pp.236-240
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• 2006

IPTG induction caused a sudden drop of alkali consumption rate during cultivation of a recombinant E. coli with ${\beta}$-galactosidase structural gene under T7 promoter on a plasmid. A series of batch cultivations showed the positive correlation of the decrease of alkali consumption and the level of expression. However, repeated IPTG induction did not cause any variation of alkali consumption rate. Supplementation of medium even at stationary phase enhanced the level of ${\beta}$-galactosidase expression. These results suggests that the drop of alkali consumption rate by IPTG induction represents the rate of expression.