• Microbiology and Biotechnology Letters
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• v.25 no.2
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• pp.144-150
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• 1997

After DNA damage, umuDC is the only SOS operon that must be induced to promote SOS mutagenesis in Escherichia coli. The recombinant plasmid pBC401 and pBC402 were constructed to fuse the lac structural genes with promoter region of umuDC operon to induce the expression of lacZ gene by DNA damage. We transformed the plasmid pBC401 and pBC402 into E. coli MC1061, lacZ deleted strain and determined the activity of $\beta$-galactosidase for various mutagen; UV, mitomycin C (MMC), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitroqunoline-1-oxide (NQO), ethyl methanesulfonate (EMS). The $\beta$-galactosidase activities of PBC401 and pBC402 for UV, MMC, and NQO were increased in proportion to expression time until 3 hours thereafter, the activities were constant or slightly decreased. The activities for MNNG and EMS were not so high as for UV, MMC, and NQO. When MNNG and EMS were treated, $\beta$-galactosidase activity of pBC402 was slightly lower than pBC401 but when UV, MMC, and NQO were treated in pBC402, $\beta$-galactosidase activity was slightly higher than in pBC401. Therefore, the pBC402 was better than the pBC401 in terms of sensitivity for frameshift mutagen. We suggest that the plasmid pBC401 and pBC402 are easy to detect mutagens which cause frameshift mutation rather than point mutation.

• Analytical Science and Technology
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• v.5 no.4
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• pp.465-469
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• 1992

An analytical procedure is presented for the quantitative determination of lactose, glucose, and galactose in the hydrolyzate of lactose by ${\beta}$-galactosidase with high-performance liquid chromatography. An Aminex HPX-87C column at $85^{\circ}C$ and refractive index detector were used to resolve lactose, glucose, and galactose in only 12 minutes with distilled and deionized water as a mobile phase. The validity of high-performance liquid chromatography as a method for the assay of ${\beta}$-galactosidase was supported by recovery experiments and comparision of results with those by ONPG method, a spectrophotometric assay. The procedure was appropriate for determination of sugars in the enzyme reaction mixture and could by applied to analysis of ${\beta}$-galactosidase activity.

• Journal of Microbiology and Biotechnology
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• v.21 no.3
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• pp.236-242
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• 2011

A psychrotrophic bacterium, Arthrobacter sp. ON14, isolated from Antarctica, was shown to exhibit a high ${\beta}$-galactosidase activity at a low temperature. A genomic library of ON14 was constructed and screened for ${\beta}$-galactosidase genes on functional plates containing 5-bromo-4-chloro-3-indolyl-${\beta}$-D-galactopyranoside (X-gal) as the substrate. Two different ${\beta}$-galactosidase genes, named as galA, galB, were found in ON14. Computational analyses of the genes revealed that the encoded protein GalA belongs to family 2 of glycosyl hydrolysases and is a cold-active protein, whereas GalB belongs to family 42 of glycosyl hydrolysases and is a mesophilic protein. Reverse transcription analyses revealed that the expression of galA is highly induced at a low temperature ($4^{\circ}C$ ) and repressed at a high temperature ($28^{\circ}C$ ) when lactose is used as the sole carbon source. Conversely, the expression of galB is inhibited at a low temperature and induced at a high temperature. The purified GalA showed its peak activity at $15^{\circ}C$ and pH 8. The mineral ions $Na^+$, $K^+$, $Mg^{2+}$, and $Mn^{2+}$ were identified as enzyme activators, whereas $Ca^{2+}$ had no influence on the enzyme activity. An enzyme stability assay revealed that the activity of GalA is significantly decreased when it is incubated at $45^{\circ}C$ for 2 h, and all its activity is lost when it is incubated at $50^{\circ}C$.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.758-761
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• 2001

In this study, the nar promoter of E. coli was characterized to see whether the nar promoter cloned onto pBBR122 can be used as an expression promoter of gram negative microbes. For this purpose, a plasmid with lacZ gene expressing ${\beta}-galactosidase$ instead of the structural genes of nar operon in a gram negative host strain(Agrobacterium.tumefaciens) was used to simplify an assay of induction of the nar promoter. The following effects were investigated to find optimal conditions: methods of inducing the nar promoter, optimal nitrate concentration, maximally inducing the nar promoter, the amount of expressed ${\beta}-galactosidase$ and induction ratio(specific ${\beta}-galactosidase$ activity after maximal induction/specific ${\beta}-galactosidase$ activity before induction). The following results were obtained from the experiments: the growth of Agrobacterium with E.coli nar promoter was not much affected by nitrate concentration in the shake-flask; induction of nar promoter was optimal when Agrobacterium was grown in the presence of 1% nitrate ion at the beginning of culture and when overnight culture was completely grown in the shake-flask before being transferred to other shake-flask; the amount of ${\beta}-galactosidase$ per cell and per medium volume was maximal when Agrobacterium was grown under aerobic condition to $OD_{600}$ of 1.7; then the nar promoter was induced under microaerobic and anaerobic condition made by lowering dissolved oxygen level(DO). After 2-3h of induction in the YEP medium selected as a main culture medium, the specific ${\beta}-galactosidase$ activity became about 17,000 Miller units in the fermentor cluture.

• Biotechnology and Bioprocess Engineering:BBE
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• v.9 no.2
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• pp.118-126
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• 2004

Recombinant E.coli ACV 1003 (recA::lacZ) releasing ${\beta}$-galactosidase by a SOS regulon system, when exposed to DNA-damaging compounds, have been used to effectively monitor endocrine disruptors. Low enzyme activity of less than 10 units/mL, corresponding to a $\mu\textrm{g}$/L(ppb) range of an endocrine disruptor (tributyl tin, bisphenol A. etc.), can be rapidly determined, not by a conventional time-consuming and tedious enzyme assay, but by an alternative interferometric biosensor. Heavily boron-doped porous silicon for application as an interferometer, was fabricated by etching to form a Fabry-Perot fringe pattern, which caused a change in the refractive index of the medium including ${\beta}$-galactosidase. In order to enhance the immobilization of the porous silicon surface, a calyx crown derivative (ProLinker A) was applied, instead of a conventional biomolecular affinity method using biotin. This resulted in a denser linked formation. The change in the effective optical thickness versus ${\beta}$-galactosidase activity, showed a linear increase up to a concentration of 150 unit ${\beta}$-galactosidase/mL, unlike the sigmoidal increase pattern observed with the biotin.

• Microbiology and Biotechnology Letters
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• v.24 no.1
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• pp.72-76
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• 1996

To investigate the expression efficiency of new transfer vector of Bombyx mori nuclear polyhedrosis virus (BmNPV), Escherichia coli lacZ gene was inserted into new transfer vector pBmKSK1, under the control of polyhedrin promoter and expressed in BmN-4 cells and larvae of silkworm, Bombyx mori. The recombinant virus containing lacZ gene was isolated from BmN-4 cells coinfected with transfer vectro pBmKSK1-LacZ and wild type BmNPV genome, and analysed by Southern blotting. The expression of ${\beta}$-galactosidase was characterized by SDS-PAGE, Western blotting and ${\beta}$-galactosidase activity assay. The results showed that the level of expression in silkworm larvae was higher than that of BmN-4 cells.

• Journal of Microbiology
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• v.45 no.6
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• pp.583-589
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• 2007

The promoter region of pyrC (dihydroorotase) gene of Escherichia coli was shown to have Fur protein binding properties by gel retardation assay. In vivo regulation of the pyrC expression was studied by measuring dihydroorotase activity and ${\beta}$-galactosidase level in the $fur^+$ and $fur^-$ genetic background. The expression of chromosomal dihydroorotase activity and ${\beta}$-galactosidase activity of pyrC-lacZ fusion plasmid was repressed to about 30% and 17%, respectively in the $fur^+$ strain compared to those in the $fur^-$ strain. Divalent ions such as $Fe^{2+}$ and $Zn^{2+}$ were not required for the repression. PyrC expression was also reduced to one half by 1 mM uracil. The effect of uracil was independent on the fur gene.

• YAKHAK HOEJI
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• v.44 no.2
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• pp.197-203
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• 2000

The enzymatic properties of $\beta$-galactosidases from Aspergillus oryzae, bovine liver and Saccharomyces pragilis have been studied using enzyme-linked lectin assay based on the RC $A_{120}$ and BS-II lectins which specifically bind to terminal galactose and GlcNAc residue, respectively. Asialofetuin, a monomeric glycoprotein with approximately 48 kDa in molecular weight, was used as a substrate. This glycoprotein contains three N-linked triantennary complex type carbohydrate chains with each of which terminating in Ga1$\beta$P1 longrightarrow4G1cNAc (74%). Their optimal pHs were 3.5 and 6.5 (A. oryzae), and 3.5~5.5 (bovine liver and S. fragilis) at 37$^{\circ}C$ during 24 hrs, and the effective concentrations were 0.9, 2.9, and 1.7 mg/ml, respectively The enzyme from A oryzae requires 100 mM N $a^{+}$ or $K^{+}$, while the enzyme from bovine liver requires $Ba^{2+}$ for activity. However all of the three $\beta$-galactosidases were inactivated by SDS and C $u^{2+}$. These results indicate that the hydrolysis of glycoprotein such as asialofetuin depends on the reaction conditions of $\beta$-galactosidases and some metal ions. ions.

• Toxicological Research
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• v.17 no.1
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• pp.65-71
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• 2001

The key targets of endocrine disruptors are nuclear hormone receptors, which bind to steroid hormones and regulate their gene transcription. A yeast-based steroid hormone receptor gene trascription assay was previously developed for the evaluation of chemicals with endocrine modulating activity. The yeast transformants used in this assay contain the human estrogen receptor along with the appropriate steroid response elements upstream of the $\beta$-galactosidase reporter gene. We tried to evaluate several natural and synthetic steroids of their potential to interact directly with the steroid receptor. Some putative endocrine disruptors, including nonylphenol, are weakly estrogenic. But the combined treatment oj these chemicals with di-(2-ethylhexyl)phthalate (DEHP) significantly increased the $\beta$-galactosidase activity in the yeast transformant. These results suggest that we also have to consider the synergistic effects of endocrine disruptors. In this study, we showed that yeast-based bioassay is a valuable tool for screening potential endocrine disruptors and quantitative determination of estrogenicity. And the possibility that the estrogen receptor binds multiple environmental chemicals adds another level of complexity to the interaction between the endocrine disruptors and the human hormone system.

• Food Science and Biotechnology
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• v.16 no.5
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• pp.778-782
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• 2007

This study assessed the effects of the antimicrobial substances sulforaphane, grapefruit seed extracts (GSE), and reuterin on the expression of Salmonella HilA and InvF virulence gene using a LacZY assay (${\beta}$-galactosidase assay) with hilA:lacZY and invF:lacZY fusion strains of Salmonella typhimurium SL1344. Salmonella was grown for 8 hr at $37^{\circ}C$ in the presence of diluted antimicrobial substances ($2\;{\mu}g/mL$ sulforaphane, $20\{\mu}g/mL$ GSE, and 0.26 mM reuterin) at concentrations that did not inhibit the cellular growth of Salmonella. Sulforaphane inhibited the expression of HilA and InvF by 50-90 and 20-80%, respectively. GSE also inhibited the expression of both genes, but to a lesser degree. Among the 3 antimicrobial substances, reuterin showed the least inhibition, which was abolished after 3-4 hr. None of the antimicrobial substances inhibited the ${\beta}$-galactosidase enzyme activity of S. typhimurium. The assay used in this study represents a very sensitive method for screening bioactive substances that inhibit the expression of virulence genes in Salmonella.