• Title/Summary/Keyword: $\beta$-galactosidase gene

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Molecular Cloning and Characterization of the ${\beta}-Galactosidase$ Gene from Bifidobacterium adolescentis Int57

  • Park, Myeong-Soo;Yoon, Hyeon-Jin;Rhim, Seong-Lyul;Ji, Geun-Eog
    • 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.

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Improving Soluble Expression of β-Galactosidase in Escherichia coli by Fusion with Thioredoxin

  • Nam, E.S.;Jung, H.J.;Ahn, J.K.
    • 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.

Molecular cloning of phospho-$\beta$-galactosidase gene of lactobacillus casei in escherichia coli (Lactobacillus casei의 phospho-$\beta$-galactosidase 유전자의 대장균내 분자클로닝)

  • 문경희;박정희;최순영;이유미;김태한;김연수;민경희
    • Korean Journal of Microbiology
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    • v.27 no.3
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    • pp.188-193
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    • 1989
  • Gene for lactose catabolism in Lactobacillus casei SW-M1 was encoded by a 60Kb metabolic plasmid. A derivative of only 10kb, pPlac 15 of recombinant plasmid, was constructed by introducing into pBR322 and was cloned into E. coli using restriction endonuclease Pst I. A 10kb insery DNA in plasmid pBR322 was identified as a gene encoded phospho-$\beta$-galactosidase by the determination of enzyme activity. Phospho-$\beta$-galactosidase was apparently expressed in E. coli. The enzyme activities of cell-free extract from transformant E. coli HB101 carrying pPLac 15 DNA were not different from that of L. casei as a donor strain on the basis of enzyme properites. However, specific activity of phospho-$\beta$-galactosidase in the cloned strain with Lac $Y^{-}$ phenotype of E. coli HB101 was lower than that in L. casei strain.

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Mapping of Gene Encoding Phospho-$\beta$-galactosidase from Lactobacillus casei and its Expression in Escherichea coli (Lactobacillus casei 의 Phospho-$\beta$-galactosidase 유전자의 지도작성과 Escherichia coli 내에서의 발현)

  • 박정희;문경희;민경희
    • Korean Journal of Microbiology
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    • v.30 no.6
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    • pp.539-545
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    • 1992
  • Recombinant plasmid pPLac15 determined both phosphoenolpyruvate-dependent phosphotransferase uptake of lactose and phospho-$\beta$-galactosidase (Moon et al., 1989). A restriction mapping of the pPLac15 was compiled with several restriction enzymes and a seriese of sub clones into pUC18 was constructed. From an analysis of the proteins produced by Escherichia coli cells of transformants containing each of the recombinant subclone plasmids, it was found that the gene for phospho-$\beta$-galactosidase in pUCI8 was expressed about 1.8-folds in E. coli.

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Cloning and Expression of Kluyveromyces fragilis $\beta$-Galactosidase Gene in Saccharomyces cerevisiae

  • Bang, Jeong-Hee;Nam, Doo-H.;Kang, Dae-Ook;Ahn, Jong-Seog;Ryu, Dewey-D.Y.
    • Journal of Microbiology and Biotechnology
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    • v.5 no.1
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    • pp.6-13
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    • 1995
  • A gene coding for the $\beta$-galactosidase (lactase) of Kluyveromyces tragilis UCD 55-55 was isolated by complementation in Escherichia coli YMC9. From the plasmid library made from Sau3A-digested chromosomal DNA, one positive clone was selected. The cloned gene for $\beta$-galactosidase was on 7.3 kilobase pair DNA fragment, and a slightly low level of $\beta$-galactosidase enzyme activity was detecied in E. coli. It was also confirmed that the cloned gene comes from K. tragilis by DNA-DNA hybridization and immunochemical blotting experiments. In order to construct a new yeast strain having the metabolic ability for lactose, the cloned gene for K. tragilis $\beta$-galactosidase was inserted in yeast vector YEp24 and YRp17, and transformed into Saccharomyces cerevisiae YNN27 and Ml-2B. The yeast transformants showed the nearly the same $\beta$-galactosidase productivity as level of K. tragilis when uninduced, but these could not utilize lactose as a sole carbon source, presumably due to the lack of lactose transport system. Nevertheless, a slightly higher ethanol productivity was achieved by these transformants than S. cerevisiae or K. tragilis, in the medium containing glucose and lactose.

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Phospho-$\beta$-galactosidase gene located on plasmid in lactobacillus casei (플라스미드에 존재하는 lactobacillus casei의 phospho-$\beta$-galactosidases 유전자)

  • 문경희;박정희;최순영;이유미;김태한;하영칠;민경희
    • 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.

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Molecular Cloning of a $\beta$-D-Galactosidase Gene from Lactococcus lactis subsp. lactis 7962

  • CHANG, HAE-CHOON;YANG-DO CHOI;HYONG-JOO LEE
    • Journal of Microbiology and Biotechnology
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    • v.6 no.6
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    • pp.386-390
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    • 1996
  • The ${\beta}$-galactosidase gene from Lactococcus lactis subsp. lactis ATCC 7962 was cloned and its enzymatic properties were characterized, with a view to assessing its potential use as a selection marker in the food-grade cloning vector. Chromosomal DNA from L. lactis subsp. lactis 7962 was cleaved with PstI and ligated into pBR322 for transformation into Escherichia coli TGl. Transformants showing ${\beta}$-galactosidase activity possessed the pBR322 plasmid containing a 10 kilobase (kb) PstI fragment and this plasmid was named pCKL11. The cloned ${\beta}$-galactosidase gene came from the chromosomal DNA of L. lactis subsp. lactis 7962 was confirmed by Southern hybridization. A restriction map of pCKL11 was constructed from the cleavage of both pCKL11 and the purified 10kb insert fraqment. The. optimum pH of the ${\beta}$-galactosidase determined with the E. coli harboring the pCKL11 was 7.0. The optimum temperature was $50^{\circ}C$, while the pI of the enzyme was 7.4. These values were the same as those of the enzyme from the parent strain.

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Molecular Cloning of $\beta$-Galactosidase from Bacillus subtilis HP-4

  • Kim, Jeong-Ho;Lee, Jae-Chang;Huh, Jeong-Won;Chung, Ki-Chul
    • Journal of Microbiology and Biotechnology
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    • v.1 no.4
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    • pp.227-231
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    • 1991
  • A gene coding for a $\beta$-galactosidase of Bacillus subtilis HP-4 was cloned in E. coli JM109 by inserting HindIII digested fragment of B. subtilis HP-4 chromosomal DNA into the site of pBR322 and selecting recombinant transformant showing blue color on X-gal plate. The recombinant plasmid, named pBG109, was found to contain the 1.4 Kbp HindIII fragment originated from B. subtilis HP-4 chromosomal DNA by Southern hybridization. The cloned gene was stably maintained and expressed in E. coli JM109 and the pBG109 encoded $\beta$-galactosidase had the same enzymatic properties as those of $\beta$-galactosidase produced by B. subtilis HP-4.

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β-Galactosidase Gene of Thermus thermophilus KNOUC112 Isolated from Hot Springs of a Volcanic Area in New Zealand: Identification of the Bacteria, Cloning and Expression of the Gene in Escherichia coli

  • Nam, E.S.;Choi, J.W.;Lim, J.H.;Hwang, S.K.;Jung, H.J.;Kang, S.K.;Cho, K.K.;Choi, Y.J.;Ahn, J.K.
    • Asian-Australasian Journal of Animal Sciences
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    • v.17 no.11
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    • pp.1591-1598
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    • 2004
  • To isolate the $\beta$-galactosidase producing thermophilic bacteria, samples of mud and water were collected from hot springs of avolcanic area near Golden Springs in New Zealand. Among eleven isolated strains, the strain of KNOUC112 produced the highest amounts of $\beta$-galactosidase at 40 h incubation time (0.013 unit). This strain was aerobic, asporogenic bacilli, immobile, gram negative, catalase positive, oxidase positive, and pigment producing. Optimum growth was at 70-72$^{\circ}C$, pH 7.0-7.2, and it could grow in the presence of 3% NaCl. The main fatty acids of cell components were iso-15:0 (30.26%), and iso-17:0 (31.31%). Based on morphological and biochemical properties and fatty acid composition, the strain could be identified as genus Thermus, and finally as Thermus thermophilus by phylogenetic analysis based on 16S rRNA sequence. So the strain is designated as Thermus thermophilus KNOUC112. A gene from Thermus thermophilus KNOUC112 encoding $\beta$-galactosidase was amplified by PCR using redundancy primers prepared based on the structure of $\beta$-galactosidase gene of Thermus sp. A4 and Thermus sp. strain T2, cloned and expressed in E. coli JM109 DE3. The gene of Thermus thermophilus KNOUC112 $\beta$-galactosidase(KNOUC112$\beta$-gal) consisted of a 1,938 bp open reading frame, encoding a protein of 73 kDa that was composed of 645 amino acids. KNOUC112$\beta$-gal was expressed as dimer and trimer in E. coli JM109 (DE3) via pET-5b.

Expression of $\beta$-Galactosidase Gene Microinjected into Xenopus Egg During Early Development (초기발생 동안 양서류 난에 미세주입된 $\beta$-galactosidase 유전자의 발현)

  • 차병직;정해문
    • The Korean Journal of Zoology
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    • v.33 no.3
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    • pp.365-372
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    • 1990
  • For the effort to produce transgenic amphibians, a plasmid DNA sequence (cytoplasmic actin promoter-linked bacterial $\beta$-galactosidase gene) was microinjected into fertilized Xenopus eggs. It appeared that the injection of 20 nl solution containing 1-2 ng of DNA was not toxic, but over 4 ng was toxic to embryonic development. The translational product of $\beta$-gal gene ($\beta$-galactosidase) had enzyme activity in all three germ layers of the embryo. Expression of the injected $\beta$-gal genes was first detected at mid-gastrula stage, and the activity persisted up to stage 43 (feeding tadpole) with decreased level of retention. However, the level of the expression was various among the injected individuals as well as each experiment. That is, $\beta$-galactosidase activities did not appear in all cells, instead a localized distribution pattern. Although other possibilities could not be omitted, this mosaic distribution of gene expression seemed to arise from unequal partition of the injected DNA into each blastomere during early cleavage.

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