• Title/Summary/Keyword: /beta-galactosidase

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Screening and Characterization of Drosophila Development Mutants Using Single P[en-lacZ] Element Mutagenesis (Drosophila single P[en-lacZ] element mutagenesis를 이용한 발생 관련 돌연변이체 작성)

  • Ha, Hye-Yeong;Lee, Heui-Jung;Park, Soon-Hee;Yoo, Mi-Ae;Lee, Won-Ho
    • Journal of Life Science
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    • v.7 no.1
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    • pp.49-58
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    • 1997
  • Single P[en-lacZ] element including 5.7 kb of engrailed upstream sequences and the E. coli lacZ fusion gene, localized on 48A in rxyho25 strain was transposed to different sites in the Drosophila genome by the jumpstart technique. From 3315 individual genetic crosses, 113 new insertion lines carrying P[en-lacZ] inserted at different sites were obtained. $\beta$-Galactosidase expression in larval tissues of 113 insertion lines were detected by X-gal staining. & among 113 lines have been indentified to be for recessive lethal mutations. Among 7 lines, the #1119 line being lethal during embryogenesis was examined about the ${\beta}$$-Galactosidase expression, nuclear behavior and cellularization pattern during embryogenesis. The P[en-lacZ] insertion lines obtained in this study could be utilized for studying structure and function of the Drosophila development-related genes.

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Expression of Heterologous Promoters in Aspersillus oryzae (Aspergillus oryzae에서의 이종 Promoter들의 발현)

  • Hahm, Young Tae;Kim, Hee Chung;Batt, Carl A.
    • KSBB Journal
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    • v.10 no.1
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    • pp.38-45
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    • 1995
  • The expression of Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase (gpdA) and trpC promoters in A. oryzae were compared using E. coli lacZ gents fusions. The specific activities of the expressed E. coli $\beta$-galactosidase in A. oryzae transformants containing the A. nidulans gpdA promoter were around 2,000 units per ug of protein. The specific activities of transformants containing the A. nidulans trpC promoter were very low, ranging from 10.5 to 52.3 units per ug of protein. These results showed that the expression of the A. nidulans gpdA promoter in A. oryzae was approximately 70 times greater than the A. nidulans trpC promoter. In western blot analysis, immunoreactive bands of a imlilar molecular weight as the E. coli $\beta$-galactosidase were observed in A. oryzae carrying the gpdA-lacZ fusion and to a lesser intensity in those carrying the tvpC-lacZ fusion. Southern analysis showed that the higher expression of the gpdA-lacZ fusion as compared to the trpC-lacZ fusion was not due a greater number of integrated plasmids.

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Effects of IAA on the Elongation and Cell Wall Glycosidase Activities in Excised Rape (Brassica napus L. cv. Yongdang) Hypocotyl Segments (유채 하배축 분절의 신장과 세포벽 분해효소의 활성에 미치는 IAA의 효과)

  • Jun, Sung-Soo
    • Journal of Plant Biology
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    • v.27 no.2
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    • pp.43-50
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    • 1984
  • Effects of IAA on the elongation and cell wall hlysocidase activities were investigated in excised rape (Brassica napus L. cv. Yongdang) hypocotyl segments. IAA promoted the elongation of rape hypocotyl segments. In rape hypocotyls, the first 10-mm segments from the hook exhibited maximal elongation and the capacity of elongation was gradually decreased with increasing distance of each 10-mm from the hook. A good correlation has been obtained between the magnitude of endogenous growth and the activities of $\alpha$, $\beta$-glucosidase and $\alpha$, $\beta$-galactosidase. However, exogenous application of IAA did not seem to enhance the tissue with IAA resulted in acidification of the incubation medium. From these data, we can conclude that IAA seems to enhance elongation of the tissue segments, at least in part, by releasing hydrogen ion into cell wall, some of which may participate in the cell wall extension process, but does not seem to trigger the activation of $\alpha$, $\beta$-glucosidase and $\alpha$, $\beta$-galactosidase.

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Purification and Characterization of $\beta$-Galactosidase from Sea Urchin, Hemicentrotus pulcherrimus (성게로부터 분리한 $\beta$-galactosidase의 정제 및 특성)

  • KIM Gyu-Hyung;KIM Yong-Tae;KIM Se-Kwon
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.31 no.5
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    • pp.637-644
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    • 1998
  • [ $\beta$ ]-Galactosidase was extracted from the internal organ of sea urchin, Hemicentrotus pulcherrimus The enzyme was purified 384.6-fold over the crude extract by the sequential chromatographic methods including DEAE-Sephadex A-25, CM-Cellulose, and Con A-Sepharose 4B affinity chromatography with a recovery $1.26\%$. The molecular weight of the purified enzyme was estimated approximately 94 kDa as monomeric term by SDS-PAGE and Sephadex G-150 gel chromatography. The maximum enzymatic activity was observed at pH 3.0 and $50^{\circ}C$ but the one was stable over the ph range or 3.0$\~$5.0 and below $37^{\circ}C$. The $K_m$ and $V_{max}$ values against PNPG (P-nitrophenyl $\beta$-D-galactopyranoside) were 15.0 mM and 214 $\mu$mole/min per mg protein, respectively. The enzymatic activity was activated by $Ba^{2+}$, but significantly inhibited by $DEP,\;Hg^{2+},\;Sn^{2+}$ and galactose.

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The Fission Yeast Gene Encoding Monothiol Glutaredoxin 5 Is Regulated by Nitrosative and Osmotic Stresses

  • Kim, Hong-Gyum;Park, Eun-Hee;Lim, Chang-Jin
    • Molecules and Cells
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    • v.20 no.1
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    • pp.43-50
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    • 2005
  • Glutaredoxin (Grx) is a small, heat-stable redox protein acting as a multi-functional glutathione (GSH)-dependent disulfide oxidoreductase. We have cloned the monothiol Grx5 gene from the genomic DNA of the fission yeast Schizosaccharomyces pombe. It has 1,904 bp, with one intron, and encodes a putative protein of 146 amino acids with a molecular mass of 16.5 kDa. Recombinant Grx5 produced functional Grx in S. pombe cells. NO-generating sodium nitroprusside (SNP, 1.0 and 2.0 mM) and potassium chloride (KCl, 0.2 and 0.5 M) increased the synthesis of ${\beta}$-galactosidase from a Grx5-lacZ fusion gene, and transcription of Grx5 was also enhanced by SNP and KCl. Synthesis of ${\beta}$-galactosidase from the Grx5-lacZ fusion was lower in Pap1-negative TP108-3C cells than in wild type KP1 cells, and when Pap1 was overproduced in KP1 cells, the level of ${\beta}$-galactosidase increased. We also found that Pap1 is involved in the induction of Grx5 by SNP and KCl. S. pombe Grx5 may play a crucial role in responses to nitrosative and osmotic stresses.

Production and Application of Galacto-oligosaccharides from Lactose by a Recombinant $\beta$-Galactosidase of Bifidobacterium infantis Overproduced by Pichia pastoris

  • Jung, Sung-Je;Lee, Byong-Hoon
    • Food Science and Biotechnology
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    • v.17 no.3
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    • pp.514-518
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    • 2008
  • After overproduction of a recombinant $\beta$-galactosidase of Bifidobacterium infantis in Pichia pastoris, a synthesis of galacto-oligosaccharides (GOS) from 36% lactose using the enzyme (170.74 U/mg) was investigated. The transgalactosylation ratio reached up to 25.2% with 83.1% conversion of initial lactose and the maximum yield of GOS was 40.6%. The GOS syrup was composed of a 13.43% galacto-oligosaccharides, 5.06% lactose, and 8.76% monosaccharides. The prebiotic effect of GOS on the growth of bifidobacteria and lactobacilli strains was investigated in vitro. The maximum growth rate of Bifidobacterium breve and Lactobacillus acidophillus in GOS syrup (5%, v/v) media were 0.49 and 0.96/hr that are higher than those in 1%(w/v) galactose and 1%(w/v) lactose containing media. However, there was no significant difference between the specific growth rates of L. acidophillus in 1%(w/v) glucose and 5%(v/v) GOS syrup. Our data showed that GOS definitely promoted the growth of B. breve ATCC $15700^T$ and L. acidophilus ATCC 33323.

Rahnella aquatilis를 이용한 Lactan gum 생산에서 탄수화물 대사

  • Na, Kun;Lee, Seong-Ho;Lee, Ki-Young
    • Microbiology and Biotechnology Letters
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    • v.24 no.4
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    • pp.493-499
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    • 1996
  • Lactan gum produced by Rahnella aquatilis is a high viscous, anionic polysaccharide and has shear thinning behaviour. Lactan gum yield and cencentration was greater on disaccharide such as lactose and sucrose than on monosaccharides such as glucose and galactose. When initial carbon source concentration was 45g/l of sucrose of lactose, the microorgnisms produced 28 g/l and 27 g/l of lactan, respectively with a yield more than 60%. $\beta$-Galactosidase, hydrolyzing lactose into galactose and glucose, was induced by lactose or galactose. When initial corbon source was 45 g/l of mixed carbon I (glucose:galactose=1:1), lactan gum concentaration was higher than that from 45 g/l of monosaccharide (glucose pf galactose) but was similar to the result from 45 g/l of lactose. Therefore, lactose hydrolysis reaction by $\beta$-galactosidase does not seem to be a rate determining step in lactan gum biosynthesis. When initial carbon source was 45 g/l of mixed carbon II (glucose:fructose=1:1). total carbon source consumption rate was slower than that from sucrose, but glucose consumption rate was faster than that from fructose.

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Construction of the Novel Baculovirus Transfer Vector Using the p10 Gene of BmNPV (BmNPV의 p10 유전자를 이용한 새로운 전이벡터 개발)

  • 강석우;진병래
    • Journal of Sericultural and Entomological Science
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    • v.39 no.2
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    • pp.180-185
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    • 1997
  • To develope the novel baculovirus transfer vector, the p10 gene was cloned from the Bombyx mori nuclear polygedrosis virus (BmNPV) vB2 strain isolated from the B. mori larvae of sericultural farms. The novel transfer vector was constructed by using the p10 gene of BmNPV vB2 strain was 210 bp. The TAAG sequence at the -71 bp of upstream from translation initiator ATG and two polyadenylation signal site at the downstream from terminator TAA were also detected in the p10 gene. The 5' and 3' flanking region of the p10 gene amplified by PCR was cloned into pBluescriptII SK(+) and then transfer vector pBm10 was construceted. The 7.9 kb pBm10 was analysed by restriction enzymes and the map was confirmed. In order to determine the expression of foreign gene of pBm10, $\beta$-galactosidase gene was inserted in the SmaI site of foreign gene cloning site of pBm10. The pBm10 containing $\beta$-galactosidase gene was cotranfected wth genomic DNA of BmNPV vB2 into BmN-4 cells. The recombinant baculovirus expressing $\beta$-galactosidase was also produced polygedra in the infected cells. The results indicated that pBm10 is functional, suggesting that in the baculovirus expression vector system, the recombinant virus produced by pBm10 was effective by oral infection for the producing recombinant proteins in in vivo expression.

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Enhanced Expression of Foreign Gene in Baculovirus-Infected Insect Cells Using a Silkworm Hemolymph (누에 체액을 이용한 외래 유전자의 발현효율 증대)

  • 우수동;김혜성
    • Journal of Sericultural and Entomological Science
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    • v.37 no.2
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    • pp.181-185
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    • 1995
  • To enhance expression of foreign gene by the novel expression vector, pBmKSK1, of Bombyx mori nuclear polyhedrosis virus, E. coli $\beta$-galactosidase gene expressing recombinant virus was infected in BmN-4 cells and various concentrations of silkworm hemolymph were added to the recombinant virus-infected BmN-4 cells containing fetal bovine serum. The expression efficiency of foreign gene was determined by $\beta$-galactosidase activity in the culture media. The results showed that the silkworm hemolymph was effective to expression of foreign gene in the BmN-4 cells, suggesting that the silkworm hemolymph could be substituted for fetal bovine serum in the BmN-4 cells to enhance expression of foreign gene.

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Development of Lactose-hydrolyzed Milk with Low Sweetness Using Nanofiltration

  • Choi, S.H.;Lee, S.-B.;Won, H.-R.
    • 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.