Permeabilization of Yeast Cells with Organic Solvents for ß-galactosidase Activity

2007 ◽  
Vol 2 (1) ◽  
pp. 34-41 ◽  
Author(s):  
Parmjit S. Panesar . ◽  
Reeba Panesar . ◽  
Ram S. Singh . ◽  
Manav B. Bera .
Keyword(s):  
Organic Solvents ◽  
Yeast Cells ◽  
Galactosidase Activity

Overlapping reading frames at the LYS5 locus in the yeast Yarrowia lipolytica

1990 ◽  
Vol 10 (9) ◽  
pp. 4795-4806
Author(s):  
J W Xuan ◽  
P Fournier ◽  
N Declerck ◽  
M Chasles ◽  
C Gaillardin
Keyword(s):  
Yarrowia Lipolytica ◽  
Stop Codon ◽  
Open Reading Frames ◽  
Yeast Cells ◽  
Lacz Gene ◽  
Galactosidase Activity ◽  
Phenotypic Effect ◽  
Frame Fusion ◽  
Beta Galactosidase ◽  
Reading Frames

Mutants affected at the LYS5 locus of Yarrowia lipolytica lack detectable dehydrogenase (SDH) activity. The LYS5 gene has previously been cloned, and we present here the sequence of the 2.5-kilobase-pair (kb) DNA fragment complementing the lys5 mutation. Two large antiparallel open reading frames (ORF1 and ORF2) were observed, flanked by potential transcription signals. Both ORFs appear to be transcribed, but several lines of evidence suggest that only ORF2 is translated and encodes SDH. (i) The global amino acid compositions of Saccharomyces cerevisiae SDH and of the putative ORF2 product are similar and that of ORF1 is dissimilar. (ii) An in-frame translational fusion of ORF2 with the Escherichia coli lacZ gene was introduced into yeast cells and resulted in a beta-galactosidase activity regulated similarly to SDH; no beta-galactosidase activity was obtained with an in-frame fusion of ORF1 with lacZ. (iii) The introduction of a stop codon at the beginning of ORF2 prevented SDH expression in yeast cells, whereas no phenotypic effect was observed when ORF1 translation was blocked.

Permeabilization of yeast cells (Kluyveromyces lactis) with organic solvents

1994 ◽  
Vol 16 (4) ◽  
pp. 340-346 ◽  
Author(s):  
M.V. Flores ◽  
C.E. Voget ◽  
R.J.J. Ertola
Keyword(s):  
Organic Solvents ◽  
Kluyveromyces Lactis ◽  
Yeast Cells

Microencapsulation of yeast cells and their use as a biocatalyst in organic solvents

2000 ◽  
Vol 49 (5) ◽  
pp. 535-543 ◽  
Author(s):  
K. D. Green ◽  
I. S. Gill ◽  
J. A. Khan ◽  
E. N. Vulfson
Keyword(s):  
Organic Solvents ◽  
Yeast Cells

scholarly journals Overlapping reading frames at the LYS5 locus in the yeast Yarrowia lipolytica.

1990 ◽  
Vol 10 (9) ◽  
pp. 4795-4806 ◽  
Author(s):  
J W Xuan ◽  
P Fournier ◽  
N Declerck ◽  
M Chasles ◽  
C Gaillardin
Keyword(s):  
Yarrowia Lipolytica ◽  
Stop Codon ◽  
Open Reading Frames ◽  
Yeast Cells ◽  
Lacz Gene ◽  
Galactosidase Activity ◽  
Phenotypic Effect ◽  
Frame Fusion ◽  
Beta Galactosidase ◽  
Reading Frames

Mutants affected at the LYS5 locus of Yarrowia lipolytica lack detectable dehydrogenase (SDH) activity. The LYS5 gene has previously been cloned, and we present here the sequence of the 2.5-kilobase-pair (kb) DNA fragment complementing the lys5 mutation. Two large antiparallel open reading frames (ORF1 and ORF2) were observed, flanked by potential transcription signals. Both ORFs appear to be transcribed, but several lines of evidence suggest that only ORF2 is translated and encodes SDH. (i) The global amino acid compositions of Saccharomyces cerevisiae SDH and of the putative ORF2 product are similar and that of ORF1 is dissimilar. (ii) An in-frame translational fusion of ORF2 with the Escherichia coli lacZ gene was introduced into yeast cells and resulted in a beta-galactosidase activity regulated similarly to SDH; no beta-galactosidase activity was obtained with an in-frame fusion of ORF1 with lacZ. (iii) The introduction of a stop codon at the beginning of ORF2 prevented SDH expression in yeast cells, whereas no phenotypic effect was observed when ORF1 translation was blocked.

scholarly journals ISOLATION AND PURIFICATION OF LACTASE FROM LACTOBACILLUS ACIDOPHILUS

2012 ◽  
Vol 15 (3) ◽  
pp. 65-72
Author(s):  
Linh Thi Van Nguyen ◽  
Dung Thi Thuy Nguyen ◽  
Lam Bich Tran
Keyword(s):  
Molecular Weight ◽  
Kinetic Parameters ◽  
Organic Solvents ◽  
Lactobacillus Acidophilus ◽  
Gel Permeation Chromatography ◽  
Galactosidase Activity ◽  
Isolation And Purification ◽  
Enzyme Recovery ◽  
Gel Permeation ◽  
Enzyme Isolation

The enzyme β-galactosidase (β-D-galactoside galactohydrolase, EC 3.2.1.23) commonly known as lactase, has important applications in the dairy industry. From culture of strain Lactobacillus acidophilus having high and stable β-galactosidase activity, the study of crude enzyme isolation were carried out by ultrasonical extraction and precipitation by neutral salts and organic solvents. Best precipitant was isopropanol with enzyme recovery 89,93%, and enzyme purity increased 4,5 folds. Further β-galactosidase purification was carried out using gel permeation chromatography on Ultrahydrogel 250 to increase purity in 14,3 folds. The molecular weight of β-galactosidase was 40 kDa. The purified enzyme had optimum activity at 40oC, pH 7 – 7,5 and kinetic parameters of Vmax and Km were 2,3 μmol/min and 0,73 mM.

Fusion of the Saccharomyces cerevisiae leu2 gene to an Escherichia coli beta-galactosidase gene

1983 ◽  
Vol 3 (4) ◽  
pp. 580-586
Author(s):  
A E Martinez-Arias ◽  
M J Casadaban
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Translation Initiation ◽  
Agar Medium ◽  
Yeast Cells ◽  
Galactosidase Activity ◽  
Chromogenic Substrates ◽  
Translation Initiation Region ◽  
Leu2 Gene ◽  
Beta Galactosidase

The promoter and translation initiation region of the Saccharomyces cerevisiae leu2 gene was fused to the Escherichia coli beta-galactosidase gene. This fusion located the control region of the leu gene and orientated its direction of expression. When the fusion was placed into yeast cells, beta-galactosidase was expressed under the same regulatory pattern as the original leu2 gene product: its synthesis was repressed in the presence of leucine and threonine. Sensitive chromogenic substrates for beta-galactosidase were used to detect expression in isolated colonies growing on agar medium. Mutant yeast cells with increased beta-galactosidase activity were identified by the color of the colonies they formed. One class of mutants obtained appeared to affect ars1 plasmid maintenance, and another class appeared to affect beta-galactoside uptake.

scholarly journals Fusion of the Saccharomyces cerevisiae leu2 gene to an Escherichia coli beta-galactosidase gene.

1983 ◽  
Vol 3 (4) ◽  
pp. 580-586 ◽  
Author(s):  
A E Martinez-Arias ◽  
M J Casadaban
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Translation Initiation ◽  
Agar Medium ◽  
Yeast Cells ◽  
Galactosidase Activity ◽  
Chromogenic Substrates ◽  
Translation Initiation Region ◽  
Leu2 Gene ◽  
Beta Galactosidase

The promoter and translation initiation region of the Saccharomyces cerevisiae leu2 gene was fused to the Escherichia coli beta-galactosidase gene. This fusion located the control region of the leu gene and orientated its direction of expression. When the fusion was placed into yeast cells, beta-galactosidase was expressed under the same regulatory pattern as the original leu2 gene product: its synthesis was repressed in the presence of leucine and threonine. Sensitive chromogenic substrates for beta-galactosidase were used to detect expression in isolated colonies growing on agar medium. Mutant yeast cells with increased beta-galactosidase activity were identified by the color of the colonies they formed. One class of mutants obtained appeared to affect ars1 plasmid maintenance, and another class appeared to affect beta-galactoside uptake.

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