beta-Galactosidase from Kluyveromyces lactis immobilized on to thiolsulfinate/thiolsulfonate supports for lactose hydrolysis in milk and dairy by-products.

1998 ◽  
Vol 12 (2) ◽  
pp. 143-148 ◽  
Author(s):  
Karen Ovsejevi ◽  
Valeria Grazu ◽  
Francisco Batista-Viera
Keyword(s):  
Kluyveromyces Lactis ◽  
Lactose Hydrolysis ◽  
Beta Galactosidase ◽  
By Products

Optimization of Acidic Labneh Whey Lactose Hydrolysis with Immobilized Beta-Galactosidase Enzyme from Kluyveromyces lactis

2011 ◽  
Vol 10 (7) ◽  
pp. 675-679 ◽  
Author(s):  
Wail- Alomari ◽  
Malik Hadadin ◽  
Ali K. Alsaed ◽  
Khalid Al-Ismail
Keyword(s):  
Kluyveromyces Lactis ◽  
Lactose Hydrolysis ◽  
Beta Galactosidase

scholarly journals Functional homology between the yeast regulatory proteins GAL4 and LAC9: LAC9-mediated transcriptional activation in Kluyveromyces lactis involves protein binding to a regulatory sequence homologous to the GAL4 protein-binding site.

1987 ◽  
Vol 7 (12) ◽  
pp. 4400-4406 ◽  
Author(s):  
K D Breunig ◽  
P Kuger
Keyword(s):  
Protein Binding ◽  
Binding Site ◽  
Transcriptional Activation ◽  
Kluyveromyces Lactis ◽  
Regulatory Protein ◽  
Binding Activity ◽  
Protein Binding Site ◽  
Regulatory Sequence ◽  
Functional Homology ◽  
Beta Galactosidase

As shown previously, the beta-galactosidase gene of Kluyveromyces lactis is transcriptionally regulated via an upstream activation site (UASL) which contains a sequence homologous to the GAL4 protein-binding site in Saccharomyces cerevisiae (M. Ruzzi, K.D. Breunig, A.G. Ficca, and C.P. Hollenberg, Mol. Cell. Biol. 7:991-997, 1987). Here we demonstrate that the region of homology specifically binds a K. lactis regulatory protein. The binding activity was detectable in protein extracts from wild-type cells enriched for DNA-binding proteins by heparin affinity chromatography. These extracts could be used directly for DNase I and exonuclease III protection experiments. A lac9 deletion strain, which fails to induce the beta-galactosidase gene, did not contain the binding factor. The homology of LAC9 protein with GAL4 (J.M. Salmeron and S. A. Johnston, Nucleic Acids Res. 14:7767-7781, 1986) strongly suggests that LAC9 protein binds directly to UASL and plays a role similar to that of GAL4 in regulating transcription.

Immobilization of ß-galactosidase from Kluyveromyces lactis on Eupergit® C and Properties of the Biocatalyst

2012 ◽  
Vol 8 (3) ◽  
Author(s):  
Graciella da Silva Campello ◽  
Renata Aguirre Trindade ◽  
Tatiane Vieira Rêgo ◽  
Janaína Fernandes de Medeiros Burkert ◽  
Carlos André Veiga Burkert
Keyword(s):  
Thermal Stability ◽  
Ionic Strength ◽  
Batch Reactor ◽  
Kluyveromyces Lactis ◽  
Immobilized Enzymes ◽  
Maximum Activity ◽  
Lactose Hydrolysis ◽  
Substrate Affinity ◽  
Burman Design ◽  
Plackett Burman Design

Abstract The main goal of this study was to investigate the immobilization of commercial ß-galactosidase from Kluyveromyces lactis (Lactozym®) on Eupergit® C. A Plackett-Burman design was proposed. The ionic strength and pH were the variables that presented significant effect (p<0.1) on immobilization. The increase in the ionic strength from 0.1 to 1.5 M and the increase in pH from 6.6 to 7.4 represented an increase of 28.56% and a reduction of 18.19% in the immobilization yield, respectively. At 25°C, pH 6.6, ionic strength of 1.5 M, immobilization for 8 h, 1 mM of divalent magnesium ion and 0.4 mL of enzyme added, reached 85% immobilization yield. The free and immobilized enzymes were characterized. pH and temperature profiles showed maximum activity at pH 6.6 and 45°C, for both free and immobilized enzymes. There was a gain in thermal stability with enzyme immobilization and there was an increase of about four times in the half-life of the immobilized derivative at 45°C (from 0.43 h to 1.78 h). This greater thermal stability was also made clear through the calculation of thermodynamic parameters (ΔH, ΔG and ΔS). Km values, 30.33 mM and 104.00 mM for free and immobilized enzymes, respectively, represented a reduction in substrate affinity after immobilization, possibly owing to stereo-conformational factors. In a batch reactor for lactose hydrolysis from cheese whey, an increase in lactose conversion with immobilization was observed at 40°C and 45°C (90.43% and 65.36%, respectively) in relation to the free enzyme (84.17% and 39.58%, respectively).

scholarly journals Constitutive expression in gal7 mutants of Kluyveromyces lactis is due to internal production of galactose as an inducer of the Gal/Lac regulon.

1997 ◽  
Vol 17 (3) ◽  
pp. 1722-1730 ◽  
Author(s):  
G Cardinali ◽  
V Vollenbroich ◽  
M S Jeon ◽  
A A de Graaf ◽  
C P Hollenberg
Keyword(s):  
Culture Medium ◽  
Kluyveromyces Lactis ◽  
Constitutive Expression ◽  
Regulatory Function ◽  
Nuclear Magnetic Resonance Analysis ◽  
Resonance Analysis ◽  
Crude Extracts ◽  
Induction Process ◽  
Beta Galactosidase ◽  
Internal Production

The induction process of the galactose regulon has been intensively studied, but until now the nature of the inducer has remained unknown. We have analyzed a delta gal7 mutant of the yeast Kluyveromyces lactis, which lacks the galactotransferase activity and is able to express the genes of the Gal/Lac regulon also in the absence of galactose. We found that this expression is semiconstitutive and undergoes a strong induction during the stationary phase. The gal1-209 mutant, which has a reduced kinase activity but retains its positive regulatory function, also shows a constitutive expression of beta-galactosidase, suggesting that galactose is the inducer. A gal10 deletion in delta gal7 or gal1-209 mutants reduces the expression to under wild-type levels. The presence of the inducer could be demonstrated in both delta gal7 crude extracts and culture medium by means of a bioassay using the induction in gal1-209 cells. A mutation in the transporter gene LAC12 decreases the level of induction in gal7 cells, indicating that galactose is partly released into the medium and then retransported into the cells. Nuclear magnetic resonance analysis of crude extracts from delta gal7 cells revealed the presence of 50 microM galactose. We conclude that galactose is the inducer of the Gal/Lac regulon and is produced via UDP-galactose through a yet-unknown pathway.

scholarly journals Absence of step changes in activity of certain enzymes during the cell cycle of budding and fission yeasts in synchronous cultures

1983 ◽  
Vol 61 (1) ◽  
pp. 339-349
Author(s):  
J. Creanor ◽  
S.G. Elliott ◽  
Y.C. Bisset ◽  
J.M. Mitchison
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Acid Phosphatase ◽  
Kluyveromyces Lactis ◽  
The Other ◽  
Linear Pattern ◽  
Synchronous Cultures ◽  
Asynchronous Control ◽  
Alpha Glucosidase ◽  
Beta Galactosidase

Synchronous cultures prepared by selection from an elutriating rotor were used to measure activity changes during the cell cycle of the following enzymes: acid phosphatase in Schizosaccharomyces pombe and Saccharomyces cerevisiae, alpha-glucosidase in S. cerevisiae and beta-galactosidase in Kluyveromyces lactis. There was no sign of step rises in activity in acid phosphatase but there were indications in S. cerevisiae of the linear pattern with rate doublings once per cycle that had been found previously in S. pombe. There was also no sign of step rises in the other two enzymes, in contrast to earlier results using different techniques. Asynchronous control cultures showed little or no perturbations after the first hour.

scholarly journals Validation and Optimization of Polyvinyl Alcohol-Functionalized Gold Nanoparticles for Producing Lactose-Free Dairy Products

2021 ◽  
Vol 37 (3) ◽  
pp. 643-647
Author(s):  
Asim Muhammed Alshanberi ◽  
Shakeel Ahmed Ansari
Keyword(s):  
Gold Nanoparticles ◽  
Polyvinyl Alcohol ◽  
Dairy Products ◽  
Biomedical Applications ◽  
Immobilized Enzyme ◽  
Kluyveromyces Lactis ◽  
Lactose Hydrolysis ◽  
Batch Reactors ◽  
Stable Matrix ◽  
Functionalized Aunps

The present study demonstrates the synthesis of lactose-free dairy items by Kluyveromyces lactis β-galactosidase bound to polyvinyl alcohol (PVA)-modified gold nanoparticles (AuNPs). The size of AuNPs was analyzed by dynamic light scattering experiment. The developed AuNPs served as a stable matrix for enzyme immobilization which was observed by obtaining 88% immobilization yield. Km and Vmax were determined for soluble and immobilized enzyme by incubating them with varying concentrations of substrate. Our findings demonstrated that immobilization leads to an increase of Km and a decline in Vmax values for the enzyme attached to PVA-functionalized AuNPs. Moreover, the enzyme conjugated to surface functionalized AuNPs displayed exceptional conversion of lactose hydrolysis in batch reactors at 40 oC in contrast to its hydrolysis at 50 oC. Hence, the developed nanosystem [β-galactosidase-(PVA-modified AuNPs)] serves as an excellent model for suggesting its application in other biomedical applications, particularly for constructing lactose based biosensors.

scholarly journals ß-galactosidase production by Kluyveromyces lactis in batch and continuous culture

2011 ◽  
Author(s):  
◽  
Elaine C. Ram
Keyword(s):  
Carbon Source ◽  
Continuous Culture ◽  
Batch Culture ◽  
Shake Flask ◽  
Kluyveromyces Lactis ◽  
Shake Flask Culture ◽  
Lactose Hydrolysis ◽  
Strictly Aerobic ◽  
Fed Batch ◽  
Cell Lysates

Kluyveromyces sp. have adapted to existence in milk due to the evolution of permeabilisation and hydrolytic systems that allow the utilisation of lactose, the sugar most abundant in milk. Lactose hydrolysis, to equimolar units of glucose and galactose, is facilitated by a glycoside hydrolase, i.e., β-galactosidase (EC 3.2.1.23). The versatility of this enzyme allows its application in numerous industrial processes, amongst the most significant of which, is its role in the alleviation of lactose intolerance, one of the most prevalent digestive ailments, globally. In this study, β-galactosidase production by Kluyveromyces lactis UOFS y-0939 was initially optimised in shake flask culture with lactose as the sole carbon source, and thereafter, production was scaled up to batch, fedbatch and continuous culture. Shake flask studies revealed optimum conditions of 30°C, pH 7 and a 10% inoculum ratio, to be most favourable for β-galactosidase synthesis, producing a maximum of 0.35 ± 0.05 U.ml-1 when cell lysates were prepared by ultrasonication with glass beads. Batch cultivation in 28.2 and 40 g.L-1 lactose revealed that elevated levels of the carbon source was not inhibitory to β-galactosidase production, as maximum enzyme activities of 1.58 and 4.08 U.ml-1, respectively, were achieved. Cell lysates prepared by ultrasonication and homogenisation were compared and homogenised cell lysates were more than 3.5 fold higher that those prepared by ultrasonication, proving homogenisation to be the superior method for cell disruption. The lactose feed rate of 4 g.L-1.h-1 in fed-batch culture operated at ± 20.4% DO, appeared to be inhibitory to biomass production, as indicated by the lower biomass productivity in fed-batch (0.82 g.L-1.h-1) than batch culture (1.27 g.L-1.h-1). Enzyme titres, however, were favoured by the low DO levels as a maximum of 8.7 U.ml-1, 5.5 fold more than that obtained in batch culture, was achieved, and would be expected to increase proportionally with the biomass. Continuous culture operated at a dilution rate of 0.2 h-1, under strictly aerobic conditions, revealed these conditions to be inhibitory to the lactose consumption rate, however, the non-limiting lactose and high DO environment was favourable for β-galactosidase synthesis, achieving an average of 8 ± 0.9 U.ml-1 in steady state.

Galactokinase encoded by GAL1 is a bifunctional protein required for induction of the GAL genes in Kluyveromyces lactis and is able to suppress the gal3 phenotype in Saccharomyces cerevisiae

1991 ◽  
Vol 11 (11) ◽  
pp. 5454-5461
Author(s):  
J Meyer ◽  
A Walker-Jonah ◽  
C P Hollenberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Kluyveromyces Lactis ◽  
Regulatory Function ◽  
Bifunctional Protein ◽  
Leloir Pathway ◽  
Gal Genes ◽  
Beta Galactosidase ◽  
Gal1 Gene ◽  
Galactokinase Activity ◽  
Negative Mutant

We have analyzed a GAL1 mutant (gal1-r strain) of the yeast Kluyveromyces lactis which lacks the induction of beta-galactosidase and the enzymes of the Leloir pathway in the presence of galactose. The data show that the K. lactis GAL1 gene product has, in addition to galactokinase activity, a function required for induction of the lactose system. This regulatory function is not dependent on galactokinase activity, as it is still present in a galactokinase-negative mutant (gal1-209). Complementation studies in Saccharomyces cervisiae show that K. lactis GAL1 and gal1-209, but not gal1-r, complement the gal3 mutation. We conclude that the regulatory function of GAL1 in K. lactis soon after induction is similar to the function of GAL3 in S. cerevisiae.

scholarly journals Identification of upstream activator sequences that regulate induction of the beta-galactosidase gene in Kluyveromyces lactis.

1987 ◽  
Vol 7 (12) ◽  
pp. 4369-4376 ◽  
Author(s):  
J M Leonardo ◽  
S M Bhairi ◽  
R C Dickson
Keyword(s):  
Gene Expression ◽  
Deletion Mutant ◽  
Kluyveromyces Lactis ◽  
Regulatory Protein ◽  
Base Sequence ◽  
Promoter Deletion ◽  
Deletion Mutations ◽  
Maximum Induction ◽  
Sequence Elements ◽  
Beta Galactosidase

Transcription of the Kluyveromyces lactis beta-galactosidase gene, LAC4, is inducible by galactose and lactose. We examined the effects of deletion mutations within the LAC4 promoter on the expression of beta-galactosidase activity. The results of these experiments indicate that at least two upstream activator sequences (UAS) mediate maximum induction by galactose. These UAS sequence elements are homologous to UAS that regulate induction of the melibiose-galactose regulon of Saccharomyces cerevisiae. We also show that a synthetic copy of one of the K. lactis UAS restores the inducibility of a deleted, noninducible LAC4 promoter. Since the uninduced or basal level of LAC4 expression was increased in several promoter deletion strains and in deletion strains carrying one or two synthetic UAS, we examined the contribution of the LAC9 positive regulatory protein to this effect. The LAC9 protein is thought to bind to UAS and activate transcription of LAC4 (L.V. Wray, M.M. Witte, R.C. Dickson, and M.I. Riley, Mol. Cell. Biol. 7:1111-1121, 1987). Our results demonstrate that LAC9 protein plays a role in setting the uninduced level of gene expression, but other factors also participate. For example, in a lac9 background a LAC4 promoter deletion mutant with two copies of a synthetic 17-base-pair UAS yields a sevenfold higher level of uninduced LAC4 expression than the same strain with one UAS. These and other data indicate that the basal level of gene expression is strongly influenced by the base sequence of the promoter.

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