scholarly journals Optimization of Saccharomyces cerevisiae α-galactosidase production and application in the degradation of raffinose family oligosaccharides

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
María-Efigenia Álvarez-Cao ◽  
María-Esperanza Cerdán ◽  
María-Isabel González-Siso ◽  
Manuel Becerra
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Food Industry ◽  
Culture Conditions ◽  
Raffinose Family Oligosaccharides ◽  
Recombinant Production ◽  
Gene Coding ◽  
Productive Process ◽  
Hydrolysis Of ◽  
Degree Of Purification

Abstract Background α-Galactosidases are enzymes that act on galactosides present in many vegetables, mainly legumes and cereals, have growing importance with respect to our diet. For this reason, the use of their catalytic activity is of great interest in numerous biotechnological applications, especially those in the food industry directed to the degradation of oligosaccharides derived from raffinose. The aim of this work has been to optimize the recombinant production and further characterization of α-galactosidase of Saccharomyces cerevisiae. Results The MEL1 gene coding for the α-galactosidase of S. cerevisiae (ScAGal) was cloned and expressed in the S. cerevisiae strain BJ3505. Different constructions were designed to obtain the degree of purification necessary for enzymatic characterization and to improve the productive process of the enzyme. ScAGal has greater specificity for the synthetic substrate p-nitrophenyl-α-d-galactopyranoside than for natural substrates, followed by the natural glycosides, melibiose, raffinose and stachyose; it only acts on locust bean gum after prior treatment with β-mannosidase. Furthermore, this enzyme strongly resists proteases, and shows remarkable activation in their presence. Hydrolysis of galactose bonds linked to terminal non-reducing mannose residues of synthetic galactomannan-oligosaccharides confirms that ScAGal belongs to the first group of α-galactosidases, according to substrate specificity. Optimization of culture conditions by the statistical model of Response Surface helped to improve the productivity by up to tenfold when the concentration of the carbon source and the aeration of the culture medium was increased, and up to 20 times to extend the cultivation time to 216 h. Conclusions ScAGal characteristics and improvement in productivity that have been achieved contribute in making ScAGal a good candidate for application in the elimination of raffinose family oligosaccharides found in many products of the food industry.

Colloid-Chemical Regularities of Reagent Wastewater Treatment of Dairies

2021 ◽  
Vol 1038 ◽  
pp. 235-241
Author(s):  
Volodymyr Andronov ◽  
Yuliya Danchenko ◽  
Yevhen Makarov ◽  
Tatyana Obizhenko
Keyword(s):  
Wastewater Treatment ◽  
Comparative Characterization ◽  
Chemical Reagents ◽  
Hydrolysis Of ◽  
Degree Of Purification

A comparative characterization of chemical reagents Al2(SO4)3, FeSO4 and FeCl3 in the process of wastewater treatment of dairies. The colloid-chemical regularities of the course of hydrolysis of chemical reagents depending on the nature, concentration and acidity of wastewater are established. It was found that the greatest degree of purification from ether-soluble substances 87-88% is provided by the addition of FeCl3 at a concentration of 150-200 mg / l at a pH of 9.5-10.

scholarly journals COLCEMID INHIBITION OF CELL GROWTH AND THE CHARACTERIZATION OF A COLCEMID-BINDING ACTIVITY IN SACCHAROMYCES CEREVISIAE

1972 ◽  
Vol 55 (2) ◽  
pp. 355-367 ◽  
Author(s):  
James E. Haber ◽  
John G. Peloquin ◽  
Harlyn O. Halvorson ◽  
Gary G. Borisy
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gel Filtration ◽  
Culture Conditions ◽  
Binding Activity ◽  
Affinity Constant ◽  
Binding Substance ◽  
Related Drug ◽  
Antimitotic Drug ◽  
The Difference

Under restricted culture conditions, the growth and division of Saccharomyces cerevisiae was inhibited by the antimitotic drug Colcemid; in contrast, the related drug colchicine had no effect. The difference in the sensitivity of yeast to these two agents was not dependent on their ability to permeate the cell but rather reflected an inherent difference in the affinity of the two drugs for a cellular-binding site. The binding moiety was characterized by gel filtration as a macromolecule of approximately 110,000 mol wt with an affinity constant for Colcemid of 0.5 x 104 liters per mole; in addition, this macromolecule was retained by diethylaminoethyl (DEAE) ion exchangers. On the basis of these properties, the Colcemid-binding substance in S. cerevisiae cells was provisionally identified as microtubule subunits.

scholarly journals Structure and activity of the Saccharomyces cerevisiae dUTP pyrophosphatase DUT1, an essential housekeeping enzyme

2011 ◽  
Vol 437 (2) ◽  
pp. 243-253 ◽  
Author(s):  
Anatoli Tchigvintsev ◽  
Alexander U. Singer ◽  
Robert Flick ◽  
Pierre Petit ◽  
Greg Brown ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Mechanisms ◽  
Site Directed Mutagenesis ◽  
Saturation Curve ◽  
Significant Activity ◽  
Active Site Residues ◽  
Living Organisms ◽  
Hydrolysis Of

Genomes of all free-living organisms encode the enzyme dUTPase (dUTP pyrophosphatase), which plays a key role in preventing uracil incorporation into DNA. In the present paper, we describe the biochemical and structural characterization of DUT1 (Saccharomyces cerevisiae dUTPase). The hydrolysis of dUTP by DUT1 was strictly dependent on a bivalent metal cation with significant activity observed in the presence of Mg2+, Co2+, Mn2+, Ni2+ or Zn2+. In addition, DUT1 showed a significant activity against another potentially mutagenic nucleotide: dITP. With both substrates, DUT1 demonstrated a sigmoidal saturation curve, suggesting a positive co-operativity between the subunits. The crystal structure of DUT1 was solved at 2 Å resolution (1 Å=0.1 nm) in an apo state and in complex with the non-hydrolysable substrate α,β-imido dUTP or dUMP product. Alanine-replacement mutagenesis of the active-site residues revealed seven residues important for activity including the conserved triad Asp87/Arg137/Asp85. The Y88A mutant protein was equally active against both dUTP and UTP, indicating that this conserved tyrosine residue is responsible for discrimination against ribonucleotides. The structure of DUT1 and site-directed mutagenesis support a role of the conserved Phe142 in the interaction with the uracil base. Our work provides further insight into the molecular mechanisms of substrate selectivity and catalysis of dUTPases.

scholarly journals Isolation and characterization of mutants which show an oversecretion phenotype in Saccharomyces cerevisiae.

Genetics ◽  
1988 ◽  
Vol 119 (3) ◽  
pp. 499-506
Author(s):  
A Sakai ◽  
Y Shimizu ◽  
F Hishinuma
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Glucose Repression ◽  
Temperature Sensitive ◽  
Cellular Functions ◽  
Isolation And Characterization ◽  
Gene Coding ◽  
Complementation Groups ◽  
Homozygous Diploid ◽  
Diploid Cells

Abstract We have isolated mutants responsible for an oversecretion phenotype in Saccharomyces cerevisiae, using a promoter of SUC2 and the gene coding for alpha-amylase from mouse as a marker of secretion. These mutations defined two complementation groups, designated as ose1 (over secretion) and rgr1 (resistant to glucose repression). The ose1 mutant produced an oversecretion of amylase by 12- to 15-fold under derepressing conditions; however, the amylase mRNA was present at nearly the same amount as it was in the parent cells. No expression of the amylase gene was detected under repressing conditions. The rgr1 mutant oversecreted amylase by 11- to 13-fold under repressing conditions by 15- to 18-fold under derepressing conditions. The rgr1 mutant showed pleiotropic effects on the following cellular functions: (1) resistance to glucose repression, (2) temperature-sensitive lethality, (3) sporulation deficieny in homozygous diploid cells, and (4) abnormal cell morphology. The rgr1 mutation was not allelic with ssn6 and cyc9, and failed to suppress snf1.

scholarly journals Mutational effects on carbapenem hydrolysis of YEM-1, a new sub-class B2 metallo-β-lactamase from Yersinia mollaretii

2020 ◽  
Author(s):  
P. S. Mercuri ◽  
R. Esposito ◽  
S. Blétard ◽  
S. Di Costanzo ◽  
M. Perilli ◽  
...  
Keyword(s):  
Genome Sequence ◽  
Catalytic Efficiency ◽  
The Other ◽  
Kinetic Characterization ◽  
Activity Profile ◽  
Gene Coding ◽  
Hydrolysis Of

ABSTRACTThe analysis of the genome sequence of Yersinia mollaretii (Y. mollaretii) ATCC 43969 indicates the presence of the blaYEM gene coding for YEM-1, a putative subclass B2 metallo-β-lactamase. The objectives of our work were to produce, purify and complete the kinetic characterization of YEM-1. Compared to the known subclass B2 metallo–β-lactamases, YEM-1 displayed a narrowest substrate profile since it is only able to hydrolyse imipenem with a high catalytic efficiency but not all the other carbapenems tested such as biapenem, meropenem, doripenem and ertapenem. A possible explanation of this peculiar activity profile is the presence of tyrosine 67 (loop L1), threonine 156 (loop L2) and serine 236 (loop L3) respectively. We showed that the substitution of Y67 broadened the activity profile of the enzyme for all carbapenems but still displayed a poor activity toward the other β-lactam classes.

scholarly journals Escherichia coli expression and characterization of α-amylase from Geobacillus thermodenitrificans DSM-465

2022 ◽  
Vol 82 ◽  
Author(s):  
A. Al-Amri ◽  
M. A. Al-Ghamdi ◽  
J. A. Khan ◽  
H. N. Altayeb ◽  
H. Alsulami ◽  
...  
Keyword(s):  
Pilot Scale ◽  
Docking Studies ◽  
Industrial Applications ◽  
Geobacillus Thermodenitrificans ◽  
E Coli ◽  
Gene Coding ◽  
Km Value ◽  
Escherichia Coli Expression ◽  
Hydrolysis Of

Abstract Alpha amylase, catalyzing the hydrolysis of starch is a ubiquitous enzyme with tremendous industrial applications. A 1698 bp gene coding for 565 amino acid amylase was PCR amplified from Geobacillus thermodenitrificans DSM-465, cloned in pET21a (+) plasmid, expressed in BL21 (DE3) strain of E. coli and characterized. The recombinant enzyme exhibited molecular weight of 63 kDa, optimum pH 8, optimum temperature 70°C, and KM value of 157.7µM. On pilot scale, the purified enzyme efficiently removed up to 95% starch from the cotton fabric indicating its desizing ability at high temperature. 3D model of enzyme built by Raptor-X and validated by Ramachandran plot appeared as a monomer having 31% α-helices, 15% β-sheets, and 52% loops. Docking studies have shown the best binding affinity of enzyme with amylopectin (∆G -10.59). According to our results, Asp 232, Glu274, Arg448, Glu385, Asp34, Asn276, and Arg175 constitute the potential active site of enzyme.

scholarly journals Influence of culture conditions on the production of extracellular polymeric substances (EPS) by Arthrospira platensis

2020 ◽  
Author(s):  
Mariana Barbalho Silva ◽  
Edwin G. Azero ◽  
Cláudia M. L. L. Teixeira ◽  
Cristina T. Andrade
Keyword(s):  
Food Industry ◽  
Extracellular Polymeric Substances ◽  
Biomass Concentration ◽  
Arthrospira Platensis ◽  
Culture Conditions ◽  
Photon Flux ◽  
Photon Flux Density ◽  
Dilute Solution ◽  
Property A

Abstract Arthrospira platensis is a cyanobacterium that exhibits a large biotechnological interest at food industry because its high protein content, pigments, lipids and carbohydrates. The extracellular polymeric substances (EPS) are co-products of secondary metabolism that present thickening or gelling property. A 3-level factorial design was used to study the combined effect of different nitrate concentrations and photon flux density (PFD) to evaluate the biomass and EPS production by Arthrospira platensis. Characterization of the EPS produced the rheological behavior were also evaluated. The best result for biomass production was obtained at condition 6 (2 g.L-1 NaNO3 and 600 µE.m-2s-1) leading a biomass concentration of 1.292 mg.L-1. Condition 1 (0.25 g.L-1NaNO3 and 200 µE.m-2s-1) produce the major EPS content (111 mg.g-1) followed by condition 9 (2 g.L-1NaNO3 and 1000 µE.m-2s-1). Rheological studies performed for the product at 5 and 10g.L-1 concentrations revealed a dilute solution behavior.

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