Purification and characterization of β-Fructosidase with inulinase activity from Aspergillus niger - 245

Aspergillus niger - 245, a strain isolated from soil samples showed good β-fructosidase activity when inoculated in medium formulated with dahlia extract tubers. The enzyme was purified by precipitation in ammonium sulphate and percolated in DEAE-Sephadex A-50 and CM-cellulose columns, witch showed a single peack in all the purification steps, maintaining the I/S ratio between 0.32 to, 0.39. Optimum pH for inulinase activity (I) was between 4.0 - 4.5 and for invertase activity (S) between 2.5 and 5.0. The optimum temperature was 60O.C for both activities and no loss in activity was observed when it was maintained at this temperature for 30 min. The Km value was 1.44 and 5.0, respectively, for I and S and Vm value 10.48 and 30.55, respectively. The I activity was strongly inhibited by Hg2+ and Ag+ and 2 x 10-3 M of glucose, but not by fructose at the same concentration. The enzyme showed an exo-action mechanism, acting on the inulin of different origins. In assay conditions total hydrolysis of all the frutans was obtained, although it has shown larger activity on the chicory inulin than that one from artichoke Jerusalem and dahlia, in the first 30 min. The obtained results suggested that the enzyme presented good potential for industrial application in the preparing the fructose syrups

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Invertase Production by Fungi, Characterization of Enzyme Activity and Kinetic Parameters

Revista de Chimie ◽

10.37358/rc.17.10.5856 ◽

2017 ◽

Vol 68 (10) ◽

pp. 2205-2208 ◽

Cited By ~ 1

Author(s):

Gabi Mirela Matei ◽

Sorin Matei ◽

Maria Pele ◽

Flavia Dumitrescu ◽

Adrian Matei

Keyword(s):

Aspergillus Niger ◽

Kinetic Parameters ◽

Invertase Activity ◽

Enzyme Concentration ◽

Maximal Velocity ◽

Aspergillus Wentii ◽

Penicillium Aurantiogriseum ◽

The Relationship ◽

Menten Constant

The present paper presents the results of research carried out on 14 fungal isolates of various origins aiming to select new efficient sources for invertase production for further biotechnological application. Aspergillus flavus presented the highest protein content and Aspergillus niger, the most intense invertase activity. The relationship between enzyme concentration and enzymatic activity at 0.25 mM mL-1sucrose as substrate assayed for successive decimal dilutions of Aspergillus niger enzyme ranging from 0.1 to 1mlLrevealed a linear correspondence between 0.1 and 0.5mL. The kinetic parameters Michaelis-Menten constant (Km) and maximal velocity (Vmax) for invertase activity of Aspergillus niger, Penicillium aurantiogriseum, Aspergillus wentii and Rhizopus stolonifer were calculated. The determination coefficients R2 calculated from Lineweaver-Burk plots presented values very close or equal to 1.

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Database mining and transcriptional analysis of genes encoding inulin-modifying enzymes of Aspergillus niger

Microbiology ◽

10.1099/mic.0.29051-0 ◽

2006 ◽

Vol 152 (10) ◽

pp. 3061-3073 ◽

Cited By ~ 43

Author(s):

Xiao-Lian Yuan ◽

Coenie Goosen ◽

Harrie Kools ◽

Marc J. E. C. van der Maarel ◽

Cees A. M. J. J van den Hondel ◽

...

Keyword(s):

Aspergillus Niger ◽

Carbon Sources ◽

Soil Fungus ◽

Invertase Activity ◽

Transcriptional Analysis ◽

Database Mining ◽

Inulinase Activity ◽

Genes Encoding ◽

Catabolite Repressor ◽

Plant Storage

As a soil fungus, Aspergillus niger can metabolize a wide variety of carbon sources, employing sets of enzymes able to degrade plant-derived polysaccharides. In this study the genome sequence of A. niger strain CBS 513.88 was surveyed, to analyse the gene/enzyme network involved in utilization of the plant storage polymer inulin, and of sucrose, the substrate for inulin synthesis in plants. In addition to three known activities, encoded by the genes suc1 (invertase activity; designated sucA), inuE (exo-inulinase activity) and inuA/inuB (endo-inulinase activity), two new putative invertase-like proteins were identified. These two putative proteins lack N-terminal signal sequences and therefore are expected to be intracellular enzymes. One of these two genes, designated sucB, is expressed at a low level, and its expression is up-regulated when A. niger is grown on sucrose- or inulin-containing media. Transcriptional analysis of the genes encoding the sucrose- (sucA) and inulin-hydrolysing enzymes (inuA and inuE) indicated that they are similarly regulated and all strongly induced on sucrose and inulin. Analysis of a ΔcreA mutant strain of A. niger revealed that expression of the extracellular inulinolytic enzymes is under control of the catabolite repressor CreA. Expression of the inulinolytic enzymes was not induced by fructose, not even in the ΔcreA background, indicating that fructose did not act as an inducer. Evidence is provided that sucrose, or a sucrose-derived intermediate, but not fructose, acts as an inducer for the expression of inulinolytic genes in A. niger.

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Characterization of human tissue carnosinase

Biochemical Journal ◽

10.1042/bj2280653 ◽

1985 ◽

Vol 228 (3) ◽

pp. 653-660 ◽

Cited By ~ 75

Author(s):

J F Lenney ◽

S C Peppers ◽

C M Kucera-Orallo ◽

R P George

Keyword(s):

High Resolution ◽

Human Tissue ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Relative Molecular Mass ◽

Human Tissues ◽

Km Value ◽

Assay Conditions ◽

Hog Kidney

Human tissue carnosinase (EC 3.4.13.3) had optimum activity at pH9.5 and was a cysteine peptidase, being activated by dithiothreitol and inhibited by p-hydroxymercuribenzoate. By optimizing assay conditions, the activity per g of tissue was increased 10-fold compared with values in the literature. The enzyme was present in every human tissue assayed and was entirely different from serum carnosinase. Highly purified tissue carnosinase had a broader specificity than hog kidney carnosinase. Although tissue carnosinase was very strongly inhibited by bestatin, it did not hydrolyse tripeptides, and thus appears to be a dipeptidase rather than an aminopeptidase. It had a relative molecular mass of 90 000, an isoelectric point of 5.6, and a Km value of 10 mM-carnosine. Two forms of kidney and brain carnosinase were separated by high-resolution anion-exchange chromatography, although only one form was detected by various electrophoretic methods. Homocarnosinase and Mn2+-independent carnosinase were not detected in human tissues, although these enzymes are present in rat and hog kidney.

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Purification and characterization of a chlorogenic acid hydrolase from Aspergillus niger catalysing the hydrolysis of chlorogenic acid

Journal of Biotechnology ◽

10.1016/j.jbiotec.2004.07.009 ◽

2005 ◽

Vol 115 (1) ◽

pp. 47-56 ◽

Cited By ~ 31

Author(s):

Michèle Asther ◽

Maria Isabel Estrada Alvarado ◽

Mireille Haon ◽

David Navarro ◽

Marcel Asther ◽

...

Keyword(s):

Aspergillus Niger ◽

Chlorogenic Acid ◽

Acid Hydrolase ◽

Purification And Characterization ◽

Hydrolysis Of

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

Brazilian Journal of Biology ◽

10.1590/1519-6984.239449 ◽

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.

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Immobilization of Naringinase from Penicillium decumbens on Chitosan Microspheres for Debittering Grapefruit Juice

Molecules ◽

10.3390/molecules24234234 ◽

2019 ◽

Vol 24 (23) ◽

pp. 4234 ◽

Cited By ~ 9

Author(s):

Joanna Bodakowska-Boczniewicz ◽

Zbigniew Garncarek

Keyword(s):

Grapefruit Juice ◽

Immobilized Enzyme ◽

Maximum Activity ◽

Chitosan Microspheres ◽

Optimum Ph ◽

Potential Applicability ◽

Km Value ◽

Hydrolysis Of ◽

Thermal Stability Of

Naringinase is an enzyme complex which exhibits α-l-rhamnosidase and β-d-glucosidase activity. This enzymatic complex catalyzes the hydrolysis of naringin (4′,5,7-trihydroxy flavanone 7-rhamnoglucoside), the main bittering component in grapefruit. Reduction of the level of this substance during the processing of juice has been the focus of many studies. The aim of the study was the immobilization of naringinase on chitosan microspheres activated with glutaraldehyde and, finally, the use of such immobilized enzyme for debittering grapefruit juice. The effect of naringinase concentration and characterization of the immobilized enzyme compared to the soluble enzyme were investigated. The maximum activity was observed at optimum pH 4.0 for both free and immobilized naringinase. However, the optimum temperature was shifted from 70 to 40 °C upon immobilization. The KM value of the immobilized naringinase was higher than that of soluble naringinase. The immobilization did not change the thermal stability of the enzyme. The immobilized naringinase had good operational stability. This preparation retained 88.1 ± 2.8% of its initial activity after ten runs of naringin hydrolysis from fresh grapefruit juice. The results indicate that naringinase immobilized on chitosan has potential applicability for debittering and improving the sensory properties of grapefruit juices.

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