β-1,3-glucan hydrolases from Euglena gracilis

Barras, D. R.; Stone, Bruce

doi:10.1016/0005-2744(69)90253-8

Cited by 52 publications

(13 citation statements)

References 23 publications

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“…The relatively slow hydrolysis of CM-pachyman is presumably due to the interference by CM substituents with productive enzyme-substrate interactions, as the rate of hydrolysis decreases as the DS increases. This type of steric hindrance has been previously noted with the endo-GNs from N. glutinosa [15] and barley [23], the exo-GN of the unicellular alga Euglena gracilis [59], and in the hydrolysis of CM-cellulose by cellulases [60]. Similarly, the highly branched structure of the yeast (1 -+3) (1 -+ 6)-fl-D-glucan [44] may prevent enzyme binding by the A. persicinum GNs.…”

Section: Discussionmentioning

confidence: 71%

Purification and characterization of three extracellular (1→3)-β-d-glucan glucohydrolases from the filamentous fungus Acremonium persicinum

Pitson

Seviour

McDougall

et al. 1995

Biochemical Journal

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Three (1-->3)-beta-D-glucanases (GNs) were isolated from the culture filtrates of the filamentous fungus Acremonium persicinum and purified by (NH4)2SO4 precipitation followed by anion-exchange and gel-filtration chromatography. Homogeneity of the purified proteins was confirmed by SDS/PAGE, isoelectric focusing and N-terminal amino acid sequencing. All three GNs (GN I, II and III) are non-glycosylated, monomeric proteins with apparent molecular masses, estimated by SDS/PAGE, of 81, 85 and 89 kDa respectively. pI values for the three enzymes are 5.3, 5.1, and 4.4 respectively. The pH optimum for GN I is 6.5, and 5.0 for GN II and III. All three purified enzymes displayed stability over the pH range 4.5-10.0. Optimum activities for GN I, II and III were recorded at 65, 55 and 60 degrees C respectively, with both GN II and III having short-term stability up to 50 degrees C and GN I up to 55 degrees C. The purified GNs have high specificity for (1-->3)-beta-linkages and hydrolysed a range of (1-->3)-beta- and (1-->3)(1-->6)-beta-D-glucans, with laminarin from Laminaria digitata being the most rapidly hydrolysed substrate of those tested. K(m) values for GN I, II, and III against L. digitata laminarin were 0.1, 0.23 and 0.22 mg/ml respectively. D-Glucono-1,5-lactone does not inhibit any of the three GNs, some metals ions are mild inhibitors, and N-bromosuccinimide and KMnO4 are strong inhibitors. All three GNs acted in an exo-hydrolytic manner, determined by the release of alpha-glucose as the initial and major product of hydrolysis of (1-->3)-beta-D-glucans, and confirmed by viscometric analysis and the inability to cleave periodate-oxidized laminarin, and may be classified as (1-->3)-beta-D-glucan glucohydrolases (EC 3.2.1.58).

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Section: Discussionmentioning

confidence: 71%

Purification and characterization of three extracellular (1→3)-β-d-glucan glucohydrolases from the filamentous fungus Acremonium persicinum

Pitson

Seviour

McDougall

et al. 1995

Biochemical Journal

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“…Though enzyme I was highly active against pNPG, its ability to hydrolyse oligo-and polysaccharides such as pachyman, yeast (1 3)-P-glucan and pustulan (Table 4) suggest that its activity is closer to that of an exo-(1 -, 3)-P-~-glucanase such as the one described by Barras & Stone (1969) than to a typical P-glucosidase. Enzyme I1 shows some peculiarities such as a broad pH range of activity, stimulation by glucuronolactone and 2-deoxy-~-glucose and mode of action, releasing laminaritetraose initially and degrading it to glucose on prolonged incubation.…”

Section: Discussionmentioning

confidence: 95%

“…When crude extracts were used, the reactions were stopped by addition of 0.5 ml 5 % (w/v) ZnSO, plus 0 -5 mlO.3 M-Ba(OH),, the reaction products being determined in the supernatants obtained after centrifugation at 2000 g for 5 min. The activity on laminarin was estimated by measuring the released glucose or equivalent reducing sugar according to Bruss & Black (1978) and Nelson (1944), respectively. Glucose and reducing sugars in the presence of SH-agents were estimated as described by Davies & Wayman (1973).…”

Section: N O T a R I Omentioning

confidence: 99%

-Glucanases from Candida albicans: Purification, Characterization and the Nature of their Attachment to Cell Wall Components

Notario

1982

Microbiology

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P-Glucanase activities were found associated with Candida albicans and their culture fluids. Mild acid treatment of the organisms led to rapid inactivation of P-glucanase activities, the degree of loss increasing with the age of the cultures; the results suggested an extracytoplasmic location of the cell-associated enzymes. Most of the P-glucanase activities were associated with the cell walls in organisms phenotypically resistant to amphotericin B methyl ester (AME).Two proteins (I and 11) exhibiting P-glucanase activity were isolated and purified by conventional procedures from cell-free extracts, cell-wall autolysates and culture fluids of C. albicans sensitive and phenotypically resistant to AME. The purified enzymes appeared homogeneous on isoelectric focusing, gel electrophoresis and ultracentrifugation, with molecular weights of 150 000 (I) and 49 000 (11). Both enzymes hydrolysed cell walls purified from AME-sensitive and phenotypically resistant organisms, but showed different substrate specificities and patterns of activity. Enzyme I1 hydrolysed (1 -, 3)-P-glucans by an endolytic mechanism releasing laminaritetraose as the initial product. Glucose was the only product released by enzyme I. The properties of the individual enzymes were unaffected by their localization or the age of the culture of the organisms.The loosening of the polysaccharide packing by ultrasonic treatment of cell walls purified from AME-resistant organisms increased the P-glucanase activities bound to the walls, but did not solubilize them. Autolysis of cell walls released 58 to 66% of their P-glucanase activity in 20 h, but no further release was attained on prolonged incubation. The amount of P-glucanase activity released by autolysis was increased by a variety of pretreatments. Diethyl pyrocarbonate inhibited Pglucanase activity and prevented autolysis. Evidence is presented indicating that interactions with lipids, polysaccharides and other cell wall proteins may be involved in the control of the activity of the cell wall-associated P-glucanases in organisms phenotypically resistant to AME. I N T R O D U C T I O NA disequilibrium between the synthesis of the cell-wall P-glucan and its breakdown by endogenous P-glucanases seems to underlie the onset and development of phenotypic resistance to amphotericin B methyl ester (AME) in Candida albicans. In resistant organisms (cultures in the stationary phase of growth) most of the P-glucanase activity was found in a latent form bound to the cell wall, and could be activated by a number of treatments (SH-reducing agents, partial enzymic digestion of the walls, reduced aeration of the cultures) which simultaneously decreased the resistance of the organisms to the antibiotic. Other t Present address:

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“…Four sequences could be identified in the transcriptome which encode candidate GT48 b-1,3-glucan synthases, three of which are closely related to each other (94% identity). 44 Both endo-and exo-b-1,3-glucan hydrolases have been biochemically identified in Euglena, 45,46 and there are many glycoside hydrolases present in the Euglena transcriptome that belong to the various GH families capable of degrading b-1,3glucans. Enzymes of the GT2 family can also participate in the biosynthesis of b-1,3and b-1,4-glycans, such as callose, chitin, and cellulose, 40 and thus also represent potential candidates for paramylon synthase.…”

Section: Carbohydrate-active Enzymesmentioning

confidence: 99%

The transcriptome of Euglena gracilis reveals unexpected metabolic capabilities for carbohydrate and natural product biochemistry

et al. 2015

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Euglena gracilis is a highly complex alga belonging to the green plant line that shows characteristics of both plants and animals, while in evolutionary terms it is most closely related to the protozoan parasites Trypanosoma and Leishmania. This well-studied organism has long been known as a rich source of vitamins A, C and E, as well as amino acids that are essential for the human diet. Here we present de novo transcriptome sequencing and preliminary analysis, providing a basis for the molecular and functional genomics studies that will be required to direct metabolic engineering efforts aimed at enhancing the quality and quantity of high value products from E. gracilis. The transcriptome contains over 30,000 protein-encoding genes, supporting metabolic pathways for lipids, amino acids, carbohydrates and vitamins, along with capabilities for polyketide and non-ribosomal peptide biosynthesis. The metabolic and environmental robustness of Euglena is supported by a substantial capacity for responding to biotic and abiotic stress: it has the capacity to deploy three separate pathways for vitamin C (ascorbate) production, as well as producing vitamin E (α-tocopherol) and, in addition to glutathione, the redox-active thiols nor-trypanothione and ovothiol.

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β-1,3-glucan hydrolases from Euglena gracilis

Cited by 52 publications

References 23 publications

Purification and characterization of three extracellular (1→3)-β-d-glucan glucohydrolases from the filamentous fungus Acremonium persicinum

Purification and characterization of three extracellular (1→3)-β-d-glucan glucohydrolases from the filamentous fungus Acremonium persicinum

-Glucanases from Candida albicans: Purification, Characterization and the Nature of their Attachment to Cell Wall Components

The transcriptome of Euglena gracilis reveals unexpected metabolic capabilities for carbohydrate and natural product biochemistry

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