Enzyme-Substrate Relationships Among β-Glucan Hydrolases

Barras, D. R.; Moore, Anne; Stone, Bruce

doi:10.1021/ba-1969-0095.ch008

Cited by 40 publications

(13 citation statements)

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“…Uronic acids were determined by the corrected carbazole test, and the sugar compositions were computed as mole per cent of the total carbohydrate (24). 3. Chromatography of fraction C on DEAE-Sephadex A-25.…”

Section: Isolationmentioning

confidence: 99%

The Structure of Plant Cell Walls

Bauer¹,

Talmadge²,

Keegstra³

et al. 1973

Plant Physiol.

360

131

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The molecular structure, chemical properties, and biological function of the xyloglucan polysaccharide isolated from cell walls of suspension-cultured sycamore (Acer pseudoplatanus) cells are described. The sycamore wall xyloglucan is compared to the extracellular xyloglucan secreted by suspension-cultured sycamore cells into their culture medium and is also compared to the seed "amyloid" xyloglucans.Xyloglucan-or fragments of xyloglucan-and acidic fragments of the pectic polysaccharides are released from endopolygalacturonase-pretreated sycamore walls by treatment of these walls with 8 M urea, endoglucanase, or 0.5 N NaOH. Some of the xyloglucan thus released is found to cochromatograph with the acidic pectic fragments on diethylaminoethyl Sephadex. The chemical or enzymic treatments required for the release of xyloglucan from the walls and the cochromatography of xyloglucan with the acidic pectic fragments indicate that xyloglucan is covalently linked to the pectic polysaccharides and is noncovalently bound to the cellulose fibrils of the svcamore cell wall.The molecular structure of sycamore xyloglucan was characterized by methylation analysis of the oligosaccharides obtained by endoglucanase treatment of the polymer. The struc. ture of the polymer is based on a repeating heptasaccharide unit which consists of 4 residues of 8-1-4-linked glucose and 3 residues of terminal xylose. A single xylose residue is glycosidically linked to carbon 6 of 3 of the glucosyl residues.In the first paper of this series (24), recently developed techniques for the methylation analysis of polysaccharides were used in order to study the structure of the primary cell wall of suspension-cultured sycamore cells and in order to characterize structurally the defined fragments released from these walls by a highly purified endopolygalacturonase. This study indicated

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Section: Isolationmentioning

confidence: 99%

The Structure of Plant Cell Walls

Bauer¹,

Talmadge²,

Keegstra³

et al. 1973

Plant Physiol.

360

131

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“…The unique action of this enzyme precludes its ability to hydrolyse repeating sequences of P1,3-or Pl,6linked glucans as in laminarin and carboxymethylcellulose, respectively. In this respect the B. subtilis enzyme behaves like that isolated from malted barley (Barras et al, 1969;Manners & Wilson, 1976). There is considerable interest in the brewing industry in enzymes produced by bacteria and fungi, and their ability to complement the enzymes present in malt (Godfrey, 1983a).…”

Section: Introductionmentioning

confidence: 99%

Cloning and Expression of a Bacillus subtilis Endo-1,3-1,4- -D-Glucanase Gene in Escherichia coli K12

Hinchliffe¹

1984

Microbiology

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EcoRI fragments of DNA from Bacillus subtilis NCIB 8565, a high producer of an endo-l,3-1,4-P-D-glucanase, were 'shot-gun' cloned in the plasmid vector pBR325. A 3.5 kb insert, carrying single restriction sites for AuaI, BglII, ClaI, PuuI and PuuII, was shown to direct the synthesis of P-glucanase in Escherichia coli K 12. Enzyme activity was demonstrated in extracellular fractions of E. coli harbouring the P-glucanase gene; however, the largest proportion ( > 50%) of total enzyme activity was periplasmic in location. P-Glucanase activity and cellular location were independent of the orientation of the 3.5 kb fragment in pBR325.

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“…Reactions were initiated by adding enzyme (1)(2)(3)(4)(5)(6)(7)(8)(9)(10) Iul) to an otherwise complete reaction mixture which had been prewarmed at 30 C for 10 min.…”

mentioning

confidence: 99%

“…The low level of activity with p-nitrophenyl-,8-D-xylopyranoside was expected. The ability of 8-glucosidases to hydrolyze fl-xylopyranosides is known, and may be due to the structural similarity of f8-xylopyranoside and ,8-glucopyranoside (6).…”

mentioning

confidence: 99%

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Host-Pathogen Interactions

Cline

Albersheim²

1981

Plant Physiol.

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The fact that fungal glucans will stimulate soybeans to accumulate phytoalexins prompted an investigation of soybean cell .8-1,3-glucanases and /3-glucosidases, as well as the ability of these enzymes to hydrolyze the fungal glucans. Several /8-1,3-glucanases and /3-glucosidases can be solubilized from the walls of suspension-cultured soybean cells by treatment with 1.0 molar sodium acetate buffer. An enzyme, which has been termed /3-glucosylase I, is the dominant /3-1,3-glucanase in the cell wall extracts.Utilizing CM-Sephadex chromatography, hydroxylapatite chromatography, and affinity chromatography, ,B-glucosylase I has been purified 71-fold, with 39% recovery, from the mixture of cell wall enzymes. The affminty chromatography column material was prepared by covalently attaching paminophenyl-1-/3-D-glucopyranoside, an analog of a /-glucosylase I substrate, to Sepharose. fl-Glucosylase I, purified by this procedure, yields a single band on isoelectric focusing gels (pH 8.9). However, the purified /3-glucosylase I yields a darkly-staining protein band at an apparent molecular weight of 69,000 and several ligtly-staining protein bands in sodium dodecyl sulfate polyacrylamide gels. Additional purification procedures fail to remove these lightly-staining protein bands. fl-Glucosylase I will hydrolyze the /8-glucan substrates, laminarin (3-linked) and llchenan (3-and 4-linked), and therefore, possesses /8-glucanase activity. Studies of the progressive hydrolysis of laminarin by /3-glucosylase I demonstrate that the enzyme hydrolyzes polysaccharide substrates in an exo manner. /3-Glucosylase I will also hydrolyze a variety of low molecular weight /8-glucosides including various /3-linked diglucosides. Thus, /8-glucosylase I also possesses /8-glucosidase activity.Several lines of evidence are presented that the /8-glucanase and the ,B-glucosidase activities exhibited by purified /8-glucosylase I preparations are catalyzed by the same enzyme. This evidence includes inhibition studies which indicate that the /-glucanase and the /8-glucosidase activities of /3-glucosylase I are catalyzed at the same active site. /8-Glucosylase I will also catalyze glucosyl transfer. This catalytic activity is responsible for the observed ability of the enzyme to synthesize di-and trisaccharides from laminarin. The disaccharides formed by /8-glucosylase I-catalyzed transglucosylation are the ,B-anomers of the 6-, 4-, 3-, and 2-linked diglucosides in the relative proportions of 101:1:1. The ability of ,B-glucosylase I to catalyze glucosyl transfer indicates that /8-glucosylase

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Enzyme-Substrate Relationships Among β-Glucan Hydrolases

Cited by 40 publications

References 0 publications

The Structure of Plant Cell Walls

The Structure of Plant Cell Walls

Cloning and Expression of a Bacillus subtilis Endo-1,3-1,4- -D-Glucanase Gene in Escherichia coli K12

Host-Pathogen Interactions

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