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. 1981 Jul;68(1):207–220. doi: 10.1104/pp.68.1.207

Host-Pathogen Interactions

XVI. PURIFICATION AND CHARACTERIZATION OF A β-GLUCOSYL HYDROLASE/TRANSFERASE PRESENT IN THE WALLS OF SOYBEAN CELLS 1

Kenneth Cline 1,2, Peter Albersheim 1,3
PMCID: PMC425917  PMID: 16661872

Abstract

The fact that fungal glucans will stimulate soybeans to accumulate phytoalexins prompted an investigation of soybean cell β-1,3-glucanases and β-glucosidases, as well as the ability of these enzymes to hydrolyze the fungal glucans. Several β-1,3-glucanases and β-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 β-glucosylase I, is the dominant β-1,3-glucanase in the cell wall extracts. Utilizing CM-Sephadex chromatography, hydroxylapatite chromatography, and affinity chromatography, β-glucosylase I has been purified 71-fold, with 39% recovery, from the mixture of cell wall enzymes. The affinity chromatography column material was prepared by covalently attaching p-aminophenyl-1-β-d-glucopyranoside, an analog of a β-glucosylase I substrate, to Sepharose. β-Glucosylase I, purified by this procedure, yields a single band on isoelectric focusing gels (pH 8.9). However, the purified β-glucosylase I yields a darkly-staining protein band at an apparent molecular weight of 69,000 and several lightly-staining protein bands in sodium dodecyl sulfate polyacrylamide gels. Additional purification procedures fail to remove these lightly-staining protein bands.

β-Glucosylase I will hydrolyze the β-glucan substrates, laminarin (3-linked) and lichenan (3- and 4-linked), and therefore, possesses β-glucanase activity. Studies of the progressive hydrolysis of laminarin by β-glucosylase I demonstrate that the enzyme hydrolyzes polysaccharide substrates in an exo manner. β-Glucosylase I will also hydrolyze a variety of low molecular weight β-glucosides including various β-linked diglucosides. Thus, β-glucosylase I also possesses β-glucosidase activity.

Several lines of evidence are presented that the β-glucanase and the β-glucosidase activities exhibited by purified β-glucosylase I preparations are catalyzed by the same enzyme. This evidence includes inhibition studies which indicate that the β-glucanase and the β-glucosidase activities of β-glucosylase I are catalyzed at the same active site. β-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 β-glucosylase I-catalyzed transglucosylation are the β-anomers of the 6-, 4-, 3-, and 2-linked diglucosides in the relative proportions of 10:1:1:1. The ability of β-glucosylase I to catalyze glucosyl transfer indicates that β-glucosylase I is biochemically more similar to previously studied β-glucosidases than to β-glucanases. This conclusion is supported by the observation that β-glucosylase I is strongly inhibited by 1,5-d-gluconolactone, an inhibitor of β-glucosidases but not of β-glucanases.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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