Purification of a fungal endo-β-glucanase with high activity on barley β-glucan

Abstract

An endo-13-glucanase was purified from a commercial enzyme preparation of fungal origin by ammonium sulphate fractionation, ion exchange chromatography, and gel filtration followed by preparative isoelectric focusing. The enzyme was homogeneous in sedimentation equilibrium analysis from which the molecular weight was determined to be 23.500 in agreement with the value 23.653 calculated on the basis of the amino acid composition. The enzyme did not contain carbohydrate and a molecular weight of 24.000 estimated by polyacrylamide gel electrophoresis in dodecyl sulphate after 2-mercaptoethanol treatment, indicated that it consisted of a single polypeptide chain. It had an isoelectric point of 4.47. The enzyme rapidly decreased the specific viscosity of barley fl-glucan with a small concomitant increase in reducing sugar. Its activity toward carboxymethyl-cellulose, acid-swollen cellulose, and a mixture of cellodextrins was low. Laminarin, carboxymethyl-pachyman, cellobiose, and p-nitrophenyl-13-D-glucoside were not hydrolyzed. The Km for hydrolysis of barley 13-glucan and carboxymethyl-cellulose was 1.8 and I 1 mg/ml, respectively, the corresponding molecular activities being 7750 and 750 equivalents of glucosidic bonds hydrolyzed per min per mole of enzyme. The products of exhaustive hydrolysis of barley !%glucan were 4.3% glucose, 4.2% disaccharide, 72.7% trisaccharide, and 18.8% tetrasaccharide, higher oligomers and polymers were absent. The enzymic activity was completely destroyed by treatment with N-bromosuccinimide but was insensitive to EDTA, sulfhydryl modifying reagents, and glucono-l,5-1actone.

Secretome analysis of Trichoderma reesei CICC41495 for degradation of arabinoxylan in malted barley

“…The assay for endo‐ β ‐glucanase was performed as for xylanase, except that the substrate was replaced by barley 1,3;1,4‐ β ‐glucan. The amount of reducing sugar produced was quantified using a standard curve constructed with glucose .…”

Immobilization of β-glucanase and studies on its degradation of barley β-glucan

“…Prior to immobilization the precipitate was redissolved in deionized water to give the final volume of the crude glucanase employed unless otherwise stated. Homogeneous endo-l,4q3-glucanase isolated from Glucanase GV-L as described elsewhere (32) was used in a single experiment.…”

“…The commercial 13-glucanase contains several enzymes capable of catalyzing the hydrolysis of 13-glucosidic bonds, but the endo-l,4-13-glucanase described elsewhere is responsible for more than half of the total activity against barley 13-glucan as determined by the release of reducing sugar (32). This enzyme has an isoelectric point around pH 4.5 (32) and thus the higher recovery of activity in the preparation of the aminolyzed nylon-13-glucanase might be due to the electrostatically favoured binding of the anionic endo-l,4-13-glucanase to the cationic support material.…”

Entrapment of chemical derivatives of glycoamylase in calcium alginate gels

“…The conditions were identical with those used for reaction with proteins and glycyl-L-leucine amide were present in a 5-16 fold molar excess with respect to sugar units. Unreacted amino compound was removed by dialysis and the amount of polymer-bound dipeptide amide was measured by amino acid analysis (27).…”

“…The protein content of gel particles was determined after lyophilization by amino acid analysis (27), norleucine being added as internal standard.…”