Novel approaches to the automated assay of β-glucanase and lichenase activity.
We report herein the development of a novel assay procedure for the measurement of β-glucanase and lichenase (EC 3.2.1.73) in crude enzyme extracts. Two assay formats based on a) a direct cleavage or b) an enzyme coupled substrate were initially investigated. The 'direct cleavage' substrate, namely 4,6-O-benzylidene-2-chloro-4-nitrophenyl-β-31-cellotriosyl-β-glucopyranoside (MBG4), was found to be the more generally applicable reagent. This substrate was fully characterised using a crude malt β-glucanase extract, a bacterial lichenase (Bacillus sp.) and a non-specific endo-1,3(4)-β-glucanase from Clostridium thermocellum (EC 3.2.1.6). Standard curves were derived that allow the assay absorbance response to be directly converted to β-glucanase/lichenase activity on barley β-glucan. The specificity of MBG4 was confirmed by analysing the action of competing glycosyl hydrolases that are typically found in malt on the substrate. Manual and automated assay formats were developed for the analysis of a) β-glucanase in malt flour and b) lichenase enzyme extracts and the repeatability of these assays was fully investigated.
Megazyme International Ireland, Bray Business Park, Southern Cross Road, Bray, County Wicklow, Ireland. Electronic address: david@megazyme.com.
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Mangan
Megazyme International Ireland, Bray Business Park, Southern Cross Road, Bray, County Wicklow, Ireland.
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Liadova
Megazyme International Ireland, Bray Business Park, Southern Cross Road, Bray, County Wicklow, Ireland.
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R
Ivory
Megazyme International Ireland, Bray Business Park, Southern Cross Road, Bray, County Wicklow, Ireland.
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BV
McCleary
D057205:Automation, Laboratory; D001412:Bacillus subtilis / Q000201:enzymology; D001426:Bacterial Proteins / Q000378:metabolism; D048013:Acetivibrio thermocellus / Q000201:enzymology; D044644:Endo-1,3(4)-beta-Glucanase / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism*; D001467:Hordeum / Q000201:enzymology; D013379:Substrate Specificity; D047071:beta-Glucans / Q000032:analysis*
D001426:Bacterial Proteins; D047071:beta-Glucans; D006026:Glycoside Hydrolases; D044644:Endo-1,3(4)-beta-Glucanase; C028020:licheninase
4,6-O-benzylidene-2-chloro-4-nitrophenyl-β-3(1)-cellotriosyl-β-glucopyranoside; Assay procedure; Colourimetric; Lichenase; MBG4; Oligosaccharides; β-glucanase