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Novel approaches to the automated assay of beta-glucanase and lichenase activity

机译：自动测定β-葡聚糖酶和地衣酶活性的新方法

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摘要

We report herein the development of a novel assay procedure for the measurement of beta-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, namely4,6-O-benzylidene-2-chloro-4-nitrophenyl-beta-3(1)-cellotriosyl-beta-glucopyranoside (MBG4), was found to be the more generally applicable reagent. This substrate was fully characterised using a crude malt beta-glucanase extract, a bacterial lichenase (Bacillus sp.) and a non-specific endo-1,3(4)-beta-glucanase from Clostridium thermocellum (EC 3.2.1.6). Standard curves were derived that allow the assay absorbance response to be directly converted to beta-glucanaseflichenase activity on barley beta-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) beta-glucanase in malt flour and b) lichenase enzyme extracts and the repeatability of these assays was fully investigated. (C) 2016 Elsevier Ltd. All rights reserved.
机译：我们在此报告了一种用于测定粗酶提取物中的β-葡聚糖酶和地衣酶（EC 3.2.1.73）的新型测定方法的开发。最初研究了基于a）直接切割或b）酶偶联底物的两种测定形式。发现“直接裂解”底物，即4，6-O-亚苄基-2-氯-4-硝基苯基-β-3（1）-纤维二糖基-β-吡喃葡萄糖苷（MBG4）是更普遍适用的试剂。使用粗麦芽β-葡聚糖酶提取物，细菌地衣酶（芽孢杆菌属）和得自热纤梭菌的非特异性内源1,3（4）-β-葡聚糖酶（EC 3.2.1.6）充分表征该底物。得出标准曲线，该曲线允许将测定的吸光度响应直接转换为对大麦β-葡聚糖的β-葡聚糖酶。通过分析通常在底物上的麦芽中发现的竞争性糖基水解酶的作用，证实了MBG4的特异性。开发了手动和自动测定形式，用于分析a）麦芽粉中的β-葡聚糖酶和b）地衣酶提取物，并对这些测定的重复性进行了全面研究。 （C）2016 Elsevier Ltd.保留所有权利。

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