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采用毛细管电泳激光诱导荧光检测预毛细酶分析法测定不同土壤中的β-葡萄糖苷酶活性。

Determination of the beta-glucosidase activity in different soils by pre capillary enzyme assay using capillary electrophoresis with laser-induced fluorescence detection.

机构信息

INQUISAL, Department of Chemistry, National University of San Luis, CONICET, Chacabuco y Pedernera, D5700BWS San Luis, Argentina.

出版信息

J Fluoresc. 2010 Mar;20(2):517-23. doi: 10.1007/s10895-009-0575-7. Epub 2009 Dec 22.

Abstract

Enzyme activities can provide indication for quantitative changes in soil organic matter (SOM). It is known that the activities of most enzymes increase as native SOM content reflecting larger microbial communities and stabilization of enzymes on humic materials. Beta-glucosidase (beta-Glu) activities have been frequently used as indicators of changes in quantity and quality of SOM. In this study we propose a simple and very sensitive method, which has lower limit of detection compared with classic spectrophotometric method with the aim of determinate the beta-Glu activity in soil samples using Fluorescein mono-beta-D-glucopyranoside (FMGlc) as a substrate. The fluorescein released by the enzymatic reaction was quantified by capillary electrophoresis-laser induced fluorescence (CE-LIF) method. The background electrolyte (BGE) consisted in 40 mM phosphate buffer, pH 6. The LOD and LOQ for fluorescein were 1.3 10(-7) mg mL(-1) and 6.4 10(-6) mg mL(-1), respectively. This work deals with the minimization of the mixture for the enzymatic reaction and with the optimization conditions of CE separation. To the best of our knowledge, this is the first time that an enzymatic activity was detected in soil using CE-LIF system.

摘要

酶活性可以为土壤有机质(SOM)的定量变化提供指示。已知大多数酶的活性随着原生 SOM 含量的增加而增加,这反映了更大的微生物群落和酶在腐殖质上的稳定性。β-葡萄糖苷酶(β-Glu)活性经常被用作 SOM 数量和质量变化的指标。在本研究中,我们提出了一种简单且非常灵敏的方法,与经典的分光光度法相比,该方法具有更低的检测限,旨在使用荧光素单-β-D-吡喃葡萄糖苷(FMGlc)作为底物来测定土壤样品中的β-Glu 活性。通过毛细管电泳-激光诱导荧光(CE-LIF)法定量测定酶反应释放的荧光素。背景电解质(BGE)由 40 mM 磷酸盐缓冲液,pH 值 6 组成。荧光素的 LOD 和 LOQ 分别为 1.3×10(-7)mg mL(-1)和 6.4×10(-6)mg mL(-1)。本工作涉及到酶反应混合物的最小化和 CE 分离条件的优化。据我们所知,这是首次使用 CE-LIF 系统在土壤中检测到酶活性。

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