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| Activity Determination of β Glucosidase by a Turn on Fluorescent Method Based on Reduction of Resazurin |
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| DOI： |
| KeyWord:β-glucosidase activity resazurin resorufin ascorbic acid fluorescence determination |
| Author | Institution |
| CHENG Xin，ZHANG Heng，MA Ji-mei |
College of Science,Huazhong Agricultural University |
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| Abstract: |
| A simple fluorescence analytical method was established for the quantitation of activity of β-glucosidase.The synthetic 2-O-β-glucopyranosyl ascorbic acid(AA-2βG) was used as the enzymatic substrate.β-Glucosidase catalyzed the hydrolysis reaction and released ascorbic acid,which would reduce non fluorescent resazurin to strongly fluorescent resorufin.Thus,β-glucosidase activity would be reflected by the fluorescent intensity variation of the system.Firstly,a validation assay was carried out to confirm the possibility to detect ascorbic acid taking advantage of reductive reaction.There was a linear relationship for the increased fluorescent intensity with the concentration of ascorbic acid in the range of 3-400 μmol/L with a correlation coefficient(r2) of 0.981,while β-glucosidase activity was successfully determined by measuring fluorescent intensity at 585 nm.Results showed that there also existed a good linear relationship between the increased fluorescent intensity and concentration of β-glucosidase in range of 1-30 U/L,with a correlation coefficient(r2) of 0.995 and a detection limit of 1.0 U/L.With the characteristics of simplicity,sensitivity and large detection range,the method could realize the quantitative detection on β glucosidase activity. |
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