Fluorescent Cellulose Microfibrils As Substrate for the Detection of Cellulase Activity

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• 作者：William Helbert ; Henri Chanzy ; Tommy Lykke Husum ; Martin Schü ; lein ; and Steffen Ernst
• 刊名：Biomacromolecules
• 出版年：2003
• 出版时间：May 2003
• 年：2003
• 卷：4
• 期：3
• 页码：481 - 487
• 全文大小：194K
• 年卷期：v.4,no.3(May 2003)
• ISSN：1526-4602

文摘

To devise a sensitive cellulase assay based on substrates having most of the physical characteristics ofnative cellulose, 5-(4,6-dichlorotriazinyl)aminofluorescein (DTAF) was used as a grafting agent to preparesuspensions of fluorescent microfibrils from bacterial cellulose. These suspensions were digested by a seriesof commercially relevant cellulases from Humicola insolens origin: cloned Cel6B and Cel 45A as well ascrude H. insolens complex. The digestion induced the release of fluorescent cellodextrins as well as reducingsugars. After adequate centrifugation, these soluble products were analyzed as a function of grafting content,digestion time, and cellulase characteristics. The resulting data allowed the grafting conditions to be optimizedin order to maximize the quantity of soluble products and therefore to increase the sensitivity of the detection.A comparison between the amount of released fluorescence and that of released reducing sugar allowed thedifferentiation between processive exo and endo cellulase activities. The casting of films of DTAF-graftedmicrofibrils at the bottom of the microwell titer plates also led to sensitive cellulase detection. As thesefilms kept their integrity and remained firmly glued to the well bottom during the digestion time, they aretailored made for a full automation of the cellulases testing.