Fluorescent cellulose microfibrils as substrate for the detection of cellulase activity
Fluorescent cellulose microfibrils as substrate for the detection of cellulase activity
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DOI：
10.1021/bm020076i
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发表时间：
2003-05-01
影响因子：
6.2
通讯作者：
Ernst, S
中科院分区：
文献类型：
--
作者：
Helbert, W;Chanzy, H;Ernst, S
为了设计一个敏感的纤维素酶测定的基础上的基板具有大多数的天然纤维素的物理特性，5-（4，6-二氯三嗪基）氨基荧光素（DTAF）被用作接枝剂，以制备悬浮液的荧光微纤维从细菌纤维素。这些悬浮液用一系列商业上相关的来自特异腐质霉来源的纤维素酶消化：克隆的Cel 6 B和Cel 45 A以及粗H. insolens复合体消化诱导荧光纤维糊精以及还原糖的释放。充分离心后，分析这些可溶性产物作为接枝含量、消化时间和纤维素酶特性的函数。所得数据允许优化接枝条件，以使可溶性产物的量最大化，从而提高检测的灵敏度。释放的荧光的量和释放的还原糖的量之间的比较允许进行性外切和内切纤维素酶活性之间的区分。在微孔滴定板的底部浇铸DTAF接枝的微纤丝的膜也导致灵敏的纤维素酶检测。由于这些薄膜在消化期间保持其完整性并牢固地粘在孔底部，因此它们是为纤维素酶测试的全自动化量身定制的。
To devise a sensitive cellulase assay based on substrates having most of the physical characteristics of native cellulose, 5-(4,6-dichlorotriazinyl)aminofluorescein (DTAF) was used as a grafting agent to prepare suspensions of fluorescent microfibrils from bacterial cellulose. These suspensions were digested by a series of commercially relevant cellulases from Humicola insolens origin: cloned Cel6B and Cel 45A as well as crude H. insolens complex. The digestion induced the release of fluorescent cellodextrins as well as reducing sugars. 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 optimized in 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 the differentiation between processive exo and endo cellulase activities. The casting of films of DTAF-grafted microfibrils at the bottom of the microwell titer plates also led to sensitive cellulase detection. As these films kept their integrity and remained firmly glued to the well bottom during the digestion time,they are tailored made for a full automation of the cellulases testing.