Fluorescent cellulose microfibrils as substrate for the detection of cellulase activity.
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.
Helbert
William
W
Station Biologique de Roscoff, UMR 1931 (CNRS and Laboratoires Goëmar), Place Georges Teissier, BP 74, 29682 Roscoff Cedex, France.
Chanzy
Henri
H
Husum
Tommy Lykke
TL
Schülein
Martin
M
Ernst
Steffen
S
D002480:Cellulase / Q000032:analysis* / Q000493:pharmacokinetics; D002482:Cellulose / Q000032:analysis* / Q000493:pharmacokinetics; D005452:Fluoresceins / Q000032:analysis* / Q000493:pharmacokinetics; D005456:Fluorescent Dyes / Q000032:analysis* / Q000493:pharmacokinetics; D020894:Microfibrils / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity
D005452:Fluoresceins; D005456:Fluorescent Dyes; C039083:5-((4,6-dichloro-1,3,5-triazin-2-yl)amino)fluorescein; D002482:Cellulose; D002480:Cellulase