Reducing end-specific fluorescence labeled celluloses for cellulase mode of action

Abstract

Cellobiohydrolases (CBH-s) attack primarily from the cellulose chain ends. It is known that CBH-s can differ in their chain end preference. Here the cellulose reducing end-specific fluorescence labeling was studied with the aim to find the most suitable labeled cellulose for easy determination of CBH-s chain end preference. Anthranilic acid (AA) was used as fluorescence label. Bacterial cellulose, amorphous Avicel and filter paper were subjected to the labeling. Suitability of modified bicincoic acid method for determination of reducing groups on cellulose was also confirmed. AA labeled celluloses were hydrolysed with family 7 and 6 CBH-s from Trichoderma reesei and Phanerochaete chrysosporium. Hydrolysis data were plotted as released label (%) versus total degradation (%) (progress curves). Most characteristic progress curves were obtained with AA labeled regenerated amorphous Avicel where the family 6 CBH-s produced clearly upward curved progress curves confirming their preference for non-reducing ends.

Processivity of Cellobiohydrolases Is Limited by the Substrate

“…PcCel7D was purified from the culture filtrate of the Pc strain K3 as previously reported (35). Enzyme concentration was determined from absorbance at 280 nm using molar extinction coefficients of 84,400 (14), 74,900 (14), 78,000, and 38,200 M Ϫ1 cm Ϫ1 for TrCel7A, PcCel7D, TrCel5A, and TrCel12A, respectively.…”

“…Celluloses-Bacterial cellulose (BC) was prepared by laboratory fermentation of the Acetobacter xylinum strain ATCC 53582 (14). Amorphous cellulose (AC) (prepared from Whatman no.…”

“…Amorphous cellulose (AC) (prepared from Whatman no. 1 filter paper) and reduced cellulose were prepared as described previously (14). For preparation of DAP-cellulose, DAP labeling was performed in 50 ml total volume.…”

“…Determination of Soluble Reducing Groups (SRGs)-SRGs were determined with a modified bicinchoninic acid (BCA) method (14,36). Supernatant (0.5 ml) from cellulose hydrolysis was mixed with 0.5 ml of BCA reagent and heated at 75°C for 30 min.…”

“…The TrCel7A counterpart in another well characterized cellulolytic organism, the white-rot fungus Phanerochaete chrysosporium (Pc) (11), is PcCel7D (formerly CBH 58) (12). PcCel7D is also a reducing end-acting CBH (13,14). A less abundant CBH produced by Tr is the non-reducing endacting TrCel6A (formerly CBH II) (15).…”