The activities of cellulases (NS50013 cellulase complex) and cellobiase
(NS50010 b-glucosidase) obtained from Novozymes North
America Inc. (Franklinton, NC) were determined to be 76.4 FPU (filter
paper unit)/ml and 283.1 CBU (cellobiase unit)/ml, respectively
(Ghose, 1987).
To evaluate the sugar yields from the pretreatments, the wet
pretreated biomass (triplicates per sample) equivalent to 1 g dry
biomass was hydrolyzed with excessive dosage of cellulase (40
FPU/g dry biomass) and cellobiase (70 CBU/g dry biomass) in
0.05 M sodium citrate buffer (pH 4.8) with a total liquid volume
of 30 ml in an Erlenmeyer flask (250 ml) in a controlled environmental
incubator shaker set (Model 25, New Brunswick Scientific).
Sodium azide (0.3% (w/v)) was added to the hydrolysis mixture to
prevent microbial growth. The hydrolysis was carried out at 55 C
and 150 rpm for 72 h after which the hydrolyzate was centrifuged
(Model 5810R, Eppendorf) at 4 C and 4000 rpm for 15 min and the
supernatant was stored at 20 C for further analysis. Enzymatic
hydrolysis of untreated biomass was conducted as a control. Enzyme
and substrate blanks both were run in the hydrolysis step
and their respective sugar concentrations were approximately
zero.