Cellulases PASC

Cellulose-cellulase incubation was made at 25 C with 1 mL of 50 mM Na-acetate of pH 5, 0.8 - 62 pM cellulase, 1.8 g/L cellulose and 8 pM BSA in 1.2-mL Pierce ImmimoWare tubes. After 30 min, the supernatant was decanted, filtered (by 0.45 p Millipore Multi Screen-HV), and assayed by PASC hydrolysis activity for free cellulase. Langmuir adsorption equation, l/[E]adsorbed = l/([E]o - [E]) = l/(No tE]) + P/No (35), was then applied to calculate apparent capacity No (in mole/mole) and adsorption constant IC. 

For a given cellulase, AvicePs ( ) was less than PASC s (j), suggesting less productive cellulase adsorption on more crystalline cellulose surface. With crystalline cellulose substrate, it could be more difficult for the cellulase to thread a cellulose chain into its active site clefl/tunnel, since more inter-chain H bonds would need to be broken in a highly cr3 talline region than in an amorphous/chain end region. 

The ( >PAsc - Avicel difference varied among cellulases, indicating differential cellulose accessibility of these cellulases, even when they were close structural/functional analogs. For instance, T. reesei CBH-I had a < i on PASC 3 times larger than that on Avicel, but H. insolens CBH-I had a i > on PASC 26 times larger dian that on Avicel. 

Table III summarizes the measured with 1 1 binary cellulase mixtures. For PASC, T. reesei CBH-I and H, insolens CBH-I mixture showed a < ) (0.062) similar to that of T. reesei CBH-I (0.062), rather than the sum (0.062 + 0.043 0.11) of the two cellulases (Table II), indicating that the two CBH-I were adsorbed at the same region (likely rich in reducing chain ends) on PASC. Similar result was seen when T. reesei CBH-I and H. insolens CBH-I were mixed at 1.4 1 ratio (()) 0.07). For Avicel, T, reesei CBH-I and K insolens CBH-I mix showed a (0.013) similar to that of T. reesei CBH-I (0.014).

Unlike processive T. reesei CBH-I s preference for non-reducing cellulose chain ends, non-processive T. reesei EG-I is believed capable of randomly act along the cellulose chain (2,13,15). Under oiu conditions, 1 1 T. reesei CBH-I and EG-I mix had a < ) of 0.082 for PASC, while 15 1 or 17 1 mix had a ( ) of 0.067. For Avicel, 1 17. reesei CBH-I and EG-I mix had a < ) of 0.015. These ( ) seemed to correspond to die ( > sums of the two cellulases, indicating different productive adsorption regions for them. However, because the ( ) of T. reesei EG-I was 15 times smaller than tiiat of T. reesei CBH-I, our experiment could not conclusively differentiate a trae siun that was 15% higher tiian ( )(CBH-I) from a ( (CBH-I) with 10% experinental error. 

Being close functional (reducing-end specific, processive) and structural (funily 7 cellulases) analogs, T. reesei and H. insolens CBH-I showed conqiarable activity on amorphous PASC however, the former was significantly more active on microcrystalline Avicel than die latter (Table I). This difference might be related to the high kinetic Avicel accessibility of T. reesei CBH-I. 

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