Mechanism of rate enhancement of wood fiber saccharification by cationic polyelectrolytes
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- Mora, S., Lu, J. & Banerjee, S. Biotechnol Lett (2011) 33: 1805. doi:10.1007/s10529-011-0647-z
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Cationic polyelectrolytes can increase the cellulase-induced hydrolysis rates of bleached wood fiber. We show that the polymer associates mainly with the amorphous region of fiber and acts principally on endoglucanase. Fiber/water partitioning of the enzyme follows a Langmuir isotherm for the untreated fiber but a Freundlich isotherm is obeyed for the polymer-treated fiber.
Cationic polyelectrolytes can increase the hydrolysis rates of both bleached wood fiber and cornstarch by more than 50% (Banerjee and Reye 2008; Reye et al. 2009). While modest by catalytic standards, the increase is, nonetheless, commercially significant. We proposed that the polyelectrolyte attaches to and neutralizes the negatively charged substrates thereby reducing the repulsion experienced by the negatively charged enzyme and increasing the binding of the enzyme to the substrate (Reye et al. 2011). Charge neutralization of a fiber by a cationic polymer is called “patching”, a process well established on fiber agglomeration (Hubbe et al. 2009).
There are several enzymes involved in the enzymatic conversion of fiber to glucose. Endoglucanases first break up the cellulose fiber into fragments. Exoglucanases then processively convert the fragments into cellobiose, which is hydrolyzed to glucose by cellobiase. The last step is comparatively rapid (Zheng et al. 2009) and is not rate-controlling for fiber hydrolysis. In this paper, we determine how a cationic polyelectrolyte affects the binding of cellulase to fiber and, in turn, the rate of fiber hydrolysis.
Materials and methods
A 2% (w/v) suspension of bleached softwood pulp (from Weyerhaeuser’s Grande Prairie mill in Alberta, Canada) was stirred with a 0.1% commercial cellulase preparation (Ctec Cellic 2, 119 FPU/ml, from Novozymes) at 220 rpm and at 50°C. A cationic polyacrylamide (c-PAM) from Eka Chemicals (XP10035, 35% charge) was added after 2 h at 250 mg polymer/l. (We have shown that the c-PAM is most effective when added after about 2 h when the fibers have shortened somewhat. Prior to that the c-PAM tends to agglomerate the fibers and reduces their surface area.) Glucose was determined with a glucose assay kit adapted to a DA3500 Discrete Analyzer from OI Corporation, College Station, TX. The average error from duplicate measurements was 4.5%.
Measurement of partition coefficients
The fiber was screened through a 28-mesh (0.6 mm) screen to remove fines so as to generate a more uniform furnish. The fines content of the screened material was 3.9%; fines are classified as fiber fragments shorter than 0.1 mm. The fiber length (length-weighted) was 2.64 mm. Both measurements were made with a Fiber Quality Analyzer from Optest, Hawkesbury, Ontario, Canada. Thus, the fiber was a polycomponent mixture distributed about an average fiber length of 2.6 mm. The fiber (1 g) was suspended in 100 ml water containing 1,000 mg c-PAM/l. It was then filtered out and resuspended in 25 ml of water. Cellulase was prepared at various concentrations in 25 ml of water and stored at 4°C. The fiber and enzyme preparations were mixed and incubated at 4°C for 1 h with continuous shaking. Previous work has shown that equilibrium is reached within 1 h (Reye et al. 2011). The samples were filtered through a 0.2 μm GHP (low protein binding) syringe filter and the protein remaining in solution was assayed with a BCA Protein Assay Kit from Pierce Protein Research Products. The protein associated with the fiber was obtained by difference. The dimensionless fiber:water partition coefficient (Kf/w) of the protein was calculated as the ratio of the amount of protein associated with fiber to that of an equal weight of water. The average deviation from duplicate measurements was less than 4%.
Results and discussion
The fines concentration rises initially because the fiber is progressively shortened by the endoglucanase. The polymer clearly increases the rate of this process and it must, therefore, have a greater effect on endoglucanase than on exoglucanase. Were it otherwise, the fines level would have fallen initially because the rate of consumption of the fines would have exceeded its rate of production. The difference in all the fiber properties reaches a maximum after 6 h (Fig. 2). The glucose concentration at 6 h is about 4,000 mg/l for the runs that included c-PAM (Fig. 1). This reflects about 20% conversion of cellulose, which is also the approximate amorphous content of a bleached softwood fiber used earlier (Lu et al. 2011). Hence, the likely reason that the polymer preferentially increases the rate of endoglucanase hydrolysis is that it associates more with the amorphous regions of fiber that are particularly susceptible to endoglucanase attack (Coughlan 1992; Zhang and Lynd 2004). Further evidence for this preferential association comes from the partitioning measurements described below.
Partitioning of enzyme between fiber and water
In conclusion, we have shown through difference plots that cationic polyacrylamides increase the rate of endoglucanase hydrolysis. The binding of the overall mixture of cellulase enzymes to fiber follows a Langmuir isotherm but switches to Freundlich in the presence of c-PAM. We interpret this to mean that the polymer associates more with the amorphous region of fiber than with the crystalline. While only a single c-PAM was used for the binding measurements, a wide variety of c-PAMs have been shown to catalyze the cellulase-mediated hydrolysis of cellulosic fiber and it is likely that the same mechanism of binding also applies to the other polymers.
This study was partly funded by the Eka Chemicals division of Akzo Nobel. We thank Novozymes for a gift of cellulase enzyme.