Cationic polyacrylamides enhance rates of starch and cellulose saccharification
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- Reye, J.T., Maxwell, K., Rao, S. et al. Biotechnol Lett (2009) 31: 1613. doi:10.1007/s10529-009-0053-y
Adding a cationic polyacrylamide (c-PAM) to either the amylase mediated hydrolysis of corn starch or the hydrolysis of wood fiber by cellulase can enhance the initial hydrolysis rates, although a rate decrease can occur under some conditions. Several c-PAMs can serve as catalysts and the same c-PAM can improve the efficiency of both amylase and cellulase. The initial amylase rate approximately doubles; the analogous cellulase hydrolysis rate increases by about 40%. c-PAMs increase the binding of enzyme to substrate.
Enzymes and other catalysts are frequently attached to inert supports in order to increase their activity or facilitate their recovery from spent mixtures. For example, polymers have been used to immobilize cellulase and glucoamylase, among other enzymes, to passive surfaces (Arica et al. 2000; Wang and Hsieh 2004). In this paper we use polymers to bind enzymes to an active surface, i.e., the surface undergoing the reaction. In many cases, this significantly increases the rate of degradation (Banerjee and Reye 2008). Two applications with implications to bioenergy production are considered: the saccharification of corn starch (Kwiatkowski et al. 2006) with a commercial amylase and the hydrolysis of the cellulose in wood pulp fiber (Perez et al. 2002) with a commercial cellulase preparation. The polymers used are industrial cationic polyacrylamides (c-PAMs), which are commonplace in water treatment (Bolto and Gregory 2007) and other applications (Yoon and Deng 2004).
Materials and methods
Strains and assays
The alpha amylase (EC 220.127.116.11) used contained 20 mg total protein/ml with an activity of 11.4 μmol glucose equivalents released per minute per milliliter of stock solution at 50°C. Mixed cellulases (prepared from Trichoderma reesei) used were Optimase CX 40L (13.2 FPU/ml, 124 mg protein/ml) and Pergalase 7547 (14.8 FPU/ml, 107 mg protein/ml). Glucose assays were done with a glucose oxidase/peroxidase assay kit. Total protein concentrations for each enzyme preparation were determined with a BCA Protein Assay Kit.
Hydrolysis of fiber
Various c-PAMs: 35% SH, PL2320, 4800 SSH, were used; the descriptors are commercial designations. The binding of cellulase to fiber was measured with and without c-PAM. Because c-PAMs bind differently to fiber fines as compared to long fiber (Hartley and Banerjee 2008), the fines were removed from a sample of bleached softwood kraft pulp with a 28-mesh screen. The remaining fiber was formed into handsheets (Tappi 2000), one set of which was treated with a solution of 200 mg 35% SH c-PAM/l for 30 min. A second set was exposed to the same volume of water for the same period. The handsheets were dried at room temperature and soaked in 1–5 g cellulase (Pergalase stock)/l at 4°C for 20 min. The protein content of the enzyme remaining in the supernatant was determined and the amount of enzyme bound to the sheet determined by difference.
Screening of polymers
Screening measurements to identify the best polymers were made with 22 commercial c-PAMs varying in charge, molecular weight, and the degree of branching. No attempt was made to optimize the dosages or the conditions; our intent was merely to rank the relative effects of the polymer.
Results and discussion
Hydrolysis of corn starch and wood fiber
Comparison of c-PAMs
In conclusion, we have shown that c-PAMs are versatile in accelerating the rate of enzyme-mediated saccharification by increasing the degree of enzyme–substrate binding. Two applications for the saccharification of biomass are provided. Both are central to the production of ethanol from biomass and have potential in reducing the dosage of the enzyme, which is a significant component of the overall cost (2) of ethanol production. The approach could have broad application in reactions of enzymes with solid or macromolecular substrates.