Biotechnology Letters

, Volume 31, Issue 10, pp 1613–1616

Cationic polyacrylamides enhance rates of starch and cellulose saccharification

Authors

  • John T. Reye
    • Institute of Paper Science and Technology, School of Chemical & Biomolecular EngineeringGeorgia Institute of Technology
  • Kendra Maxwell
    • Institute of Paper Science and Technology, School of Chemical & Biomolecular EngineeringGeorgia Institute of Technology
  • Swati Rao
    • Institute of Paper Science and Technology, School of Chemical & Biomolecular EngineeringGeorgia Institute of Technology
  • Jian Lu
    • Institute of Paper Science and Technology, School of Chemical & Biomolecular EngineeringGeorgia Institute of Technology
    • Institute of Paper Science and Technology, School of Chemical & Biomolecular EngineeringGeorgia Institute of Technology
Original Research Paper

DOI: 10.1007/s10529-009-0053-y

Cite this article as:
Reye, J.T., Maxwell, K., Rao, S. et al. Biotechnol Lett (2009) 31: 1613. doi:10.1007/s10529-009-0053-y

Abstract

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.

Keywords

AmylaseCellulosePolymerc-PAMBindingRate

Introduction

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 3.2.1.1) 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

A typical effect of a c-PAM on the amylase induced hydrolysis of corn starch in water is shown in Fig. 1. The substrate is rate-limiting under these conditions of high enzyme load. The rate of glucose generation increases with increasing c-PAM concentration up to 100 mg/l, but a higher polymer dose of 1,000 mg/l inhibits the rate.
https://static-content.springer.com/image/art%3A10.1007%2Fs10529-009-0053-y/MediaObjects/10529_2009_53_Fig1_HTML.gif
Fig. 1

Effect of a linear c-PAM (35% SH) on the efficiency of amylase on 10 g corn starch/l at 47°C. The values in the plot represent c-PAM in mg/l. The enzyme stock was added at 10 g/l

An analogous plot of the hydrolysis of bleached hardwood fiber to glucose by 1 g cellulase (Optimase CX 40L)/l is presented in Fig. 2. Here values for dissolved total organic carbon (TOC) generated from the dissolution of fiber are presented. Fiber dissolution leads initially to a variety of polysaccharides and our intent was to collectively determine all the dissolved organics derived from the fiber. The rise in TOC is much more pronounced in the presence of 1,000 mg c-PAM (PL2320, 38% active ingredient)/l. The results of binding studies illustrated in Fig. 3 show that the polymer increases the binding of enzyme to fiber. c-PAMs are used in industry to flocculate particulate matter. It is possible that the c-PAM binds the enzyme to the substrate in a geometry that promotes their interaction. The reaction is inhibited if the geometry is unfavorable. The c-PAM also accelerates the rate of fiber length reduction as shown in Fig. 4. The effect is small but significant, and demonstrates that the c-PAM affects more than one enzyme present in the cellulase preparation. As expected, c-PAM did not break down the fiber in the absence of the enzyme.
https://static-content.springer.com/image/art%3A10.1007%2Fs10529-009-0053-y/MediaObjects/10529_2009_53_Fig2_HTML.gif
Fig. 2

Effect of c-PAM (35% SH) on the efficiency of 1 g cellulase (Optimase CX 40L)/l on bleached hardwood fiber

https://static-content.springer.com/image/art%3A10.1007%2Fs10529-009-0053-y/MediaObjects/10529_2009_53_Fig3_HTML.gif
Fig. 3

Effect of 1,000 mg (PL 2320, 38% active ingredient) c-PAM/l on the binding of cellulase (expressed as volume of stock enzyme solution to volume of water) to bleached softwood kraft fiber

https://static-content.springer.com/image/art%3A10.1007%2Fs10529-009-0053-y/MediaObjects/10529_2009_53_Fig4_HTML.gif
Fig. 4

Effect of 500 mg c-PAM (4800 SSH)/l on the degradation of 10 g bleached hardwood fiber/l by 10 g Pergalase/l

Comparison of c-PAMs

Several members of the c-PAM family are effective in catalyzing both the corn starch and fiber applications albeit to varying degrees. A set of commercial c-PAMs were screened for their effect on the performance of amylase on corn starch and of cellulose on fiber. Glucose yields from corn starch and fiber were each normalized to the maximum yield for each case. The results are presented in Fig. 5. The controls are at the point (0,0) where no polymer is present; the best overall polymer would have a coordinate of (1,1). Rate measurements made in some case showed that 6 h is at the high end of the linear part of the curve, so the glucose yields correspond approximately to the initial rates. It is striking that except for three instances where there was no effect, the c-PAMs provide a clear benefit for the corn starch application. For the fiber work, five of the polymers tested were inhibitory; they have negative values on the abscissa. Our main conclusion is that the effect of the c-PAM is non-specific in that it boosts the performance of two completely different systems.
https://static-content.springer.com/image/art%3A10.1007%2Fs10529-009-0053-y/MediaObjects/10529_2009_53_Fig5_HTML.gif
Fig. 5

Effect of various c-PAMs on the efficiency of amylase on corn starch and on cellulase on bleached softwood pulp fiber. The substrate (corn starch or cellulose) was added at 10 g substrate/l; enzyme (cellulase or amylase) was also added at 10 g enzyme stock/l. The mixture was shaken at 50°C for 6 h. c-PAMs were applied at 500 and 100 mg of polymer/l of solution for the cellulase and amylase applications, respectively

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.

Copyright information

© Springer Science+Business Media B.V. 2009