(460c) Enzymatic Hydrolysis of Afex Treated Corn Stover by Cellulolytic and Hemicellulolytic Synergistic Enzyme Cocktails

The identification of a suitable cocktail of cellulases and hemicellulases to hydrolyze residual glucan and xylan becomes pertinent in the case of lower severity pretreatments (e.g. ammonia fiber expansion or AFEX). A fundamental understanding of enzyme synergy in the hydrolysis of pretreated biomass requires a high-throughput combinatorial enzyme screening approach for lignocellulosic biomass.

Standard enzymatic hydrolysis protocols for cellulosic enzyme systems have several inherent disadvantages including long analysis times, excessive reagent and substrate usage, high labor inputs and non-realistic substrates (e.g. filter paper, purified xylans, and chromogenic substrates). The choice of an optimum enzyme cocktail depends largely on the substrate characteristics rather than standard enzyme-activities that are currently measured. Screening multi-enzyme systems directly on pretreated lignocellulosics would be a better way of identifying optimum synergistic cocktails of enzymes. The automated 96-well BCRL microplate method is a rapid hydrolytic assay technique (Chundawat et al., 2008, Biotech Bioeng, 99, 6:1281-1294) that is currently employed at our lab.

Commercially available cellulase (Spezyme® CP) was purified to isolate cellobiohydrolase (CBH) I, CBH II and Endoglucanase (EG) I. Ternary mixtures of purified cellulases were used to hydrolyze AFEX treated corn stover to determine optimum ratio of CBH I: CBH II: EG I. It was found that CBH II does not have significant synergistic interaction with either CBH I or EG I. AFEX corn stover was also hydrolyzed by several mixtures of hemicellulases and other accessory enzymes from Genencor and Novozyme in the presence of well defined cellulase cocktails (e.g. Accellerase® and purified CBH I/EG I/beta-glucosidase) to maximize synergistic hydrolysis yields with minimum enzyme loading (0.5-30 mg/gm glucan). Individual protein components for several commercial enzymes were identified using a high-throughput proteomics (LC-MS/MS) approach. Purified Aspergillus nidulans beta-xylosidase and xylanase were also isolated to develop a rational, minimal enzyme cocktail for AFEX treated corn stover. Several proprietary cellulases and hemicellulases from Verenium Corporation were also screened on AFEX corn stover.

The overall goal of the current project is to develop an enzyme cocktail specifically tailored for AFEX treated lignocellulosics to help reduce total protein loading employed during enzymatic hydrolysis.