Monday, April 30, 2007

Development and validation of an automated 96-well microplate hydrolytic assay for AFEX treated lignocellulosics using commercial enzyme mixtures & synergistic fungal enzymes

Shishir P.S. Chundawat1, Balan Venkatesh1, Subashini Nagendran2, Holly A. Gunter1, Jonathan D. Walton2, and Bruce E. Dale1. (1) Biomass Conversion Research Laboratory, Department of Chemical Engineering and Material Science, Michigan State University, 2527 Engineering building, E. Lansing, MI 48824, (2) MSU-DOE Plant Research Laboratory, Michigan State University, E. Lansing, MI 48824

Standard enzymatic hydrolysis protocols for cellulosic enzyme systems have several inherent disadvantages including high throughput time, extensive reagent usage, labor intensive and non-realistic substrates (i.e. filter paper, purified xylans, and chromogenic substrates). Recent research has indicated that the choice of an appropriate enzyme preparation depends more on the characteristics of the substrate rather than on standard enzyme-activities measured. Therefore, the need for a rapid hydrolysis assay based on lignocellulosic substrates becomes apparent.

The automated 96-well microplate method is a rapid hydrolytic assay technique (essentially a scaled down version of the NREL LAP 009 protocol) developed at our laboratory. The critical bottlenecks addressed in the development of this technique are: reproducible substrate delivery, minimization of evaporation losses, hydrolysis time period, rapid sugar assay and high throughput. A suitable method for delivery of substrate was through dispensing a biomass slurry suspension, using a robotic liquid handling workstation. Delivery parameters were optimized for varying substrate slurry concentrations. The microplate method was optimized for hydrolysis of avicel and AFEX treated corn stover using a mixture of commercial cellulase and β-glucosidase. The method was validated and statistical analysis on the data was performed. We also screened several commercially available cellulases, xylanases and beta-glucosidases under a wide range of total enzyme concentrations for the hydrolysis of AFEX pretreated biomass. Further screening of new fungal cellulase systems that aid synergistic hydrolysis of AFEX treated biomass was also accomplished using the microplate assay technique.

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