Enzymatic hydrolysis of lignocelluloses Cloning, expression, and characterization of β-glucosidases Annette Sorensen 1,2, Peter S. Lübeck 1, Mette Lübeck 1 and Birgitte K. Ahring 1,2. 1. Section for Sustainable Biotechnology, Copenhagen Institute of Technology, Aalborg University, Denmark. 2. Center for Biotechnology and Bioenergy, Washington State University, Richland, WA Introduction The key to future success for the 2nd generation bioethanol production is efficient and optimized use of the lignocellulosic biomasses through an environmentally friendly technology.  Lignocelluloses are mainly composed of hemicelluloses, cellulose, and lignin. One important step is enzymatic hydrolysis of the pretreated lignocellulose composed cell wall.  A combination of several enzymes is required to obtain monomeric sugars.  For cellulose degradation, the main actors involved are endoglucanases, cellobiohydrolases, and β-glucosidases (Fig.1). In this project, focus is on finding new β-glucosidases with high activity and efficiency. Cloning of β-glucosidase genes Aspergilli produces several intracellular and extracellular β-glucosidases. Here, the secreted ones are of main interest. Methods Alignment of known Aspergilli β-glucosidase genes. Identification of conserved regions for degenerate primer design (Fig.4). Genome walking to obtain full length genes (Fig.5). Results One full length β-glucosidase gene has been cloned. It belongs to the same group of β-glucosidases as the ones used in the initial alignment (Fig.6). Current and future work  Cloning of more β-glucosidase genes from a selected Aspergillus sp. strain is in progress.  Overexpression of the cloned genes in Trichoderma with the aim of purification of the different enzymes  Specific activity measurements of the proteins expressed to compare the different β-glucosidases from the same Aspergillus sp. Fig. 3. Results of screening, β-glucosidase activity measured as units per mg secreted protein. The best β-glucosidase producers were generally identified as Apsergilli strains. Fig. 6. Alignment of amino acid sequence of known β-glucosidases vs cloned Aspergillus sp β-glucosidase. Color codes: Yellow: identical, Blue: conservative, Light green: block of similar, Dark green: weakly similar, Black: non-similar. Fig. 4. An example of an alignment of known Aspergilli β-glucosidases used to identify conserved regions for the design of degenerate primers. Fig. 2. Simple screening for β- glucosidase activity on cellulase inducing media. Screening Unidentified fungi from nature have been isolated and screened for β-glucosidase activity:  Liquid fermentation on cellulase inducing media (Fig.2).  pNPG assay for β-glucosidase activity determination.  Pierce BCA assay for protein determination. Results: Generally, Aspergilli strains performed best (Fig.3). The identity of strains of interest was determined by sequen- cing gDNA regions of ITS, calmodulin, and beta-tubulin. Fig. 5. An example of genome walking. The arrow suggests the pcr product to be cloned and sequenced. Fig. 1. Enzymatic hydrolysis of cellulose; the synergistic action of the different cellulolytic enzymes [1] References: [1] Lynd et al, (2002) Microbiology and Molecular Biology Reviews 66 pp Contact: