Enzymatic hydrolysis of lignocelluloses
Cloning, expression, and characterization of β-glucosidases
Annette Sorensen1,2, Peter S. Lübeck1, Mette Lübeck1 and Birgitte K. Ahring1,2.
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.
Screening
Fig. 2. Simple screening for βglucosidase activity on
cellulase inducing media.
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 sequencing gDNA regions of ITS, calmodulin, and beta-tubulin.
Fig. 1. Enzymatic hydrolysis of
cellulose; the synergistic action of the
different cellulolytic enzymes [1]
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).
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.
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. 5.
An example of
genome walking.
The arrow suggests
the pcr product to be
cloned and sequenced.
Fig. 4. An example of an alignment of known Aspergilli β-glucosidases used to
identify conserved regions for the design of degenerate primers.
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.
References: [1] Lynd et al, (2002) Microbiology and Molecular Biology Reviews 66 pp 506-578
Contact: aso@bio.aau.dk