(490z) Heterologous Expression and Characterization of Three Trichoderma Reesei Cellulose Hydrolases in Kluyveromyces Lactis

Three Trichoderma reesei cellulose hydrolases ? two exoglucanases Cel7A and Cel6A, and one endoglucanase Cel7B ? were cloned into the Kluyveromyces lactis yeast expression system (New England BioLabs, Cat. #E1000S). One particular exoglucanase, Cel6A, was expressed and secreted to the cell culture medium at high concentrations (~10 ug/ml) and possesses an apparent molecular weight similar to the native enzyme as determined by SDS-PAGE. Protein production for Cel6A was scaled-up to five liter batch growth in a Bioflow 3000 bioreactor. The protein was concentrated and purified from the cell culture supernatant by ammonium sulfate precipitation and re-suspension in 20mM Tris-Cl, pH 7.4, followed by anion exchange chromatography. Research is ongoing to determine catalytic activity for Cel6A on a variety of substrates. Cel7A and Cel7B appear to be produced and secreted with a wide range of glycosylation in a continuous distribution. This will be verified by deglycosylation with Endo H followed by SDS-PAGE. Purification and catalytic activity determination of the recombinant Family 7 enzymes will be performed in a similar manner to that for Cel6A.

A high-throughput screening assay is being developed for rapid semi-quantitative characterization of cellulose hydrolases. Future work will be done to create a library of mutant enzymes by error-prone PCR, and to screen for improved performance under industrially relevant conditions. Additionally, an investigation into the differences between Family 6 and Family 7 glycosyl hydrolases which cause the different observed glycosylation patterns in K. lactis is desirable.