Improving the activity of endoglucanase I (EGI) from Saccharomyces cerevisiae by DNA shuffling

Abstract

To enhance the specific endo-β-1,4-glucanase activity (EGI) of three mixed substances: Trichoderma sp. (Trichoderma reesei, Trichoderma longibrachiatum, and Trichoderma pseudokoningii), we optimized the efficiency of the encoding gene, eg1, using DNA shuffling and Saccharomyces cerevisiae INVSc1 as a host. One variant, SEGI₈ (the high activity protein encoded by eg1 gene using DNA shuffling) was found through screening approximately 1000 variants. The extracellular enzyme work of SEGI₈ reached its highest level when cultured for 96 h, at 50 °C and a pH of 5.6, akin to the wild types. Clone harboring of the best mutant SEGI₈ (selected via first-round mutagenesis) produced 169–257% more activity than transformants with three wild-type EGIs. Mutant SEGI₈ produced by the clone showed broad pH stability (5.6–6.6) while performing comparable thermotolerance (60–70 °C) compared to wild EGIs; it also showed a high homology of 95%, 96%, and 97%, identical to the EGI of T. reesei, T. longibrachiatum, and T. pseudokoningii, respectively. Analysis of the sequences indicated that four point mutations causing amino acid substitution (V10A/G100S/F192L/S318G) were replaced. The major structure of the active site in SEGI₈ was comprised of loop regions, with the same location as β-helix in NTEGI (the protein encoded by eg1 genes in T. reesei). It has been suggested that the mutation S318G might affect substrate binding and improved actions based on an analysis of the predicted 3D structural-modeling of SEGI₈. Overall, this novel system could facilitate cellulose activity and create a foundation for constructing an efficient cellulose degradation system.