Engineering Of Volvariella Volvacea Glycoside Hydrolase Family 5 Endoglucansel For Improved Thermostability

Enzymatic hydrolysis of cellulose by microorganisms is a key step in the global carbon cycle. Effective degradation of lignocellulose by cellulases, triggering bioconversion of cellulose to simple sugars, applied to produce biomass energy—bioethanol.In this study, we employed two methods to engineer Glycoside hydrolase family 5 EndoglucanaseI(EGI) from the straw edible mushroom Volvariella volvacea for improved thermostability:directed evolution and site-directed mutagenesis. We successfully constructed the episomal plasmid pGAPZαB-PARS 1 containing Pichia autonomous replication sequencel for application in the directed evolution of EGI.Directed evolution provides a practical means of tailoring biocatalysts as irrational desigan. Error-prone PCR was carried out to generate egI's molecular diversity of mutations, following digestion and ligation with episomal plasmid pGAPZaB-PARS 1 to construct a mutant lialiry in Pichia pastoris. Screening for improved thermostability mutants by Congo red plate assay overlaid with a CMC molten agar at 60℃after pretreatment at a high temperature 70℃for 30min. we obtained 6 mutants with improved thermo stability, EG8-1-1,EG8-1-2,EG8-4-1,EG8-5-1,EG9-7-1 and EG9-8-2. The mutants EG8-4-1,EG8-5-1,EG9-7-1 and EG9-8-2 lack a 52 amino acid peptide at the C-terminus compared with EGI. All 6 mutants had a distinguished improved thermostability with a optimum temperature 65℃on substrate CMC versus EGI 55℃. The 80℃half-life of mutants is not lower than 2hr while EGI loses it's activity completely 5min in the same condition.Site-directed mutagenesis belongs to rational design, which is an approach of alternative to directed evolution. We identified the two active sites E192 and E301 of EGI using homology modeling. Structure-guided consensus method was employed to determine EGI sites of site-directed mutagenesis through comparison with 3 GH5 thermostable EGs from Thermoascus aurantiacus,Talaromyces emersonii and Penicillium brasilianum using both crystal structures and sequence-alignment.4 mutants EG-SD1,EG-SD2,EG-SD3 and EG-SD4 performed a distinguished improved thermostability similar to the result of directed evolution, EG-SD1,EG-SD2 and EG-SD3 had a optimum temperature 65℃on substrate CMC while EG-SD4 50℃lower than EGI.