Directed Evolution And Screening Of Trichoderma Endoglucanase Variants With High Acitvity

Cellulose is the most abundant renewable bioresource produced in the biosphere. Enzymatic degradation of cellulose is achieved by the synergistic action of three cellulase enzyme groups:endoglucanases, exoglucanases and β-glucosidases.Endoglucanases I, the important component of cellulase complex, is composed of catalytic domain, linker and cellulose binding domain.However, the low enzymatic activity limited the applification in industry recently. We constructed and screened S.cerevisiae mutant library by DNA shuffling of three wild egl genes from T.reesei, H.pseudokoningii and T.longibrachiatum. The results were as follows:1. Endoglucansae I gene was cloned from Trichoderma reesei, Hypocrea pseudokoningii and Trichoderma longibrachiatum respectively. Results of sequence analysis indicate that T. reeseiegl1is1507bp long, the others two are all1566bp. All of them are composed of3exons and2introns as reported. The deduced amino acid sequence of the T. reesei egl is459aa, and the others are all461aa. There is22aa signal peptide sequence from the N-terminate of three coding proteins.2. The egl gene with no introns was obtained by overlap PCR in T. reesei. pYEa-Reglwas constructed by inserting the encoding sequence without the signal peptide into the S.cerevisiae secretion vector pYEa containing a singal peptide. Recombinant pYE-Regl plasmid containing own signal peptide was as control. The two plasmids were then transformed into S.cerevisiae respectively. After inducing, pYEa-Regl transformants has enzymatic activity. However, there was no clear activity in control.It is noticed that a signal peptide facinitated secretory expression of mature peptide endoglucanases I.3. The egl gene with no introns was obtained by overlap PCR in H.pseudokoningii and T.longibrachiatum. The recombinant pYEa-Pegl and pYEa-Legl plasmid was constructed and then transformed into S.cerevisiae respectively. Reconbinant transformants all produced active enzyme.4. The mutant library was construced by DNA shuffling of three wild egl genes encoding the mature peptide. After screening, variant with high activity was obtained and named New8. Extracellular enzyme activity of New8approached the highest level (1.97U/mL) when cultured for96h, which is about1.9times than the average of the wild EGI. The optimum temperature and pH were50℃and5.6respectively, the same as the wild EGI. The expression was confirmed by a protein band in the SDS-PAGE, which is a little larger than predicted EGI. 5. The gene New8egl, about1320bp long, was subcloned and sequencing. It is94%,95%and96%identical to T.reesei、T.longibrachiatum and H.pseudokoningii egl. And the deduced amino acid sequence is about95%,96%and97%homologous to the wild EGI. Analysis of the sequences indicated that there were114recombination sites, and four extra mutations had been introduced, caused amino acid substitution. VIOA and F192L didn’t change the hydrophobicity. G100S and S318G changed the hydrophilicity. S318G located near the activity center in the catalytic domain by tertiary structure prediction.In summary, three wild eg1genes were cloned from T.reesei, H.pseudokoningii and T.longibrachiatum. S.cerevisiae secretory expression system for EGI was established and used for DNA shuffling of three wild eg1genes. A new eg1gene encoding higher enzymatic activity EGI was obtain by screening. This system should facilitate the improvement of other cellulase (CBH and BG) and lay the foundation for consturcting efficient cellulose degradation system.