Protein Engineering And Expression Of The Endoglucanase Ⅱ From Trichoderma Reesei

At present,lignocellulose-degrading enzymes have been widely used in many industries such as textile,dyeing,pulp and paper,detergent,food.Therefore,they have huge market potentials.However,the cellulases derived from fungi have their highest activity at acidic pH.Once the pH value was above 6,their activities are greatly reduced and this largely limits the applications of fungal cellulases under neutral or alkaline condition,therefore,one of the goals in protein engineering for fungal cellulases is to increase the catalytic efficiency of these enzymes at higher pH.Saccharomyces cerevisiae is a good host for directed evolution because of its high expression and transformation efficiency.Many fungal cellulases had been expressed in S.cerevisiae and many recombinant proteins were obtained. EndoglucanaseⅡ(EGⅡ)accounts for most of the endoglucanase activity produced by Trichoderrna reesei.Lack of EGⅡproduction reduces the endoglucanase activity in the culture supernatant by as much as 55%.In the former work of our lab,the gene of EGⅡwas obtained and expressed in S.cerevisiae;and screened a mutant whose pH optimum has some increase by directed evolution.We investigated the enzyme characters of rEGⅡand enhanced some characters of enzyme by directed evolution.The relationship of enzyme structure and pH optimum was elucidated.Modifying the protein can not only produce enzymes used in alkaline industry,but also enhance our knowing on protein's structure and function relation.The main results of the thesis are as follows:1.Assays of the glycosylation influence on rEGⅡEndoglucanaseⅡof T.reesei was expressed in S.cerevisiae and then purified. Two components(C1 and C2)of recombinant EGⅡwith different glycosylation were obtained and stained with the activity staining after SDS-PAGE.Purified C1 had a larger molecular mass(57 kDa)than that of the native EGⅡproduced by T.reesei(48 kDa)due to the different extents of asparagines-linked glycosylation.There was no significant difference in enzymatic activity between the C1 and the native-EGⅡfrom T.reesei.C1 treated with Endoglycosidase H had a molecular mass of 54 kDa and retained about 88%of its original activity.Unpurified C2 was larger form of hyperglycosylation proteins.Its molecular mass was larger than 85 kDa till up to 200 kDa.It still retained activity regardless of its magnitude molecular mass.With increased glycosylation extent of the enzyme components(C2＞C1＞native Cel5A), the pH range of activity become wider,and thermal stability become higher.2.Assays of the influence of lost N-glyeosylation at N124 on the mutant enzyme.Mutagenesis at N124 site was accomplished with PCR overlap extension method. The mutants of N124D and N124H were obtained.The two mutants had the same part of C1 and C2 as the wild-type rEGⅡ.The molecular mass of C1 was 54 KDa,the same as that of treated by Endo H of wild-type.We assumed that lost of N-glycosylation site at N124 attributed the decrease in the molecular mass of mutants. However,there were not much different at the enzyme activity,pH optima and the resistance to the temperature between the recombinant mutants and wild-type enzyme.3.The investigation of the role of the site 342 of EGII in catalytic efficiency and pH optima by site saturation mutagenesis.The role of the site 342 of EGⅡfrom T.reesei in catalytic efficiency and pH optima was investigated by site saturation mutagenesis.The mutations can be divided into three separate classes according to their amino acids features.When Asn342 was substituted by hydrophobic and non-polar amino acids,most variants for example N342V,N342I and M342L exhibited an up shift in pH optimum and their catalytic efficiency was similar to that of wild-type at their optimal pH.N342R variant showed a pH optimum at 6.2.N342K variant did not bring the up-shift of pH optimum although K and R are both amino acids carrying positive charges.The molecular model of EGⅡindicated that theN342 located at the C-terminus of one ofα-helix. Hydrophobic side chains and more H-bonds might cause the helix more rigid,which might have affected the stability and activity of enzyme at higher pH.4.Modifying the rEGⅡby error-prone PCR and DNA shuffling and some mutants with enhancing enzyme characters were obtained.The catalytic efficiency and pH optimum of T.reesei endo-β-1,4-glucanaseⅡwas improved by directed evolution including the random mutagenesis and two-rounds of DNA shuffling.Enzyme variants were purified and characterized. N39R/L218H/W276R/N342T had a pH optimum of 6.0～6.2 and had improved catalytic efficiency(1.4-fold of kcat/Km)at pH 6.2.Variants L218H and Q139R/N342T had more than 4.5-fold higher activity in reactions compared with the wild type at pH 7.0 although their pH optimum changed not much.The relationship between the structures and the activities of the variants were analyzed by modeling the structures of the endoglucanaseⅡvariants.More stable helixes,changed electrostatic interactions between the catalytic residues and substrates may explain the higher activities and higher pH optima of the variants.5.The bioinformation of endoglucanses in glycoside hydrolases family 5 were gathered and assayed.The amino acids sequence and optimum pH information of endoglucanses in glycoside hydrolases family 5 were gathered.The influence of amino acids composition to optimum pH was investigated.The relationship of the content of Arg plus His residues and the content of Cys plus Tyr residues with optimum pH was investigated:with the increase of content of Arg plus His residues,the optimum pH increased;with the increase of content of Cys plus Tyr residues,the optimum pH decreased.The information of seven endoglucanases whose optimum pH and 3D structure known were collected and made a comparison with the EGⅡfrom T.reesei: although they have the same hydrolysis mechanism and their 3D structure are similar, their optimum pH are absolutely different.6.Effective protocols of formation,regeneration and transformation of the protoplasts of Aspergillu-niger N593 were established.The results showed that the mycelia incubated for 14 hours were most suitable for protoplast release.0.6 M KCl was the most effective osmotic stabilizer.When the mixture solution of 3%0 snailase -4.5%0 cellulase -1.5%o lysozyme with the hydrolysis temperature and time for the release of protoplast were 32℃and 2 hours, the protoplasts yield was 10~6～10~7。The regeneration percentage was 45%when protoplasts were cultured with double layer medium.The transfomation value was about 30 cell/μg plasmid when auto-replication plasmid ANEp7 was used as transformation vector and the improved transformation reagent was used.7.The EGⅡfrom T.reesei expressed in A.niger was investigated.The carrying the AMA1 sequence of auto-replicated vector ANEp7 was constructed.The ANEp7-eg2 that carrying the eDNA of EGⅡfrom T.reesei was transformed into the A.niger N593.The lost rate of auto-replicated vector ANEp7 in transformants and whether the EGⅡhas been expressed in A.niiger were investigated. The lost rate of auto-replicated vector is very high:the lost rate could be 100%if used the spores of the third culture:The integrated express vector ANIp-eg2 was constrcted. However,we could not obtain its transformants.Improve the quality of protoplasts and transformation condition should be the next objective of our work.