Gene Excavation And Molecular Engineering Of Glycoside Hydrolyase Amily 5 Cellulase From Fungi

Cellulases from microbiology can convert cellulose into fermentable sugar,which has been widely used in biofuel,animal feed,food,paper and various other types of production industries.It is of great significance to discover novel cellulases with potential applications from microbial genome and improve their performance through molecular modification.In this study,a few cellulases and swollenin protein were cloned from Talaromyces leycettanus JCM12802,Bispora sp.MEY-1,Bispora antennata CBS 126.38,Gloeophyllum trabeum CBS900.73,Stegonsporium opalus CBS 125034,and expressed successfully.Based on structural analysis of different proteins,through methodologies including half-barrel fusion,secondary structure unit replacement.site mutation and molecular dynamics analysis system.we studied the relationships between the structures and the functions of specific amino acids in GH5 cellulases.The stability and catalytic mechanism of GH5 cellulases were further clarified.Seven GH5 cellulase genes from the above 5 strains were successfully cloned and expressed.The results of enzymatic characterization indicated that the seven recombinant proteins(TlCel5A,TlCel5B,BsCel5A,BsCel5B,GtCel5,SoCel5,BaCel5)showed maximum activity within pH 3.0-5.0 and 50-800 C.TlCel5A and BsCel5B which belong to thermophilic cellulases showed maximum activity at around 80? and good thermostability at 70?.TICel5B has an optimal pH of 3.0 and showed good acid resistance.In addition,seven recombinant proteins showed highest activity against to Barley glucan,Lichenan and CMC-Na.BsCel5B has high cellulase(1164 U/mg)and mannase activity(2203 U/mg)which is much higher than that of other reported cellulases.SoCel5 with optimal temperature at 60? showed sequence similarity of 51%to TeEgl5A with optimal temperature at 90? from Talaromyces emersonii CBS394.64.Based on the analysis of secondary structure,TeEgl5A and SoCel5 were divided into 10 different modules.after which 10 hybrid proteins from H1 to H10,were built.Two thermophilic proteins,H8 and H9 were obtained.Characterization analysis showed that H8 and H9 had an optimal temperature of 80? and 70?,respectively.The half-life was further measured at 55?,H8 and H9 extended half-lives(t1/2)155-650 folds relative to that of the mesophilic parent SoCel5,In addition,the specific activities of H8 and H9 were 720 U/mg and 920 U/mg respectively,both higher than those of the two parents(350 U/mg for SoCel5 and 600 U/mg for TeEgl5A).Molecular dynamics(MD)simulations suggested that the hydrogen bond network around Arg52 and the hydrophobic packing of the interface between a2 and ?3 play an important role in the thermal stability of thermophilic enzymes.The N-terminal half-barrel structure(??)i-4 and(??)1-3 of TeEgl5A were substituted into the corresponding region of BaCel5,creating two hybrid enzymes,BaCel5127and BaCel5167.BaCel5127 and BaCel5167 showed similar enzymatic properties but improved catalytic performance.They had increased specific activities and catalytic efficiencies by 1.8-6.7-fold.The underlying mechanism was analyzed by molecular docking and MD simulation.The analysis suggested that the introduction of the N-terminal segment of TeEgl5A significantly changed the overall configuration of the catalytic channels of BaCel5127 and BaCel5167,which also were more conducive to the substrate binding and the product release.Besides,the distance between two catalytic residues was significantly reduced,which was conducive to the hydrolysis of substrates.In addition,more hydrogen bonds were present in BaCel5127 and BaCel5167,promoting the binding of enzyme and substrate.Through this study,it was proven that the N-terminal half-barrel structure of TeEgl5A had an impactful influence on the catalytic activity of cellulases,and the catalytic efficiency of BaCel5 was also successfully improved.The saturated mutation of Asn233 of GtCel5 was studied to explore its influence on the catalytic performance of GH5 cellulase.Asn233 is involved in forming the hairpin structure on loop 6.By analyzing the properties of the mutants,it was found that activity of N233G and N233A mutants on the substrate was improved by 26-70%compared with that of the wild type.The reverse mutations at the same site were constructed in the other two GH5 cellulase,TeEgl5A and SoCel5.The activity of the mutant from high to low was Gly>Ala>Asn,which was consistent with the result of GtCel5.MD simulation suggested that due to the introduction of Ala and Gly,a stronger hydrogen bond formed between the substrate and 233 site,and the binding energy were also lower than that of the wild type,providing support for the study on the mechanism of loop region affecting catalytic activity.We cloned a swollenin gene with cellulase activity from T.leycettanus and expressed it successfully,obtaining the recombinant protein TlSWO.TlSWO showed its degradation ability on cellulose substrates that contained ?1,4-bond,with optimal activity pH and operating temperature at 4.0 and 60?,respectively.When using pretreated corn stover(PCS)as the substrate,the synergistic effect reaches optimal value when the mixing ratio of TlSWO and the endoglucase EGI which derived from Trichoderma longibrachiatu was 1.2:0.8.When using phosphoric acid swelling microcrystalline cellulose(PASC)and highly crystallized cellulose(CNC)as the substrates,TlSWO and CBHI which derived from Trichoderna reesei showed a significant synergistic effect,and the conversion rate of substrate was improved by 9.6%and 30%,respectively.This study has provided us with numerous discoveries that introduced new understanding of fungal swollenin protein.