Characterization Of Thermophilic Endoglucanase And Functional Study Of Related Cellulose Binding Module

Cellulases play a key role in the biotransformation process of bio-ethanol.Thus it is of great interest and significance to study the mechanism of enzymaticdegradation of insoluble biomass. Characterization of thermostable and thermoactivecellulases from thermophilic bacteria and investigation of their structure-functionrelationship will not only shed light on the structure base of stability and catalyticmechanism at high temperature, but also provide us with new enzymes for potentialindustrial applications. This thesis reports the functional research of theendo-cellulase and its related CBM from Caldicellulosiruptor bescii.The cellulase gene （CbCelA） from C. bescii consists of five modules, whichare designated CbCel9A, three CBMs （CBM3c-CBM3b-CBM3b） and CbCBH48A.This thesis focuses on this gene and get the following results:（1） Successful cloned,expressed and purified the truncated enzymes. Four truncated enzymes whichcontained GH9module and0-3CBMs were constructed and functionallyoverexpressed in E. coli BL21-CodonPlus （DE3）–RIL, respectively. The proteinswere designated CbCel9A, CbCel9A_CBM, CbCel9A₂CBM and CbCel9A₃CBM.The recombined proteins were purified after the heat-treated and nickel affinitychromatography.（2） the enzymes with different numbers of CBMs had activity athigh temperature. The optimum temperature of enzymes are about95-100oC andthermal stability maintained a good situation even at85oC for50min, however, theoptimum temperature of CbCel9A is45-50oC, and thermal stability analysis showedthat it only remained20%activity after20min incubation at60oC.（3） The optimalsubstrate was the sodium carboxymethyl cellulose （CMC） for soluble saccharideswhich was a typical characteristics of endo-cellulase. The activity of truncatedenzymes towards microcrystalline cellulose Avicel was in an order of CbCel9A₃CBM>CbCel9A₂CBM>CbCel9A_CBM>CbCel9A;（4） The enzymeshad adsorption and processivity on filter paper and Avicel. The adsorption andprocessive ratio of the recombinant enzymes on filter paper showed the same orderabove.（5） The major oligosaccharides in the hydrolysis of the RAC were glucose,cellobiose and cellotriose. The activity of CbCel9A towards cellotriose was lowerthan the others.（6） CbCel9A_CBM and CbCel9A₂CBM synergistically workedwith cellobiohydrolase CbCbh48A from C. bescii and β-glucosidase CbBgl1A ondegradation of filter paper, and generated sole glucose. Based on the above results, theCBM3c adjacent to CbCel9A plays critical roles in maintaining themoactivity andthermostability of CbCel9A, and has positive effects on the adsorption of insolublesubstrate. The CBMs are conductive to the adsorption and processive ratio ofCbCel9A on the insoluble substrates; The enzyme has synergy with both CbCBH48Aand FnCel5A, and can hydrolyze the Filter paper efficiently. The result revealed thefunction of CbCel9A and its related CBM, and the enzymes we investigated here arevery valuable for further research and potential application.