Functional And Synergistic Studies Of Thermophilic Cellulases From Caldicellulosiruptor Bescii

In view of rising prices of crude oil due to increasing fuel demands, the need foralternative sources of bioenergy inresponse to decreasing of non-renewable resourcesand climate change is expected to increase sharply in the coming years.Lignocellulosic biomass, as the most abundant renewable source of organic matter onthe earth, has been identified as the prime source of biofuels and other value-addedproducts. To initiate the production of industrially important products from cellulosicbiomass, bioconversion of the cellulosic components into fermentable sugars isnecessary, whereas cellulase is the key element in this process. Thermostable enzymeshave been studied for their abilities to hydrolyze lignocellulosic materials since theyhave several potential advantages compared to their mesophilic counterparts, such asbetter hydrolytic efficiency and stability at higher temperatures. Screening novelthermostable cellulase and detailed study into the biochemical and catalytic propertiesof them, together with an improved understanding of the synergistic mechanisms inthe presence of other cellulase components is important to assess their full potentialfrom both fundamental and applied standpoints. Hearein we report cloning andoverexpression of processive endoglucanase, cellobiohydrylase and β-glucosidasefrom thermophile Caldicellulosiruptor bescii, we systematically determined thesubstrate specificities, product specificities and synergistic effects of the enzymes.A bifunctional enzyme designated CbCelA from C. bescii is a modular protein, ithas a glycoside hydrolase family9（GH9） module at the N-terminus and a GH48module at the C-terminus. Located between the two modules are threecarbohydrate-binding modules （CBMs）, one of which belongs to CBM3c subfamily,other two modules are repeated one belongs to CBM3b subfamily. To dissect function of the each module, several truncated forms （CbCel9A₃cbm，CbCel9A₂cbm,CbCel9A₁cbm, CbCel9A, CbCbh48A,2cbm_CbCbh48A, CBM3c and CBM3b）were constructed. The biochemical properties were described by determiningsubstrate and product specificities, ability of adsorption on cellulose and synergisticeffects with other cellulases. The results suggested that the GH9domain （CbCel9A）was a processive endoglucanase with high activity on insoluble cellulose, whereasGH48（CbCbh48A） was a cellobiohydrolase. It possessed very low activity towardscellulose but could synergistically work with CbCel9A₁cbm or other cellulase tohydrolyze substrate. The CBM3c module was very important to the activity andthermostability of CbCel9A, meanwhile, this protein helped CbCel9A partially bind tocellulose. The CBM3b module could adsorb to cellulose, resulting in increasedconcentration of enzymes on surface of cellulose, thereby enhancing the activities ofCbCel9A₁cbm and CbCbh48A.Bacterial β-glucosidase （BGL） is a major component of the cellulase system andis responsible for the hydrolysis of cellobiose and short chain oligosaccharides intoglucose. In an effort to obtain new thermostable BGLs desirable for the hydrolysis ofcellulose, herein we report a novel recombinant BGL from the thermophile C. bescii（CbBgl1A）. Enzyme properties of CbBgl1A were determined by analyses of substratespecificity, catalytic kinetics, structure modeling, along with synergistic effects withother cellulase components. We found that CbBgl1A could hydrolyze a range ofcellooligosaccharides, β-diglycosides, as well as aryl-β-glycosides at high temperature.It was resistance to both high concentrations of substrate and product, and its catalysiswas highly synergistic with exocellulases （cellobiohydrolases） and processiveendocellulase in the hydrolysis of cellulose.To further understand the synergistic mechanisms of cellulases, CbCel9A₃cbmand CbCbh48A, together with another thermalstable endoglucanase FnCel5A whichwas previously characterized in our lab, were selected to form a minimal set ofbacterial cellulases for bioconversion of cellulose. In addition, fixed amounts ofCbBgl1A were added to eliminate product inhibition.21reactions containing differentratios of three cellulases and fixed amounts of CbBgl1A were studied on disordered high-accessibility regenerated amorphous cellulose （RAC）. Processive endoglucanaseCbCel9A₃cbm was the most important for high cellulose digestibility, whereasCbCbh48A contributed the least. The optimal ratio for maximum cellulosedigestibility （81.9%） was60:20:20（CbCel9A₃cbm: CbCbh48A: FnCel5A） after24h incubation at60oC. The digestibility improved along with temperature increase.After2h incubation at75oC, the best mixture for maximum digestibility （46%） was80:0:20, which was2.82-fold improvement than that of CbCel9A alone.