Synergistic Effects Of Swollenin And Endoglucanase On The Degradation Of Crystalline Cellulose

Lignocellulose is the most abundant organic substances on the earth, and also a kind of renewable resources. Thus, lignocellulose plays an important role in the sustainable development of industrial society. To date, how to effectively and cheaply hydrolyse the lignocellulose is still a key problem in the production of biofuel, since the tight construction of lignocellulose will limit the access of cellulase hydrolases. Swollenin is a kind of protein which can destroy the hydrogen bond network between the polysaccharide chains and further decompose the tight and ordered construction of lignocellulose. After the treatment of swollenin, the cellulose hydrolases can easily reach and hydrolyse the polysaccharide chains, which would be a new route to realize the transformation of lignocellulose into biofuel.In this paper, the synergistic effects of swollenin from Trichoderma asperellum and endocellulase FnCel5A on the degradation.of crystalline cellulose Avicel were systematically investigated.The swollenin from Trichoderma asperellum (TasSWO) was first reported by Yariv Brotman. The TasSWO protein contains a secretion signal peptide of 19-amino acid with a predicted cleavage site between amino acids 19 and 20 (SignalP 3.0), yielding a mature protein of 477 amino acids. Smart-architecture domain analysis predicts a CBD between amino acids 24 and 57, and an expansin domain (pollen allergen 1 domain) at amino acids 381 to 471. BLAST search with two-thirds (amino acids 170–477) of the C-terminal sequence showed low homology to an EXPB-like protein precursor from tomato and EXB10 from maize. The theoretical molecular weight and isoelectric point of TasSWO were 52184.87Da and 4.90, respectively.In the present study, a recombinant E.coli BL21 CodonPlus(DE3) RIL with pET-11a-tas swo plasmid were constructed and over-expressed in the form of inclusion body. Then we used the denaturant solution, which contained 6 M guanidine hydrochloride and 10 mM DTT, to dissolve the inclusion body protein. After the inclusion bodies were solubilized by high concentration of denaturing agents, refolding was performed by the controlled remove of excess denaturant. Through the comparison of efficiency of three refolding methods (dilution, dialysis and column chromatography), we determined the chromatographic process, because it was easily automated using commercially available preparative chromatography systems, and can often be combined with simultaneous partial purification. Using Avicel as the solid support, the effects of temperature, velocity and the concentration of guanidine hydrochloride and DTT were optimized to obtain the highest refolding efficiency. The results showed that 22 oC, 20 mL/h of the velocity, 2.5 M guanidine hydrochloride and 7 mM DTT were beneficial for the refolding. The absorption amount of denatured TasSWO on Avicel was 4 mg/g, and after refolding, there was only 0.6 mg TasSWO adhered to the TasSWO. The refolding TasSWO could be thoroughly eluted from the Avicel by glycol, and then used for further research.Endocellulase FnCel5A has been cloned and expressed from Fervidobacterium nodosum Rt17-B1, an extremely thermophilic, glycolytic anaerobic bacterium. FnCel5A had a high activity towards endo-β-1,4-glucan. However, no activity towards Avicel was observed. Using CMC as the substrate, the specific activity was 440 U/mg in citrate acid-NaH2PO4 buffer (pH 5.2) at 83 oC. For the synergistic effect of TasSWO and FnCel5A on the crystalline cellulose Avicel, the optimal temperature and pH was 45 and 5.0, respectively. Therefore, the synergistic effect of TasSWO and FnCel5A on Avicel provided a new route to produce reducing sugar, and also a new method to define TasSWO's swelling activity. Since TasSWO had the swelling activity and no hydrolytic activity on Avicel, in the presence of FnCel5A, the amount of reducing sugar produced would be a quantitative index, which represented the swelling activity of TasSWO. The swelling activity of TasSWO was 0.374U/mg towards Avicel in citrate acid-NaH2PO4 buffer (pH 5.0) at 45 oC.In the thesis, the synergistic effects of swollenin TasSWO and endocellulase FnCel5A on the crystalline cellulose degradation was systematically investigated. The characterization and function of swollenin TasSWO could be analyzed in the research, and moreover, we could determine the activity of destroying crystalline cellulose Avicel. The research provided a new route for degrading the natural crystalline cellulose in the biomass, and also an effective quantitative method of the swollenin TasSWO for its degradation activity.