Expression And Purification Of Trichoderma Reesei Swollenin In Aspergullus Oryzae

The swollenin protein is similar to plant expansin, it can act on cellulose materials and cause loose structure, promote cellulase to decopositon cellulose despite the lack of hydrolytic activity itself. The protein was named swollenin and the gene was named swol due to its ability to swell cotton fibers.Cellulose is the most abundant forms of carbohydrate in nature and represents potential feedstocks for the production of chemicals to replace petroleum. On a commercial scale, the conversion of cellulosic biomass requires the use of cellulase that makes the process more costly. Accordingly, the activity of swollenin and plant expansin is very helpful to cellulose decomposition by cellulase despite the lack of hydrolytic activity themselves. However, it is difficult to express plant expansin in other spieces, only purification can not meet the industrial need, swollenin have an advantage in that it can be expressed heterologously in a different microorganism host, and thus the expression level can be improved through microorganism fermentation. Most of the previous works on swollenin mainly focused on the investigation of swollenin activity, little information about the effects of medium composition and expression conditions on the productivity of swollenin protein is available.The swol gene encodes a polypeptide of 493 amino acid residues, the bio-informatics software analysis result indicates that the predicted molecular weight and pI of the protein was 51.5 kD and 4.8, respectively. The swollenin protein has three mainly domain as follows: cellulose binding domain, expansin-EG 45 domain and expansin-CBD.The secondary structure of swollenin is mainlyβ-sheet and randon coil, especially the structure of CBD is the most loosy. We successfully expressed the swollenin protein in recombinant Aspergillus oryzae, with regard to the swollenin bioactivity, investigations on the effects of culture compisiton (such as carbon source and nitrogen source) and culture conditions development were presented here. The optimal conditions were determined as follows: cultivation in the medium with glycerin and peanut as the carbon and nitrogen source, respectively, with the 1.5×107 spores in 40mL culture, at pH 5.6, and expression for 88 h. Under the above optimal conditions, the highest productivity (50mg/L) of swollenin was achieved. Swollenin in cultural supetnatant was purified by affinity chromatography and cationic exchange chromatography, but the swollenin purified by affinity chromatography did not show synergistic activity, the probable reason is that the protein had been denatured; the soluble swollenin proteins were then successfully recovered from the cultural supernatant by ammonium sulfate precipitation and cationic exchange chromatography. The purity and overall recovery of the swollenin was 95.1% and 53.2% after purification, respectively. 26.6 mg swollenin could be purified from 1 L fermentation medium. Finally, the synergistic activity of purified swollenin was investigated, and the protein exhibited a significant synergistic effect on cellulose hydrolysis with cellulase. When a mixture containing 0.06FPU cellulase and 300μg of swollenin per g of filter paper was evaluated, the released reducing sugar yield was 81.7% higher than that of control.