The development of recombinant fungal enzyme cocktails for the hydrolysis of cellulosic waste products

[摘要] ENGLISH ABSTRACT: Biofuels, such as bioethanol, provide an alternative, environmentally friendly transportation fuel. Renewable energy sources, such as lignocellulosic material, are therefore being explored for the production of biofuels, since they offer an attractive and sustainable source for bioconversion processes. However, the major obstacle in the use of lignocellulosic biomass is its recalcitrant nature, which decreases the enzyme accessibility to cellulose and thus affects the overall hydrolysis process. Current commercial enzyme cocktails are not yet sufficient to promote hydrolysis on an industrial scale, thus hampering biofuel production.A number of cellulase enzymes are needed to act in synergy to obtain complete hydrolysis of lignocellulosic material. The enzymatic hydrolysis of cellulose requires the synergistic action of three cellulase enzymes namely endoglucanases, exoglucanases and β-glucosidases. However, cellulolytic organisms do not produce significant amounts of ethanol, whereas strong fermentative organisms don't produce enzymes for cellulose hydrolysis. A need has therefore arisen to develop recombinant technologies to obtain maximum production of cellulolytic enzymes that can be used (exogenously) in combination with a fermentative organism.Paper sludge is a lignocellulosic waste material that is generated in large quantities by the pulp and paper industry. Non-hazardous paper sludge can be converted to fermentable sugars, which can then be fermented to bioethanol. Biological conversion of paper sludge requires no pre-treatment, making it an ideal substrate for industrial use. The development of enzyme cocktails for efficient hydrolysis of paper sludge is therefore important in the pursuit of second-generation bioethanol production.A recombinant cellulase enzyme cocktail tailored for the degradation of paper sludge was developed using cellulases from recombinant Aspergillus niger and Saccharomyces cerevisiae strains. The recombinant strains were cultured and their supernatants used to develop an enzyme cocktail based on activity ratios. The core cellulases in the optimal cocktail included a cellobiohydrolase I, cellobiohydrolase II, endoglucanase and β-glucosidase. The enzyme cocktails were subsequently evaluated on triticale, Avicel and wheat bran.The activities (in Filter Paper Units) for the final cocktails were 0.7 and 0.45 for the CbhI:CbhII:EgA:Bgl1 and CbhI:CbhII:EgA:Bgl2 cocktails, respectively. The optimum enzyme ratio (based on protein concentrations) for the CbhI:CbhII:EgA:Bgl1 cocktail was 7.4:6.6:1:208 and 7.4:6.6:1:41 for the CbhI:CbhII:EgA:Bgl2 cocktail. Overall, hydrolysis with the Bgl2 cocktail allowed for longer incubation times and an improved degree of saccharification when the enzyme concentration was doubled. Comparison of paper sludge hydrolysis results with those from Avicel hydrolysis highlight the need to tailor enzyme cocktails based on natural substrates.Two industrial amylolytic S. cerevisiae yeast strains were compared in an SSF (10% wheat bran) process, using the Bgl2-cocktail. The maximum ethanol yield produced by S. cerevisiae S2[TLG, SFA] and S. cerevisiae MH1000[TLG, SFA], in the presence of the 1x enzyme cocktail, was 5.72 g.l-1 and 5.45 g.l-1, respectively. This study demonstrated that the addition of the recombinant cellulase cocktail improved the ethanol yields by 8.69% in the SSF process and that the S. cerevisiae S2[TLG, SFA] and MH1000[TLG, SFA] strains efficiently converted starch to ethanol.To our knowledge, this is the first report of the use of individual enzymes from recombinant strains, for the hydrolysis of paper sludge and wheat bran. This study has provided insight into the hydrolysis of cellulosic materials, using recombinant cellulase cocktails. The knowledge obtained could be applied in optimising lignocellulose hydrolysis, for efficient sugar release and ultimately improving ethanol production by recombinant yeast strains. This study also demonstrates the potential of using agricultural and industrial wastes as lignocellulosic feedstocks for biofuels production.

[发布机构] Stellenbosch University