Browsing by Author "Van Wyk, Niel"

Now showing 1 - 3 of 3
  • Thumbnail Image
    Item
    Comparative secretome analysis of Trichoderma asperellum S4F8 and Trichoderma reesei Rut C30 during solid-state fermentation on sugarcane bagasse
    (BioMed Central, 2013-11) Marx, Isa Jacoba; Van Wyk, Niel; Smit, Salome; Jacobson, Daniel; Viljoen-Bloom, Marinda; Volschenk, Heinrich
    Background: The lignocellulosic enzymes of Trichoderma species have received particular attention with regard to biomass conversion to biofuels, but the production cost of these enzymes remains a significant hurdle for their commercial application. In this study, we quantitatively compared the lignocellulolytic enzyme profile of a newly isolated Trichoderma asperellum S4F8 strain with that of Trichoderma reesei Rut C30, cultured on sugarcane bagasse (SCB) using solid-state fermentation (SSF). Results: Comparison of the lignocellulolytic enzyme profiles of S4F8 and Rut C30 showed that S4F8 had significantly higher hemicellulase and β-glucosidase enzyme activities. Liquid chromatography tandem mass spectrometry analysis of the two fungal secretomes enabled the detection of 815 proteins in total, with 418 and 397 proteins being specific for S4F8 and Rut C30, respectively, and 174 proteins being common to both strains. In-depth analysis of the associated biological functions and the representation of glycoside hydrolase family members within the two secretomes indicated that the S4F8 secretome contained a higher diversity of main and side chain hemicellulases and β-glucosidases, and an increased abundance of some of these proteins compared with the Rut C30 secretome. Conclusions: In SCB SSF, T. asperellum S4F8 produced a more complex lignocellulolytic cocktail, with enhanced hemicellulose and cellobiose hydrolysis potential, compared with T. reesei Rut C30. This bodes well for the development of a more cost-effective and efficient lignocellulolytic enzyme cocktail from T. asperellum for lignocellulosic feedstock hydrolysis.
  • Thumbnail Image
    Item
    Expression and characterization of exoglucanases in Saccharomyces cerevisiae
    (Stellenbosch : University of Stellenbosch, 2010-03) Van Wyk, Niel; Van Zyl, Willem Heber; Den Haan, Riaan; University of Stellenbosch. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: Currently a world-wide tendency exists to shift away from relying on fossil fuels as a primary energy source and to focus on sustainable, environmentally-friendly alternatives. Ethanol is one such alternative and shows potential to replace petroleum as a transport fuel. Plant biomass, deemed a renewable energy source, can be converted to ethanol. The process of conversion via biologicallymediated events is problematic mainly due to the recalcitrance of the chief components of plant biomass namely cellulose, hemicellulose and lignin towards enzymatic degradation. A concept of consolidated bioprocessing (CBP) aims to make the process of bioconversion of plant biomass to ethanol cost-effective. For such a bioconversion, a biocatalyst is needed which can depolymerize the complex carbohydrates i.e. the cellulose and hemicellulose to their respective monomers for concurrent fermentation to ethanol. Saccharomyces cerevisiae shows potential as a candidate CBPbiocatalyst due to its high ethanol productivity, general robustness and amenability to genetic manipulation. However, S. cerevisiae does not possess the ability to break down the abovementioned carbohydrates. This study attempted to address certain aspects of yeast strain development for CBP. Genes encoding cellulases responsible for major crystalline cellulose hydrolysis i.e. exoglucanases were expressed in S. cerevisiae and the recombinant proteins were characterized. Further work involved exploring different ways of increasing the cellulolytic capability of recombinant S. cerevisiae. Both the cel9A of Thermobifida fusca and Npcel6A of Neocallimastix patriciarum were functionally expressed in S. cerevisiae. Expression of cel9A enabled S. cerevisiae to grow on phosphoric acid swollen cellulose reaching an aerobic growth rate (mMAX) of 0.088 h-1. This is the first report of S. cerevisiae growing on such a substrate while producing only one heterologous protein. An increase in the cellulolytic capability of recombinant S. cerevisiae was observed when cel9A was coexpressed with Trcel6A, cel7A and cel7B of Trichoderma reesei. These results proved that the synergy between cellulases can contribute towards increasing the cellulolytic capability of recombinant S. cerevisiae. NpCel6A has the highest reported individual activity on a crystalline cellulose substrate. However, expression of Npcel6A by S. cerevisiae resulted in lower levels of exoglucanase activity on Avicel of 0.540±0.062 mU/gDCW compared to the recombinant S. cerevisiae strains that produces Cel6A of T. reesei (4.101±0.243 mU/gDCW). This observation could be ascribed to glycosylation of the catalytic domain of NpCel6A. The replacement of the carbohydrate-binding module (CBM) and asparagine-rich linker of NpCel6A with the CBM and serine/threonine-rich linker of TrCel6A resulted in a decrease in recombinant cellulolytic activity produced by S. cerevisiae. In contrast, when the CBM and linker of NpCel6A were appended to the N-terminus of the catalytic domain of TrCel6A, significantly higher levels of cellulase activity were observed when produced by S. cerevisiae. This observation was largely attributed to the difference in glycosylation of the linkers. These results showed the value of domain swapping for obtaining increased cellulase secretion by S. cerevisiae. The native S. cerevisiae genes PSE1 and SOD1, were individually overexpressed in the S. cerevisiae strain producing NpCel6A, Cel3A of Saccharomycopsis fibuligera and Cel7B of Trichoderma reesei. The DDI1 gene of S. cerevisiae was also disrupted in the strain producing NpCel6A. In all cases, transformants were identified which displayed higher levels of cellulase activity compared to the original strain. This demonstrated the potential of S. cerevisiae to be considered as a “chassis”- strain that can, with the help of metabolic engineering, produce more recombinant cellulases. The swelling factor protein called swollenin, a contributor in the disruption of the crystallinity of cellulose, was co-expressed with cel9A and Npcel6A individually in S. cerevisiae. Even though functionality of swollenin was confirmed, no noteworthy increase in the levels of cellulase activity was observed for recombinant strains. The recombinant yeast strains generated during this study represent significant progress towards developing S. cerevisiae as a CBP organism.
  • Thumbnail Image
    Item
    Identification of the gene for β-fructofuranosidase from Ceratocystis moniliformis CMW 10134 and characterization of the enzyme expressed in Saccharomyces cerevisiae
    (BioMed Central, 2013-11) Van Wyk, Niel; Trollope, Kim M.; Steenkamp, Emma T.; Wingfield, Brenda D.; Volschenk, Heinrich
    Background: β-Fructofuranosidases (or invertases) catalyse the commercially-important biotransformation of sucrose into short-chain fructooligosaccharides with wide-scale application as a prebiotic in the functional foods and pharmaceutical industries. Results: We identified a β-fructofuranosidase gene (CmINV) from a Ceratocystis moniliformis genome sequence using protein homology and phylogenetic analysis. The predicted 615 amino acid protein, CmINV, grouped with an existing clade within the glycoside hydrolase (GH) family 32 and showed typical conserved motifs of this enzyme family. Heterologous expression of the CmINV gene in Saccharomyces cerevisiae BY4742Δsuc2 provided further evidence that CmINV indeed functions as a β-fructofuranosidase. Firstly, expression of the CmINV gene complemented the inability of the Δsuc2 deletion mutant strain of S. cerevisiae to grow on sucrose as sole carbohydrate source. Secondly, the recombinant protein was capable of producing short-chain fructooligosaccharides (scFOS) when incubated in the presence of 10% sucrose. Purified deglycosylated CmINV protein showed a molecular weight of ca. 66 kDa and a Km and Vmax on sucrose of 7.50 mM and 986 μmol/min/mg protein, respectively. Its optimal pH and temperature conditions were determined to be 6.0 and 62.5°C, respectively. The addition of 50 mM LiCl led to a 186% increase in CmINV activity. Another striking feature was the relatively high volumetric production of this protein in S. cerevisiae as one mL of supernatant was calculated to contain 197 ± 6 International Units of enzyme. Conclusion: The properties of the CmINV enzyme make it an attractive alternative to other invertases being used in industry.