Production of carbohydrases by fungus Trichoderma reesei grown on soy-based media

Trichoderma reesei RUT-C30 was cultivated in shaker flasks and pH-controlled, agitated batch fermentations to study the effects of soy-based media on the production of cellulase, xylanase, and pectinase (polygalacturonase) for the purposes of soybean polysaccharide hydrolysis. Growth on defatted soybean flour as sole nitrogen source was compared to the standard combination of ammonium sulfate, proteose peptone, and urea. Carbon source effect was also examined for a variety of substrates, including lactose, microcrystalline cellulose (Avicel), citrus pectin, soy molasses, soy flour hydrolysate, and soybean hulls (both pretreated and natural). Flask study results indicated exceptional enzyme induction by Avicel and soybean hulls, while citrus pectin, soy molasses, and soy flour hydrolysate did not promote enzyme production. Batch fermentation experiments reflected the flask system results, showing the highest cellulase and xylanase activities for systems grown with Avicel and soybean hulls at near-neutral pH levels, and the highest polygalacturonase activity resulting from growth on lactose and soybean hulls at lower pH levels, 4.0 to 4.5. Soy polysaccharides were hydrolyzed with fermentation enzyme broths of varying enzyme composition and concentration. Enzyme mixtures with larger proportions of cellulase and polygalacturonase showed slightly higher sugar release. Hydrolysis was incomplete in all experiments, solubilizing roughly one-third of the polysaccharides to oligomeric products. Additionally, methods for estimation of cell concentration via cell DNA extraction and cell wall chitin hydrolysis and a modified intracellular protein analysis procedure were investigated, showing high levels of error and interference by soy protein from the growth media. Finally, a consistent and reliable assay for polygalacturonase activity was developed and verified.