Morphology and enzyme production of Trichoderma reesei Rut C-30 are affected by the physical and structural characteristics of cellulosic substrates

Abstract

The industrial production of cellulolytic enzymes is dominated by the filamentous fungus Trichoderma reesei (anamorph of Hypocrea jecorina). In order to develop optimal enzymatic cocktail, it is of importance to understand the natural regulation of the enzyme profile as response to the growth substrate. The influence of the complexity of cellulose on enzyme production by the microorganisms is not understood. In the present study we attempted to understand how different physical and structural properties of cellulose-rich substrates affected the levels and profiles of extracellular enzymes produced by T. reesei. Enzyme production by T. reesei Rut C-30 was studied in submerged cultures on five different cellulose-rich substrates, namely, commercial cellulose Avicel® and industrial-like cellulosic pulp substrates which consist mainly of cellulose, but also contain residual hemicellulose and lignin. In order to evaluate the hydrolysis of the substrates by the fungal enzymes, the spatial polymer distributions were characterised by cross-polarisation magic angle spinning carbon-13 nuclear magnetic resonance (CP/MAS (13)C-NMR) in combination with spectral fitting. Proteins in culture supernatants at early and late stages of enzyme production were labeled by Tandem Mass Tags (TMT) and protein profiles were analysed by liquid chromatography-tandem mass spectrometry. The data have been deposited to the ProteomeXchange with identifier PXD001304. In total 124 proteins were identified and quantified in the culture supernatants, including cellulases, hemicellulases, other glycoside hydrolases, lignin-degrading enzymes, auxiliary activity 9 (AA9) family (formerly GH61), supporting activities of proteins and enzymes acting on cellulose, proteases, intracellular proteins and several hypothetical proteins. Surprisingly, substantial differences in the enzyme profiles were found even though there were minor differences in the chemical composition between the cellulose-rich substrates.

Impact of the supramolecular structure of cellulose on the efficiency of enzymatic hydrolysis

“…In a previous study by our group, enzyme production during the filamentous fungus T. reesei growth on cellulose-rich pulps isolated from softwood and NCC-OPHS-ND was compared with growth of the fungus on Avicel. It was found that enzyme production by the filamentous fungus T. reesei showed the highest protein production when Avicel was used as a carbon and energy source for the fungus and that protein production levels were significantly lower when cellulose-rich pulps or NCC-OPHS-ND were used as carbon sources [ 36 ]. This observation led us to hypothesize that there may exist a relation between the structural properties of the substrate used for the fungal growth and the capacity of the produced enzymes to hydrolyse the same substrate.…”

Applying Taguchi design and large-scale strategy for mycosynthesis of nano-silver from endophytic Trichoderma harzianum SYA.F4 and its application against phytopathogens

“…The assay was performed in 96-well microtitre plates in duplicates for each sample. The protein concentration was defined as the amount of extracellular proteins measured per L of fungal supernatant515253. All analyses were done in triplicate in a temperature-controlled incubator.…”

Visualization of structural changes in cellulosic substrates during enzymatic hydrolysis using multimodal nonlinear microscopy

“…The Fibres were stored in the never-dried state at 4°C until use. AT fibre was produced from the Fibre, according to the procedure described previously by prolonged hydrolysis with hydrochloric acid at elevated temperature (95-100°C) for 17 h (Peciulyte et al 2014), and stored in excess deionized water in a glass bottle with a lid at room temperature. Avicel Ò PH-101 was purchased as a powdered microcrystalline cellulose (Fluka BioChemika, Ireland).…”