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Origin of initial burst in activity for Trichoderma reesei endo-glucanases hydrolyzing insoluble cellulose.

ABSTRACT: The kinetics of cellulose hydrolysis have long been described by an initial fast hydrolysis rate, tapering rapidly off, leading to a process that takes days rather than hours to complete. This behavior has been mainly attributed to the action of cellobiohydrolases and often linked to the processive mechanism of this exo-acting group of enzymes. The initial kinetics of endo-glucanases (EGs) is far less investigated, partly due to a limited availability of quantitative assay technologies. We have used isothermal calorimetry to monitor the early time course of the hydrolysis of insoluble cellulose by the three main EGs from Trichoderma reesei (Tr): TrCel7B (formerly EG I), TrCel5A (EG II), and TrCel12A (EG III). These endo-glucanases show a distinctive initial burst with a maximal rate that is about 5-fold higher than the rate after 5 min of hydrolysis. The burst is particularly conspicuous for TrCel7B, which reaches a maximal turnover of about 20 s(-1) at 30 °C and conducts about 1200 catalytic cycles per enzyme molecule in the initial fast phase. For TrCel5A and TrCel12A the extent of the burst is 2-300 cycles per enzyme molecule. The availability of continuous data on EG activity allows an analysis of the mechanisms underlying the initial kinetics, and it is suggested that the slowdown is linked to transient inactivation of enzyme on the cellulose surface. We propose, therefore, that the frequency of structures on the substrate surface that cause transient inactivation determine the extent of the burst phase.

SUBMITTER: Murphy L

PROVIDER: S-EPMC3256860 | biostudies-other | 2012 Jan

REPOSITORIES: biostudies-other

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Origin of initial burst in activity for Trichoderma reesei endo-glucanases hydrolyzing insoluble cellulose.

Murphy Leigh L Cruys-Bagger Nicolaj N Damgaard Heidi Delcomyn HD Baumann Martin J MJ Olsen Søren Nymand SN Borch Kim K Lassen Søren Flensted SF Sweeney Matt M Tatsumi Hirosuke H Westh Peter P

The Journal of biological chemistry 20111122 2

The kinetics of cellulose hydrolysis have long been described by an initial fast hydrolysis rate, tapering rapidly off, leading to a process that takes days rather than hours to complete. This behavior has been mainly attributed to the action of cellobiohydrolases and often linked to the processive mechanism of this exo-acting group of enzymes. The initial kinetics of endo-glucanases (EGs) is far less investigated, partly due to a limited availability of quantitative assay technologies. We have ...[more]

PMID: 22110134

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Project description:BackgroundOn-site enzyme production using Trichoderma reesei can improve yields and lower the overall cost of lignocellulose saccharification by exploiting the fungal gene regulatory mechanism that enables it to continuously adapt enzyme secretion to the substrate used for cultivation. To harness this, the interrelation between substrate characteristics and fungal response must be understood. However, fungal morphology or gene expression studies often lack structural and chemical substrate characterization. Here, T. reesei QM6a was cultivated on three softwood substrates: northern bleached softwood Kraft pulp (NBSK) and lodgepole pine pretreated either by dilute-acid-catalyzed steam pretreatment (LP-STEX) or mild alkaline oxidation (LP-ALKOX). With different pretreatments of similar starting materials, we presented the fungus with systematically modified substrates. This allowed the elucidation of substrate-induced changes in the fungal response and the testing of the secreted enzymes' hydrolytic strength towards the same substrates.ResultsEnzyme activity time courses correlated with hemicellulose content and cellulose accessibility. Specifically, increased amounts of side-chain-cleaving hemicellulolytic enzymes in the protein produced on the complex substrates (LP-STEX; LP-ALKOX) was observed by secretome analysis. Confocal laser scanning micrographs showed that fungal micromorphology responded to changes in cellulose accessibility and initial culture viscosity. The latter was caused by surface charge and fiber dimensions, and likely restricted mass transfer, resulting in morphologies of fungi in stress. Supplementing a basic cellulolytic enzyme mixture with concentrated T. reesei supernatant improved saccharification efficiencies of the three substrates, where cellulose, xylan, and mannan conversion was increased by up to 27, 45, and 2800%, respectively. The improvement was most pronounced for proteins produced on LP-STEX and LP-ALKOX on those same substrates, and in the best case, efficiencies reached those of a state-of-the-art commercial enzyme preparation.ConclusionCultivation of T. reesei on LP-STEX and LP-ALKOX produced a protein mixture that increased the hydrolytic strength of a basic cellulase mixture to state-of-the-art performance on softwood substrates. This suggests that the fungal adaptation mechanism can be exploited to achieve enhanced performance in enzymatic hydrolysis without a priori knowledge of specific substrate requirements.

Dataset Information

Origin of initial burst in activity for Trichoderma reesei endo-glucanases hydrolyzing insoluble cellulose.

Publications

Origin of initial burst in activity for Trichoderma reesei endo-glucanases hydrolyzing insoluble cellulose.

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