Abstract
BackgroundAs a green alternative for the production of transportation fuels, the enzymatic hydrolysis of lignocellulose and subsequent fermentation to ethanol are being intensively researched. To be economically feasible, the hydrolysis of lignocellulose must be conducted at a high concentration of solids, which results in high concentrations of hydrolysis end-products, cellobiose and glucose, making the relief of product inhibition of cellulases a major challenge in the process. However, little quantitative information on the product inhibition of individual cellulases acting on cellulose substrates is available because it is experimentally difficult to assess the hydrolysis of the heterogeneous polymeric substrate in the high background of added products.ResultsThe cellobiose and glucose inhibition of thermostable cellulases from Acremonium thermophilum, Thermoascus aurantiacus, and Chaetomium thermophilum acting on uniformly 14C-labeled bacterial cellulose and its derivatives, 14C-bacterial microcrystalline cellulose and 14C-amorphous cellulose, was studied. Cellulases from Trichoderma reesei were used for comparison. The enzymes most sensitive to cellobiose inhibition were glycoside hydrolase (GH) family 7 cellobiohydrolases (CBHs), followed by family 6 CBHs and endoglucanases (EGs). The strength of glucose inhibition followed the same order. The product inhibition of all enzymes was relieved at higher temperatures. The inhibition strength measured for GH7 CBHs with low molecular-weight model substrates did not correlate with that measured with 14C-cellulose substrates.ConclusionsGH7 CBHs are the primary targets for product inhibition of the synergistic hydrolysis of cellulose. The inhibition must be studied on cellulose substrates instead of on low molecular-weight model substrates when selecting enzymes for lignocellulose hydrolysis. The advantages of using higher temperatures are an increase in the catalytic efficiency of enzymes and the relief of product inhibition.
Highlights
As a green alternative for the production of transportation fuels, the enzymatic hydrolysis of lignocellulose and subsequent fermentation to ethanol are being intensively researched
This approach inevitably results in high concentrations of the hydrolysis end-products cellobiose and glucose, and it has been proposed that the endproduct inhibition of cellulases is rate limiting for lignocellulose hydrolysis in high-solid conditions [5]
Ki is a fundamental parameter of enzyme kinetics that is directly related to the thermodynamic stability of the enzymeinhibitor complex
Summary
As a green alternative for the production of transportation fuels, the enzymatic hydrolysis of lignocellulose and subsequent fermentation to ethanol are being intensively researched. The hydrolysis of lignocellulose must be conducted at a high concentration of solids, which results in high concentrations of hydrolysis end-products, cellobiose and glucose, making the relief of product inhibition of cellulases a major challenge in the process. To decrease the water consumption and reduce the costs of equipment and distillation, the hydrolysis of lignocellulose must be conducted at a high concentration of solids This approach inevitably results in high concentrations of the hydrolysis end-products cellobiose and glucose, and it has been proposed that the endproduct inhibition of cellulases is rate limiting for lignocellulose hydrolysis in high-solid conditions [5]. The end-product inhibition can be relieved in a simultaneous saccharification and fermentation process, where the fermenting organism is added in parallel with hydrolytic enzymes, but one drawback is the need for different conditions for optimal hydrolysis and fermentation. A process concept using high temperature liquefaction with thermostable enzymes preceding simultaneous saccharification and fermentation has been developed [7], and this has triggered the search for novel thermostable enzymes [8,9]
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