Hypocrea jecorina (formerly Trichoderma reesei) Cel7A has a catalytic domain (CD) and a cellulose-binding domain (CBD) separated by a highly glycosylated linker. Very little is known of how the 2 domains interact to degrade crystalline cellulose. Based on the interaction energies and forces on cello-oligosaccharides computationally docked to the CD and CBD, we propose a molecular machine model, where the CBD wedges itself under a free chain end on the crystalline cellulose surface and feeds it to the CD active site tunnel. Enzyme-substrate interactions produce the forces required to pull cellulose chains from the surface and also to help the enzyme move on the cellulose chain for processive hydrolysis. The energy to generate these forces is ultimately derived from the chemical energy of glycosidic bond breakage.
"The binding of xylo-oligosaccharides with DP 8 – 10 is expected to mimic the binding of the cellulose chain to the active site of TrCel7A, resulting in competitive inhibition. In contrast, despite the strong binding of cellobiose to the product sites (+1/+2) of TrCel7A [52,54,55], the cellulose chain can still bind to the substrate sites (from −7 to −1), and this predicts non-competitive inhibition [23,31,32]. The results of our previous studies of the inhibition of TrCel7A under single-turnover and steady-state conditions suggested that cellobiose might be a mixed-type inhibitor of TrCel7A acting on cellulose. "
[Show abstract][Hide abstract] ABSTRACT: As 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.
The 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.
GH7 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.
Biotechnology for Biofuels 07/2013; 6(1):104. DOI:10.1186/1754-6834-6-104 · 6.04 Impact Factor
"It has been proposed that the hydrolase domain and adjacent CBM of CelZ structurally modify the substrate by freeing cellulose chains to make them accessible to other cellulases (Riedel and Bronnenmeier, 1999). In the Hypocrea jecorina Cel7A hydrolase, the CBM and the catalytic domain are proposed to function as a molecular machine (Mulakala and Reilly, 2005) where the CBM is a wedge to pry cellulose chains from the crystalline cellulose surface. The CBM then functions as a Brownian ratchet to pass the freed chain to the GH domain for cleavage. "
[Show abstract][Hide abstract] ABSTRACT: Summary Microbial cellulose degradation is a central part of the global carbon cycle and has great potential for the development of inexpensive, carbon-neutral biofuels from non-food crops. Clostridium phytofermentans has a repertoire of 108 putative glycoside hydrolases to break down cellulose and hemicellulose into sugars, which this organism then ferments primarily to ethanol. An understanding of cellulose degradation at the molecular level requires learning the different roles of these hydrolases. In this study, we show that interspecific conjugation with Escherichia coli can be used to transfer a plasmid into C. phytofermentans that has a resistance marker, an origin of replication that can be selectively lost, and a designed group II intron for efficient, targeted chromosomal insertions without selection. We applied these methods to disrupt the cphy3367 gene, which encodes the sole family 9 glycoside hydrolase (GH9) in the C. phytofermentans genome. The GH9-deficient strain grew normally on some carbon sources such as glucose, but had lost the ability to degrade cellulose. Although C. phytofermentans upregulates the expression of numerous enzymes to break down cellulose, this process thus relies upon a single, key hydrolase, Cphy3367.
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