Product inhibition of cellulases studied with 14C-labeled cellulose substrates

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.

Engineering Neurospora crassa for Improved Cellobiose and Cellobionate Production

“…Cellobiose is a known inhibitor of cellulases, although there is much debate as to the mechanism of inhibition due to the complexity of the cellulase mixtures and hydrolysis of cellulose (44). Previous studies have shown that cellobiose is more inhibitory to CBHs than to EGs and BGLs and that the presence of CDH can alleviate the competitive inhibition of cellobiose on CBHs by oxidizing cellobiose to cellobionate (45,46). Should the rate of cellobiose conversion to cellobionate be increased by means such as improving CDH production in the F5 ⌬ace-1 ⌬cre-1 ⌬ndvB strain, more cellobiose could be directed to cellobionate, which can possibly relieve the inhibition of cellobiose on cellulase.…”

Development and validation of a stochastic molecular model of cellulose hydrolysis by action of multiple cellulase enzymes

“…The observation of relatively lower cellobiose inhibition towards CBH II was also reported in a comprehensive study on cellobiose inhibition using 14 C-labeled cellulose substrates, conducted by Teugjas and Väljamäe (2013). In that study, it was reported that enzymes from glycoside hydrolase (GH) family 7 were most sensitive to cellobiose inhibition followed by family 6 CBHs and endoglucanases (EGs).…”

Produção e caracterização bioquímica de enzimas lignocelulolíticas fúngicas e sua aplicação na sacarificação de biomassa lignocelulósica

“…De acordo com essa classificação, as glicosil hidrolases são divididas em 133 famílias, e as β-glucosidases estão reunidas nas famílias 1 e 3, com representantes nas famílias 5, 9, 30 e 116 (Teugjas & Väljamäe, 2013). Na família 1 estão incluídas as β-glucosidases de arqueobactérias, mamíferos e plantas, sendo algumas também originárias de fungos.…”

“…niger apresentou uma produção de 508,0 U g -1 , quando cultivado em farelo de trigo suplementado com sais minerias (Qian et al, 2012 Os valores de pH e temperatura encontrados para a maioria das β-glucosidases de fungos mesófilos estão na faixa de 4,0 a 6,5 e 50 a 65°C, respectivamente (Sorensen et al, 2013). No entanto, uma β-glucosidase produzida por Fomitopsis palustres apresentou temperatura ótima de 70°C, enquanto as β-glucosidases de P. brasilianum, A. niger (N188) e Phanerochaete chrysosporium apresentaram temperatura ótima de apenas 22°C (Sorensen et al, 2013;Teugjas & Väljamäe, 2013). Desse modo, os valores de pH e temperatura ótimos encontrados para a β-glucosidase de C. graminicola são semelhante aos descritos para as enzimas de outros fungos mesófilos.…”

“…Similarmente ao observado para a enzima de C. graminicola, diversos estudos mostraram que o pH ótimo de atividade para a maioria das β-glucosidases fúngicas está na faixa de 4,0-6,0 ( Bathia et al, 2002;Issam et al, 2003;Amouri and Gargouri, 2006;Korotkova et al, 2009;Teugjas Väljamäe, 2013).…”