Synergistic enhancement of cellobiohydrolase performance on pretreated corn stover by addition of xylanase and esterase activities (original) (raw)
Related papers
Biotechnology for Biofuels, 2011
Background We and other workers have shown that accessory enzymes, such as β-glucosidase, xylanase, and cellulase cofactors, such as GH61, can considerably enhance the hydrolysis effectiveness of cellulase cocktails when added to pretreated lignocellulosic substrates. It is generally acknowledged that, among the several factors that hamper our current ability to attain efficient lignocellulosic biomass conversion yields at low enzyme loadings, a major problem lies in our incomplete understanding of the cooperative action of the different enzymes acting on pretreated lignocellulosic substrates. Results The reported work assessed the interaction between cellulase and xylanase enzymes and their potential to improve the hydrolysis efficiency of various pretreated lignocellulosic substrates when added at low protein loadings. When xylanases were added to the minimum amount of cellulase enzymes required to achieve 70% cellulose hydrolysis of steam pretreated corn stover (SPCS), or used to...
Implications of cellobiohydrolase glycosylation for use in biomass conversion
Biotechnology for Biofuels, 2008
The cellulase producing ascomycete, Trichoderma reesei (Hypocrea jecorina), is known to secrete a range of enzymes important for ethanol production from lignocellulosic biomass. It is also widely used for the commercial scale production of industrial enzymes because of its ability to produce high titers of heterologous proteins. During the secretion process, a number of post-translational events can occur, however, that impact protein function and stability. Another ascomycete, Aspergillus niger var. awamori, is also known to produce large quantities of heterologous proteins for industry. In this study, T. reesei Cel7A, a cellobiohydrolase, was expressed in A. niger var. awamori and subjected to detailed biophysical characterization. The purified recombinant enzyme contains six times the amount of N-linked glycan than the enzyme purified from a commercial T. reesei enzyme preparation. The activities of the two enzyme forms were compared using bacterial (microcrystalline) and phosphoric acid swollen (amorphous) cellulose as substrates. This comparison suggested that the increased level of N-glycosylation of the recombinant Cel7A (rCel7A) resulted in reduced activity and increased non-productive binding on cellulose. When treated with the N-glycosidase PNGaseF, the molecular weight of the recombinant enzyme approached that of the commercial enzyme and the activity on cellulose was improved.
Applied Biochemistry and Biotechnology, 2002
It is commonly observed that the rate of enzymatic hydrolysis of solid cellulose substrates declines markedly with time. In this work the mechanism behind the rate reduction was investigated using two dominant cellulases of Trichoderma reesei: exoglucanase Cel7A (formerly known as CBHI) and endoglucanase Cel7B (formerly EGI). Hydrolysis of steam-pretreated spruce (SPS) was performed with Cel7A and Cel7B alone, and in reconstituted mixtures. Throughout the 48-h hydrolysis, soluble products, hydrolysis rates, and enzyme adsorption to the substrate were measured. The hydrolysis rate for both enzymes decreases rapidly with hydrolysis time. Both enzymes adsorbed rapidly to the substrate during hydrolysis. Cel7A and Cel7B cooperate synergistically, and synergism was approximately constant during the SPS hydrolysis. Thermal instability of the enzymes and product inhibition was not the main cause of reduced hydrolysis rates. Adding fresh substrate to substrate previously hydrolyzed for 24 h with Cel7A slightly increased the hydrolysis of SPS; however, the rate increased even more by adding fresh Cel7A. This suggests that enzymes become inactivated while adsorbed to the substrate and that unproductive binding is the main cause of hydrolysis rate reduction. The strongest increase in hydrolysis rate was achieved by adding Cel7B. An improved model is proposed that extends the standard endo-exo synergy model and explains the rapid decrease in hydrolysis rate. It appears that the processive action of Cel7A becomes hindered by obstacles in the lignocellulose substrate. Obstacles created by disordered cellulose chains can be removed by the endo activity of Cel7B, 42 Eriksson, Karlsson, and Tjerneld Applied Biochemistry and Biotechnology Vol. 101, 2002 which explains some of the observed synergism between Cel7A and Cel7B. The improved model is supported by adsorption studies during hydrolysis.
Applied and Environmental Microbiology, 2011
ABSTRACTSeveral members of the glycoside hydrolase 61 (GH61) family of proteins have recently been shown to dramatically increase the breakdown of lignocellulosic biomass by microbial hydrolytic cellulases. However, purified GH61 proteins have neither demonstrable direct hydrolase activity on various polysaccharide or lignacious components of biomass nor an apparent hydrolase active site. Cellobiose dehydrogenase (CDH) is a secreted flavocytochrome produced by many cellulose-degrading fungi with no well-understood biological function. Here we demonstrate that the binary combination ofThermoascus aurantiacusGH61A (TaGH61A) andHumicola insolensCDH (HiCDH) cleaves cellulose into soluble, oxidized oligosaccharides. TaGH61A-HiCDH activity on cellulose is shown to be nonredundant with the activities of canonical endocellulase and exocellulase enzymes in microcrystalline cellulose cleavage, and while the combination of TaGH61A and HiCDH cleaves highly crystalline bacterial cellulose, it do...
BioResources, 2022
Enzymatic saccharification of corn stover can be enhanced by partially replacing commercial enzymes with bacterial crude enzyme extracts. Thus, in this study, three bacteria (Bacillus sp. A0, Bacillus sp. CH20S1, and Exiguobacterium sp. AS2B) were cultured in a media with corn stover as the substrate to produce crude enzyme extract and saccharify corn stover. The cultural conditions were monitored and optimized to maximize CMCase and xylanase activity in the crude enzyme extracts. After 72 h of hydrolysis of corn stover with diluted crude enzymes (DCE) from the three strains, reducing sugars ranging from 48.2 to 71.7 mg g-1 were released from non-pretreated and pretreated corn stover. Furthermore, the maximum reducing sugars of 316 and 321 mg g-1 were observed when 12 and 4 FPU g-1 of commercial cellulase were added to the DCE of the CH20S1 strain, respectively. It was shown that an effective combination of bacterial DCE with commercial enzymes could achieve higher saccharification ...
Biochemical Engineering Journal, 2010
Trichoderma reesei and Aspergillus awamori enzymes were concentrated, pooled and assessed for the hydrolysis of steam-pretreated sugarcane bagasse. The enzyme profile of T. reesei gave (IU/L): 1700 FPA, 20,000 CMCase, 340 β-glucosidase and 12,600 xylanase. FPA and CMCase activities that were 4-fold higher than those of A. awamori (420 and 4900 IU/L, respectively). However the β-glucosidase and xylanase activities were 134- and 6-fold lower than those of A. awamori (45,600 and 79,100 IU/L, respectively). Furthermore, A. awamori produced ferulic acid esterase (160 IU/L) which acts synergistically with cellulolytic–xylanolytic enzymes in the hydrolysis of lignocellulosic materials. The FPA and CMCase activities in the T. reesei–A. awamori blends were enhanced synergistically by 2-fold. Moreover, the hydrolytic effectiveness of the blends was superior to the use of unblended T. reesei or A. awamori enzymes, under corresponding conditions (10 FPU/g bagasse, 20 g bagasse/L and 50 °C). Hydrolysis experiments, presenting either 20 or 200 g/L bagasse, resulted in 3.9 or 40 g glucose/L, respectively. These values corresponded to 41% cellulose hydrolysis within 6 or 24 h, respectively. A. awamori enzymes hydrolyzed 91% (1.7 g/L xylose) of the residual xylan in the bagasse within 6 h in experiments presenting 20 g/L bagasse.
Enzyme and Microbial Technology, 2012
Recycling of enzymes during biomass conversion is one potential strategy to reduce the cost of the hydrolysis step of cellulosic ethanol production. Devising an efficient enzyme recycling strategy requires a good understanding of how the enzymes adsorb, distribute, and interact with the substrate during hydrolysis. We investigated the interaction of individual Trichoderma reesei enzymes present in a commercial cellulase mixture during the hydrolysis of steam-pretreated corn stover (SPCS). The enzyme profiles were followed using zymograms, gel electrophoresis, enzyme activity assays and mass spectrometry. The adsorption and activity profiles of 6 specific enzymes Cel7A (CBH I), Cel7B (EG I), Cel5A (EG II), Xyn 10 (endo-1,4--xylanase III), Xyn 11 (endo-xylanase II), and -glucosidase were characterized. Initially, each of the enzymes rapidly adsorbed onto the SPCS. However, this was followed by partial desorption to an adsorption equilibrium where the Cel7A, Cel7B, Xyn 10, and -glucosidase were partially adsorbed to the SPCS and also found free in solution throughout the course of hydrolysis. In contrast, the Cel5A and Xyn 11 components remained primarily free in the supernatant. The Cel7A component also exhibited a partial desorption when the rate of hydrolysis leveled off as evidenced by MUC zymogram and SDS-PAGE. Those cellulase components that did not bind to the substrate were generally less stable and lost their activities within the first 24 h when compared to enzymes that were distributed in both the liquid and solid phases. Therefore, to ensure maximum enzyme activity recovery, enzyme recycling seems to be most effective when short-term rounds of hydrolysis are combined with the recovery of enzymes from both the liquid and the solid phases and potentially enzyme supplementation to replenish lost activity.
2021
Enzymatic saccharification of corn stover can be enhanced by partially replacing commercial enzymes with bacterial crude enzyme extracts. Three bacteria, Bacillus sp. A0, Bacillus sp. CH20S1, and Exiguobacterium sp. AS2B, were cultured in a media with corn stover as the substrate. The cultural conditions were monitored and optimized to maximize CMCase and xylanase activity in the crude enzyme extracts. After 72 h of hydrolysis of corn stover with diluted crude enzymes (DCE) from the three strains, reducing sugars were released from non-pretreated and pretreated corn stovers. Values of the released sugars ranged from 48.23–71.69 mg g− 1, which were lower than those released by commercial cellulase (100–400 mg g− 1). The synergistic effects were observed when 12 FPU g− 1 and 4 FPU g− 1 of commercial cellulase were added to the DCE of the CH20S1 strain producing 315.90 mg g− 1 and 320.65 mg g− 1 reducing sugars, respectively. It was shown that an effective combination of bacterial DCE ...
Applied Biochemistry and Biotechnology
We evaluated various agricultural lignocellulosic biomass and variety of fungi to produce cellulolytic enzymes cocktail to yield high amount of reducing sugars. Solid-state fermentation was performed using water hyacinth, paddy straw, corn straw, soybean husk/tops, wheat straw, and sugarcane bagasse using fungi like Nocardiopsis sp. KNU, Trichoderma reesei, Trichoderma viride, Aspergillus flavus, and Phanerochaete chrysosporium alone and in combination to produce cellulolytic enzymes. Water hyacinth produced (U ml −1) endoglucanase (51.13) and filter paperase (0.55), and corn straw produced (U ml −1) β-glucosidase (4.65), xylanase (113.32), and glucoamylase (41.27) after 7-day incubation using Nocardiopsis sp. KNU. Production of cellulolytic enzymes was altered due to addition of various nitrogen sources, metal ions, vitamins, and amino acids. The maximum cellulolytic enzymes were produced by P. chrysosporium (endoglucanase; 166.32 U ml −1 and exoglucanase; 12.20 U ml −1), and by T. viride (filter paperase; 1.57 U ml −1). Among all, co-culture of T. reesei, T. viride, A. flavus, and P. chrysosporium showed highest β-glucosidase (17.05 U ml −1). The highest xylanase (1129 U ml −1) was observed in T. viride + P. chrysosporium co-culture. This study revealed the dependency on substrate and microorganism to produce good quality enzyme cocktail to obtain maximum reducing sugars.
Cellulase-poor xylanases produced by Trichoderma reesei RUT C-30 on hemicellulose substrates
Applied Microbiology and Biotechnology, 1992
Hemicellulose components from industrial viscose fibre production are characterized by a lower cellulose content than commercial xylan and the presence of a carboxylic acid fraction originating from the alkaline degradation of carbohydrates during the process. This substrate, after neutralization, can be used by Trichoderma reesei RUT C-30 for the production of cellulase-poor xylanases, useful for the pulp and paper industry. The yields of xylanase ranged up to almost 400 units/ml, with a ratio of carboxymethylcellulase/xylanase of less than 0.015. This crude xylanase enzyme mixture was shown to be superior to that obtained on beech-wood xylan when used for bleaching and, particularly, upgrading of hard-wood chemical pulp by selective removal of the xylan components. Biochemical studies indicate that the low cellulase production by T. reesei grown on these waste hemicelluloses is the result of a combination of at least three factors: (a) the comparatively low content of cellulose in these hemicellulosic wastes, (b) the inhibitory action of the carboxylic acid fraction present in the hemicellulosic wastes on growth and sporulation of T. reesei, and (c) the use of a mycelial inoculum that is unable to initiate the attack on the cellulose components within the carbon source.