Contrasted enzymatic cocktails reveal the importance of cellulases and hemicellulases activity ratios for the hydrolysis of cellulose in presence of xylans (original) (raw)
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Biotechnology for Biofuels, 2013
Background: On-site cellulase production using locally available lignocellulosic biomass (LCB) is essential for cost-effective production of 2 nd -generation biofuels. Cellulolytic enzymes (cellulases and hemicellulases) must be produced in fed-batch mode in order to obtain high productivity and yield. To date, the impact of the sugar composition of LCB hydrolysates on cellulolytic enzyme secretion has not been thoroughly investigated in industrial conditions.
Background: High enzyme loading is a major economic bottleneck for the commercial processing of pretreated lignocellulosic biomass to produce fermentable sugars. Optimizing the enzyme cocktail for specific types of pretreated biomass allows for a significant reduction in enzyme loading without sacrificing hydrolysis yield. This is especially important for alkaline pretreatments such as Ammonia fiber expansion (AFEX) pretreated corn stover. Hence, a diverse set of hemicellulases supplemented along with cellulases is necessary for high recovery of monosaccharides. Results: The core fungal cellulases in the optimal cocktail include cellobiohydrolase I [CBH I; glycoside hydrolase (GH) family 7A], cellobiohydrolase II (CBH II; GH family 6A), endoglucanase I (EG I; GH family 7B) and β-glucosidase (βG; GH family 3). Hemicellulases tested along with the core cellulases include xylanases (LX1, GH family 10; LX2, GH family 10; LX3, GH family 10; LX4, GH family 11; LX5, GH family 10; LX6, GH family 10), β-xylosidase (LβX; GH family 52), α-arabinofuranosidase (LArb, GH family 51) and α-glucuronidase (LαGl, GH family 67) that were cloned, expressed and/or purified from different bacterial sources. Different combinations of these enzymes were tested using a high-throughput microplate based 24 h hydrolysis assay. Both family 10 (LX3) and family 11 (LX4) xylanases were found to most efficiently hydrolyze AFEX pretreated corn stover in a synergistic manner. The optimal mass ratio of xylanases (LX3 and LX4) to cellulases (CBH I, CBH II and EG I) is 25:75. LβX (0.6 mg/g glucan) is crucial to obtaining monomeric xylose (54% xylose yield), while LArb (0.6 mg/g glucan) and LαGl (0.8 mg/g glucan) can both further increase xylose yield by an additional 20%. Compared with Accellerase 1000, a purified cocktail of cellulases supplemented with accessory hemicellulases will not only increase both glucose and xylose yields but will also decrease the total enzyme loading needed for equivalent yields. Conclusions: A diverse set of accessory hemicellulases was found necessary to enhance the synergistic action of cellulases hydrolysing AFEX pretreated corn stover. High glucose (around 80%) and xylose (around 70%) yields were achieved with a moderate enzyme loading (~20 mg protein/g glucan) using an in-house developed cocktail compared to commercial enzymes.
Biotechnology for biofuels, 2011
Background: High enzyme loading is a major economic bottleneck for the commercial processing of pretreated lignocellulosic biomass to produce fermentable sugars. Optimizing the enzyme cocktail for specific types of pretreated biomass allows for a significant reduction in enzyme loading without sacrificing hydrolysis yield. This is especially important for alkaline pretreatments such as Ammonia fiber expansion (AFEX) pretreated corn stover. Hence, a diverse set of hemicellulases supplemented along with cellulases is necessary for high recovery of monosaccharides. Results: The core fungal cellulases in the optimal cocktail include cellobiohydrolase I [CBH I; glycoside hydrolase (GH) family 7A], cellobiohydrolase II (CBH II; GH family 6A), endoglucanase I (EG I; GH family 7B) and β-glucosidase (βG; GH family 3). Hemicellulases tested along with the core cellulases include xylanases (LX1, GH family 10; LX2, GH family 10; LX3, GH family 10; LX4, GH family 11; LX5, GH family 10; LX6, GH family 10), β-xylosidase (LβX; GH family 52), α-arabinofuranosidase (LArb, GH family 51) and α-glucuronidase (LαGl, GH family 67) that were cloned, expressed and/or purified from different bacterial sources. Different combinations of these enzymes were tested using a high-throughput microplate based 24 h hydrolysis assay. Both family 10 (LX3) and family 11 (LX4) xylanases were found to most efficiently hydrolyze AFEX pretreated corn stover in a synergistic manner. The optimal mass ratio of xylanases (LX3 and LX4) to cellulases (CBH I, CBH II and EG I) is 25:75. LβX (0.6 mg/g glucan) is crucial to obtaining monomeric xylose (54% xylose yield), while LArb (0.6 mg/g glucan) and LαGl (0.8 mg/g glucan) can both further increase xylose yield by an additional 20%. Compared with Accellerase 1000, a purified cocktail of cellulases supplemented with accessory hemicellulases will not only increase both glucose and xylose yields but will also decrease the total enzyme loading needed for equivalent yields. Conclusions: A diverse set of accessory hemicellulases was found necessary to enhance the synergistic action of cellulases hydrolysing AFEX pretreated corn stover. High glucose (around 80%) and xylose (around 70%) yields were achieved with a moderate enzyme loading (~20 mg protein/g glucan) using an in-house developed cocktail compared to commercial enzymes.
Industrial Crops and Products, 2018
Considering the growing importance of biofuels in Brazil and throughout the world, and taking into account that glycerol is a surplus byproduct of biodiesel production, the present study aimed to evaluate the production of cellulases and xylanases by Peniciliium echinulatum, mutant strain S1M29, using different concentrations of glycerol as a carbon source in combination or not with cellulose. Experimental use of 50% glycerol and 50% cellulose showed promising FPA activity, however, this was only approximately 50% of that obtained by previous studies employing only cellulose as a carbon source. More relevant was the fact that the enzyme broth prepared with 50% glycerol and 50% cellulose proved to be efficient in the hydrolysis of lignocellulosic biomasses, especially for elephant grass pretreated by steam explosion.
Enhancement of fermentable sugar yields by α-xylosidase supplementation of commercial cellulases
Biotechnology for Biofuels, 2013
Background: Although α-linked xylose is a major constituent of the hemicelluloses of land plants, few secreted α-xylosidases have been described from fungi or bacteria. AxlA of Aspergillus niger is a secreted α-xylosidase that was earlier shown to promote the release of free glucose (Glc) and xylose (Xyl) from substrates containing α-linked xylose, including isoprimeverose (IP), the heptasaccharide subunit of pea xyloglucan (XG), and tamarind XG. Results: The utility of AxlA for enhancing release of free Glc and Xyl in combination with commercial enzyme cocktails from dicotyledonous and monocotyledonous plants was examined. Without AxlA supplementation, a mixture of CTec2 and HTec2 (both of which are derived from T. reesei) did not release significant levels of Glc from pea XG or tamarind XG. This is consistent with their lack of detectable α-xylosidase activity using model substrates. On alkaline hydrogen peroxide-pretreated corn stover, supplementation of CTec2/HTec2 (at a loading of 2.5 mg/g glucan) with AxlA (at a loading of 8 mg/g glucan) increased Glc yields from 82% to 88% of the total available Glc and increased Xyl yields from 55% to 60%. AxlA supplementation also improved Glc yields from corn stover treated with the commercial cellulase Accellerase 1000. The AxlA enhancement was not a general protein effect because bovine serum albumin or bovine gamma-globulin at similar concentrations did not enhance Glc yields from corn stover in response to CTec2/HTec2. Supplementation of CTec2/HTec2 with AxlA did not enhance Glc release from pretreated green or etiolated pea tissue. However, AxlA did enhance Glc and Xyl yields compared to CTec2/HTec2 alone from another dicotyledonous herbaceous plant, Chenopodium album (lamb's quarters). Conclusion: Supplementation of commercial cellulase cocktails with AxlA enhances yields of Glc and Xyl from some biomass substrates under some conditions, and may prove useful in industrial lignocellulose conversion.
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.
Applied Microbiology and Biotechnology, 2016
The hydrolysis of xylans, one of the main classes of carbohydrates that constitute lignocellulosic biomass, requires the synergistic action of several enzymes. The development of efficient enzymatic strategies for hydrolysis remains a challenge in the pursuit of viable biorefineries, particularly with respect to the valorisation of pentoses. The approach developed in this work is based on obtaining and characterising hemicellulasic cocktails from Thermobacillus xylanilyticus after culturing this bacterium on the hemicellulose-rich substrates wheat bran and wheat straw, which differ in their chemistries. The two obtained cocktails (WSC and WBC, for cocktails obtained from wheat straw and wheat bran, respectively) were resistant to a broad range of temperature and pH conditions. At 60°C, both cocktails efficiently liberated pentoses and phenolic acids from wheat bran (liberating more than 60, 30 and 40 % of the total xylose, arabinose and ferulic acid in wheat bran, respectively). They acted to a lesser extent on the more recalcitrant wheat straw, with hydrolytic yields of more than 30 % of the total arabinose and xylose content and 22 % of the ferulic acid content. Hydrolysis is associated with a high rate of sugar monomerisation. When associated with cellulases, high quantities of glucose were also obtained. On wheat bran, total glucose yields were improved by 70 % compared to the action of cellulases alone. This improvement was obtained by cellulase complementation either with WSC or with WBC. On wheat straw, similar levels of total glucose were obtained for cellulases alone or complemented with WSC or WBC. Interestingly, the complementation of cellulases with WSC or WBC induced an increase in the monomeric glucose yield of more than 20 % compared to cellulases alone.
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.
Cellulases: From Lignocellulosic Biomass to Improved Production
Energies
Cellulases are enzymes that are attracting worldwide attention because of their ability to degrade cellulose in the lignocellulosic biomass and transform it into highly demanded bioethanol. The enzymatic hydrolysis of cellulose by cellulases into fermentable sugars is a crucial step in biofuel production, given the complex structure of lignocellulose. Due to cellulases’ unique ability to hydrolyze the very recaltricant nature of lignocellulosic biomass, the cellulase market demand is rapidly growing. Although cellulases have been used in industrial applications for decades, constant effort is being made in the field of enzyme innovation to develop cellulase mixtures/cocktails with improved performance. Given that the main producers of cellulases are of microbial origin, there is a constant need to isolate new microorganisms as potential producers of enzymes important for biofuel production. This review provides insight into current research on improving microbial cellulase productio...