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[Display omitted] • Application of CDH biosensor for productive binding capacity determination. • LPMO pretreatment increased the productive binding capacity of Tl CBHI on cellulose. • Both C1 and C4 LPMO activity was beneficial for the reducing end cellulase. • Non-enzymatic decrystallization phase following the LPMO activity. Cellobiohydrolases are crucial for cellulose breakdown, but their efficiency on crystalline cellulose is hampered by limited access to single chain ends to initiate hydrolysis. As a result, they depend on enzymes like lytic polysaccharide monooxygenases (LPMOs), which directly target the crystalline cellulose surface. This study investigated how LPMO pretreatment affected the productive binding capacity of a Trichoderma longibrachiatum cellobiohydrolase, Tl CBHI, on crystalline cellulose by applying an amperometric cellobiose dehydrogenase biosensor. After the 24-hour of LPMO pretreatment, the productive binding capacity of Tl CBHI significantly increased in all reactions. However, with a shorter 5-hour LPMO pretreatment, minimal to no effect on productive binding capacity was observed. Of note, all LPMO reactions were inactivated around this time point. This delayed LPMO effect suggests that the improved binding capacity for cellulases does not directly result from cellulose chain cleavage by LPMOs but rather from the cellulose decrystallization following the oxidative cleavage. [ABSTRACT FROM AUTHOR]

Because cellulase was the main enzyme used in bioconversion of lignocellulose, it was a valid way to reduce the hydrolysis cost by increasing the adsorption and hydrolysis efficiency of cellulase. In this study, modified cellobiohydrolases (CBHs) and endoglucanases (EGs) were constructed. Two engineered cellulases CBH- Tr CBM V27E,P30D,Link1 and EG- Tr CBM V27E,P30D,Link1 well-performed during hydrolysis. Compared to wild-type enzymes, EG- Tr CBM V27E,P30D,Link1 had relatively less adsorption ability to lignin and greater affinity to cellulose, especially Avicel. However, for CBH- Tr CBM V27E,P30D,Link1 , the hydrolysis manner was changed and in favor to hydrolysis process, although the adsorption properties were unexpected. It suggested that various binding conformations of polysaccharide on CBMs hypothetically resulted in different functions of CBMs, including binding ability, processive and digestive properties on fiber surface. Fusion of T. r- CBM V27E,P30D,Link1 to cellulase, both CBH and EG, gave the destruction ability of enzyme and increased the accessible surface of substrate to cellulase, enhanced the adsorption and hydrolysis efficiency of cellulase. [ABSTRACT FROM AUTHOR]

Commercial cellulase preparations for lignocellulose bioconversion are mainly produced by the fungus Trichoderma reesei . The maximum cellulose conversion of T. reesei cellulase mixture was 15%–20% higher than that of Penicillium oxalicum in the hydrolysis of corncob residue and Avicel. Nevertheless, both preparations hydrolyzed more than 92% of cellulose in NaOH-mercerized Avicel. When added to Avicel hydrolysis residue that was less reactive to P. oxalicum cellulases, cellobiohydrolase I (CBH I) from T. reesei resulted in a higher cellulose conversion than its homologous proteins from P. oxalicum and Aspergillus niger at the same protein loadings. Further domain exchange experiment attributed the high hydrolytic efficiency of T. reesei CBH I to its inter-domain linker and cellulose-binding domain. The results in part explained the superior performance of T. reesei cellulases on the degradation of native crystalline cellulose, and highlighted the important role of cellulose-binding region in determining the degree of hydrolysis by cellulases. [ABSTRACT FROM AUTHOR]

Crude enzyme extracts typically contain a broad spectrum of enzyme activities, most of which are redundant to those naturally produced by the rumen microbiome. Identification of enzyme activities that are synergistic to those produced by the rumen microbiome could enable formulation of enzyme cocktails that improve fiber digestion in ruminants. Compared to untreated barley straw, Viscozyme® increased gas production, dry matter digestion (P < 0.01) and volatile fatty acid production (P < 0.001) in ruminal batch cultures. Fractionation of Viscozyme® by Blue Native PAGE and analyses using a microassay and mass-spectrometry revealed a GH74 endoglucanase, GH71 α-1,3-glucanase, GH5 mannanase, GH7 cellobiohydrolase, GH28 pectinase, and esterases from Viscozyme® contributed to enhanced saccharification of barley straw by rumen mix enzymes. Grouping of these identified activities with their carbohydrate active enzymes (CAZy) counterparts enabled selection of similar CAZymes for downstream production and screening. Mining of these specific activities from other biological systems could lead to high value enzyme formulations for ruminants. [ABSTRACT FROM AUTHOR]

Non-productive adsorption of cellulase onto lignin restricted the movement of cellulase and also hindered the cellulase recycling in bioconversion of lignocellulose. In this study, effect of temperature and pH on adsorption and desorption of cellobiohydrolase (CBH) on lignin and its possible mechanism were discussed. It found that pH value and temperature influenced the adsorption and desorption behaviors of CBH on lignin. Different thermodynamic models suggested that the action between lignin and CBH was physical action. More CBH was adsorbed onto lignin, but lower initial adsorption velocity was detected at 50 °C comparing with 4 °C. Elevating pH value could improve desorption of cellulase from lignin. The changes of hydrophobicity and electric potential on protein surface may partially explain the impact of environmental conditions on the adsorption and desorption behaviors of CBH on lignin, and comparing to electrical interaction, the hydrophobicity may be the dominating factor influencing the behaviors. [ABSTRACT FROM AUTHOR]

[Display omitted] • Distinct pretreated substrate can be used as inducer for specific enzyme production. • Formulation of cocktails with distinct components improved the hydrolysis efficiency. • 100% glucose and xylose of ultrafine grinding treated corn stover were obtained. In order to optimize the composition of enzyme cocktail for improving the hydrolytic efficiency of lignocellulose, different substrates were tested as inducers for producing lignocellulolytic enzymes by Trichoderma harzianum EM0925 in this study. As results, ultrafine grinding or steam explosion pretreated substrates can induce T. harzianum EM0925 to secret holo lignocellulolytic enzymes; acid treated substrate can induce cellobiohydrolase; while alkali or sodium chlorite treated substrates can induce β-xylosidase specifically. Furthermore, the combination of enzyme cocktails with different hydrolysis characteristics can further improve the hydrolysis efficiency, since 100% yields of glucose and xylose were obtained simultaneously from ultrafine grinding treated corn stover at low enzyme dosage (1.2 mg proteins/g substrate). This study for the first time demonstrated an effective solution that specific-pretreated substrates can be used as inducers for specific enzyme production by T. harzianum , which provided new idea and potential strategy for the construction of highly-efficient lignocellulolytic enzyme cocktails. [ABSTRACT FROM AUTHOR]

[Display omitted] • Low temperature severely limits rice straw-silage processing. • Novel cold-active cellobiohydrolase Vv CBHI-I from V. volvacea was isolated. • Vv CBHI-I exhibited outstanding cellobiohydrolase activity at 10–30 °C. • Vv CBHI-I greatly improved saccharification efficiency of cellulase complex at 10 °C. • A flexible structure and fewer disulfide bonds explain Vv CBHI-I cold acclimation. Low ambient temperature poses a challenge for rice straw-silage processing in cold climate regions, as cold limits enzyme and microbial activity in silages. Here, a novel cold-active cellobiohydrolase (Vv CBHI-I) was isolated from Volvariella volvacea , which exhibited outstanding cellobiohydrolase activity at 10–30 °C. The crude cellulase complex in the Vv CBHI-I-expressing transformant T1 retained 50% relative activity at 10 °C, while the wildtype Trichoderma reesei showed <5% of the activity. Vv CBHI-I greatly improved the saccharification efficiency of the cellulase complex with pretreated rice straw as substrate at 10 °C. In rice straw silage, pH (<4.5) and lactic acid content (>4.6%) remained stable after 15-day ensiling with the cellulase complex from T1 and Lactobacillus plantarum. Moreover, the proportions of cellulose and hemicellulose decreased to 29.84% ± 0.15% and 21.25% ± 0.26% of the dried material. This demonstrates the crucial potential of mushroom-derived cold-active cellobiohydrolases in successful ensiling in cold regions. [ABSTRACT FROM AUTHOR]

Mycothermus thermophilus (Syn. Scytalidium thermophilum / Humicola insolens ), a thermophilic fungus, is being reported to produce appreciable titers of cellulases and hemicellulases during shake flask culturing on cellulose/wheat-bran/rice straw based production medium. The sequential and differential expression profile of endoglucanases, β-glucosidases, cellobiohydrolases and xylanases using zymography was studied. Mass spectrometry analysis of secretome (Q-TOF LC/MS) revealed a total of 240 proteins with 92 CAZymes of which 62 glycosyl hydrolases belonging to 30 different families were present. Cellobiohydrolase I (17.42%), β glucosidase (8.69%), endoglucanase (6.2%), xylanase (4.16%) and AA9 (3.95%) were the major proteins in the secretome. In addition, carbohydrate esterases, polysaccharide lyases, auxiliary activity and a variety of carbohydrate binding modules (CBM) were identified using genomic database of the culture indicating to an elaborate genetic potential of this strain for hydrolysis of lignocellulosics. The cellulases from the strain hydrolyzed alkali treated rice straw and bagasse into fermentable sugars efficiently. [ABSTRACT FROM AUTHOR]

[Display omitted] • The CBP yeast strains were engineered to benefit conversion of cellulosic biomass. • Overexpression of MIG1 spsc01 affected expression of proteins related to cell wall. • Engineering MIG1 spsc01 , UTH1 and YPG1 are novel strategies for improving CBP. • Disruption of YGP1 with overexpressing SED5 elevated CBH activity of 2.2-fold. • CBP yeast with improved cellulase production facilitates economic bioproduction. Sustainable bioproduction using carbon neutral feedstocks, especially lignocellulosic biomass, has attracted increasing attention due to concern over climate change and carbon reduction. Consolidated bioprocessing (CBP) of lignocellulosic biomass using recombinant yeast of Saccharomyces cerevisiae is a promising strategy for lignocellulosic biorefinery. However, the economic viability is restricted by low enzyme secretion levels. For more efficient CBP, MIG1 spsc01 isolated from the industrial yeast which encodes the glucose repression regulator derivative was overexpressed. Increased extracellular cellobiohydrolase (CBH) activity was observed with unexpectedly decreased cell wall integrity. Further studies revealed that disruption of CWP2, YGP1 , and UTH1, which are functionally related to MIG1 spsc01 , also enhanced CBH secretion. Subsequently, improved cellulase production was achieved by simultaneous disruption of YGP1 and overexpression of SED5 , which remarkably increased extracellular CBH activity of 2.2-fold over the control strain. These results provide a novel strategy to improve the CBP yeast for bioconversion of carbon neutral biomass. [ABSTRACT FROM AUTHOR]

[Display omitted] • Efficient lignocellulases were heterologously expressed. • Addition of lignocellulases promoted the humification efficiency of composting. • Revealing the novel microbial functions during organic matter transformation. • Providing direction for functional studies of non-culturable or refractory microbes. This study aimed to reveal the potential effects of exogenous lignocellulases addition on the composting efficiency and microbial communities. The lignocellulases addition at the mesophilic phase (MEP) greatly expedited the substrate conversion and the rise of temperature at the initial stage, driving the early arrival of thermophilic phase (THP), caused by the positive effects of Sphingobacterium and Brevundimonas. When being added at the THP, the potential functions and interactions of microbial communities were stimulated, especially for Thermobispora and Mycothermus , which prolonged the duration of the THP and expedited the humic acid formation. Simultaneous addition (MEP and THP) significantly altered the microbial community succession and activated the microbes that contributed to the lignocellulases secretion, exhibiting the highest cellobiohydrolase (36.19 ± 3.25 U· g−1 dw) and xylanase (47.51 ± 3.32 U·g−1 dw) activity at the THP. These findings provide new strategies that can be effectively utilized to improve the efficiency and quality of composting. [ABSTRACT FROM AUTHOR]

The cellulolytic and hemicellulolytic enzymes of Trichoderma reesei comprise one of the best characterised enzyme systems involved in lignocellulose degradation. In this paper, swollenin (SWOI), a protein recognised based on its sequence similarity with plant expansins, has been characterised. SWOI and its catalytic domain were subjected to analysis of their hydrolytic activity on different soluble carbohydrate polymers. By measuring the production of reducing ends, zymogram-, and viscosity analysis, SWOI was shown to have activity on substrates containing β-1,4 glucosidic bonds, i.e. carboxymethyl cellulose, hydroxyethyl cellulose and β-glucan. The formation of oligosaccharides from β-glucan was analysed by HPLC and showed cellobiose as the main reaction product. SWOI was also able to hydrolyse soluble cello-oligosaccharides and the products formed were all consistent with SWOI cleaving a cellobiose unit off the substrate. In conclusion, the T. reesei swollenin showed a unique mode of action with similarities with action of both endoglucanases and cellobiohydrolases. [ABSTRACT FROM AUTHOR]

A new strain of rumen fungus was isolated from Bos taurus , identified and designated Orpinomyces sp.Y102. A clone, celC7 , isolated from the cDNA library of Orpinomyces sp.Y102, was predicted to encode a protein containing a signal peptide (Residues 1–17), an N-terminal dockerin-containing domain, and a C-terminal cellobiohydrolase catalytic domain of glycoside hydrolase family 6. CelC7 was insoluble when expressed in Escherichia coli . Deletion of 17 or 105 residues from the N-terminus significantly improved its solubility. The resulting enzymes, CelC7(-17) and CelC7(-105), were highly active to β-glucan substrates and were stable between pH 5.0 and 11.0. CelC7(-105) worked as an exocellulase releasing cellobiose and cellotriose from acid-swollen Avicel and cellooligosaccharides, and displayed a V max of 6321.64 μmole/min/mg and a K m of 2.18 mg/ml to barley β-glucan. Further, the crude extract of CelC7(-105) facilitated ethanol fermentation from cellulose. Thus, CelC7(-105) is a good candidate for industrial applications such as biofuel production. [ABSTRACT FROM AUTHOR]

• Classical and integrated system biology tools to develop hyper-cellulolytic strain. • Comparative analysis of wild and developed strain at proteome and transcriptional level. • Efficient hydrolysis of acid pre-treated rice straw slurry by developed enzyme. • Spiking of developed enzyme with LPMO from M. thermophiloides from enhanced hydrolysis. The objective of this study was to develop thermophilic fungus Rasamsonia emersonii using integrated system biology tools (genomics, proteomics and transcriptional analysis) in combination with classical strain breeding approaches. Developed hyper cellulolytic mutant strain M36 showed endoglucanase (476.35 U/ml), β-glucosidase (70.54 U/ml), cellobiohydrolase (15.17 U/ml), FPase (4.89 U/ml) and xylanase (485.21 U/ml) on cellulose/gram flour based production medium. Comparison of the expression profile at proteome and transcriptional level of the developed strain and wild type parent gave detailed insight into the up-regulation of different CAZymes including glycosyl hydrolases (GH5, GH6, GH7, GH3, GH10) and auxiliary enzymes (lytic polysaccharide monooxygenase, swollenin) at system level. Furthermore, the potential of lignocellulolytic enzyme produced by the developed strain and custom designed cocktail spiked with heterologously expressed lytic polysaccharide monooxygenase from Mycothermus thermophiloides were analyzed for the hydrolysis of biorefinery relevant unwashed pretreated rice straw slurry (PRAJ and IOCL) @17% substrate loading rate. [ABSTRACT FROM AUTHOR]

Highlights: [•] CBH was not required to efficiently hydrolyze amorphous IL-treated cellulose. [•] CBH activity was necessary to hydrolyze BM-treated bagasse. [•] Low CBH loads can hydrolyze IL-treated sugarcane bagasse. [•] A crystalline residue was obtained after hydrolysis of the BM-treated substrates. [•] IL greatly increased the specific surface area of both substrates, while BM did not. [ABSTRACT FROM AUTHOR]

Highlights: [•] Temperature increased inhibition arising from unproductive enzyme adsorption. [•] Inhibition became evident at different temperatures for different enzymes. [•] TeCel7A-CBM1 was most lignin-tolerant among the studied enzymes. [•] TrCel7A was least lignin-tolerant among the studied enzymes. [Copyright &y& Elsevier]

The cbh1 strong promoter was employed to over-express the cbh2 gene for enhancing cellobiohydrolase (CBH) production in Trichoderma reesei because CBH II component has higher specific activity than CBH I and is an important component in cellulase. The recombinant plasmid pCAMBIA1300-hph-PsCT containing strong expression cassette was constructed and transformed into T. reesei via optimized Agrobacterium -mediated transformation, producing 324 positive T. reesei transformants for the two steps of screening. Ten fast-growing T. reesei transformants were selected, amongst which C10 was found to have the highest filter paper activity 28.92 ± 2.45 IU/mL, 4.3-fold higher than that of ZU-02, 6.71 ± 0.79 IU/mL. C10 also has the highest cellobiohydrolase activity 122.44 ± 7.42 U/mL, 5.4 times higher than that of ZU-02, 22.49 ± 2.27 U/mL. The cellulase from C10 performed better (93.06 ± 2.83%) than the one from ZU-02 in enzymatic hydrolysis because the exo–exo-synergism played a role. [ABSTRACT FROM AUTHOR]

Highlights: [•] Fusarium verticillioides present at least one cellulase multienzyme complex. [•] Multienzyme complex contain two endoglucanases, one cellobiohydrolase and one xylanase. [•] Cellulase multienzyme complex and free endoglucanase are highly thermostable. [Copyright &y& Elsevier]

Abstract: The aim of this study was to compare the hydrolysis performances of four lignocellulolytic complexes from commercial or laboratory origin and produced either by solid-state fermentation or by submerged fermentation. To evaluate their potential, saccharification tests were performed on cellulose, as model substrate, and wheat bran, as lignocellulosic substrate, using either the same filter paper unit or the same amount of protein to introduce these enzymatic complexes. A great difference was observed for the laboratory enzymatic complex produced by solid-state fermentation, which has shown a greater efficiency of cellobiohydrolase on cellulose and better conversion capacity on wheat bran, probably due to the presence of side activities. This comparison has proved that solid-state fermentation could be a promising technology to overcome the biomass recalcitrance and lower the cost of conversion step. [Copyright &y& Elsevier]

Abstract: The impact of pectinases in the hydrolysis of fresh, steam-exploded and ensiled hemp was investigated and the synergy between cellulases, pectinases and xylanase in the hydrolysis was evaluated. About half; 59.3% and 46.1% of pectin in the steam-exploded and ensiled hemp, respectively, could be removed by a low dosage of pectinases used. Pectinases were more efficient than xylanase in the hydrolysis of fresh and ensiled hemp whereas xylanase showed higher hydrolytic efficiency than the pectinase preparation used in the hydrolysis of steam-exploded hemp. Clear synergistic action between cellulases and xylanase could be observed in the hydrolysis of steam-exploded hemp. Supplementation of pectinase resulted in clear synergism with cellulases in the hydrolysis of all hemp substrates. Highest hydrolysis yield of steam-exploded hemp was obtained in the hydrolysis with cellulases and xylanase. In the hydrolysis of ensiled hemp, the synergistic action between cellulases and pectinases was more obvious for efficient hydrolysis. [Copyright &y& Elsevier]

Abstract: The effect of enzyme loading under static and agitated conditions was investigated. Enzymatic hydrolysis of 10w/v% de-lignified cellulose slurry such as filter paper, avicel and pulp was conducted under agitated (120rpm) and static condition, and the enzyme loading ranging from 1.2 to 120mg-protein/g-dry substrate. Under the agitated condition, the final sugar concentration decreased with the decreasing enzyme loading. Under the static condition, the final sugar concentration was maintained even if the enzyme loading was decreased. The above phenomenon was caused by a rapid precipitation of cellobiohydrolase 2 (CBH2) under the agitated condition, which was not observed under the static condition. The hydrolysis experiments using enzymes containing different ratios of cellobiohydrolase 1 (CBH1) and CBH2 under the static condition suggested that preservation of CBH2 and its synergism with CBH1 is essential for static condition’s characteristics, and for efficient hydrolysis of cellulose. [Copyright &y& Elsevier]

Abstract: Hemicelluloses have been found to be physical barriers in the hydrolysis of cellulose, and prevent the access of enzymes to cellulose surface. In addition, soluble hemicelluloses may strongly inhibit the cellulase activity. In this work, birchwood xylan clearly inhibited the enzymatic hydrolysis of wheat straw, Avicel and nanocellulose by cellulases. Hydrolysis efficiencies of cellobiohydrolase I (CBHI, from Thermoascus aurantiacus), cellobiohydrolase II (CBHII, from Trichoderma reesei) and endoglucanase II (from T. aurantiacus) were clearly inhibited by birchwood xylan, respectively. The strongest inhibitory effect of birchwood xylan was observed on the hydrolysis of Avicel by CBHI and CBHII, as a dramatically decreased formation of the main product, cellobiose. After additions of soluble and insoluble oat spelt xylan, cleaved cellobiose units by CBHI from cellulose chain decreased from 8 to 4 and 6, respectively. The results in this work demonstrated that xylans clearly inhibited the hydrolysis efficiencies of both endoglucanase and cellobiohydrolase. [Copyright &y& Elsevier]

Abstract: A basidiomycetous fungus, identified as Pholiota adiposa SKU0714 on the basis of morphological and phylogenetic analyses, was found to secrete efficient lignocellulose degrading enzymes. The strain showed maximum endoglucanase, cellobiohydrolase and β-glucosidase activities of 26, 32 and 39U/mL, respectively and also secreted xylanase, laccase, mannanase, and lignin peroxidase with activities of 1680, 0.12, 65 and 0.41U/mL, respectively when grown with rice straw as a carbon source. Among the various plant biomasses tested for saccharification, poplar biomass produced the maximum amount of reducing sugar. Response surface methodology was used to optimize hydrolysis parameters. A maximum saccharification yield of 83.4% (667mg/g-substrate), the highest yield from any plant biomass, was obtained with Populus biomass after 24h of hydrolysis. P. adiposa was proven to be a good choice for the production of reducing sugars from cellulosic biomass. [Copyright &y& Elsevier]

Abstract: The effects of xylo-oligosaccharides (XOS) and xylose on the hydrolytic activities of cellulases, endoglucanase II (EGII, originating from Thermoascus aurantiacus), cellobiohydrolase I (CBHI, from T. aurantiacus), and cellobiohydrolase II (CBHII, from Trichoderma reesei) on Avicel and nanocellulose were investigated. After the addition of XOS, the amounts of cellobiose, the main product released from Avicel and nanocellulose by CBHI, decreased from 0.78 and 1.37mg/ml to 0.59 and 1.23mg/ml, respectively. During hydrolysis by CBHII, the amounts of cellobiose released from the substrates were almost cut in half after the addition of XOS. Kinetic experiments showed that xylobiose and xylotriose were competitive inhibitors of CBHI. The results revealed that the strong inhibition of cellulase by XOS can be attributed to the inhibitory effect of XOS especially on cellobiohydrolase I. The results indicate the necessity to totally hydrolyze xylo-oligosaccharides into the less inhibitory product, xylose, to increasing hydrolytic efficiency. [Copyright &y& Elsevier]

Abstract: A lipase gene (Lip) of the Aspergillus niger was de novo synthesized and expressed in the Trichoderma reesei under the promoter of the cellobiohydrolase I gene (cbh1). RNAi-mediated gene silencing was successfully used to further improve the recombinant lipase production via down-regulation of CBHI which comprised more than 60% of the total extracellular proteins in T. reesei. The gene and protein expression of CBHI and recombinant lipase were analyzed by real-time PCR, SDS–PAGE and activity assay. The results demonstrated that RNAi-mediated gene silencing could effectively suppress cbh1 gene expression and the reduction of CBHI could result in obvious improvement of heterologous lipase production. The reconstructed strains with decreased CBHI production exhibited 1.8- to 3.2-fold increase in lipase activity than that of parental strain. The study herein provided a feasible and advantageous method of increasing heterologous target gene expression in T. reesei through preventing the high expression of a specific endogenenous gene by RNA interference. [Copyright &y& Elsevier]

Abstract: Deactivation extents of cellobiohydrolase, endoglucanase, and a total cellulase mixture (Spezyme CP) were studied independently as functions of incubating time and mixing intensity. It was found that the decrease in total cellulase activity was more strongly related to deactivation of cellobiohydrolase 1 (CBH1) than endoglucanase. The mass-averaged shear in orbiting flasks at 50, 150, and 250rpm was quantified by computational fluid dynamics and was two-orders smaller than shear in typical stirred tanks. Endoglucanase activity did not change significantly with mixing speed, but CBH1 and total cellulase activities were 10–25% higher at 250rpm compared to the lower speeds after a 24-h incubation. Total deactivation due to mechanical mixing (∼20%) may be too low to account for all the rate reduction during cellulose hydrolysis. Thermal deactivation was independent of enzyme concentration while deactivation due to mechanical stress decreased when cellulase loading increased over 0.15filterpaperunit/ml. [Copyright &y& Elsevier]

Abstract: An acidic Cel6A, cellobiohydrolase (CBH) II, was purified from Penicillium decumbens and designated as PdCel6A. The deduced internal amino acid sequence of the novel CBH has a high degree of sequence identity with the CBH II from Aspergillus fumigatus. Surprisingly, PdCel6A exhibits characteristics comparable to that of CBH I, as well as CBH II. Similar to CBH I, the novel CBH has a specific activity of 1.9IU/mg against p-nitrophenyl-β-d-cellobioside. The enzyme retains about 80% of its maximum activity after 4h of incubation at pH 2.0. Using delignified corncob residue as the substrate, ethanol concentration increased by 20% during simultaneous saccharification and fermentation when supplemented with low doses of PdCel6A (0.2mg/g substrate). To our knowledge, this is the first report involving a CBH I-like CBH II. The present paper provides new insight into the role of CBH II in cellulose degradation. [Copyright &y& Elsevier]

Abstract: To improve the β-glucosidase yield and total cellulase activity of Trichoderma reesei, extracellular β-glucosidase (BGLI) was overexpressed under the control of the modified four-copy cbh1 promoter. Three transformants B2, B12 and B15 with successful integration of the bgl1 gene expression cassette were obtained, which exhibited 3.7-, 2.0- and 1.8-fold increase in β-glucosidase activity than the parental strain RUT C30, respectively. The filter paper activities of the productive transformants B12 and B15 were improved by up to 130% and 55%, respectively. Saccharification of corncob residue with the crude enzyme dosages showed that the reducing sugar yields of B12 (5.59mg/ml) and B15 (4.80mg/ml) were 29% and 11% higher than that of RUT C30 (4.34mg/ml), respectively. The present results proved that the modified four-copy cbh1 promoter was a useful tool for improving the cellulase activity of T. reesei, and the engineering strains developed in this study could be potentially used as promising cell factories for β-glucosidase or cellulase production. [ABSTRACT FROM AUTHOR]

Abstract: The glycoside hydrolase family 9 cellulase (Cel9) from Clostridium phytofermentans has a multi-modular structure and is essential for cellulose hydrolysis. In order to facilitate production and purification of Cel9, recombinant Cel9 was functionally expressed in Escherichia coli. Cel9 exhibited maximum activity at pH 6.5 and 65°C on carboxymethyl cellulose in a 10-min reaction period. The hydrolysis products on regenerated amorphous cellulose (RAC) were cellotetraose (a major product), cellotriose, cellobiose and glucose, and 71–80% of the reducing sugars produced by Cel9 were in soluble form, suggesting that Cel9 was a processive endoglucanase. The highest synergy between C. phytofermentans Cel9 and C. phytofermentans cellobiohydrolase Cel48 on Avicel was about 1.8 at a ratio of about 1:5. Cel9 alone was sufficient to solublize filter paper while Cel48 was not; however, it enhanced the solublization process along with Cel9 synergistically. This study provided useful information for understanding of the cellulose hydrolysis mechanism of this cellulolytic bacterium with potential industrial importance. [Copyright &y& Elsevier]

• Effect of electron donors of LPMO (AscA and GalA) on cellulases action was studied. • Different responses of cellulases to AscA and GalA were observed. • The hydrolytic action of cellulases could be fueled by AscA, but not GalA. • AscA showed a special boosting effect on Cel7A, but not Cel5A and Cel3A. • The cellulose saccharification could be optimized by adjusting the dosage of AscA. Ascorbic acid (AscA) and gallic acid (GalA) are common electron donors and their boosting effect on lytic polysaccharide monooxygenases (LPMO) has been studied extensively. However, their influence on cellulase hydrolytic action has been ignored. In this work, the effect of AscA and GalA on cellulases hydrolytic action was evaluated. It was found that AscA could increase the hydrolysis of cellulose by cellulases, while GalA showed no effect on cellulases' hydrolytic action. The effect of AscA differed for the monocomponent cellulases: it showed a special boosting effect on cellobiohydrolase, rather than endoglucanase and β-glucosidase. This promoting effect could be another mechanism behind the boosting effect of the AscA-driven LPMO system on cellulose saccharification. These findings thus advance the understanding of the role of electron donors on cellulose saccharification and offer important clues on how to evaluate the feasibility of electron donors from a new perspective. [ABSTRACT FROM AUTHOR]

[Display omitted] • Cellobiose and vanillin are the most severe inhibitors to cellulase. • Different binding sites of major inhibitors on CBH I lead to negative cooperation. • Negative synergistic inhibition results in more β-glucosidase requirement. • Combined β-glucosidase and PEG addition is the feasible strategy. Soluble compounds produced during the enzymatic hydrolysis of lignocelluloses hampers cellulose conversion. Cellobiose and vanillin most severely inhibited the effect of cellobiohydrolase I. A concentration-dependent negative cooperative effect was found between cellobiose and vanillin. The combined inhibitory effect was about 83.5% of the cellobiose and 88.1% of the vanillin when their concentration was 20 mg/ml. However, the negative synergy could be eliminated by excessive enzyme loading. Differences in their binding sites on the catalytic domain of cellobiohydrolase I lead to negative synergistic inhibition, which should be considered in devising strategies to alleviate this effect. Combined β-glucosidase and PEG addition at an appropriate dose was feasible to balance cost and hydrolytic efficiency. To achieve efficient hydrolysis, especially at high solid concentrations, it is important to understand the synergistic inhibition between these inhibitors. [ABSTRACT FROM AUTHOR]

• Novel enzyme cocktails from Penicillium and Aspergillus species were developed. • Native and recombinant basic cellulases and auxiliary enzymes were pooled. • Sugarcane, soybean and oil palm solid residues were efficiently saccharified. • Enzyme pool choice depended on substrate, pretreatment strategy and targeted sugars. The aim of this study was to develop optimized enzyme cocktails, containing native and recombinant purified enzymes from five fungal species, for the saccharification of alkali- and acid-pretreated sugarcane bagasse (SCB), soybean hulls (SBH) and oil palm empty fruit bunches (EFB). Basic cellulases were represented by cellobiohydrolase I (CBH) and endo -glucanase II (EG) from Penicillium verruculosum and β-glucosidase (BG) from Aspergillus niger. Auxiliary enzymes were represented by endo -xylanase A (Xyl), pectin lyase (PNL) and arabinoxylanhydrolase (AXH) from Penicillium canescens , β-xylosidase (BX) from Aspergillus japonicus , endo -arabinase (ABN) from A. niger and arabinofuranosidase (Abf) from Aspergillus foetidus. Enzyme loads were 5 mg protein/g dry substrate (basic cellulases) and 1 mg/g (each auxiliary enzyme). The best choice for SCB and EFB saccharification was alkaline pretreatment and addition of Xyl + BX, AXH + BX or ABN + BX + Abf to basic cellulases. For SBH, acid pretreatment and basic cellulases combined with ABN + BX + Abf or Xyl + BX performed better than other enzyme preparations. [ABSTRACT FROM AUTHOR]

• A ternary enzyme mixture was optimized for the hydrolysis of six cellulosic materials. • The optimal composition of cellulases was affected by pretreatment strategy. • The requirement of critical enzyme was greater during high-solids hydrolysis. Optimization of the composition of cellulase mixtures is an effective strategy to improve their hydrolytic efficiency and reduce protein demand during enzymatic degradation of lignocelluloses. In this study, the mixture design method was used to optimize the ratios of endoglucanase II (EG II), cellobiohydrolase I (CBH I) and β-glucosidase I (BG I) from Penicillium oxalicum in an artificial cellulase mixture for the hydrolysis of six different cellulosic materials. The optimal composition of enzyme mixture was distinctly different among not only cellulosic materials with different pretreatment methods but hydrolyses at different solids concentrations. CBH I was most critical for the hydrolysis of two acid-pretreated materials, probably due to its strong adsorption on lignin. A higher proportion of EG II was needed for the hydrolysis of ammonium sulfite pretreated wheat straw. The requirements of specific cellulase components were more pronounced at high solids concentrations, highlighting the importance of considering solids loading when optimizing cellulase cocktails. [ABSTRACT FROM AUTHOR]