Cellulases and Hemicellulases

Cellulases and hemicellulases are the main industrial sources from different microorganisms used to depolymerise plant biomass to simple sugars that are converted to chemical intermediates and biofuels, such as ethanol. Cellulases are formed adaptively, and several positive (xyr1, Ace2, HAP2/3/5) and negative (Ace1, Cre1) components involved in this regulation are now known. In this review, we summarise current knowledge about how cellulase biosynthesis is regulated, and outline recent approaches and suitable strategies for facilitating the targeted improvement of cellulase production by genetic engineering. Trichoderma reesei is the preferred organism for producing industrial cellulases. However, a more efficient heterologous expression system for enzymes from different organism is needed to further improve its cellulase mixture. In addition those optimizations of the promoter and linker for hybrid genes can dramatically improve the efficiency of heterologous expression of cellulase genes.

The cellulase and hemicellulase genes of the filamentous fungus Trichoderma reesei have been shown to be under carbon catabolite repression mediated by the regulatory gene cre1. In this study, strains were constructed in which the cre1 gene was either completely removed or replaced by a truncated mutant variant, cre1-1, found previously in the Rut-C30 mutant strain with enhanced enzyme production capability. The T. reesei transformants with either deletion or truncation of cre1 had clearly altered colony morphology compared with the parental strains, forming smaller colonies and fewer aerial hyphae and spores. Liquid cultures in a medium with glucose as a carbon source showed that the transformants were derepressed in cellulase and hemicellulase production. Interestingly, they also produced significantly elevated levels of these hydrolytic enzymes in fermentations carried out in a medium inducing the hydrolase genes. This suggests that cre1 acts as a modulator of cellulase and hemicellulase gene expression under both noninducing and inducing conditions. There was no phenotypic difference between the ⌬cre1 and cre1-1 mutant strains in any of the experiments done, indicating that the cre1-1 gene is practically a null allele. The results of this work indicate that cre1 is a valid target gene in strain engineering for improved enzyme production in T. reesei.

Ribonucleic acid interference (RNAi) inhibits the expression of target genes in a sequence-specific manner, and shows potential for gene knockdown in filamentous fungi, in which the locus-specific gene knockout occurs in low frequency. In this study, the function of the repressor of cellulase expression I (ACEI) was verified in Trichoderma koningii (T. koningii) YC01 through RNAi, and ace1silenced strains with improved cellulase productivity were obtained. An expression cassette that transcribed the interfering double-stranded RNA (dsRNA) of ace1 was constructed and transformed into T. koningii, and the transformants, in which the expression of ace1 was successfully silenced, were selected. As a result of the ace1 gene silencing, the expression levels of the main cellulase and xylanase genes were elevated, and the enhanced production of total proteins, cellulase, and xylanase was observed in the cultivation. In addition, the downregulation of ace1 resulted in an increasing expression of xyr1, but no clear variation in the expression of cre1, which suggested that ACEI acted as a repressor of the xyr1 transcription, but was not involved in the regulation of the cre1 expression. The results of this work indicate that ace1 is a valid target gene for enhancing enzyme production in T. koningii, and RNAi is an appropriate tool for improving the properties of industrial fungi.

To investigate whether enzyme production can be enhanced in the Trichoderma reesei industrial hyperproducer strain RUT C30 by manipulation of cellulase regulation, the positive regulator Xyr1 was constitutively expressed under the control of the strong T. reesei pdc promoter, resulting in significantly enhanced cellulase activity in the transformant during growth on cellulose. In addition, constitutive expression of xyr1 combined with downregulation of the negative regulator encoding gene ace1 further increased cellulase and xylanase activities. Compared with RUT C30, the resulting transformant exhibited 103, 114, and 134 % greater total secreted protein levels, filter paper activity, and CMCase activity, respectively. Surprisingly, strong increases in xyr1 basal expression levels resulted in very high levels of CMCase activity during growth on glucose. These findings demonstrate the feasibility of improving cellulase production by modifying regulator expression, and suggest an attr...

Xylanase treatment can be an environmentally friendly way to improve the formability and drainability of chemi-mechanical pulp (CMP). Improvements in xylanase treatment efficiency are possible with application of an ultrasonic wave via the cavitation effect. Results showed that the specific surface area (SSA) of the combined treated pulp increased by 14.6% at an ultrasonic treatment of 30 min and xylanase dosage of 10 U/g, in comparison to xylanase treatment alone. Also, the drainability of xylanase-treated pulp increased from 450 to 500 mL, and it further increased to 775 mL with ultrasonic-assisted xylanase treatment. Morphological characterization of pulps showed an enhanced fibrillation for the combined treatment, as shown by scanning electron microscope (SEM) images. In addition, the dimensions of treated fibers were negligibly affected.

Trichoderma reesei is a paradigm for the regulation and industrial production of plant cell wall-degrading enzymes. Among these, five xylanases, including the glycoside hydrolase (GH) family 11 XYN1 and XYN2, the GH10 XYN3, and the GH30 XYN4 and XYN6, were described. By genome mining and transcriptome analysis, a further putative xylanase, encoded by xyn5, was identified. Analysis of xyn5 from the genome-sequenced reference strain T. reesei QM6a shows that it encodes a non-functional, truncated form of XYN5. However, non-truncated orthologues are present in other genome sequenced Trichoderma spp., and sequencing of xyn5 in other T. reesei wild-type isolates shows that they harbor a putative functional xyn5 allele. In silico analysis and 3D modeling revealed that the encoded XYN5 has significant structural similarities to xylanases of the GH11 family, including a GH-typical substrate binding groove and a carboxylate pair in the active site. The xyn5 of wild-type strain TUCIM1282 was recombinantly expressed in a T. reesei strain with a (hemi)cellulase-free background and the corresponding protein purified to apparent homogeneity. The pH and temperature optima and the kinetic parameters of the purified XYN5 were pH 4, 50°C, and V max = 2646 nkat/mg with a K m of 9.68 mg/ml. This functional xyn5 allele was used to replace the mutated version which led to an overall increase of the xylanolytic activity. These findings are of particular importance as GH11 xylanases are of high biotechnological relevance, and T. reesei is one of the main industrial producers of such lignocellulose-degrading enzymes.

Gamba grass (Andropogon gayanus), a forage biomass, was used as carbon source for cellulases production by Fusarium verticillioides. A Plackett Burman design was employed to evaluate the effects of different factors over enzymatic production and two different media composition, M1 and M2, were found. In M1 media, endoglucanase and cellobiase activities were 6.5 and 0.39 U/mL, respectively. By another hand, the activities of endoglucanase and cellobiase in M2 conditions were 2.6 and 6.8 U/mL, respectively. Blends of these two extracts were tested on a sugarcane bagasse saccharification and M1 presented the best performance, indicating that higher ratios endoglucanase/cellobiase (12:1) improved sugarcane bagasse hydrolysis. Different dosages of protein (10, 20 and 40 mg/g of dry biomass) were tested intending to improve the hydrolysis performance of M1 extract. Saccharification rates of 43.4 and 73.1% were observed for glucan and xylan fractions respectively when 40 mg/g was used. Some non-productive factors such as enzyme adsorption, sugar inhibition and thermostability were evaluated to get a better understanding about saccharification bottlenecks related to Fusarium verticillioides enzymatic extract. This paper showed, for the first time, that gamba grass can be an efficient inductor for cellulase production in the endophytic fungus Fusarium verticillioides. It was also showed that, in Fusarium verticillioides, the induction of enzymes specialized on plant biomass degradation is highly dependent on carbon source concentration. Endoglucanase was mostly induced in middle concentrations while cellobiase is induced in high concentrations.

Enzymatic hydrolysis is an essential step in the lignocellulosic biomass treatment for the conversion of cellulose and hemicellulose into fermentable sugars. In the present study, a physical steam explosion (200°C) and chemical alkaline pre-hydrolysis (10% v/v) were applied to hemp (Cannabis sativa L.) biomass, to verify the effectiveness of different pretreatment processes, in order to increase enzymatic hydrolysis and the final sugar yield. The enzymatic degradations were carried out also utilizing extremophilic microorganisms, exploited as a source of (hemi)cellulases to be used as components of the enzymatic cocktail applied in the bioconversion into fermentable sugars for production of second generation ethanol and high value products. Alkaline pretreatment showed to be superior over the physical method with respect to the rate of enzymatic hydrolysis, with the obtainment of an hydrolysis yields of 90% xylose and 40% glucose, respectively.

Due to its capacity to produce large amounts of cellulases, Trichoderma reesei is increasingly being investigated for second-generation biofuel production from lignocellulosic biomass. The induction mechanisms of T. reesei cellulases have been described recently, but the regulation of the genes involved in their transcription has not been studied thoroughly. Here we report the regulation of expression of the two activator genes xyr1 and ace2 , and the corepressor gene ace1 , during the induction of cellulase biosynthesis by the inducer lactose in T. reesei QM 9414, a strain producing low levels of cellulase (low producer). We show that all three genes are induced by lactose. xyr1 was also induced by d -galactose, but this induction was independent of d -galactose metabolism. Moreover, ace1 was carbon catabolite repressed, whereas full induction of xyr1 and ace2 in fact required CRE1. Significant differences in these regulatory patterns were observed in the high-producer strain RUT C...

Gamba grass (Andropogon gayanus), a forage biomass, was used as carbon source for cellulases production by Fusarium verticillioides. A Plackett Burman design was employed to evaluate the effects of different factors over enzymatic production and two different media composition, M1 and M2, were found. In M1 media, endoglucanase and cellobiase activities were 6.5 and 0.39 U/mL, respectively. By another hand, the activities of endoglucanase and cellobiase in M2 conditions were 2.6 and 6.8 U/mL, respectively. Blends of these two extracts were tested on a sugarcane bagasse saccharification and M1 presented the best performance, indicating that higher ratios endoglucanase/cellobiase (12:1) improved sugarcane bagasse hydrolysis. Different dosages of protein (10, 20 and 40 mg/g of dry biomass) were tested intending to improve the hydrolysis performance of M1 extract. Saccharification rates of 43.4 and 73.1% were observed for glucan and xylan fractions respectively when 40 mg/g was used. Some non-productive factors such as enzyme adsorption, sugar inhibition and thermostability were evaluated to get a better understanding about saccharification bottlenecks related to Fusarium verticillioides enzymatic extract. This paper showed, for the first time, that gamba grass can be an efficient inductor for cellulase production in the endophytic fungus Fusarium verticillioides. It was also showed that, in Fusarium verticillioides, the induction of enzymes specialized on plant biomass degradation is highly dependent on carbon source concentration. Endoglucanase was mostly induced in middle concentrations while cellobiase is induced in high concentrations.

Low cost and high efficiency cellulolytic cocktails can consolidate lignocellulosic ethanol technologies. Sugarcane bagasse (SCB) is a low cost agro-industrial residue, and its use as a carbon source can reduce the costs of fungi cultivation for enzyme production. Chrysoporthe cubensis grown under solid state fermentation (SSF) with wheat bran has potential to produce efficient enzymatic extracts for SCB saccharification. This fungus was grown under submersed fermentation (SmF) and SSF with in natura SCB, pretreated with acid or alkali and with others carbon sources. In natura SCB induced the highest carboxymethylcellulase (CMCase), xylanase, β-xylosidase, α-galactosidase and mannanase activities by C. cubensis under SSF. In natura and washed SCB, inducers of enzyme production under SSF, did not induce high cellulases and hemicellulases production by C. cubensis in SmF. The C. cubensis enzymatic extract produced under SSF with in natura SCB as a carbon source was more efficient for ...

h i g h l i g h t s C. cubensis and P. pinophilum enzyme extracts were optimally blended at 50:50. Enzyme synergy attained nearly 80% for filter paper activity. Glucan and xylan conversions of NaOH-pretreated sugarcane bagasse reached 63% and 93%. Hydrolysis at 50°C increased glucan and xylan conversions by 16-20% compared to 45°C.

Growth in population and thereby increased industrialization to meet its requirement, has elevated significantly the demand for energy resources. Depletion of fossil fuel and environmental sustainability issues encouraged the exploration of alternative renewable eco-friendly fuel resources. Among major alternative fuels, bio-ethanol produced from lignocellulosic biomass is the most popular one. Lignocellulosic biomass is the most abundant renewable resource which is ubiquitous on our planet. All the plant biomass is lignocellulosic which is composed of cellulose, hemicellulose and lignin, intricately linked to each other. Filamentous fungi are known to secrete a plethora of biomass hydrolyzing enzymes. Mostly these enzymes are inducible, hence the fungi secrete them economically which causes challenges in their hyperproduction. Biomass’s complicated structure also throws challenges for which pre-treatments of biomass are necessary to make the biomass amorphous to be accessible for t...

Cellulases and hemicellulases are two important classes of enzymes produced by filamentous fungi and secreted into the cultivation medium. The production of these enzymes is under carbon catabolite repression (CCR), a general mechanism that prevents their synthesis in the presence of a preferred carbon source such as glucose. CRE1 causes the repression of transcription of cellulase and xylanase encoding genes. This study describes the isolation and cloning of a partial sequence of glucose repressor creI gene ...

Optimization of alkaline pretreatment of sugarcane bagasse for consolidated bioprocessing fermentation by the cellulose-fermenting fungus Phlebia sp. MG-60 was studied. The lignin and xylan contents of bagasse were decreased and ethanol production from each pretreated sugarcane bagasse by MG-60 was increased in an alkaline concentration-dependent manner. The fungus produced cellulase and xylanase rapidly over 120 h. When this fungus was cultured with 20 g L À1 of sugarcane bagasse pretreated with NaOH (0.8 wt%, 121 C, 60 min), 4.5 g L À1 ethanol was produced, equivalent to 210 mg ethanol per gram of the original untreated bagasse after 240 h fermentation, giving ethanol yields of 65.7% of the theoretical maximum. These data suggest that Phlebia sp. MG-60 is a potential candidate for ethanol production from alkali-pretreated bagasse in a single bioreactor, without enzymatic or chemical hydrolysis.

Background: In addition to its outstanding cellulase production ability, Trichoderma reesei produces a wide variety of valuable secondary metabolites, the production of which has not received much attention to date. Among them, sorbicillinoids, a large group of hexaketide secondary metabolites derived from polyketides, are drawing a growing interest from researchers because they exhibit a variety of important biological functions, including anticancer, antioxidant, antiviral, and antimicrobial properties. The development of fungi strains with constitutive, hyperproduction of sorbicillinoids is thus desired for future industry application but is not well-studied. Moreover, although T. reesei has been demonstrated to produce sorbicillinoids with the corresponding gene cluster and biosynthesis pathway proposed, the underlying molecular mechanism governing sorbicillinoid biosynthesis remains unknown. Results: Recombinant T. reesei ZC121 was constructed from strain RUT-C30 by the insertion of the gene 12121-knockout cassette at the telomere of T. reesei chromosome IV in consideration of the off-target mutagenesis encountered during the unsuccessful deletion of gene 121121. Strain ZC121, when grown on cellulose, showed a sharp reduction of cellulase production, but yet a remarkable enhancement of sorbicillinoids production as compared to strain RUT-C30. The hyperproduction of sorbicillinoids is a constitutive process, independent of culture conditions such as carbon source, light, pH, and temperature. To the best of our knowledge, strain ZC121 displays record sorbicillinoid production levels when grown on both glucose and cellulose. Sorbicillinol and bisvertinolone are the two major sorbicillinoid compounds produced. ZC121 displayed a different morphology and markedly reduced sporulation compared to RUT-C30 but had a similar growth rate and biomass. Transcriptome analysis showed that most genes involved in cellulase production were downregulated significantly in ZC121 grown on cellulose, whereas remarkably all genes in the sorbicillinoid gene cluster were upregulated on both cellulose and glucose. Conclusion: A constitutive sorbicillinoid-hyperproduction strain T. reesei ZC121 was obtained by off-target mutagenesis, displaying an overwhelming shift from cellulase production to sorbicillinoid production on cellulose, leading to a record for sorbicillinoid production. For the first time, T. reesei degraded cellulose to produce platform chemical compounds other than protein in high yield. We propose that the off-target mutagenesis occurring at the telomere region might cause chromosome remodeling and subsequently alter the cell structure and the global gene expression pattern of strain ZC121, as shown by phenotype profiling and comparative transcriptome analysis of ZC121. Overall, T. reesei ZC121 holds great promise for the industrial production of sorbicillinoids and serves as a good model to explore the regulation mechanism of sorbicillinoids' biosynthesis.

Sorbicillinoids are a diverse group of yellow secondary metabolites that are produced by a range of not closely related ascomycetes, including , , and . They share a similarity to the name-giving compound sorbicillin, a hexaketide. Previously, a conserved gene cluster containing two polyketide synthases has been identified as the source of sorbicillin, and a model for the biosynthesis of sorbicillin in has been proposed. In this study, we deleted the major genes of interest of the cluster in , namely , , and . Sor1 is the homolog of SorA, which is the first polyketide synthase of the proposed biosynthesis pathway. Sor3 is a flavin adenine dinucleotide (FAD)-dependent monooxygenase, and its homolog in , SorC, was shown to oxidize sorbicillin and 2',3'-dihydrosorbicillin to sorbicillinol and 2',3'-dihydrosorbicillinol, respectively, . Sor4 is an FAD/flavin mononucleotide-containing dehydrogenase with an unknown function. We measured the amounts of synthesized sorbicill...

Due to its capability to secrete large quantities of plant biomass degrading enzymes (PBDE), is widely applied for industrial purposes. In nature, expression of PBDE is efficiently regulated in this fungus. Several factors involved in this regulatory network have been identified. However, most of them are transcription factors. Long noncoding RNAs (lncRNAs) emerged as common players acting on epigenetic or transcriptional regulation in several eukaryotic organisms. To date, no lncRNA has been described in filamentous fungi. A lncRNA termed was identified in QM9414. In this study, it was characterized and evidence for its regulatory impact on cellulase expression was provided. Interestingly, different versions of were identified in different strains (namely, QM6a, QM9414, and Rut-C30), varying in terms of RNA length. Remarkably, considerable longer variants of this lncRNA are present in hypercellulolytic strains compared to the wild-type strain QM6a. Based on these results, a correla...

Trichoderma reesei is known as a good producer of industrial proteins but has hitherto been less successful in the production of therapeutic proteins. In order to elucidate the bottlenecks of heterologous protein production, human α-galactosidase A (GLA) was chosen as a model therapeutic protein. Fusion partners were designed to compare the effects of secretion using a cellobiohydrolase I (CBHI) carrier and intracellular production using a gamma zein peptide from maize (ZERA) which accumulates inside the endoplasmic reticulum (ER). The two strategies were compared on the basis of expression levels, purification performance, enzymatic activity, bioreactor cultivations, and transcriptional profiling. Constructs were cloned into the cbh1 locus of the T. reesei strain Rut-C30. The secretion and intracellular strains produced 20 mg/l and 636 mg/l of GLA respectively. Purifications of secreted product were accomplished using Step-Tactin affinity columns and for intracellular product, a me...

Background: Due to the intact structure of lignocellulosic biomass, pretreatment was a prerequisite to improve the enzymatic hydrolysis by disrupting the recalcitrant lignocellulose and increasing the accessibility of cellulose to enzyme. In this study, an alkaline hydrogen peroxide (AHP) pretreatment of sugarcane bagasse with various loadings of H 2 O 2 (1.25-6.25 wt%) at temperatures of 60-160 °C was proposed to degrade hemicellulose/lignin and improve the enzymatic digestibility. Results: It was found that increasing H 2 O 2 loadings during pretreatment lead to the enhancement of substrate digestibility, whereas the alkali (only NaOH)-pretreated solid generated higher glucose yield than that pretreated under AHP pretreatment with lower loading of H 2 O 2. This enhancement of enzymatic digestibility was due to the degradation of hemicellulose and lignin. Furthermore, Tween 80 was added to promote enzymatic digestibility, however, the increased yields were different with various substrates and hydrolysis time. The highest glucose yield of 77.6% was obtained after pretreatment at 160 °C for 60 min with 6.25% H 2 O 2 and the addition of Tween 80, representing 89.1% of glucose in pretreated substrate. Conclusions: This study demonstrated that the AHP pretreatment could greatly enhance the enzymatic saccharification. The addition of Tween 80 played remarkable performances in promoting the glucose yield during enzymatic hydrolysis by stabilizing and protecting the enzyme activity. This study provided an economical feasible and gradual process for the generation of glucose, which will be subsequently converted to bioethanol and bio-chemicals.

Lignin is complex polymer compound contained in woody plant tissue. In sugar extraction and bioethanol production from lignocellulosic materials, it is necessary to remove lignin due to its recalcitrant nature. The present work aimed to study the effect of alkali pretreatment on removal of lignin from sugarcane bagasse using sodium hydroxide. Two solid loading conditions (20 %w/v and 10 %w/v) were used in this study. For 20 % loading condition, base concentrations were varied at four levels and autoclaving time was varied at 3 levels, and the experiments were performed in 12 runs for this loading rate. For 20 % w/v solid loading, the concentrations of sodium hydroxide were 0.5 wt%, 1.0 wt%, 1.5 wt%, and 2.0 wt% at 121 °C for 10 min, 30 min and 60 min. For 10 % loading condition, the levels of each factor were increased. The base concentrations were varied at six levels and autoclaving time was varied at 5 levels, and the experiments were also performed, resulting in 30 runs for this...

Trichoderma sp. is extensively applied as a beneficial fungus for the management of plant diseases, plant growth promotion, induced resistance, and plays an important role in global sustainable agriculture. This study aimed to enhance the production of microbial xylanase in high titer from the endophytic fungus Trichoderma harzianum kj831197.1, and the cloning of xylanase genes in E. coli DH5α using a pUC19 vector. A combination of glucose, 0.1 mM, Tween 80 with lactose, and 2 mM galactose combined with malt extract boostedthe enzyme production. Xylanase production was maximized at a pH of 5.0, temp. of 30 °C, and agitation of 150 rpm in the presence of malt extract and bagasse as the best nitrogen source and waste, respectively, using submerged fermentation. The molecular weight of highly purified xylanase was 32 KDa, identified using SDS-PAGE. The xylanase gene of T. harzianum kj831197.1 was screened in fungal DNA using definite primers specified in the gene bank database. The ide...

Background: Development of efficient methods for production of renewable fuels from lignocellulosic biomass is necessary to maximize yields and reduce operating costs. One of the main challenges to industrial application of the lignocellulosic conversion process is the high costs of cellulolytic enzymes. Recycling of enzymes may present a potential solution to alleviate this problem. In the present study enzymes associated with the insoluble fraction were recycled after enzymatic hydrolysis of pretreated sugarcane bagasse, utilizing different processing conditions, enzyme loadings, and solid loadings. Results: It was found that the enzyme blend from Chrysoporthe cubensis and Penicillium pinophilum was efficient for enzymatic hydrolysis and that a significant portion of enzyme activity could be recovered upon recycling of the insoluble fraction. Enzyme productivity values (g glucose/mg enzyme protein) over all recycle periods were 2.4 and 3.7 for application of 15 and 30 FPU/g of glucan, representing an increase in excess of ten times that obtained in a batch process with the same enzyme blend and an even greater increase compared to commercial cellulase enzymes. Conclusions: Contrary to what may be expected, increasing lignin concentrations throughout the recycle period did not negatively influence hydrolysis efficiency, but conversion efficiencies continuously improved. Recycling of the entire insoluble solids fraction was sufficient for recycling of adhered enzymes together with biomass, indicative of an effective method to increase enzyme productivity.

Background: Development of efficient methods for production of renewable fuels from lignocellulosic biomass is necessary to maximize yields and reduce operating costs. One of the main challenges to industrial application of the lignocellulosic conversion process is the high costs of cellulolytic enzymes. Recycling of enzymes may present a potential solution to alleviate this problem. In the present study enzymes associated with the insoluble fraction were recycled after enzymatic hydrolysis of pretreated sugarcane bagasse, utilizing different processing conditions, enzyme loadings, and solid loadings. Results: It was found that the enzyme blend from Chrysoporthe cubensis and Penicillium pinophilum was efficient for enzymatic hydrolysis and that a significant portion of enzyme activity could be recovered upon recycling of the insoluble fraction. Enzyme productivity values (g glucose/mg enzyme protein) over all recycle periods were 2.4 and 3.7 for application of 15 and 30 FPU/g of glucan, representing an increase in excess of ten times that obtained in a batch process with the same enzyme blend and an even greater increase compared to commercial cellulase enzymes. Contrary to what may be expected, increasing lignin concentrations throughout the recycle period did not negatively influence hydrolysis efficiency, but conversion efficiencies continuously improved. Recycling of the entire insoluble solids fraction was sufficient for recycling of adhered enzymes together with biomass, indicative of an effective method to increase enzyme productivity.

h i g h l i g h t s C. cubensis and P. pinophilum enzyme extracts were optimally blended at 50:50. Enzyme synergy attained nearly 80% for filter paper activity. Glucan and xylan conversions of NaOH-pretreated sugarcane bagasse reached 63% and 93%. Hydrolysis at 50°C increased glucan and xylan conversions by 16-20% compared to 45°C.

h i g h l i g h t s C. cubensis and P. pinophilum enzyme extracts were optimally blended at 50:50. Enzyme synergy attained nearly 80% for filter paper activity. Glucan and xylan conversions of NaOH-pretreated sugarcane bagasse reached 63% and 93%. Hydrolysis at 50°C increased glucan and xylan conversions by 16-20% compared to 45°C.

Enzymatic hydrolysis is an essential step in the lignocellulosic biomass treatment for the conversion of cellulose and hemicellulose into fermentable sugars. In the present study, a physical steam explosion (200°C) and chemical alkaline pre-hydrolysis (10% v/v) were applied to hemp (Cannabis sativa L.) biomass, to verify the effectiveness of different pretreatment processes, in order to increase enzymatic hydrolysis and the final sugar yield. The enzymatic degradations were carried out also utilizing extremophilic microorganisms, exploited as a source of (hemi)cellulases to be used as components of the enzymatic cocktail applied in the bioconversion into fermentable sugars for production of second generation ethanol and high value products. Alkaline pretreatment showed to be superior over the physical method with respect to the rate of enzymatic hydrolysis, with the obtainment of an hydrolysis yields of 90% xylose and 40% glucose, respectively.

The importance of enzymes as biotechnological catalysts for paper industry is now recognized. In this study, five cellulase formulations were used for fibre modification. The number of PFI revolutions decreased by about 50% while achieving the same freeness value (decrease in CSF by 200 mL) with the enzymatic pretreatment. The physical properties of handsheets were modified after enzymatic pretreatment followed by PFI refining. A slight decrease in tear strength was observed with enzymes C1 and C4 at pH 7 while the most decrease in tear was observed after C2, C3, C5 treatments. C1 and C4 which had xylanase activity improved paper properties, while other enzymes had a negative impact. Therefore, the intricate balance between cellulolytic and hemicellulolytic activity is the key to optimizing biorefining and paper properties. It was also observed that C1 impact was pH dependent, which supports the importance of pH in developing an enzymatic strategy for refining energy reduction.

The pulp and paper industry has started applying new, ecologically sound technology (biotechnology) in its manufacturing processes. Many interesting enzymatic applications have been proposed. Implemented technologies tend to change the existing industrial process as little as possible. Enzymes have great potentials in solving many problems associated with the use of recycled fiber, especially related to deinking, drainability, hornification, refining, and stickies. Based on the promising results of mill-scale trials, several mills in the world have started using enzymes for deinking. The potentials of cellulase enzymes have also been demonstrated for reducing the energy requirement in pulp refining, improving the machine runnability and stickies control when using recycled fiber. They have the important benefits in that they can be considered a "green" product. They are natural occurring compounds with little adverse impact on the environment. This paper deals with the imp...

Obtaining low cost lignocellulolytic enzymes and efficient biomass pretreatment are key to increase the competitiveness of second-generation ethanol in comparison with fossil fuels. The enzymatic cocktail produced by the Chrysoporthe cubensis fungus as well as the mixture prepared with the cocktails of the Chrysoporthe cubensis and Penicillium pinophilum fungi have already proven to be efficient for hydrolyzing biomass pretreated with alkali. In this study, they were evaluated in saccharification of sugarcane bagasse pretreated with dilute acid or hot water at 121°C using an enzyme loading equal to 8 filter paper units per gram of biomass. The most promising results were obtained from the hydrolysis of biomass pretreated with hot water by the C. cubensis-P. pinophilum enzymes blend. In this condition, the glucose and xylose production were 25.2 g.L-1 and 4.6 g.L-1 , respectively, that resulted in the conversion of 68% of glucan and 23% of xylan in only 48 hours. This study shows that the hydrothermal pretreatment is a promising alternative to improve the enzymes performance, produced by the fungi C. cubensis and P. pinophilum, in the sugarcane bagasse hydrolysis without the need of chemical compounds, generally used in the acid and alkali pretreatments. Furthermore, the hydrothermal pretreatment for 60 min allowed all cocktails applied to convert the cellulose efficiently with only 24 h of saccharification, which contributes to the energy savings employed in the process. HIGHLIGHTS  C. cubensis-P. pinophilum enzyme blend resulted in high levels of synergy  Mild hydrothermal pretreatment reduced the hemicellulases requirement in hydrolysis  The best results were obtained by the enzymatic blend in H60-SCB saccharification 2 De Albuquerque, M.F.G.; et al.

Enzymatic hydrolysis is an essential step in the lignocellulosic biomass treatment for the conversion of cellulose and hemicellulose into fermentable sugars. In the present study, a physical steam explosion (200°C) and chemical alkaline pre-hydrolysis (10% v/v) were applied to hemp (Cannabis sativa L.) biomass, to verify the effectiveness of different pretreatment processes, in order to increase enzymatic hydrolysis and the final sugar yield. The enzymatic degradations were carried out also utilizing extremophilic microorganisms, exploited as a source of (hemi)cellulases to be used as components of the enzymatic cocktail applied in the bioconversion into fermentable sugars for production of second generation ethanol and high value products. Alkaline pretreatment showed to be superior over the physical method with respect to the rate of enzymatic hydrolysis, with the obtainment of an hydrolysis yields of 90% xylose and 40% glucose, respectively.

The cellulase production with Penicillium nalgiovense S11 on wheat pollard was enhanced using substrate pretreatments, i.e.: (i) mechanic process by Wiley milling, (ii) reducing sugars removal by water soaking, and (iii) chemical pretrcatment by 0.5% NaOH soaking at 100°C. The enzyme production stated as enzyme activities of all prctrcated substrates were higher than the untreated substrate. Although soaking with water showed significant increase in enzyme activities, the highest CMCase (EC 3.2.1.4), FPase (filter papcrase) and p-glucosidase (EC 3.2.1.21) were observed on NaOH pretreated pollard. The NaOH prctreatment also enhanced the enzyme production by increasing substrate concentration from 2 to 4%. The op timal incubation time in the cellulase production on 4% NaOH-pretrcated pollard was observed on the fifth day. Add ition of 250 ppm glucose also increased the enzyme activities. The optimal treatments increased the specific activities of CMCase, FPase, and |3-glucosidase into...

Aims: The escalating demands for traditional fossil fuels with unsecured deliverance and issues of climate change compel the researchers to develop alternative fuels like bioethanol. This study examines the prospect of biofuel production from high carbohydrate containing lignocellulosic material, e.g. sugarcane bagasse through biological means. Methodology and Results: Cellulolytic enzymes were collected from the culture filtrate of thermotolerant Trichoderma viride grown on variously pre-treated sugarcane bagasse. CMCase and FPase enzyme activities were determined as a measure of suitable substrate pre-treatment and optimum condition for cellulolytic enzyme production. The highest CMCase and FPase activity was found to be 1.217 U/ml and 0.109 U/ml respectively under the production conditions of 200 rpm, pH 4.0 and 50 °C using steamed NaOH treated bagasse as substrate. SEM was carried out to compare and confirm the activity of cellulolytic enzymes on sugarcane bagasse. Saccharificat...

The trial to investigate hormonal and sugar changes in tree peony buds associated with dormancy was conducted in the fi eld at the Beijing Forestry University Experimental Site in China during autumn, winter and spring seasons (2009/2010 and 2010/2011), the periods of dormancy development and release. The experimental design was randomized complete block with three replications. The hormone and sugar levels were determined using the enzyme-linked immunosorbent assay (ELISA) technique and spectrophotometer, respectively. Winter temperature accumulated abscisic acid (ABA) and sugars in tree peony buds which most likely induced dormancy. Spring temperature, on the other hand, degraded ABA and sugars, and accumulated gibberellic acid (GA3) that possibly released dormancy in tree peony buds indicating that environmental temperature was the key regulator of hormone and sugar levels that infl uenced bud dormancy and growth. The results suggest that accumulation of ABA, GA3 and sugars in bu...

Pretreated water hyacinth was used as sole carbon source for the production of cellulase enzyme by Rhizopus oryzae PR 7 MTCC 9642 in both liquid state (LSF) and solid state fermentation (SSF) that was measured by the FPase activity. To maximize the FPase production, the critical parameters like substrate concentration, cultivation temperature and pH on enzyme production were optimized using response surface methodology using Central Composite Design (CCD). The LSF was found to be better than SSF for the production of FPase. The best preferred combination for highest FPase activity from LSF was with substrate concentration 1.25%,pH 7.32 and temperature 25.25 °C. Estimated optimum conditions for FPase production from SSF was a combination of substrate concentration of 0.5%, pH 6, temperature 18°C. Under the optimized cultivation condition, the strain synthesized 123 U/ml and 48U/ml FPase from LSF and SSF respectively and the highest production was achieved within only 48 hours of cult...

The enzymatic digestibility of sugarcane bagasse was greatly increased by alkali (NaOH)-peracetic acid (PAA) pretreatment under mild conditions. The effects of several factors affecting the pretreatment were investigated. It was found that when bagasse was pre-pretreated by 10% (based on initial dry materials) NaOH with 3:1 liquid-to-solid ratio at 90 • C for 1.5 h and further delignified by 10% peracetic acid (based on initial dry materials) at 75 • C for 2.5 h, the yield of reducing sugars reached 92.04% by enzymatic hydrolysis for 120 h with cellulase loading of 15 FPU/g solid. Compared with acid and alkali pretreatment, alkali-PAA pretreatment could be conducted under milder conditions and was more effective for delignification with less carbohydrates being degraded in the pretreatment process. Alkaline stage played an important role for partial delignification, swelling fibers and subsequently reducing PAA loading. No loss of cellulase activity (FPA) was observed in the liquid phase for alkali-PAA pretreated bagasse after enzymatic hydrolysis for 120 h.

Sugarcane bagasse (SB) and sugarcane trash (SCT) containing 30% hemicellulose content are the waste from the sugarcane industry. Hemicellulose being heterogeneous, more complex, and less abundant than cellulose remains less explored. The optimized conditions for the pretreatment of SB and SCT for maximizing the delignification are soaking in aqueous ammonia (SAA), 18.5 wt %, followed by heating at 70°C for 14 h. The optimization of hydrolysis of SAA pretreated (ptd) SB and SCT by the Box−Behnken design in the first step of saccharification by xylanase (CtXyn11A) and α-L-arabinofuranosidase (PsGH43_12) resulted in the total reducing sugar (TRS) yield of xylooligosaccharides (TRS (XOS)) of 93.2 mg/g ptd SB and 85.1 mg/g ptd SCT, respectively. The second step of saccharification by xylosidase (BoGH43) gave the TRS yield of 164.7 mg/g ptd SB and 147.2 mg/g ptd SCT. The high-performance liquid chromatography analysis of hydrolysate obtained after the second step of saccharification showed 69.6% xylan-to-xylose conversion for SB and 64.1% for SCT. This study demonstrated the optimization of the pretreatment method and of the enzymatic saccharification by recombinant xylanolytic enzymes, resulting in the efficient saccharification of ptd hemicellulose to TRS by giving 73.5% conversion for SB and 71.1% for SCT. These optimized conditions for the pretreatment and saccharification of sugarcane waste can also be used at a large scale.

In search of native cellulase-producing microorganisms, collected from lignocellulosic waste in Ecuatorian Andes, Amazon region, and in Antarctica, 17 sporulating strains were found. These strains and the cocktails obtained from them were used in assays to measure glucose concentration produced from carboxymethylcellulose, crystalline cellulose and filter paper units (FPU). The obtained values were that only the cocktail from Bacillus sp. collected at a “panela” artisan factory produced glucose concentrations that might be of interest, after the cellulases degraded all substrates: carboxymethylcellulose, crystalline cellulose and filter paper. The amount of glucose liquor obtained in the best assay was measured in HPLC, producing 3.45 mg/ml of glucose after the action of endo-beta-glucanase and 1.8 mg/ml of glucose in presence of exo-glucanase. The amount of filter paper units produced in vitro were 0.0073 FPU. However, the study found that, since the Payback Period for the investme...

The hypersecreting mutant Trichoderma reesei RUT-C30 (ATCC 56765) is one of the most widely used strains of filamentous fungi for the production of cellulolytic enzymes and recombinant proteins, and for academic research. The strain was obtained after three rounds of random mutagenesis of the wild-type QM6a in a screening program focused on high cellulase production and catabolite derepression. Whereas RUT-C30 achieves outstanding levels of protein secretion and high cellulolytic activity in comparison to the wild-type QM6a, recombinant protein production has been less successful. Here, we bring together and discuss the results from biochemical-, microscopic-, genomic-, transcriptomic-, glycomic-and proteomic-based research on the RUT-C30 strain published over the last 30 years.

The cellulase production with Penicillium nalgiovense S11 on wheat pollard was enhanced using substrate pretreatments, i.e.: (i) mechanic process by Wiley milling, (ii) reducing sugars removal by water soaking, and (iii) chemical pretrcatment by 0.5% NaOH soaking at 100°C. The enzyme production stated as enzyme activities of all prctrcated substrates were higher than the untreated substrate. Although soaking with water showed significant increase in enzyme activities, the highest CMCase (EC 3.2.1.4), FPase (filter papcrase) and pglucosidase (EC 3.2.1.21) were observed on NaOH pretreated pollard. The NaOH prctreatment also enhanced the enzyme production by increasing substrate concentration from 2 to 4%. The optimal incubation time in the cellulase production on 4% NaOH-pretrcated pollard was observed on the fifth day. Addition of 250 ppm glucose also increased the enzyme activities. The optimal treatments increased the specific activities of CMCase, FPase, and |3-glucosidase into 60, 4, and 198 times, respectively, as compared to the specific activities on 2% unpretreated pollard.

The effectiveness of several commercial enzymes has been evaluated for energy savings in beating and refining. A number of unbleached kraft pulps viz. softwood, bamboo and mixed pulp (60% waste corrugated kraft cuttings and 40% unbleached softwood pulp) were treated with the enzymes. With softwood pulp, the maximum reduction in beating time was found to be 25% in the case of hemicellulase 'Amano' 90 whereas in the case of Bleachzyme F, Irgazyme 4OS, Pulpzyme HC and Cartazyme HS 10, the reduction in beating time was of the order of 17-22%. The enzyme-treated pulp retained the required strength properties except in the case of hemicellulase 'Amano' 90, where the strength properties of the pulp were slightly affected. With bamboo pulp and mixed pulp, treatment with Bleachzyme F and hemicellulase 'Amano' 90, reduced the beating time by about 18% and 15%, respectively. The strength properties of the pulp were not found to be affected.