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The food enzyme cellulase (4‐(1,3;1,4)‐β‐d‐glucan 4‐glucanohydrolase; EC 3.2.1.4) is produced with the non‐genetically modified Penicillium funiculosum strain Lzc35 by Danisco US Inc. The cellulase is intended to be used in distilled alcohol production, baking and brewing processes. Since residual amounts of total organic solids (TOS) are removed by distillation, dietary exposure was only calculated for baking and brewing processes. Based on the proposed maximum use levels, dietary exposure to the food enzyme–TOS was estimated to be up to 0.416 mg TOS/kg body weight (bw) per day. Genotoxicity tests did not raise a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 84 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 200. Similarity of the amino acid sequence of the food enzyme to those of known allergens was searched and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood is considered to be low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Lytic polysaccharide monooxygenases (LPMOs) are crucial industrial enzymes required in the biorefinery industry as well as in the natural carbon cycle. These enzymes, known to catalyze oxidative cleavage of glycosidic bonds, are produced by numerous bacterial and fungal species to assist in the degradation of cellulosic biomass. In this study, we annotated and performed structural analysis of an uncharacterized LPMO from Penicillium funiculosum (PfLPMO9) based on computational methods in an attempt to understand the behavior of this enzyme in biomass degradation. PfLPMO9 exhibited 75% and 36% sequence identity with LPMOs from Thermoascus aurantiacus (TaLPMO9A) and Lentinus similis (LsLPMO9A), respectively. Furthermore, multiple fungal genetic manipulation tools were employed to simultaneously overexpress LPMO and cellobiohydrolase I (CBH1) in a catabolite-derepressed strain of Penicillium funiculosum, PfMig188 (an engineered variant of P. funiculosum), to improve its saccharification performance toward acid-pretreated wheat straw (PWS) at 20% substrate loading. The resulting transformants showed improved LPMO and CBH1 expression at both the transcriptional and translational levels, with ∼200% and ∼66% increases in ascorbate-induced LPMO and Avicelase activities, respectively. While the secretome of PfMig88 overexpressing LPMO or CBH1 increased saccharification of PWS by 6% or 13%, respectively, over the secretome of PfMig188 at the same protein concentration, the simultaneous overexpression of these two genes led to a 20% increase in saccharification efficiency over that observed with PfMig188, which accounted for 82% saccharification of PWS under 20% substrate loading. IMPORTANCE Enzymatic hydrolysis of cellulosic biomass by cellulases continues to be a significant bottleneck in the development of second-generation biobased industries. While increasing efforts are being made to obtain indigenous cellulases for biomass hydrolysis, the high production cost of this enzyme remains a crucial challenge affecting its wide availability for efficient utilization of cellulosic materials. This is because it is challenging to obtain an enzymatic cocktail with balanced activity from a single host. This report describes the annotation and structural analysis of an uncharacterized lytic polysaccharide monooxygenase (LPMO) gene in Penicillium funiculosum and its impact on biomass deconstruction upon overexpression in a catabolite-derepressed strain of P. funiculosum. Cellobiohydrolase I (CBH1), which is the most important enzyme produced by many cellulolytic fungi for saccharification of crystalline cellulose, was further overexpressed simultaneously with LPMO. The resulting secretome was analyzed for enhanced LPMO and exocellulase activities and the corresponding improvement in saccharification performance (by ∼20%) under high substrate loading using a minimal amount of protein.

Lytic polysaccharide monooxygenases (LPMOs) are crucial industrial enzymes required in the biorefinery industry as well as in natural carbon cycle. These enzymes known to possess auxiliary activity are produced by numerous bacterial and fungal species to assist in the degradation of cellulosic biomass. In this study, we annotated and performed structural analysis of an uncharacterized thermostable LPMO from Penicillium funiculosum (PfLPMO9) in an attempt to understand nature of this enzyme in biomass degradation. PfLPMO9 exhibited 75% and 36% structural identity to Thermoascus aurantiacus (TaLPMO9A) and Lentinus similis (LsLPMO9A), respectively. Analysis of the molecular interactions during substrate binding revealed that PfLPMO9 demonstrated a higher binding affinity with a ΔG free energy of -46 k kcal/mol when compared with that of TaLPMO9A (−31 kcal/mol). The enzyme was further found to be highly thermostable at elevated temperature with a half-life of ∼88 h at 50 °C. Furthermore, multiple fungal genetic manipulation tools were employed to simultaneously overexpress this LPMO and Cellobiohydrolase I (CBH1) in catabolite derepressed strain of Penicillium funiculosum, PfMig188, in order to improve its saccharification performance towards acid pretreated wheat straw (PWS) at 20% substrate loading. The resulting transformants showed ∼200% and ∼66% increase in LPMO and Avicelase activities, respectively. While the secretomes of individually overexpressed LPMO and CBH1-strains increased saccharification of PWS by 6% and 13%, respectively, over PfMig188 at same enzyme concentration, the simultaneous overexpression of these two genes led to 20% increase in saccharification efficiency over PfMig188, which accounted for 82% saccharification of PWS at 20% substrate loading.ImportanceEnzymatic hydrolysis of cellulosic biomass by cellulases continues to be a significant bottleneck in the development of second-generation bio-based industries. While efforts are being intensified at how best to obtain indigenous cellulase for biomass hydrolysis, the high production cost of this enzyme remains a crucial challenge confronting its wide availability for efficient utilization of cellulosic materials. This is because it is challenging to get an enzymatic cocktail with balanced activity from a single host. This report provides for the first time the annotation and structural analysis of an uncharacterized thermostable lytic polysaccharide monooxygenase (LPMO) gene in Penicillium funiculosum and its impact in biomass deconstruction upon overexpression in catabolite derepressed strain of P. funiculosum. Cellobiohydrolase I (CBH1) which is the most important enzyme produced by many cellulolytic fungi for saccharification of crystalline cellulose was further overexpressed simultaneously with the LPMO. The resulting secretome was analyzed for enhanced LPMO and exocellulase activities with the corresponding improvement in its saccharification performance at high substrate loading by ∼20% using a minimal amount of protein.

Co-culture of fungal strains Penicillium janthinellum EMS-UV-8 (E), Penicillium funiculosum strain P (P) and Aspergillus sp. strain G (G) and blending of their crude cellulase were evaluated for improvements in cellulase activities as well as for enhanced hydrolysis of dilute acid pretreated wheat straw (PWS). The blending of crude enzymes of P and E enhanced the hydrolysis of PWS more effectively due to synergism in cellulolytic enzyme activities. Here, three types of blends were made on the basis of equal FPUs, equal protein content or fixed volume containing different proportions of individual enzymes, the former blend hydrolyzed 42.6% of PWS due to the 98%,62%, 64% and 34% synergistic enhancement in endo-glucanase, cellulase (FPU), β-glucosidase and xylanase activities, respectively. Hydrolysis at 10% solid loading of PWS in roller bottle reactor with this blend further enhanced hydrolysis yield to 74% within 24 h, which was much better than the corresponding hydrolysis yields of individual (38.1% by E and 61.5% by P) or the commercial enzyme (62.3%). This study proved that synergistic blends of cellulases from two Penicillium spp. are cost-effective tools for efficient wheat straw hydrolysis for on-site biofuel production.

Aim To carry out a comparative study of cellulase and xylanase activities of 16 endophytic and soil Penicillium funiculosum strains. Methods The objects of the study were 16 endophytic and soil P. funiculosum strains from culture collection of microscopic fungi of Department of Physiology and Taxonomy of Micromycetes of D.K. Zabolotny Institute of Microbiology and Virology of NASU. Cellulase and xylanase activities were determined by enzyme activity zone on solid agar media with soluble cellulose (Na-CMC) and hemicellulose (xylan) and by enzymatic index (EІ). Results All investigated P. funiculosum strains, regardless of the isolation place, can hydrolyze Na-CMC and beech xylan. Enzymatic index was studied to characterize cellulolytic and xylanolytic activities of P. funiculosum strains from different econiches for the frst time. It was established that cellulase activity of endophytic P. funiculosum strains was medium and soil ones – from low to high. Endophytic strains showed low and medium xylanase activity, while the soil strains had only high activity. Conclusions It was established that endophytic and soil P. funiculosum strains were able to hydrolyze Na-CMC and beech xylan, while endophytes characterized by medium cellulase activity, and soil strains had from low to high level of this activity. Xylanase activity of endophytes was low, but soil isolates – only high one. Obtained experimental data might be the basis for explaining of endophytic life style of P. funiculosum in the plant, as well as in screening of producers of hydrolytic enzyme complexes for biotechnological purposes.

Poly(ethylene glycol) (PEG 4000) and bovine serum albumin (BSA) were investigated with the purpose of evaluating their influence on enzymatic hydrolysis of sugarcane bagasse. Effects of these supplements were assayed for different enzymatic cocktails (Trichoderma harzianum and Penicillium funiculosum) that acted on lignocellulosic material submitted to different pretreatment methods with varying solid (25 and 100 g/L) and protein (7.5 and 20 mg/g cellulose) loadings. The highest levels of glucose release were achieved using partially delignified cellulignin as substrate, along with the T. harzianum cocktail: increases of 14 and 18 % for 25 g/L solid loadings and of 33 and 43 % for 100 g/L solid loadings were reached for BSA and PEG supplementation, respectively. Addition of these supplements could maintain hydrolysis yield even for higher solid loadings, but for higher enzymatic cocktail protein loadings, increases in glucose release were not observed. Results indicate that synergism might occur among these additives and cellulase and xylanases. The use of these supplements, besides depending on factors such as pretreatment method of sugarcane bagasse, enzymatic cocktails composition, and solid and protein loadings, may not always lead to positive effects on the hydrolysis of lignocellulosic material, making it necessary further statistical studies, according to process conditions.

Enzymatic hydrolysis of lignocellulosic biomass is limited by economic considerations arising from enzyme production costs and specific activities. The effect of six hemicellulases on raw sugarcane bagasse and two types of pretreated sugarcane bagasse was investigated using experimental designs. Our strategy was successful for developing more efficient and less expensive enzymatic mixture, and also revealed that hemicellulase mixtures with multiple activities could be less effective than expected. In this study, only two hemicelulases, the endo-1,4-xylanases (GH11) from Penicillium funiculosum (XynC11/CAC15487) and the feruloyl esterase (CE1) from Clostridium thermocellum (CtFAE/ATCC27405), effectively broke-down hemicellulose from pretreated sugarcane bagasse (up to 65%), along with the production of xylooligosaccharides (XOS). Our results also demonstrated that GH11 and CE1 can improve biomass saccharification by cellulases. Treatment with these two enzymes followed by a commercial cellulase cocktail (Accellerase®1500) increased saccharification of pretreated lignocellulose by 24%. Collectively, our data contributes to the rational design of more efficient and less expensive enzyme mixtures, targeting the viable production of bioethanol and other biorefinery products.

Glycoside Hydrolase Family 7 cellobiohydrolases (GH7 CBHs) catalyze cellulose depolymerization in cellulolytic eukaryotes, making them key discovery and engineering targets. However, there remains a lack of robust structure–activity relationships for these industrially important cellulases. Here, we compare CBHs from Trichoderma reesei (TrCel7A) and Penicillium funiculosum (PfCel7A), which exhibit a multi-modular architecture consisting of catalytic domain (CD), carbohydrate-binding module, and linker. We show that PfCel7A exhibits 60% greater performance on biomass than TrCel7A. To understand the contribution of each domain to this improvement, we measure enzymatic activity for a library of CBH chimeras with swapped subdomains, demonstrating that the enhancement is mainly caused by PfCel7A CD. We solve the crystal structure of PfCel7A CD and use this information to create a second library of TrCel7A CD mutants, identifying a TrCel7A double mutant with near-equivalent activity to wild-type PfCel7A. Overall, these results reveal CBH regions that enable targeted activity improvements., Cellobiohydrolases (CBHs) are critical for natural and industrial biomass degradation but their structure–activity relationships are not fully understood. Here, the authors present the biochemical and structural characterization of two CBHs, identifying protein regions that confer enhanced CBH activity.

A partial peptide sequence of β-glucosidase isoform (Bgl4) of Penicillium funiculosum NCL1 was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The cDNA (bgl4) encoding Bgl4 protein was cloned from P. funiculosum NCL1 RNA by consensus RT-PCR. The bgl4 gene encoded 857 amino acids that contained catalytic domains specific for glycoside hydrolase family 3. The cDNA was over-expressed in Pichia pastoris KM71H and the recombinant protein (rBgl4) was purified with the specific activity of 1,354.3 U/mg. The rBgl4 was a glycoprotein with the molecular weight of ~130 kDa and showed optimal activity at pH 5.0 and 60 °C. The enzyme was thermo-tolerant up to 60 °C for 60 min. The rBgl4 was highly active on aryl substrates with β-glucosidic, β-xylosidic linkages and moderately active on cellobiose and salicin. It showed remarkably high substrate conversion rate of 3,332 and 2,083 μmol/min/mg with the substrates p-nitrophenyl β-glucoside and cellobiose respectively. In addition, the rBgl4 showed tolerance to glucose concentration up to 400 mM. It exhibited twofold increase in glucose yield when supplemented with crude cellulase of Trichoderma reesei Rut-C30 in cellulose hydrolysis. These results suggested that rBgl4 is a thermo- and glucose-tolerant β-glucosidase and is a potential supplement for commercial cellulase in cellulose hydrolysis and thereby assures profitability in bioethanol production.

Background There is an urgent requirement for second-generation bio-based industries for economical yet efficient enzymatic cocktail to convert diverse cellulosic biomass into fermentable sugars. In our previous study, secretome of Penicillium funiculosum NCIM1228 showed high commercial potential by exhibiting high biomass hydrolyzing efficiency. To develop NCIM1228 further as an industrial workhorse, one of the major genetic interventions needed is global deregulation of cellulolytic genes to achieve higher enzyme production. Mig1 orthologs found in all yeast and filamentous fungi are transcriptional regulators that maintain carbon homeostasis by negatively regulating genes of secondary carbon source utilization. Their disruption has long been known to be beneficial for increasing the production of secreted enzymes for alternate carbon source utilization. Results Upon detailed genotypic and phenotypic analysis, we observed that NCIM1228 harbors a truncated yet functional allele of homolog of a well-known catabolite repressor, Mig1. Alleviation of carbon repression in NCIM1228 was attained by replacing functional Mig1134 allele with null allele Mig188. P. funiculosum having Mig188 null allele showed better growth characteristics and 1.75-fold better glucose utilization than parent strain. We also showed that visibly small colony size, one of the major characteristics of CCR disruptant strains in filamentous fungi, was not due to retarded growth, but altered hyphal morphology. CCR-disrupted strain PfMig188 showed profuse branching pattern in terminal hyphae resulting in small and compact colonies with compromised filamentous proliferation. We further observed that basal level expression of two major classes of cellulases, namely, cellobiohydrolase and endoglucanase, was regulated by Mig1134 in NCIM1228, whereas other two major classes, namely, xylanases and β-glucosidase, were only marginally regulated. Finally, CCR disruption in P. funiculosum NCIM1228 led to prolonged cellulase induction in production medium resulting in twofold increased cellulase activity than the parent strain with maximum secreted protein titer being > 14 g/l. Conclusions CCR-disrupted P. funiculosum showed better growth, enhanced carbon source utilization, profuse branching pattern in terminal hyphae, and higher cellulase activity than parent strain. Our findings are particularly important in shedding light on important functions performed by Mig1 in addition to its role as negative regulator of alternate carbon source utilization in filamentous fungi. Electronic supplementary material The online version of this article (10.1186/s13068-018-1011-5) contains supplementary material, which is available to authorized users.

Aim: Lignocelluloytic enzymes are the largest class of hydrolase enzyme which utilizes the plant biomass to produce renewable sources. Hence practices for larger production of these enzymes at lower cost received much attention for industrial use. Hence this paper deals with expression and purification of cellobiohydrolase gene from Penicillium funiculosum NCL1. Methods & Results: A cellobiohydrolase gene, cbhII of Penicillium funiculosum NCL1 was cloned and expressed in Pichia pastoris X33. Two exons of the cbhII gene were amplified separately and fused by overlap extension PCR. The fused product was cloned in yeast expression vector pPICZαA and expressed in P. pastoris under the control of the AOX1 promoter. P. pastoris transformants expressing recombinant cellobiohydrolase were selected on CMC agar plate and their ability to produce the cellobiohydrolase was evaluated in flask cultures. P. pastoris X33 (pPICbh6) efficiently secreted the recombinant cellobiohydrolase into the medium and produced the cellobiohydrolase activity (5 U/ml) after 96 h of growth. The recombinant cellobiohydrolase produced by P. pastoris (pPICBH6) showed maximum activity at pH 4.0 and temperature 50&degC and higher specificity in hydrolysis of filter-paper.

Filamentous fungi respond to the need to secure utilisable carbon from their growth milieu by secreting unique extracellular proteins depending upon the types of polymeric substrates. We have here profiled the variations in the secretome pattern of a non-model hypercellulolytic fungus – Penicillium funiculosum, grown in minimal media containing four different polymeric cellulase inducers, i.e., Avicel, wheat bran, ammonium-pretreated wheat straw and Avicel & wheat bran, and glucose over its early and late log phases of growth. Of the 137 secreted proteins validated at 1% FDR, we identified the quantified proteins in three clusters as early, persistently or lately expressed. The type of carbon substrate present in the culture media significantly affected the levels of cellulolytic enzymes expression by the fungus. The top abundant proteins quantified in the secretome for Avicel and wheat bran were cellobiohydrolaseI [GH7-CBM1], cellobiohydrolaseII [GH6-CBM1], β-glucosidase [GH3], arabinofuranosidase [GH51] and β-xylosidase [GH3], with bicupin being highest in case of wheat straw. Our results further suggested that the fungus secreted the extracellular proteins in waves, such that the initial responders act to hydrolyse the composite substrates in the culture environment before the second wave of proteins which tend to be more tailored to the specific substrate in the cultivating media. Biological significance In this article, we have comprehensively examined the dynamics of the secretome of a non-model hypercellulolytic fungus produced in response to model and composite cellulase inducers. Our study has provided additional insights into how the fungus enzyme machinery responds to the presence of different polymeric cellulase inducers over the two different growth phases (early growth and late growth phase). The comprehensive typing and quantification of the different proteins present in the secretomes of the cellulolytic fungal strains in response to diverse nutrient sources hold many prospects in understanding the fungus unique enzyme machinery and dynamics for the downstream biotechnological applications.

In the present study, we have characterized high molecular weight multi-enzyme complexes in two commercial enzymes produced by Trichoderma reesei (Spezyme CP) and Penicillium funiculosum (Accellerase XC). We successfully identified 146–1000 kDa complexes using Blue native polyacrylamide gel electrophoresis (BN-PAGE) to fractionate the protein profile in both preparations. Identified complexes dissociated into lower molecular weight constituents when loaded on SDS PAGE. Unfolding of the secondary structure of multi-enzyme complexes with trimethylamine (pH >10) suggested that they were not a result of unspecific protein aggregation. Cellulase (CMCase) profiles of extracts of BN-PAGE fractionated protein bands confirmed cellulase activity within the multi-enzyme complexes. A microassay was used to identify protein bands that promoted high levels of glucose release from barley straw. Those with high saccharification yield were subjected to LC–MS analysis to identify the principal enzymatic activities responsible. The results suggest that secretion of proteins by aerobic fungi leads to the formation of high molecular weight multi-enzyme complexes that display activity against carboxymethyl cellulose and barley straw.

The objective of the present study was to transform “Waste to Food” using enzymes to recover value-added food ingredients from biomass. Six commercial cellulases were screened to generate proof of concept that enzymes are selective and efficient catalysts for opening of lime peel biomass to recover pectin. The most efficient enzyme preparation was Laminex C2K derived from Penicillium funiculosum which, during 4 h treatment at pH 3.5, 50 °C, released pectin with similar yield (23% w/w), molecular weight (69 kDa), and functional properties e.g. gelling, stabilization of acidified milk drinks and viscosity as the classically acid-extracted pectins (8 h treatment at 70 °C, pH

Penicillium funiculosum NCL1, a filamentous fungus, produced significantly higher levels of β-glucosidase. The effect of initial pH, incubation temperature, and different carbon sources on extracellular β-glucosidase production was studied in submerged fermentation. At 30 °C with initial pH 5.0, enzyme production was increased by 48-fold upon induction with paper mill waste, as compared to commercial cellulose powder. In zymogram analysis, four isoforms of β-glucosidases were observed with wheat bran whereas a minimum of one isoform was observed with other carbon sources. A major β-glucosidase (Bgl3A) with the apparent molecular weight of ~120 kDa, induced by paper mill waste, was purified 19-fold to homogeneity, with a specific activity of 1,796 U/mg. Bgl3A was a monomeric glycoprotein with 29% of neutral carbohydrate content. It showed optimum activity at pH 4.0 and 5.0, optimum temperature at 60 °C, and exhibited a half-life of 1 h at 60 °C. K m of Bgl3A was found to be 0.057 mM with p-nitrophenyl β-d-glucoside and V max was 1,920 U/mg. The purified enzyme exhibited glucose tolerance with a K i of 1.5 mM. Bgl3A readily hydrolyzed glucosides with β-linkage. Bgl3A activity was enhanced (156%) by Zn2+ and was not affected by other metal cations and reagents. The supplementation of Bgl3A (5 U/mg) with Trichoderma reesei cellulase complex (5 FPU/mg) resulted in about 70% of enhanced glucose production, which emphasizes the industrial importance of Bgl3A.

In this study, we investigated the enzymatic hydrolysis of pretreated sugarcane bagasse using eight different enzymatic blends obtained from concentrated crude enzyme extracts produced by Penicillium funiculosum and Trichoderma harzianum as well as from the extracts in combination with a commercial enzymatic cocktail. The influence of different levels of biomass delignification, degree of crystallinity of lignicellulose, composition of enzymatic activities and BSA on enzymatic hydrolysis yields (HYs) was evaluated. Our X-ray diffraction studies showed that crystallinity of lignocellulose is not a key determinant of its recalcitrance toward enzymatic hydrolysis. In fact, under the experimental conditions of our study, an increase in crystallinity of lignocellulosic samples resulted in increased glucose release by enzymatic hydrolysis. Furthermore, under the same conditions, the addition of BSA had no significant effect on enzymatic hydrolysis. The most efficient enzyme blends were obtained by mixing a commercial enzymatic cocktail with P. funiculosum or T. harzianum cellulase preparations (HYs above 97%) followed by the concentrated extract of P. funiculosum alone (HY = 88.5%). Increased hydrolytic efficiencies appeared to correlate with having an adequate level of both β-glucosidase and xylanase activities in the blends.

Cellulase from Penicillium funiculosum was immobilized on functionalized MCF (Meso Cellular Foam) silica by imine bond formation followed by reduction using NaBH4. The specific activities of free and immobilized enzyme were measured for hydrolysis of soluble carboxymethyl cellulose (CMC). The highest activity of MCF immobilized and native enzyme was obtained at optimum pH 5 and 4.5 respectively. Kinetic parameters, Michaelis–Menten constant (Km) and maximum reaction velocity (Vmax), were calculated as Km = 0.025 × 10−2 mg/mL, Vmax = 5.327 × 10−3 U/mg for the free enzyme and Km = 0.024 × 10−2 mg/mL, Vmax = 9.794 × 10−3 U/mg for MCF immobilized enzyme respectively. The reusability of immobilized enzymes showed that 66% of its activity is retained even after 15 cycles. The availability of polar groups (–NH–, –OH) and large pore size of surface modified MCF could be electrostatically stabilizing the cellulase. Functionalized MCF was found to be a promising material for stabilizing cellulase with 16.4 wt% loading of enzyme.

Plant cell wall degrading enzymes are given importance due to the development of efficient biomass degradation methods and the conversion of sugars to valuable products like butanol, amino acids and utilizable forms such as ethanol and methane. Plant biomass is an abundant renewable resource with cellulose and hemicellulose comprising about 40-50% of plant cell. The rate-limiting step in biomass degradation is the conversion of the cellulose and hemicellulose polymers to sugars. Cellulase enzyme has many potent commercial applications in industries. It is commonly produced as an extra cellular enzyme in fungi, bacteria and insects, however fungal enzymes are amongst the most extensively studied and characterized. Trichoderma reesei is the most commonly used organism for the commercial production of cellulase. An attempt has been made in the present study to improve the yield by using a mixed culture of Trichoderma reesei and Penicillium funiculosum on low cost substrates, corn fibre and sugarcane bagasse. The results show that the growth of T. reesei and P. funiculosum, production rate and cellulase activity was high only when sugarcane bagasse was used as substrate than that of corn fibre. Also the yield of cellulase enzyme and its activity were greater in the case of mixed culture than pure cultures of T. reesei and P. funiculosum.

The presence of endo-1,4-beta-D-glucanase, cellobiohydrolase, and beta-glucosidase activities in a multi-enzymatic complex system from Penicillium funiculosum was investigated. The interesting feature of these enzymes is their synergistic action for the hydrolysis of the native cellulose into glucose units. Both endo-1,4-beta-D-glucanase and cellobiohydrolase showed broader pH activity profiles, with pH optima of 4.0 and 4.0-5.0, respectively. However, beta-glucosidase activity showed a narrow pH-activity profile, with an optimum pH of 4.5. The different cellulolytic activities were stable in the acidic pH range of 2.5-6.0 and showed a similar optimal temperature of 60 degrees C. Although beta-glucosidase has shown a close catalytic efficiency as that of endo-1,4-beta-D-glucanase, its thermal stability was lower. However, the thermal stability profile of cellobiohydrolase was close to that of endo-1,4-beta-D-glucanase. The results also revealed the presence of high levels of endo-1,3-1,4-beta-D-glucanase, endo-1,3-beta- d-glucanase, and pectinase activities in the multi-enzymatic cellulolytic complex system. Moreover, the investigated multi-enzymatic complex system was effective in degrading the nonstarch polysaccharides of soybean meal.

Lignocellulosic materials represent a very important and promising source of renewable biomass. In order to turn them into fermentable sugars, synergism among the different enzymes that carry out bioconversion of these materials is one of the main factors that should be considered. Experimental mixture design was performed to optimize the proportion of enzymes produced by native strains of Trichoderma harzianum IOC 3844, Penicillium funiculosum ATCC 11797, and Aspergillus niger ATCC 1004, resulting in a proportion of 15, 50, and 35%, respectively. This mixture was able to hydrolyze 25 g/L of pretreated sugarcane bagasse with 91% of yield after 48 h of enzymatic reaction. Synergism along the hydrolysis process, besides the influence of lignin, hemicellulose, and solids loading, were also studied. Response surface methodology (RSM) based on Central Composite Rotatable Design was used to optimize solids and protein loadings to increase glucose release and enzymatic hydrolysis yield. The optimum solid and protein loadings established with RSM were 196 g/L and 24 mg/g cellulose, respectively, and under these conditions (94.1 ± 8) g/L of glucose were obtained, corresponding to a hydrolysis yield of 64%. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1222-1229, 2016.

Two recombinant ferulic acid esterases from Penicillium funiculosum produced in Aspergillus awamori were evaluated for their ability to improve the digestibility of pretreated corn stover. The genes, faeA and faeB, were cloned from P. funiculosum and expressed in A. awamori using their native signal sequences. Both enzymes contain a catalytic domain connected to a family 1 carbohydrate-binding module by a threonine-rich linker peptide. Interestingly, the carbohydrate binding-module is N-terminal in FaeA and C-terminal in FaeB. The enzymes were purified to homogeneity using column chromatography, and their thermal stability was characterized by differential scanning microcalorimetry. We evaluated both enzymes for their potential to enhance the cellulolytic activity of purified Trichoderma reesei Cel7A on pretreated corn stover.

Optimal conditions for preparing of chitooligosaccharides from chitosan with cellulase was researched by response surface methodo-logy, Penicillium funiculosum derived cellulase was most effective for chitooligosaccharides production as the point of hydrolyzing activity and commercial utility. The result which measures the change of degrading ratio at time course, 10 hr reaction showed a exponential increase and after that time degrading ratio was not changed. The optimal conditions determined by response surface methodology with central composite design of total 26 species were of chitosan, 143 U enzyme, 49 of reaction temperature, 13.2 hr of reaction time and pH 3.8. Major chitooligosaccharides produced from chitiosan on optimal conditions were dimer and trimer.

Carbon catabolite repression (CCR) is a regulatory mechanism which negatively regulates genes of for ancillary carbon source utilization. It is mediated by Mig1 orthologues, which are Zn finger transcriptional repressors. We studied the effect of CCR disruption in Penicillium funiculosum NCIM1228, a hypercelluloytic ascomycete. Upon phylogenetic analysis of fungal genomes, Mig1 presence across all taxa of kingdom fungi revealed its conserved role in catabolite repression. Also it was found to constitute distinct clade from industrially important cellulase producing fungi like Trichoderma reesei and Aspergillus sp. It shared the clade with other highly evolved fungi T. cellulolyticus and P. marneffei and represented more recent radiations of evolutionary conserved catabolite repressor Mig1. Genotypic analysis showed that NCIM1228 harbors a truncated yet functional allele of Mig1. Mig1 orthologue of NCIM1228 has a non-sense mutation at 134th amino acid position, making a large C-terminal portion of Mig1 (415aa) dispensable for carbon repression. NCIM1228 was grown in presence of allyl alcohol to check the phenotypic effect. NCIM1228 showed sensitivity to allyl alcohol as compared to Penicillium funiculosum (Pf). Deleting active Zn finger domain made NCIM1228 completely sensitive to allyl alcohol. Surprisingly, the deletion showed small and compact colonies with compromised filamentous proliferation while the dry mycelial weight didn’t change when grown on 0.5% glucose supplemented with 2% avicel. Using microscopy, we unraveled that PfΔMig1 showed reduced aerial hyphae and profuse branching pattern in terminal hyphae resulting in compact colonies. We further observed more than two-fold (7.6 FPU/ml) higher FPU in production media than NCIM1228 under similar condition.

Twenty-three kinds of commercially available enzymes were subjected to the screening test for xylanolytic activities, namely, endo-β-xylanase, β-xylosidase, α-arabinofuranosidase and α-glucuronidase.The majority of the enzymes contained some of the activities. The above enzymes were also submitted to the hydrolyzable test on the arabinoglucuronoxylan of corn hull. Among them, Cellulase C-0901 (Sigma Chemicals) originating from Penicillium funiculosum, although lacking in a-glucuronidase, degraded the xylan very well, and released 80.5% of arabinose and 60.8% of xylose based on the component sugars of the xylan. On the other hand, Cellulase 'Onozuka' R-10 (Yakult Co.) originating from Trichoderma viride, although containing all of the xylanolytic activities, decomposed hardly any of the arabinoglucuronoxylan. According to these results, commer cially available enzymes contained high and various xylanolytic activities. However, the intensities of enzyme activity and hydrolyzable ability differed remarkably in the degradation of arabinogluc uronoxylan. In addition, Cellulase C-0901 was most suitable among them for the enzymatic hydrolysis of the xylan.

Different used paper materials and mixtures thereof were saccharified with Penicillium funiculosum cellulase. Non-similar biodegradation patterns were concluded to be operating as well as declining bioconversion efficiencies with increasing biodegradation. Biowaste mixtures were less effectively biodegraded indicating the importance of separating biowaste into distinctive materials prior to developing it as a resource of bioproduct synthesis.

Used paper, a potential resource of alternative energy, can be recycled but mostly it forms a significant component of solid waste. Used office paper, foolscap paper, filter paper and newspaper have been treated with cellulase from Trichoderma reesei and Penicillium funiculosum to bioconvert their cellulose component into sugars. Both non-pretreated and pretreated paper was incubated successively with the two cellulases during four consecutive incubation periods of 1 h each. The amount of sugars released during this sequential treatment was compared with the total sugar produced during a 4 h period of continuous incubation with each enzyme system independently. Pre-treatment milling of paper proved to effectively increase the sugar formation under all incubation conditions. Successive incubation with the two enzyme systems of both non-pretreated and pretreated paper materials was more efficient than the corresponding continuous bioconversion. The highest relative sugar yield was experienced during successive treatment of pretreated materials when T. reesei cellulase initiated the degradation. However, maximum bioconversion of pretreated newspaper was obtained when P. funiculosum initiated degradation. Pretreated foolscap paper was the most susceptible substrate with maximum bioconversion when exposed to both forms of successive cellulase treatment.

Various used paper materials have been exposed to the action of cellulases from Penicillium funiculosum, Trichoderma reesei, Trichoderma viride and Aspergillus niger. A 2 h incubation period showed cellulase from T. viride the most active except for office paper that was maximally degraded by A. niger cellulase. Cellulase mixtures increased saccharification while sequential treatment with cellulases from T. reesei and P. funiculosum increased biodegradation at values between 15% and 190%. The maximum increase of saccharification (190%) was obtained when T. reesei cellulase initiated the sequential treatment of newspaper relative to the sole action of P. funiculosum cellulase on this non-pretreated and pretreated material.

Paper materials, filter paper, foolscap paper, newspaper and office paper contribute largely towards organic-based waste. These materials were incubated with cellulase from Penicillium funiculosum. In order to increase the extent of glucose formation from the cellulose components, all were treated prior to enzymatic-catalyzed hydrolysis. Homogenization, heat and ultrasonification were treatments applied, followed by incubation with different cellulase concentrations. Foolscap paper showed the highest susceptibility to saccharification, followed by filter paper, newspaper and office paper. Homogenization of all materials before treatment with different cellulase concentrations resulted in an increased glucose formation, but newspaper was the only cellulosic material exhibiting an increased degree of saccharification after all the applied pretreatments and hydrolysis with the different cellulase concentrations.

Used paper products contribute largely towards organic-based waste produced and dumped by the world population. Cellulose, a structural component of paper materials, can be hydrolysed into glucose by a multi-component enzyme system called cellulase. Cellulase from Penicillium funiculosum and Trichoderma reesei were applied in the saccharification of paper products such as foolscap paper, filter paper, newspaper and office paper as well as microcrystalline cellulose. Foolscap paper showed the strongest susceptibility towards enzymatic hydrolysis with both enzymes. With an enzyme concentration of 10.0 mg/mL for each cellulase system the strongest synergistic action was observed at a combination of 1:1 (m/m) during saccharification of all cellulose materials. The individual enzyme performance as well as their synergistic actions showed different rates of hydrolysis during degradation of the investigated cellulose substrates.

El objetivo de este estudio fue estandarizar un método enzimático para medir la digestibilidad de forrajes empleando la enzima celulasa proveniente del hongo Penicillium funiculosum y compararlo con el método de digestibilidad in vitro de Tilley y Terry (1963). Para la estandarización de la técnica se utilizó como sustrato forrajes de digestibilidad in vitro conocida: alfalfa y paja de avena; y para la comparación entre métodos se utilizó forrajes de diferente calidad provenientes de varias zonas del Perú. La concentración óptima enzima-sustrato hallada fue de 200 y 250 mg, respectivamente. Los valores de digestibilidad del método enzimático fueron estadísticamente menores que los del método in vitro, y el coeficiente de correlación fue de 0.987 (p, The objective of this study was to standardize the enzymatic procedure for estimating the digestibility of forages using the fungal cellulase Penicillium funiculosum, and its comparison with the Tilley and Terry (1963) in vitro digestibility method. The wellknown in vitro digestibility of alfalfa and oat straw were used for the standardization of the technique. Different quality forages from various locations of Peru were used for the comparison between methods. The best enzyme-substrate concentrations were 200 and 250 mg respectively. Both methods were statistically different, being the enzymatic digestibility values lower than the in vitro method ones. The correlation coefficient between methods was 0.987 (p

Treatment of beaten bleached kraft pulp with cellulase preparations can increase the pulp drainability, and pretreatment of unbeaten pulp with cellulases can improve the pulp beatability. To determine the enzyme(s) responsible for these changes in pulp properties, a bleached hardwood kraft pulp was treated with Trichoderma reesei cellulase (Celluclast™) before and after laboratory beating of pulp samples. By fractionating the Celluclast cellulases and evaluating the effectiveness of enzyme components in changing the pulp freeness, an endoglucanase (EG II) was found to play the key role in both the pulp drainability and beatability changes. In contrast, a cellobiohydrolase (CBH I), a major cellulase protein in the Celluclast cellulases, had no effect on either the pulp drainability or beatability. The drainage increase was closely correlated with the endoglucanase activity (assayed as CMCase) in three different cellulase preparations from T. reesei, Aspergillus niger, and Penicillium funiculosum. On the other hand, the beatability enhancement effected by these enzymes had no correlation with their CMCase activities.

The secretome of Penicillium funiculosum contains two family GH7 enzymes, one of which (designated XynA) has been described as a xylanase. This is unusual because it is the only xylanase in family GH7, which is mainly composed of cellobiohydrolases and endoglucanases, and also because XynA is highly similar to the cellobiohydrolase I from Talaromyces emersonii and Trichoderma reesei (72 and 65 % identity, respectively). To probe this enigma, we investigated the biochemical properties of XynA, notably its activity on xylans and β-d-glucans. A highly pure sample of XynA was obtained and used to perform hydrolysis tests on polysaccharides. These revealed that XynA is 100-fold more active on β-1,4-glucan than on xylan. Likewise, XynA was active on both 4-nitrophenyl-β-d-lactopyranoside (pNP-β-d-Lac) and 4-nitrophenyl-β-d-cellobioside (pNP-cellobiose), which shows that XynA is principally an exo-acting type 1 cellobiohydrolase enzyme that displays 5.2-fold higher performance on pNP-cellobiose than on pNP-β-d-Lac. Finally, analyses performed using cellodextrins as substrate revealed that XynA mainly produced cellobiose (C2) from substrates containing three or more glucosyl subunits, and that C2 inhibits XynA at high concentrations (IC50 C2 = 17.7 μM). Overall, this study revealed that XynA displays typical cellobiohydrolase 1 activity and confirms that the description of this enzyme in public databases should be definitively amended. Moreover, the data provided here complete the information provided by a previous proteomics investigation and reveal that P. funiculosum secretes a complete set of cellulose-degrading enzymes.

Renewable energy would become more topical as natural energy resources are depleted. Also a major global issue is the extent of solid waste generation of which biowaste is the main component and development of its bio-energy potential can address issues such as renewable energy and environmental pollution, simultaneously. Used paper is a primary component of biowaste with cellulose its dominant structural biopolymer. Various used paper materials have been treated with a cellulose hydrolyzing enzyme from Penicillium funiculosum to bioconvert their cellulose component into sugars such as glucose that could be utilized as feedstock for the synthesis of biodegradable products such as bio-ethanol. These paper materials exhibited nonsimilar susceptibilities toward this cellulase enzyme and consequently different sugar releasing and bioconversion efficiency patterns were obtained when changing quantities of each paper were treated with a fixed amount of enzyme. When a fixed amount of each paper material was trea...

An in vitro method for the determination of digestibility of grass hay was developed by using a combination of three commercial cellulases (Aspergillus niger, Tricholerma virile, and Penicillium funiculosum) showing activities of β-1,4-glucanase, β-1,4-xylanase, β-1,3-glucanase, mixed linkage enzymes, and esterases hydrolyzing ferulic acid and p-coumaric acid esterified to methyl arabinofuranoside. For individual enzymes, hydrolysis of glucose from mature timothy was proportional to their β-1,4-glucanase activities, but the hydrolysis of xylose was limited by the phenolic acid esterase activity

The present study aimed at maximizing cellulase production by Penicillium funiculosum using sequential experimental design methodology for optimizing the concentrations of nitrogen sources. Three sequential experimental designs were performed. The first and the second series of experiments consisted of a 2(4) and a 2(3) factorial designs, respectively, and in the third one, a central composite rotational design was used for better visualizing the optimum conditions. The following nitrogen sources were evaluated: urea, ammonium sulfate, peptone, and yeast extract. Peptone and ammonium sulfate were removed from the medium optimization since they did not present significant statistical effect on cellulase production. The optimal concentrations of urea and yeast extract predicted by the model were 0.97 and 0.36 g/L, respectively, which were validated experimentally. By the use of the desirability function, it was possible to maximize the three main enzyme activities simultaneously, which resulted in values for FPase of 227 U/L, for CMCase of 6,917 U/L, and for beta-glucosidase of 1,375 U/L. These values corresponded to increases of 3.3-, 3.2-, and 6.7-folds, respectively, when compared to those obtained in the first experimental design. The results showed that the use of sequential experimental designs associated to the use of the desirability function can be used satisfactorily to maximize cellulase production by P. funiculosum.