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PfMig188, a catabolically derepressed engineered strain of the hyper-cellulolytic fungus Penicillium funiculosum NCIM1228, was investigated for the efficacy of its secretome for biomass saccharification. An inexpensive version of media containing microcrystalline cellulose, wheat bran and soya protein was optimized for producing a high-quality secretome from the PfMig188 strain in both shake flasks and in a 20-L bioreactor. The activities of four classes of core cellulolytic enzymes required for saccharification in the PfMig188 secretome, namely, cellobiohydrolase (Avicelase activity), endoglucanase (CMCase activity), β-glucosidase (PNPGase activity) and xylanase (xylanase activity), were found to be 2.29 U/mL, 28.24 U/mL, 150 U/mL and 76 U/mL, respectively. The saccharification potential of the PfMig188 secretome was evaluated on rice straw and sugarcane bagasse pretreated with nitric acid and/or ammonium hydroxide. Saccharification performed using a 15 % (w/v) biomass load and a 3% (w/w) enzyme load released >100 g/L sugar in the hydrolysate, irrespective of the type of biomass and pre-treatment, with >80 % hydrolysis. Furthermore, the presence of lignin in nitric acid-pretreated biomass only marginally affected saccharification. Overall, the results demonstrated that the PfMig188 secretome, having relatively broad substrate specificity, is a viable and efficient substitute for T. reesei-based secretomes for diverse biomass saccharification.

The article reports the chemical composition, antioxidant, six key enzymes inhibitory and antimicrobial activities of two solvent extracts (water and methanol) of leaves and stem bark of Uapaca togoensis. For chemical composition, methanol extract of stem bark exhibited significant higher total phenolic (129.86 mg GAE/g) and flavanol (10.44 mg CE/g) contents. Methanol extract of leaves and water extract of stem bark showed high flavonoids (20.94 mg RE/g) and phenolic acid (90.40 mg CAE/g) content, respectively. In addition, HPLC-ESI-TOF-MS analysis revealed that U. togoensis was rich in procyanidins. The methanol and water extracts of stem bark had overall superior antioxidant activity; however, only methanol extract of stem bark showed higher inhibition of cholinesterase (AChE: 2.57 mg GALAE/g; BChE: 4.69 mg GALAE/g), tyrosinase (69.53 mg KAE/g) and elastase (2.73 mmol CE/g). Potent metal chelating ability was showed by water extract of leaves (18.94 mg EDTAE/g), higher inhibition of amylase was detected for water extracts of leaves (0.94 mmol ACAE/g) and stem bark (0.92 mmol ACAE/g). The tested extracts have shown wide-spectrum antibacterial properties and these effects have shown to be more effective against Aspergillus ochraceus, Penicillium funiculosum, Trichoderma viride, Bacillus cereus, Escherichia coli and Pseudomonas aeruginosa. The results revealed that the antioxidant, enzyme inhibitory and antimicrobial activities depended on the extraction solvents and the parts of plant. Bioinformatics analysis on the 17 major compounds showed modulation of pathway associated with cancer. In brief, U. togoensis might be valuable as potential source of natural agents for therapeutic application.

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.

The fungus Penicillium purpurogenum grows on a variety of natural carbon sources and secretes a large number of enzymes which degrade the polysaccharides present in lignocellulose. In this work, the gene coding for a novel endoxylanase has been identified in the genome of the fungus. This gene (xynd) possesses four introns. The cDNA has been expressed in Pichia pastoris and characterized. The enzyme, XynD, belongs to family 10 of the glycoside hydrolases. Mature XynD has a calculated molecular weight of 40,997. It consists of 387 amino acid residues with an N-terminal catalytic module, a linker rich in ser and thr residues, and a C-terminal family 1 carbohydrate-binding module. XynD shows the highest identity (97%) to a putative endoxylanase from Penicillium subrubescens but its highest identity to a biochemically characterized xylanase (XYND from Penicillium funiculosum) is only 68%. The enzyme has a temperature optimum of 60 °C, and it is highly stable in its pH optimum range of 6.5-8.5. XynD is the fourth biochemically characterized endoxylanase from P. purpurogenum, confirming the rich potential of this fungus for lignocellulose biodegradation. XynD, due to its wide pH optimum and stability, may be a useful enzyme in biotechnological procedures related to this biodegradation process.

The conversion path and the complex procedure of products separation elaborated for ketophosphonates and hydroxyphosphonates formed during the biotransformations of aminophoshonates are reported. Penicillium funiculosum (Thom 3), Geotrichum candidum (6593), Beauveria bassiana (271B) were applied for bioconversion of a racemic mixture of the phosphonic analogue of isoleucine (IleP), whereas P. funiculosum (Thom 3), F. oxysporum (DSM 12646) and Trigonopsis variabilis (DSM 70714) were used for transformation of the phosphonic analogue of valine (ValP). The biocatalytic processes were analysed to confirm and to compare the path and the selectivity of the biological conversion of aminophosphonates with one (ValP) and two stereogenic centers (IleP). The efforts undertaken allowed to demonstrate, that applied biocatalysts act in two steps. A racemic mixture of substrates was bioconverted via oxidative deamination with formation of ketones, which were subsequently bioreduced in the next step of the proces...

This study reports on successful photodynamic inactivation of planktonic and biofilm cells of Candida albicans using Rose Bengal (RB) in combination with biogenic gold nanoparticles synthesized by the cell-free filtrate of Penicillium funiculosum BL1 strain. Monodispersed colloidal gold nanoparticles coated with proteins were characterized by a number of techniques including SEM-EDS, TEM, UV-Vis absorption and fluorescence spectroscopy, as well as Fourier transform infrared spectroscopy to be 24 ± 3 nm spheres. A Xe lamp (output power of 20mW, delivering intensity of 53 mW cm-2 ) was used as a light source to study the effects of RB alone, the gold nanoparticles alone and the RB-gold nanoparticle mixture on the viability of C. albicans cells. The most effective reduction in the number of planktonic cells was found after 30 min of Xe lamp light irradiation (95.4 J cm-2 ) and was 4.89 log10 that is 99.99% kill for the mixture of RB with gold nanoparticles compared with 2.19 log10 or 99.37% for RB alone. The biofilm cells were more resistant to photodynamic inactivation, and the highest effective reduction in the number of cells was found after 30 min of irradiation in the presence of the RB-gold nanoparticles mixture and was 1.53 log10 , that is 97.04% kill compared with 0.6 log10 or 74.73% for RB. The probable mechanism of enhancement of RB-mediated photodynamic fungicidal efficacy against C. albicans in the presence of biogenic gold nanoparticles is discussed leading to the conclusion that this process may have a multifaceted character.

Mangrove forests exemplify a multifaceted ecosystem since they do not only play a crucial ecological role but also possess medicinal properties. Methanolic, ethyl acetate and aqueous leaf and bark extracts were prepared using homogenizer-assisted extraction (HAE), infusion and maceration (with and without stirring). The different extracts were screened for phytochemical profiling and antioxidant capacities in terms of radical scavenging (DPPH, ABTS), reducing potential (CUPRAC, FRAP), total antioxidant capacity and chelating power. Additionally, R. racemosa was evaluated for its anti-diabetic (&alpha, amylase, &alpha, glucosidase), anti-tyrosinase and anti-cholinesterase (AChE, BChE) activities. Additionally, antimycotic and antibacterial effects were investigated against Eescherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Listeria monocytogenes, Enterobacter cloacae, Bacillus cereus, Micrococcus luteus, Staphylococcus aureus, Aspergillus fumigatus, Aspergillus niger, Trichoderma viride, Penicillium funiculosum, Penicillium ludwigii and Penicillium verrucosum. Finally, based on phytochemical fingerprint, in silico studies, including bioinformatics, network pharmacology and docking approaches were conducted to predict the putative targets, namely tyrosinase, lanosterol-14-&alpha, demethylase and E. coli DNA gyrase, underlying the observed bio-pharmacological and microbiological effects. The methanolic leave and bark extracts (prepared by both HAE and maceration) abounded with phenolics, flavonoids, phenolic acids and flavonols. Results displayed that both methanolic leaf and bark extracts (prepared by HAE) exhibited the highest radical scavenging, reducing potential and total antioxidant capacity. Furthermore, our findings showed that the highest enzymatic inhibitory activity recorded was with the tyrosinase enzyme. In this context, bioinformatics analysis predicted putative interactions between tyrosinase and multiple secondary metabolites including apigenin, luteolin, vitexin, isovitexin, procyanidin B, quercetin and methoxy-trihydroxyflavone. The same compounds were also docked against lanosterol-14&alpha, demethylase and E. Coli DNA gyrase, yielding affinities in the submicromolar&ndash, micromolar range that further support the observed anti-microbial effects exerted by the extracts. In conclusion, extracts of R. racemosa may be considered as novel sources of phytoanti-oxidants and enzyme inhibitors that can be exploited as future first-line pharmacophores.

This study investigates the stress-mitigating effects of endophytic Penicillium funiculosum LHL06 on soybean roots via modulation of physio-biochemical, molecular, and proteomic responses to combined heavy metal (Ni, Cu, Pb, Cr, and Al) toxicity. Preliminary screening revealed that LHL06 can tolerate and remediate combined heavy metal contamination in its media and upregulate gibberellins (GA1, GA3, GA4, GA7 and GA9) and indole-3-acetic acid (IAA) production. Inoculation of LHL06 resulted in marked reduction of metals uptake in roots and shoots by downregulating heavy metal ATPase genes (GmHMA13, GmHMA14, GmHMA19) and GmMATE1 compared to non-inoculated plants; in turn, this decreased abscisic acid and jasmonic acid levels. Moreover, triggering of free amino acid metabolism in LHL06-inoculated roots significantly upregulated expression of stress-related proteins (glutathione S-transferase L3, isoflavone reductase-like, chalcone isomerase A, NAD(P)H dehydrogenase (quinone), FQR1-like 1 isoform X2, and Peroxidase 3) to combat metals toxicity. Compared to non-inoculated-plants, LHL06-inoculated-plants exhibited higher antioxidant activity and transcript accumulation of glutathione S-transferase (GmGST8 and GmGST3), G6PDH, and GmSOD1[Cu-Zn], which decreased metal-induced reactive oxygen species. Therefore, LHL06-inoculation remediate combined metal contamination in soil, activate signaling network of stress-responsive hormones and antioxidant systems for promoting growth and tolerance, and reduce metal-accumulation, thereby making plants safer for consumption.

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.

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.

The article deals with testing and evaluating the mould resistance of carbide lime by-product from deposits in comparison with the effect of reference conventional slaked lime. The mould resistance of limes was tested on a mixture of mould such as Gliocladium virens, Paecilomyces variotii, Penicillium funiculosum, Chaetomium globosum, and Aspergillus niger as well as evaluated according to the method stated in the technical standard STN 724310: 1993. The evaluating scale for mould resistance is from 0 up to 5 in the fungal growth degree (FGD). The FGD value 0 stands for no fungal growth comes about and construction materials and products have the microbiostatic effect against filamentous fungi. The experiments confirm the microbiostatic effect against mould of carbide lime by-product from deposits as well as reference conventional slaked lime; however, the microbicidal effect against mould has not been confirmed at all; the inhibiting area-zone-did not form on the agar growth media around the tested samples. Application of carbide lime by-product from deposits ensures increased mould resistance of construction materials with the microbiostatic effect against mould in accordance with technical standard STN 724310: 1993. Web of Science 73 10 2582 2575

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 β-glucosidase cDNA belonging to glycoside hydrolase family 1 was cloned from RNA isolated from lactose grown cellulolytic fungus, Penicillium funiculosum NCL1 by reverse-transcriptase polymerase chain reaction. The gene (bgl6) contained an open reading frame of 1470 bp that encoded 490 amino acids. bgl6 gene was overexpressed in Escherichia coli BL21-CodonPlus and the recombinant enzyme (rBgl6) was purified. The rBgl6 was a monomer with the molecular weight of 52 kDa. The enzyme was optimally active at pH 6.0, 50 °C and stable in the wide range of pH and temperature. rBgl6 showed higher activity with pNPG, pNPGal cellobiose and lactose, while it showed substantial activity on pNPX. When the substrate was pNPG, the enzyme showed glucose tolerance at a concentration of 500 mM. rBgl6 exhibited enhanced activity with metal cations Ca2+, Mn2+, Mg2+ and Zn2+, while it showed stability with DTT and EDTA. The enzyme showed Km values of 0.125 and 0.25 mM, whereas Vmax of 256 and 166 μmol/mg with pNPG and cellobiose respectively. rBgl6 possessed transglycosylation activity and it converted glucose into gentiobiose, a rare oligosaccharide with greater prebiotic value. The yield of gentiobiose was 0.57 mg/ml for 5 mg/ml of glucose. These results indicated that rBgl6 could be a valuable candidate for the enzymatic synthesis of gentiobiose.

The aim of this work was to optimize the growth conditions and continuous production of the enzyme using free and immobilized cells of inulinase by Penicillium funiculosum. The highest yield of enzyme (163.5U/mL) was obtained when the culture was incubated at 27oC and 200 rpm for 96h in a fermentation medium containing both inulin and peptone as sole carbon and nitrogen source, respectively. When the cells of the P. funiculosum were immobilized on different carriers, especially linen fibers, their production ability was successfully maintained for seven successive batches. When the fermentation was carried out using inulin juice prepared from Jerusalem artichoke tubers (in place of pure inulin), inulinase production could be sustained till the second cultivation batch of the P. funiculosum immobilized on linen fibers, yielding 122 U/mL enzyme. Results proved the feasibility of using crude inulin juice as a simple and economic carbon source for the production of inulinase.

We investigated the antimicrobial, cytotoxicity, skin irritation, and ion elution behaviors of glass doped with silver ions with respect to its application to electronic equipment such as phones and tablet screens. The microbes tested were Escherichia coli, Staphylococcus aureus, and Penicillium funiculosum. AgNO3 powder was spread on both sides of aluminosilicate glass, and it was heated to 250-280°C for 10min. Under optimized heating conditions (260°C, 10min), the antimicrobial activity of ion-exchanged glass against bacteria and fungi was over 99.9% after 24 weeks. The glass failed to irritate the skin of experimental animals and was considered non-cytotoxic. The maximum amount of Ag ions that were eluted from the ion-exchanged glass into drinking water was measured at 0.037±0.003μgL(-1), an amount which is several orders of magnitude below the standard limit of 0.1mgL(-1) in drinking water. Ag ion-exchanged glass had characteristics suitable for use as a display screen, such as a light transmittance of 90% and a surface roughness of 0.704nm. Our findings suggest that glass doped with silver ions is more hygienic than non-doped glass is, and should be applied to display screens and glassware.

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.

This work aimed to characterize compositional and bioactive features of Aloe vera leaf (fillet, mucilage, and rind) and flower. The edible fillet was analysed for its nutritional value, and all samples were studied for phenolic composition and antioxidant, anti-inflammatory, antimicrobial, tyrosinase inhibition, and cytotoxic activities. Dietary fibre (mainly mannan) and available carbohydrates (mainly free glucose and fructose) were abundant macronutrients in fillet, which also contained high amounts of malic acid (5.75 g/100 g dw) and &alpha, tocopherol (4.8 mg/100 g dw). The leaf samples presented similar phenolic profiles, with predominance of chromones and anthrones, and the highest contents were found in mucilage (131 mg/g) and rind (105 mg/g) extracts, which also revealed interesting antioxidant properties. On the other hand, the flower extract was rich in apigenin glycoside derivatives (4.48 mg/g), effective against Pseudomonas aeruginosa (MIC = 0.025 mg/mL and MBC = 0.05 mg/mL) and capable of inhibiting the tyrosinase activity (IC50 = 4.85 mg/mL). The fillet, rind, and flower extracts also showed a powerful antifungal activity against Aspergillus flavus, A. niger, Penicillium funiculosum, and Candida albicans, higher than that of ketoconazole. Thus, the studied Aloe vera samples displayed high potential to be exploited by the food or cosmetic industries, among others.

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.

The optimization of growth conditions for the production of inulinase by Penicillium funiculosum cells were studied as well as the continuous production of the enzyme using immobilized cells. The highest amount of enzyme (163.5U/mL) was obtained when the producing cells were incubated for 96 hours at 27 o C and 200 rpm in a fermentation medium containing both inulin and peptone as sole carbon and nitrogen sources respectively. However, when the cells of the tested microorganism were adsorbed on different carriers, especially linen fibers, their production ability was also successfully maintained, to different extends, for seven successive batches. Moreover, commercially pure inulin is very expensive in only small quantities, this fermentation medium was later substituted by a crude inulin solution obtained from Jerusalem artichoke tubers ( Helianthus tuberosus) . The crude inulin juice was able to sustain inulinase production during the second batch cultivation of the P. funiculosum , immobilized by their adsorption on linen fibers, in a satisfactory level of about 122U/mL. Moreover, the use of the previously mentioned crude inulin preparation was also compared to the use of either complete or minimal media, composed solely of 1% pure inulin. The method, adopted in this study for inulinase production, is simple, economic, time saving, non-toxic to the microorganism and the loaded linen pads are reusable.

Our previous studies indicated that extracellular glycoproteins produced by some white-rot and brown-rot basidiomycetous fungi reduce Fe(III) to Fe(II) and O2 to H2O2 and produce hydroxyl radicals. The continuous generation of hydroxyl radicals requires a constant supply of O2 and an electron donor for the reduction of oxidized forms of the glycoproteins to the reduced forms. However, electron donors for this reaction, such as NADH, have not been identified. In this study, the amounts of the extracellular pyridine coenzymes, NAD(＋) and NADH, were measured in agar cultures of four white-rot fungi, one brown-rot fungus, and three soft-rot fungi. The sums of NAD(＋) and NADH detected in wood-containing cultures of all five basidiomycetes were greater than those in glucose cultures. The amounts of NAD(＋) were higher than those of NADH in all wood-containing cultures except that of Irpex lacteus, and NAD(＋) was greater than NADH in all glucose cultures except that of Fomitopsis palustris. Significant amounts of pyridine coenzymes were present in glucose and wood-containing cultures of the three soft-rot fungi. The non-wood-degrading fungus, Penicillium funiculosum, did not produce NAD(＋) or NADH in either glucose or wood-containing cultures. The extracellular pyridine coenzyme levels were relatively high compared to the rates of extracellular hydroxyl radical generation in wood-degrading fungal cultures. Thus, white-, brown-, and soft-rot fungi produce pyridine coenzymes that could serve as electron donors for the production of hydroxyl radicals during wood degradation.

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.

Glucose oxidase (GOx) from Penicillium funiculosum 46.1 was purified using step-by-step ultrafiltration and it was characterized by spectrophotometric and spectrofluorometric methods. It was shown that spectra of GOx produced by P. funiculosum are typical for flavoproteins. Absorption spectrum has distinct peaks at 380 and 457 nm, excitation spectrum at 373 and 447 nm, and emission spectrum at 530 and 562 nm. The pH correlation of enzyme activity and catalytic characteristics in various buffer systems (phosphate (pH 5.0-9.0), citrate (pH 3.0-5.0), citrate-phosphate (pH 3.0-9.0), and universal (pH 3.0-9.0)) were registered. It was determined that the GOx is the most efficiently interacting with substrate (glucose) in phosphate buffer at pH 7.0 with k cat/K m = 21,825 M(-1) s(-1). Interaction of several different redox mediators (9,10-phenantroline-5,6-dione, 9,10-phenanthrenequinone, N-methylphenazonium methyl sulfate, ferrocene, ferrocenecarboxylic acid, α-methylferrocenemethanol, ferrocenecarboxaldehyde) with GOx from P. funiculosum was investigated by evaluation of the difference in fluorescence emission intensity of FAD(oxidized) and FADH2(reduced) forms. It was found that 9,10-phenantroline-5,6-dione and 9,10-phenanthrenequinone are the best redox mediators for this type of GOx.

Several fungal species namely: Aspergillus niger , Aspergillus parasiticus , Penicillium funiculosum , Trigonopsis variabilis and two different strains of Fusarium oxysporum were tested toward racemic mixtures of following phosphonic acids: 1-amino-2-methylpropanephosphonic acid, 1-aminophenylmethanephosphonic acid and 1-amino-2-phenylethanephosphonic acid. Application of F. oxysporum strain (UW1) allowed obtaining optically pure S -isomer of 1-aminophenylmethanephosphonic acid and R -isomer of 1-amino-2-phenylethanephosphonic acid, whereas biotransformation of racemic mixture of 1-amino-2-methylpropanephosphonic acid with F. oxysporum strain (DSM 12646) cells resulted in obtaining pure enantiomer of R -configuration.

The cytogenetic characterization of Centaurea solstitialis L. (Asteraceae) showed a chromosome number of 2n = 16. Karyotype is composed by four pairs of metacentric, two pairs of submetacentric and two pairs of subtelocentric chromosomes. Physical mapping of two rDNA probes revealed two loci of 35S and one locus of 5S rRNA genes. Chromomycin fluorochrome banding revealed that all rDNA loci were GC rich. The genome size (2C-value) of 1.95 pg classes this species in the group of very small genomes. Chemical composition of C. solstitialis volatile oil (VO) from Croatia, studied with gas chromatography–mass spectrometry showed dominant components as it follows: hexadecanoic acid, α-linolenic acid, germacrene D and heptacosane. Antioxidant capacity, measured by ferric reducing power assay and 2,2-diphenyl-1-picrylhydrazyl methods, as well as inhibition of acetyl- and butyrylcholinesterase of VO was lower comparing to a standard solutions. Volatile oil tested with disc diffusion method showed good inhibitory potential against Pseudomonas aeruginosa, Escherichia coli and all tested fungi: Candida albicans, Penicillium funiculosum and Aspergillus fumigatus. The microdilution method showed best activity against Chronobacter sakazakii and A. fumigatus.

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.

Hyaluronidase (HAase) activity was detected in the culture supernatants of Penicillium purpurogenum and Penicillium funiculosum. The HAase from Penicillium spp. (HAase-P) was a hyaluronate 4-glycanohydrolase, which catalyzed the endolytic hydrolysis of the β-1,4 glycosidic linkage, as do vertebrate HAases. The gene encoding HAase-P was cloned and expressed in Escherichia coli. According to homology analyses of the deduced amino acid sequences, HAase-P is not classified into any of the known HAase groups, but belongs to glycoside hydrolase family 16, which includes endo-β-1,3(4)-glucanase. Regarding the substrate specificities, no chondroitinase and glucanase activities were detected. Judging from homology analyses and enzymatic properties, HAase-P seems to be a new type of HAase.

The full-length cDNA gene that encodes an acidophilic endo-1,4-β- xylanase XynI of Aspergillus usamii E001 was amplified by rapid amplification of cDNA 3′ and 5′ ends (RACE) using the total RNA as template and then cloned onto the pUCm-T vector, followed by sequencing. The cloned cDNA is 881 bp in length including 5′ and 3′ non-encoding regions, as well as a 678 bp of open reading frame (ORF) which encodes an E001 XynI of 188 amino acid residues together with a signal peptide of 37 amino acid residues. The homologies of E001 XynI with xylanases of Aspergillus niger, Aspergillus kawachii, Emericella nidulans and Penicillium funiculosum are 97.8, 92.0, 74.6 and 60.5%, respectively. From a BLAST search result, we concluded that E001 XynI belongs to the glycoside hydrolase family 11. Its three-dimensional structure was predicted using http://swiss-model.expasy.org/on-line programs based on that of the P. funiculosum xylanase (1TE1B) from the family 11. In addition, the complete DNA gene xynI encoding E001 XynI was cloned from the genomic DNA of A. usamii E001 by conventional PCR and ligation-mediated PCR amplification. The cloned xynI is 1,206 bp in length, composed of a promoter region, a 68 bp of intron and two exons when compared with the cDNA of E001 XynI.

A multi-enzymatic system from Penicillium funiculosum displayed alpha-L-arabinofuranosidase, endo-1,4-beta-D-xylanase, beta-D-xylosidase and endo-1,3-1,4-beta-D-glucanase activities at high levels over a wide acidic pH range of 2.0 to 5.5. Moreover, the pH stability was particularly extended over the wide range of pH of 2.0 to 8.0 with endo-1,3-1,4-beta-D-glucanase and endo-1,4-beta-D-xylanase; however, alpha-L-arabinofuranosidase and beta-D-xylosidase exhibited higher stability in the pH range of 2.0 to 5.5. The results indicate that the optimal temperature of alpha-L-arabinofuranosidase (65 degrees C) and beta-D-xylosidase (70 degrees C) as well as their thermal stability were higher than those of endo-1,3-1,4-beta-D-glucanase (60 degrees C) and endo-1,4-beta-D-xylanase (50 degrees C). Although V(maxapp) of beta-D-xylosidase and endo-1,4-beta-D-xylanase was higher than that of alpha-L-arabinofuranosidase and endo-1,3-1,4-beta-D-glucanase, respectively, their catalytic efficiency was lower. High levels of ferulolyl esterase, alpha-D-galactosidase, beta-D-mannosidase and endo-1,4-beta-D-mannanase activities were also detected in the multi-enzymatic system. The overall features of the multi-enzymatic system from P. funiculosum reveal its potential for degrading and modifying plant cell walls from a variety of food and feedstuffs.

When grown on arabinoxylan as the sole carbon source, the cereal phytopathogen Fusarium graminearum expresses four xylanases. Cloning and heterologous expression of the corresponding xylanase encoding genes and analysis of general biochemical properties, substrate specificities and inhibition sensitivities revealed some marked differences. XylA and XylB are glycoside hydrolase family (GH) 11 xylanases, while XylC and XylD belong to GH10. pH and temperature for optimal activity of the enzymes were between 6.0 and 7.0 and 40 °C, respectively. Interestingly, XylC displayed remarkable pH stability as it retained most of its activity even after pre-incubation at pH 1.0 and 13.0 for 120 min at room temperature. All xylanases hydrolysed xylotetraose, xylopentaose and xylohexaose, but to different extents, while only XylC and XylD hydrolysed xylotriose. The two GH10 xylanases released a higher percentage of smaller products from xylan and xylo-oligosaccharides than did their GH11 counterparts. Analysis of kinetic properties revealed that wheat arabinoxylan is the favoured XylC substrate while XylA and XylB prefer sparsely substituted oat spelt xylan. XylC and XylD were inhibited by xylanase inhibiting protein (XIP), while XylA and XylB were sensitive to Triticum aestivum xylanase inhibitor (TAXI). Because of its pH stability and preference for arabinoxylan, XylC is a valuable candidate for use in biotechnological applications.

The crystal structure of poly(3-hydroxybutyrate) (PHB) depolymerase PhaZ7 purified from Paucimonas lemoignei was determined at 1.90 A resolution. The structure consists of a single domain with an alpha/beta hydrolase fold in its core. The active site is analogous to that of serine esterases/lipases and is characterized by the presence of a catalytic triad comprising Ser136, Asp242, and His306. Comparison with other structures in the Protein Data Bank showed a high level of similarity with the Bacillus subtilis lipase LipA (RMSD, 1.55 A). Structural comparison with Penicillium funiculosum PHB depolymerase, the only PHB depolymerase whose structure is already known, revealed significant differences, resulting in an RMSD of 2.80-3.58 A. The two enzymes appear to utilize different types of solvent-exposed residues for biopolymer binding, with aliphatic and hydroxyl residues used in P. funiculosum PHB depolymerase and aromatic residues in PhaZ7. Moreover, the active site of P. funiculosum PHB depolymerase is accessible to the substrate in contrast to the active site of PhaZ7, which is buried. Hence, considerable conformational changes are required in PhaZ7 for the creation of a channel leading to the active site. Taken together, the structural data suggest that PhaZ7 and P. funiculosum PHB depolymerase have adopted different strategies for effective substrate binding in response to their diverse substrate specificity and the lack of a substrate-binding domain.

MS/MS techniques are well customized now for proteomic analysis, even for non-sequenced organisms, since peptide sequences obtained by these methods can be matched with those found in databases from closely related sequenced organisms. We used this approach to characterize the protein content of the “Rovabio™ Excel”, an enzymatic cocktail produced by Penicillium funiculosum that is used as feed additive in animal nutrition. Protein separation by bi-dimensional electrophoresis yielded more than 100 spots, from which 37 proteins were unambiguously assigned from peptide sequences. By one-dimensional SDS-gel electrophoresis, 34 proteins were identified among which 8 were not found in the 2-DE analysis. A third method, termed ‘peptidic shotgun’, which consists in a direct treatment of the cocktail by trypsin followed by separation of the peptides on two-dimensional liquid chromatography, resulted in the identification of two additional proteins not found by the two other methods. Altogether, more than 50 proteins, among which several glycosylhydrolytic, hemicellulolytic and proteolytic enzymes, were identified by combining three separation methods in this enzymatic cocktail. This work confirmed the power of proteome analysis to explore the genome expression of a non-sequenced fungus by taking advantage of sequences from phylogenetically related filamentous fungi and pave the way for further functional analysis of P. funiculosum.

Aims: To study the growth inhibitory properties of a series of phytosphingosine (PHS) and phytoceramide (PHC) analogues. Methods and Results: A panel of two yeast (Candida albicans and Saccharomyces cerevisiae) and six moulds (Aspergillus repens, Aspergillus niger, Penicillium chrysogenum, Cladosporium cladosporioides, Arthroderma uncinatum and Penicillium funiculosum) has been used in this study. A series of new PHS and PHC analogues differing at the sphingoid backbone and the functional group at C1 position were synthesized. Conclusions: Among PHS analogues, 1-azido derivative 1c, bearing the natural d-ribo stereochemistry, showed a promising growth inhibitory profile. Among PHC analogues, compound 12, with a bulky N-pivaloyl group and a Z double bond at C3 position of the sphingoid chain, was the most active growth inhibitor. Minimal inhibitory concentration values were in the range of 23–48 μmol l−1 for 1c and 44–87 μmol l−1 for 12. Significance and Impact of the Study: Only scattered data on the antifungal activity of phytosphingolipids have been reported in the literature. This is the first time that a series of analogues of this kind are tested and compared to discern their structural requirements for antifungal activity.

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.

The rheological behaviour of cellulose fermentation broths of Penicillium funiculosum culture has been investigated. The shear stress-shear rate behaviour is explained satisfactorily by a power law model, although there appear to be small yield stresses, particularly at high substrate concentrations.

A cDNA encoding Penicillium funiculosum P(3HB) depolymerase (PhaZPfu) was cloned from a cDNA library. This cDNA contained a 1,020-bp open reading frame (ORF) that encoded 339 amino acids. Edman degradation of PhaZPfu indicated that 20 amino acids from the N terminus function as a signal peptide. Homology analysis revealed that PhaZPfu lacks linker and substrate-binding domains, both of which are observed in bacterial P(3HB) depolymerases. This may account for a weak binding affinity of PhaZPfu to the P(3HB) surface.

The quest for cheaper and better enzymes needed for the efficient hydrolysis of lignocellulosic biomass has placed filamentous fungi in the limelight for bioprospecting research. In our search for efficient biomass degraders, we identified a strain of Penicillium funiculosum whose secretome demonstrates high saccharification capabilities. Our probe into the secretome of the fungus through qualitative and label-free quantitative mass spectrometry based proteomics studies revealed a high abundance of inducible CAZymes and several nonhydrolytic accessory proteins. The preferential association of these proteins and the attending differential biomass hydrolysis gives an insight into their interactions and clues about possible roles of novel hydrolytic and nonhydrolytic proteins in the synergistic deconstruction of lignocellulosic biomass. Our study thus provides the first comprehensive insight into the repertoire of proteins present in a high-performing secretome of a hypercellulolytic Penicillium funiculosum, their relative abundance in the secretome, and the interaction dynamics of the various protein groups in the secretome. The gleanings from the stoichiometry of these interactions hold a prospect as templates in the design of cost-effective synthetic cocktails for the optimal hydrolysis of biomass.

Polymers and copolymers of horseradish root peroxidase (HRP) and Penicillium funiculosum 46.1 glucose oxidase (GO) have been synthesized and their catalytic properties have been characterized (free and immobilized forms of each enzyme were studied). The cooxidation reaction of phenol and 4-aminoantipyrin (4-AAP), performed in an aqueous medium in the presence of equimolar amounts of GO and HRP, was characterized by effective KM and kcat of 0.58 mM and 20.9 s−1 (for phenol), and 14.6 mM and 18.4 s−1 (glucose), respectively. The catalytic efficiency of polymerization products (PPs) of GO (GO-PPs) depended on the extent of their aggregation. The combinations GO + HRP-PP and HRP + GO-PP, as well as the copolymer HRP*-GO-PP, proved promising as reagents for enzyme-based analytical systems. When adsorbed on aluminum hydroxide gels, GO-PPs exhibited higher catalytic activity than the non-polymeric enzyme. Maximum retention of GO-PP activity on the inorganic carrier was observed in the case of GO-PP copolymers with an activated HRP. Polymerization of HRP in the presence of a zinc hydroxide gel, paralleled by HRP-PP immobilization onto the gel, increased both the activity of the enzyme and its operational stability.

Different methods of immobilization of extracellular glucose oxidase (GO) from Penicillium funiculosum 46.1 on gels of aluminum or zinc hydroxides have been compared. GO from the culture liquid filtrate (CLF) associated with Zn(OH)2 but not Al(OH)3 gels. Preparation of samples of immobilized GO does not require isolation of the enzyme (CLF may be used). GO immobilized on Zn(OH)2 gels from CLF was 1.6 times more efficient in catalyzing D-glucose oxidation than the enzyme contained in the original culture liquid. Crosslinking of gel-adsorbed CLF proteins affected the properties of GO adversely and to a considerable extent. Various means of polymerization and immobilization of GO isolated from CLF have been studied. Optimum results were obtained when GO polymeric products were pre-synthesized in solution, followed by adsorption to Al(OH)3 but not Zn(OH)2 gels. The catalytic efficiency of GO immobilized on a Zn(OH)2 gel was significantly lower than that of the enzyme associated with Al(OH)3.

Pen II [(2S,2' R,3R,3' E,4E,8E)-1-O-(β-D-glucopyranosyl)-N-(2'-hydroxy-3'-octadecenoyl)-9-methyl-4,8-sphingadienine], the major component of the cerebrosides isolated from Penicillium funiculosum A-1 as the fruiting inducer against Schizophyllum commune, was synthesized by starting from D-glucose, L-serine, (2R,3E)-2-(tert-butyldiphenylsilyloxy)-3-octadecenoic acid, and (E)-6-methyl-5-pentadecen-1-yne.

Filamentous fungi produce a range of carbohydrate-degrading enzymes, in particular hemicellulases, that are often exploited by the baking, brewing and feed industries. Controlled xylan degradation is a key component in several industrial processes, in which the efficiency of xylanases (endo-1,4-β-D-xylan hydrolases) depends on both their substrate specificity and their susceptibility to inhibitors present in the plant material. The three major xylanases present in a commercial hemicellulase product derived from a Penicillium funiculosum fermentation broth were characterized with respect to their substrate specificity and interaction with three known xylanase inhibitor proteins (XIP-I, TAXI I and TAXI II) present in wheat flour. XYNA (48kDa) and XYNB (22kDa) have significant sequence similarity with family 7 cellobiohydrolases and family 11 xylanases respectively (previous studies), while XYND (36kDa) has similarity with family 10 xylanases (this paper). All three enzymes hydrolysed various heteroxylan substrates, including birchwood xylan and soluble wheat arabinoxylan, at 30 ◦ C and pH 5.5. XYNA was strongly inhibited by XIP-I, TAXI I and TAXI II (Ki = 106, 46 and 46nM respectively); XYNB was only significantly inhibited by TAXI I (Ki = 165nM). In each case the inhibition of XYNA and XYNB was competitive. XYND was only inhibited by XIP-I but, in contrast to XYNA and XYNB, the apparent inhibition was dependent on the order that xylanase, inhibitor and substrate were added to the incubation mixture; the inhibition was weak when incubations were initiated by addition of xylanase (to a mixture of substrate and XIP-I), but was very strong when enzyme and XIP-I were pre-incubated before addition of substrate. These findings demonstrate that the susceptibility of individual xylanases to inhibitor proteins present in cereals is likely to be a critical factor in determining their efficiency in various commercial applications.  2004 Society of Chemical Industry

The regioselectivity of 20 extracellular β- N -acetylhexosaminidases of fungal origin was screened in the reverse hydrolysis with 2-acetamido-2-deoxy- d -glucopyranose. Most of the enzymes used yielded 2-acetamido-2-deoxy-β- d -glucopyranosyl-(1→4)-2-acetamido-2-deoxy- d -glucopyranose ( 3 ) and 2-acetamido-2-deoxy-β- d -glucopyranosyl-(1→6)-2-acetamido-2-deoxy- d -glucopyranose ( 4 ). So far unknown product of enzymatic condensation, 2-acetamido-2-deoxy-β- d -glucopyranosyl-(1→3)-2-acetamido-2-deoxy- d -glucopyranose ( 2 ) was synthesised using the β- N -acetylhexosaminidases from Penicillium funiculosum CCF 1994, P. funiculosum CCF 2325 and Aspergillus tamarii CCF 1665. Addition of salts ((NH 4 ) 2 SO 4 or MgSO 4 (0.1–1.0 M)) to the reaction increased the yields and also enhanced the β- N -acetylhexosaminidase regioselectivity.

The main parameters of growth and glucose oxidase production by the mutant Penicillium funiculosum strains BIM F-15.3, NMM95.132, and 46.1 were studied. The synthesis of extracellular glucose oxidase by these strains was constitutive and occurred following the phase of exponential growth. The mutant strains also synthesized extracellular invertase and cell-associated catalase and glucose oxidase. The syntheses of invertase, the cell-associated enzymes, and extracellular glucose oxidase were found to be maximum between 14 and 18 h, between 48 and 52 h, and by the 96th hour of cultivation, respectively. Among the mutants studied, P. funiculosum 46.1 showed the maximal rates of growth and glucose oxidase synthesis.

A procedure for purification of extracellular glucose oxidase (GO, EC 1.1.3.4) from a filtrate of culture liquid (CLF) of the fungus Penicillium funiculosum 46.1 has been developed using alluvial quartz sand as an adsorbent. Modifying the sand by changing the charge and polarity did not lead to a significant increase in its adsorption capacity towards GO. The effectiveness of sand and aluminum oxide used as adsorbents for GO isolation from CLF has been compared. Glucose oxidase isolated from CLF by adsorption on sand exhibited a greater catalytic activity than enzyme preparations obtained by column chromatography on CLF. Glucose oxidase from P. funiculosum 46.1 was adsorbed on sand more effectively than on aluminum oxide. It is concluded that sand may be used for fractionation of partially purified GO.