Background
Sugarcane bagasse (SCB) is an abundant feedstock for second-generation bioethanol production. This complex biomass requires an array of carbohydrate active enzymes (CAZymes), mostly from filamentous fungi, for its deconstruction to monomeric sugars for the production of value-added fuels and chemicals. In this study, we evaluated the repertoire of proteins in the secretome of a catabolite repressor-deficient strain of Penicillium funiculosum, PfMig188, in response to SCB induction and examined their role in the saccharification of SCB.
Results
A systematic approach was developed for the cultivation of the fungus with the aim of producing and understanding arrays of enzymes tailored for saccharification of SCB. To achieve this, the fungus was grown in media supplemented with different concentrations of pretreated SCB (0–45 g/L). The profile of secreted proteins was characterized by enzyme activity assays and liquid chromatography–tandem mass spectrometry (LC–MS/MS). A total of 280 proteins were identified in the secretome of PfMig188, 46% of them being clearly identified as CAZymes. Modulation of the cultivation media with SCB up to 15 g/L led to sequential enhancement in the secretion of hemicellulases and cell wall-modifying enzymes, including endo-β-1,3(4)-glucanase (GH16), endo-α-1,3-glucanase (GH71), xylanase (GH30), β-xylosidase (GH5), β-1,3-galactosidase (GH43) and cutinase (CE5). There was ~ 122% and 60% increases in β-xylosidase and cutinase activities, respectively. There was also a 36% increase in activities towards mixed-linked glucans. Induction of these enzymes in the secretome improved the saccharification performance to 98% (~ 20% increase over control), suggesting their synergy with core cellulases in accessing the recalcitrant region of SCB.
Conclusion
Our findings provide an insight into the enzyme system of PfMig188 for degradation of complex biomass such as SCB and highlight the importance of adding SCB to the culture medium to optimize the secretion of enzymes specific for the saccharification of sugarcane bagasse.
The antidiabetic potentials of Heliotropium indicum L. leaf aqueous (HILA) extract used for the management of diabetes by Traditional Medicinal Practitioners (TMPs) in Nigeria was assessed. Alloxan (ALX)-induced hyperglycaemic rats were orally administered with known folkloric dosage of 30 and 75 mg/kg b. wt. of HILA extract, once a day, for 14 days. Fasting blood glucose (FBG) levels were monitored and pancreatic histology was examined. Net hepatic glycogen (GLY) concentration and lipid profiles were also determined. Prior to treatment, ALX-induced hyperglycaemia (>250 mg/dL) was established in rats. Oral administration of 30 and 75 mg/kg b. wt. HILA extract to diabetic rats for 14 days caused significant reduction in FBG to baseline values observed in non-diabetic conditions. Treatment with HILA extract also showed improvement in lipid abnormalities observed in hyperglycaemic condition, levels of triglyceride, total cholesterol and LDL-cholesterol were significantly reduced and HDL-cholesterol increased resulting in improved artherogenic index. Hepatic GLY concentration was significantly increased in diabetic rat treated with the extract. Histological examinations showed degenerated and sparse pancreatic islets β-cells in non-treated diabetic rat, whereas microscopy of treated rats showed mild to normal architecture with enriched β-cells. Preliminary phytochemical profiling of the extract revealed the presence of alkaloids (2.54 mg/g), saponins (0.28 mg/g), phenols (0.04 mg/g) and anthraquinones (0.01 mg/g). Results from this study revealed that the aqueous leaf extract of H. indicum possesses not only antihyperglycaemic, but also antidyslipidemic activities, that may prove to be of clinical importance in the management of diabetes and associated secondary complications.
The aim of this research work is to evaluate the pharmacological potentials of the mushroom Ganoderma lucidium grown in Federal Capital Territory Abuja, Nigeria. Phytochemical, antioxidant and antibacterial properties of ethanolic extract were carried out. The phytochemical analysis of the ethanolic extract revealed the presence of pharmacological constituents such as: steroids, triterpenoid, carbohydrate, cardiac glycosides and glycosides .The antibacterial activity of the extract was conducted using well agar diffusion method on four clinical bacterial isolate which are identified using various standard biochemical tests. The extract shows activity on: Escherichia coli (12mm), Klebsiella pneumonia (12mm), Proteus mirabilis (13mm) and Streptococcus spp (14mm) at 1000mg/ml respectively .The antioxidant potentiality of the extract was also evaluated using the stable radical 1,1- Diphenyl-l-pienyl hydroxyl (DPPH) and the IC50 of the standards and extract was obtained at 0.06, 0.13, and 0.23 respectively.
Enzymatic lignocellulosic biomass conversion to bioethanol is dependent on efficient enzyme systems with β-glucosidase as a key component. In this study, we performed in-depth profiling of the various β-glucosidases present in the genome of the hypercellulolytic fungus;Penicillium funiculosumusing genomics, transcriptomics, proteomics and molecular dynamics simulation approaches. Of the eight β-glucosidase genes identified in theP. funiculosumgenome, three were found to be extracellular, as evidenced by presence of signal peptides and mass spectrometry. Among the three secreted β-glucosidase, two belonged to the GH3 and one belonged to GH1 families. Modelled structures of these proteins predicted a deep and narrow active site for the GH3 β-glucosidases (PfBgl3A andPfBgl3B) and a shallow open active site for the GH1 β-glucosidase (PfBgl1A). The enzymatic assays indicated thatP. funiculosumsecretome showed high β-glucosidase activities with prominent bands on 4-methylumbelliferyl β-D-glucopyranoside (MUG) zymogram. To understand the contributory effect of each of the three secreted β-glucosidases (PfBgls), the corresponding gene was deleted separately and the effect of the deletion on β-glucosidase activity of the secretome was examined. Although not the most abundant β-glucosidase,PfBgl3A was found to be the most significant one as evidenced by a 42 % reduction in β-glucosidase activity in the ΔPfBgl3A strain. To improve the thermostability, two mutants ofPfBgl3A were designed with the help of molecular dynamics (MD) simulation and were expressed in Pichia pastoris for evaluation. ThePfBgl3A mutant (Mutant A) gave 1.4 fold increase in the half-life (T1/2) of the enzyme at 50 °C.
The production of second-generation fuels from lignocellulosic residues such as sugarcane bagasse (SCB) requires the synergistic interaction of key cellulose-degrading enzymes and accessory proteins for their complete deconstruction to useful monomeric sugars. Here, we recombinantly expressed and characterized unknown GH5 xylanase from P. funiculosum (PfXyn5) in Pichia pastoris, which was earlier found in our study to be highly implicated in SCB saccharification. The PfXyn5 has a molecular mass of ~ 55 kDa and showed broad activity against a range of substrates like xylan, xyloglucan, laminarin and p-nitrophenyl-β-d-xylopyranoside, with the highest specific activity of 0.7 U/mg against xylan at pH 4.5 and 50 °C. Analysis of the degradation products of xylan and SCB by PfXyn5 showed significant production of xylooligosaccharides (XOS) with a degree of polymerization (DP) ranging from two (DP2) to six (DP6), thus, suggesting that the PfXyn5 is an endo-acting enzyme. The enzyme synergistically improved the saccharification of SCB when combined with the crude cellulase cocktail of P. funiculosum with a degree of synergism up to 1.32. The PfXyn5 was further expressed individually and simultaneously with a notable GH16 endoglucanase (PfEgl16) in a catabolite-derepressed strain of P. funiculosum, PfMig188, and the saccharification efficiency of the secretomes from the resulting transformants were investigated on SCB. The secretome of PfMig188 overexpressing Xyn5 or Egl16 increased the saccharification of SCB by 9% or 7%, respectively, over the secretome of PfMig188, while the secretome of dual transformant increased SCB saccharification by ~ 15% at the same minimal protein concentration.
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