Dataset Information

A Thermostable Aspergillus fumigatus GH7 Endoglucanase Over-Expressed in Pichia pastoris Stimulates Lignocellulosic Biomass Hydrolysis.

ABSTRACT: In the context of avoiding the use of non-renewable energy sources, employing lignocellulosic biomass for ethanol production remains a challenge. Cellulases play an important role in this scenario: they are some of the most important industrial enzymes that can hydrolyze lignocellulose. This study aims to improve on the characterization of a thermostable Aspergillus fumigatus endo-1,4-?-glucanase GH7 (Af-EGL7). To this end, Af-EGL7 was successfully expressed in Pichia pastoris X-33. The kinetic parameters K_m and V_max were estimated and suggested a robust enzyme. The recombinant protein was highly stable within an extreme pH range (3.0-8.0) and was highly thermostable at 55 °C for 72 h. Low Cu²⁺ concentrations (0.1-1.0 mM) stimulated Af-EGL7 activity up to 117%. Af-EGL7 was tolerant to inhibition by products, such as glucose and cellobiose. Glucose at 50 mM did not inhibit Af-EGL7 activity, whereas 50 mM cellobiose inhibited Af-EGL7 activity by just 35%. Additionally, the Celluclast^® 1.5L cocktail supplemented with Af-EGL7 provided improved hydrolysis of sugarcane bagasse "in natura", sugarcane exploded bagasse (SEB), corncob, rice straw, and bean straw. In conclusion, the novel characterization of Af-EGL7 conducted in this study highlights the extraordinary properties that make Af-EGL7 a promising candidate for industrial applications.

SUBMITTER: Vianna Bernardi A

PROVIDER: S-EPMC6540056 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

A Thermostable Aspergillus fumigatus GH7 Endoglucanase Over-Expressed in Pichia pastoris Stimulates Lignocellulosic Biomass Hydrolysis.

Vianna Bernardi Aline A Kimie Yonamine Deborah D Akira Uyemura Sergio S Magnani Dinamarco Taisa T

International journal of molecular sciences 20190507 9

In the context of avoiding the use of non-renewable energy sources, employing lignocellulosic biomass for ethanol production remains a challenge. Cellulases play an important role in this scenario: they are some of the most important industrial enzymes that can hydrolyze lignocellulose. This study aims to improve on the characterization of a thermostable Aspergillus fumigatus endo-1,4-β-glucanase GH7 (Af-EGL7). To this end, Af-EGL7 was successfully expressed in Pichia pas ...[more]

PMID: 31067833

Similar Datasets

Project description:BACKGROUND: Recently, the increased demand of energy has strongly stimulated the research on the conversion of lignocellulosic biomass into reducing sugars for the subsequent production, and ?-glucosidases have been the focus because of their important roles in a variety fundamental biological processes and the synthesis of useful ?-glucosides. Although the ?-glucosidases of different sources have been investigated, the amount of ?-glucosidases are insufficient for effective conversion of cellulose. The goal of this work was to search for new resources of ?-glucosidases, which was thermostable and with high catalytic efficiency. RESULTS: In this study, a thermostable native ?-glucosidase (nBgl3), which is secreted by the lignocellulose-decomposing fungus Aspergillus fumigatus Z5, was purified to electrophoretic homogeneity. Internal sequences of nBgl3 were obtained by LC-MS/MS, and its encoding gene, bgl3, was cloned based on the peptide sequences obtained from the LC-MS/MS results. bgl3 contains an open reading frame (ORF) of 2622 bp and encodes a protein with a predicted molecular weight of 91.47 kDa; amino acid sequence analysis of the deduced protein indicated that nBgl3 is a member of the glycoside hydrolase family 3. A recombinant ?-glucosidase (rBgl3) was obtained by the functional expression of bgl3 in Pichia pastoris X33. Several biochemical properties of purified nBgl3 and rBgl3 were determined - both enzymes showed optimal activity at pH 6.0 and 60°C, and they were stable for a pH range of 4-7 and a temperature range of 50 to 70°C. Of the substrates tested, nBgl3 and rBgl3 displayed the highest activity toward 4-Nitrophenyl-?-D-glucopyranoside (pNPG), with specific activities of 103.5 ± 7.1 and 101.7 ± 5.2 U mg-1, respectively. However, these enzymes were inactive toward carboxymethyl cellulose, lactose and xylan. CONCLUSIONS: An native ?-glucosidase nBgl3 was purified to electrophoretic homogeneity from the crude extract of A. fumigatus Z5. The gene bgl3 was cloned based on the internal sequences of nBgl3 obtained from the LC-MS/MS results, and the gene bgl3 was expressed in Pichia pastoris X33. The results of various biochemical properties of two enzymes including specific activity, pH stability, thermostability, and kinetic properties (Km and Vmax) indicated that they had no significant differences.

Project description:Background:Liquid hot water (LHW) pretreatment has been considered as one of the most industrially viable and environment-friendly methods for facilitating the transformation of lignocelluloses into biofuels through biological conversion. However, lignin fragments in pretreatment hydrolysates are preferential to condense with each other and then deposit back onto cellulose surface under severe conditions. Particularly, lignin tends to relocate or redistribute under high-temperature LHW pretreatment conditions. The lignin residues on the cellulose surface would result in significant nonproductive binding of cellulolytic enzymes, and therefore negatively affect the enzymatic conversion (EC) of glucan in pretreated substrates. Although additives such as bovine serum albumin (BSA) and Tween series have been used to reduce nonproductive binding of enzymes through blocking the lignin, the high cost or non-biocompatibility of these additives limits their potential in industrial applications. Results:Here, we firstly report that a soluble soy protein (SP) extracted from inexpensive defatted soy powder (DSP) showed excellent performance in promoting the EC of glucan in LHW-pretreated lignocellulosic substrates. The addition of the SP (80 mg/g glucan) could readily reduce the cellulase (Celluclast 1.5 L®) loading by 8 times from 96.7 to 12.1 mg protein/g glucan and achieve a glucan EC of 80% at a hydrolysis time of 72 h. With the same cellulase (Celluclast 1.5 L®) loading (24.2 mg protein/g glucan), the ECs of glucan in LHW-pretreated bamboo, eucalyptus, and Masson pine substrates increased from 57%, 54% and 45% (without SP) to 87%, 94% and 86% (with 80 mg SP/g glucan), respectively. Similar effects were also observed when Cellic CTec2, a newer-generation cellulase preparation, was used. Mechanistic studies indicated that the adsorption of soluble SP onto the surface of lignin residues could reduce the nonproductive binding of cellulolytic enzymes to lignin. The cost of the SP required for effective promotion would be equivalent to the cost of 2.9 mg cellulase (Celluclast 1.5 L®) protein (or 1.2 FPU/g glucan), if a proposed semi-simultaneous saccharification and fermentation (semi-SSF) model was used. Conclusions:Near-complete saccharification of glucan in LHW-pretreated lignocellulosic substrates could be achieved with the addition of the inexpensive and biocompatible SP additive extracted from DSP. This simple but remarkably effective technique could readily contribute to improving the economics of the cellulosic biorefinery industry.

Project description:BACKGROUND:Lignocellulosic biomass, is a great resource for the production of bio-energy and bio-based material since it is largely abundant, inexpensive and renewable. The requirement of new energy sources has led to a wide search for novel effective enzymes to improve the exploitation of lignocellulose, among which the importance of thermostable and halotolerant cellulase enzymes with high pH performance is significant. RESULTS:The primary aim of this study was to discover a novel alkali-thermostable endo-?-1,4-glucanase from the sheep rumen metagenome. At first, the multi-step in-silico screening approach was utilized to find primary candidate enzymes with superior properties. Among the computationally selected candidates, PersiCel4 was found and subjected to cloning, expression, and purification followed by functional and structural characterization. The enzymes' kinetic parameters, including Vmax, Km, and specific activity, were calculated. The PersiCel4 demonstrated its optimum activity at pH?8.5 and a temperature of 85?°C and was able to retain more than 70% of its activity after 150?h of storage at 85?°C. Furthermore, this enzyme was able to maintain its catalytic activity in the presence of different concentrations of NaCl and several metal ions contains Mg2+, Mn2+, Cu2+, Fe2+ and Ca2+. Our results showed that treatment with MnCl2 could enhance the enzyme's activity by 78%. PersiCel4 was ultimately used for enzymatic hydrolysis of autoclave pretreated rice straw, the most abundant agricultural waste with rich cellulose content. In autoclave treated rice straw, enzymatic hydrolysis with the PersiCel4 increased the release of reducing sugar up to 260% after 72?h in the harsh condition (T?=?85?°C, pH?=?8.5). CONCLUSION:Considering the urgent demand for stable cellulases that are operational on extreme temperature and pH conditions and due to several proposed distinctive characteristics of PersiCel4, it can be used in the harsh condition for bioconversion of lignocellulosic biomass.

Project description:BackgroundAspergillus niger, along with many other lignocellulolytic fungi, has been widely used as a commercial workhorse for cellulase production. A fungal cellulase system generally includes three major classes of enzymes i.e., β-glucosidases, endoglucanases and cellobiohydrolases. Cellobiohydrolases (CBH) are vital to the degradation of crystalline cellulose present in lignocellulosic biomass. However, A. niger naturally secretes low levels of CBH. Hence, recombinant production of A. niger CBH is desirable to increase CBH production yield and also to allow biochemical characterisation of the recombinant CBH from A. niger.MethodsIn this study, the gene encoding a cellobiohydrolase B (cbhB) from A. niger ATCC 10574 was cloned and expressed in the methylotrophic yeast Pichia pastoris X-33. The recombinant CBHB was purified and characterised to study its biochemical and kinetic characteristics. To evaluate the potential of CBHB in assisting biomass conversion, CBHB was supplemented into a commercial cellulase preparation (Cellic® CTec2) and was used to hydrolyse oil palm empty fruit bunch (OPEFB), one of the most abundant lignocellulosic waste from the palm oil industry. To attain maximum saccharification, enzyme loadings were optimised by response surface methodology and the optimum point was validated experimentally. Hydrolysed OPEFB samples were analysed using attenuated total reflectance FTIR spectroscopy (ATR-FTIR) to screen for any compositional changes upon enzymatic treatment.ResultsRecombinant CBHB was over-expressed as a hyperglycosylated protein attached to N-glycans. CBHB was enzymatically active towards soluble substrates such as 4-methylumbelliferyl-β-D-cellobioside (MUC), p-nitrophenyl-cellobioside (pNPC) and p-nitrophenyl-cellobiotrioside (pNPG3) but was not active towards crystalline substrates like Avicel® and Sigmacell cellulose. Characterisation of purified CBHB using MUC as the model substrate revealed that optimum catalysis occurred at 50 °C and pH 4 but the enzyme was stable between pH 3 to 10 and 30 to 80 °C. Although CBHB on its own was unable to digest crystalline substrates, supplementation of CBHB (0.37%) with Cellic® CTec2 (30%) increased saccharification of OPEFB by 27%. Compositional analyses of the treated OPEFB samples revealed that CBHB supplementation reduced peak intensities of both crystalline cellulose Iα and Iβ in the treated OPEFB samples.DiscussionSince CBHB alone was inactive against crystalline cellulose, these data suggested that it might work synergistically with other components of Cellic® CTec2. CBHB supplements were desirable as they further increased hydrolysis of OPEFB when the performance of Cellic® CTec2 was theoretically capped at an enzyme loading of 34% in this study. Hence, A. niger CBHB was identified as a potential supplementary enzyme for the enzymatic hydrolysis of OPEFB.

Project description:BackgroundFilamentous fungi are among the most powerful cellulolytic organisms in terrestrial ecosystems. To perform the degradation of lignocellulosic substrates, these microorganisms employ both hydrolytic and oxidative mechanisms that involve the secretion and synergism of a wide variety of enzymes. Interactions between these enzymes occur on the level of saccharification, i.e., the release of neutral and oxidized products, but sometimes also reflected in the substrate liquefaction. Although the synergism regarding the yield of neutral sugars has been extensively studied, further studies should focus on the oxidized sugars, as well as the effect of enzyme combinations on the viscosity properties of the substrates.ResultsIn the present study, the heterologous expression of an endoglucanase (EG) and its combined activity together with a lytic polysaccharide monooxygenase (LPMO), both from the thermophilic fungus Myceliophthora thermophila, are described. The EG gene, belonging to the glycoside hydrolase family 5, was functionally expressed in the methylotrophic yeast Pichia pastoris. The produced MtEG5A (75 kDa) featured remarkable thermal stability and showed high specific activity on microcrystalline cellulose compared to CMC, which is indicative of its processivity properties. The enzyme was capable of releasing high amounts of cellobiose from wheat straw, birch, and spruce biomass. Addition of MtLPMO9 together with MtEG5A showed enhanced enzymatic hydrolysis yields against regenerated amorphous cellulose (PASC) by improving the release not only of the neutral but also of the oxidized sugars. Assessment of activity of MtEG5A on the reduction of viscosity of PASC and pretreated wheat straw using dynamic viscosity measurements revealed that the enzyme is able to perform liquefaction of the model substrate and the natural lignocellulosic material, while when added together with MtLPMO9, no further synergistic effect was observed.ConclusionsThe endoglucanase MtEG5A from the thermophilic fungus M. thermophila exhibited excellent properties that render it a suitable candidate for use in biotechnological applications. Its strong synergism with LPMO was reflected in sugars release, but not in substrate viscosity reduction. Based on the level of oxidative sugar formation, this is the first indication of synergy between LPMO and EG reported.

Dataset Information

A Thermostable Aspergillus fumigatus GH7 Endoglucanase Over-Expressed in Pichia pastoris Stimulates Lignocellulosic Biomass Hydrolysis.

Publications

A Thermostable <i>Aspergillus fumigatus</i> GH7 Endoglucanase Over-Expressed in <i>Pichia pastoris</i> Stimulates Lignocellulosic Biomass Hydrolysis.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets