scholarly journals LPMO AfAA9_B and Cellobiohydrolase AfCel6A from A. fumigatus Bost Enzymatic Saccharification Activity of Cellulase Cocktail

2020 ◽  
Author(s):  
Taisa Magnani Dinamarco ◽  
Aline Vianna Bernardi ◽  
Luis Eduardo Gerolamo ◽  
Paula Fagundes de Gouvêa ◽  
Deborah Kimie Yonamine ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Lignocellulosic Biomass ◽  
Filamentous Fungi ◽  
Biochemical Parameters ◽  
Enzymatic Saccharification ◽  
Fermentable Sugars ◽  
Optimal Temperature ◽  
High Turnover ◽  
Lytic Polysaccharide Monooxygenase ◽  
Polysaccharide Monooxygenase

Cellulose is the most abundant polysaccharide in lignocellulosic biomass, where it is interlinked with lignin and hemicellulose. Bioethanol can be produced from biomass. Because breaking down biomass is difficult, cellulose-active enzymes secreted by filamentous fungi play an important role in degrading recalcitrant lignocellulosic biomass. We characterized a cellobiohydrolase (AfCel6A) and lytic polysaccharide monooxygenase LPMO (AfAA9_B) from A. fumigatus after they were expressed in Pichia pastoris and purified. The biochemical parameters suggested that the enzymes were stable; the optimal temperature was ~60 °C. Further characterization revealed high turnover numbers (kcat of 147.9 s-1 and 0.64 s-1, respectively). Surprisingly, when combined, AfCel6A and AfAA9_B did not act synergistically. Association of AfCel6A and AfAA9_B inhibits the activity of AfCel6A, an outcome that needs to be further investigated. However, addition of AfCel6A or AfAA9_B boosts the enzymatic saccharification activity of a cellulase cocktail and the activity of cellulase Af-EGL7. The supplementation of an enzymatic cocktail with AfCel6A or AfAA9_B boosted the yield of fermentable sugars from complex substrates, especially sugarcane exploded bagasse, by up to 95%. The synergism between the cellulase cocktail and AfAA9_B is enzyme- and substrate-specific, which suggests a specific enzymatic cocktail for each biomass.

scholarly journals LPMO AfAA9_B and Cellobiohydrolase AfCel6A from A. fumigatus Boost Enzymatic Saccharification Activity of Cellulase Cocktail

2020 ◽  
Vol 22 (1) ◽  
pp. 276
Author(s):  
Aline Vianna Bernardi ◽  
Luis Eduardo Gerolamo ◽  
Paula Fagundes de Gouvêa ◽  
Deborah Kimie Yonamine ◽  
Lucas Matheus Soares Pereira ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Aspergillus Fumigatus ◽  
Lignocellulosic Biomass ◽  
Biochemical Parameters ◽  
Enzymatic Saccharification ◽  
Fermentable Sugars ◽  
Optimal Temperature ◽  
High Turnover ◽  
Lytic Polysaccharide Monooxygenase ◽  
Polysaccharide Monooxygenase

Cellulose is the most abundant polysaccharide in lignocellulosic biomass, where it is interlinked with lignin and hemicellulose. Bioethanol can be produced from biomass. Since breaking down biomass is difficult, cellulose-active enzymes secreted by filamentous fungi play an important role in degrading recalcitrant lignocellulosic biomass. We characterized a cellobiohydrolase (AfCel6A) and lytic polysaccharide monooxygenase LPMO (AfAA9_B) from Aspergillus fumigatus after they were expressed in Pichia pastoris and purified. The biochemical parameters suggested that the enzymes were stable; the optimal temperature was ~60 °C. Further characterization revealed high turnover numbers (kcat of 147.9 s−1 and 0.64 s−1, respectively). Surprisingly, when combined, AfCel6A and AfAA9_B did not act synergistically. AfCel6A and AfAA9_B association inhibited AfCel6A activity, an outcome that needs to be further investigated. However, AfCel6A or AfAA9_B addition boosted the enzymatic saccharification activity of a cellulase cocktail and the activity of cellulase Af-EGL7. Enzymatic cocktail supplementation with AfCel6A or AfAA9_B boosted the yield of fermentable sugars from complex substrates, especially sugarcane exploded bagasse, by up to 95%. The synergism between the cellulase cocktail and AfAA9_B was enzyme- and substrate-specific, which suggests a specific enzymatic cocktail for each biomass by up to 95%. The synergism between the cellulase cocktail and AfAA9_B was enzyme- and substrate-specific, which suggests a specific enzymatic cocktail for each biomass.

scholarly journals Eco-friendly additives in acidic pretreatment to boost enzymatic saccharification of hardwood for sustainable biorefinery applications

2021 ◽  
Author(s):  
Qiulu Chu ◽  
Wenyao Tong ◽  
Shufang Wu ◽  
Yongcan Jin ◽  
Jinguang Hu ◽  
...  
Keyword(s):  
Lignocellulosic Biomass ◽  
Enzymatic Saccharification ◽  
Fermentable Sugars ◽  
Acidic Pretreatment

Pretreatment of renewable lignocellulosic biomass is essential to produce fermentable sugars and biofuels in a sustainable biorefinery. However, lignin repolymerization during pretreatment was reported to intensify the lignin inhibition on...

scholarly journals Biochemical, structural insights of newly isolated AA16 family of Lytic Polysaccharide Monooxygenase (LPMO) from Aspergillus fumigatus and investigation of its synergistic effect using biomass.

2020 ◽  
Author(s):  
Musaddique Hossain ◽  
Subba Reddy Dodda ◽  
Bishwajit Singh Kapoor ◽  
Kaustav Aikat ◽  
sudit mukhopadhyay
Keyword(s):  
Aspergillus Fumigatus ◽  
Lignocellulosic Biomass ◽  
Three Dimensional ◽  
Cellulose Hydrolysis ◽  
Inducible Promoter ◽  
Dimensional Structure ◽  
Biomass Hydrolysis ◽  
Lytic Polysaccharide Monooxygenase ◽  
Intronless Gene ◽  
Polysaccharide Monooxygenase

The efficient conversion of lignocellulosic biomass into fermentable sugar is a bottleneck for the cheap production of bio-ethanol. The recently identified enzyme Lytic Polysaccharide Monooxygenase (LPMO) family has brought new hope because of its boosting capabilities of cellulose hydrolysis. In this report, we have identified and characterized a new class of auxiliary (AA16) oxidative enzyme LPMO from the genome of a locally isolated thermophilic fungus Aspergillus fumigatus (NITDGPKA3) and evaluated its boosting capacity of biomass hydrolysis. The AfLPMO16 is an intronless gene and encodes the 29kDa protein. While Sequence-wise, it is close to the C1 type of AaAA16 and cellulose-active AA10 family of LPMOs, but the predicted three-dimensional structure shows the resemblance with the AA11 family of LPMO (PDB Id: 4MAH). The gene was expressed under an inducible promoter (AOX1) with C-terminal His tag in the Pichia pastoris. The protein was purified using Ni-NTA affinity chromatography, and we studied the enzyme kinetics with 2,6-dimethoxyphenol. We observed polysaccharides depolymerization activity with Carboxymethyl cellulose (CMC) and Phosphoric acid swollen cellulose (PASC). Moreover, the simultaneous use of cellulase cocktail (commercial) and AfLPMO16 enhances lignocellulosic biomass hydrolysis by 2-fold, which is highest so far reported in the LPMO family.

scholarly journals Bioreactor and Bioprocess Design Issues in Enzymatic Hydrolysis of Lignocellulosic Biomass

Catalysts ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 680
Author(s):  
Giuseppe Olivieri ◽  
René H. Wijffels ◽  
Antonio Marzocchella ◽  
Maria Elena Russo
Keyword(s):  
Lignocellulosic Biomass ◽  
Enzymatic Saccharification ◽  
Product Inhibition ◽  
Cellulolytic Enzymes ◽  
Main Process ◽  
Bioprocess Design ◽  
Operating Modes ◽  
Biomass Saccharification ◽  
Starting Point ◽  
The Impact

Saccharification of lignocellulosic biomass is a fundamental step in the biorefinery of second generation feedstock. The physicochemical and enzymatic processes for the depolymerization of biomass into simple sugars has been achieved through numerous studies in several disciplines. The present review discusses the development of technologies for enzymatic saccharification in industrial processes. The kinetics of cellulolytic enzymes involved in polysaccharide hydrolysis has been discussed as the starting point for the design of the most promising bioreactor configurations. The main process configurations—proposed so far—for biomass saccharification have been analyzed. Attention was paid to bioreactor configurations, operating modes and possible integrations of this operation within the biorefinery. The focus is on minimizing the effects of product inhibition on enzymes, maximizing yields and concentration of sugars in the hydrolysate, and reducing the impact of enzyme cost on the whole process. The last part of the review is focused on an emerging process based on the catalytic action of laccase applied to lignin depolymerization as an alternative to the consolidated physicochemical pretreatments. The laccases-based oxidative process has been discussed in terms of characteristics that can affect the development of a bioreactor unit where laccases or a laccase-mediator system can be used for biomass delignification.

scholarly journals A Lytic Polysaccharide Monooxygenase with Broad Xyloglucan Specificity from the Brown-Rot Fungus Gloeophyllum trabeum and Its Action on Cellulose-Xyloglucan Complexes

2016 ◽  
Vol 82 (22) ◽  
pp. 6557-6572 ◽  
Author(s):  
Yuka Kojima ◽  
Anikó Várnai ◽  
Takuya Ishida ◽  
Naoki Sunagawa ◽  
Dejan M. Petrovic ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Dynamic Viscosity ◽  
Brown Rot ◽  
Primary Cell Wall ◽  
Gloeophyllum Trabeum ◽  
Lytic Polysaccharide Monooxygenase ◽  
Content Type ◽  
Brown Rot Fungus ◽  
Sensitive Method ◽  
Polysaccharide Monooxygenase

ABSTRACTFungi secrete a set of glycoside hydrolases and lytic polysaccharide monooxygenases (LPMOs) to degrade plant polysaccharides. Brown-rot fungi, such asGloeophyllum trabeum, tend to have few LPMOs, and information on these enzymes is scarce. The genome ofG. trabeumencodes four auxiliary activity 9 (AA9) LPMOs (GtLPMO9s), whose coding sequences were amplified from cDNA. Due to alternative splicing, two variants ofGtLPMO9A seem to be produced, a single-domain variant,GtLPMO9A-1, and a longer variant,GtLPMO9A-2, which contains a C-terminal domain comprising approximately 55 residues without a predicted function. We have overexpressed the phylogenetically distinctGtLPMO9A-2 inPichia pastorisand investigated its properties. Standard analyses using high-performance anion-exchange chromatography–pulsed amperometric detection (HPAEC-PAD) and mass spectrometry (MS) showed thatGtLPMO9A-2 is active on cellulose, carboxymethyl cellulose, and xyloglucan. Importantly, compared to other known xyloglucan-active LPMOs,GtLPMO9A-2 has broad specificity, cleaving at any position along the β-glucan backbone of xyloglucan, regardless of substitutions. Using dynamic viscosity measurements to compare the hemicellulolytic action ofGtLPMO9A-2 to that of a well-characterized hemicellulolytic LPMO,NcLPMO9C fromNeurospora crassarevealed thatGtLPMO9A-2 is more efficient in depolymerizing xyloglucan. These measurements also revealed minor activity on glucomannan that could not be detected by the analysis of soluble products by HPAEC-PAD and MS and that was lower than the activity ofNcLPMO9C. Experiments with copolymeric substrates showed an inhibitory effect of hemicellulose coating on cellulolytic LPMO activity and did not reveal additional activities ofGtLPMO9A-2. These results provide insight into the LPMO potential ofG. trabeumand provide a novel sensitive method, a measurement of dynamic viscosity, for monitoring LPMO activity.IMPORTANCECurrently, there are only a few methods available to analyze end products of lytic polysaccharide monooxygenase (LPMO) activity, the most common ones being liquid chromatography and mass spectrometry. Here, we present an alternative and sensitive method based on measurement of dynamic viscosity for real-time continuous monitoring of LPMO activity in the presence of water-soluble hemicelluloses, such as xyloglucan. We have used both these novel and existing analytical methods to characterize a xyloglucan-active LPMO from a brown-rot fungus. This enzyme,GtLPMO9A-2, differs from previously characterized LPMOs in having broad substrate specificity, enabling almost random cleavage of the xyloglucan backbone.GtLPMO9A-2 acts preferentially on free xyloglucan, suggesting a preference for xyloglucan chains that tether cellulose fibers together. The xyloglucan-degrading potential ofGtLPMO9A-2 suggests a role in decreasing wood strength at the initial stage of brown rot through degradation of the primary cell wall.

scholarly journals Recent Advances in Screening Methods for the Functional Investigation of Lytic Polysaccharide Monooxygenases

2021 ◽  
Vol 9 ◽  
Author(s):  
Damao Wang ◽  
Yanping Li ◽  
Yuting Zheng ◽  
Yves S. Y. Hsieh
Keyword(s):  
Copper Ions ◽  
Biomass Conversion ◽  
Screening Methods ◽  
Lytic Polysaccharide Monooxygenase ◽  
Indirect Measurements ◽  
Glycosidic Bonds ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases ◽  
Functional Investigation ◽  
Polysaccharide Monooxygenase

Lytic polysaccharide monooxygenase (LPMO) is a newly discovered and widely studied enzyme in recent years. These enzymes play a key role in the depolymerization of sugar-based biopolymers (including cellulose, hemicellulose, chitin and starch), and have a positive significance for biomass conversion. LPMO is a copper-dependent enzyme that can oxidize and cleave glycosidic bonds in cellulose and other polysaccharides. Their mechanism of action depends on the correct coordination of copper ions in the active site. There are still difficulties in the analysis of LPMO activity, which often requires multiple methods to be used in concert. In this review, we discussed various LPMO activity analysis methods reported so far, including mature mass spectrometry, chromatography, labeling, and indirect measurements, and summarized the advantages, disadvantages and applicability of different methods.

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