| 1. |
- Anasontzis, George E, 1980
(författare)
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Biomass modifying enzymes: From discovery to application
- 2012
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Ingår i: Oral presentation at the Chalmers Life Science AoA conference.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- It has now been realized that the road towards the bio-based economy is a one-way street, leaving gradually the oil-based technology and driving slowly towards a more sustainable society. The current non-biodegradable hydrocarbon fuels and plastics will be replaced by new products which will derive from natural and renewable resources. The synthesis of such biofuels and biochemicals is still challenged by the difficulties to cost efficiently degrade lignocellulosic material to fermentable sugars or to isolate the intact polymers. Biomass degrading and modifying enzymes play an integral role both in the separation of the polymers from the wood network, as well as in their subsequent modification, prior to further product development.Our group interests focus on all levels of applied enzyme research of biomass acting enzymes: Discovery, assay development, production and application. Relevant examples will be provided: What is our strategy for discovering novel microorganisms and enzymes from the tropical forests and grasslands of Vietnam? How do we design novel real-world assays for enzyme activity determination? Which are the bottlenecks in the enzymatic cellulose hydrolysis? How enzymes can be used to produce high added value compounds from biomass?
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| 2. |
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| 3. |
- Jansson, Ronnie, et al.
(författare)
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Functionalized silk assembled from a recombinant spider silk fusion protein (Z-4RepCT) produced in the methylotrophic yeast Pichia pastoris
- 2016
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Ingår i: Biotechnology Journal. - : Wiley-VCH Verlagsgesellschaft. - 1860-6768 .- 1860-7314. ; 11:5, s. 687-699
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Tidskriftsartikel (refereegranskat)abstract
- Functional biological materials are a growing research area with potential applicability in medicine and biotechnology. Using genetic engineering, the possibility to introduce additional functions into spider silk-based materials has been realized. Recently, a recombinant spider silk fusion protein, Z-4RepCT, was produced intracellularly in Escherichia coli and could after purification self-assemble into silk-like fibers with ability to bind antibodies via the IgG-binding Z domain. In this study, the use of the methylotrophic yeast Pichia pastoris for production of Z-4RepCT has been investigated. Temperature, pH and production time were influencing the amount of soluble Z-4RepCT retrieved from the extracellular fraction. Purification of secreted Z-4RepCT resulted in a mixture of full-length and degraded silk proteins that failed to self-assemble into fibers. A position in the C-terminal domain of 4RepCT was identified as being subjected to proteolytic cleavage by proteases in the Pichia culture supernatant. Moreover, the C-terminal domain was subjected to glycosylation during production in P. pastoris. These observed alterations of the CT domain are suggested to contribute to the failure in fiber assembly. As alternative approach, Z-4RepCT retrieved from the intracellular fraction, which was less degraded, was used and shown to retain ability to assemble into silk-like fibers after enzymatic deglycosylation.
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| 4. |
- McKee, Lauren S., et al.
(författare)
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A GH115 alpha-glucuronidase from Schizophyllum commune contributes to the synergistic enzymatic deconstruction of softwood glucuronoarabinoxylan
- 2016
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Ingår i: Biotechnology for Biofuels. - : BioMed Central. - 1754-6834. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- Background: Lignocellulosic biomass from softwood represents a valuable resource for the production of biofuels and bio-based materials as alternatives to traditional pulp and paper products. Hemicelluloses constitute an extremely heterogeneous fraction of the plant cell wall, as their molecular structures involve multiple monosaccharide components, glycosidic linkages, and decoration patterns. The complete enzymatic hydrolysis of wood hemicelluloses into monosaccharides is therefore a complex biochemical process that requires the activities of multiple degradative enzymes with complementary activities tailored to the structural features of a particular substrate. Glucuronoarabinoxylan (GAX) is a major hemicellulose component in softwood, and its structural complexity requires more enzyme specificities to achieve complete hydrolysis compared to glucuronoxylans from hardwood and arabinoxylans from grasses. Results: We report the characterisation of a recombinant alpha-glucuronidase (Agu115) from Schizophyllum commune capable of removing (4-O-methyl)-glucuronic acid ((Me) GlcA) residues from polymeric and oligomeric xylan. The enzyme is required for the complete deconstruction of spruce glucuronoarabinoxylan (GAX) and acts synergistically with other xylan-degrading enzymes, specifically a xylanase (Xyn10C), an alpha-l-arabinofuranosidase (AbfA), and a beta-xylosidase (XynB). Each enzyme in this mixture showed varying degrees of potentiation by the other activities, likely due to increased physical access to their respective target monosaccharides. The exo-acting Agu115 and AbfA were unable to remove all of their respective target side chain decorations from GAX, but their specific activity was significantly boosted by the addition of the endo-Xyn10C xylanase. We demonstrate that the proposed enzymatic cocktail (Agu115 with AbfA, Xyn10C and XynB) achieved almost complete conversion of GAX to arabinofuranose (Araf), xylopyranose (Xylp), and MeGlcA monosaccharides. Addition of Agu115 to the enzymatic cocktail contributes specifically to 25 % of the conversion. However, traces of residual oligosaccharides resistant to this combination of enzymes were still present after deconstruction, due to steric hindrances to enzyme access to the substrate. Conclusions: Our GH115 alpha-glucuronidase is capable of finely tailoring the molecular structure of softwood GAX, and contributes to the almost complete saccharification of GAX in synergy with other exo- and endo-xylan-acting enzymes. This has great relevance for the cost-efficient production of biofuels from softwood lignocellulose.
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| 5. |
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| 6. |
- Shin, Jae Ho, 1987, et al.
(författare)
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Molecular docking and linear interaction energy studies give insight to α, β-reduction of enoate groups in enzymes
- 2018
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Production of adipic acid from renewable sources has been gaining attention in an attempt to move from an oil-based economy to a biobased economy. Metabolic engineering allows microorganisms to produce useful chemicals using renewable resources as carbon sources. We target a theoretical metabolic pathway that relies on conversion of L-lysine to adipic acid. One of the enzymatic steps in this conversion pathway is an α, β-reduction of an unsaturated bond in an enoate moiety and no aerobic enzymes have been identified to specifically make this conversion on 6-amino-trans-2-hexenoic acid. We evaluated Escherichia coli NemA, and Saccharomyces pastorianus Oye1 (Old Yellow Enzyme 1) for their potenstial capability to carry out the desired α, β-reduction. Here, we build homology models for E. coli NemA and perform molecular docking studies of trans-2-hexenoic acid and trans-2-hexenal to the candidate enzyme models. Ligand-enzyme binding stability is assessed by molecular dynamics (MD) simulations. Additionally, linear energy calculations were used to investigate binding stability in solution environment. Here, we propose that NemA and Oye1, both belonging to the Old yellow enzyme family, have large enough catalytic pocket for accommodating enoate moieties but not enough stability to carry out the α, β-reduction. Protein engineering of both NemA and Oye1 would be necessary for these enzymes to perform the targeted reactions efficiently. The results shown in this study provides a useful insight to α, β-reduction reaction potentially crucial in bio-based production of adipic acid.
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| 7. |
- Skoog, Emma, 1983, et al.
(författare)
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Biobased adipic acid – The challenge of developing the production host
- 2018
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Ingår i: Biotechnology Advances. - : Elsevier BV. - 0734-9750. ; 36:8, s. 2248-2263
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Forskningsöversikt (refereegranskat)abstract
- Adipic acid is a platform chemical, and is the most important commercial dicarboxylic acid. It has been targeted for biochemical conversion as an alternative to present chemical production routes. From the perspective of bioeconomy, several kinds of raw material are of interest including the sugar platform (derived from starch, cellulose or hemicellulose), the lignin platform (aromatics) and the fatty acid platform (lipid derived). Two main biochemical-based production schemes may be employed: (i) direct fermentation to adipic acid, or (ii) fermentation to muconic or glucaric acid, followed by chemical hydrogenation (indirect fermentation). This review presents a comprehensive description of the metabolic pathways that could be constructed and analyzes their respective theoretical yields and metabolic constraints. The experimental yields and titers obtained so far are low, with the exception of processes based on palm oil and glycerol, which have been reported to yield up to 50 g and 68 g adipic acid/L, respectively. The challenges that remain to be addressed in order to achieve industrially relevant production levels include solving redox constraints, and identifying and/or engineering enzymes for parts of the metabolic pathways that have yet to be metabolically demonstrated. This review provides new insights into ways in which metabolic pathways can be constructed to achieve efficient adipic acid production. The production host provides the chassis to be engineered via an appropriate metabolic pathway, and should also have properties suitable for the industrial production of adipic acid. An acidic process pH is attractive to reduce the cost of downstream processing. The production host should exhibit high tolerance to complex raw material streams and high adipic acid concentrations at acidic pH.
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| 8. |
- Gullfot, Fredrika, 1967-
(författare)
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Synthesis of xyloglucan oligo- and polysaccharides with glycosynthase technology
- 2009
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Xyloglucans are polysaccharides found as storage polymers in seeds and tubers, and as cross-linking glycans in the cell wall of plants. Their structure is complex with intricate branching patterns, which contribute to the physical properties of the polysaccharide including its binding to and interaction with other glycans such as cellulose. Xyloglucan is widely used in bulk quantities in the food, textile and paper making industries. With an increasing interest in technically more advanced applications of xyloglucan, such as novel biocomposites, there is a need to understand and control the properties and interactions of xyloglucan with other compounds, to decipher the relationship between xyloglucan structure and function, and in particular the effect of different branching patterns. However, due to the structural heterogeneity of the polysaccharide as obtained from natural sources, relevant studies have not been possible to perform in practise. This fact has stimulated an interest in synthetic methods to obtain xyloglucan mimics and analogs with well-defined structure and decoration patterns. Glycosynthases are hydrolytically inactive mutant glycosidases that catalyse the formation of glycosidic linkages between glycosyl fluoride donors and glycoside acceptors. Since its first conception in 1998, the technology is emerging as a useful tool in the synthesis of large, complex polysaccharides. This thesis presents the generation and characterisation of glycosynthases based on xyloglucanase scaffolds for the synthesis of well-defined homogenous xyloglucan oligo- and polysaccharides with regular substitution patterns.
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| 9. |
- Hong, Kuk-ki, 1976, et al.
(författare)
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Metabolic Engineering of Saccharomyces cerevisiae: A Key Cell Factory Platform for Future Biorefineries
- 2012
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Ingår i: Cellular and Molecular Life Sciences. - : Springer Science and Business Media LLC. - 1420-9071 .- 1420-682X. ; 69:16, s. 2671-2690
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Forskningsöversikt (refereegranskat)abstract
- Metabolic engineering is the enabling science of development of efficient cell factories for the production of fuels, chemicals, pharmaceuticals, and food ingredients through microbial fermentations. The yeast Saccharomyces cerevisiae is a key cell factory already used for the production of a wide range of industrial products, and here we review ongoing work, particularly in industry, on using this organism for the production of butanol, which can be used as biofuel, and isoprenoids, which can find a wide range of applications including as pharmaceuticals and as biodiesel. We also look into how engineering of yeast can lead to improved uptake of sugars that are present in biomass hydrolyzates, and hereby allow for utilization of biomass as feedstock in the production of fuels and chemicals employing S. cerevisiae. Finally, we discuss the perspectives of how technologies from systems biology and synthetic biology can be used to advance metabolic engineering of yeast.
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| 10. |
- Sunner, Hampus, 1981, et al.
(författare)
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Fungal Ferulic Acid Esterases – Specificity and Phylogeny
- 2009
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Ingår i: Italic5 Science and Technology of Biomasses Proceedings Book, M Orlandi, C Crestine (Ed.). Italic5/COST conference, Sept 1-4 2009, Varenna, Italy.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Ferulic Acid Esterases (FAE) is a large heterogeneous group of enzymes with activity on esters of hydroxy- and metoxy- substituted cinnamic acid derivatives, such as ferulic acid. These ester bonds occur in the cell walls of plants and are especially common in grasses. As little systematic knowledge has been collected about this group of enzymes and only a few enzymes have been biochemically characterised to date, we have explored the phylogeny of FAEs using bioinformatic tools. We can conclude that the known Ferulic Acid Esterases belong to several evolutionary distant groups, two of which have dozens of highly related sequences, and a few groups with no members other than the known enzyme. The phylogeny also suggests certain similarities of substrate specificity within groups and proposes enzymes, whose biochemical characterisation would be especially informative for our understanding of the FAE families.
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