Enzyme production and immobilization in mesoporous materials

Enzymes display high reactivity and selectivity under normal conditions, but may suffer from denaturation in industrial applications. A strategy to solve this limitation is to immobilize enzymes [1]. Mesoporous silica materials (MPS) have become a common choice as support to immobilized enzymes. MPS offer unique properties such as high enzyme loading and tunable pore size [2].Feruloyl esterase (FAE) is a subclass (EC. 3.1.1.73) of carboxylic ester hydrolases. They catalyze the hydrolysis of ester linkages in plant cell walls materials releasing ferulic acid and other hydroxycinnamic acids [3]. They are also examples of FAEs used for esterification and transesterification [4].From the genomes of Aspergillus glacus and Aspergillus oryzae, some putative FAE were identified. Among them, five were selected for further investigation in order to find a suitable enzyme for catalyzing the reaction of interest. The selected genes were quite distant in an evolutionary tree.The five putative FAEs were cloned into Pichia pastoris and produced by fed-batch fermentation. They were then purified either by IMAC columns or by ion-exchange chromatography. Their activity was assessed against a range of substrate to screen for FAE, tannase and other esterase activities. When the type of the respective enzyme activity was determined, some of them were further characterized. Five new enzymes were recombinantly produced and purified. Their activity type was determined and some of them were immobilized.Enzymes produced in sufficient quantities and having a good free activity were further investigated by immobilization. The selected support for immobilization was mesoporous silica particles (MPS). The conditions of immobilization were investigated and the activity once immobilized was tested and compared to the free one to gain insights on what happens during the immobilization of enzymes. Results were compared to those obtained with a commercially available FAE (E-FAERU, Megazyme).References.[1] Hudson S.; Cooney J.; Magner E., Angew. Chem. Int. Ed. 2008, 47, 8582-8594. [2] Carlsson N.; Gustafsson H.; Th\uf6rn C.; Olsson L.; Holmberg K.; \uc5kerman B. Advances in Colloid and Interface Science 2014, 204, 339-360.[3] Topakas E.; Vafiadi C.; Christakopoulos P. Process Biochemistry 2007, 42, 497-509.[4] Th\uf6rn C.; Gustafsson H.; Olsson L. Journal of molecular Catalysis B: Enzymatic 2011, 72, 57-6

A GH115 alpha-glucuronidase from Schizophyllum commune contributes to the synergistic enzymatic deconstruction of softwood glucuronoarabinoxylan

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 α-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 α-l-arabinofuranosidase (AbfA), and a β-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 α-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.Lauren S. McKee, Hampus Sunner, George E. Anasontzis, Guillermo Toriz, Paul Gatenholm, Vincent Bulone, Francisco Vilaplana and Lisbeth Olsso

Enzyme production and immobilization in mesoporous materials

Enzymes display high reactivity and selectivity under normal conditions, but may suffer from denaturation in industrial applications. A strategy to solve this limitation is to immobilize enzymes [1]. Mesoporous silica materials (MPS) have become a common choice as support to immobilized enzymes. MPS offer unique properties such as high enzyme loading and tunable pore size [2].Feruloyl esterase (FAE) is a subclass (EC. 3.1.1.73) of carboxylic ester hydrolases. They catalyze the hydrolysis of ester linkages in plant cell walls materials releasing ferulic acid and other hydroxycinnamic acids [3]. They are also examples of FAEs used for esterification and transesterification [4].From the genomes of Aspergillus glacus and Aspergillus oryzae, some putative FAE were identified. Among them, five were selected for further investigation in order to find a suitable enzyme for catalyzing the reaction of interest. The selected genes were quite distant in an evolutionary tree.The five putative FAEs were cloned into Pichia pastoris and produced by fed-batch fermentation. They were then purified either by IMAC columns or by ion-exchange chromatography. Their activity was assessed against a range of substrate to screen for FAE, tannase and other esterase activities. When the type of the respective enzyme activity was determined, some of them were further characterized. Five new enzymes were recombinantly produced and purified. Their activity type was determined and some of them were immobilized.Enzymes produced in sufficient quantities and having a good free activity were further investigated by immobilization. The selected support for immobilization was mesoporous silica particles (MPS). The conditions of immobilization were investigated and the activity once immobilized was tested and compared to the free one to gain insights on what happens during the immobilization of enzymes. Results were compared to those obtained with a commercially available FAE (E-FAERU, Megazyme).References.[1] Hudson S.; Cooney J.; Magner E., Angew. Chem. Int. Ed. 2008, 47, 8582-8594. [2] Carlsson N.; Gustafsson H.; Th\uf6rn C.; Olsson L.; Holmberg K.; \uc5kerman B. Advances in Colloid and Interface Science 2014, 204, 339-360.[3] Topakas E.; Vafiadi C.; Christakopoulos P. Process Biochemistry 2007, 42, 497-509.[4] Th\uf6rn C.; Gustafsson H.; Olsson L. Journal of molecular Catalysis B: Enzymatic 2011, 72, 57-6