Enzyme production and immobilization in mesoporous materials
Conference poster, 2014
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 . 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 .
Feruloyl esterase (FAE) is a subclass (EC. 22.214.171.124) of carboxylic ester hydrolases. They catalyze the hydrolysis of ester linkages in plant cell walls materials releasing ferulic acid and other hydroxycinnamic acids . They are also examples of FAEs used for esterification and transesterification .
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).
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