Immobilized l-aspartate ammonia-lyase from Bacillus sp. YM55-1 as biocatalyst for highly concentrated l-aspartate synthesis
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l-Aspartate ammonia-lyase from Bacillus sp. YM55-1 (AspB, EC 188.8.131.52) catalyzes the reversible conversion of l-aspartate (Asp) into fumarate and ammonia with a high specific activity toward the substrate. AspB was expressed in Escherichia coli and partially purified by heat precipitation and saturation with ammonium sulfate reaching purification factor of 7.7 and specific activity of 334 U/mg of protein. AspB was immobilized by covalent attachment on Eupergit® C (epoxy support) and MANA-agarose (amino support), and entrapment in LentiKats® (polyvinyl alcohol) with retained activities of 24, 85 and 63 %, respectively. Diffusional limitations were only observed for the enzyme immobilized in LentiKats® and were overcome by increasing substrate concentration. Free and immobilized AspB were used for the synthesis of aspartate achieving high product concentration (≥450 mM) after 24 h of reaction. Immobilized biocatalysts were efficiently reused in 5 cycles of Asp synthesis, keeping over 90 % of activity and reaching over 90 % of conversion in all the cases.
KeywordsEnzymatic l-aspartate synthesis l-Aspartate ammonia-lyase immobilization Eupergit® C MANA-agarose LentiKats®
This work was supported by the Spanish MICINN (project CTQ2011-28398-CO2-01 and EngBiocat ERA-IB project EUI2008-03615) and by Generalitat de Catalunya (Research group 2009SGR281). The Department of Chemical Engineering of UAB constitutes the Biochemical Engineering Unit of the Reference Network in Biotechnology of the Generalitat de Catalunya (XRB). Authors thank c-LEcta (Leipzig, Germany) for its collaboration in providing the recombinant E. coli strain. Max Cárdenas-Fernández thanks the financial support of a predoctoral grant from AECID (Spanish MAEC).
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