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Homologous expression of the feruloyl esterase B gene from Aspergillus niger and characterization of the recombinant enzyme
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International audience. The faeB gene encoding the feruloyl esterase B (FAEB) was isolated from Aspergillus niger BRFM131 genomic DNA. The faeB gene, with additional sequence coding for a C-terminal histidine tag, was inserted into an expression vector under the control of the gpd promoter and trpC terminator and expressed in a protease deficient A. niger strain. Homologous overproduction allows to reach an esterase activity of 18 nkat mL−1 against MCA as substrate. The improvement factor was 16-fold higher as compared to the production level obtained with non-transformed A. niger strain induced by sugar beet pulp. The corresponding secretion yield was estimated to be around 100 mg L−1. Recombinant FAEB was purified 14.6-fold to homogeneity from an 8-day-old culture by a single affinity chromatographic step with a recovery of 64%. SDS–PAGE revealed a single band with a molecular mass of 75 kDa, while under non-denatured conditions, native enzyme has a molecular mass of around 150 kDa confirming that the recombinant FAEB is a homodimer. The recombinant and native FAEB have the same characteristics concerning temperature and pH optima, i.e., 50 °C and 6, respectively. In addition, the recombinant FAEB was determined to be quite stable up to 50 °C for 120 min. Kinetic constants for MCA, MpCA, and chlorogenic acid (5-O-caffeoyl quinic acid) were as follows: Km: 0.13, 0.029, and 0.16 mM and Vmax: 1101, 527.6, and 28.3 nkat mg−1, respectively. This is the first report on the homologous overproduction of feruloyl esterase B in A. niger.
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