Homologous expression of the feruloyl esterase B gene from Aspergillus niger and characterization of the recombinant enzyme

摘要

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., 50degreesC and 6, respectively. In addition, the recombinant FAEB was determined to be quite stable up to 50degreesC for 120 min. Kinetic constants for MCA, MpCA, and chlorogenic acid (5-O-caffeoyl quinic acid) were as follows: K-m : 0.13, 0.029, and 0.16 mM and V-max: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. (C) 2004 Elsevier Inc. All rights reserved.