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

Anthony Levasseur, Isabelle Benoit, Michèle Asther, Marcel Asther, Eric Record

    Research output: Contribution to journal/periodicalArticleScientificpeer-review

    45 Citations (Scopus)

    Abstract

    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 degrees C and 6, respectively. In addition, the recombinant FAEB was determined to be quite stable up to 50 degrees 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.

    Original languageEnglish
    Pages (from-to)126-33
    Number of pages8
    JournalProtein Expression and Purification
    Volume37
    Issue number1
    DOIs
    Publication statusPublished - Sept 2004

    Keywords

    • Aspergillus niger
    • Carboxylic Ester Hydrolases
    • Cloning, Molecular
    • Enzyme Stability
    • Fungal Proteins
    • Hydrogen-Ion Concentration
    • Isoenzymes
    • Recombinant Proteins
    • Temperature

    Fingerprint

    Dive into the research topics of 'Homologous expression of the feruloyl esterase B gene from Aspergillus niger and characterization of the recombinant enzyme'. Together they form a unique fingerprint.

    Cite this