Gene overexpression and biochemical characterization of the biotechnologically relevant chlorogenic acid hydrolase from Aspergillus niger.
The full-length gene that encodes the chlorogenic acid hydrolase from Aspergillus niger CIRM BRFM 131 was cloned by PCR based on the genome of the strain A. niger CBS 513.88. The complete gene consists of 1,715 bp and codes for a deduced protein of 512 amino acids with a molecular mass of 55,264 Da and an acidic pI of 4.6. The gene was successfully cloned and overexpressed in A. niger to yield 1.25 g liter(-1), i.e., 330-fold higher than the production of wild-type strain A. niger CIRM BRFM131. The histidine-tagged recombinant ChlE protein was purified to homogeneity via a single chromatography step, and its main biochemical properties were characterized. The molecular size of the protein checked by mass spectroscopy was 74,553 Da, suggesting the presence of glycosylation. ChlE is assembled in a tetrameric form with several acidic isoforms with pIs of around 4.55 and 5.2. Other characteristics, such as optimal pH and temperature, were found to be similar to those determined for the previously characterized chlorogenic acid hydrolase of A. niger CIRM BRFM 131. However, there was a significant temperature stability difference in favor of the recombinant protein. ChlE exhibits a catalytic efficiency of 12.5 x 10(6) M(-1) s(-1) toward chlorogenic acid (CGA), and its ability to release caffeic acid from CGA present in agricultural by-products such as apple marc and coffee pulp was clearly demonstrated, confirming the high potential of this enzyme.
Benoit
Isabelle
I
UMR 1163 INRA/Université de Provence de Biotechnologie des Champignons Filamenteux, IFR-IBAIM, Universités de Provence et de la Méditerranée, ESIL, 163 avenue de Luminy, Case Postale 925, 13288 Marseille Cedex 09, France.
Asther
Michèle
M
Bourne
Yves
Y
Navarro
David
D
Canaan
Stéphane
S
Lesage-Meessen
Laurence
L
Herweijer
Marga
M
Coutinho
Pedro M
PM
Asther
Marcel
M
Record
Eric
E
D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D001709:Biotechnology / Q000379:methods*; D002726:Chlorogenic Acid / Q000378:metabolism*; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014170:Transformation, Genetic; D015854:Up-Regulation
D005656:Fungal Proteins; D011994:Recombinant Proteins; D002726:Chlorogenic Acid; D006867:Hydrolases