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Applied and Environmental Microbiology, September 2007, p. 5624-5632, Vol. 73, No. 17
0099-2240/07/$08.00+0     doi:10.1128/AEM.00374-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Gene Overexpression and Biochemical Characterization of the Biotechnologically Relevant Chlorogenic Acid Hydrolase from Aspergillus niger{triangledown}

Isabelle Benoit,1 Michèle Asther,1 Yves Bourne,2 David Navarro,1 Stéphane Canaan,3 Laurence Lesage-Meessen,1 Marga Herweijer,4 Pedro M. Coutinho,2 Marcel Asther,1 and Eric Record1*

UMR-1163, INRA de Biotechnologie des Champignons Filamenteux, IFR86-BAIM, Universités de Provence et de la Méditerranée, ESIL, 163 avenue de Luminy, CP 965, 13288 Marseille Cedex 09, France,1 UMR 6098, Architecture et Fonction des Macromolécules Biologiques, CNRS/Universités de Provence et de la Méditerranée, 163 avenue de Luminy, CP 932, 13288 Marseille Cedex 09, France,2 UPR 9025, CNRS Laboratoire d'Enzymologie Interfaciale et de la Physiologie de la Lipolyse, 31 chemin Joseph Aiguier, 13402 Marseille Cedex 20, France,3 DSM Food Specialties, P.O. Box 1, 2600 MA Delft, The Netherlands4

Received 16 February 2007/ Accepted 30 June 2007

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 106 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.

* Corresponding author. Mailing address: 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. Phone: 33 (0)4 91 82 86 07. Fax: 33 (0)4 91 82 86 01. E-mail: eric.record{at}esil.univmed.fr

{triangledown} Published ahead of print on 13 July 2007.

Applied and Environmental Microbiology, September 2007, p. 5624-5632, Vol. 73, No. 17
0099-2240/07/$08.00+0     doi:10.1128/AEM.00374-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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