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Applied and Environmental Microbiology, December 2005, p. 8132-8140, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8132-8140.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Construction of Engineered Bifunctional Enzymes and Their Overproduction in Aspergillus niger for Improved Enzymatic Tools To Degrade Agricultural By-Products

UMR 1163 INRA de Biotechnologie des Champignons Filamenteux, IFR86-BAIM, Universités de Provence et de la Méditerranée, ESIL, 163 Ave. de Luminy, Case Postale 925, 13288 Marseille Cedex 09, France,¹ Department of Microbiology, Quality of Life, Zeist, The Netherlands,² Bioénergétique et Ingéniérie des Protéines, Centre National de la Recherche Scientifique, IBSM, 13402 Marseille, France,³ Université de Provence, 3 Place Victor Hugo, Marseille, France⁴

Received 18 May 2005/ Accepted 2 August 2005

Two chimeric enzymes, FLX and FLXLC, were designed and successfully overproduced in Aspergillus niger. FLX construct is composed of the sequences encoding the feruloyl esterase A (FAEA) fused to the endoxylanase B (XYNB) of A. niger. A C-terminal carbohydrate-binding module (CBM family 1) was grafted to FLX, generating the second hybrid enzyme, FLXLC. Between each partner, a hyperglycosylated linker was included to stabilize the constructs. Hybrid proteins were purified to homogeneity, and molecular masses were estimated to be 72 and 97 kDa for FLX and FLXLC, respectively. Integrity of hybrid enzymes was checked by immunodetection that showed a single form by using antibodies raised against FAEA and polyhistidine tag. Physicochemical properties of each catalytic module of the bifunctional enzymes corresponded to those of the free enzymes. In addition, we verified that FLXLC exhibited an affinity for microcrystalline cellulose (Avicel) with binding parameters corresponding to a K_d of 9.9 x 10^–8 M for the dissociation constant and 0.98 µmol/g Avicel for the binding capacity. Both bifunctional enzymes were investigated for their capacity to release ferulic acid from natural substrates: corn and wheat brans. Compared to free enzymes FAEA and XYNB, a higher synergistic effect was obtained by using FLX and FLXLC for both substrates. Moreover, the release of ferulic acid from corn bran was increased by using FLXLC rather than FLX. This result confirms a positive role of the CBM. In conclusion, these results demonstrated that the fusion of naturally free cell wall hydrolases and an A. niger-derived CBM onto bifunctional enzymes enables the increase of the synergistic effect on the degradation of complex substrates.