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

Content Provider	PubMed Central
Author	Levasseur, Anthony Navarro, David Punt, Peter J. Jean-pierre, Belaïch Asther, Marcel Record, Eric
Copyright Year	2005
Abstract	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 Kd of 9.9 × 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.
Related Links	http://dx.doi.org/10.1128/aem.71.12.8132-8140.2005
Ending Page	8140
Page Count	9
Starting Page	8132
File Format	PDF
ISSN	00992240
e-ISSN	10985336
Journal	Applied and Environmental Microbiology
Issue Number	12
Volume Number	71
Language	English
Publisher	American Society for Microbiology
Publisher Date	2005-12-01
Access Restriction	Open
Rights Holder	American Society for Microbiology
Subject Keyword	Biotechnology Food Science Ecology Applied Microbiology and Biotechnology Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Ecology Food Science Applied Microbiology and Biotechnology Biotechnology

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