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Enzymatic extraction of ferulic acid from agricultural by-products


Key words Fusion proteins, bifunctional enzymes, ferulic acid, biomass degradation, fungal enzymes, Aspergillus niger, by-products, vanillin
Latest version 2012/10/18
Completed by INRA - IATE

How does it work?

Primary objective To improve the extraction of ferulic acid from agricultural by-products.

Ferulic acid (4-hydroxy-3-methoxy-cinnamic acid) is a very attractive phenolic compound found as the most abundant hydroxycinnamic acid in the plant world. For instance, ferulic acid can be used as an antioxidant or can be transformed by microbial conversion into “natural” vanillin. The latter is a valuable flavouring used in the food, and an antioxidant compound for cosmetic and pharmaceutical industries.

Working principle Ferulic acid can be found in vegetable material including agricultural by-products (maize and wheat brans and wheat straw, but could be also from sugar beet, rice bran, apple marc, coffee marc, coffee by-products, all the trees used for paper industries, olive mill wastewater); it is chemically linked to the compounds (generally toa xylan chain) of the plant cell walls.

Enzymes are biochemical tools that catalyze the cleavage reactions between the compounds of the cell walls, consequently releasing the ferulic acid (4). This ability can be improved by associating two or more degrading enzymes (1, 2, 3, 6). For this technology, engineered multifunctional plant cell-wall degrading enzymes have been designed and can be produced in the extracellular medium of fungus (Aspergillus niger), thus facilitating the purification of these enzymes with a high yield.

The first one of these multifunctional enzymes is composed of three parts: two fungal enzymes and a linker. Both enzymes are proteins coming from the fungus Aspergillus niger: the feruloyl esterase A (FAEA) and the xylanase B (XYNB). The linker between both enzymes is a hyperglycosylated peptide. The fusion of the three parts results in a bifunctional enzyme called FLX with increased efficiency for the ferulic acid release.

The second one is the same as FLX but merged with a fourth part, a carbohydrate binding module (CBM, 8). The latter is a binder coming from another enzyme of Aspergillus niger: cellobiohydrolase B (CBHB). The resulting fusion enzyme called FLXLC is even more efficient than FLX because the fourth part (CBM) is responsible for a longer and closer contact of the substrate with the catalytic domain of the fusion enzyme (the substrate is cellulose; in fact, the CBM is a module that fixes the cellulose, but as the xylan is close to cellulose, it can help xylanase to act on xylan chains). Moreover, in some cases, CBM can also alter the cellulose microfibril structure, improving more the yield of extraction.

When production is carried out in 5 litres fermenters, the production yield for the fusion enzymes reaches 2 g/litre, which is quite good for pure protein.

The ferulic acid extraction yields can reach 100 % for wheat bran and 7 % for maize bran.

Additional effects Added-value on by-products.

Environmentally friendly process in comparison to chemical processes usual for the ferulic acid extraction.

Important process parameters The extraction process is performed by the multifunctional enzymes in water buffered at pH 6.0, for 4 hours at temperatures up to 45 °C.

The temperature of 50 °C should not be reached, because the fusion protein starts to separate (thermal degradation of the linker).

Important product parameters

What can it be used for?

Products Among agricultural by-products, maize and wheat brans are potential substrates according to their high amounts of ferulic acid in the cell wall, i.e. 3 % and 1 % (w\w), respectively.
Operations separation
Solutions for short comings Production of natural compounds for the food, cosmetic and pharmaceutical industries.

Use of by-products.

Use of environmentally friendly processes (7).

What can it NOT be used for?

Products non-vegetable products
Operations Any other than ferulic acid extraction for these specific enzymes FLX and FLXLC
Other limitations not known
Risks or hazards no


Maturity This technology is currently available only at lab scale.

The extraction yields for ferulic acid are variable depending on the material.

Modularity /Implementation The technology can be carried out in classical biotechnology equipment.

The possibilities of batch and/or continuous extraction process should be studied.

Consumer aspects not known
Legal aspects IPatent: WO2007/019949 (A1)

International deposit : 2005/08/12 PCT/EP/2006/007370.

Please check local legislation

Environmental aspects The use of these enzymatic tools allows working at atmospheric pressure, low temperature, in water.

Further Information

Institutes INRA - BCF, CNRS, IFP New Energies, TNO Institute
Companies Currently, the patent is not exploited by any company and no fusion protein is industrially produced by a company. For example, vanillic acid enzymatically obtained from ferulic acid is produced by SAF-ISIS (Lesaffre group).
References (1) Levasseur, A., Navarro, D., Punt, P., Belaich, J.P., Asther, M., Monot, F., Record, E. : Fusion proteins between plant cell-wall degrading enzymes, and their uses. WO2007/019949 (A1)

(2) Levasseur A., Navarro D., Punt P.J., Belaïch J-P., Asther M., Record E. (2005) Construction of engineered bifunctional enzymes and their overproduction in Aspergillus niger for improved enzymatic tools to degrade agricultural by-products. Applied and Environmental Microbiology, 71:8132-8140.

(3) De Vries R., Kester H. C. M., Poulsen C. H., Benen J. A. E., and Visser. J., 2000. Synergy between enzymes from Aspergillus involved in the degradation of plant cell wall polysaccharides. Carbohydr. Res. 327: 401-410.

(4) Faulds, C. B., and Williamson G., 1995. Release of ferulic acid from wheat bran by a ferulic acid esterase (FAE-III) from Aspergillus niger. Appl. Microbiol. Biotechnol. 463:1082-1087.

(5) Levasseur A., Asther M., and Record E., 2005. Overproduction and characterization of xylanase B from Aspergillus niger. Can. J. Microbiol. 51:177-183.

(6) Levasseur, A., Pagès S., Fierobe H. P., Navarro D., Punt P. J., Belaïch J. P., Asther M. and Record E., 2004. Design and production in Aspergillus niger of a chimeric protein associating a fungal feruloyl esterase and a clostridial dockerin domain. Appl. Environ. Microbiol. 70:6984-6991.

(7) Record, E., Asther M., Sigoillot C., Pagès S., Punt P. J., Delattre M., Haon M., Van den Hondel C.A., Sigoillot J.C., Lesage-Meessen L., and Asther M., 2003. Overproduction of the Aspergillus niger feruloyl esterase for pulp bleaching application. Appl. Microbiol. Biotechnol. 62:349-355.

(8) Tomme, P. N., Gilkes R., Miller C. M. Jr., Warren A. J. and Kilburn D. G., 1994. An internal cellulose-binding domain mediates adsorption of an engineered bifunctional xylanase/cellulase. Protein Eng. 7:117-123.

The extraction process is performed by the multifunctional enzymes in water buffered at pH 6.0, for 4 hours at temperatures up to 45 °C. The temperature of 50 °C should not be reached, because the fusion protein starts to separate (thermal degradation of the linker).warning.png"The extraction process is performed by the multifunctional enzymes in water buffered at pH 6.0, for 4 hours at temperatures up to 45 °C. The temperature of 50 °C should not be reached, because the fusion protein starts to separate (thermal degradation of the linker)." cannot be used as a page name in this wiki.

Fermentors 2.1.3 biological separation biotechnology Researchers from INRA - UMR 1163 (Biotechnology of filamentous fungi): E. Record, Anthony Levasseur WikiSysop :Template:Review document :Template:Review status

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Created by Hte inra on 21 June 2011, at 11:19