Progress 10/01/03 to 09/30/04
Outputs
The research develops an enzymatic method to recover ferulic acid from agriculture residues as precursors for natural vanillin production. To this end, a collection of bacteria and fungal strains were screened for novel ferulic acid esterase activities. Two fungal strains and a bacterium strain showing promising results in the preliminary screening were followed upon closely. The FAE activities from these strains were confirmed. Conditions for FAE production in both strains were optimized. Using a known FAE producer as positive control, the novel FAEs found from this work were carefully studied. One of the fungal FAE was purified to homogeneity and its property was fully characterized. The purified protein is being analyzed for its N-terminal sequence. Overexpression of this enzyme is in E. coli or another suitable host will be pursued as soon as the sequence data is made available. The other two newly discovered FAEs are being purified using chromatography. An
invention disclosure is being prepared for the enzymes discovered so far. A publication on the discovery, purification and characterization of one of the fungal enzyme is being prepared. New fungal enzymes are significantly different from a commercial Aspergillus FAE. In particular, both new fungal enzymes are capable of liberating ferulic acid from corn bran whereas Aspergillus enzyme is not. Medium compositions and induction conditions are important for the yield of FAE. Simple ammonium salts lead to best yield of FAE as compared to complex nitrogen source. Esterified ferulic acid (in bonded form) is necessary to induce the production of FAE. These results are in sharp contrast with that of Aspergillus niger, where FAE production requires the presence of free ferulic acid in the growth medium. The two FAEs showed highest activity on a synthetic substrate, methyl ferulate, and liberated the ferulic acid more effectively from corn bran than oat spelt xylan. However, the positive
control, the Aspergillus FAE showed no activity on corn bran and very little activity on oat spelt xylan. This result indicates that the two FAEs are suitable for releasing ferulic acid from corn bran. The pH optimum of the purified FAE activity was at 6.5-7.5, but it retains more than 80% of its activity after 12 h of storage between pH 4.0 and 10.0. Thus the enzyme is more stable in acidic or alkaline conditions than most of other fungal FAEs. The high acid and base stability of FAE makes it useful in many biotechnological applications. Optimal temperature of the crude FAE was at 45-50 degree of celcius, which is in the range of FAEs from other mesophilic fungi. However, most of FAE activity was lost after 30 min incubation at 55 degree of celcius at pH 7.0 The FAE from one fungal strain was purified using a procedure which involved ammonium sulfate precipitation, anion exchange, and cation exchange chromatography. This procedure purifies the enzyme to its homogeneity. The molecular
mass of the purified FAE was found to be 31 kD as determined by SDS-PAGE electrophoresis. The purified FAE exhibits similar pH and temperature optima, and pH and temperature stabilities to that of the crude enzyme.
Impacts
The newly discovered enzymes will allow a viable strategy to recover ferulic acid from agriculture residues for vanillin production. Particularly, the enzymes discovered are capable of liberating ferulic acid from corn bran with the assistance of xylanase. It will be useful in derive ferulic acid from an inexpensive, abundant renewable source. Besides for vanillin production, the FAE could find other applications. It can be used with xylanase in production of xylo-oligosaccharides. It is useful in degradation of plant cell wall by breaking the crosslinkings between polysaccharides chains. It could play a very important role in complete hydrolysis of lignocellulose (hemicellulose), in removal of lignin in pulp and paper making process, in bleaching by reducing the chlorine used in the process thereby reducing its adverse effect on environments. FAE also found use in human and animal nutritions.
Publications
- No publications reported this period
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