Handbook of Vanilla Science and Technology

Alkaline, acid, and enzymatic treatments hydrolyze the ester bond. Alkaline and acid hydrolyses performed at higher temperatures (85-100°C) effectively break the covalent bond. The alkaline hydrolysis of corn cobs yields about 1.1 g/l of ferulic acid and 2.1 g/l of p-coumaric acid (Torre et al. 2008). An inexpensive method to enrich and partially purify ferulic acid is an alkaline hydrolysis coupled with adsorption on active powdered charcoal (Ou et al. 2007). Even though efficient, the chemical hydrolyses are not considered natural processes under the current US and EC regulations.

An enzymatic treatment of autoclaved plant material presents an economically feasible route to natural ferulic acid. Several microbial enzymes were identified as efficient in releasing ferulic acid from plant cell walls. Enzymes such as feruloyl esterase, cinnamoyl ester hydrolases, xylanases, and carbohydrases have been investigated for their capacity to hydrolyze phenolic precursors from variety of matrixes. Feruloyl esterase has been associated with a number of microorganisms, such as Aspergillus niger, Talaromyces stipitatus, Fusarium oxysporum, Humicola insolens, Streptomyces avermitilis, etc. Two different feruloyl esterases (FAEA and FAEB) from A. niger effectively released ferulic and caffeic acids from apple marc, coffee pulp, wheat straw, maize husk, and sugar beet pulp (Benoit et al. 2006). Besides conventional batch fermentation, feruloyl esterase can be also produced by microencapsulated Lactobacillus fermentum 11976. Alginate encapsulated L.fermentum produced feruloyl esterase to de-esterify ethyl ferulate with a yield of 45.59% of ferulate (Bhathena et al. 2007).

Once a free ferulic acid is available, it is converted to vanillin in a series of enzymatic reactions. Enzymes involved in the biotransformation are typically induced by ferulic acid.

Five different catabolic pathways of ferulic acid have been proposed:

I non-β-oxidative deacetylation (CoA-dependent)

II β-oxidative deacetylation (CoA-dependent)

III non-oxidative decarboxylation

IV CoA-independent deacetylation

V side-chain reduction

19.2.1.1 Non-β-oxidative deacetylation (CoA-dependent)

The genes encoding enzymes needed for non- β-oxidative deacetylation were identified and their activities confirmed (Gasson et al. 1998; Narbad and Gasson 1998, Figure 19.2). This pathway utilizes three enzymatic steps. It requires two enzymes, 4-hydroxycinnamate-CoA ligase (fcs) (4-CL) and 4-hydroxycinnamate CoA-hydratase/lyase (HCHL)(ech). First, the 4-CL transforms ferulic acid to feruloyl-SCoA. 4-CL also catalyzes the reaction with 4-coumaric and caffeic acids, yielding 4-hydroxybenzaldehyde and protocatechuic aldehyde, respectively (Mitra et al. 1999). HCHL catalyzes hydration of feruloyl-SCoA into a transient intermediate 4-hydroxy-3-methoxyphenyl-p-hydroxypropionyl-SCoA and subsequently cleaves the side chain in a retro-aldol fashion into vanillin and acetyl-SCoA (Gasson et al. 1998; Narbad and Gasson 1998). The third gene associated with fcs and ech encoding vanillin-oxidoreductase is induced by ferulic acid (Gasson et al. 1998). The genes and enzymes involved in the corresponding reactions have been characterized in P. fluorescens AN103 (Gasson et al. 1998), Pseudomonas sp. HR199 (Overhage et al. 1999), Streptomyces setonii (Muheim and Lerch 1999), Amycolatopsis sp. HR167 (Achterholt et al. 2000), Delftia acidovorans (Plaggenborg et al. 2001), and Pseudomonas putida KT2440 (Plaggenborg et al. 2003). Identification and characterization of the genes coding for these enzymes offer new opportunities for metabolic engineering and for the construction of recombinant strains.

Fig. 19.2 Non-β-oxidative deacetylation (CoA-dependent).

19.2.1.2 β-oxidative deacetylation (CoA-dependent)