Method for preparing para-hydroxystyrene by biocatalytic decarboxylation of para-hydroxycinnamic acid in a biphasic reaction medium

Abstract

A biocatalytic method for preparing para-hydroxystyrene from para-hydroxycinnamic acid is described. The method uses an enzyme source having para-hydroxycinnamic acid decarboxylase activity to catalyze the decarboxylation of para-hydroxycinnamic acid in a biphasic reaction medium to produce para-hydroxystyrene, which is extracted into the organic phase of the biphasic reaction medium. The method results in a high yield of para-hydroxystyrene due to the decreased exposure of the enzyme source to the inhibitory product. The product is readily recovered from the extractant, or may be chemically derivatized directly in the extractant before recovery.

1 . A process for producing para-hydroxystyrene comprising:
 a) providing an enzyme source having para-hydroxycinnamic acid decarboxylase activity, said enzyme source comprising a polypeptide having the amino acid sequence as set forth in SEQ ID NO:2 or SEQ ID NO:4;   b) contacting said enzyme source with para-hydroxycinnamic acid in a biphasic reaction medium comprising an aqueous phase and an extractant, said extractant being a water-immiscible organic solvent selected from the group consisting of toluene, methyl decanoate, 2-undecanone, dichloromethane, hexane, 2-decanol, 4-decanol, 3-decanone, 4-decanone, 1-nonanol, 2-nonanol, 2-heptanol and mixtures thereof, to form para-hydroxystyrene which is extracted into the extractant of the biphasic reaction medium;   c) separating the extractant from the aqueous phase; and   d) optionally, recovering the para-hydroxstyrene from the extractant.   
     
     
         2 . A process according to  claim 1  wherein the enzyme source having para-hydroxycinnamic acid decarboxylase activity is selected from the group consisting of: purified enzyme, cell-free extract, wildtype host cells, recombinant host cells, treated wildtype host cells, and treated recombinant host cells. 
     
     
         3 . A process according to  claim 2  wherein the wildtype host cell is selected from the group consisting of  Lactobacillus plantarum  and  Bacillus subtilis.    
     
     
         4 . A process according to  claim 2  wherein the recombinant host cell is selected from the group consisting of bacteria, yeasts, plant cells, and algae. 
     
     
         5 . A process according to  claim 4  wherein the recombinant host cell is selected from the group consisting of  Escherichia, Salmonella, Bacillus, Lactobacillus, Acinetobacter, Streptomyces, Methylobacter, Rhodococcus, Pseudomonas, Rhodobacter, Synechocystis, Aspergillus  and  Arthrobotrys.    
     
     
         6 . A process according to  claim 4  wherein the recombinant host cell is selected from the group consisting of  Saccharomyces, Zygosaccharomyces, Kluyveromyces, Candida, Hansenula, Debaryomyces, Pichia, Mucor , and  Torulopsis.    
     
     
         7 . A process according to  claim 4  wherein the recombinant host cell is selected from the group consisting of soybean, rapeseed, pepper, sunflower, cotton, corn, tobacco, alfalfa, wheat, barley, oats, sorghum, rice,  Arabidopsis , cruciferous vegetables, melons, carrots, celery, parsley, tomatoes, potatoes, strawberries, peanuts, grapes, grass seed crops, sugar beets, sugar cane, beans, peas, rye, flax, hardwood trees, softwood trees, and forage grasses. 
     
     
         8 . A process according to  claim 1  wherein the enzyme source is immobilized. 
     
     
         9 . A process according to  claim 1  wherein the extractant is present in the biphasic reaction medium in an amount from about 5% to about 70% by volume. 
     
     
         10 . A process according to  claim 1  wherein the extractant is present in the biphasic reaction medium in an amount from about 20% to about 50% by volume. 
     
     
         11 . A process according to  claim 1  wherein the extractant is separated from the aqueous phase by use of a gravity settler, a centrifuge, or a hydrocyclone. 
     
     
         12 . A process according to  claim 1  wherein the enzyme source is recovered from the aqueous phase of the biphasic reaction medium after the separating of step (c) for reuse. 
     
     
         13 . A process according to  claim 12  wherein the enzyme source is recovered from the aqueous phase using a method selected from the group consisting of filtration, ultrafiltration, nanofiltration, and centrifugation. 
     
     
         14 . A process according to  claim 1  wherein the recovering of step (d) is accomplished by means selected from the group consisting of evaporation, distillation, adsorption by resins, and adsorption by molecular sieves. 
     
     
         15 . A process according to  claim 1  wherein after step (d), the extractant is optionally added back to the biphasic reaction medium. 
     
     
         16 . A process according to  claim 1  wherein the aqueous phase after step (c) is optionally added back to the biphasic reaction medium. 
     
     
         17 - 41 . (canceled)