Patent
This invention provides an Amycolatopsis sp. strain (zhp06), and a method of using the whole cell preparation of the strain for vanillin production. The strain was deposited in China Center for Type Culture Collection on Jul. 26, 2011 with the number of CCTCC NO: M 2011265. Under high concentrations of ferulic acid substrate, the vanillin production by this method can reach more than 10 g/L. The molar conversion rate of ferulic acid is more than 50% and the purity of vanillin is from 80% to 95%. The advantage of this invention includes: repeated use of biocatalyst cells, mild biotransformation condition, low environmental pollution, short production cycle, high product purity and simple purification procedure. It has a great potential for industrial applications.
1. A method for vanillin production using cells of Amycolatopsis sp. strain (zhp06) or its mutant strains, comprising the steps of:
(a) addition of Amycolatopsis sp. zhp06 cells isolated from a standard cell culture to a substrate solution comprising about 3-40 g/L ferulic acid to form a biotransformation solution, whereby the ferulic acid is converted to vanillin using said Amycolatopsis sp. zhp06 cells as a biocatalyst; and
(b) purification of vanillin from said biotransformation solution after 5-12 hours of said conversion reaction of ferulic acid to vanillin, wherein said purification is performed by adding 5-50% (w/v) of macroporous absorbent resin to said biotransformation solution, wherein vanillin is extracted from the solution using said absorbent resin which is then eluted with acetic ether or alcohol at 35-40° C. to produce an eluate solution containing vanillin; and wherein said eluate solution is dried by anhydrous sodium sulfate and further dried by vacuum evaporation to produce a concentrated extract containing 200-230 g/L of vanillin; and subsequently, said concentrated extract is settled at 4° C. for vanillin crystallization thereby purifying vanillin from the biotransformation solution.
2. The method according to claim 1 , wherein the standard cell culture comprises a fermentation medium containing 5-15 g/L carbon source, 1-20 g/L yeast powder, 1-10 g/L Na 2 HPO 4 .10H 2 O, 0.1-2 g/L KH 2 PO 4 , 0.1-0.5 g/L NaCl, 0.2-0.5 g/L MgSO 4 .7H 2 O, 0.01-0.1 g/L CaCl 2 .2H 2 O, 0.05-0.5 g/L ferulic acid, and wherein said fermentation medium is sterilized at 121° C. for 20 min.
3. The method according to claim 2 , wherein the carbon source is selected from glucose, starch, sucrose, fructose and maltose.
4. The method according to claim 1 , wherein said substrate solution contains 3-40 g/L ferulic acid, 1-10 g/L Na 2 HPO 4 .10H 2 O, 0.1-0.5 g/L KH 2 PO 4 (pH 7.5), and wherein said conversion of ferulic acid to vanillin was performed at 28-45° C. for 20-70 hours.
5. The method according to claim 1 , wherein a weight-to-volume ratio (w/v) of the wet weight (g) of said Amycolatopsis sp. zhp06 cells and the volume (ml) of said substrate solution is from 1:2 to 1:20.
6. The method according to claim 1 , further comprising recycling said Amycolatopsis sp. zhp06 cells from the biotransformation solution and adding said cells into a fresh biotransformation solution comprising ferulic acid for another vanillin production.
7. The method according to claim 1 , wherein said Amycolatopsis sp. zhp06 cells were isolated from the standard cell culture by centrifugation.
8. The method of claim 1 , wherein the macroporous absorbent resin is HZ-16 or HZ-802.
9. The method of claim 1 , wherein said eluate solution is dried by anhydrous sodium sulfate for 12-15 hours.