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 producing vanillin comprising:
a. adding 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;
b. after said conversion of ferulic acid to vanillin, extracting the vanillin from the biotransformation solution to produce a concentrated extract containing 200-230 g/L of vanillin, wherein the extracting is performed by adding 5-50% (w/v) of macroporous absorbent resin to said biotransformation solution and then eluting the vanillin from the resin; and
c. crystallizing the vanillin from the concentrated extract thereby producing purified vanillin using said Amycolatopsis sp. zhp06 cells.
2. The method of 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, and 0.05-0.5 g/L ferulic acid.
3. The method of claim 2 , wherein the carbon source is selected from glucose, starch, sucrose, fructose and maltose.
4. The method of claim 2 , wherein said fermentation medium is sterilized at 121° C. for 20 min.
5. The method of claim 1 , wherein said substrate solution contains 3-40 g/L ferulic acid, 1-10 g/L Na 2 HPO 4 .10H 2 O, and 0.1-0.5 g/L KH 2 PO 4 having pH 7.5-7.9.
6. The method of claim 1 , wherein said conversion of ferulic acid to vanillin is performed at 28-45° C. for 20-70 hours.
7. The method of 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.
8. The method of claim 1 , wherein after 5-12 hours of said conversion of ferulic acid to vanillin, the vanillin is extracted and eluted from the resin with acetic ether or alcohol to, and wherein the eluent is dried to produce the concentrated extract.
9. The method of claim 8 , wherein the eluent is dried by anhydrous sodium sulfate and is vacuum evaporated to produce the concentrated extract.
10. The method of claim 8 , wherein the macroporous absorbent resin is HZ-16 or HZ-802.
11. The method of claim 10 , wherein said eluent is dried by anhydrous sodium sulfate for 12-15 hours.
12. The method of claim 1 , further comprising recycling said Amycolatopsis sp. zhp06 cells from the biotransformation solution and adding said cells into a fresh biotransformation solution comprising the ferulic acid for another vanillin production.
13. The method of claim 1 , wherein said Amycolatopsis sp. zhp06 cells is isolated from the standard cell culture by centrifugation.
14. A method for producing vanillin comprising:
a. adding Amycolatopsis sp. zhp06 cells to a substrate solution comprising about 3-40 g/L ferulic acid to form a first biotransformation solution, whereby the ferulic acid is converted to vanillin using said Amycolatopsis sp. zhp06 cells as a biocatalyst;
b. after said conversion reaction of ferulic acid to vanillin, recycling said Amycolatopsis sp. zhp06 cells from the first biotransformation solution; and
c. adding the recycled Amycolatopsis sp. zhp06 cells into a second biotransformation solution comprising ferulic acid for another vanillin production.