Enzymatic production of glycolic acid
View Patent ↗Various methods are provided for the enzymatic production of glycolic acid from glycolonitrile. These methods include: 1) use of Acidovorax facilis 72W nitrilase mutants having improved nitrilase activity for converting glycolonitrile to glycolic acid, and 2) methods to improve catalyst stability and/or productivity. The methods to improve catalyst stability/productivity include use of reaction stabilizers, running the reactions under substantially oxygen free conditions, and controlling the concentration of substrate in the reaction mixture.
1. An isolated nucleic acid molecule encoding a polypeptide having nitrilase activity, said polypeptide having an amino acid sequence of SEQ ID NO: 6 with at least one amino acid substitution, or both amino acid substitutions, selected from the group consisting of:
a) a substitution at amino acid residue 168 with methionine or threonine; and
b) a substitution at amino acid residue 201 with glutamine, glycine, histidine, lysine, asparagine, serine, alanine, cysteine, or threonine; wherein said polypeptide provides at least a 1.5-fold increase in nitrilase activity relative to the nitrilase activity of the Acidovorax facilis 72W nitrilase when converting glycolonitrile to glycolic acid under identical reaction conditions.
2. The isolated nucleic acid molecule of claim 1 encoding an amino acid sequence of SEQ ID NO: 8, 10, 12, 14, 16, 18, 20, 22, 24, 28, or 30.
3. The isolated nucleic acid molecule of claim 2 having a nucleic acid sequence of SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21, 23, 27, or 29.
4. A chimeric gene comprising the isolated nucleic acid molecule of claim 1 operably linked to a suitable regulatory sequence.
5. An expression cassette comprising the chimeric gene of claim 4 .
6. A transformed host cell comprising the expression cassette of claim 5 .
7. The transformed host cell of claim 6 wherein said host cell is Comamonas sp., Corynebacterium sp., Brevibacterium sp., Rhodococcus sp., Azotobacter sp., Citrobacter sp., Enterobacter sp., Clostridium sp., Klebsiella sp., Salmonella sp., Lactobacillus sp., Aspergillus sp., Saccharomyces sp., Zygosaccharomyces sp., Pichia sp., Kluyveromyces sp., Candida sp., Hansenula sp., Dunaliella sp., Debaryomyces sp., Mucor sp., Torulopsis sp., Methylobacteria sp., Bacillus sp., Escherichia sp., Pseudomonas sp., Rhizobium sp., or Streptomyces sp.
8. The transformed host cell of claim 7 wherein said host cell is Escherichia coli.
9. The transformed host cell of claim 8 wherein the host cell is E. coli MG1655 having international depository number ATCC 47076 or E. coli FM5 having international depository number ATCC 53911.