Enzymatic Synthesis and Purification

Chemicals, biochemicals, buffers, solvents, and the components for Luria-Bertani (LB) media were obtained from sources reported elsewhere.¹³ The syntheses of 2-hydroxy-2,4-pentadienoate (2) is reported elsewhere.^{9 (link)} The 5-methyl derivative (6) was generated from 5-(methyl)-2-hydroxymuconate¹⁴ following the procedure used to produce 2.^{9 (link)} The Phenyl Sepharose 6 Fast Flow, DEAE-Sepharose resins, and the prepacked PD-10 Sephadex G-25 columns were obtained from GE Healthcare (Piscataway, NJ). The Econo-Column chromatography columns and Freeze ‘N Squeeze units were obtained from Bio-Rad Laboratories, Inc. (Hercules, CA). Enzymes and reagents used for molecular biology procedures were obtained from New England Biolabs, Inc. (Ipswich, MA). 5-(carboxymethyl)-2-Hydroxymuconate isomerase (CHMI),¹⁵ 4-oxalocrotonate tautomerase (4-OT)^{16 (link),17 (link)}, 4-OD/VPH and 4-OD/E106QVPH from P. putida mt-2 were purified by procedures reported elsewhere with some modifications.^{9 (link)} The 4-OD/VPH complex from L. cholodnii SP-6 was purified similarly to the P. putida mt-2 complex.^{9 (link)} Activities were determined using previously described assays.^{9 (link),15 –17 (link)}