Prepacked pd 10 sephadex g 25 columns

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)}

Chemicals, biochemicals, buffers, and solvents were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO), Fisher Scientific Inc. (Pittsburgh, PA), Fluka Chemical Corp. (Milwaukee, WI), or EMD Millipore, Inc. (Billerica, MA). Reagents for molecular biology manipulations were obtained from sources listed in the text. trans-o-Hydroxybenzylidenepyruvate was synthesized as described elsewhere.^{5 (link)} The prepacked PD-10 Sephadex G-25 columns were obtained from GE Healthcare (Piscataway, NJ). The Econo-Column^® chromatography columns were obtained from BioRad (Hercules, CA). The Ni-NTA resin was purchased from Fisher Scientific (Pittsburgh, PA). The Amicon stirred cell concentrators and the ultrafiltration membranes (10,000 Da, MW cutoff) were purchased from EMD Millipore Inc. Oligonucleotide primers were synthesized by Sigma-Aldrich.