L-Tryptophan → D-Tryptophan

Participants

Reaction Steps

Total protein assays were done using a Pierce BCA kit (Rockford, Ill.). Tryptophan racemase assays with the mutant enzyme were performed in triplicate using the wild-type enzyme prepared in the same manner as a positive control. The assays contained per mL: 30 mM L-tryptophan, 50 mM potassium phosphate pH8, 10 μM PLP, and approximately 200 μg of racemase protein in a cell free extract. Zero, 20 minute, 1 hour, and overnight samples were collected, treated with 2% formic acid, filtered, and diluted 1:10 for analysis using the chiral amino acid method described in Example 1. At the 20 minute time point, the Y396C mutant produced approximately 264 ppm D-tryptophan, as compared to 761 ppm for the wild-type enzyme. At 1 hour, the mutant produced an average of 444 ppm D-tryptophan, as compared to 1110 ppm for the wild-type enzyme. The overnight samples contained 2300 ppm D-tryptophan, whereas the wild-type enzyme had reached equilibrium by producing approximately 3000 ppm D-tryptophan.

Provenance & Evidence