Enhancement of stability of L-tryptophan dehydrogenase from Nostoc punctiforme ATCC29133 and its application to L-tryptophan assay.

Microbial NAD(+)-dependent L-tryptophan dehydrogenase (TrpDH, EC1.4.1.19), which catalyzes the reversible oxidative deamination and the reductive amination between L-tryptophan and indole-3-pyruvic acid, was found in the scytonemin biosynthetic pathway of Nostoc punctiforme ATCC29133. The TrpDH exhibited high specificity toward L-tryptophan, but its instability was a drawback for L-tryptophan determination. The mutant enzyme TrpDH L59F/D168G/A234D/I296N with thermal stability was obtained by screening of Escherichia coli transformants harboring various mutant genes, which were generated by error-prone PCR using complementation in an L-tryptophan auxotroph of E. coli. The specific activity and stability of this mutant enzyme were higher than those of the wild type enzyme. We also revealed here that in these four mutation points, the two amino acid residues Asp168 and Ile296 contributed to increase the enzyme stability, and the Leu59, Ala234 residues to increase its specific activity. Growth of the strain harboring the gene of above 4 point mutated enzyme was accelerated by the enhanced performance. In the present study, we demonstrated that TrpDH L59F/D168G/A234D/I296N was available for determination of L-tryptophan in human plasma.