反应详情

The best mutants identified from o-methoxyphenol oxidation by the nylon membrane assay and colorimetric assay, T4MO TmoA I100 L, G103A, G103A/A107S, and G103S, were further examined by HPLC and found to make different regioselective products from o-methoxyphenol (FIG. 16 and FIG. 18); HPLC also was used to corroborate the initial 3-methoxycatechol formation rates determined by the colorimetric assay and to confirm the possible product identification based on the nylon membrane assay and catechol colorimetric assay. G103A synthesized 3-methoxycatechol 6 times faster than wild-type T4MO (FIG. 16) and had an increased regiospecificity for 3-methoxycatechol formation (about 52.3%). Notably, G103A/A107S produced primarily 3-methoxycatechol whose synthesis rate was more than about 7 times faster than that of wild-type (FIG. 16). The additional mutation A107S makes this enzyme yield about 30% more 3-methoxycatechol than that of G103A mutant; G103A/A107S produced about 82.5% 3-methoxycatechol after the accumulation of these two beneficial mutations. I100L was found to have nearly unchanged regiospecificity for o-methoxyphenol oxidation and produced predominantly 4-methoxyresorcinol (about 73%). The second major product, 3-methoxycatechol was made at a rate nearly 4 times faster than wild-type T4MO. The specific activities for 3-methoxycatechol formation by HPLC analysis corroborated those of the colorimetric assay (FIG. 16). In contrast, two mutants, G103S and G103S/A107T, produced methoxyhydroquinone as a major product (about 80% and about 35% respectively) from o-methoxyphenol whereas wild-type T4MO produced methoxyhydroquinone only in trace amounts (FIG. 16). Since methoxyhydroquinone auto-oxidizes easily in aqueous solutions, care was taken for analyzing this compound. HPLC confirmed the initial product identification for these two mutants via the nylon membrane and catechol colorimetric assay. G103S synthesized methoxyhydroquinone at a 5-fold faster rate and with an about 47-fold increase in the percentage of methoxyhydroquinone formation relative to wild-type T4MO (FIG. 16); however, the o-methoxyphenol oxidation rate decreased about 10-fold relative to wild-type T4MO. G103S/A107T produced methoxyhydroquinone at the same rate as the wild-type while changing its regiospecificity. To confirm that the activity of the mutants were not improved because of some artifact of growth or change in expression, growth rates, toluene oxidation, and total cellular protein profiles via SDS-PAGE were measured. Using SDS-PAGE, the expression levels of these mutants were compared with wild-type T4MO and found to remain approximately constant except for T4MO G103S/A107G which had about 5-fold reduced expression levels compared to wild-type T4MO; two T4MO subunit polypeptides TmoA (55 KDa) and TmoE (36 kDa) were seen on the gel for both the mutant and wild-type enzymes. Variant I100L grew nearly at the same rate as the wild-type (about 1.34±0.01/h vs. about 1.14±0.03/h in LB kanamycin medium). Among the six mutants (T4MO TmoA I100 L, G103A, G103A/A107S, G103S, G103S/A107T, and G103S/A107G), the toluene oxidation rates varied from about 13% to about 166% of wild-type T4MO activity (FIG. 14), indicating that the six mutants were active with toluene as a substrate even though the mutations created regiospecific changes in toluene oxidation (FIG. 14). These results also show that the changes in enzyme activity with the saturation mutants are not due to differences in protein expression; for example, if the increases in the activity for 3-methoxycatechol and methoxyhydroquinone synthesis seen with G103A/A107S were due to expression differences, toluene oxidation and o-methoxyphenol oxidation would have been enhanced proportionally; instead, this variant had about 1.5-fold increase in toluene oxidation but about 2-fold decrease in o-methoxyphenol oxidation. Similarly, G103S had about 10-fold less activity for o-methoxyphenol oxidation but about 1.5-fold increase in toluene oxidation relative to wild-type T4MO. In contrast, G103S/A107G had about 13% of wild-type toluene oxidation activity and about 4% of wild-type o-cresol oxidation activity (below), which is probably due to its about 5-fold reduced protein expression.

1. customNotably, G103A/A107S produced primarily