The Research Of Tyrosine Hydroxylase: Expression, Purification And Activity

Tyrosine hydroxylase is a kind of enzyme that using L-Tyr as the substrate, catalyzing the ortho-hydroxylation of L-tyrosine to L-DOPA. Tyrosine hydroxylase is the rate limiting enzyme in the synthesis biological materials, such as catecholamines dopamine, epinephrine and norepinephrine, and plays a very important role on the body’s nervous system. Therefore, it has a extensive value of medical research. Most of animal and human tyrosine hydroxylase put the tetrahydrobiopterin as its cofactor, only a few bacteria tyrosine hydroxylase put heme as its cofactor. Such as in this issue the tyrosine hydroxylase puts heme b as its cofactor. The enzyme is from S.refuineus that as the rate limiting enzyme in the biosynthesis of Anthramycin.In this study we used molecular biological methods constructed the fusion expression vector of pET-28a-orfl3, pET-orfl3-ELP60 and pET-28a-ELP60-orfl3. We optimized the induction conditions of the expression such as inducing temperature, inducer concentration, induction time and loading volume. We found the optimal expression conditions of the enzyme. To facilitate easy purification, in the study we introduced a Elastin-like protein tag ELP60 as the purification tag. We found that when the ELP60 was on the C terminal of Orfl3 it has a higher activity than the ELP60 was on the N terminal of Orfl3, and it purification effect is relatively high. Therefore, in the active exploration test we used Orfl3-ELP60 protein as the interest protein.In the catalytic process the tyrosine hydroxylase needs H2O2 as a donor to provide hydroxyl. In order to get H2O2 sequentially, we introduced D-amino acid oxidase (DAAO) to provide H2O2. DAAO can catalyzed D-amino acid to produced H2O2 and a-keto acid. The addition of DAAO promotes the conversion of L-Tyr, and can be completed in a short time. In order to make the maximum L-Tyr conversions, different concentration ratio of the two substrates L-Tyr and D-Ala were studied. And found that when the concentration of D-Ala is lower than the concentration of L-Tyr, L-Tyr cannot get a higher conversion in a short period of time, and when D-Ala and L-Tyr concentration of the same, or higher than the concentration of L-Tyr, the L-Tyr transformation can be reach more than 90% in a short period of time.