Modification Of L-Tyrosine Hydroxylase And Double Enzyme System Mediated By Intein

L-tyrosine hydroxylase plays a very important role in biology,it catalyzes the conversion of L-tyrosine to L-dopa,the lack of tyrosine hydroxylase will lead to damage the synthesis of dopamine,adrenaline and norepinephrine.The special properties of the elastin-like polypeptides is that allow the solubility to vary depending on the ambient temperature,and this property is still effective after it is connected with other proteins,we use it to improve the protein's solubility,stability,beyond that it provide a method of purification.In this study,L-tyrosine hydroxylase is a class of heme dependent peroxidase,it origins from orf gene of Streptomyces refuineus subsp..In this paper,we used the pET28a-HDP vector and pET28a-HDP-ELP vector of our laboratory constructed to transform E.coli BL21.In the condition of 25 ?,0.4 mM IPTG,200 rpm,inducing 10 h lead to efficient expression.HDP protein was successfully purified by Ni-NTA column,HDP-ELP was purified by the ELP ITC cycle.After investigation,it was found that the solubility of HDP-ELP and HDP at 90 min was about 93%and 63%respectively;The results of CD spectra show that the addition of ELP does not affect the structure of the HDP,and it will improve its tolerance to hydrogen peroxide;The L-Tyr conversion rate of HDP-ELP and HDP catalyzing after 90 min was about 70.3%and 21.1%respectively;In different temperature and pH environment,HDP-ELP showed very stable enzyme activity.Except L-Tyr is the substrate of HDP,hydrogen peroxide also is necessary for catalytic,therefore,this paper introduces the D-amino acid oxidase to catalyze D-alanine and the production of hydrogen peroxide provides H2O2 for HDP.The pETDuet-HDP-IN-Ic-DAAO vector was successfully constructed by the method of genetic engineering.After transforming E.coli BL21,in the condition of 25 ?,0.4 mM IPTG,200 rpm,inducing 10 h lead to efficient expression of HDP-DAAO.HDP-DAAO was successfully purified by Ni-NTA chromatography.Comparing the enzyme activity of HDP-DAAO with H2O2 and D-Ala,the conversion rate of L-Tyr was 52.8%and 66.3%respectively;The specific activity of DAAO of HDP-DAAO was higher than that of single enzyme;the results of CD spectra showed that the construction of HDP-DAAO fusion protein had no effect on the structure of HDP;The fluorescence quenching experiments showed that the stability of HDP-DAAO fusion protein was better than that of HDP.