Research And Modification Of D-Amino Acid Oxidase And Its Dual Enzyme Catalysis System

It is said that the establishment of an efficient multi-enzymatic system has been one of the hotspots of biological engineering, which has a very important value to improve the catalytic system. In this study the multi-enzymatic systems, such as D-Amino acid oxidase and Vitreoscilla hemoglobin complex enzymatic system, D-Amino acid oxidase, Vitreoscilla hemoglobin and Tyrosine hydroxylase complex enzymatic system, are constructed by the methods of direct integration and Intein-mediated fusion. We hope that it can provide useful reference value for multi-enzymatic system to study these complex enzymatic systems and be widely used in industrial applications.Firstly, using the knowledge of molecular biology and genetic engineering means, we successfully used two different ways to constructe fusion protein expression vector, such as pET28a-DAAO-VHb? pETDuet-1-VHb-IN-Ic-DAAO. Secondly we studied and determined the optimal induction conditions of the expression strain from induction time, induction temperature, the concentration of IPTG, the number of liquid volume. Then the expression strain grown under the optimal induction conditions and the cells were collected. Thirdy the His tag can be utilized to purify recombinant protein. Finally, the recombinant protein p28-DV and DU-DV exhibited a better enzymatic activity in aqueous solutions than DAAO, and these enzymatic activities are about 1.8 times and 2.13 times that of DAAO. The circular dichroism was measured. The results demonstrated that that DU-DV retained the a-helix content with a high percentage than that of p28-DV. The result of enzymatic activity is consistent with that of the CD spectra measurements.By using the same method, we successfully constructed fusion protein expression vector, such as pET28a-DAAO-VHb-TH?pETDuet-1-TH-IN-IC-DAAO-VHb?pETDuet-l-VHb-TH-IN-IC-DAAO. We determined the optimal induction conditions. The fusion protein of DU-TDV was not successfully expressed. The others had grown and the cells were collected. The His-tag can be utilized to purify protein. The combined enzymes DU-VTD has he activity of catalytic substrate of tyrosinase. In contrast, the combined enzymes p28-DVT was lost the activity.In summary, the different ways of constructing vector will not only affect the expression of the protein, but also affect the activity of combined enzymes. Compared with the two technologies, the method of intein-mediated fusion was more conducive to building a dual-enzyme enzymatic system.