Elastin Proteins Modified R-ω-transaminase Used For Catalytic R-phenylethylamine

R-co-transaminase is a kind of enzyme that refers to selectively catalytic R-amine generate transamination reaction which the substrate or product must have no a-amino acids or α-keto acids.5’-pyridoxal phosphate (PLP) assisted to complete the whole amino exchange catalytic process. R-co-transaminase is widely researched and applied in the resolution of raceme and synthesis of chiral amine. Started with R-co-transaminase genes from Arthrobacter sp. KNK 168,we constructed several expression vectors to express and purify,then explored the multiple enzyme system in the application of catalytic R-phenylethylamine.First of all, we constructed expression vector of pET28a-RTA through genetic method. In order to use more simple and low-cost method for purification, ELP was introduced as a kind of elastic protein purification tag.So we constructed expression vector of pET28a-10ELP- RTA. Compared with enzyme activity, we found the single enzyme RTA enzyme activity is very similar with that of 10ELP-RTA. May safely draw the conclusion that ELP did not affect enzyme activity of RTA. Besides, the two proteins have similar secondary structures in the results of circular dichroism spectrum.Then, in order to move forward the balance of reaction under the condition of lack of pyruvic acid, using DAAO of converting the by-product D-Ala to pyruvic acid, so as to realize pyruvic acid cycle, DAAO is introduced in the reaction system to build a double enzyme reaction system. Results from the conversion:RTA single enzyme conversion rate is very low, and the double enzyme system of 13 h conversion rate as high as 99.6%.With a laser confocal microscope,we noticed that for the single enzyme system, in the reaction media, ELP-RTA self-assembled and formed enzyme clusters of micrometer size, and the substrate, (R)-1-phenylethylamine, also formed droplets of micrometer size.Intimate contact of the enzyme clusters and the substrate droplets provided a microenvironment of high substrate concentration close to the enzyme,facilitating the diffusion of substrate molecules into the active sites. For the two-enzyme system, ELP-RTA and ELP-fusion D-amino acid oxidase assembled to form two-enzyme complexes, forming clusters with a size much larger size than that of single enzymes which promoted the whole conversion greatly.Finally, using CAT, H2O2 generated by DAAO can be into H2O and O2, intein mediated RTA-DAAO and CAT-DAAO double enzyme splicing system was apllied to explore three enzyme catalysis system RTA-DAAO and CAT-DAAO effects on conversion under the condition of 30 mM R-phenethylamine and 2 mM pyruvate sodium. As can be seen from the results, the conversion rate was very fast in presence of CAT.