The Construction Of Mutation Libraries And Screening Method Of α-N-acetylgalactosaminidase

Alpha-a-N-acetylgalactosaminidase belongs to an important family of enzymes that can be used to degrade surface antigen on the surface of A-type red blood cells to eliminate the antigen-antibody reaction. It can help to relieves difficulties associate with typing and crossmatching in blood transfusion. The best activity of a-N-acetylgalactosaminidase was selected and the recombinant plasmid was constructed. With the expression and purification of a-N- acetylgalactosaminidase, its enzyme kinetics were measured. The key site of the enzyme was mutated by PCR directed mutagenesis. We hoped to get the higher efficiency of a-N-acetylgalactosaminidase through high-throughput screening. The main objectives are as follows:1. The construction ofa-N-acetylgalactosaminidaseThe coding region of an a-N-acetylgalactosaminidase (A4) was PCR amplified from genomic DNA of Chryseobacterium meningosepticum and subcloned into pET24a-A4 for overexpression His-A4. The overexpressed His-A4 enzyme was purified using affinity chromatography and has activity that is comparable with literature reported using a conventional method with an artificial substrate. To better measure the activity of a-N-acetylgalactosaminidase in real application, we established a novel method that directly use the surface antigen of red blood cell as substrate and use ELISA to detect the un-cleaved antigen. The activity of His-A4 was evaluated in the new ELISA method and was demonstrated to be able to decrease the blood cell surface antigen-antibody reaction in concentration- and time-dependent fashion.2. The screening of a-N-acetylgalactosaminidase mutation librariesThe major obstacle to use glycosidase in the preparation of universal blood is their low enzymatic activity. We hoped to get the higher efficiency enzyme by directed mutagenesis and high-throughput screening. The site of amino acid were 96,98,179,181,225 and 228 separately. We screened about 2,400 recombinant bacteria. The results showed that 179,181,225 and 228 these four sites are the key sites for the enzyme because the activity of a-N-acetylgalactosaminidase has been lost after the mutation. We got the four recombinant bacterias. Their activities were comparable to the wild bacteria. The mechanism of the changing enzyme activity should be further studied.