Clone And Expression Analysis Of The Amylosucrase Genes From Neisseria Polysaccharea

Amylosucrase fron Neisseria polysaccharea is a transglucosidase from family13of the glycoside-hydrolases that synthesizes an insoluble amyloselike polymer from sucrose in the absence of any primer, such as expensive ADP or UDP. Amylosucrase can synthesis starch, oligosaccharides and glycoconjugate depending on the receptor. It’s the first time that purpose polysaccharide can be synthesised for the discovery of the enzyme by the time increasing the degree of polymerization and molecular weight of the starch molecules. It provides a basis for the treatment of polysaccharide drugs such as inflammation, tumor vaccines and antivirals. Therefore, amylosucrase will be a simple, convenient, and effective glucosyl transferase and supply a broad space for development in the industry,. In this study, designing a pair of oligonucleotide primers according to the conserved sequence of sort of Neisseria. The AS of the Amylosucrase genes which from the whole genome of Neisseria Polysaccharea ATCC43768were cloned through TD-PCR. The recombinant plasmid pET-AS was constructed by inserting gene AS into expression vector pET-28a and then transformed into Escherichia coli BL21. The recombinant strain was induced by IPTG to express the high active amylosucrase and then inhance the yields of AS through response surface. The contents and results are as follows:1. Amylosucrase genes were amplified by PCR technology from Neisseria polysaccharea and cloned to plasmid pMD-18T. Sequencing analysis showed that the AS consisted of1911bp, encoding a protein of637amino acids. The nucleotide sequence of AS exhibited96%homology with ATCC85322, the amino acid sequence exhibited97%. And the amino acide sequence deduced from ORF shows homologies with Neisseria mucosa C102(86%)、Neisseria macacae(86%)、Neisseria sp (85%) respectively.2. The recombinant plasmid was transformed into E.coli.BL21(DE3) strain. Then induced by IPTG. Through optimizing the induce condition, the optimized condition is concentration of IPTG for0.8mM, induction time for5h.3. By applying response surface methodology to optimize the fermentation conditions, The optimized condition is temperature35℃, pH6.5,,rotate speed190rpm, inoculums1.9%. The activity increased to21.9higher compared to Neisseria polysaccharea. It is concluded that study on the key enzyme in the biosynthetic pathways of methionine is an effective way to enhance the methionine yields.