Cloning And Expression Of A-glucosidase Gene From Thermotoga Neapolitana

Alpha-glucosidase (EC3.2.1.20), also named as a-transglucosidase. It could release the glucose by incising the biberation of α-glucose from the non-reducing end of oligosaccharides, it also tranfer the free glucose residues to another sugar substrate as the form of a-1,6glycosidic bond. As we known, a-glucosidase plays a key role in the production process of Iso-maltooligosaccharide. Thernotoga neapolitana is a kind of heat-resistant and ancient bacteria, it can produce a variety of heat-resistant enzymes, such as a-glucosidase, xylanase and cellulase, and these enzymes have broad prospects in the range of industrial to the food industry. It’s very difficult to achieve mass production of the heat-resistant enzymes, because of the thermotoga neapolitana should cultured in high-temperature and anaerobic environments. In this thesis, we propose to complete the cloning and expression of a-glucosidase gene from Thermotoga neapolitana DSM4359in the Escherichia coli, all these works will made a good foundation of its application.1. Cloning and expression of a-glucosidase gene fromT.neapolitana DSM4359At the base of the a-glucosidase gene sequences of T.neapolitana DSM4359obtained from Gene Bank, in combination with the expression vector pET32a (+), the specific primers were designed using the Primer5. About1.4kb DNA fragment was obtained by PCR from genomic DNA using the specific primers, then proceed the TA cloning and sequencing. The target gene fragment and expression vector was cleaved with BamH I and Xho I and ligated using T4DNA ligase, after transformed into competent cells E.coli DH5a, transformants pET32a-tnag were gained. The recombinants was digested by the BamH I and Xho I too,5.9kb and1.4kb fragment were obtained respectively, which were similar to the size of the expression vector pET32a (+) and the target gene, it primarily confirmed that the clone of a-glucosidase gene is correct. Recombinants were sequenced by Shanghai Sangon Company, the sequence is as same as the original sequence, it is also proveed that the transformant DNA was the gene of a-glucosidase. The gene’s open reading frame is1443bp, could encoding480amino acids. The recombinants pET32a-tnag was transferred into the E.coli Rosseta to obtained the engineered bacteria R-tnag, after6hours of0.8mmol/L IPTG induction at30℃, it was observed that the total secreted protein in cluture migrated as the main bands of80KDa on SDS-PAGE.2. The purification of recombinant a-glucosidase and characterization of the pure enzymeThe a-glucosidase expressed in E. coli has a with a6×His tag at the carboxy-terminal, it also could bear the heat treatment. First, the recombinant enzyme was heated for20minutes at65℃, and then using Ni-NTA affinity column to pure the recombinant enzyme once more, it was observed that a single, clear and pure enzyme bands on SDS-PAGE. Considered the pNPG as the substrate, the optimum temperature of a-glucosidase was80℃and has a good temperature stability. The optimum pH value was5.0and good pH stability from4.0to6.0. Fe3+, Fe2+and EDTA can greatly activate the enzyme activity, and Ag+and Al3+can significantly inhibit the activity, and SDS, Ca2+and Ni2+can slightly inhibit the enzyme. At80℃and pH5.0, the Km and Vmax of α-glucosidase were1.4387mmol/L and2.0734mmol/L·min respectively.