Cloning And Expression Of Dextransucrase Gene From Leuconostoc Mesenteroides

Dextran is one of the glucans primarily consisting ofα(1, 6) glucosidic bond. It is soluble and can be used as plasma substitute, ironcarrier or anti-coagulant, giving many industrial applications in pharmaceutical, food and feed fields. Dextransucrase (EC 2.4.1.5) is one of glucansucrase (glucosyltransferase EC 2.4) which can transfer glucosyl units from sucrose to synthesize dextran. Dextransucrases are existed in many strains of Streptococcus and Leuconostoc and some tobacoo cells. Its expression in native strains leads to enzyme production concomitantly with the dextran synthesis. Addition to the low yields, it is hard to purifiy the enzyme from ropy dextran. For the structural and functional study or for the future production and application, it is necessary to construct the recombinant strains for the high-level expression of the dextransucrase.In this paper, the gene encoding dextransucrase was amplified by PCR with the template of genomic DNA of Leuconostoc mesenteroides CGMCC 1.544, and cloned into expression vector pET28a(+), then the whole gene of dextransucrase were sequenced. The recombinant pET28a-dsrX was transformed into the expression host—E.coli BL21(DE3). SDS-PAGE analysis suggested that dsrX gene was expressed as a recombinant protein of about 170 kDa, its production was 24.5% of the total cell protein computed by the BandScan software. The expression conditions of recombinant protein in E.coli BL21(DE3) and the method of SDS-PAGE analysis were optimized. The maximum activity of recombinant protein was up to 8.8U/ml. The optimum pH, temperature and substrate concentration for both enzymes of native and recombinant were 5.4, 30℃and 10%. A comparison of the recombinant enzyme and the native enzyme revealed similarities in their properties. The stability of temperature and pH of the recombinant enzyme was a little weaker than the native one, so the recombinant dextransucrase was somewhat unstable. TLC and HPAEC analysis showed the recombinant enzyme could produce the dextran just like the native one. And the primary purification of recombinant protein and Western Blotting had been done.