The Clone And Recombinant Expression Of Fructosyltransferase Code Gene From Aspergillus Oryzae

In this paper, we used molecular cloning techniques to obtain fructosyltransferase genefrom an Aspergillus oryzae strain that yielded higher fructosyltransferase. We found that thesequence of this gene differ from that in the database by sequence ansignment. The gene wasisolated and purified, then cloned into the Escherichia coli prokaryotic expression system,andPichia pastoris eukaryotic expression system respectively to achieve the recombinantexpression of fructosyltransferase protein in cellular or extracellular, which lay the frameworkfor further improving the enzyme production and large-scale fructosyltransferase production.We first extracted total RNA from a A. oryzae strain SBB201by Trizol method, and thengained cDNA by RT-PCR. We designed specific primer pairs P-PEASY-S/P-EEASY-A andP-pPICZαB-A/P-pPICZαB-S according to the sequence of the fructosyltransferase gene ofAspergillus oryzae from GenBank，and amplified a1782bp target gene. Then, this gene waslinked to the prokaryotic vector PMD-20T and obtained the recombinant plasmid of the targetgene, which were named DH5α/PMD20-Ftranse-bend and DH5α/PMD20-Ftranse-E/Xend.The result of sequence alignment showed that the simaliarity between this gene sequence andthe online date was98.93%.Based on the recombinant strain DH5α/PMD20-Ftranse-bend, we inserted the targetgene into the prokaryotic expression vector pEASY-E1and then transformed recombinantplasmid into E.coli strain. The new recombinant strain was namedBL21-DE3/pEASY-Ftranse-bend. IPTG was added into the medium of the recombinant strainto induce the expression of objective protein which was detected subsequently by SDS-PAGEgel electrophoresis..The result showed that the recombinant strain could produce targetproteins whose molecular weight was64KDa. By optimizing the induction condition, wedetermined that the expression amount of the recombinant protein was highest when theconcentration of IPTG was1.0μmol/mL and the induction temperature was25℃. We usedhistidine6-tag to purify the target protein and got the enzyme with highly purified.By sourceconversion reaction, we determined that the activity of the recombinant enzyme was59.0U/g.Based on the recombinant strain DH5α/PMD20-Ftranse-E/Xend, the target gene was linked to eukaryotic expression vector pPICZαB, and transformed into P. pastoris X-33byelectroporation.The new recombinant was named X-33/pPICZαB-Ftranse-E/Xend.Therecombinant strain could be cultured in the methanol media for its Mut+Phenotype, andexpressed target protein after methanol induction.The activity of the enzyme in thefermentation broth could reach to14.9U/mL.