JOURNAL OF LIGHT INDUSTRY

CN 41-1437/TS  ISSN 2096-1553

Volume 35 Issue 3
May 2020
Article Contents
WEI Tao, ZHAO Caimeng, JIA Weiwei, et al. Expression and purification of fructosyl transferase AoFT in Pichia pastoris and study on its enzymatic properties[J]. Journal of Light Industry, 2020, 35(3): 1-10. doi: 10.12187/2020.03.001
Citation: WEI Tao, ZHAO Caimeng, JIA Weiwei, et al. Expression and purification of fructosyl transferase AoFT in Pichia pastoris and study on its enzymatic properties[J]. Journal of Light Industry, 2020, 35(3): 1-10. doi: 10.12187/2020.03.001 shu

Expression and purification of fructosyl transferase AoFT in Pichia pastoris and study on its enzymatic properties

  • Received Date: 2019-12-12
  • Using genetic engineering techniques,the recombinant plasmid pPICZαA-AoFT was linearized,and then electroporated into Pichia pastoris X33 to construct recombinant Pichia pastoris engineering strain pPICZαA-AoFT/X33 and achieve the recombinant fructosyl transferase AoFT in Pichia pastoris.Furthermore,the enzymatic properties of the purified fructosyl transferase AoFT were studied.The results showed that the pPICZαA-AoFT/X33 multiplied rapidly with the increase of fermentation time.The OD600 value of the cell reached a peak at 120 h,and the total protein concentration of the extracellular protein in the fermentation broth was 1.64 mg/mL.The specific activity of the purified fructosyl transferase AoFT enzyme was 71.4 U/mg,and the yield was 70.6%.The peptide N-glycosidase F digestion reaction confirmed that the purified fructosyl transferase AoFT had some N-glycosylation sites.The optimal reaction temperature of fructosyl transferase AoFT was 45 ℃,and the optimal buffer system was sodium acetate buffer (pH value was 4.5~6.0).Metal ions Mn2+ and Ca2+ could activate fructosyl transferase AoFT,while Al3+,Fe2+,Ni2+,Zn2+ and EDTA could inhibit fructosyl transferase AoFT.Surfactants (SDS,Tween-20 and Triton X-100) had little effect on the activity of fructosyl transferase AoFT,while the reductant DTT had obvious inhibition effect on the activity of fructosyl transferase AoFT.The low concentration of PMSF had no significant effect on the activity of fructosyl transferase,but the high concentration could reduce the activity.The activity of fructosyl transferase AoFT showed strong resistance to organic solvents (ethanol,DMSO,dimethylformamide,n-butanol,n-hexane and toluene).
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