Cloning of 1– fructan: fructan fructosyltransferase gene and expression of recombinant 1– fructan: fructan fructosyltransferase in yeast

Abstract

Fructosyltransferases (FTases) were important enzymes for fructo-oligosaccharides (FOS) synthesis. Recently, FOS was considered as a potential prebiotic in food industry.  In particularly, FOS with short chains was used as a new alternative sweetener. The production of recombinant 1- fructan: fructan fructosyltransferase (1- FFT) in yeast system was conducted in this research. The 1- fructan: fructan fructosyltransferase gene (1- fft) cloned from 105 days old tuber of Kaentawan ( Helianthus tuberosus L.) was sub cloned to expression vector, pPICZαB by adding Pst I and Sac II sites. The cloned gene was successfully transformed to yeast Pichia pastoris X-33 by lithium chloride transformation method. The yeast transformant; P. pastoris X- 33 PF1 showed the ability to produce recombinant 1- FFT. The enzyme activity at 3.57 and 3.33 unit/L was determined in cell and culture medium, respectively. It was also found that FOS was synthesized when recombinant 1- FFT was incubated with 1- kestose and synthesized FOS as substrates. The synthesis of FOS was not detected when sucrose was used as substrate of recombinant 1- FFT.