Here, sequences encoding two XETs from Gerbera hybrida and Betula pendula are reported. The encoded proteins, which are 51% identical at the amino acid level, were expressed in the yeast Pichia pastoris in secreted form with the aid of mating factor alpha signal sequence.
XET production in shake flask cultivations was better at 22 °C than at 30 °C. Both the yield of protein of expected molecular mass and the XET activity improved at the lower temperature. Under all cultivation conditions studied, higher amounts of XET from B. pendula (BXET) were expressed than XET from G. hybrida (GXET).
Both XET enzymes were produced in 16 l fed-batch bioreactor cultures. GXET was produced in methanol-limited fed-batch cultivation in minimal medium, and BXET in temperature-limited fed-batch (TLFB) in minimal or complex medium.
Production was highest in TLFB in complex medium. BXET was purified from the culture filtrate and characterized. Based on the specific activity of the purified protein, 60–70 mg l−1 BXET was produced in the TLFB in complex medium.
- Xyloglucan endotransglycosylase (XET)
- Pichia pastoris
- Heterologous expression
- Bioreactor cultivation
- Gerbera hybrida
- Betula pendula