Expression of xyloglucan endotransglycosylases of Gerbera hybrida and Betula pendula in Pichia pastoris.
The plant enzyme xyloglucan endotransglycosylase (XET; EC 2.4.1.207, xyloglucan:xyloglucosyl transferase) participates in selective modification of plant cell walls during cell growth. XETs are potential catalysts in various applications. 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 degrees C than at 30 degrees 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 16l 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.
Toikkanen
Jaana H
JH
VTT, Technical Research Centre of Finland, P.O. Box 1000, FI-02044 VTT, Finland. jaana.toikkanen@vtt.fi
Niku-Paavola
Marja-Leena
ML
Bailey
Michael
M
Immanen
Juha
J
Rintala
Eija
E
Elomaa
Paula
P
Helariutta
Yrjö
Y
Teeri
Teemu H
TH
Fagerström
Richard
R
D000595:Amino Acid Sequence; D019659:Asteraceae / Q000201:enzymology* / Q000235:genetics; D029662:Betula / Q000201:enzymology* / Q000235:genetics; D018533:Biomass; D019149:Bioreactors; D001709:Biotechnology / Q000379:methods*; D018929:Cell Culture Techniques; D003470:Culture Media; D005374:Filtration; D015870:Gene Expression; D016695:Glycosyltransferases / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000432:Methanol / Q000378:metabolism; D008969:Molecular Sequence Data; D010843:Pichia / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D003470:Culture Media; D016695:Glycosyltransferases; C073693:xyloglucan endotransglycosylase; D000432:Methanol