Production of poplar xyloglucan endotransglycosylase using the methylotrophic yeast Pichia pastoris.
The gene XET16A encoding the enzyme xyloglucan endotransglycosylase (XET) from hybrid aspen (Populus tremula x tremuloides Mich) was transformed into Pichia pastoris GS115 and the enzyme was secreted to the medium. The influence of process conditions on the XET production, activity, and proteolytic degradation were examined. Inactivation of XET occurred in the foam, but could be decreased significantly by using an efficient antifoam. Rich medium (yeast extract plus peptone) was needed for product accumulation, but not for growth. The proteolytic degradation of the enzyme in the medium was substantially decreased by also adding yeast extract and peptone to the glycerol medium before induction with methanol. Decreasing the fermentation pH from 5.0 to 4.0 further reduced the proteolysis. The specific activity was further improved by production at 15 degrees C instead of 22 degrees C. In this way a XET production of 54 mg/L active enzyme could be achieved in the process with a specific activity of 18 Unit/mg protein after a downstream process including centrifugation, micro- and ultrafiltration, and ion exchange chromatography.
Bollok
Monika
M
Department of Biotechnology, Royal Institute of Technology, KTH, Alba Nova University Center, Roslagstullsbacken 21, S-106 91, Stockholm, Sweden.
Henriksson
Hongbin
H
Kallas
Asa
A
Jahic
Mehmedalija
M
Teeri
Tuula T
TT
Enfors
Sven-Olof
SO
D018929:Cell Culture Techniques / Q000379:methods*; D004789:Enzyme Activation; D016695:Glycosyltransferases / Q000096:biosynthesis* / Q000235:genetics; D000432:Methanol / Q000378:metabolism*; D010843:Pichia / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D032107:Populus / Q000201:enzymology* / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis
D011994:Recombinant Proteins; D016695:Glycosyltransferases; C473049:xyloglucan - xyloglucosyltransferase; D000432:Methanol