Heterologous expression of the hydrophobin FcHyd5p from Fusarium culmorum in Pichia pastoris and evaluation of its surface activity and contribution to gushing of carbonated beverages.
The class II hydrophobin FcHyd5p from Fusarium culmorum was heterologously expressed by Pichia pastoris. Transcription of the recombinant gene was confirmed by RT-PCR and expression of FcHyd5p was demonstrated using SDS-PAGE and immuno-staining with 6 x His-tag antibodies. FcHyd5p was purified and concentrated by dialysis, isoelectric focussing and the use of ultra filtration. It was demonstrated that FcHyd5p is able to change the hydrophopic properties of surfaces rendering them wettable after coating with the supernatant of recombinant P. pastoris cultures. Furthermore, due to its surface activity, FcHyd5p was able to stabilise air bubbles in aqueous solutions. The supernatant of a culture medium containing a FcHyd5p producing P. pastoris clone remained turbid for 24h and the foam stable for more than 72 h after the treatment with a homogeniser, whereas the liquid of the wild type strain clarified after 10 min and the foam disintegrated after 2h. Finally it was demonstrated, that FcHyd5p can induce spontaneous overfoaming of carbonated liquids, referred to as gushing. Addition of 2 mg freeze-dried culture supernatant from a FcHyd5p producing P. pastoris clone resulted in a lost volume of 252 ml +/- 20 per 500 ml of beer (50%) and 179 ml +/- 7 per 330 ml of carbonated water (54%), respectively. Neither untreated beer/water, nor beer/water treated with freeze-dried culture supernatant from the wild type strain showed any gushing. Furthermore, addition of 215 microg highly purified FcHyd5p resulted in a lost volume of 77 ml +/- 40 per 500 ml beer (15%).
Stübner
Matthias
M
Lehrstuhl für Technische Mikrobiologie, Technische Universität München, Weihenstephaner Steig 16, 85350 Freising, Germany.
Lutterschmid
Georg
G
Vogel
Rudi F
RF
Niessen
Ludwig
L
D001515:Beer; D002253:Carbonated Beverages / Q000382:microbiology*; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005516:Food Microbiology; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D005670:Fusarium / Q000235:genetics*; D015870:Gene Expression; D005800:Genes, Fungal; D057927:Hydrophobic and Hydrophilic Interactions; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014867:Water
D005656:Fungal Proteins; D011994:Recombinant Proteins; D014867:Water