bioreactor studies of heterologous protein production by ...

cerevisiae. Yeast FY 178 ceb, which secreted a-peptide, were immobilVcd in Ca-alginate
gel and a fermentation was performed in nornelective media in a column bioreactor.
Experiments showed bat both the plesmid stability and a-peptide productivity were
enhanced by immobilization. Wa& and Gainer (1989) deScnid another immobilized S.
cerevisiae system. The yeast used was the Mc16 strain S. cerevisiae containing the
piasmid vector pMA230 (2 prn based); the piasmïd encoded for the amylase. The enzyme
was secreted into the culture medium. They used the beads of gelatin as the carrier and
covalently coupled the yeast ceils to the carier surface using glutaraldehyde. The beads
were suspended in a fluidhed bed bioreactor for continuous culture using a selective
minunal medium. The immobüized œU system was examinai at a dilution rate below
washout to see if the attached population retained the plasmid while the free ail
population gradually lost it. After 50 hous of continuons culture, the fiee œil population
began to show plasmid loss. but the attached œlls nmained stable. Furthemore. the
plasrnid instability in free ceb varied with the dilution rate, greater plasrnid los was seen
at Iower dilution rates. For the attached œil population, no such variation was observed.
However, no sound explanations for these results wen offend. Later, they extended their
work by using the same yeast strah but a dinerent plasmid p520. which expressed a wheat
amylase (Walls and Gainer, 1991). The sune immobilization method was used, but a
nonselective complex medium was employed In this case, continuous suspension culture
was unstable and fapidly lost the plasmid. The plasmid stabilîty improved greatly upon
Unmobilization, and a near constant M o n of piasmidi=ontainllig cells was maintained
during continuons culture. Relative to 6ree suspension, irnmobilizaton improved protein