The effect of thermal deactivation on the properties and processing characteristics of E. coli.
Recombinant microorganisms are often employed to produce proteins of commercial significance. It is important to render such organisms non-viable before culture fluids are released from the fermenter. Thermal deactivation is a superficially attractive option because of its simplicity. The effects of such a thermal deactivation step at the end of a recombinant fermentation are reported in this study. In particular, the consequences of this treatment for down-stream process operations, namely homogenisation and centrifugation, are analysed and discussed. Homogenisation efficiency was adversely affected by a simple treatment whereby cells are raised to 65 degrees C from stationary phase. Cell debris size was also significantly increased. These changes could be partially explained by an increase in fractional peptidoglycan crosslinkage and a decrease in mean cell length. A simple process modification removed these detrimental effects. Specifically, the addition of 15 g of glucose 15 mins prior to thermal deactivation enhances downstream processing. Disruption efficiency is increased above that for stationary-phase cells and the resultant cell debris size is significantly reduced, theoretically aiding inclusion body purification. This novel process modification demonstrates that thermal deactivation may be employed to prevent the release of viable recombinant organisms while providing a broth with desirable processing characteristics. It also emphasises the need to optimise any bioprocess as an interconnected sequence of units.
Collis
M A
MA
Department of Chemical Engineering, University of Adelaide, Australia.
O'Neill
B K
BK
Thomas
C J
CJ
Middelberg
A P
AP
D019149:Bioreactors; D001709:Biotechnology; D015169:Colony Count, Microbial; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D005285:Fermentation; D006358:Hot Temperature; D008970:Molecular Weight; D010457:Peptidoglycan / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis; D012449:Safety; D013242:Sterilization
D010457:Peptidoglycan; D011994:Recombinant Proteins