Factors determining more efficient large-scale release of a periplasmic enzyme from E. coli using lysozyme.
Large scale use of lysozyme for periplasmic release has been impeded by the cost of the pure enzyme and its subsequent presence as a contaminant in later downstream processing steps. In this paper, we discuss the use of lysozyme for pilot scale recovery of a periplasmic enzyme from E. coli. The effects of concentration of sucrose, lysozyme and cells on periplasmic enzyme release were examined. Lysozyme concentration can be reduced 5-fold from previous reports and a reduction in sucrose concentration from 20 to 15% (w/v) allows an improvement in centrifugal harvesting by reducing viscosity. High levels of release were still achieved using this technique and further improvements in yield were obtained by optimising other components of the releasing mixture. Results show that some release is still achieved in circumstances where no lysozyme use is possible. Results also indicate that a substantial proportion (up to 70%) of lysozyme remains bound to the cellular debris after its action and is removed with this material.
Pierce
J J
JJ
Department of Chemical and Biochemical Engineering, University College London, UK.
Turner
C
C
Keshavarz-Moore
E
E
Dunnill
P
P
D004926:Escherichia coli / Q000201:enzymology*; D009113:Muramidase / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000096:biosynthesis*; D013535:Suspensions; D013997:Time Factors; D000516:alpha-Amylases / Q000096:biosynthesis* / Q000378:metabolism
D011994:Recombinant Proteins; D013535:Suspensions; D000516:alpha-Amylases; D009113:Muramidase