Integrated development of an effective bioprocess for extracellular production of penicillin G acylase in Escherichia coli and its subsequent one-step purification.
An integrated bioprocess for effective production and purification of penicillin G acylase (PAC) was developed. PAC was overexpressed in a genetically engineered Escherichia coli strain, secreted into the cultivation medium, harvested, and purified in a single step by anion-exchange chromatography. The cultivation medium developed in this study had a sufficiently low conductivity to allow direct application of the extracellular fraction to the anion-exchange chromatography column while providing all of the required nutrients for sustaining cell growth and PAC overexpression. It was contrived with the purposes of (i) providing sufficient osmolarity and buffering capacity, (ii) minimizing ionic species to facilitate the binding of extracellular proteins to anion-exchange media, and (iii) enhancing PAC expression level and secretion efficiency. Employing this medium recipe the specific PAC activity reached a high level at 871 U/g DCW, of which more than 90% was localized in the extracellular medium. In addition, the osmotic pressure and induction conditions were found to be critical for optimal culture performance. The formation of inclusion bodies associated with PAC overexpression tended to arrest cell growth, leading to potential cell lysis. Clarified culture medium was applied to a strong anion-exchange (Q) column and PAC was purified by non-retentive separation, where most contaminant proteins bound to the chromatographic media with PAC being collected as the major component in the flow-through fraction. After removing the contaminant oligopeptides using ultrafiltration, purified PAC with a specific activity of 16.3 U/mg was obtained and the overall purification factor for this one-step downstream purification process was up to 3 fold.
Department of Chemical Engineering, University of Waterloo, 200 University Avenue West, Waterloo, Ontario N2L 3G1, Canada.
Valerie
V
Orr
Jeno
J
Scharer
Murray
M
Moo-Young
C Howie
CH
Honeyman
Drew
D
Fenner
Lisa
L
Crossley
Shing-Yi
SY
Suen
C Perry
CP
Chou
D001709:Biotechnology / Q000379:methods*; D015153:Blotting, Western; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005818:Genetic Engineering / Q000379:methods; D002479:Inclusion Bodies / Q000378:metabolism; D007218:Industrial Microbiology / Q000379:methods*; D010399:Penicillin Amidase / Q000096:biosynthesis* / Q000302:isolation & purification*; D014462:Ultrafiltration
D010399:Penicillin Amidase