Maximization of uricase production in a column bioreactor through response surface methodology-based optimization

Area of Science:

• Biotechnology
• Enzyme Engineering
• Industrial Microbiology

Background:

• Uricase (EC 1.7.3.3) is a crucial oxidoreductase enzyme with significant medical applications in diagnostics and therapeutics.
• Efficient large-scale production of active uricase is essential for meeting clinical and research demands.

Purpose of the Study:

• To optimize recombinant uricase production from E. coli BL21 in a bubble column bioreactor (BCB).
• To determine the optimal culture conditions for maximizing uricase enzyme activity.

Main Methods:

• Utilized Plackett-Burman design to identify key variables affecting uricase activity from eight factors.
• Employed response surface methodology (RSM) with central composite design for optimizing inoculum size, IPTG concentration, and initial pH.
• Monitored uricase activity, optical density at 600 nm (OD600), and final pH as response variables.
• Purified recombinant uricase using Ni-NTA agarose affinity chromatography.

Main Results:

• Identified inoculum size (3% v/v), IPTG concentration (0.54 mM), and initial pH (6.0) as critical optimization parameters.
• Achieved a maximum uricase activity of 5.84 U·ml−1 and a final OD600 of 3.42 under optimized conditions.
• Successfully purified 165 ± 1.5 mg of uricase from a 600 ml cell culture.

Conclusions:

• Bubble column bioreactors (BCBs) are highly effective for the large-scale production of recombinant uricase.
• The optimized conditions provide a robust protocol for maximizing enzyme yield and activity.