Recombinant l‐asparaginase production using Pichia pastoris (MUT^s strain): establishment of conditions for growth and induction phases

Abstract

BACKGROUND: L-asparaginase (ASNase), a biopharmaceutical enzyme used in the treatment of childhood lymphoid malignancies, is commercially produced from Escherichia coli and Erwinia chrysanthemi. However, it causes severe adverse effects due to allergenic prokaryotic epitopes on the protein surface. ASNase II from Saccharomyces cerevisiae can be a promising alternative source of this enzyme. In this study, conditions to produce ASNase from S. cerevisiae expressed in Pichia pastoris have been investigated in shake flasks and 3 L-bioreactor. We evaluated if medium composition, concentration of carbon source (i.e. glycerol), growth time, concentration of inducer (i.e. methanol), temperature and initial pH influenced both biomass and ASNase expression. RESULTS: Biomass of around 53 g L-1 and ASNase volumetric activity of 710 U L-1 were achieved using the buffered glycerol-complex medium (BMGY) containing 40 g L-1 glycerol, with induction after 141 h using 3.0% (v/v) methanol, at 20°C and initial pH 6.0. CONCLUSION: The experiments performed in shake flasks were scalable to a 3 L-bioreactor, suggesting that this bioprocess could be scaled-up for industrial production.

Optimizing medium composition with wastewater from Coffea arabica processing to produce single‐cell protein using Candida sorboxylosa

“…Biomass was measured as DCW using the protocol detailed by Pillaca‐Pullo et al 24 . Pre‐weighed dry Falcon tubes containing 40 ml cell suspension were centrifuged at 3320 × g for 20 min at room temperature.…”

“…Measurement of biomass, sugar, total protein, and SCP yield Biomass was measured as DCW using the protocol detailed by Pillaca-Pullo et al 24 Pre-weighed dry Falcon tubes containing 40 ml cell suspension were centrifuged at 3320 × g for 20 min at room temperature. The supernatants were discarded, the pellets were washed twice with distilled water, and the tubes containing the cell pellets were dried at 50°C in an oven until a constant weight was achieved.…”

Effects of Oxygen Transference on Protease Production by Rhodotorula mucilaginosa CBMAI 1528 in a Stirred Tank Bioreactor

“…In the case of enzyme production, large-scale production is preferred using bioreactors. However, this production is first established at the laboratory level to reach a larger scale under equal or improved yield [ 46 ]. The stage for establishing conditions can be carried out in shake flasks or bioreactors, despite the fact that the physical and biological factors would be different in these systems.…”

“…The maximum specific growth rate ( µ max ) (2) and the substrate-to-cell conversion factor ( Y X/S ) (3) were calculated according to equations reported by Pillaca-Pullo et al [ 46 ]: where X f is the cell concentration during the exponential phase, X 0 is the initial cell concentration, and t f and t 0 are the final and initial time, respectively. X max is the maximum cell concentration, X 0 is the initial cell concentration, and S 0 and S f are the initial and final glucose concentration, respectively.…”

Reusing wastewater from Coffea arabica processing to produce single‐cell protein using Candida sorboxylosa: Optimizing of culture conditions

“…Biomass was determined by measuring the dry cell weight (DCW) after evaporating the water content, following the method described by Pillaca‐Pullo et al 25 Briefly, pre‐weighed dry Falcon tubes were filled with 40 mL of fermented medium and centrifuged at 3320 g for 20 min at room temperature. The supernatant was discarded, and the tubes containing the cell pellets were dried at 50°C until a constant weight was obtained.…”