L-Asparaginase (ASNase) is used in the treatment of acute lymphoblastic leukemia, being produced and commercialized only from bacterial sources. Alternative Saccharomyces cerevisiae ASNase II coded by the ASP3 gene was biosynthesized by recombinant Pichia pastoris MUTs under the control of the AOX1 promoter, using different cultivation strategies. In particular, we applied multistage fed-batch cultivation divided in four distinct phases to produce ASNase II and determine the fermentation parameters, namely specific growth rate, biomass yield, and enzyme activity. Cultivation of recombinant P. pastoris under favorable conditions in a modified defined medium ensured a dry biomass concentration of 31 gdcw.L−1 during glycerol batch phase, corresponding to a biomass yield of 0.77 gdcw.gglycerol-1 and a specific growth rate of 0.21 h−1. After 12 h of glycerol feeding under limiting conditions, cell concentration achieved 65 gdcw.L−1 while ethanol concentration was very low. During the phase of methanol induction, biomass concentration achieved 91 gdcw.L−1, periplasmic specific enzyme activity 37.1 U.gdcw-1, volumetric enzyme activity 3,315 U.L−1, overall enzyme volumetric productivity 31 U.L−1.h−1, while the specific growth rate fell to 0.039 h−1. Our results showed that the best strategy employed for the ASNase II production was using glycerol fed-batch phase with pseudo exponential feeding plus induction with continuous methanol feeding.

Background Coffee production is an important agro‐industrial activity in several countries worldwide. However, during wet processing of coffee beans, substantial quantities of wastewater effluents are generated and discharged into nearby water systems, negatively impacting ecosystems. A compelling biotechnological alternative involves the use of nutrients contained in wastewater (e.g., sugars, proteins, and salts) to produce value‐added molecules–such as single‐cell protein (SCP) using the yeast Candida sorboxylosa–with high potential for use as animal feed. Results Yeast isolated from the feces of ring‐tailed coatis showed a positive assimilation of reducing sugars (glucose, mannose, and fructose). Efficient reuse of coffee wastewater (CWW) for SCP production was achieved via the statistical design of experiments (DoE) technique. The bioprocess was standardized in a medium culture composed of 87.5% (v/v) CWW, and of 1.38 and 7.24 g/L yeast extract (YE) and ammonium sulfate [(NH4)2SO4], respectively, resulting in a high SCP yield of approximately 38%. Conclusion The reuse of CWW shows potential as a low‐cost component for SCP production that can ultimately reduce the environmental impact of wastewater effluent by circumventing its release into river systems. The bioprocess developed in this study presented certain advantages, including high volumes of CWW with only YE and (NH4)2SO4 supplementation, and without requiring additional components to correct the pH of the medium. Therefore, it is highly feasible to apply this bioprocess in close proximity to coffee‐processing agrobusinesses, thus generating an alternative source of income for Peruvian coffee‐producing communities. © 2022 Society of Chemical Industry (SCI).

Highlights A protease from a psychrotolerant yeast was characterized. Protease production was dependent on temperature and medium composition. Mass spectrometry analysis indicated that the protein belongs to the pepsin family. We propose that the enzyme reported here could be Rodothorulapepsin.

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.