Production of extracellular aspartic protease in submerged fermentation with Mucor mucedo DSM 809

Fungal milk-clotting enzymes have gained value as bovine Chymosin substitutes in the cheese industry. In this work, the effects of culture conditions on the production of extracellular milk clotting enzymes from Mucor mucedo DSM 809 in submerged fermentation were studied. The maximum activity was observed after 48 h of cultivation at 24°C in Erlenmeyer flasks. The optimized initial pH and shaking speed for enzyme production were 4.5 and 220 rpm, respectively. Glucose at a concentration of 1% (w/v) was the best carbon source for the production of enzyme among the carbohydrates examined (glucose, fructose, lactose, maltodextrin). On the other hand casein at a concentration of 0.5% (w/v) was the selected nitrogen source in the media formulation. Under optimized conditions enzyme levels reached 130 SU per ml fermentation broth. The inoculum type and size has also affected biomass production and the biosynthesis of the enzyme. The preferred method was the inoculation of the culture media with spores at a total load of 6x105 spores per flask.Key words: Milk clotting enzyme, Aspartic protease, Mucor mucedo, Sub-merged fermentation

LYTAG-driven purification strategies as a key to integrate and intensify the downstream processing of monoclonal antibodies

Monoclonal antibodies (mAbs) are currently the most important class of recombinant protein therapeutics in the biotechnological and biopharmaceutical industry with more than 250 mAbs currently undergoing clinical trials. High titer producing cultures and complex mixtures containing high cell densities, together with an increasing growing demand for highly pure mAbs is making recovery and purification processes hot targets for improvement and opens important technological challenges in mAbs manufacturing platforms. This work explores the use of an affinity dual ligand based on a choline binding polypeptide tag (LYTAG) fused with the synthetic antibody Z domain (LYTAG-Z) as a tool to integrate and optimized the downstream processing of mAbs. Upon addition of this ligand to an animal cell culture broth, antibody-LYTAG-Z complexes are formed which can be easily captured and separated from host cell impurities by affinity partitioning in aqueous two-phase systems (ATPS) composed of polyethylene glycol –PEG, as PEG molecules have the ability to binding to the choline binding sites of LYTAG. Integration of clarification and primary mAbs recovery was successfully accomplished using a system composed of 6% PEG 3350 Da and 7% dextran 500,000 Da in which an extraction yield of 89% and a clarification higher than 95% were achieved. IgG-rich phases were further processed by chromatography, using three different strong anion exchange matrices charged with quaternary methyl amines (a choline analogue) – CIMmultus QA, HiTrap Q FF and gPore NW Q. A two-elution method was developed for the separation of the antibody-LYTAG-Z complexe, allowing simultaneous purification of the antibody and recovery of the ligand. The process was successfully scale-up 10000 times allowing a global antibody recovery of 70% with a purity of 89% and enabling 100% cell removal

Outcomes from elective colorectal cancer surgery during the SARS-CoV-2 pandemic

This study aimed to describe the change in surgical practice and the impact of SARS-CoV-2 on mortality after surgical resection of colorectal cancer during the initial phases of the SARS-CoV-2 pandemic

Affinity chromatography with pseudobiospecific ligands on high-performance supports for purification of proteins of biotechnological interest

High-performance affinity matrices were obtained by attaching pseudobiospecific ligands to hollow-fibre membranes. The neutral protease contained in FlavourzymeTM was purified to homogeneity with Yellow 4R-HE affinity hollow-fibre membranes. Immobilisation of Red HE-3B allowed purification of a milk-clotting enzyme obtained by solid-state culture of Mucor bacilliformis. Copper immobilisation through iminodiacetic acid allowed fractionation of Biocon Bioconcentrated PlusTM to separate the pectinesterase-containing fraction. The productivity of the developed processes - 1900, 94 and 750 U/ml.min, respectively - was 10- to 15-fold higher than that achieved with the same ligands immobilised on agarose-based soft gels, mainly due to the shortening of the purification processes