RESUMO
A laboratory exercise for undergraduate advanced students of enzymology and biocatalysis is presented. Since enzyme assays can be quenched or continuous, this experiment compares the performance of two quenching agents for lactase, in a continuous setup. Enzymatic activity of ß-galactosidase (Aspergillus oryzae) was determined based on the release of 4-nitrophenol from 4-nitrophenyl ß-D-galactopyranoside using a microplate reader. Sodium carbonate and borate buffer were tested as quenching agents, and experimental control was the unstopped assay. Based on released 4-nitrophenol, enzyme activity, and rate constant k, the students could assess the performance of each termination agent. The experiment promotes disciplinary and transversal competencies, including research-based learning, critical thinking, and introduce the students to high-throughput techniques that are common in the research and development environment.
Assuntos
Boratos , Lactase , Carbonatos , Humanos , Laboratórios , beta-GalactosidaseRESUMO
Antibodies for therapeutic use are being continuously approved and their demand has been steadily growing. As known, the golden standard for monoclonal antibody (mAb) purification is Protein A affinity chromatography, a technology that has gained high interest because of its great performance and capabilities. The main concerns are the elevated resins costs and their limited lifetime compared to other resins (e.g. ion exchange chromatography). Great efforts have been carried out to improve purification conditions, such as resin characterization and designing alkali/acid stable resins with a longer lifetime. Modification of Protein A ligands and alternative formats such as monoliths membranes and microshperes have been tested to increase the purification performance. New technology has been proposed to improve the large-scale separation; in addition, alternative ligands have been suggested to capture mAbs instead of Protein A ligand; however, most of the information is locked by pharmaceutical companies. This mini review summarizes and describes the advances, results, and impact on the Protein A chromatography purification processing.
Assuntos
Anticorpos Monoclonais/isolamento & purificação , Cromatografia Líquida/métodos , Animais , Anticorpos Monoclonais/química , Cromatografia Líquida/tendências , Humanos , Proteína Estafilocócica A/químicaRESUMO
An efficient cold-mechanical/sonic-assisted extraction technique was developed for extraction of genipin from genipap (Genipa americana) peel. Ultrasound assisted extraction (285 W, 24 kHz) was performed at 5, 10 and 15 °C for 5, 10 and 15 min. After cold-extraction, genipin was separated from pectin and proteins by aid of fungal pectinesterase. The maximum yield of non-cross-linked genipin was 7.85±0.33 mg/g, at 10 °C for 15 min by means of ultrasound extraction. The protein amount in extracts decreased in all samples. If mechanical process is combined with ultrasound assisted extraction the yield is increased by 8 times after the pectinesterase-assisted polyelectrolyte complex formation between pectic polysaccharides and proteins, avoiding the typical cross-linking of genipin. This novel process is viable to obtain non-cross-linked genipin, to be used as a natural colorant and cross-linker in the food and biotechnological industries.