Chapter 3 enzyme production and purification 20141009

ENZYME PRODUCTION Surface and submerged fermentation techniques  Surface = enzyme produced on the surface of a solid medium  Submerged = the mould or bacterium producing enzyme is grown throughout a liquid medium 04/06/2023 by TDLV ENZYME PRODUCTION 04/06/2023 by TDLV ENZYME PRODUCTION Removal of Whole Cells Collect enzyme (extracellualar/intracellular enzyme) Concentration Purification Characterization 04/06/2023 by TDLV Enzymes and Sources • Proteases – Overproducing strains of Bacillus, Aspergillus, Rhizopus, and Mucor – From Animal pancreas, Plants • Pectinases – Aspergillus niger • Lactases – Yeast and Aspergillus • Lipases – Certain strains of yeast and fungi • Glucose isomerase – Flavobacterium arborescens or Bacillus coagulans 04/06/2023 by TDLV Removal of Whole Cells • Centrifugation – 5000 g for 15 for cells – 10 000 g for 45 for cell debris  High capital and running costs • Filtration: membrane filters (0.1 -10 μm) 04/06/2023 by TDLV Removal of Whole Cells • Removal of nucleic acids – Nucleic acids increases viscosity of cellular homogenate  difficult to process – Methods: precipitation (by polyethylenimine) or treatment with nucleases • Removal of lipids – Removal: Glass wool or a cloth of very fine mesh size 04/06/2023 by TDLV Cell Disruption For Intracellular enzyme Animal cells (no Cell Wall): – Potter homogenizer – Osmotic shock – Freeze-thaw cycles Plant cells (CW): – The Waring blender 04/06/2023 by TDLV Cell Disruption Microbial cells (CW): 04/06/2023 by TDLV Concentration In laboratory scale, Concentration by: • • • • • Ultrafiltration Precipitation Ion-exchange chromatography Dialysis (using semi-permeable membrane) Freeze drying 04/06/2023 by TDLV Concentration by precipitation 04/06/2023 by TDLV 10