細胞破碎技術 Cell Rupture 國立宜蘭大學 食品科學系 馮臨惠

Separation Processes Selection of Operations in Separation Processes Chemical, Physical, and Biochemical Concepts in Isolation and Purification of Proteins

Selection of Operations in Separation Processes Defining Final Product Characterization of Starting Material Selecting of Separation Sequence Purification Process and Unit Operations Protein Recovery Protein Purification

Protein separation techniques

Protein Recovery Cell separation Cell disruption and debris separation (for intracellular proteins only) Concentration

Cell disruption Different types of cells (e.g. microbial, animal and plant) produce proteins either intracellular or extracellular For recovering intracellular proteins, the cells have to be disrupted

Disruption of microorganisms Release of intracellular proteins Release of intracellular enzymes Batch and continuous flow cell disruption Degree of disruption Reproducibility Practical application

The ideal technology for cell Disruption Maximum release of the product of interest No mechanical or thermal denaturation of the product during disruption Minimal release of proteases which may degrade the product Minimal release of particulates or soluble contaminants that may influence downstream processing

The desired attributes of mechanical cell disruption equipment Low capital and operating costs Could be Sterilized May be cleaned in place (CIP) May be validated for cGMP requirements Scalable (scale up) May be automated

Process Design Considerations Disruption Kinetics Operating Pressure Energy Requirements Protein Denaturation Valve Design Cell Physiological Factors Scale up Enzyme Release Application

Disruption and homogenization of cells Different techniques: shearing, grinding, sonication, French pressure cell disruption, depending of cell types Isolation of organelles; solubilization of membranes, excreted proteins

Kinetics of Homogenizers Rm log ＝ kN Rm － R Rm ＝ maximium protein release or enzyme activity R ＝ measured protein release or enzyme activity after N passes k ＝ rate constant (1/s) N ＝ number of passes

Mechanical disruption of cell Homogenizers Principle of operation Influence of pressure Influence of valve design Influence of temperature Influence of cell concentration

Bead Miller Disruption of microorganisms Solubilization of protein, first-order process Schutte et al., (1986) Rm log ＝ LnD ＝ kNt Rm － R Rm ＝ maximium protein release or enzyme activity R ＝ measured protein release or enzyme activity after N passes k ＝ rate constant (1/s) N ＝ number of passes t ＝ mean residence time (s) per pass

Operational Parameters Agitator speed Feed rate Size and density of beads Beads loading Cell concentration Temperature

Methods of permeabilizing cells Chemical permeabilization of cells Mechanical permeabilization of cells Enzymatic permeabilization of cells Other permeabilization Techniques (Pulse)

Chemical permeabilization of various host cells Gram negative microorganisms Gram positive microorganisms Yeast Plant cells Mammalian cells

Pulsed Electric Fields processing Mechanisms of Microbial Inactivation Figure. Schematic diagram of reversible and irreversible breakdown (a) cell membrane with potential V'm (b) membrane compression (c) pore formation with reversible breakdown (d) large area of the membrane subjected to irreversible breakdown with large pore (Zimmermann, 1986)  

Pulsed Electric Fields processing Mechanisms of Microbial Inactivation Electroporation( 電穿孔、電擊通透 ) (Vega-Mercado, 1996) 

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