lecture 7&8

An Introduction to Bio separations
Bio separation is the name given to the practice of purifying biological products on large-scale, using fundamental aspects of engineering and scientific principles. The end goal of bio separation is to refine molecules, cells and parts of cells into purified fractions.
Bio separation study the processes used to recover, isolate, predicate and polish products made by/of biological materials
Biological products can be separated and purified depending upon the following characteristics: density, diffusivity, electrostatic charge, polarity, shape, size, solubility and volatility.
Bio separation or downstream processing: - refers to the recovery, isolation, purification and polishing of products synthesized by biotechnological processes (e.g. r-DNA technology, conventional microbial fermentation, enzyme technology, and hybridism technology).
Differences between Bio separation and Chemical Separation
Although bio separation is based on traditional chemical separation processes, they do differ in significant ways. This is because the materials being purified and separated in bio separation are biological substances rather than the synthetic chemicals used in traditional techniques. As such, substances such as proteins, carbohydrates and nucleic acids are not suitable for the rigors of traditional techniques like packed-bed adsorption and evaporation.
Often, the desired final product is only found in very minute quantities in the starting substance from which they are refined. Because of this, vast quantities of dilute product streams must undergo processing in order to obtain a small amount of pure product. Meanwhile, there are often unwanted impurities in the starting substance, which have similar genetic makeup to the desired product, thus making separation very difficult.
Because biological products are more apt to degradation than chemical ones, this rules out the use of many common organic solvents in bio separation, since they have a tendency to act as a catalyst for degradation. Furthermore, many biological substances are unstable when heated and as such have to be handled in sub-ambient temperatures.
Bio separation Techniques
Many different techniques by which bio separation can be achieved – however, there is none currently works effectively on their own. This is because bio separation requires a combination of high resolution (also known as selectivity) with high throughput (also called productivity). As you will, notice in the table of techniques below, not even one combines those two traits.
One of the more commonly used methods of achieving bio separation is through the deployment of a RIPP scheme (Recovery, Isolation, Purification, and Polishing). This technique will first utilize one of the low-resolution methods from the left column above to achieved recovery and isolation of the desired product. Then, one of the high resolution methods from the right column will purify the product and “polish” it. Polishing can refer to sterilization, removal of contaminants and any other final processing steps before it is packaged into a marketable form.
Common stages of bio separations
1-Removal of solids
2-Isolation (volume reduction)
3-Purification
4- Polishing
Unit operations:-
A-Removal of solids: Filtration, Centrifugation, Sedimentation
B-Isolation (volume reduction): Adsorption, Extraction
C-Purification: Chromatography. Electrophoresis, precipitation
D- Polishing: Crystallization, Drying
Methods of bios products extraction:
The importance of the selection the ideal extraction:
Enzymes and active proteins are the most important biological products and are most sensitive to the extraction circumstances, so the goal of these methods are:
1- Getting the enzyme with highest activity
2- Keeping the enzyme with highest stability grades
3-High extraction selectivity increase the purification efficiency
M.Os enzymes extraction:
This point should be priority for the extraction of the enzymes from the M.O:
(1)Type of enzymes: the micro enzymes are classified according to their production nature in to two types: (a) constitutive enzymes, is the enzymes which produce by the cells always with good quantity during their growth stages like Glycolysis enzymes and the respiratory chain enzymes. While the other type is the inducible enzymes, the enzymes which produce only when the basic reaction substance is present in the growth media.
Principles methods of the M.Os cell lysis:
1-Mechanical lysis method: it’s characterized by the flowing which make it the beast.
-No need for chemical substances
-Decrease the differences among the strains
-Economically good
While the disadvantages are:
a-The high temperature produced by the mechanical action which need control
b-Surface denaturation and foaming which should be controlled to prevent the surface distortion and allow oxidation
c-High mechanical action lysis cause to form fine insoluble particles which affect the purification process later.
d-DNA releasing which cause high viscosity.
The most important mechanical lysis are three:
1-Agitation with abrasive 2-Liquid shear lysis 3-Sonication
2-The enzymes lysis: the enzymes lysis characterized by
1-Determenation of denaturation
2-Can be used without the determination of the cells
3-There is some selectivity in the production of cellular components
The disadvantages: the lysis efficiency depending on many substances interfere with purification steps, the success of the lysis determined by the following:
1-The differences among the types and strains
2-The growth media
3-The cells situation during the lysis(frozen or fresh)
4-protease suppressers presence
5-The buffer solution presence
6-Cells concentration in the suspension
7-Osmotic pressure of the buffer solution
8-The growth phase when the cells collected
The enzymes, which used in the cell lysis are:
-Lysozymes enzyme for G+ve bacteria, lysostaphin for S.aureus - Lysozymes enzyme for G-ve bacteria
-Glucuronidase, Zymolyase, Glusulase for yeast lysis.
Plant enzyme extraction: we should follow the following for the extraction design
1-Plant age
2-The plant parts (leaves, stems, roots, flours, seeds)
3-The seasonal changes for the desired enzymes activity in the selected part or the tissue as a source of enzyme
4-Position of the enzyme in the cell (membrane, cytoplasm, mitochondria or nucleus)
5-The capacity for the probably effects of some cellular components and the determination of the necessary principles to decrease them, like the vacuoles which contain the in organic components and the middle compounds for the Tri carbonate, water lysis enzymes especially the protease and the phenol compounds.
We should follow the following in the extraction process:
1-Using buffer solutions with pH and buffering capacity can withstand the organic acids, which collect in the holes
2-Reduction agent should be used like ascorbic acid for deoxidizing the oxidizing enzymes produced
3-Using the chelating agents like EDTA for pulling the multi equivalent ions and suppressing the oxidizing enzymes
4-Using of some insolub