CHAPTER 1

1. INTRODUCTION

PROCESS INTEGRATION

Biotherapeutics especially monoclonal antibodies, recombinant proteins, and small molecules derived from plant and microbes (e.g. antibiotics, alkaloids), has emerged as a hottest approach in targeting a number of diseases. The demand for new and effective biotherapeutics production has resulted in new technologies/approaches/strategies associated for expression, upstream and downstream processing. Downstream processing accounts for 60 to 80% of cost of production. Therefore, there is need for development of new and innovative approaches/strategies for downstream processing of biotherapeutics. Process integration is emerging as a leading theme in modern bioprocessing and integrated processes are required to reduce the number of processing steps, to simplify handling, to shorten the time a sensitive product has to be in contact with harsh conditions with an overall aim to reduce total processing cost. Strategies such as effective sequencing and dovetailing of operations to benefit overall processing i.e. correct scheduling of process steps and coupling of the processes such as chromatography and crystallization, achievement of more than one objective in one operational step using expanded bed, fluidized bed, liquid solid circulating fluidized bed and creative combination of two or more processes in one (new) unit operation to benefit the manufacture i.e. integration of purification and transformation steps, and in situ adsorption to enhance yield and molecular integrity of the product has been developed and applied for large scale economic production of biotherapeutics. The concept of affinity bio-chromatography coupled with membrane filtration/crystallization/precipitation was developed an applied for production of plasma proteins (IgG Transferrin, Human Serum albumin, Fibrinogen etc.), whey proteins (Lactoferin, lactoperoxidase, albumin, globulin etc.), egg white proteins

(lysozyme and conalbumin), monoclonal antibodies from cell culture fluids, natural products (artemisinin, Scopoletin, polyphenols etc.), antibiotics (Adipoly-7-ADCA and vancomycin) and growth hormones (hCG and HMG) with 30% cost improvement and more than 80% recovery without compromising pharmacopoeia purities. Final stages of downstream processing including crystallization/precipitation and drying (such as freeze drying, supercritical spray-drying and spray freeze drying) has been evaluated for preserving potency with increased stability and ease of formulation of biotherapeutics. The strategies developed have various advantages and can be effective over conventional ways of large scale production of biotherapeutics.

Process Intergration Flow Sheet

Schematic representation of the process intergration worl performed

1.1 Lysozyme Lysozyme is an enzyme also known as N-acetyl muramoyl hydrolase is widely distributed in many plants and animals. It was discovered in 1922 by Alexander flemming. Lysozyme was the first enzyme whose three dimensional structure at the atomic level was determined. It consist of a cleft which resembles the active site of the enzyme the place where long polymers (polysaccharides) found in the cell wall of bacteria and fungi are cut into small sub units. One lysozyme is made up of single polypeptide chain consisting of 129 amino acid residues with molecular weight of 14,300 KDa and contains 4 disulphide bonds. It has an

isoelectric point (PI) of 11.0. it contains 18(+) ve and 11(-)ve charged groups. It is almost inactive in absence of salts. Lysozyme activity was found at above pH 10. 1.2 Alternative Names 1,4-N-acetylmuramidase, L-7001, N,O-diacetylmuramidase, PR1-Lysozyme, Globulin Gl, Globulin G, Lysozyme g, Mucopeptide N-acetamuramolhydrolase, Mucopeptide glucohydrolase and Muramidase Source Lysozyme is present in the mucosal secretion such as saliva and tears, human milk and mucus. In high concentration, about 3% from all proteins Lysozyme is present in chicken egg-white. This enzyme is only effective against gram positive bacteria cells. Gram negative bacteria and yeast are completely resistant to lysing by it. It is also present in cytoplasmic granules of polymorhonuclear neutrophil (PMN) 1.3 Function The enzyme function by attacking peptidoglycans (found in the cell walls of bacteria especially Gram-positive bacteria) and hydrolyzing the glycosidic bond that connects Nacetylmuramic acid with the fourth carbon atom of N-acetyl glucosamine.It does it by binding to the peptidoglycan molecule in the binding site within the prominent cleft between its two domains. This causes the substrate molecule to adopt a straight confirmation similar to that of the transition state. According to Philips-Mechanism, the lysozyme binds to a hexasacchraide. The lysozyme then distorts the 4th sugar in hexasacchraide (the D ring )into a half-chair confirmation in this stressed state the glycosidic bond is easily broken.

The amino acid side chains glutamic acid( Glu35) and Aspartate 52 (Asp52) have been found to be critical to the activity of this enzyme. Glu35 acts as a proton donor to the glycosidic bond, cleaving the c-o bond in the substrate, while Asp52 acts as a nucleophile to generate a glycosyl enzyme intermediate. The glycosyl enzyme intermediate then reacts with the water molecule, to give the product of hydrolysis and leaving the enzyme unchanged.

1.4 Structure The primary structure of egg white lysozyme is a single polypeptide chain of 129 amino acids. There are 4 pairs of cysteins that form disulphide bridges between positions.

6 and 127 30 and 115

64 and 80

76 and 94( counting from N-terminal lysine). These cross bridges shows that the polypeptide is not a straight chain. Rather the chains fold to allow these cysteins to be close to each other.

1.5 Application of Lysozyme.

1.5.1 Applications of Hen Egg White Lysozyme in wine production.

Hen egg white lysozyme has been employed in many food applications as a natural preservative in wine production. Growth of certain Lactic acid bacteria(LAB) may cause sluggish fermentation, reduce wine quality and even result in unmarketable wines due to the production of Acetic acid, Biogenic amines and other undesirable compounds. In the reported lab scale wine making studies, various strains of wine spoilage LAB were inoculated into sterile grape juice. Hen egg white lysozyme, at dosages of 0, 125, 250 and 500 ppm, was added into the samples during wine making to inhibit the growth of inoculated LAB. Lysozyme can be useful for wine malers to prevent the spoilage of wines by LAB.

1.5.2 Uses of Lysozyme in food processing industry.

The use of lysozyme in food processing industry is connected primarily with its application as a natural preservative. The enzyme is widely used as a preservative for meat, fish and their products, for milk and dairy products as well as for fruits and vegetables.

1.5.3 Uses of Lysozyme in Pharmaceutical industry.

A method for treating or preventing a disease including administering a pharmaceutical composition containing lysozyme dimmer to human or animal recipient in an amount effective for non specific stimulation of immune system or regenerative mechanism of human or animal body, and where in the disease is selected from the group consisting of leukemia, hair growth disorder, a fish disease and a bee disease and successfully treated. In addition, the method can be used for immunodulating a humoral response in a human or animal subject to immunosupression, such as positively affecting the primary humeral response of immunized antigen after immunosupression. The pharmaceutical industry uses this enzyme in the manufacture of adjuvant drugs for antibiotics (to destroy micro organisms) and analgesics (pain relief) in viral and bacterial infections, in the treatment of leukemia and neoplastic (to treat extra growth cells like cancer) diseases. Lysozyme is also used as diagnostic agent, being an indicator of the occurrence and progression of pathological changes in human and animals. The dimeric form of lysozyme has been used in the treatment of bacterial and viral animal diseases A drug produced on the basis of lysozyme dimmer shows immune stimulating and immune corruptive activity

Reverse Micelle

Reverse micelle extraction is a novel technology of liquid-liquid extraction, which has received immense attention for isolation and purification of proteins, recently. It has been extensively studied with model proteins, such as cytochrome, C, ribonuclease, myoglobin, bovine serum, albumin etc. reverse micellar extraction (RME) is an attractive liquid-liquid extraction technique for down stream processing of biomolecules.

In a non-polar solvent, it is the exposure of the hydrophilic head groups to the surrounding solvent that is energetically unfavourable, giving rise to a water-in-oil system. In this case the hydrophilic groups are sequestered in the micelle core and the hydrophobic groups extend away from the centre. These inverse micelles are proportionally less likely to form on increasing headgroup charge, since hydrophilic sequestration would create highly unfavorable electrostatic interactions.

Reverse micelles provide mild separation conditions for enzyme recovery in an active form. The number of entrapped enzymes in reverse micelles that have been investigated has been incrasing since studied the catiliticactivity of phospholipase A2 in etheryl solution of phosphatidylcholine.

A micelle is an aggregate of surfactant molecules dispersed in a liquid colloid. A typical micelle in aqueous solution forms an aggregate with the hydrophilic "head" regions in contact with surrounding solvent, sequestering the hydrophobic single tail regions in the micelle centre. This phase is caused by the insufficient packing issues of single tailed lipids in a bilayer. The difficulty filling all the volume of the interior of a bilayer, while accommodating the area per head group forced on the molecule by the hydration of the lipid head group leads to the formation of the micelle. This type of micelle is known as a normal phase micelle (oil-in-water micelle). Inverse micelles have the headgroups at the centre with the tails extending out (water-in-oil micelle). Micelles are approximately spherical in shape. Other phases, including shapes such as ellipsoids, cylinders, and bilayers are also possible. The shape and size of a micelle is a function of the molecular geometry of its surfactant molecules and solution conditions such as surfactant concentration, temperature, pH, and ionic strength. The process of forming micellae is known as micellization and forms part of the phase behavior of many lipids according to their polymorphism.

Application

 Reverse micelles provide mild separation conditions for enzyme recovery in an active form. Micelle formation is essential for the absorption of fat soluble vitamins and complicated lipids within the human body. Bile salts formed in the liver and secreted by the gall bladder allow micelles of fatty acids to form. This allows the absorption of complicated lipids (eg lecithin) and lipid soluble vitamins (A, D, E and k) within the micelle by the small intestine, they can act as emulsifiers that will allow a compound normally insoluble (in the solvent being used) to dissolve. Reverse micelles bring mild and effective micro environment in organic solvents that contain bio-molecules, which have attracted immense attention for application in the isolation of proteins, protein refolding and enzymatic reaction.

The application of reverse micelle for bio separation has attracted considerable attention because the technique is considered to be potentially useful in downstream processing for a large-scale separation of bio-molecules from fermentation mixtures.

SURFACTANTS

Surfactants are wetting agents that lower the surface tension of a liquid, allowing
easier spreading, and lowering of the inter facial tension between two liquids, the term surfactant is a blend of surface active agent. Surfactants are usually organic compounds that are amphiphilic, meaning they contain both hydrophobic groups and hydrophilic groups. Therefore, they are soluble in both organic solvents and water. The term surfactant was coined by antara products in 1950. Surfactant reduce the surface tension of water by adsorbing at the liquid-gas interface. They also reduce the interfacial tension between oil and water by adsorbing at the liquid-liquid interface. Many surfactant can also assemble in the bulk solution into aggregates. Example of such aggregates is vesicles and micelles. The concentration at which surfactants begin to form micelles is known as the critical micelle concentration (CMC). When micelles form in water, their tails form a core that can encapsulate an oil droplet, and their (ionic/polar) heads form an outer shell that maintains favorable contact with water. When surfactants assemble in oil, the aggregate is referred to as a reverse micelle. In a reverse micelle, the heads are in the core and the tails maintain favorable contact with oil. Surfactants are also often classified into four primary groups; anionic, cationic, non-ionic and zwitter ionic (dual charge).

Applications and sources

Detergents Fabric softeners Emulsions Paints Adhesives Inks Anti-fogs Ski waxes, snowboard wax Deinking of recycled papers, in flotation, washing and enzymatic processes Laxatives Agrochemical formulations Herbicides (some) Insecticides Quantum dot coatings Biocides (sanitizers) Cosmetics: Shampoos Hair conditioners (after shampoo) Toothpastes Spermicides (nonoxynol-9) Firefighting Pipelines, liquid drag reducing agent Alkali Surfactant Polymers (used to mobilize oil in oil wells) Ferrofluids Leak Detectors

Classification

According to the composition of their tail

The tail of surfactants can be:

A hydrocarbon chain: aromatic hydrocarbons (arenes), alkanes (alkyl), alkenes, cycloalkanes, alkyne-based; An alkyl ether chain:

Ethoxylated surfactants: polyethylene oxides are inserted to increase the hydrophilic character of a surfactant; Propoxylated surfactants: polypropylene oxides are inserted to increase the lipophilic character of a surfactant;

A fluorocarbon chain: fluorosurfactants; A siloxane chain: siloxane surfactants

A surfactant can have one or two tails, these are called double-chained.

According to the composition of their head A surfactant can be classified by the presence of formally charged groups in its head. A nonionic surfactant has no charge groups in its head. The head of an ionic surfactant carries a net charge. If the charge is negative, the surfactant is more specifically called anionic; if the charge is positive, it is called cationic. If a surfactant contains a head with two oppositely charged groups, it is termed zwitterionic. Some commonly encountered surfactants of each type include:

Ionic

Anionic: based on permanent anions (sulfate, sulfonate, phosphate) or pH-dependent anions (carboxylate):

Sulfates:

Alkyl sulfates: ammonium lauryl sulfate, sodium lauryl sulfate (SDS, sodium dodecyl sulfate, another name for the compound); Alkyl ether sulfates: sodium laureth sulfate, also known as sodium lauryl ether sulfate (SLES), sodium myreth sulfate; Docusates: dioctyl sodium sulfosuccinate; Sulfonate fluorosurfactants: perfluorooctanesulfonate (PFOS), perfluorobutanesulfonate; Alkyl benzene sulfonates; Alkyl aryl ether phosphate Alkyl ether phosphate Alkyl carboxylates: Fatty acid salts (soaps): sodium stearate; Sodium lauroyl sarcosinate; Carboxylate fluorosurfactants: perfluorononanoate, perfluorooctanoate (PFOA or PFO)

Sulfonates:

Phosphates:

Carboxylates:

Cationic: based on:

pH-dependent primary, secondary or tertiary amines: primary amines become positively charged at pH < 10, secondary amines become charged at pH < 4:

Octenidine dihydrochloride; Alkyltrimethylammonium salts: cetyl trimethylammonium bromide (CTAB) a.k.a. hexadecyl trimethyl ammonium bromide, cetyl trimethylammonium chloride (CTAC); Cetylpyridinium chloride (CPC);

Permanently charged quaternary ammonium cation:

Polyethoxylated tallow amine (POEA); Benzalkonium chloride (BAC); Benzethonium chloride (BZT); 5-Bromo-5-nitro-1,3-dioxane; Dimethyldioctadecylammonium chloride Dioctadecyldimethylammonium bromide (DODAB)

Zwitterionic (amphoteric): based on primary, secondary or tertiary amines or quaternary ammonium cation with:

Sulfonates:

CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate); Sultaines: cocamidopropyl hydroxysultaine; Amino acids Imino acids Betaines: cocamidopropyl betaine;

Carboxylates:

Phosphates: lecithin

Nonionic

Fatty alcohols:

Cetyl alcohol,

Stearyl alcohol, Cetostearyl alcohol (consisting predominantly of cetyl and stearyl alcohols), Oleyl alcohol Octaethylene glycol monododecyl ether, Pentaethylene glycol monododecyl ether;

Polyoxyethylene glycol alkyl ethers (Brij): CH3(CH2)1016(O-C2H4)125OH:

Polyoxypropylene glycol alkyl ethers: CH3(CH2)1016(O-C3H6)125OH; Glucoside alkyl ethers: CH3(CH2)1016(O-Glucoside)13OH:

Decyl glucoside, Lauryl glucoside, Octyl glucoside;

Aqueous Two Phase System

Aqueous biphasic systems (ABS) or aqueous two phase systems are clean alternatives for traditional organic-water solvent extraction systems. ABS are formed when 2 polymers, one polymer and one kosmotropic salt, or two salts (one chaotropic salt and the other a kosmotropic salt) are mixed together at appropriate concentrations or at a particular temperature. The two phases are mostly composed of water and non volatile components, thus eliminating volatile organic compounds. They have been used for many years inbiotechnological applications as non-denaturing and benign separation media. Recently, they have been used for metal ion separations, environmental remediation, metallurgical applications and as a reaction media. Other systems that form aqueous biphases are: PEG sodium carbonate or PEG and phosphates, citrates or sulfates. Aqueous biphasic systems are used during downstream processing mainly in biotechnological and chemical industries.

The Two Phases

It is a common observation that when oil and water are poured into the same container, they separate into two phases or layers, because they are immiscible. In general, aqueous (or water-based) solutions, being polar, are immiscible with non-polar organic solvents (chloroform, toluene, hexane etc) and form a two-phase system. However, in an ABS, both immiscible components are water-based. The formation of the distinct phases is affected by the pH, temperature and ionic strength of the two components, and separation occurs when the amount of a polymer present exceeds a certain limiting concentration (which is determined by the above factors).

PEGsalt system

The "upper phase" is formed by the more hydrophobic polyethylene glycol (PEG), which is of lower density than the "lower phase," consisting of the more hydrophilic and denser salt solution. Although PEG is inherently denser than water, it occupies the upper layer. This is believed to be due to its solvent 'ordering' properties, which excludes excess water, creating a low density water environment. The degree of polymerisation of PEG also affects the phase separation and the partitioning of molecules during extraction.

Applications

ABS is an excellent method to employ for the extraction of proteins/enzymes and other labile biomolecules from crude cell extracts or other mixtures. Most often, this technique is employed in enzyme technology during industrial or laboratory production of enzymes.

They provide mild conditions that do not harm or denature unstable/labile biomolecules. The interfacial stress (at the interface between the two layers) is far lower (400-fold less) than water-organic solvent systems used for solvent extraction, causing less damage to the molecule to be extracted.

The polymer layer stabilizes the extracted protein molecules, favouring a higher concentration of the desired protein in one of the layers, resulting in an effective extraction. Specialised systems may be developed (by varying factors such as temperature, degree of polymerisation, presence of certain ions etc ) to favour the enrichment of a specific compound, or class of compounds, into one of the two phases. They are sometimes used simultaneously with ion-exchange resins for better extraction.

Separation of the phases and the partitioning of the compounds occurs rapidly. This allows the extraction of the desired molecule before endogenous proteases can degrade them.

These systems are amenable to scale-ups, from laboratory-sized setups to those that can handle the requirements of industrial production. They may be employed in continuous protein-extraction processes.

Specificity may be further increased by tagging ligands specific to the desired enzyme, onto the polymer. This results in a preferential binding of the enzyme to the polymer, increasing the effectiveness of the extraction. One major disadvantage, however, is the cost of materials involved, namely highpurity dextrans employed for the purpose. However, other low-cost alternatives such as less refined dextrans, hydroxypropyl starch derivatives and high-salt solutions are also available.

Ultrafiltration

This article relies largely or entirely upon a single source. Please help improve this article by introducing appropriate citations to additional sources. Ultrafiltration (UF) is a variety of membrane filtration in which hydrostatic pressure forces a liquid against a semipermeable membrane. Suspended solids and solutes of high molecular weight are retained, while water and low molecular weight solutes pass through the membrane. This separation process is used in industry and research for purifying and concentrating macromolecular (103 - 106 Da) solutions, especially protein solutions. Ultrafiltration is not fundamentally different from microfiltration, nanofiltration or gas separation, except in terms of the size of the molecules it retains. Ultrafiltration is applied in cross-flow or dead-end mode and separation in ultrafiltration undergoes concentration polarization.

Ultrafiltration systems eliminate the need for clarifiers and multimedia filters for waste streams to meet critical discharge criteria or to be further processed by wastewater recovery systems for water recovery. Efficient ultrafiltration systems utilize membranes which can be submerged, back-flushable, air scoured, spiral wound UF/MF membrane that offers superior performance for the clarification of wastewater and process water.

Spiral wound module:

Consists of large consecutive layers of membrane and support material rolled up around a tube. Maximizes surface area. Less expensive, however, more sensitive to pollution.

Tubular membrane:

The feed solution flows through the membrane core and the permeate is collected in the tubular housing. Generally used for viscous or bad quality fluids. System is not very compact and has a high cost per m2 installed

Hollow fiber membrane:

The modules contain several small (0.6 to 2 mm diameter) tubes or fibers. The feed solution flows through the open cores of the fibers and the permeate is collected in the cartridge area surrounding the fibers. The filtration can be carried out either inside-out or outside-in.

Pressurized system or pressure-vessel configuration:

TMP (transmembrane pressure) is generated in the feed by a pump, while the permeate stays at atmospheric pressure. Pressure-vessels are generally standardized, allowing the design of membrane systems to proceed independently of the characteristics of specific membrane elements. Immersed system: Membranes are suspended in basins containing the feed and open to the atmosphere. Pressure on the influent side is limited to the pressure provided by the feed column. TMP is generated by a pump that develops suction on the permeate side. Ultrafiltration, like other filtration methods can be run as a continuous or batch process.

Applications

Dialysis and other blood treatments

Concentration of milk before making cheese Fractionation of proteins Clarification of fruit juice Recovery of vaccines and antibiotics from fermentation broth Laboratory grade water purification Wastewater treatment Drinking water disinfection (including removal of viruses) Removal of endocrines and pesticides combined with Suspended Activated Carbon pretreatment

3. MATERIALS AND METHODS 3.1 Materials Hen egg used in the study was purchased from the local market

S.no 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

Chemicals AOT (Sodium di-2-ethyl hexyl sulfosuccinate) Cetyl konium choride Isooctane Glycine A.R KCL KH2PO4 K2HPO4 Sodium carbonate Copper sulphate Sodium potassium tartarate Nutrient agar Mueller Hinton agar Tri sodium citrate Acrylamide Bis acrylamide SDS Tris HCL Ammonium per sulphate TEMED Tris base Glycerol Beta mercapto ethanol Bromophenol blue Methanol Acetic acid Coomassie brilliant blue

Laboratory Sources SIGMA SIGMA SIGMA HIMEDIA HIMEDIA HIMEDIA HIMEDIA HIMEDIA HIMEDIA HIMEDIA HIMEDIA HIMEDIA HIMEDIA HIMEDIA HIMEDIA HIMEDIA HIMEDIA HIMEDIA HIMEDIA HIMEDIA HIMEDIA HIMEDIA HIMEDIA HIMEDIA HIMEDIA HIMEDIA

3.2 Methods

3.2.1 Crude preparation

The egg white was separated from the yolk by cracking the shell and poured into the beaker. The egg white was diluted with phosphate buffer (Ph 7). The mixture is centrifuged at 5000rpm for 10minutes and the supernatant is collected. This supernatant is stored at 4oC overnight and again it is diluted using phosphate buffer (ph 7). This mixture is centrifuged at 8000rpm for 10minutes and the supernatant is collected which is rich in lysozyme.

3.2.1 Crude preparation

The egg white was separated from the yolk by cracking the shell and poured into the beakder. The egg white was diluted with 5 volumes of 20mm phosphate buffer at (pH8.6) and sheared the mixture. The mixture was centrifuged at 5000rpm for 20mins. The supernatant is collected and stored overnight at 40C. the stored overnight culture is again diluted in the ratio 3:1 using phosphate buffer and centrifuged at 5000rpm at 20mins. The supernatant is collected and it is used for the experimental procedure.

Pre treatment and centrifugation

The homogenized sample was then centrifuged for 6000rpm for 20 minutes. The supernatant was collected separately and stored at 4oC.

3.3 Reverse micellar extraction system

3.3.1 Formation of micelle into reverse micellar

The extraction if lysozyme is carried out using reverse micelle extraction. When a charged molecule of surfactant dissolved in organic phase reverse micelle is formed charged

molecule may cationic, anionic, neutral. When AOT-sodiumdi (2-ethyl hexyl) sulfo succinate dissolved in iso octane reverse micelle is formed. Hydrophilic head present outside will come to inner core, while the hydrophobic tail will protect outside. The hydrophilic head absorbs the enzyme present in the enzyme solution.

3.3.2 Forward extraction

Different concentration of AOT and Cetyl konium chloride (50mnm, 100mm, 150mm,200mm) is dissolved in 5ml of iso-octane. This solution is said to be reverse micellar solution. It is maintained at room temperature. Equal volume of sample is taken and ph is maintained below pI value (5-4.5).Reverse micellar solution is added to the sample and mixed well. The sample is agitated for 15 minutes and kept for incubation time of 30 minutes. Then the sample was centrifuged at 6000 rpm for 20 minutes. Two phased are formed. Top phase is transferred separately.

3.3.3 Backward extraction

5ml of 100mm KCI is added to the top phase. The sample is agitated for 15 minutes and kept for incubation time of 30 minutes. Then the sample is centrifuged at 6000 rpm for 20 minutes two phases are formed. Both the phases are estimated under Lowry etal.

3.4 Characteristic study

Various factors affecting the enzymes activity is taken considered into account. Some factors are optimized for high enzyme activity. Increase or decrease in any of the factors will leads to the decline in enzyme activity. The various parameters optimized in enzyme are pH, concentration of AOT, Cetyl konium chloride and KCI in reverse micellar extraction.

3.4.1 Effect of AOT and pH in forward extraction

The surfactant concentration is responsible for formation of reverse micelle AOT (sodium-di-2 ethyl hexyl sulfo succinate) is an anionic surfactant whose concentration ahould not exceed high levels. Concentration of AOT is varied as 50, 100 , 150 and 200 mM. pH of the samples since positive charge is implemented for ionic attraction pH of the samples is varied from 3 to 5 with increments units of 0.4 NaCl is added for ionic migration in the sample. The positive charge in the sample gets migrated to entire solution and ionic attraction takes place. The concentration of NaCl is varied ranging from 0.25 to 1mM.

3.4.2 Effect of pH and KCI in backward extraction

KCI is used in backward extraction for the removal of enzyme from reverse micellar phase. KCI is added to the top phase negative ions are mostly migrated since removal of the enzyme is favoured when divalent cations concentration is more. The concentration of KCI is varied as 0.25,0.50,0.75 and IM.

3.5 Estimation of Total Protein (Lowry s et al)

The protein content of the sample was estimated by the following the procedure opted by Lowry et al.

Principle

It is the most commonly used method for determination of protein in cell free extract because of its high sensitivity and quantities as a s20 kg of proteins can be measured the co-NH-(peptide bond) in polypeptide chain reacts with copper sulphate in Alakaline medium to give a blue colored complex. In addition tyrosine and trptophan residues of protein cause reduction of the phosohomolybdate and phosphotungstate components of the folin ciocaultaue reagent to give bluish product which contribute towards enhancing the sensitivity of this method.

Reagents

1. Reagent A: 2. Reagent B: 3. Reagent C:

2% of sodium carbonate 1% of copper sulphate in 1% potassium sodium tartarate. To 49ml of reagent A, 1ml of reagent B was added

4. Reagent D:

Folin-fiocalteau was diluted in the ratio 1:1(v/v)

Procedure:

Suitable aliquots of BSA solution were taken in the range of 0.2 to 1.0 mg/ml for the calibration curve.

0.3 ml of the enzyme extract was taken and made up to 1 ml using distilled water. Then 3 ml Lowry stock solution was added to the aliquots and protein sample and allowed to stand for 10 minutes

0.3 ml of folin phenol reagent was added and allowed to stand for 45 minutes Absorbance was read at 570nm.

Aqueous Two Phase Extraction

Methodology:

Aqueous two-phase system (ATPS):

a)To choose the most appropriate PEG/ salt system and to optimize their respective concentrations for effective isolation of proteins from the crude.

b) To separate the two phases and determine the protein recovery in the top phase using Lowrys method and recycle the bottom phase to enhance the recovery in the separation process.

c) The proteins concentrated in the top phase are obtained in purified form are analysed for lysozyme content.

Forward Extraction
The sample was diluted to 3:1 ratio using phosphate buffer(pH-7)

The optimized % composition of PEG/ salt was weighed.

PEG was added to 5ml of sample taken in a test tube and was subjected to uniform intervals mixing to ensure complete homogenization.

The weighed salt was added and vortexed well.

It was incubated for 20 mins. A distinct two phase formation was observed and the proteins were concentrated in the PEG rich top phase.

 The sample was centrifuged at 8000rpm for 15 mins to allow complete phase separation.

rich

The two phases were separated and backward extraction was performed for the PEG

protein concentrated top phase.

Backward Extraction
Backward extraction was performed using 10% NaCl solution

This solution was added ( volume equal to the PEG rich top phase) to the top phases. Its pH was adjusted to 9 using phosphate buffer.

Incubate for 20 mins and centrifuge at 5000 rpm for 10 mins.

The proteins are concentrated to the aqueous bottom phase from the PEG region.

The two phases are separated and the aqueous phase was desalted by running it in Gel Filtration Chromatography.

The desalted extract was subjected to SDS-PAGE to analyse for the presence of lysozyme.

The protein recovered is determined by Lowrys method.

Prior to the extraction process, the PEG/salt system was charecterised percentage composition of PEG 6000 and percentage composition of Na2SO4 was optimized, effect of pH was studied and graphs were plotted for each optimization studies.

Salting out method

Gel Filtration Chromatography Gel filtration chromatography (sometimes referred to as molecular sieve chromatography) is a method that separates molecules according to their size and shape. The separation of the components in the sample mixture, with some exceptions, correlates with their molecular weights. In these cases, gel filtration can be used as an analytical method to determine the molecular weight of an uncharacterized molecule. Gel filtration is also an important preparative technique since it is often a chromatographic step in the purification of proteins, polysaccharides and nucleic acids.

The basic components of the gel filtration experiment are the matrix, chromatography column and the elution buffer. The matrix is the material in the column that is actually the separation medium. It is the stationary phase of the chromatography.

The column is a tube with a frit and elution spout fitted at the bottom. The frit is a membrane or porous disk that supports and retains the matrix in the column but allows water and dissolved solutes to pass. The elution buffer is the mobile phase of the chromatography and flows through the matrix and out of the column. The column, with the matrix and applied sample, is developed by the elution buffer. This means that the molecules in the sample are carried by the flow of buffer into the matrix where they are gradually separated. The separated zones of molecules then flow out of the column where they are collected for analysis.

Filling the chromatography column with matrix is referred to as packing. The packed matrix is called the bed and the volume it occupies is termed the bed volume. It is very important not to allow the bed to run dry. Otherwise, cracks and fissures develop and the matrix has to be removed and repacked. The gel filtration matrix consists of microscopic beads that contain pores and internal channels. The larger the molecule, the more difficult it is for it to pass through the pores and penetrate the beads. Larger molecules tend to flow around and in between the beads. The total volume of buffer between the beads is the void volume. Smaller molecules tend to spend more time in the maze of channels and pores in

the bed. Consequently, the larger, higher molecular weight molecules are eluted from the column before smaller molecules. Larger molecules take the faster, more direct path that involves less time in the beads. This is somewhat analogous to finding your way out of a complicated maze or simply walking around the outside of the maze and avoiding the whole situation entirely.

Molecules can have the same molecular weight but radically different shapes. Molecules with a more compact shape, such as a sphere, will penetrate the beads more easily than those having an elongated shape, like a rod. Therefore, a rod-like molecule will elute before a spherical one of the same molecular weight.

There are many different types of gel filtration matrices. The spectrum of molecular weights the matrix is capable of separating is called the fractionation range. For example, consider a matrix that has a fractionation range (in molecular weight) of 1000 to 100,000 daltons. Molecules with an average molecular weight of 1000 or less will not be separated from each other since they all penetrate the beads completely and with equal efficiency. These molecules take the maximum volume of buffer for elution, which is equal to one bed volume.

The bed volume is equal to the volume of the beads plus the void volume. Molecules in the range of 1000 to 100,000 daltons will enter the beads with varying efficiencies and be partially or completely separated from one another. Molecules greater than 100,000 daltons will not enter the beads and be eluted in the void volume. Note that in this example, any number of different molecules having molecular weights of 100,000 daltons or greater will all elute at the same time since they are not sieved by the matrix. The partially or completely separated zones of molecules that are eluted from the column are called peaks. A peak consists of an increasing and decreasing concentration gradient of molecules.

This experiment includes columns which will be pre-packed with the appropriate matrix for separation of the sample mixture. The sample in this experiment contains a mixture of orange and blue molecules. The orange dye has a molecular weight of 452. The blue is a polymer of glucose with an average molecular weight of 2,000,000 daltons, and has a rodlike shape. The fractionation range of the matrix is 1000 to 5000 daltons.

APPLICATIONS

Biochemical applications

In general, SEC is considered a low resolution chromatography as it does not discern similar species very well, and is therefore often reserved for the final "polishing" step of purification. The technique can determine the quaternary structure of purified proteins that have slow exchange times, since it can be carried out under native solution conditions, preserving macromolecular interactions. SEC can also assay protein tertiary structure, as it measures the hydrodynamic volume (not molecular weight), allowing folded and unfolded versions of the same protein to be distinguished. For example, the apparent hydrodynamic radius of a typical protein domain might be 14 and 36 for the folded and unfolded forms, respectively. SEC allows the separation of these two forms, as the folded form will elute much later due to its smaller size.

Ultrafiltration

Apparatus Specification

Mid gee ultrafiltration system-it contains feed reservoir, permeate collector, peristaltic pump, filter catridge and pressure display unit. The filter module of 0.026m2 surface area and pore diameter of 0-2mm is used. A protein suspension is pumped at high speed through the module so that it crosses the membrane at right angkes to the direction of flow. The material retained and filtered is returned to the reservoir tank and the filtrate (permeate) collected separately. The trans membrane pressure (TMP) is regulated by adjusting the pressure at the entry and exit of the apparatus Trans Membrane Pressure = ( pressure at entry point + pressure at exit point ) / 2 Filtrate Pressure

Methodology

The aueous phase obtained at the end of both lle methods is subjected to purification
ultrafiltration technique.

Since egg is rich in several other proteins a severe contamination is observed in the
aqueous phase obtained from which lysozyme has to be purified.

Therefore ultra filtration helps to selectively isolate lysozyme and it is a highly effective
separating technique as it does not affect the structure and function of the enzyme.

Molecular weight of lysozyme is 14.3 KDa.

Different modules assigned with specific Nominal Molecular Weight Cut Off (NMWCO) is used and containant proteins are separated from the desired one.

We used 10KDa, 30KDa, 50KDa poly carbonate modules to purify the sample. The transmembrane pressure and average permeate flow rate was maintained at 1 bar and 3 ml/ min, respectively.

The phase containing the proteins of both extraction processes was initially concentrated using 10KDa membrane and the retentate was collected. The second ultra filtration is performed on this retentate using 30KDa membrane module. Now the permeate was collected and checked for protein content. This permeate is expected to contain lysozyme in a purified and concentrated form and this to be verified by performing suitable assay technique. It was found that the bulk volume was reduced to almost half and the desired proteins were obtained in a purified and concentrated form.

3.8.2

CONFORMATIONAL STUDY

SDS PAGE (Poly Acrlamide Gel Electrophoresis)

SDS-PAGE is the most eidely used methodfor qualitatively analyzing any protein mixture, monitoring protein purity and to determine their molecular

weight. It is based on the separation of protein according to their size and hen locating them binding to dye.

Sodium Dodecyl Sulfate (SDS) is an anionic detergent that binds to each gram of protein, giving denaturation. In the presence of excess 5 Ds that binds to the protein a constant negative charge unit mass. As a result protein SDS complexes moves towards the anode during electro phoresis and owing to molecular serving properties of the polyacrylamide gel get separated based on the molecular weight. Since the principle of this technique is separation of proteins of known molecular weight on the same gel as unknown protein molecular weight of the unknown protein can be determined.

Mobility of the protein in SDS gel electrophoresis is expressed as relative mobility (RM) with respect to the tracing dye, bromophenol blue. RM= distance migrated by protein / distance migrated by tracking dye.

Cross linked polyacrylamide gets are formed by copolymerization of acrylamide monomer and a cross linking agent N, N- Methlylene bis acrylamide. This reaction is catalyzed by N,N,N,N- Tetramethlylene diamine (TEMED) and initiated by APS (Ammonium Per Sulphate) porosity of the gel is determined by the amount of acrylamide and biscarylamide mix used. Low percentage gel gave high pore sizes thereby offering less resistance to passage of larger molecules, while higher percentage gels favor for separating of smaller molecules.Most proteins separation is carried out using gels ranging from 5-15% acrylamide.

Material

Separating gel (12%)

Acryl amide (30%)

5.8 ml

(30g acrylamide +0.8g bis acrylamide+100ml distilled water)

1.5m Tris Hcl (PH8.8) 10% SDS Deionized water 1% APS TEMED

3.75 ml 0.3 ml 4.25 ml 380l 30 l

Stacking gel (4.5%)

Acryl amide (30%) 0.5 m Tris Hcl (PH 6.8) 10% SDS Deionized water 10% APS TEMED

1.5 ml 2.5 ml 0.1ml 5.85 ml 50 l 20 l

Tank buffer

5 Tris base Glycine SDS Distilled water

1.5gm 7.2 gm 0.5gm 100ml

SDS Page (loading dye)

0.5 m Tris Hcl (PH 6.8) 10% SDS Glycerol B- Mercaptoethanol 1% Bromophenol blue

2.0ml 3.2 ml 1.6 ml 0.8ml 0.4 ml

Staining solution

Methanol Acetic acid Distilled water Coomassie brilliant blue

40 ml 10 ml 50 ml 0.1%

Destaining solution

Methanol Acetic Acid Distilled water

40 ml 10 ml 50 ml

Acrylamide (30 % of acryl amide 0.8 % of bis acrylamide)

Acrylamide gel was prepared by dissolving 30g of acrylaide and 0.8g of bisacrylamide in 60 ml of distilled water. When the acrylamide was completely dissolved , the double distilled water was added to make up to the solution to 100 ml.

Resolving gel preparation (1.5 M Tris base buffer-PH 8.8)

Resolving gel was prepared by dissolving 18.2g of tris base in 80,ml of double distilled water and adjusted the PH to 8.8 with I n HCI and made up to 100 ml with double distilled water.

Staking gel preparation (0.5 Tris base buffer PH6.8)

Stacking gel was prepared by dissolving 6.1g of Tris base in 80 ml of double distilled water and adjusted to PH to 6.8 with 1 N HCI and made up to 100ml with double distilled water.

10% Ammonium per sulphate solution (APS)

APS was prepares by dissolving by 1g of ammonium per sulphate in 10 ml of double distilled water and cover with aluminium foil.

TEMED Electrode buffer SDS-PAGE-Sample loading buffer Glass plates & spacers Vertical gel electrophoresis apparatus Double distilled water

Procedure

Two clean plates with three Teflon spacers maje a single cassette. Separating gel was prepared by according the gien table and mixed thoroughly. After swirling to mix, we simply pour the solution into the space occupied by the cassettes up to volumes of glass plates. Then it was allowed to polymerize for 15 minutes at room temperature. After polymerization, stacking gel was prepared as given in the table. Stacking gel was mixed thoroughly and poured along the edge of the space markers until the height of the solution is 1 cm from the top of the plate. Then the plastic comb was immediately inserted into the top of the gel during polymerization, from indentations in the gel that serve as sample wells. The sample was prepared by mixing the given protein sample with equal volume of SDS-PAGE sample loading buffer. To denature the protein, sample mixture was heated at 40c for 2 mins. The marker protein also prepared in the same manner. After polymerization the spacers and comb were removed carefully. Then the plate was clamped into place between two reservoirs. 20l of sample and dye mixture were loaded into each well. Then the lower and upper electrophoresis reservoirs were filled with electrode buffer.The voltage was applied until the tracking dye reaches the bottom of the plate. To detect the protein bands in electrophoresis gels, the slab gel was removed and stained.