READY RECKONER FOR DOWN STREAM PROCEESING PRACTICALS

M.ABISHEK.,M.Tech(Biotech)., R.JANANI.,M.Sc.,M.Phil (Biochemistry).,

S.AISHWARYA.,M.Tech(Biotech).,

UI MEDIA PUBLICATIONS-INDIA READY RECKONER FOR DOWN STREAM PROCESSING PRACTICALS

ISBN Pending

UI PUBLICATIONS-INDIA

No part of this work may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, or recording or otherwise, without written permission from the publisher, with the exception of any material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work.

Product Liability: The Publisher cannot guarantee the accuracy of any information about biochemical medical values or text or any other related terms contained in this book. In every individual case the user must check such information by consulting the relevant literature.

This is copyright of an Product of UI Publications-India

Product of UI Publications-India

PREFACE

Downstream processing implies manufacture of a purified product fit for a specific use, generally in marketable quantities, while analytical bio separation refers to purification for the sole purpose of measuring a component or components of a mixture, and may deal with sample sizes as small as a single cell. Downstream processing (DSP) involves multistage unit operations after upstream processes that improve the quality of the final product in terms of concentration and purity. One of the most important objectives of the DSP, in addition to maximizing product recovery, is to reduce the cost of production. Downstream processing refers to the recovery and purification of biosynthetic products, particularly pharmaceuticals, from natural sources such as animal or plant tissue or fermentation broth, including the recycling of salvageable components and the proper treatment and disposal of waste.

The five stages are: (1) Solid-Liquid Separation (2) Release of Intracellular Products (3) Concentration (4) Purification by Chromatography and (5) Formulation. The purpose of downstream processing is to isolate, purify and concentrate the previously synthesized drug substance or other product from the complex bulk matrix. Downstream processing may also include formulation activities,signifyingthetransitionfromdrugsubstancetodrugproduct (DP).Fermentation factors affecting downstream processing include the properties of microorganisms, particularly morphology, flocculation characteristics, size and cell wall rigidity.

This book “Ready Reckoner for Down Stream Processing Practical’s” will give immense Knowledge to all Graduates of Biotech and Chemical Engineering Students.

Thanks & Regards,

1. ENZYME IMMOBILIZATION ENTRAPMENT IN ALGINATE GEL

PRINCIPLE:

Alginate, commercially available as alginic acid, sodium salt commonly called sodium alginate. It is a linear polysaccharide normally isolated from many strains of marine, brown seaweed and algae. Thus the name alginate. The copolymer consists of two uronic acids. They are D-mannuronic acid and L-glucoronic acid.

Because it is the skeletal component of algae it has nice property of being strong and it is flexible.

Alginic acid is either water soluble or insoluble depending on the associated salt. The salts of sodium, other alkali metals and ammonia are soluble. Where as the polyvalent cations, example calcium or water insoluble, with the exception of magnesium. The alginate polymer itself is anionic (negative charge) over all. The Polyvalent cations bind to the polymer whenever there are two neighboring glucoronic acid manures.

Thus polyvalent cations responsible for cross linking of different polymer of molecule and different parts of same polymer chain. The process of gelation, simply the exchange of calcium ions for sodium ions, is carried out and are relatively mild conditions. Because the method is based on availability of glucoronic acid residues, which will not vary once given a batch of alginate. The molecular permeability does not depend on the immobilization conditions. Rather, the pore size is controlled by the choice of starting material.

The ionically linked gel structure is thermastable over the range of 0-100 C. Therefore heating wills not the gel. However, the gel can be easily redissolved by immersing the alginate gel in a solution containing a high concentration of sodium, potassium, or magnesium.

Maintaining sodium: calcium 25:1 will help avoid gel destabilization. Infact, it is recommended by alginate vendors. To include 3mM calcium ions in the substrate medium. On the other hand, intrate or phosphate, pH buffer cannot be effectively used without destabilizing the alginate gel.

2Na (Alginate) + Ca++ → Ca (alginate) 2+2Na+

Alginate is currently widely used food, pharmaceutical, textile, paper products. The properties of alginate utilized in these products are thickening stabilizing, gel forming and film forming. Alginate polymers isolated from different alginate sources varying properties. Different algae or for that matter different part of the same algae, yield alginate of different monomer composition and arrangements. There may be sections of homopolymeric blocks of only one type of monomer. (M-M-M), (G-G-G), or there may be section of alternating monomers (M-G-M-G). Different types of alginate selected for each application on the basis of molecular weight and relative composition of D-mannuronic acid and D-glucoronic acid. For example, thickening function (viscosity properly depends mainly on molecular weight of the polymer where as gelation (affinity for cation) closely related to the glucoronic acid content. Thus high glucoronic acid, results in stronger gel.

REAGENTS AND INSTRUMENTS:-

a) Equipment:Beakers

Weigh balance

Graduated cylinder

Pipette

Syringe

b

) Reagents:-

Alginic Acid

Sodium salt or alginate

Calcium chloride

Enzyme

PROCEDURE:-

 Dissolve 30g of sodium alginate in 1liter of make a solution of 3%(sodium alginate solution is best prepared by adding the powder to agitated water, rather then vice versa, to avoid the formation of clumps. Prolonged the stirring may be necessary to

be achieve the complete dissociation of sodium alginate. After sodium alginate is completely dissolved leave the solutions undisturbed for 30 min to eliminate the air bubble that can be later entrapped and cause the beads to float0.

 Mix approximately 0.015g of enzyme with10ml of 3% (wt) sodium alginate solution. (The concentration of sodium alginate can be vary between 6-12% depending on desired constant (although not necessary the beads may be harder by mixing some amines in sodium alginate solution and cross linking with glutaraldehyde)

 The beads are formed by tripping the polymer solution from height of approximately 20cm into an excess 100ml of stirred 0.2M calcium chloride solution with a syringe and needle at room temperature.

 The bead size can be controlled by pump pressure and needle gauge. The typical hypodermic needle produces beads of 0.5-2mM in diameter.

 Other steps can be obtained by using a molds whose walls is permeable.

 Calcium chloride ions to leave the beads in the calcium solution to precursor for 0.53 hours.

RESULT:

POSSIBLE VIVA QUESTIONS:

1. Define Enzyme immobilization technique

2. Classify Enzyme immobilization techniques

3. What are the applications of enzyme immobilization techniques?

4.Give examples for covalent linking immobilization techniques

5. Define shelf life of enzymes.

AIM:

2. CITRIC ACID PRODUCTION

To produce citric acid by Aspergillus niger

PRINCIPLE:

Citric acid is one of the organic acids that are produced by micro organisms. It is one of intermediate components of cellular regulatory circuit named TCA cycle. Under controlled conditions it is released into the medium i.e., hypothetical additional of Mg2+ ions in form of MgSO4 in the medium enables the release of citric acid in the medium.

Aspergillus niger is an opportunistic pathogen that causes Aspergillosis at certain conditional when immune system become weak. Aspergillus niger is commonly fund in the decayed fruits and vegetables like onion, potato in form black spores due to the presence of pigment virgin.

MATERIALS REQUIED:

1. Decayed onion was used as a source of Aspergillus niger.

2. Czabkdex-Dox agar/nutrient agar medium.

3. Production medium.

Sucrose – 125 to 150g/l.

NH4NO3 – 2.0 to 2.5g/l.

KH2PO4 – 0.752 to 1.0g/l.

Mgso4.7H20 – 0.20 to 0.25g/l

4. 1N NaOH.

5.Bromothymol blue – 40mg Which is dissolved in 95% ethanol (50ml) and make upto 100ml with distilled water?

PROCEDURE:

STEP –I: Preparation of inoculum

The decade portion of onion containing spore which was scraped and inoculate into agar slants for 3-4 days go to obtain a well sporulated slant.

STEP-II: Inoculation into production

1. The production media (50ml was prepared and sterilized).

2. From the seed culture the spores are scraped and inoculated in the medium using a loop.

3. It is then incubated at room temperature for 2-3 days in an shaker at 1500rpm. The initial pH of the medium is 7-4. After citric acid production the pH of the medium is reduced to 2

STEP-III:

After the growth of the organism the contents are filtered using a filter paper to remove the fungal biomass, cell debris and impurities.

STEP-IV:

Estimation of citric acid production

1. Volumetric analysis is carried out to confirm the presence of citric acid and find the normality.

2. 1ml of bromothymol blue is added to the filter ate which acts as an indicator and is titrated against NaOH (1N).

3. The color change from yellow to blue which indicates the end point and confirms the presence of citric acid.

RESULT:

POSSIBLE VIVA QUESTIONS:

1. How are Citric acid produced by fermentation technique?

2. What is the name of the organism used for Citric acid production?

3. Give the industrial applications of citric acid.

4. Define inoculum.

5. Give the composition of fermentation medium used in citric acid production?

3. EXTRACTION AND PURIFICATION OF α-AMYLASE

AIM :

To purify amylase by dialysis method.

PRINCIPLE:

What are amylases?

Amylases are enzymes that break down starch or glycogen.

Amylases are produced by a variety of living organisms, ranging from bacteria to plants and humans. Bacteria and fungi secrete amylases to the outside of their cells to carry out extracellular digestion. When they have broken down the insoluble starch, the soluble end products such as (glucose or maltose) are absorbed into their cells.

Amylases are classified based on how they break down starch molecules

i. α-amylase (alpha-amylase) - Reduces the viscosity of starch by breaking down the bonds at random, therefore producing varied sized chains of glucose

ii. ß-amylase (Beta-amylase) - Breaks the glucose-glucose bonds down by removing two glucose units at a time, thereby producing maltose

iii. Amyloglucosidase (AMG) - Breaks successive bonds from the non-reducing end of the straight chain, producing glucose

Many microbial amylases usually contain a mixture of these amylases.

Why bother about amylases?

Humans exploit microbial amylases for the following purposes:

1. High Fructose Corn syrup preparation

2. Additives to detergents for removing stains

3. Saccharification of starch for alcohol production

4. Brewing

What organisms are responsible for amylase production?

Although many microorganisms produce this enzyme, the ones most commonly used for their industrial production are Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquifaciens and Aspergillus niger

Materials Required:

• Autoclave or pressure cooker

• Hot Plate or Microwave oven

• Nutrient Agar powder

• Potato Dextrose Agar powder

• Soluble starch

• Weighing scales

• Shaker

• Spectrophotometer or colorimeter

• Water bath (Temperature controlled

• Hand trowel or disposable spoons

• Sterile pipettes (One each of 10 mL, 5 mL and 1 mL)

• Pipette pumps

• Six bottles of sterile water, containing 90 mL each

• Sterile Glass Petri dishes or Pre-sterilized disposable Petri-dishes

• Wire loop

• Dissecting needle or Cork borer

• Bunsen burner and matches

• Glass spreader

• 95% ethanol.

• Dialysis tube

PROCEDURE :

LEVEL 1 :

Isolation of Amylase producers from the environment

The soil contains a rich deposit of both bacteria and fungi, which produce amylases. Starch hydrolyzing fungi or bacteria could be isolated from the soil, foods or could be purchased. Buying saves time and ensures a high yielding strain. However, isolating could be fun, and constitutes an additional lab.

Isolation Procedure

i. Select a moist part in a wood or park (only in warm climates, during the warm season in colder climates)

EITHER: Sweep off the debris from the top of the soil, use a hand trowel to collect a sample of the top soil (about 100 grams), into a "Poly or Ziploc " bag

OR:

Bury a cut piece of cut potato about four inches deep, and cover with soil. After about 8 days, dig the potato out, scrape the soil off and take it to the lab in a "Poly or Ziploc" bag. (This is called baiting)

i. Suspend about 10 grams of either soil or rotten potato, in 90 mL sterile distilled water, mix properly

ii. Pipette 10 mL of the above and transfer to another 90 mL of water

iii. Dilute further in two more 90 mL sterile water blanks

• For fungi, spread 0.1 mL from the dilutions on Potato Dextrose Agar plates (fortified with 0.1 mg/mL streptomycin sulfate) with a glass spreader. (The glass spreader is quickly sterilized by dipping in 95% ethanol and putting in

the flame, so that the alcohol burns off) Incubate at room temperature for about 3 days

• For bacteria, spread 0.1 mL of the diluted samples on Nutrient Agar plates containing 1 % w/v soluble starch and incubate at 30ºC for 24 hours

iv. Starch-producing colonies will have an area of clearing around them.

v. Confirm by flooding plates with Gram's iodine. Parts of the plant still containing starch will stain ink-black.

Transfer distinguishable, amylase-producing fungi to fresh plates of Potato Dextrose agar containing 1 % starch, using a sterilized dissecting needle. For bacteria, streak on a fresh plate of Nutrient Agar containing 1 % starch.

Transfer your isolated amylase-producing fungi to Potato Dextrose Agar slants, and the bacteria to Nutrient Agar. Allow bacteria to grow for 24 hours and fungi to grow for 72hours, then store in the refrigerator until needed.

LEVEL 2 :

Set up for fungal amylase production

1. Pour 10 mL of sterile distilled water on the slant containing fungal spores

2. Scrape with a wire loop to loosen the spores

3. Inoculate medium with 0.5 mL spore suspension of fungi

Medium composition (grams per liter)

20

(Distribute in 30-40 mL of medium into 50 mL Erlenmeyer flasks and sterilize by autoclaving at 121ºC for 15 minutes) Allow to cool down to room temperature

Incubate with shaking (If shaker is available) for 72 hours. 200 rev/min is adequate if grown on a shaker. However, it possible to obtain a good yield with intermittent shaking with hand if a shaker is not available.

Set up for Bacterial Amylase production

1. Grow bacteria in Nutrient Agar, or Tryptic Soy agar slants

2. Add a loopful of bacterial culture into the amylase production medium

Medium for bacterial amylase production (g/1)

Bacteriological Peptone 6

MgSO4 ·7H 2 O 0.5

KCI 0.5

Starch 1

Mix and distribute in 30-40 mL volumes into 100 mL Erlenmeyer flasks Sterilize by autoclaving at 121ºC for 15 minutes .

EXTRACTION OF ENZYME FROM FUNGAI :

It is very easy to remove the fungal mycelium from the enzyme production medium. Pour the whole content of the flask containing the growing fungus through a funnel fitted with Whattman number 1 filter paper. The filtrate contains the crude amylase.

EXTRACTION OF ENZYME FROM BACTERIA :

You will need a high speed, refrigerated centrifuge for this. Pour the bacterial culture into centrifuge tubes, and spin for 20 minutes at 5000 rpm. Decant the supernatant, which is the crude enzyme extract.

Dialysis (Optional)

Dialysis will remove residual sugars from the enzyme mixture

i. Tie one end of a dialysis tube

ii. Pour enzyme mixture into dialysis tube

iii. Tie the other end securely

iv. Put dialysis tube in distilled water in a beaker

v. Change the water several time for 24hours

vi. Pour crude enzyme into clean Universal bottles or screw cap tubes and store in a freezer at about 0ºC until needed

Demonstration of Enzyme Activity

i. Pipette 1 mL of culture extract "enzyme" into a test tube

ii. Add 1 mL of 1% soluble starch in citrate-phosphate buffer (pH6.5)

iii. Incubate in a water bath at 40 ° C for 30minutes

iv. Set up a blank consisting of 2mL of the enzyme extract that has been boiled for 20 minutes (boiling inactivates the enzyme), added to the starch solution and treated with the same reagent as the experimental tubes.

v. Stop the reaction by adding 2 mL of *DNS reagent (1.0 g of 3, 5, dinitrosalicyclic acid, 20 mL of NaOH and 30 grams of sodium potassium tartarate in 100 mL)

vi. Boil for 5 minutes

vii. Cool and add 20 mL of distilled water

viii. Determine color intensity at 540 nm

A concentration glucose calibration curve is used to convert color to reducing sugar equivalent.

Enzyme activity may be defined as the amount of glucose produced per mL in the reaction mixture per unit time.

RESULT :

Thus, Amylase Enzyme is extracted & Purified by Dialysis method and confirmed by Enzyme activity tests.

POSSIBLE VIVA QUESTIONS:

1. Define Enzyme.

2.How are enzyme extracted?

3. What are the conformational tests for Enzymes?

4.List some sources of amylase.

5. Define Dialysis.

4. TO ESTIMATE THE DETERMINATION OF ACTIVITY OF ENZYME

α- AMYLASE

AIM:

To estimate the enzyme activity of α- amylase by DNSA (Dinitrosalicylic acid) method.

PRINCIPLE:

The substrate starch gels are broken down by the action of enzyme alpha amylase to form dextrin, limit dextrin and ultimately the monosaccharide glucose. Starch is the homopolysaccharide of glucose containing 20-30% straight chain amylase residues and 7080% branched chain amylopectin residues. The linkage in amylase is an alpha-1, 4 glycosidic linkage. Amylopectin posses the same basic chain attached by alpha -1,4 glycosidic linkage like that of amylase, but has in addition, many side chains attached to the basic chain by alpha-1,6 glycosidic linkages.

Alpha-amylase is an enzyme found in the digestive tract of the animals which hydrolyzes the linear amylase chain by attracting alpha-1, 4 linkage at random to produce maltose and ultimately glucose.

The reducing sugar glucose converts nitro salicylate under the alkaline condition to alkaline condition to amino nitro salicylate, an orange yellowish compound that has absorption, maximum at 540 nm. The activity of alpha-amylase is directly proportional to the concentration of glucose.

REAGENTS USED:

 Starch 2%

 Amylase enzyme

 Buffer (6-Phosphate buffer)

 DNSA Reagent

 Rochelle’s salt (4%) (Sodium Potassium Tartarate)

 Glucose stock (1 mg/ml)

PROCEDURE: I. STANDARD GLUCOSE CURVE:

 A series of test tubes containing different standard solutions of glucose such as 200 micro grams, 400 micro gram of glucose were taken according to the observation table.

 The volume of each tube was made up to ml with distilled water. 1 ml DNSA reagent was added to each test tube.

 The tubes were heated in the water bath for 10 minutes at 100 degree centigrade, and then 1ml of Rochelle’s salt was added to each tube.

 Absorbance was taken at 540 nm against reagent blank using colorimeter

 A standard curve was plotted with glucose concentration along x-axis a d optical density on Y-axis.

ESTIMATION OF GLUCOSE CONCENRATION

 A series of 4 test tubes as per the observation table.

 In each of the 4 test tube, starch buffer and amylase was added according to the observation table. Amylase addition incubation was done for 20 minutes.

 After pre incubation period of 35 minutes, 1ml of DNSA was added to each tube.

 Test tubes were incubated in water bath for 10 minutes at 100 degree centigrade followed by the addition of 1 ml of Rochelle’s salt to each test tube.

 Light vortexing is done and absorbance was noted at 540 nm against the reagent blank using colorimeter.

 From the graph standard curve, the concentration corresponding to the optical density is noted and the enzyme activity was calculated by using the formula,

Enzyme Activity = Concentration of glucose formed×dilution factor

Incubation Time

STANDARD: 1

Glucose

Glucose Concentration

Distilled water

Dinitrosalicilic acid

Rochelle’s salt

Optimum density at 570 nm

Incubate in water bath for 10 minutes

TABLE: 2

PARTICULARS BLANK

Starch (ml)

Buffer (ml)

Amylase (ml)

Incubation for 5 minutes

DNSA (ml)

Incubation for 30 minutes

Incubation for 10 minutes in boiling water bath

Rochelle’s salt (ml)

Distilled water (ml)

Optimum density at 570nm

RESULT:

The unit of enzyme activity per ml is ______________ kg/ml/min

POSSIBLE VIVA QUESTIONS:

1. Define enzyme activity.

2. Define substrate.

3. What are other possible methods to determine enzyme activity?

4.Why we are using standard glucose concentration curve in case of enzyme determination?

5. What are the compositions of Rochelle’s salt?

AIM:

5. STUDY OF ENZYME KINETICS

To study enzyme kinetics with the help of Michalies menton equation.

PRINICIPLE:

Enzyme being the aim metabolic regulator of any biology achieves living system plays the most role in studies of BC and MB and to get a detail views of properties of individual enzyme, enzyme kinetics is mentatory.

The factor that affects enzyme catalysis is

1) Temperature

2) pH

3) Enzyme concentration

4) Substrate concentration

MM equation was put forward consuming that in all enzyme catalyzed reactions, the rate limiting step is the break down of Emax complex to give the product and free enzyme K1 K3

The equation states, VO = Vmax(s)

Km + (S)

Vmax is an index of catalytic efficiency of an enzyme. In other words, it represents the specificity of action of a given enzyme towards similar subject.

In other words Km and Vmax represents the kinetic date of enzyme catalytic reaction.

Product of UI Publications-India

E+S ↔ ES → E+P K2

MATERIALS REQUIRED:

Enzyme solution, substrate solution, buffer, DNS, Rochelle’s salt

PROCEDURE:

The substrate, buffer, enzyme are being an as per table. In every case, the tubes were incubated for 30 minutes. Then after incubation 1 ml of DNS is added to all the tubes and heated on boiling water bath for 10minutes.

After cooling, 1 ml of Rochelle’s salt was added and 5ml of distilled water was added.

The intensity was measured at 570nm.

Using the given formula ,Enzyme Activity is calculated ,

Enzyme Activity = Concentration of glucose formed×dilution factor Incubation Time

the by using graph, MM equation is plotted.

Glucose

Glucose Concentration

Distilled water

Dinitrosalicilic acid

Rochelle’s salt

Optimum density at 570 nm

Incubate in water bath for 10 minutes

TABLE: 2

PARTICULARS BLANK

Starch (ml)

Buffer (ml)

Amylase (ml)

Incubation for 5 minutes

DNSA (ml)

Incubation for 30 minutes

Incubation for 10 minutes in boiling water bath

Rochelle’s salt (ml)

Distilled water (ml)

Optimum density at 570nm

RESULT:

Thus, from graph, the Vmax and Km value is calculated,

1) Vmax =

2) Km =

POSSIBLE VIVA QUESTIONS:

1. Define Enzyme kinectics.

2. What is the application of enzyme kinectics?

3. Define enzymes?

4. Is enzymes play a vital role in Biopharma industries?

5. How are Biocatalysts produced from Beneficial Microbes?

AIM:

6. PRECIPITATION OF PROTEIN BY AMMONIUM SULPHATE USING CHICKPEA

To precipitate the protein by using ammonium sulphate salt from chickpea.

PRINCIPLE:

Edible legumes provide readily available and economical source of protein for much of the world’s population.

The protein starved conditions of inhabitants of tropical Africa and other parts of the world.

The aim of the experiment is to precipitate protein by using ammonium sulphate salt from chick pea.

Ammonium sulfate precipitation is a method of protein purification by altering solubility of proteins.

It is a specific case of a more general technique known as salting out. Ammonium sulphate is commonly used as its solubility is so high that all salt solutions with high ionic strength are allowed.

The solubility of protein varies according to the ionic strength of the solution, and hence according to the salt concentration. Two distinct effects are observed.

1) At low salt concentration the solubility of protein increases with increasing salt concentration (i.e. increasing ionic strength), an effect termed “Salting in”.

2) As the salt concentration is increased further the solubility of protein will be decreases. At sufficiently increased ionic strength the protein will be completely precipitated from the solution (or), this effect termed as “Salting out”).

The precipitated protein was confirmed by biuret, ninhydrin qualitative tests comparing with control that doesn’t have ammonium sulphate salt.

PROCEDURE:

PROTEIN EXTRACTION:

1. Chick pea, Leupin, Lendil seed, protein was solubilized using 50g of chick pea suspended in 1litre of distilled water at pH 7.0.

2. Grind it with the help of mortar and pestle by using extraction buffer.

3. Distilled water-25°C-pH 7.0.

4. The pH was adjusted with 0.1M NaOH, pH adjusted to 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, and 12.0.

5. pH was maintained for 60 minutes with agitation. Then the suspension was centrifuged at 6000rpm at 20°C for 30 minutes and supernatants were stored at 4°C for future use.

SDS EXTRACTION FOLLOWED BY AMMONIUM SULPHATE:

Grind the chick pea into powder with the help of mortar and pestle by using extraction buffer 0.175M Tris Hcl, pH 8.8, 5% SDS,15%glycerol directly into the mortar and continue the grinding for additional 30 seconds.

PHENOL EXTRACTION FOLLOWED BY AMMONIUM SULPHATE PRECIPITATION:

Grind the chick pea into powder with the help of mortar and pestle by using extraction buffer 2.5ml of Tris, pH 8.8 buffered phenol, 2.5ml of extraction buffer (0.1M Tris Hcl pH 8.8, 10mM EDTA, 0.4% 2-mercaptoethanol, 0.9M sucrose) and continue the grinding for the addition of 30 seconds

AMMONIUM SULPHATE PRECIPITATION:

1. Protein precipitation by ammonium sulphate was carried out in which protein solution was placed in a beaker kept in ice and put over a magnetic stirrer (stirring was started slowly). 61.2g of solid ammonium sulphate were added in 100ml of solution.

2. Then pH of the solution was adjusted with 0.1M NaOH (The pH 7.0). The sample was centrifuged for 15 minutes at 7000rpm, 20°C.

3. Supernatant was decanted off then the protein pellets were resuspended in desired volume of buffer and residual ammonium sulphate was removed by filter paper.

CONFIRMATIONAL TEST:

NINHYDRIN TEST:

Add 2-5 drops of Ninhydrin to 1ml of test solution and keep it for 5 minutes in boiling water path and observe the color purple, pink, indicates the presence of protein.

BIURET TEST:

Add 0.5 ml of NaOH to 1ml of test solution and mix well. Then add 2-5 drops of copper sulphate and observe pink or violet colour which indicates the presence of protein.

RESULT:

POSSIBLE VIVA QUESTIONS:

1. Define Precipitation.

2. What are the methods involved in precipitation of proteins?

3. What are the conformational tests for proteins?

4.Why precipitation method is carried out for separation of proteins?

5. What are the techniques involved in protein production by large scale?

7. LEACHING

AIM:

To plot the theoretical and actual recovery versus solvent feed ratio

APPARATUS:

1. Measuring jar

2. Conical flask

3. Burette

4. Pipette

5. Stirrer

PRINCIPLE :

Leaching is a process of the removal of solute or solvent from a solid by the use of liquid solvent. It originally refers to the perforation of liquid through a fixed bed but it is also used for mean solid-liquid extraction generally. The presence of a solid phase distinguishes it from liquid extraction. TABULATION:

MODEL CALCULATION

Volume of NaOH×Normality of NaOH

Normality of Extract = ---------------------------------------------------------

Volume of Extract

Amount of solvent×100

Theoretical Calculation =

Amount of Solvent +Amount of Feed

Amount of solvent×100

Theoretical Calculation =

Amount of Solvent +Amount of Feed

Normality of extract × Equivalent weight of Oxalic acid × Volume of solvent

Actual Recovery = 1000

Actual Recovery × 100

Percentage of actual recovery = ------------------------------

TABULATION 2:

S.NO THEORETICAL RECOVERY ACTUAL RECOVERY SOLVENT FEED RATIO

PROCEDURE :

Two steps that are involved in solid-liquid extraction are:-

1. Contact of solid and solvent to effect transfer of solutes to the solvent.

2. Separation of the resulting solution from the residual solid.

1) About 45g of sand, 5g of oxalic acid and 100ml of distilled water taken in five beakers. About 50ml of water was added to the first beaker and stirred well and allowed to settle. From the supernatant solution 5ml if pipetted out and titrated against 0.2N NaoH.

2) About 45g of casein, 5g of glacial acid and 100ml of distilled water taken in five beakers. About 50ml of water was added to the first beaker and stirred well and allowed to settle. From the supernatant solution 5ml if pipetted out and titrated against 0.2N NaoH.

PREPARATION OF CASEIN:

Taking milk in a beaker and boiling in a waterbath, meanwhile adding drops of acetic acid in to it and stir continuously till casein get separated.then allow to cool and filtered with filter paper and air dried.

Then 100ml of water was taken in the second beaker and the same procedure was followed. Likewise, 150ml, 200ml, 250ml of distilled water were added to third, fourth and fifth beaker respectively and the same procedure were repeated for both 1 and 2.

From this, actual recovery, theoretical recoveryand solvent feed ratio were calculated. Agraphof %actualrecoveryand %theoretical recoveryversussolvent feedrationis plotted.

CALCULATION:

RESULT:

The theoretical , actual recovery versus solvent feed ratio was calculated and the graph was drawn.

POSSIBLE VIVA QUESTIONS :

1. Define Leaching.

2. Define casein.

3. How are leaching plays a role in separation of two immiscible solvents?

4. What is called Theoretical recovery?

5.What is called Actual recovery?

AIM :

To determine the area of the thickness and study the setteling characteristics of calcium carbonate slurry of concentration 50 kg / m3 for the feed ratio 2.5 tons / hr.

THEORY:

When dilute slurry is settled by gravity into a clean fluid and slurry of higher concentration the process is called as sedimentation or thickening. This is done thickener which is made of cylindrical vessel within which there is a flat bottomed having a central shaft set with a slurry resolving arm.

FORMULA:

CoZo

Ci = kg/m3

Zi

Where

Co = Initial concentration kg/m3

Zo =n Initial at time t = 0

Ci = Concentration of height Zi

Zi = Height level at t = I (in cm) (obtained from graph) FCo

Cu = Concentration of solid at Zo (cm)

A = Area of thickness in m2

F = Volumetric feed rate

dH

V = (slope obtained from graph)

dt CoZo

Cu = kg/m3

Zu

PROCEDURE:

50g of calcium carbonate is prepared in 1 litre of water with different heights of interference between clear clear liquid and settled solids and the time taken to reach each height are noted and tabulated.

A graph was plotted between height and time tangents are shown in various points and their slope intersections of y – axis are noted.

Values of Ci is found using the formula

CoZo

Ci = kg/m3 Zi

The slope which is find from the graph gives the values of V / (1 / Ci – 1 / Cu) is calculated. Then maximum area of thickness for the feed is calculated.

TABULATION: 2

RESULT:

The area of thickness is found to be = -----------------------

POSSIBLE VIVA QUESTIONS :

1. Define Sedimentation

2. Define Settling time.

3. What are the applications of sedimentation Techniques ?

4. Is centrifugal force plays a role in sedimentation, if so, How?

5.What did you observed from this experiment?

AIM:

To draw the binodal curve for the ternary system and find its concentration.

APPRATUS REQUIRED:

Micro Burette, Pipette, Conical flask.

THEORY:

Liquid liquid extraction called solution extraction is the separation of constituents liquid solution by contact with another soluble or partially soluble liquid. In all such operation, the solution which is to be extracted is called the 'feed'. The liquid solution which is to be fed and contacted is solvent. The solvent rich product of the operation is the extract and the residual liquid from which all the solute has been removed is raffinate. The most common type of system in extraction is that of three liquid in which onepairissoluble.TypicalexamplesareWater(A),Benzene(B),Aceticacid(C).Theliquid ‘c’ is completely dissolved in A and B only limited to extent each other.

The binodal curve is a solubility curve indicating the change in A and B rich phase upon the acetic acid. Any mixture inside the curve will be heterogeneous, the line joining the equilibrium composition of the extract and the raffinate phase lines is called the “Tie line”. There are infinite numbers of lives in 2 phases (Heterogeneous region). They are rarely parallel to change in slope slowly in one direction, the last if the point where A rich and B solubility curve is called “Plait Point”. It is ordinarily not the maximum volume on solution curve.

PROCEDURE:

TO DRAW A BINODAL CURVE:

The binodal curve extract and raffinate curve. To draw raffinate curve, different mixtures of water and acetic acid we prepared in conical flask by taking 10ml of water in each of the flask and adding 2, 4, 6, 8, 10 and 12ml of acetic acid respectively.

The mixture curve titrated separately with benzene added drop by drop through a micro burette till turbidityoccurs. Shaken well during titration. All this point, the volume of benzene and acetic acid were converted into weights and weight fractions. The raffinate phase curve is drawn on a triangular graph.

In the same way, for the extract curve different mixtures of benzene and acetic acid were prepared in conical flask by taking 10ml of benzene in each of the flask and adding 2, 4, 6, 8, 10 and 12ml of acetic acid respectively. The mixtures were titrated separately with water was done for raffinate curve.

To get the solubility of A in B and B in A i.e., to get the end point of the binodal curve. 10Ml of B was taken and titrated against A till the turbidity occurs and vive versa. The readings are tabulated, calculations are made and reported.

TABULATION 1- RAFFINATE PHASE

TABULATION 2- EXTRACTS PHASE

STANDARD VALUES:

Density of water = Kg/ml

Density of acetic acid = Kg/ml

Density of benzene = Kg/ml

CALCULATION:

BINODAL CURVE:

Weight = Volume × Density Individual Weight

Weight% = × 100 Total weight

Weight ( 2H O) =

Weight (AA) =

Weight ( 6c 6H ) =

Weight Fraction=

Weight% =

RESULT:

The binodal curve for the given system is drawn and the triangular graph was plotted.

POSSIBLE VIVA QUESTIONS:

1. Define Extraction.

2. Define Binodal curve.

3. What is called tie-line?

4. In what way extraction technique plays a role in Bioseperations?

5. Is homogeneous mixtures can be separated by this technique, if so, Explain?

AIM:

To isolate the genomic DNA from Bacteria using centrifugation.

PRINCIPLE:

Centrifugation is a process that involves the use of the centrifugal force for the separation of the mixtures, used in industry and laboratory settings. The rate of centrifugation is specified by the acceleration applied to the sample, typically measured in revolution per minute (RPM) or g. The particles settling velocity in centrifugation is a function of their size and shape, centrifugal acceleration, volume fraction of solids present, density difference between the particle and liquid and the viscosity. The centrifuge works using the sedimentation principle, where centripetal acceleration is used to evenly distribute the substances (usually present in a solution for small scale applications) of grater and lesser density. Applications for centrifugation are many and may include sedimentation of cells and viruses, separation of sub cellular organelles and isolation of macromolecules such as DNA,RNA proteins or lipids.

REAGENTS REQUIRED:

1. Solution I :

50mM glucose

25mM Tris HCL (pH 8.0)

10mM EDTA (pH 8.0)

solution I can be prepared in batches of 100ml. Autoclave for 156 minutes on Liquid cycle. Store at 4° C.

 10% SDS

Dissolve 10g of SDS (Sodium Dodecyl Sulfate) in 80 ml of distilled water and adjust the volume is made to 100ml with water.

6. 3M sodium acetate (pH 5.2 and 7.0)

Dissolve 40.82g of sodium acetate trihydrate (CH3COONa.3H2O; M.W. 136.08) in 80ml of water. Adjust the pH to 5.2 with glacial acetic acid or adjust the pH to 7.0 with dilute acetic acid. Adjust the volume to 100 ml with water.

5. Phenol

PROCEDURE:

 Spin down 5ml of saturated cell culture in eppendorff tube.

 Resuspend the pellet in 500µl of ice cold solution I. Incubate on ice for 10 minutes.

 Add 50µl of 10% SDS buffer. Incubate at 37° C for 5 – 10 min until clear and viscous.

 Add 550µl of phenol (freshly equilibrated with an equal volume of 0.3M Sodium acetate).

 Mix gently by inversion and centrifuge at 4° C for 15 min.

 Transfer the top layer to a new tube and repeat step 5

 Transfer top layer to eppendorff and one – tenth volume of 3 M Sodium acetate.

 Spin for 3 minutes and transfer the supernatant.

 Add 2 volumes of 100% ethanol and mix by inverting.

 Cool sample at-80° C for 5 min.

 Centrifuge for 15 minutes at 4° C.

 Remove and discard the supernatant.

 Vacuum dry the pellet and resuspend in 50 – 100µl of water.

RESULT:

Thus the Genomic DNA from bacteria was isolated using centrifugation.

POSSIBLE VIVA QUESTIONS :

1. Define Centrifugation.

2. How are centrifugal force plays a role in Bioseperations?.

3. Compare gravity force and centrifugal force.

4. Is centrifugal filtration plays a role in Bioseperation?

5.What is called centrifugal decantation?

About the Authors:

Mr. M.Abishek M.Tech (Biotech).,is currently working as Assistant Professor in Biotech Department, ROEVER Engineering College, Perambalur, Tamilnadu- India. He has International and National Experience around 15 years in Industry and Academics. He has Published more than dozen research Papers in Peer Reviewed Journals.

Mrs. R.Janani M.Sc.,M.Phil., (Biochemistry).,is currently working as Director in Bliss Academy,Trichy, Tamilnadu- India. She has International and National Experience around 13 years in Industry and Academics. She has Published more than seven research Papers in Peer Reviewed Journals.

Ms. S.Aiswarya M.Tech (Biotech).,is currently working as Assistant Professor in Biotech Department, ROEVER Engineering College, Perambalur, Tamilnadu- India. She has excellent track records in Academics. UI Media Publications-India

2023-24 Edition

Product of UI Publications-India