Estimation of Enzymes | Biotechnology

In this article we will discuss about the estimation of various enzymes:- 1. Catalase 2. Peroxidase (Pod) 3. Polyphenol Oxidase (PPO) 4. Indol Acetic Acid Oxidase 5. Xylanase 6. Cellulases 7. Lipase 8. Nitrate Reductase 9. Nitrite Reductase 10. Aflatoxin 11. Phenolics 12. Ergot Alkaloids.

Catalase:

This enzyme catalyses

(1) Decomposition of H₂O₂ to give water and oxygen.

(2) Oxidation of H donors e.g. methanol, formic acid, phenol-with one molecule of per­oxide.

Principle:

Solution of hydrogen peroxide absorbs U.V. light which can be measured between 230— 250 nm. When H₂O₂ is decomposed by cata­lase, the absorption decreases with time. From this decrease, enzyme activity can be found out. However, this method is applicable only for enzymes which do not absorb strongly at 230-250 nm.

Requirements:

1. Enzyme extract of plant tissue.

2. Phosphate buffer 0.067 (M) (pH 7.0) Dissolve 3.522 g of KH₂PO₄ and 7.268 g Na₂HPO₄.2H₂O in distilled water. Make up the volume to one litre.

3. H₂O₂ – Phosphate buffer.

Dilute 0.16 ml of H₂O₂ (10% W/V) to 100 ml with phosphate buffer. Prepare fresh. The absorbance of the solution should be 0.5 at 240 nm. with 1 cm light path.

4. Plant tissue.

5. Homogenizer.

6. Cuvettes.

7. Pipettes.

8. Cold room (4°C).

9. Glass marking pencil.

10. Spectronic 20.

11. Tubes.

Procedure:

1. Homogenise 1 g of plant tissue with phos­phate buffer (assay buffer diluted 10 times) at 1°-4°C and centrifuge. Add cold phosphate buffer and stir sediment. Al­low to stand in cold with intermittent shaking and repeat extraction one or two times. The whole process should not take more than 24 hrs. Combine all supernatants and use.

2. Prepare experimental cuvettes with 3 ml H₂O₂-P04 buffer. Add 0.01-0.4 ml sample and mix this with a flattened glass rod. Along with this, prepare control cuvettes with enzyme.

3. Note the time ‘t’ required for decrease in absorbance at 240 nm from 0.45 to 0.4. Use this value for calculation. If ‘t’ is more than 60 sec. use more concen­trated solutions of sample and repeat the experiment.

Calculation:

For 1 g tissue homogenised in 20 ml, diluted to l to 10 volume with water and from this 0.01 ml which was taken for assay, the absorb­ance at 240 nm decreased from 0.45 to 0.4 in 13.9 sec.

17 / 13.9 = 1.22 units in the assay mixture or

1.22 × 10/0.01 = 1220units/ml extract in 2.44 × 10⁴ units/g tissue.

Concentration of H₂O₂ can be calculated us­ing the extinction coefficient 0.036 per µ mole/ ml.

Peroxidase (Pod):

Peroxidase occurs in animals and higher plants and catalyses the dehydrogenation of large number of organic compounds like phenols, aromatic amines, hydroquinones etc. POD includes a group of specific enzymes like NAD-peroxidase, NADP peroxidase, fatty acid peroxidase etc.

Principle:

Guaiacol, the substrate is oxidised by POD resulting in more than one compound depend­ing on the conditions of reaction. The rate of formation of the dehydrogenation products is a measure of the enzyme activity which can be measured spectrophotometrically at 436 nm.

Requirements:

1. Guaiacol 20 mM solution:

Dissolve 240 mg guaiacol in distilled water and make up the volume to 100 ml. This can be frozen and stored for several months.

2. Enzyme:

1 g of horse radish (fresh) is ground in a mortar and pestle (pre cooled) with 3 ml of 0.1 (M) PO4 buffer (pH 7). Centri­fuge homogenate at 5°C for 15 minutes. Supernatant is kept on ice till use. It should be used within 2-3 hrs for assay.

3. H₂0₂ solution (12.3 mM=0.042%):

Dilute 0.14 ml of 30% to H₂O₂100 ml with distilled water. The extinction of this solution should be 0.485 at 240 nm. Freshly prepared solution should be used every time.

4. PO₄ buffer 0.1 (M) (pH 7.0)

5. Cuvettes.

6. Pipettes.

7. Spectrophotometer.

Procedure:

1. Pipette out 0.1 ml enzyme extract + 3 ml buffer solution (Bring buffer solution to 25°C before assay) + 0.05 ml guaiacol solution + 0.03 ml H₂O₂solution in a cuvette and mix well.

2. Place the cuvette in the spectrophoto­meter and wait till the absorbance has increased by 0.05.

3. Start a stop watch and note time (t) re­quired in minutes to increase the absorb­ance by 0.1.

Calculation:

Since the extinction coefficient of the dehydrogenation product of guaiacol at 436 nm under the specified conditions is 6.39 per micromolecule, the enzyme ac­tivity per litre of extract is Enzyme activity units/litre

Polyphenol Oxidase (PPO):

These are generally assumed to be single en­zymes with broad specificity, though there are reports of the presence of more than one en­zyme in certain tissues. PPOs are copper pro­teins that catalyse the aerobic oxidation of certain phenolic substrates to quinones which are autooxidised to brown pigments, the melanins. The PPO comprises of catechol oxidase and laccase and each individual enzyme tends to catalyse the oxidation of one particular phe­nol or phenolic compound more readily than others.

They are important with regard to appearence, palatability and use of fresh fruits, vegetables, mushrooms etc. since they have these enzymes. They are also important with regard to defence mechanism in plants against pests and diseases.

Principle:

Quinones generated through enzymatic oxi­dation of 4-methylcatechol (catechol oxidase) and 1, 4 hydroxybenze (laccase) reacts with the intensely yellow 2-nitro-5-thiobenzoic acid (TNB) with an absorption maximum at 412 nm to yield colourless adducts. This decrease in the absorbance of yellow colour due to en­zyme activity is measured on a spectropho­tometer.

Requirements:

1. 2-nitro-5-thiobenzoic acid-(TNB): 30 mg of sodium borohydride is added to a suspension of Ellman’s reagent [i.e. 5, 5-dithiobis (2-nitrobenzoic acid): 10 mg in 100 ml distilled water] within 1 hr, the disulphide is quantitatively reduced to the intensely yellow, water soluble thiol. Store at 4°C and is stable for one week.

2. Quinine solutions:

In a 50 ml volumetric flask dissolve 4- methyl-1, 2-benzoquinone in double dis­tilled water by bubbling nitrogen gas un­til quinone is dissolved completely.

P-berizoquinone solution also is prepared in the same way. Both solutions are sta­ble for 30 minutes and within this time spectrophotometric assay should be car­ried out.

3. Citrate-P0₄ buffer 0.2 (M) (pH 6.0).

4. Substrate solution. 4-methylcatechol (2 mM) for catechol oxidase assay. Quinol (1, 4-dihydroxy- benzene, 2 mM) for laccase assay.

5. Enzyme extract:

Homogenise acetone powder from fro­zen mushroom/fruit (25 g) using a blender in two successive 100 ml aliquots of cold acetone. By Buchner filtration through Whatman No. 1 filter paper col­lect the homogenate of both grindings. Air dry the homogenate until it is free of acetone smell. Weigh the dry powder and store it in freezer in cold containers.

For crude enzyme, mix 100 g of acetone powder with 2.5 ml of 0.2(M) citrate PO4 buffer (pH 6.0), + 1 ml of 1% Triton X- 100 + 6.5 ml distilled water + 500 mg polyamide. Shake for 1 hour and filter. The filtrate is used as enzyme source.

6. Pipettes.

7. Cuvetts.

8. Spectrophotometer.

Procedure:

1. Pipette out 1.4 ml citrate 0.1 (M) PO4 buffer (pH 6.0), 0.5 ml of TNB and 1 ml of the substrate solution. Add 0.1 ml of enzyme preparation (reaction initiated) and immediately note down the absorb­ance at 412 nm in an already set spec­trophotometer.

2. Record the decrease in absorbance at 30 sec. intervals.

Plot a graph.

Read change in absorbance per minute from the linear part of the curve.

Calculate the enzyme units.

Units in the test = K X (Absorbance/min) where K=0.272 for catechol oxidase. K= 0.242 for laccase. One unit of catechol oxidase or laccase is defined as the enzyme which transforms 1 µ, mol of dihydricphenol to 1 µ, mol of quinone per minute under conditions of assay. One unit is equivalent to the consumption of 1 µ mol of TNB.

Indol Acetic Acid Oxidase:

This is one of the commonly occurring plant auxin which takes part in controlling several phases of plant growth and differentiation. Levels of IAA are, in turn, regulated via syn­thesis, binding, esterification and enzyme degradation. Catabolic degradation of IAA to 3-methylene oxindole is done by IAA oxidase.

Principle:

IAAO activity is determined by measuring residual IAA after dark incubation with shak­ing at 30°C. IAA is determined by Salkowski reaction. Since monophenols act as cofactors of IAAO and o- and p-dihydroxy phenols and polyphenols act as inhibitors of this enzyme, the monophenolic compound, para-coumaric acid is added in the enzyme assay for activa­tion.

Requirements:

1. Para-coumaric acid solution: 25 mg of p-coumaric acid in 50 ml distilled water.

2. 0.071 (M) P0₄ buffer, pH 6.2.

3. IAA solution—10 mg IAA in 40 ml dis­tilled water.

4. Manganese chloride solution. 118 mg. MnCl₂.4H₂0 in 20 ml distilled water.

5. 5(M) Perchloric acid.

6. Ferric nitrate 0.1 (M). Dissolve 24.18 g in 100 ml distilled water.

7. Plant tissue.

8. Acetone.

9. Blender.

10. Buchner funnel.

11. Enzyme extract:

Homogenise 25 g of plant tissue in a blender and prepare acetone powder in two successive 100 ml aliquots of cold acetone. Filter through Whatman No. 1 in a Buchner funnel and collect the homogenate, air dry until free of acetone smell. Weigh the powder and store in freezer in cold containers.

12. Grind 1 g of acetone powder in two suc­cessive 20 ml aliquots of 25 mM PO₄ buffer (pH 6.2) in a chilled mortar in ice bath. Filter through Buchner funnel in Whatman No. 1 fil­ter after each grinding. Combine filtrates, di­lute to 50 ml with PO₄ buffer.

Procedure:

1. Pipette out in a test tube

PO₄ buffer (pH 6.2) 2 ml

P. coumaric acid: 1 ml

MnCl₂.4H₂O: 1 ml

Enzyme extract: 2 ml

in the order given above.

2. Add 4 ml IAA solution to start the reac­tion.

3. Incubate in dark with shaking at 30°C,

4. Withdraw 2 ml of the mixture after 0 and 50 min of incubation and add 5.2 ml perchloric acid and 0.5 ml ferric nitrate solution and dilute to 10 ml with distilled water.

5. Incubate reaction mixture in dark for 60 min. Measure absorbance at 535 nm.

6. Estimate protein content in the enzyme extract by Lowry et al, method.

Express enzyme activity as micromole IAA oxidised/min/mg protein.

Xylanase:

Xylan is a polymer of xylose units.

Xylose occur in two forms in nature:

(i) associated with glucuronic acid in polyuronide hemicellulose and

(ii) found as simple polymerised chain of pentose units.

The most abundant hemicellulose polysaccharide serving as an integral part of the cellulosic architecture of plants is xylan.

The main chain of xylan is composed of D-xylose and its structure is variable from lin­ear 1, 4-P linked polyxylose chains. Complete break-down of branched acetyl xylan needs several enzymes. The best known are endo-1, 4-(3-xylanase which attacks the polysaccha­ride backbone and (3-xylosidase which hydro­pses xylooligosaccharides to D-xylose.

Principle:

In the presence of substrate, the organism pro­duces the enzymes which releases xylose that can be estimated by DNSA method.

Requirements:

1. Modified medium:

Wheat bran: 50g

Yeast extract: 1.0g

Cellobiose: 1.0g

Asparagine: 0.5g

Citric acid: 0.1g

Nicotinic acid: 0.04g

MnS0₄  0.04

Distilled water: 100.00ml

2. Cultures of Pullularia pullulan Aspergillus niger A.flavus.

3. PDA slants.

4. 0.05 (M) Citrate buffer pH 4.8.

(Citric acid-1.92 g/100 ml… A

Sodium citrate- 2.94 g/100 ml….B.

Add 46 ml of A to 54 ml of B.

Xylan substrate-1 gxylan in 1 (N) NaOH (approx. 20 ml; until it dissolves), make final volume to 100 by adding distilled water after adjusting pH 6 with 1 (N) HCl.

Enzyme:

Incubate culture at 29°-30°C for 5-8 days on rotary shaker (150 rpm) with 50 ml medium. Filter the culture.

Procedure:

1. To 0.9 ml xylan substrate solution add 0.1 ml enzyme solution and incubate for 30 minutes at 50°C.

2. Centrifuge and from supernatant esti­mate reducing sugar xylose by dinitro­salicylic acid method and measure OD at 540 nm.

3. Prepare a standard xylose curve and find out the amount of xylose produced in the medium.

1 unit of xylanase activity is defined as the amount of enzyme releasing re­ducing sugar powder equivalent to 1 µ mol of xylose/minute.

Cellulases (C₁ AND C_x):

There are three major enzymes involved in the hydrolysis of crystalline cellulose:

(i) Endo-β- 1,4 glucanase (C_x-cellulase)

(ii) Exo-β-1,4 glucanase (C₁-cellulase)

(iii) β-glucosidase (Cellobiase)

Hydrolysis of native cellulose starts with exo-β-1,4-glucanase (C₁ enzyme) which splits up alternate bonds from the non-reducing end of cellulose chain, yielding cellobiose mol­ecules. Endo-β-1,4-glucanase (C_x enzyme) will not attack native cellulose but is distin­guished by the mechanism of its attack on carboxy methyl cellulose. β-glucosidase hy­dropses cellobiose which is an inhibitor of exoglucanase. Native cellulose like filter paper cotton etc. can be hydrolysed only by organ­isms that produce C₁-cellulase (exo- glucanase).

(i) Assay of C_x cellulase (Endo-β-1,4- Glucanase)

Viscometric Method:

Principle:

C_x enzyme acts on carboxymethyl cellulose (CMC) and the β-1,4-glucosidic bonds are randomly hydrolysed leading to the viscosity of CMC being reduced which is measured using an Oswald viscometer.

Requirements:

1. Carboxy methyl cellulose solution (0.5%) is prepared by dissolving 0.5 g sodium carboxymethyl cellulose in hot water and adjusting the pH to 6.0.

2. Citrate-P0₄ buffer 0.1 (M) (pH 6.0).

3. Enzyme.

4. Chloramphenicol-cyclohexamide so­lution.

In 20 ml distilled water dissolve 25 mg each of chloramphenicol and cyclohexamide.

5. Oswald viscometer.

6. Pipettes (graduated).

7. Test tubes.

8. Incubator (37°C).

9. Water bath.

10. Centrifuge.

Procedure:

1. Pipette out 3 ml of carboxymethyl cellu­lose + 1 ml of citrate-P04 buffer in a test tube and add 1 ml, of enzyme ex­tract.

2. Add 0.1 m chloramphenicolcyclohexa- mide solution to prevent contamination by microbes and incubate at 37°C for 16 hr.

3. Heat in boiling water for 3 min. Cool and centrifuge for 20 min.

4. Run a control of denatured enzyme (heat the enzyme extract for 3 min in boiling water for denaturing).

5. Measure the viscosity of 5 ml portions of control and test supernatant on an Oswald viscometer.

6. The reduction (%) in viscosity is propor­tional to cellulase activity.

where V= % loss in viscosity.

TO = flow time in seconds of zero time.

T= flow time after incubation and

T_H2O = flow time of water.

(ii) Assay of C₁-cellulase (β-1, 4-glucanase) Colorimetic-DNSA Method:

The production of reducing sugar glu­cose due to cellulase activity is meas­ured by DNSA method.

Lipase:

Triglycerides are hydrolysed by lipase to release free fatty acids and glycerol.

Lipases hydrolyse stored oil during germination of oil seeds like castor, sunflower ground nut etc. and provide the required energy for growth and carbon skeleton for synthesis of new compounds.

Principle:

The amount of fatty acid released in unit time is measured by the quantity of NaOH required to maintain constant pH. The milliequivalent of alkali consumed is taken as a measure of the activity of lipase.

Requirements:

1. 0.1 (N) NaOH.

2. Mortar and pestle.

3. Ice cold acetone.

4. Substrate:

2 ml of clear vegetable oil-pH 7.0 Stir well with 25 ml distilled water along with 100 mg bile salts (sodium taurocholate) till an emulsion is formed. Emulsification can be hastened by addition of 2 g of gum arabic.

5. Enzyme source:

Germinating ground­nut. Grind 5 g of ground nut seeds (germinat­ing) in a mortar and pestle with twice the vol­ume of ice cold acetone. Filter and wash the powder successively with acetone, acetone:ether (1:1) and ether. Air dry acetone powder in a refrigerator. Extract 1 g of pow­der in 20 ml ice cold water or a suitable buffer. Centrifuge at 15000 rpm for 10 min and use supernatant as enzyme source.

6. 50 mM P0₄ buffer (pH 7.0).

7. Beaker.

8. Magnetic stirrer cum hot plate.

9. pH meter.

10. Stop watch.

11. Burette.

12. Mortar and pestle.

13. Ground nut seeds.

14. Acetone.

15. Ether.

Procedure:

1. 20 ml of substrate + 5 ml PO₄ buffer (pH 7.0) in a 500 ml beaker and place it on a magnetic stirrer cum heater and stir contents slowly maintaining temperature at 35°C.

2. Dip electrodes of a pH meter in the re­action mixture.

3. Note pH and adjust to pH 7.0.

4. Add 0.5 ml of enzyme extract and note pH immediately and set the timer on. This pH is at zero time.

5. At intervals of 10 minutes or when pH drops by about 0.2 unit add 0.1 (N) NaOH to bring the pH to the initial value. Con­tinue titration for 30-60 min. and note the value of alkali consumed.

Enzyme activity can be defined as the amount of enzyme which releases one milliequivalent of free fatty acid per minute per gram of the sample. Specific activity is expressed as milliequivalents/min/mg protein.

Activity (meq/min/g. sample) =

Vol. of alkali consumed × strength of alkali / wt.of sample in g × time in minutes.

Nitrate Reductase:

In the biological process, assimilatory reduc­tion of nitrate by plants occur where a highly oxidised form of inorganic N₂ is reduced to nitrite and then to ammonia.

NO^–₃ + H₂ □ NO^–₂+ H₂O

This system has nitrate reductase and ni­trite reductase which catalyse stepwise re­duction of nitrate to nitrite and then to NH3.

Based on the specificity of electron donor, two major types of nitrate reductase occur:

(i) Ferredoxin dependent nitrate reductase (blue-green algae).

(ii) Pyridine nucleotide dependent nitrate reductase (higher plants).

Principle:

For the reduction of nitrate to nitrite, nitrate reductase is capable of utilising the reduced form of pyridine nucleotides, flavins or benzyl viologen as electron donors. Plants mostly have NADH dependent nitrate reductase and hence nitrate reductase activity in plants can be measured by following the oxidation of NAD (P)H at 340 nm. However, nitrate re­ductase activity is usually measured by colorimetric determination of nitrite produced.

Requirements:

1. 0.1 (M) potassium phosphate buffer (pH 7.5)

2. NADH 2 mM: Dissolving 14 mg NADH disodium salt in 100 ml distilled water.

3. Potassium nitrate 0.1(M)-Dissolve 1.01 g KNO₃ in 100 ml distilled water.

4. N-(l-naphthyl) Ethylenediamine Dihydrochloride, 0.02%-Dissolve 20 mg in 100 ml distilled water.

5. Sulphanilamide 1% (WAO-Dissolve 1 g in 100 ml 2.4(N) HCl.

6. Standard solution of potassium ni­trate 0.01(M)- Dissolve 851 mg in 100 ml distilled water. Dilute 10 ml of this to 100 ml-working standard solution.

7. Enzyme extract

Homogenize 1 g of fresh plant material in 6 ml of medium containing 1mM EDTA, 1-25 mM cysteine and 25 mM potassium phosphate adjusted to pH 8.8 with KOH. Filter through 4 layers of cheese cloth, centrifuge for 15 minutes. Decant supernatant through glass wool and use for assays. Extraction should be carried out under ice cold conditions.

8. Fresh plant material.

9. Tubes.

10. Pipettes.

11. Spectrophotometer.

12. Mortar and pestle.

Procedure:

1. Pipette out 0.5 ml P0₄ buffer (pH 7.5) + 0.2 ml KN0₃ + 0.4 ml NADH + 0.7 ml distilled water.

2. Add 0.2 ml of enzyme extract (to initiate reaction).

3. Set up a control in the same way but with 0.2 ml distilled water instead of enzyme.

4. Incubate both tubes at 30°C for 15 minute.

5. Rapidly add 1 ml of sulphanilamide + 1 ml of naphthyl ethylenediamine reagent to terminate reaction. Wait for 10 minutes and measure absorbance at 540 nm.

6. To prepare standard graph pipette out dif­ferent known aliquots of standard solu­tion of KNO₃ into a series of tubes and make up the volume to 2 ml by adding distilled water. Add 1 ml of sulphanilamide followed by 1 ml naph­thyl ethylenediamine reagent, wait for 10 minutes and measure absorbance at 540 nm.

Enzyme activity is expressed as micromole nitrite produced/min/mg pro­tein or g fresh tissue.

Nitrite Reductase:

Nitrate reductase directly reduces nitrite to ammonia.

This enzyme is present in both eukaryotes and prokaryotes. It accepts electrons from photosynthetically reduced ferredoxin but not from reduced pyridine nucleotides.

Principle:

Reduced methyl viologen is used as electron donor and the disappearence of nitrite is meas­ured.

Requirements:

1. Test tubes, pipettes blender, vortex mixer.

2. Sodium nitrite solution (43.2 mg. in 20 ml distilled water).

3. Tris-HCl Buffer 0.5(M) (pH 7.5).

4. Leaves.

5. Methyl viologen solution: 60.1 me­thyl viologen in 20 ml distilled water.

6. Sodium dithionite-bicarbonate solu­tion: 250 mg each Na₂S₂O₄ + NaHCO₃ in 10 ml distilled water.

7. Sulphanilamide

8. Naphthyl ethylenediamine

9. Enzyme extract.

Homogenise leaves (10 g) in 100 ml with Tris-HCl buffer (pH 7.5) in a warring blender for 3 minutes (high speed). Fil­ter through eight layers of cheese cloth, use filtrate as enzyme source. Extraction should be carried out in cold (4°C) room.

Procedure:

Reaction mixture:

6.25 ml Tris-HCl buffer + 2 ml NaN0₂ solution + 2 ml methyl viologen + 14.75 ml distilled water.

1. Pipette out 1.5 ml of reaction mixture + 0.3 ml enzyme preparation into a tube along with blank without enzyme.

2. Add 0.2 ml of freshly prepared dithionite -sodium bicarbonate solution to start re­action and incubate at 30°C for 15 min.

3. Stop the reaction by vigorously shaking on a vortex mixer until blue colour dis­appears.

4. Use a 20 µl aliquot for nitrite determina­tion.

5. Terminate reaction by adding 1 ml sulphanilamide followed by 1 ml naphthyl ethylenediamine reagent.

6. Wait for 10 minutes and measure absorb­ance at 540 nm.

7. Prepare standard graph with NaN0₂ as in the previous experiment [Different aliquots + distilled water (make up vol­ume to 2 ml) add 1 ml sulphanilamide + 1 ml naphthyl ethylenediamine-wait for 10 minutes and measure absorbance at 540 nm].

The enzyme activity is measured as µM of nitrite reduced/min/mg protein.

Aflatoxin:

Aflatoxin is an organic compound which ex­ists as B₁ and B₂ and M₁ and M₂ forms.

Among these B₁ is the most potent one. They are produced by species of Aspergillus es­pecially A. flavus.

Quantification of aflatoxin:

Requirements:

1. Culture of Aspergillus flavus- 5 days old.

2. Beaker, Erlenmeyer flask, separating funnel.

3. Water bath, shaker, centrifuge.

4. TLC, silica gel ‘G’, Whatman No.l filter paper, U.V. lamp.

5. Chemicals

Acetone, chloroform, methanol, lead ac­etate (20%), anhydrous sodium sulphate and anhydrous NaCl.

6. Standard of aflatoxin B₁ solution.

a. Stock solution: 4 mg Aflatoxin B₁/100 ml chloroform.

b. Working solution: Dilute 1 ml of stock solution to 10 ml with chloroform.

c. 5 microliters of working solution con­tain 0.02 microgram of aflatoxin B1.

Procedure:

1. Take 50 g of Aspergillus flavus culture from broth. Mix with 250 ml of 70% aque­ous acetone and keep it on a shaker (1200 rpm) for 60 minutes in a water bath.

2. Filter into 500 ml beaker and evaporate on a water bath to 140 ml.

3. Cool and add 20 ml of 20% lead acetate and 60 ml of water.

4. Centrifuge at 10000 rpm for 10 minutes, three times or double filter through Whatman No. 1 filter paper.

5. Transfer filtrate to a separating funnel and add 50 ml chloroform, shake well.

6. Collect lower chloroform layer and pass it successively through anhydrous NaCl and sodium sulphate.

7. Collect liquid in an Erlenmeyer flask and evaporate to dryness-crude extract.

8. Dissolve the crude extract in a known volume (0.5 ml) of chloroform.

9. Spot various aliquots of the standard (0.02, 0.04, 0.06, 0.08, 0.12 and 0.2 micrograms) and the unknown sample on a silica gel (0.02 mm) plate.

10. Develop plates in Chloroform: methanol mixture (95:5).

11. Compare spots under U.V. light (blue flourescence).

12. Calculate the amount of aflatoxin as parts per million (ppm) in the unknown sam­ple as follows:

Phenolics:

Estimation of Phenols Swain and Hillis (1959) Method.

Principle:

Phenols react with phosphomolybdic acid present in folin phenol reagent in an alkaline medium and develop blue coloured complex.

Requirements:

1. Folin-phenol reagent.

2. Sodium bicarbonate-saturated solu­tion in distilled water.

3. Standard tannic acid solution-Dis­solve 10 mg of tannic acid in little amount of 95% ethanol and make up the volume to 100 ml with distilled water (=100 µg/ ml).

4. Sample (Uromyces infected part)

5. Tubes and stand.

6. Graduated pipettes.

7. Mortar and pestle.

8. 80% ethanol.

9. Spectronic 20.

Procedure:

1. Grind 1 gm of rust infected plant (Uromyces hobsoni) in a mortar and pestle with 80% ethanol.

2. Take 0.1, 0.2, 0.3, 0.4, 0.5 ml of stand­ard tannic acid and 0.5 ml of the sample extract in tubes. Take one blank with 6 ml distilled water.

3. Add distilled water to make up the vol­ume to 6 ml and label all tubes.

4. Add 1.0 ml of folin phenol reagent to all tubes, keep for two or three minutes.

5. Add 1.0 ml of sodium bicarbonate solu­tion and keep for 1-4 hours.

6. Blue colour developed is read at 725 nm plot a graph.

7. Calculate the amount of phenol in the unknown sample.

Ergot Alkaloids:

The alkaloids are produced in the sclerotia of the ergot fungi Claviceps paspali and C. purpurea parasitizing, Paspalam and rye re­spectively. The sclerotia have over 100 com­pounds falling under ten groups of substances viz. amino acids, simple bases like betaine, clavine, tyrosine, histidine, ergotic acid, ergothiomine, cadavarine, putrescine, isoamy- lamine and tyramine. Besides about 25-30% fats and oils have also been reported to be present.

There are three groups of ergot alkalids:

1. Ergotamine group: ergotamine, ergosine and corresponding isomers.

2. Ergotoxine group: ergocornine, ergocristine, ergocryptine and their isomers.

3. Ergometrine and its isomers.

Claviceps paspali has (i) Ergine, Isoergine (ii) Lysergic acid, hydroxy ethylamide. C.purpurea and C. paspali has (iii) Er­gometrine + Ergometrimine. C. purpurea has (iv) Ergosecaline, Ergosecalinine (v) Ergot­amine, Ergotaminine (vi) Ergosine, Ergosinine (vii)Ergocristine, Ergo-cristinine (viii)Ergo- cornine, Ergo-corninine (ix)Ergo-cryptine, Ergocryptinine.

Production Large scale production will be successful de­pending on (a) proper selection of fungal strain (b) selection of suitable medium and (c) con­tainer in which inoculum has to be multiplied and transported to fields.

Selection of strain:

1. Select sclerotia of virulent strain. Every year new cultures should be made.

2. Surface sterilize sclerotia with 1/1000 HgCl₂ (5 minutes) wash several times with sterile distilled water.

3. Remove central portion of stroma with a sterile needle/scalpel and inoculate on potato dextrose agar (composition given under fungi) and incubate at room tem­perature (28°C) for 10-14 days.

4. In die mean time, soak wheat or rye and oats (1.5 g) in 125 ml tap water for 8-12 hours. Drain off water and fill 500 ml milk bottles/flasks and autoclave under 15 lb pressure for one hour.

5. Make suspension of conidia from 10-14 days old culture on PDA and inoculate the rye and oats seeds.

6. Incubate at 15°-25°C for one month.

7. Make conidial suspension and use for inoculation of ear heads in field.

Inoculation apparatus:

This consists of good quality heat resist­ant glass aspirator of 4 litre capacity with an outlet at the bottom which is con­nected to a heat resistant 1.5 to 2 metre rubber tubing. This rubber tubing is con­nected to a metallic delivery unit. This delivery unit for bottles consist of a me­tallic nozzle with long sprout of stainless steel, copper or brass 25-30 cm long and with a basal controlling unit.

8. Pass contents of the (spawn) bottle with conidia through a wire screen to sepa­rate grains (rye & oats).

9. Inoculate the plants in plots after emer­gence of spike with this spore suspen­sion (3200 spores/ml).

10. For inoculation, dip a sponge rubber cov­ered board, catch the spikes in between it and a needle studded board and press each spike in between them.

11. Harvest sclerotia after 6-8 weeks of in­oculation.

12. Dry them in shade, otherwise alkaloid will get transformed from active to inactive form.

13. Powder dry sclerotia in a mortar and pestle.

14. Add 2 ml of 4% tartaric acid dissolved in an analar grade methanol, for each 50 mg of sclerotia powder in the tube.

15. Heat the tube in a water bath upto al­most boiling stage only but not boiling.

16. Remove the tube and shake vigorously for 30 seconds.

17. Repeat heating and shaking thrice and add 2 ml of aqueous 10% zinc acetate solution and shake vigorously (150-175 shakes/minute) for 10 minutes.

18. Centrifuge at 2500 rpm for 3 minutes.

19. Remove 1 ml of the supernatant to a clean dry tube and add 2 ml of modified Van Urk reagent containing paradi-methyl amine-benzaldehyde (PDAB).

20. Allow the mixture to stand for 30 min­utes (till colour develops).

21. Measure absorbance at 590 nm.

22. Keep for 6 hours at room temperature (28°C) and take a second reading.

23. Convert OD to % of alkaloid with the help of a standard curve for authentic (standard) samples.

P. S. Do not expose reaction mixture to strong day light.