ASPARTOKINASE Introduction Nitrogen is essential for plant growth and development, and is often a limiting factor for high

productivity (Mauad et al., 2003; Medici et al., 2004). Amino acids are the major nitrogen containing compounds of plants and are the building-blocks of proteins (Helm et al., 2004). The essential amino acids lysine, threonine, methionine and isoleucine are derived from aspartic acid in a strongly regulated metabolic pathway (Lefvre et al., 2002; Toro et al., 2003). Aspartate kinase (AK, EC 2.7.2.4) is the first enzyme of the aspartate family pathway and is required for the synthesis of the amino acid end products (Azevedo et al., 1997). AK catalyses the phosphorylation of aspartate to form b-aspartyl phosphate, with the accompanying hydrolysis of ATP (Azevedo, 2002). AK has been identified, isolated and partially purified from a wide range of economically important crops, including some high-lysine mutants (Azevedo et al., 2003; 2004a 2000b; Ferreira et al., 2004). Several lines of evidence have indicated that plants generally possess at least two or three different isoenzymes of AK. The distribution of the AK isoenzymes may vary among the plant species. In general, the lysine-sensitive AK isoenzyme has been shown to represent approximately 80% of the total AK activity in almost all plant species tested so far (Azevedo & Lea, 2001). Homoserine dehydrogenase (HSDH, EC 1.1.1.3) catalyzes the first reaction that is unique for threonine, methionine, and isoleucine biosynthesis, which converts aspartate semialdehyde into homoserine in the presence of the coenzymes NADH or NADPH (Azevedo et al., 1997). Two isoenzymes of HSDH, one sensitive and the other resistant to threonine inhibition have been detected in plants (Azevedo & Lea, 2001). The existence of a bifunctional AK-HSDH isoenzyme sensitive to feedback inhibition by threonine, has been confirmed by the analysis of genes encoding the enzymes from several plant species (Muehlbauer et al., 1994). We have previously studied AK from a wide range of plant species and noted a considerable variability in its action and stability during isolation, purification and characterization. Maize is one of the major crops worldwide (Pinto et al., 2003) and in this report we have analyzed AK from maize tissues callus cell cultures and optimized the assay for the determination of AK activity from maize. Due to the nature of such a study, the experiments were carried out twice and the means of three repetitions for each assay were taken into consideration. As each optimal assay condition was established, it was immediately adopted as standard in subsequent experiments. In higher plants and bacteria the amino acids lysine, homoserine, threonine, isoleucine, and methionine are derived from aspartic acid (6-8, 13, 19). The first
1

reaction of the branched pathway is: Mg2+ L-Aspartate + ATP < i L-Aspartyl-phosphate + ADP and is catalyzed by aspartokinase (ATP: L-aspartate 4-phosphotransferase, EC 2.7.2.4). This enzyme has been extensively studied in bacteria but has not been definitely detected in plants. The enzymes from bacteria show considerable diversity, especially in their regulatory properties. In E. coli, there are three aspartokinases; one of which is inhibited by lysine, another by threonine, and the third shows no feedback regulation but is repressed by methionine (14, 18). Other bacteria possess only one aspartokinase, but this is regulated in a variety of ways. The enzyme from Rhodopseudomonas spheroids is inhibited by aspartic semialdehyde (5), whereas the enzyme from Rhodospirillum rubrum is inhibited by threonine (3). In other bacteria such as Rhodopseudomonas capsulatus (3, 4), Brevibacterium flavum (17), and some Bacillus species (10, 15), the enzyme is inhibited by the concerted action of lysine and threonine. There have been a number of attempts to detect this enzyme in plants, but it has never been positively identified. Webster and Varner (21) have reported an aspartylhydroxamate forming system in extracts of lupine, pea, and wheat germ, and there were recent attempts to find this enzyme in extracts of maize roots (2) and Marchantia (9). The present investigation shows the presence of aspartokinase in wheat germ and demonstrates that the higher plant enzyme is subjected to feedback regulation by lysine and threonine in a concerted manner. AMMONIUM SALT PRECIPITATION Introduction: Ammonium sulphate is a method for protein purification by altering the solubility of protein (NH4)2SO4 precipitation is a simple and effective means of fractionating protein. it is based on the fact that at high salt concentration the natural tendency of protein not to aggregate is overcome, since the surface charges are neutralized Charge neutralized means that protein will tend to find together, from large complexes and hence are easy to precipitate out by mild centrifugation Since each protein will start to aggregate at a characteristic salt concentration, than approach provides a simple way of enriching for particular protein in a mixture and is used SALTING OUT Increase in the salt concentration implies that there is less and less water available to solubilize protein finally ,protein starts to precipitate when there are not sufficient water molecules to interacts with protein molecule, then phenomenon of protein precipitation in the presence of excess salt is know as salting out SALTING IN At Low concentration, the presence of salt stabilizer the various charged groups on a protein molecule, then attracting protein into the solution and enhancing the solubility of protein, then phenomenon commonly know as salting in
2

FRACTIONATION: The precipitated proteins is collected and categorized to the concentration of salt solution at which it is formed. This partial collection of the separated protein pellet called Fractionation For example the atraction of the precipitated protein collected between 20 and 21% of salt saturation is commonly referred to as the 20-21% fraction Salting out is thought to work by dehydrating the environment

Ammonium sulphate is used for the precipitation of the protein. Different concentrations of salt is used for the precipitation of the protein, as the protein is get precipitated at a particular concentration of salt .By adding salt , the protein molecules dissolve as the ionic concentration of water and protein increase (salting in). By adding more salt , the water molecules which bound to the proteins make bond with salt, resulting all the protein molecules free from water .The free proteins makes bond with each other and get precipitated out (salting out ).Ammonium salt is used as it is 99.99% purified and it does not affect the proteins , easily available and dissolvable . DIALYSIS In biochemistry, dialysis is the process of separating molecules in solution by the difference in their rates of diffusion through a semi permeable membrane, such as dialysis tubing. Dialysis is a common laboratory technique, and operates on the same principle as medical dialysis. Typically a solution of several types of molecules is placed into a semi permeable dialysis bag, such as a cellulose membrane with pores, and the bag is sealed. The sealed dialysis bag is placed in a container of a different solution, or pure water. Molecules small enough to pass through the tubing (often water, salts and other small molecules) tend to move into or out of the dialysis bag, in the direction of decreasing concentration. Larger molecules (often proteins, DNA, or polysaccharides) that have dimensions significantly greater than the pore diameter are retained inside the dialysis bag. One common reason for using this technique would be to remove the salt from a protein solution. The technique will not distinguish between proteins effectively. Dialysis of Proteins After a protein has been ammonium sulfate precipitate and taken back up in buffer at a much greater protein concentration than before precipitation, the solution will contain a lot of residual ammonium sulfate which was bound to the protein. One way to remove this excess salt is to dialyze the protein against a buffer low in salt concentration.

This graphic illustrates the dialysis process. First, the concentrated protein solution is placed in dialysis bag with small holes which allow water and salt to pass out of the bag while protein is retained. Next the dialysis bag is placed in a large volume of buffer and stirred for many hours (16 to 24 hours), which allow the solution inside the bag to equilibrate with the solution outside the bag with respect to salt concentration. When this process of equilibration is repeated several times (replacing the external solution with low salt solution each time), the protein solution in the bag will reach a low salt concentration:

The graphic illustrates the complete dialysis process, except for it suggests you do this with distilled water. Really you want to do this process with buffer to prevent the protein from denaturing due to the fact that distilled or deionized water is too low in salt and may have an undesirable pH for your protein, which may cause it to denature. In fact, dialysis is a good way to exchange the buffer the protein is in at the same time you get rid of excess salt. After ammonium sulfate precipitation contains a mixture of buffers as well as excess salt. So we use the buffer we want for the next step in the purification, which is ion-exchange chromatography, as the external solution during dialysis. After the 3 step dialysis process where the protein solution is dialyzed against the starting buffer for the ion-exchange chromatography step, not
5

only will the salt be removed but the protein will now be in the buffer needed for the next step and ready to go. Sometimes, proteins will precipitate during the dialysis process and you will need to centrifuge the solution after dialysis to remove any particles which would interfere with the next step - such as ionexchange chromatography where particles would clog the column and prevent the chromatography step from working. In addition, you may lose enzyme activity during dialysis. So it is a good idea to keep some of your protein solution as a sample before it is put in the dialysis bag so that it can be assayed for enzyme activity before and after dialysis.

Review of Literature

Lysine, threonine, methionine and isoleucine are synthesized from aspartate in a branched pathway in higher plants. Aspartate kinase plays a key role in the control of the aspartate pathway. The enzyme is very sensitive to manipulation and storage and the hydroxamate assay normally used to determine aspartate kinase activity has to be altered according to the plant species and tissue to be analyzed. In this study an optimized assay for the determination of aspartate kinase in maize plants is used by using callus cell cultures. In this assay it has found that the concentration of ATP/Mg and temperature were critical for enzyme activity and 35C temperature was shown to be the optimum temperature for aspartate kinase activity. (Ferreira R et al., 2005) Renato Rodrigues Ferreira1; Ariane Vendemiatti2; Priscila Lupino Grato2; Peter John Lea3; Ricardo Antunes Azevedo2 Sci. Agric. (Piracicaba, Braz.), v.62, n.2, p.184-189, Mar./Apr. 2005

An aspartate kinase-deficient mutant of Thermus thermophilus, AK001, was constructed. The mutant strain did not grow in a minimal medium, suggesting that T. thermophilus contains a single aspartate kinase. Growth of the mutant strain was restored by addition of both threonine and methionine, while addition of lysine had no detectable effect on growth. To further elucidate the lysine biosynthetic pathway in T. thermophilus, lysine auxotrophic mutants of T. thermophilus were obtained by chemical
6

mutagenesis. For all lysine auxotrophic mutants, growth in a minimal medium was not restored by addition of diaminopimelic acid, whereas growth of two mutants was restored by addition of aaminoadipic acid, a precursor of lysine in biosynthetic pathways of yeast and fungi. A BamHI fragment of 4.34 kb which complemented the lysine auxotrophy of a mutant was cloned. Determination of the nucleotide sequence suggested the presence of homoaconitate hydratase genes, termed hacA and hacB, which could encode large and small subunits of homoaconitate hydratase, in the cloned fragment. Disruption of the chromosomal copy of hacA yielded mutants showing lysine auxotrophy which was restored by addition of a-aminoadipic acid or a-ketoadipic acid. All of these results indicated that in T. thermophilus, lysine was not synthesized via the diaminopimelic acid pathway, believed to be common to all bacteria, but via a pathway using a-aminoadipic acid as a biosynthetic intermediate.

JOURNAL OF BACTERIOLOGY, 0021-9193/99/$04.0010 Mar. 1999, p. 17131718 Vol. 181, No. 6 Copyright 1999, American Society for Microbiology. All Rights Reserved. Aspartate Kinase-Independent Lysine Synthesis in an Extremely Thermophilic Bacterium, Thermus thermophilus: Lysine Is Synthesized via a-Aminoadipic Acid Not via Diaminopimelic Acid NOBUYUKI KOBASHI, MAKOTO NISHIYAMA,* AND MASARU TANOKURA* Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan Received 28 September 1998/Accepted 23 December 1998

MATERIALS REQUIRED: Ammonium sulphate Protein sample Magnetic stirrer

7

 PROCEDURE:

Beaker Centrifuge Micro pipette Tris buffer

The supernatant after cell disruption was taken in a beaker. The volume of the sample was measured. 30% Ammonium sulphate was weighed. The beaker containing the sample was placed in the magnetic stirrer. While rotating Ammonium sulphate was added slowly to the sample. After adding Ammonium salt completely, the solution was transferred into the centrifuge tube. Centrifugation 15000 rpm for 10 min was done. The protein precipitate was collected and to that Tris buffer was added . These protein samples were taken for the purification by dialysis method.

OBSERVATION: The precipitated protein samples were taken for the dialysis process. PREPARATION OF DIALYSIS TUBE
1) Boil the tubing on a sitre plate (preferably in the hood) in a 4L volume of 2% (w/v) sodium

bicarbonate and 1mM EDTA pH 8.0 2) Rise the tubing in distilled water thoroughly 3) Boil for 10 minutes in 1mM EDTA (pH 8.0)
4) Allow tubing to cool then store it in freezer at 4oC with tubing submerged.

5) Before use wash out tubing with distilled water 6) Tie one end of the tube with thread and pour the protein the tube 7) Close the other end also tightly with the thread 8) Submerge the tube in the buffer and place the magnetic bead and place it on the magnetic stirrer 9) Change the buffer for every 1 hour and allow the bag in the buffer for 2 hours. 10) Collect the purified protein from the bag after the dialysis is completed. 11) The purified and collected enzyme is further purified and screed by ion exchange chromatography AFFINITY CHROMATOGRAPHY Introduction: Chromatography is a separation method that exploits the differences in partitioning behavior between
8

a mobile phase and a stationary phase to separate the components in a mixture. Components of a mixture may be interacting with the stationary phase based on charge, relative solubility or adsorption. The Russian botanist Mikhail Tswett is credited with original development of separation technique that we now recognize as a form of chromatography. In 1903 he reported the successful separation of a mixture of plant pigment using a column of calcium carbonate and in the process became the first scientist to recognize that chlorophyll was not a single compound. Modern chromatographic techniques take a multiple forms, the majority of which can be automated an adapted to deal with large or very small amounts of substances to be separated and purified. The Greek word chromo in chromatography means color in English and refers both to Tswet's name that is literally translated from Russian as color and to the color of the plant pigments he was separating at that time.

In 1952 Archer John Porter Martin and Richard Laurence Millington Synge were awarded the Chemistry Nobel Prize "for their invention of partition chromatography. Basically all chromatographic techniques systems consist of the stationary phase, which may be solid, gel, liquid or a solid/liquid mixture that is immobilized, and the mobile phase which may be liquid or gaseous and which flows over or through the stationary phase. The choice of stationary and mobile phases is made so that the compounds to be separated have different distribution coefficients. This may be achieved by setting up: a) An adsorption equilibrium between a stationary solid phase and a mobile liquid phase(adsorption chromatography, hydrophobic interaction chromatography)
b) A partition equilibrium between a stationary phase and mobile liquid or gas phase( partition

chromatography, reversed-phase liquid chromatography, ion-pair chromatography, chiral chromatography, gas-liquid chromatography) Affnity Chromatography Set Up

Columns: The glass column used should have a means of supporting the stationary phase as near to the base of the column as possible in order to minimize the dead space below the column support in which post column mixing of separated analyte could occur. Commercial column columns posses either a porous glass plate fused onto the base of the column or a suitable device for supporting a replaceable nylon net, which in turn support the stationary phase. A cheaper alternative to these commercial column supports is to use a small plug of glass wool together with a minimal amount of quartz sand or glass beads.

AIM: To separate the bacteriocins from the crude.

PRINCIPLE OF AFFINITY CHROMATOGRAPHY: This form of chromatography relies on the attraction between oppositely charged particles. Many biological materials, for eg. Amino acids and proteins have ionizable groups and the fact that they may carry a net positive or negative charge can be utilized in separating mixture of such a compounds. The net charge executed by these compounds is dependant on the pKa and on the pH of the solution in accordance with the Henderson Hasselbalch equation. Ion exchange separations are carried out mainly in columns packed with an ion exchanger. There are two types of ion exchanger, namely, Cation and Anion Exchangers.

10

Cation exchangers possess negatively charged groups and these will attract positively charged cations. These exchangers are also called acidic ion exchange materials because their negative charges result from the ionization of acidic groups. Carboxy methyl cellulose is a cation exchanger, here CM-cellulose is most applicable for the separation of proteins which are positively charged at around pH 4-5. Anion exchangers have positively charged groups that will attract negatively charged anions. The term basic ion exchange material is also used to describe this exchanger, as positive charges generally results from the association of the protons with basic groups. The most frequently used are Diethylaminoethyl (DEAE)-cellulose which is anion exchanger. The DEAE group, -CH2H5NH(C2H5)2,is highly positive charge at pH 6-8, so DEAE- cellulose is most useful for the chromatography of protein which are negatively charged in this range. Elution of the proteins from the columns may be brought about by changes in either salt concentration of salt (e.g. NaCl) increases, protein is displaced from DEAE by Cl- ions and from CMC-cellulose by the cation Na+ The ion exchange mechanism is thought to be composed of five distinct steps: Diffusion of the ion to the exchanger surface occurs very quickly in homogenous solution. Diffusion of the ion to the matrix structure of the exchanger to the exchange site is dependant upon the degree of cross linkage of the exchanger and the concentration of the solution. This process is thought to be the feature that controls the rate of the whole ion exchange process.
11

Exchange of ions at the exchange site. This is thought to occur instantaneously and to be an equilibrium process.

APPARATUS REQUIRED: Column chromatography Test tubes Beaker Pipette Distilled water

CHEMICALS REQUIRED:

Stationary phase DEAE cellulose Mobile phase Nacl in 25mM Tris buffer pH 8.0.

PREARATION OF STATIONARY PHASE:

Take 3 gm of DEAE cellulose.

Put in to the 10 ml D W.

12

Make slurry.

PREPARATION OF MOBILE PHASE: Tris Buffer: 50 mM pH 8.8 Dissolve 0.6gm of Tris Hcl in 50ml distill water. Make up to the volume up to 100ml by adding distill water.
ELUENTS BUFFER PREPARATION:

Take 10 test tubes and mark it as 1 to 8.

Add 40ml Tris buffer to each tube.

Add calculated weights of Nacl to the test tubes to give a series of concentration gradients.

SL NO 01 02 03 04 05 06 07 08 1. 2. 3. 4.

Tris Buffer(ml) 40 40 40 40 40 40 40 40

NaCl (gm) 0.058 0.117 0.175 0.234 0.290 0.350 0.406 0.468

concentration 25mM 50mM 75mM 100mM 125mM 150mM 175mM 200mM

PROCEDURE:

Packing the column

The column was taken & washed properly. To the column DEAE-cellulose slurry was loaded up to 10cm. Leave the column undisturbed for one hour. After settling DEAE cellulose formed the column matrix. 5. MATRIX : Length 5cm; diameter 0.5 cm. Washing the column
1. To the column 10 ml of 50 mM TRIS buffer pH 8.8 was loaded.

2. The buffer was eluted from the column. 3. This process was repeated twice.
13

4. Washing with buffer provides better flow rates. Flow rate determination The volume of sample in ml eluting from the column per minute is flow rate.
1. To the column 10 ml of 50 mM TRIS buffer pH 8.8 was loaded.

2. The buffer was eluted from the column. 3. The volume of the buffer to elute in 1 min was measured. 4. Flow rate of the column was1.25 ml/min. Loading the sample Volume of sample 10 ml.
1. 10 ml of sample was poured in column using a pipette.

2. Sample was eluted from the column. 3. Effluent was poured back in the column and reluted. 4. Process was repeated thrice to ensure all the 5. Proteins present in the dialysis sample binds with DEAE Cellulose.

ELUTION Washing with buffer

1. 20 ml 0f 50 mM tris buffer pH 8.8 was applied to the column.

2. This was done to remove the loosely bound peptides from the column 3. The elutants were collected in fractions of 2 ml each and there absorbance was determined at 280 nm. 4. There was no absorbance at 280 nm. Applying counterions (salt gradients)
1. 40 ml of 25 mM NaCl in 50 mM tris pH 8.8 was applied to the column. The effluent

2.
3.

4. 5.

was collected in fractions of 2 ml. Similarly remaining gradients were applied into the column one by one. The elutants were collected in fractions of 2 ml each and there absorbance was determined at 280 nm. Absorbance at 280nm was recorded for 150 mM NaCl gradient. For remaining gradients no absorbance was measured at 280nm.

Fraction number O.D at 280 nm (150 mM NaCl)

14

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

0.010 0.024 0.036 0.076 0.163 0.214 0.182 0.139 0.101 0.075 0.063 0.059 0.051 0.042 0.032 0.028 0.011 0.007 0.006 0.004

17 18 19 20

0.011 0.007 0.006 0.004

15

OBSERVATION: Bacteriocins eluted at the concentration of 150 mM NaCl.

ESTIMATION OF PROTEIN (BACTERIOCINS) BY LOWRYS METHOD AIM: To estimate the concentration of the BSA protein (Standard). PRINCIPLE: In these method peptide bonds of proteins reacts with copper under alkaline conditions to produce Cu+ ions. Cu+ ions react with FC reagent, phosphomolybdotungstate is reduced to hetero phosphomolybdenium blue by the copper-catalyzed oxidation of aromatic amino acids. The reaction results in a strong blue color, which depends partly on the tyrosine and tryptophan content. This method is sensitive down to about 0.01mg of protein/ml and is best used on solution with concentration in the range0.01-1.0mg/ml of protein REAGENTS REQUIRED:
1. Na2 CO3

2. NaOH 3. Copper sulphate

4. Sodium potassium tartarate

5. BSA protein 6. FC reagent PREPARATION OF REAGENTS:

16

Reagent A: 1% Na2CO3in 0.1N NaOH Take 0.4gm NaOH IN 50ml distill water and dissolve it. Then add 1gm Na 2CO3 to the mixture and maintain the volume upto 100ml. Reagent B: 0.5% CuSO4 in 1% NaKT Dissolve 0.05gm copper sulphate in 10ml of distills water and after dissolving completely add 0.1gm sodium potassium tartarate to it. Reagent C: Take 50ml of solution A and mix with 1ml of solution B. 1mg / ML BSA solution: Take 10mg (0.01gm) BSA protein and dissolve it in 10ml distill water. Fc reagent: Take 2ml of Fc reagent and make it 1:1 dilute by adding 2ml distill water.

Procedure: Take a series of test tubes and label them as 1 to 5. Then add different concentrations of standard between 0.2 and 1.00 to the test tubes. Then make up the test tubes to 1ml by using buffer. Then add 4ml of Reagent C to all the test tubes. Incubate the test tubes for 10 minutes at room temperature. Add 0.5ml FC Reagent to all the tubes.

OBSERVATION: A standard graph or curve is obtained by taking different concentrations of BSA protein and its O.D at 660nm.

17

GRAPH

Sl.No

Vol of ion Vol of exchange sample distilled water 1ml 0.9ml 0.8ml 0.7ml 0.6ml 0.5ml 0.4ml 0.3ml 0.2ml

Vol of reagent C

Incubation at room temp.

Vol of FC OD reagent values at 660nm 0.5ml 0.5ml 0.5ml 0.5ml 0.5ml 0.5ml 0.5ml 0.5ml 0.5ml 0.00 0.15 0.30 0.45 0.50 0.77 1.15 1.47 2.03

Blank 1 2 3 4 5 6 7 8

nil 0.1 0.2 0.3 0.4 0.5ml 0.6ml 0.7ml 0.8ml

5ml 5ml 5ml 5ml 5ml 5ml 5ml 5ml 5ml

30 min 30 min 30 min 30min 30min 30min 30min 30min 30min

OBSERVATION: The concentration of purified bacteriocin is found to be 200g/ml.

Molecular weight determination of bacteriocin in SDS-PAGE: The separation of macromolecules in an electric field is called electrophoresis. A very common method for separating proteins by electrophoresis uses a discontinuous polyacrylamide gel as a support medium and sodium dodecyl sulfate (SDS) to denature the proteins. This method is called sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
18

The most commonly used system is also called the Laemmli method after U.K. Laemmli, who was the first to publish a paper employing SDS-PAGE in a scientific study. SDS (also called lauryl sulfate) is an anionic detergent, meaning that when dissolved its molecules have a net negative charge within a wide pH range. A polypeptide chain binds amounts of SDS in proportion to its relative molecular mass. The negative charges on SDS destroy most of the complex structure of proteins, and are strongly attracted toward an anode (positively-charged electrode) in an electric field. Polyacrylamide gels restrain larger molecules from migrating as fast as smaller molecules. Because the charge-to-mass ratio is nearly the same among SDS-denatured polypeptides, the final separation of proteins is dependent almost entirely on the differences in relative molecular mass of polypeptides.

PRINCIPLE: There are three important "current-carrying" anions in SDS PAGE: 1) Glycine from the Tris-Glycine buffer found in the buffer above the stacking gel and below the resolving gel. 2) Chloride ions from the Tris-Cl buffer in the sample buffer, the stacking gel, and the resolving gel. 3) SDS/protein molecule. When the electric field is applied a "current-carrying" anion race through the gel is initiated. The

mobility of each "current-carrying" anion in the electric field is proportional to its charge/mass ratio; and the SDS-protein molecule all with the same charge/mass ratio are sieved by their size in the resolving gel matrix: small protein-molecule moves the fastest while larger ones sieve more slowly through the gel matrix. After a set amount of time (usually an hour or two), the protein micelles will have differentially migrated through the gel with the smaller proteins traveling farther down through the gel than the larger ones, which remain closer to the top of the gel. Thus proteins may be separated roughly according to size and molecular weight. Stacking occurs as a result of the differential rate of migration of the protein-micelles in the presence and absence of chloride ion "clouds" that initially surround and shield the SDS/protein micelles. To achieve this clearing of the chloride cloud, the titrate-ability of the glycine anion is employed. When the electric field is turned on, glycine, in the running buffer at pH 8.3 is slightly negatively charged and as such it carries the current in the buffer until it enters the sample buffer, pH 6.8, where the

19

glycine becomes neutral as the amino group becomes totally protonated and the carboxyl group remains de-protonated. The chloride ions in the sample buffer and the gels create a "cloud" through which the SDS/proteins micelles can migrate only relatively slowly in the electric field. It is as if the chloride ions shield the micelle strings from experiencing the full force of the applied electric field (they don't move very fast). However, the chloride ions in the sample buffer and the gel buffers carry the current in these parts of the system initially, and start their migration toward the positive electrode upon the application of the electric field. As the fastest moving species in the mix, the chloride ions, clear from the top of the sample buffer moving toward the positive electrode at the bottom of the gel, the slower moving SDS/protein micelles are left "out of the cloud of chloride". The entering glycine changes from negatively charged to neutral upon entering the pH 6.8 environment, leaving the protein-micelles unshielded so they now move faster toward the positive electrode than the micelles still in the chloride cloud lower in the sample buffer or in the gel. By the time the loaded sample reaches the resolving gel, the protein-micelles have managed to form a nice tight band < 1mm wide. This accounts for one component of the stacking phenomenon. The other components result from the slowing of the micelles upon encountering the various buffer-gel interfaces.

SDS POLYACRYLAMIDE GEL ELECTROPHORESIS APPARATUS

Preparing Polyacrylamide Gels:

20

A gel of given acrylamide concentration separates proteins effectively within a characteristic range. A typical gel of 7% acryl amide composition nicely separates polypeptides with molecular mass between 45kDa to 200 kDa. Polypeptides below the cutoff of around 45 kDa do not resolve. A denser gel, say 14%T, usually resolves all of the smallest polypeptides in a mix. Cassettes: We use casting stands to prepare the mini-slab gels. Two clean plates with two teflon spacers make a single cassette. We stack the cassettes upright in the stand with the bottoms of the cassettes tight to the bottom of the stand, using modeling clay to seal a thick acrylic cover in place against the last cassette to make a water-tight chamber. Using a well-former (comb) as a template, we mark a fill line about a centimeter below the bottom of the comb for the height of the first (separating) gel solution.

Polymerization of Polyacrylamide Gel: Acryl amide polymerizes spontaneously in the absence of oxygen, so the polymerization process involves complete removal of oxygen from the solution. Polymerization is more uniform if the mix is de-gassed to remove much of the dissolved oxygen, by placing it under a vacuum for 5 minutes or so before polymerization. When the bifunctional and polyfunctional units are combined or by crosslinking polymers from bifunctional monomers. Cross linkages can form from covalent bonds or through weak forces such as hydrogen bonds, Van Der Waals forces or hydrophobic or ionic interactions. Acryl amide and bis-acrylamide are dissolved in water together with polymerization initiators (Ammonium per sulfate) and catalyst (TEMED) TEMED reacts with Ammonium persufate to leave unpaired valence electrons (per sulfate free radical) which combines with acrylamide to form polyacrylamide gel. Once the catalysts TEMED are added, polymerization may occur quickly, thus it is necessary to have the casting stand completely ready and to have the overlay solution ready to go. After swirling to mix, we simply pour the solution into the space occupied by the cassettes. The
21

cassettes will self-level eventually, but leveling can be hurried along by adding solution to selected cassettes with a Pasteur pipette. Excess solution can be removed by tipping the apparatus and pulling off the excess with a pipette, so that the final level is at the fill mark.

PREPARATION OF ACRYLAMIDE 3gm Acryl amid 0.03gm Bis Acryl amide 10ml distilled water SOLUTION 1[10ml] 10%SDS SEPARATING GEL BUFFER 0.85mM Tris HCl pH 8.8 STOCKING GEL BUFFER 0.6mM Tris HCl pH 6.8

[stock solution]

ELECTROD BUFFER 200ml 0.05 mM TrisHCl 0.192 Glycine 10%SDS distil water 200ml APS: Prepare 5% Ammonium Per Sulphate dissolve in distilled water [this solution has to be

prepared freshly before use] TEMED: Commercially available SEPARATING GEL Stock Acryl amide 3.3ml
22

Separating gel buffer 2.0ml

10% SDS Distilled water TEMED APS

0.2ml 4.5ml 15-30l 150-250 l

STACKING GEL Stock Acrylamide Stacking gel bufffer Distilled water 10%SDS TEMED APS 1ml 1ml 3.5ml 0.1ml 15l 70

SAMPLE BUFFER Stacking buffer 10%SDS Glycerol Mercaptoethanol Distilled water 2.5ml 4.0ml 2.0ml 1.0ml 0.5ml

STAINING SOLUTIONS [100ML] 2gm coomasie blue 500ml methanol 100ml glacial acetic acid 400ml distill water 0.04ml [per 20ml] 10.0ml 2.0ml 8.0ml

DESTAINING SOLUTION [100ML]

23

Methanol Glacial acetic acid Distilled water

50ml 10ml 10ml

PROCEDURE:

All the vertical gel apparatus was washed by spirit. The gel casting tray was prepared by using the 3 spacer and join the glass by silica gel. After casting the vertical gel apparatus, separating gel was loaded (up to 5cm long) between the glass plates and allowed to solidified. Stacking gel was poured on the top of the separating gel. The comb was inserted immediately into the stacking gel and allowed the gel to set. Protein sample with the sample buffer was taken in the ratio 1:1. The sample was boiled for 2minutes in the water bath. After settling of the stacking gel the comb was removed slowly. Water molecules were dried from the wells by whatmann filter paper. The bottom spacer was removed and the gel slab was placed in the buffer in the vertical gel apparatus. The sample was loaded into the wells. Powers of 50 volts was supplied to the apparatus and allows the sample to run in the gel. The tracking dye bromophenol blue when reaches at the bottom of the gel the current was turned off. The gel was removed slowly from between the glass plates and put in coomassieve blue staining solution for overnight. The gel was washed in the destaining solution till a clear back ground comes. Only the stained proteins were visible as blue colour bands.

Biochemical characterization of purified pectinase: EFFECT OF pH ON ACTIVITY OF PECTINASE: O.D at 550 nm

24

The effect of the pH on the purified pectinase was examined at various pH values ranging from pH 3.0 to pH 9.0. Sl.No Volume of pectin Blank 1 0.0 ml 0.1ml Volume Volume Incubation of buffer 0.9ml 0.9ml (pH-3) 2 0.1ml 0.1ml 0.9ml (pH-4) 3 0.1ml 0.1ml 0.9ml (pH-5) 4 0.1ml 0.1ml 0.9ml (pH-6) 5 0.1ml 0.1ml 0.9ml (pH-7) 6 0.1ml 0.1ml 0.9ml (pH-8) 7 0.1ml 0.1ml 0.9ml (pH-9) 8 0.1ml 0.1ml 0.9ml (pH-10) 50C 4ml 5-15min 0.5ml 50C 4ml 5-15min 0.5ml 50C 4ml 5-15min 0.5ml 50C 4ml 5-15min 0.5ml 50C 4ml 5-15min 0.5ml 50C 4ml 5-15min 0.5ml 50C 4ml 5-15min 0.5ml 50C 50C 10 minutes enzyme 0.1ml 0.1ml Volum e DNS 4ml 4ml Water 100C 5-15min 5-15min Vol. of at Na k tartrate 0.5ml 0.5ml

1% of

of bath

Procedure: Take test tubes and then label them properly. Then add 0.1ml of standard solutions to the test tubes. Then add 0.1ml of enzyme to the all test tubes. Incubate the test tubes for10 minutes at 50C. Then make up the test tubes to 1 ml by using different pH 3.0 to 10.0 buffer solutions. Then add 4 ml of DNS reagent to all the test tubes.
25

 Maintain the tube blank as same as, instead of standard add distilled water to adjust the colorimeter to zero. Keep the test tubes in hot water bath at 100C for 10 minutes.
Then cool the tubes and add 0.5 ml of NaK tartarate to all the tubes.

Then take the O.D values by using colorimeter at 550nm. Then plot the graph between concentration Vs O.D values.

EFFECT OF TEMPERATURE ON ACTIVITY OF PECTINASE: The effect of temperature on the purified pectinase was determined at various temperatures ranging from 10 to 90 C OD at 550nm

Sl.No

Volume of pectin

Volume Volume Incubation Volu of buffer 0.9ml 0.9ml 0.9ml 0.9ml 0.9ml 0.9ml 0.9ml 0.9ml 0.9ml 0.9ml 0C 10C 20C 30C 40C 50C 60C 70C 80C 90C enzyme 0.1ml 0.1ml 0.1ml 0.1ml 0.1ml 0.1ml 0.1ml 0.1ml 0.1ml 0.1ml DNS 4ml 4ml 4ml 4ml 4ml 4ml 4ml 4ml 4ml 4ml

Water 100C 5-15min 5-15min 5-15min 5-15min 5-15min 5-15min 5-15min 5-15min 5-15min 5-15min

Vol. of at Na k tartrate 0.5ml 0.5ml 0.5ml 0.5ml 0.5ml 0.5ml 0.5ml 0.5ml 0.5ml 0.5ml

1% of

me of bath

Blank 1 2 3 4 5 6 7 8 9

0.0ml 0.1ml 0.1ml 0.1ml 0.1ml 0.1ml 0.1ml 0.1ml 0.1ml 0.1ml

Procedure: Take test tubes and then label them properly. Then add 0.1ml of standard solutions to the test tubes. Then add 0.1ml of enzyme to the all test tubes.
26

 Incubate the test tubes for10 minutes at 0C to 80C. Then make up the test tubes to 1 ml by using buffer solution. Then add 4 ml of DNS reagent to all the test tubes. Maintain the tube blank as same as, instead of standard add distilled water to adjust the colorimeter to zero. Keep the test tubes in hot water bath at 100C for 10 minutes.
Then cool the tubes and add 0.5 ml of NaK tartarate to all the tubes.

Then take the O.D values by using colorimeter at 550nm.

Then plot the graph between concentration Vs O.D values..

EFFECT OF SUBSTRATE CONCENTRATION ON ACTIVITY OF AMYLASE: Sl.No Volume of Starch Volume Volume Incubation of buffer (pH8.0) Blank 1 2 3 4 0.0 ml 0.1ml 0.2ml 0.3ml 0.4ml 0.1ml 0.1ml 0.1ml 0.1ml 0.1ml 1.0ml 0.9ml 0.7ml 0.5ml 0.6ml 50C 50C 50C 50C 50C 4ml 4ml 4ml 4ml 4ml
27

Volum e DNS

Water 100C

Vol. of at Na k tartrate

1% of enzyme

of bath

5-15min 5-15min 5-15min 5-15min 5-15min

0.5ml 0.5ml 0.5ml 0.5ml 0.5ml

5 6 7 8 9

0.5ml 0.6ml 0.7ml 0.8ml 0.9ml

0.1ml 0.1ml 0.1ml 0.1ml 0.1ml

0.5ml 0.4ml 0.3ml 0.2ml 0.1ml

50C 50C 50C 50C 50C

4ml 4ml 4ml 4ml 4ml

5-15min 5-15min 5-15min 5-15min 5-15min

0.5ml 0.5ml 0.5ml 0.5ml 0.5ml

Procedure: Take test tubes and then label them properly. Then add 0.1ml to 0.8 of standard solutions to the test tubes. Then add 0.1ml of enzyme to the all test tubes. Incubate the test tubes for10 minutes at 50C. Then make up the test tubes to 1 ml by using different pH 7.0 buffer solutions. Then add 4 ml of DNS reagent to all the test tubes. Maintain the tube blank as same as, instead of standard add distilled water to adjust the colorimeter to zero. Keep the test tubes in hot water bath at 100C for 10 minutes.
Then cool the tubes and add 0.5 ml of NaK tartarate to all the tubes.

Then take the O.D values by using colorimeter at 550nm. Then plot the graph between concentration Vs O.D values.

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