EP1705987A2

EP1705987A2 - Proteins of nutraceutical and biotherapeutic potential

Info

Publication number: EP1705987A2
Application number: EP04787519A
Authority: EP
Inventors: V. M.; c/o Avestha Gengraine Tec. Pvt Ltd PATELL
Original assignee: Avestha Gengraine Technologies Pvt Ltd
Current assignee: Avesthagen Ltd
Priority date: 2003-09-24
Filing date: 2004-09-17
Publication date: 2006-10-04
Also published as: WO2005027632A2; WO2005027631A2; EP1708558A4; WO2005027631A3; EP1708558A2

Abstract

The present invention relates to the isolation of a nucleic acid sequences from Indian traditional medicinal plants, the products of which, confer protection to plants, animals and human beings against environmental stress like salinity, drought and high temperature and improves oxidative stress tolerance. They are also found to act as useful molecules in the pharmaceutical industry on account of their antioxidant properties. Another aspect of the invention relates to the isolation of a nucleic acid sequences from Indian traditional medicinal plants, the products of which can be used novel biotherapeutics.

Description

PROTEINS OF NUTRACEUTICAL AND BIOTHERAPEUTIC POTENTIAL

Field of the Invention

The present invention relates to the isolation of a nucleic acid sequences from Indian traditional medicinal plants, the products of which, confer protection to plants, animals and human beings against environmental stress like salinity, drought and high temperature and improves oxidative stress tolerance. They are also found to act as useful molecules in trie pharmaceutical industry on account of their antioxidant properties. Another aspect of the invention relates to the nucleic acid sequences from Indian traditional medicinal plants, the products of which can be used as nutraceuticals, biotherapeutics and functional food supplement.

Background of the Invention

Plants possess an array of compounds which have anti oxidant properties and which are believed to play a significant role in protection against abiotic and biotic stress, a prominent one of which is glutathione ascorbic acid (Vitamin C), phenolic isoflavanoid compounds and carotenoids. The reduced forms of these compounds together with anti oxidant enzymes are believed to scavenge the reactive oxygen species and other products of oxidative reaction. Several reduction oxidation cycles that scavenge the reactive oxygen species in different sub cellular compartments involve these enzymes and anti-oxidants.

Glutathione is considered as a key component of anti oxidant defenses in most aerobic organisms including plants (Foyer et. al., 1997). There are two forms of glutathione peroxidase enzyme in plants, namely, the chloroplastic glutathione peroxidase and the cytosolic glutathione peroxidase. The latter is believed to have two functions, as a glutathione peroxidase and as a glutathione S transferase. Literature has revealed that over expression of ascorbate peroxidase isolated from barley showed enhanced thermo tolerance in Arabidopsis thaliana. The transgenic plants were significantly more tolerant to heat stress as compared with the wild type (Shi WM et al., 2001).

Expression analysis of the promoter GUS-fusion on transgenic plants revealed that the gene is under strict developmental control, becomes transcriptionally active in cells which apparently undergo mechanical stimulation generated during different phases of growth and this gene may constitute a basic mechanism for deployment of antioxidative defences in plants which act to limit the deleterious effects of hydrogen peroxide during normal plant development (Gadea J., et al., 1999).

Transgenic tobacco plants that over express ascorbate peroxidase either in the cytoso or chloroplastic compartments also show reduced damage following either methyl viologen exposure or photo oxidative treatment and transgenic plants that express increased levels of Glutathione reductase have elevated pools of ascorbate and GSH. Though still preliminary, these results clearly indicate that alterations in the expression of enzymes involved in Reactive Oxygen Intermediates scavenging can have significant metabolic effects and provide the hope that this strategy can be used to develop crop plants with increased stress tolerance (Allen R.D. et al., 1997).

Increase in hydrogen peroxide levels are known to be associated with leaves of higher plants, under adverse environmental conditions such as ultra violet radiation, temperature and increased light intensity. Expression of catalase in plants alleviates the problems associated with higher levels of hydrogen peroxide.

Photooxidative stress imposed by paraquat treatment under light illumination in transgenic tobacco plants expressing catalase from E. coli targeted to the chloroplast showed increased tolerance to drought stress at high light intensity. There are reports of this molecule's presence in pharmaceutical compositions for use in the treatment of cancer, arthrosis, hepatitis, lipomatosis, uricaemia and hypercholestarolemia and as an anti ageing cosmetic.

Catalases are present in certain animal, plant and microorganism cells, and are the required enzymes for these cells to survive in the aerobic environment. At present almost all the catalases available are obtained by purification from such cells, and among which the catalase derived from bovine livers, in particular, has been the most preferred one for the above mentioned purposes. However, in 1990, a chronic viral disease known as BSE (bovine spongiform encephalopathy) out-broke in European cattle herds. Recent studies have shown that human may be infected with this disease [Dellar and Lacey 1991 Nutr. Health (Bicester) 7:117-134; Dealler and Lacey 1990 Food Microbiol. 7:253-280). It arouses interests in finding non-mammalian- derived catalases, such as those derived from microorganism sources, instead of bovine liver catalases for the industrial applications.

WO0136454 patent application describes the method of solving the problem of counteracting the destructive oxidative effect of elevated levels of ROS and free radicals by providing peptide compounds that stimulate (i. e., upregulate) expression of genes encoding antioxidative enzymes, such as superoxide dismutase (SOD) and/or catalase (CAT), to reduce, eliminate, or prevent an undesirable elevation in the levels of ROS and free radicals in cells and tissues, and to restore age-related reduction of constitutive antioxidative enzymes. Furthermore, the peptide compounds of this invention may have antioxidative activity independent of their ability to stimulate expression of genes encoding antioxidative enzymes.

JP10179167 patent describes cold-resistant enzyme that comprises a cold- resistant catalase including a specific amino acid sequence, imparting resistance to low temperatures to plants such as rice plant by introduction of its gene and is useful in physiological decomposition of hydrogen peroxide used in bleaching or washing.

Reactive oxygen species are produced either from light-dependent energy conversion or by chemical electron transfer to molecular oxygen in the metabolism of aerobic organisms. ROS in turn oxidize susceptible biomolecules, and, subsequently alkyl hydroperoxides are formed. In plants the chloroplast is particularly prone to oxidative damage by photosynthetic oxygen production and activation. Despite the presence of elaborate enzymatic and nonenzymatic antioxidative defense mechanisms, ROS escape from detoxification and oxidize organic compounds such as proteins, nucleic acids, terpenoids, and fatty acids to the respective peroxides. In addition, alkyl hydroperoxides are formed by enzymatic reactions in chloroplasts, e.g. lipoxygenase catalyzes peroxidation of fatty acids and other desaturated organic biomolecules, such as carotenoids. Detoxification of alkyl hydroperoxides is important because they can act as long-distance mediators of oxidative damage by oxidizing other biomolecules and initiating radical chain reactions. For example, proteins and membrane lipids are oxidized, which results in degradation, loss of membrane function, and death of the organism. Apparently detoxification of alkyl hydroperoxides is indispensable but yet not understood in plants. (Protective Function of Chloroplast 2 Cysteine Peroxiredoxin in Photosynthesis. Evidence from Transgenic Arabidopsis. Plant Physiology, April 1999, Vol. 119, pp. 1407-1414)

Objects and Summary of the invention:

The first object of the invention relates to the isolation of a nucleic acid sequences from Indian traditional medicinal plants, the products of which, • confer protection to plants, animals and human beings against environmental stress like salinity, drought and high temperature and improves oxidative stress tolerance.

Another object of the invention relates to the use of these proteins as important molecules in the pharmaceutical industry, on account of their antioxidant properties.

Yet another object of the invention is isolation of a nucleic acid sequences from Indian traditional medicinal plants, the products of which can be used novel biotherapeutics. Brief description of the Figures:

Seq ID. No 1: Nucleic acid sequence of Glutathione peroxidase isolated from Terminalia arjuna.

Seq ID. No 2: Nucleic acid sequence of Ascorbate peroxidase isolated from Terminalia arjuna.

Seq ID. No 3: Nucleic acid sequence of Catalase isolated from Terminalia arjuna.

Seq ID. No 4: Nucleic acid sequence of Cysteine-2-peroxidase isolated from Gloriosa superba.

Seq ID. No 5: Nucleic acid sequence of Glutamate decarboxylase RASI variety of rice seedlings under environmental stress conditions.

Seq ID. No 6: Nucleic acid sequence of thionine from RASI variety of rice seedlings under environmental stress conditions.

Seq ID. No 7: Nucleic acid sequence of Arabinoxylan furanohydohydrolases RASI variety of rice seedlings under environmental stress conditions.

Seq ID. No 8: Nucleic acid sequence of Methionine aminopeptidase isolated from Gloriosa superba.

Seq ID. No 9: Nucleic acid sequence of microsomal signal peptidase isolated from coleoptile of

Gloriosa superba.

Seq ID. No. 10: Nucleic acid sequence of S- Adenosylmethionine Synthetase isolated from

Terminalia arjuna.

Seq ID. No. 11 : Nucleic acid sequence of Beta glucosidase isolated from Terminalia arjuna.

Detailed description of the Invention

Biological organisms generate harmful reactive oxygen species (ROS) and various free radicals in their cells and tissues in the course of normal metabolic. The most reactive and, therefore, toxic ROS and free radicals include the superoxide anion (ou"), singlet oxygen, hydrogen peroxide (H202), lipid peroxides, peroxinitrite, and hydroxyl radicals. Even a relatively small elevation in ROS or free radical levels in a cell can be damaging.

Likewise, a release or increase of ROS or free radicals in extracellular fluid can jeopardize the surrounding tissue and result in tissue destruction and necrosis. Indeed, hydrogen peroxide, which is somewhat less reactive than the superoxide anion, is a well known, broad spectrum, antiseptic compound. In eukaryotes, a major source of superoxide anion is the electron transport system during respiration in the mitochondria. The majority of the superoxide anion is generated at the two main sites of accumulation of reducing equivalents, i. e., the ubiquinone-mediated and the NADH dehydrogenase-mediated steps in the electron transport mechanism. Hydrogen peroxide is generated metabolically in the endoplasmic reticulum, in metal-catalyzed oxidations in peroxisomes, in oxidative phosphorylation in mitochondria, and in the cytosolic oxidation of xanthine.

Accordingly, an elevation of ROS and free radicals has been linked with the progression and complications developed in many diseases, drug treatments, traumas, and degenerative conditions, including age-related oxidative stress induced damage, Tardive dyskinesia, Parkinson's disease, Huntington's disease, degenerative eye diseases, septic shock, head and spinal cord injuries, Alzheimer's disease, ulcerative colitis, human leukemia and other cancers, and diabetes.

Plants, which express genes in either or both sub cellular compartments, have revealed increased glutathione content in the leaves and/or in the developing fruit. The increased anti oxidant capacity resulting from sustained, elevated glutathione content is expected to be beneficial to the plant including resistance to a number of potentially damaging oxidative events, arising from both, biotic and abiotic stress.

The methods and vectors of the present invention may play a significant role in increasing tolerance to abiotic and biotic stress, enhance plant capacity for anti oxidant regeneration and leading to improvements in water use and nutrient uptake.

According to the present invention, there is provided a method of manipulating the oxidative status of a plant, wherein the genes are differentially expressed under the control of different promoters. In the alternative, the construct also includes one gene encoding an enzyme involved in the redox cycling of glutathione between its reduced and oxidized forms. Preferably the gene encoding an enzyme involved in the redox cycling of glutathione is a gene encoding the glutathione peroxidase. The nucleic acid construct will most preferably comprise of the glutathione peroxidase gene. The alteration in the oxidative status may be assessed by comparing with a plant in which the nucleic acid has not been introduced. Preferably the construct is comprised within the vector. Wherever reference is made to genes encoding an anti oxidant enzyme, capable of reducing oxidized glutathione, it should be understood that except where the context demands otherwise, variants, both, artificial and natural, may be used as long as the variant forms retain the ability to encode a polypeptide with an appropriate corresponding enzymatic capability. Variants may used to alter the oxidative stress resistance characteristics of the Terminalia arjuna plant. In the alternative they may include a sequence which interferes with the expression or activity of the polypeptide, namely, the sense or anti sense suppression.

Variants may be naturally occurring nucleic acids or they may be artificial. They may include orthologues, alleles, isoalleles or homologues of the gene. The variants may include only a distinctive part or fragment corresponding to a portion of the relevant gene, encoding at least functional parts of the polypeptide. The vectors will be stable and capable of modifying the production and/or redox cycling of the glutathione in organisms in which they are expressed.

Homology may be at the nucleotide sequence and/or at the amino acid sequence level, around 70% homology. Homology may be over the full length of the relevant sequence.

Changes to a sequence may produce a derivative by way of addition, insertion, deletion or substitution of one or more nucleotides in the nucleic acid leading to the addition, deletion, insertion or substitution of one or more amino acids in the encoded polypeptide. Such changes may alter sites required for post translation alteration in the encoded polypeptide. Altering the primary structure of a polypeptide may not significantly alter the activity of that peptide since the side chain of the amino acid which is inserted into the sequence may be able to form similar bonds and contacts as the side chain of the amino acid which has been substituted. Substitutions to regions of a peptide which are not critical in determining its confirmation because they may not significantly alter the three dimensional structure of the peptide. In regions that are critical in determining the confirmation or activity of the peptide, such changes may confer advantageous properties on the peptide, e.g. altered stability or specificity. Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences including promoter sequences, polyadenylation sequences, enhancer sequences, marker genes, and other appropriate sequences. Shuttle vector refers to a DNA vehicle capable, naturally or by design, of replication in two different host organisms, e.g. higher plant, yeast or fungal cells.

The nucleic acid in a vector is under the control of and operably linked to two appropriate promoters or other regulatory elements for transcription in a host cell such as a plant cell, operably linked to a different promoter. The vector may be a bi-functional expression vector which functions in multiple hosts and in the case of the genomic DNA, it may contain its own promoter or other regulatory elements and in the case of cDNA, it may be under the control of an appropriate promoter or other regulatory elements for expression in the host cell.

Promoter is a sequence of nucleotides from which the transcription may be initiated of DNA operably linked downstream. Operably linked means, joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter. DNA operably linked to a promoter is under transcriptional initiation regulation of the promoter. In a preferred embodiment, at least one of the promoters is an inducible promoter and the expression refers to the 'switching on' or increase in response to an applied stimulus. The nature of the stimulus varies between promoters. Expression from any inducible promoter is increased in the presence of the correct stimulus.

If desired, select genetic markers may be used in the construct, namely, those that may be used to confer select phenotypes such as resistance to antibiotics or herbicides.

In a further aspect of this invention, there is disclosed a host cell containing a heterologous construct, especially a plant cell. The term heterologous is used broadly to indicate that the gene/sequence of the nucleotide have been introduced into the said cell of the plant or an ancestor thereof using genetic engineering, i.e., by human intervention. A heterologous gene may replace an equivalent, endogenous gene, i.e., one which performs normally the same or similar function or the inserted sequence may be additional to the endogenous gene or other sequence. Nucleic acid heterologous to a plant cell may be non naturally occurring in cells of that type, variety or species and may comprise a coding sequence of or derived from a particular type, variety or species. The nucleic acid sequence could also be placed within a cell in which it or a homologue is found naturally but where the nucleic acid sequence is linked and/or adjacent nucleic acid sequence which does not occur naturally within the cell or cells of that type or specie or variety of plant, such as operably linked to one or more regulatory sequences such as a promoter sequence, for control of expression.

The host cell, namely the plant cell, is preferably transformed by the construct, i.e. the construct becomes established within the cell altering one or more of the cell's characteristics and hence the phenotype, e.g., with reference to the anti oxidant capacity due to the elevated glutathione content.

Nucleic acid can be introduced into plant cells by using any suitable technology.

The invention also encompasses a host cell, especially a plant cell transformed with a vector and the gene under the control of different promoters to enable differential expression.

In addition to the regenerated plant, the invention also covers the following, namely, clone of the plant, seed or hybrid progeny and descendants. The invention also provides a plant propagule from the plant, i.e., any part which may be used in reproduction or propagation, sexual or asexual including cuttings, seed etc. It also provides for any part of the plant including the plant cell heterologus to the gene involved in the cycling of the glutathione between the reduced and the oxidized form.

The plant involved in the present invention has been found to have improved root weight and development in comparison to control plants, enabling improved water and nutrient uptake. Further, the plant has been found to have enhanced glutathione level.

In plants, ascorbate peroxidase (APX) is another important peroxide-detoxifying enzyme. The expression of APX is rapidly induced in response to stresses that result in the accumulation of reactive oxygen species. The steady-state level of transcripts encoding cytosolic APX is dramatically induced during the hypersensitive response of plants infected with virus. Tolerance to low temperature and oxidative stress has been demonstrated for plants having increased ascorbate peroxidase activities. In general, ascorbate peroxidase has been suggested as a particularly important antioxidant enzyme in helping plants survive oxidative stress.

Peroxidases prevent oxidative damage following anoxia (i.e. oxygen deprivation) in plants (Amor et al. (2000) FEBS Lett. 477:175-180). Anoxia followed by reoxygenation causes extensive damage to cellular components through the generation of reactive oxygen intermediates.. However, anoxia pretreatment protected soybean (but not fibroblasts) again peroxide concentrations that induced programmed cell death in normoxic cells. This protection involved an increase in the expression of alternative oxidase (AOX) and peroxidases. Ascorabate peroxidases have also been shown to play a role in protecting against oxidative stress (Wang et al. (1999) Plant Cell Physiol. 40:725-32). The expression of the peroxisomal ascorbate peroxidase APX3 was demonstrated to protect tobacco leaves from oxidative stress damage caused by aminotriazole.

Exposure to environmental stress results in the formation of oxidative compounds in plants. To prevent damage, these compounds must be rapidly removed. In plant leaves, the enzyme, ascorbate peroxidase, may play a pivotal role in preventing the increase in hydrogen peroxidase levels during environmental stress (Charles Caldwell et al., 1998).

Ascorbate peroxidase is of potential value in plant biotechnology programmes, by assisting in developing crop plants with increased stress tolerance. This gene could constitute a basic mechanism for deployment of antioxidative defences in plants.

These enzymes function in concert to detoxify ROS and free radicals. SOD is present in virtually all oxygen-respiring organisms where its major function is the dismutation (breakdown) of superoxide anion to hydrogen peroxide. Hydrogen peroxide, itself, is a highly reactive and oxidative molecule, which must be further reduced to avoid damage to cells and tissues. In the presence of the appropriate electron acceptors (hydrogen donors), CAT catalyzes the further reduction of hydrogen peroxide to water. In the presence of reduced glutathione (GSH), GSH-Px also mediates reduction of hydrogen peroxide to water by a separate pathway. Catalase present in the peroxisomes of nearly all aerobic cells, serves to protect the cell from the toxic effects of hydrogen peroxide by catalyzing its decomposition into molecular oxygen and water without the production of free radicals. The mechanism of catalysis is not fully elucidated, but the overall reaction is as follows:

2 H₂O₂ ~ 2 H₂0 + O₂ The protein exists as a dumbbell-shaped tetramer of four identical subunits (220,000 to 350,000 kD). Each monomer contains a heme prosthetic group at the catalytic center. Catalase monomers from certain species (e.g. cow) also contain one tightly bound NADP per subunit. This NADP may serve to protect the enzyme from oxidation by its H₂O substrate.

Catalase was one of the first enzymes to be purified to homogeneity, and has been the subject of intense study. The enzyme is among the most efficient known, with rates approaching 200,000 catalytic events/second/subunit (near the diffusion-controlled limit). Catalase structure from many different species has been studied by X-ray diffraction. Although it is clear that all catalases share a general structure, some differ in the number and identity of domains. In this display, beef liver catalase will be used as a model for catalase structure. It will then be compared to catalase structure from a fungus, Penicillium vitale.

Proposed Mechanism of Catalase

The chemistry of catalase catalysis has not been precisely solved yet, but the following, which is similar to the mechanism of cytochrome c peroxidase, has been proposed. The catalytic process is thought to occur in two stages:

H₂O₂ + Fe(III)-E - H₂O +O=Fe(IV)-E (1) H₂O₂ + O=Fe(IV)-E - H₂O + Fe(III)-E (2)

where Fe-E represents the iron center of the heme attached to the rest of the enzyme (E).

Peroxide, upon entering the heme cavity, is severely sterically hindered and must interact with His⁷⁴ and Asn¹⁴⁷ . It is in this position that the first stage of catalysis takes place. Transfer of a proton from one oxygen of the peroxide to the other, via His⁷⁴, elongates and polarizes the O-O bond, which eventually breaks heterolytically as a peroxide oxygen is coordinated to the iron center. This coordination displaces water and forms Fe(IV)=O plus a heme radical. The radical quickly degrades in another one electron transfer to rid of the radical electron, leaving the heme ring unaltered. During the second stage, in a similar two electron transfer reaction, Fe(IV)=O reacts with a second hydrogen peroxide to produce the original Fe(III)-E, another water, and a mole of molecular oxygen.

The heme reactivity is enhanced by the phenolate ligand of Tyr³⁵⁷ in the 5th iron ligand position , which may aid in the oxidation of Fe(III) to Fe(IV) and the removal of an electron from the heme ring. The efficiency of catalase may, in part, be due to the interaction of His⁷⁴ and Asn¹⁴⁷ with reaction intermediates. This mechanism is supported by experimental evidence indicating modification of His⁷⁴ with 3-amino-l,2,4-triazole inhibits the enzyme by hindering substrate binding.

Procedure of the isolation of Glutathione peroxidase, Ascorbate peroxidase, Catalase, S- Adenosylmethionine Synthetase and Beta glucosidase gene sequence ( Seq ID. No. 1, 2, 3, 10 & 11)

1. Total RNA was extracted from mature leaves of Terminalia arjuna plant.

2. Subsequently, RNA was isoated from the total RNA.

3. A cDNA library was constructed with the aid of the GIBCOBRL Superscript Plasmid System with the Gateway Technology for the cDNA Synthesis and Cloning kit.

4. The clones were screened and selected for sequencing and subjected to a data-base search to ascertain their identity.

5. Functional details of the clones were collected.

Procedure for isolation of 2-Cys Peroxiredoxin, Methionine aminopeptidase and microsomal signal peptidase gene sequence ( Seq ID. No. 4, 8 & 9)

1. Total RNA was extracted from the 13 day old actively growing apical region of the Gloriosa superba plant following the Trizol protocol (Life Technologies). 2. Subsequently, the mRNA was isolated from the total RNA following Oligotex mRNA Batch Protocol (Genetix)

3. A cDNA library was constructed by making use of the GIBCOBRL Superscript Plasmid System with the Gateway Technology for the cDNA Synthesis and Cloning Kit.

4. The cDNA clones obtained were screened and selected for sequencing and subjected to a data base search in order to ascertain their identity.

5. Functional details of the clones were collected.

Procedure for isolation of Glutamate decarboxylase, Thionine and Arabinoxylan furanohydohydrolases gene sequences ( Seq ID. No. 5, 6 & 7)

1. mRNA purification was performed by first, isolating high quality total RNA from 6 day old RASI seedlings and, subsequently by isolating mRNA from total RNA using oligo (dT) cellulose in a filter syringe by making use of a double purification method. 2. mRNA was converted into first and second strand cDNA followed by Sal I adapter addition, Not I digestion, cDNA vector ligation and transformation to obtain the cDNA library. 3. The superscript plasmid system with Gateway for cDNA cloning and synthesis was employed throughout. 4. The clones obtained were picked, digested using Not I and Sal I enzymes, to obtain the inserts and these were further sequenced and checked for homology. 5. The sequencing of the selected clones was done on ABI Prism, 377, DNA Sequencer (Perkin Elmer).

Claims

Claims 1. Nucleic acid sequences Seq ID. No. Ito 11, isolated from plants, the protein products of which, confer protection to against environmental stress like salinity, drought and high temperature, improves oxidative stress tolerance and can be used as nutraceuticals, biotherapeutics and functional food supplement.

2. Nucleic acid sequences Seq ID. No. 1, isolated from Terminalia arjuna, the protein products of which improves oxidative stress tolerance.

3. Nucleic acid sequences Seq ID. No. 2, isolated from Terminalia arjuna, the protein products of which improves oxidative stress tolerance.

4. Nucleic acid sequences Seq ID. No. 3, isolated from Terminalia arjuna, the protein products of which enhances the capability of a host to detoxify reactive oxygen species.

5. Nucleic acid sequences Seq ID. No. 4, isolated from Gloriosa superba, the protein products of which improves oxidative stress tolerance.

6. Nucleic acid sequences Seq ID. No. 5, the protein products of which used as nerve inhibitory mediator and other biotherapeutic applications.

7. Nucleic acid sequences Seq ID. No. 6, the protein products of which has anitfungal activity.

8. Nucleic acid sequences Seq ID. No. 7, the protein products of which are known to activate the Natural killer cell which is said to increase an individuals immune power through oral administration, thereby controlling the growth of cancerous cells.

9. Nucleic acid sequences Seq ID. No. 8, the protein products of which is useful as a bioenhancer and bioactivator facilitator together with a therapeutically effect amount of combinations of nutraceuticals, antibiotics, anti-infectives and anti-cancer agents across all.

10. Nucleic acid sequences Seq ID. No. 9, the protein products of which are found to be associated with the diagnosis, prevention and treatment of cancer.

11. Nucleic acid sequences Seq ID. No. 10, the protein products of which are useful in the treatment of various diseases, tumour, HIV infection and immunological diseases.

12. Nucleic acid sequences Seq ID. No. 11, the protein products of which can be used as xenobiotics.

13. A claim as in claim 1, wherein the proteins are effective in the treatment of age-related diseases like amyloidosis, acute pancreatitis, arthritis, cancer, inflammatory bowel disease, senile dementia, retinal degeneration and senile cataract.

14. A claim as in Claims 1 to 4, wherein, the proteins can form a major source of new therapeutics and nutraceuticals

15. A claim as in claims 1 to 4, wherein the formulation containing the proteins can be administered orally, parenterally, intravenously, and intradermal administration.

16. A claim as in claim 7, wherein the dosage can be administered in the form of tablet, capsule, powder, pill, solution, syrup, suspension, emulsion, and granules.