Title of Invention

A PROCESS FOR ISOLATING BETA GLUCOSIDASE FROM TERMINALIA ARJUNA AND USES THEREOF

Abstract

The present invention relates to the isolation of a nucleic acid sequence, the product of which, modulates the lignin content in plants. It exhibits increased activity in the presence of pathogens. The invention also relates to the generation of a cDNA clone from the pool of mRNA, isolated from the mature leaves of Terminalia arjuna, the identity of which, has been found to be beta glucosidase and the product of which leads to alteration of the lignin content in plants and enhances the aroma in fruits and vegetables.

Full Text

In one embodiment, transformation vectors may be constructed to over-express the coniferin beta- glucosidase enzyme ("sense" orientation). Enhanced lignin synthesis may be achieved by introducing such vectors into plants- Examples of the application of this approach to modify plant phenotypes include U.S. Pat. No. 5,268,526, "Overexpression of Phytochrome in Transgenic Plants", U.S. Pat. No. 4,795,855, "Transformation and Foreign Gene Expression in Woody Species", and U.S. Pat. No. 5,443,974 (over-expression of stearoyl-ACP desaturase gene). Alternatively, such over-expression vectors may be used to suppress coniferin beta-glucosidase enzyme activity through sense-suppression, as described in U.S. Pat. Nos. 5,034,323 and 5,283,184, both entitled "Genetic Engineering of Novel Plant Phenotypes". US patent 5,973,228 describes a cDNA molecule encoding coniferin beta-glucosidase is disclosed. This enzyme catalyzes one of the last steps in the synthesis of lignin in plants. Plants having modified lignin content may be produced by transformation with this cDNA (or parts of the cDNA), for example, in either sense or antisense orientation. The invention includes methods of altering-lignin content in plants using this cDNA, as well as transformed plants, such as conifers, having modified lignin content. US patent 5,997,913 describes a process for expressing extracellular .beta.-glucosidase in a filamentous fungus by expressing a fungal DNA sequence encoding enhanced, deleted or altered .beta-glucosidase in a recombinant host microorganism is disclosed. Recombinant fungal cellulase compositions containing enhanced, deleted or altered expression of .beta-glucosidase is also disclosed. Description Beta-Glucosidases occurs in all three domains (Eukarya, Archaea, and Bacteria) of living organisms and play a key role in many biological processes, which make them a suitable target for protein engineering to address the problems of biomass production in agriculture and forestry, as well as biomass conversion in biotechnology. The following selected cases illustrate the importance of enzyme in plants. 1. Defense: Plants are anchored to the soil and generally cannot hide or escape from pests and environmental stresses. Consequently, plants have evolved defense mechanisms against pests based on storing and releasing toxic chemicals. These defense chemicals are typically 6-glucosides in monocots and dicots and 8-glucosinolates in certain dicots. 6-glucosidic substrates and 8-glucosidase are stored in different subcellular or tissue compartments. Damage to cells and tissues by pests brings the enzyme and substrate together, leading to the hydrolysis of substrate and release of bitter and toxic aglycones and their breakdown products (e.g., thiocyanates, isothiocyanates, nitriles, terpenoid alkaloids, saponins, hydroxymates, benzaldehydes, HCN). These substances then deter herbivores and inhibit the entry, growth and spread of phytopathogens, serving as a built-in pest control system. For example, it has been shown that environmental stresses ranging from chewing insects, nematodes and phosphate starvation to cold as well as specific treatments (e.g., jasmonic acid) induces 8-glucosidase genes in Arabidopsis thaliana while NaCl suppresses. In a recent study of the transcriptional profile of Arabidopsis thaliana during systemic acquired resistance (SAR), it is showed that transcription of the 6-glucosidase gene psr3.1 was elevated nearly 8-fold within 48 hours after infection with the oomycete Peronospora parasitica. Another Arabidopsis thaliana 6-glucosidase gene (T209.120) has been shown to be associated with growth arrest and senescence in cell cultures and mature plants and is also reported to be cold inducible. Although the precise role of these genes in stress, defense and senescence response is not known, two (psr3.1 and F19K6.15) encode 8-glucosidases with a C-terminal ER retention signal and may be involved in signal transduction by activating another protein or nonprotein component in the ER via deglycosylation. These genes are potential targets for engineering enhanced crop protection and reducing or eliminating the need for costly and environmentally undesirable pesticides. 2. Food Processing and Quality Enhancement: There are several hundred different 6-glucosidic flavor precursor identified from plants whose aglycones are products of mevalonate or shikimate pathways. Obviously, there are B-glucosidases in source plant tissues that hydrolyze these flavor precursors. Thus, in each case, there is need for isolating and characterrizing the specific enzyme that hydrolyzes a 8-glucoside whose aglycone moiety is of interest to food quality and processing. Such biochemical data are crucial to making practical decisions as to whether or not enzymes from host plants or other sources should be added to drinks and beverages before, during or after processing to enhance flavor, aroma and other quality factors. Likewise, such data are essential for targeting enzymes with desirable properties for overproduction in transgenic microbial or plant hosts and improvement of their catalytic properties and stability for specific uses by genetic engineering. Another aspect of B-glucosidases that pertain to food processing and quality is that edible portions of some plants contain compartmentalized 6-glucosidase-B-glucoside systems that produce toxic aglycones and/or HCN when tissue is macerated during preparation or by chewing. This is exemplified by cassava roots and leaves, lima beans and flax seed. Of these, cassava is a food staple in tropical regions of Africa, Asia and South America, consumption reaches about 1 kg/per capita/day in some parts of Africa (e.g., Congo). It contains the cyanogenic 8-glucoside linamarin and the corresponding A-glucosidase linamarase. When consumed raw, cyanide poisoning can occur depending on the amount ingested, where symptoms are difficulty in breathing, paralysis, convulsion, coma and even death. Cooking inactivates the enzyme and eliminates the possibility of cyanogenesis. Similar symptoms can arise when bitter almonds are eaten and ingested without roasting. The myrosinase-glucosinolate (or B-thioglucosidase-8-thioglucoside) system, which occurs in cruciferous vegetables (e.g., mustard, cabbage, kale, broccoli, rapeseed, horseradish, etc.), has also importance for food quality and processing because the aglycone moiety and its breakdown products from enzymatic hydrolysis of glucosinolates are responsible for bitter, pungent taste and aroma associated with these vegetables, as well as the processed foods and relishes that include them (10). The distinct flavor associated with glucosinolates comes primarily from isothiocyanates and is believed to have evolved to serve as a repellent against microorganisms and herbivores. Glucosinolates and their breakdown products may impart undesirable flavors to milk, meat and eggs when farm animals graze on cruciferous plants or when their feed includes seed meals from such plants. Besides ingestion of large amount of cruciferous vegetables is thought to cause endemic goiter in humans, as well as toxicity in laboratory animals. Similarly, claims have been made on anti-carcinogenic effects of glucosinolates and their breakdown products in humans. Although the precise mechanism of action is not clear, studies on rodents showed that raw or cooked cruciferous vegetables (e.g., cabbage, broccoli, cauliflower and turnip) increased aryl hydrocarbon hydroxylase activity (11). 3. Biomass Conversion: Polysaccharides, specifically cellulose, are the most abundant substances in the biosphere (~5xio10 tons produced/year) and are potential renewable sources of chemicals and fuels. Moreover, about 40% of typical municipal garbage includes newspaper and other paper products. Hydrolysis of cellulose using inorganic acids and high temperature is not ecologically sound and economically feasible. An enzyme (cellulase) complex, secreted by cellulolytic organisms, can hydrolyze cellulose to glucose, thus presenting itself as a suitable model for industrial processes that need to be developed (12). The complex includes three enzymes: an endoglucanase, an exoglucanase (cellobiohydrolase) and a 8-glucosidase. The rate- limiting step in cellulose degradation is the one that is catalyzed by A-glucosidase, which hydrolyzes cellobiose and other small cellodextrins to glucose. Therefore, cellulosic biomass degradation and cellulose conversion to glucose at industrial scale by using microorganisms or isolated cellulase complex hinge upon increasing the rate of enzymatic reactions and overcoming product inhibition. Some plant 8-glucosidases are specifically implicated in cellulose or cell wall metabolism during germination and growth as they hydrolyze cellobiose, as well as other disaccharides and oligosaccharides resulting from cell wall catabolism (13,14). Such B-glucosidases are potential targets for engineering to use in the degradation of cellulosic biomass by the cellulase complex. 4. Lignin Biosynthesis and Paper Quality: Lignin is the second most abundant substance in the biosphere and its major precursor, coniferyl alcohol, is derived from coniferin (4-O-coniferyl glucoside) after hydrolysis by 6-glucosidase (15), suggesting that some plant 6-glucosidase isoforms are involved in lignin biosynthesis. This makes the enzyme a suitable target for improving wood strength and quality for paper production. 5. Growth and Development: There is circumstantial evidence that 8-glucosidases are involved in growth and development by releasing active hormones from phytohormone glucoconjugates, another potential function for some plant 8-glucosidases. If indeed this function could be unequivocally shown, it opens the door to engineering the enzyme for regulating and improving plant growth and development to enhance productivity. 6. Secondary Plant Metabolism: Many secondary plant biochemical pathways (e.g., phenyl propanoid metabolism) use 8-glucosides as precursors, intermediates or synthesize them as end products. Some of these substances may be glycosylated to enhance solubility or may be deglycosylated as part of degradation pathway. Very little is known about the fate and function of many B-glucosides. These compounds must have a function and be hydrolyzed by B-glucosidases exhibiting unique substrate specificities and tissue and cellular localization, providing further clues to the existence of a 44-member multigene family encoding B-glucosidase in Arabidopsis thaliana. Aromatic substances are produced in the green tissues of plants and are conveyed from there to the flowers or fruits of the plants. At the proper moment, the enzyme beta glucosidase is released, which serves to break the bonding between the molecules of sugar and the aromatic substances, thus releasing the latter into the air. In flowers this occurs when the flower is ready for pollination, with the smell serving to attract insects that will bear pollen to other plants. In fruits, the aroma is meant to attract animals, which will spread its seeds. However in many vegetables and fruits, there are aromatic substances that remain bound and thus are not released at all. The introduction of the beta glucosidase enzyme can enable these plants to reach their full aromatic potential (Dr. Oded Shoseyov et al., 2001). Beta glucosidase is of potential value in plant biotechnology programmes for modulation of the lignin content in plants which would help in the production of fuel alcohol from cellulose. The technology could be utilised to increase the aroma in wine as well as in vegetables and fruits. Procedure 1. Total RNA was extracted from mature leaves of Terminalia arjuna plant. 2. Subsequently, mRNA 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 and one cDNA clone designated as cDTaML03B02, was predicted to be a homologue of Dihydroflavanol-4 reductase. We claim 1. A method of engineering enhanced crop protection and reducing or eliminating the need for costly and environmentally undesirable pesticides by expressing beta-glucosidase nucleotide sequenceand thereby generating transgenic plants pest/insect resistance. 2. A method of engineering transgenic plants having enhanced aroma and flavour. 3. A claim as in claim 1 & 2, wherein the transgenic plants could apply to all varieties of plants. 4. A claim as in claim 3, wherein, beta-glucosidase enzyme can be engineered for regulating and improving plant growth and development to enhance productivity. 5. A claim as in claim 1 & 3, wherein, the transgenic plant expressing beta-glucosidase, show increased production of lignin, imparting improved wood strength and quality for paper production. 6. A claim as in claim 1, whereby beta-glucosidase enzyme plays a useful role in food, agricultural and pharmaceutical industry.

Documents:

732-CHE-2003 CORRESPONDENCE OTHERS 23-10-2009.pdf

732-che-2003 abstract 29.07.2009.pdf

732-che-2003 claims 29.07.2009.pdf

732-che-2003 correspondence others 29 .07.2009.pdf

732-che-2003 description(complete) 29.07.2009.pdf

732-che-2003 drawings 29.07.2009.pdf

732-che-2003 form-1 29.07.2009.pdf

732-che-2003-abstract.pdf

732-che-2003-claims.pdf

732-che-2003-description(complete).pdf

732-che-2003-form 1.pdf