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INTRODUCTION Proteins, indispensable a for class of life. complex They are organic molecules, bio-polymers of are simpler compounds known as am:l.no acids. Amino acids possess amino group attached to the carbon atom lying next to the carboxyl oC. - group. Due to the presence of at least one asymmetric carbon atom amino acids, except glycine, exist in two isomeric forms, d and 1 of which only the 1-forms are found in proteins. discovered in 1806 and 1810 are asparagine and amino acids cystine, The 1st two respectively. There are 23 amino acids which may be seen to occur frequently in protein hydrolyzates. Besides these there are more than 170 amino acids known to occur in living systems (Meister, 1965). Animals including man are unable to synthesize some amino acids called "essential amino acids" from simple ingredients. These essential amino acids are isoleucine, phenylalanine, threonine, leucine, lysine, tryptophan and valine. methionine, All these amino acids must be present in requisite quantities in the ingested food materials. The absence or shortage of any one of these amino acids in the food may cause malnutrition and protein deficiency (Karlson, 1969). Population in developing countries like India, are mainly dependent on vegetable diet. It is known that the vegetable proteins are deficient in essential amino acid content. For this reason, sole dependence on vegetable diet leads to the appearance of symptoms of protein deficiency. An inadequate supply of balanced protein is the main reason for high rate of sickness and death in developing countries (Fontanel, 1972). It is reported that nausea, dizziness deficiency. and Methionine hypersensitivity deficiency results observed in in hepatic lysine and renal damage. Apart from their use as food supplement amino acids are also used in pharmaceutics, herbicide) and industry. importance as therapeutic cosmetic, Amino agents, agriculture acids, taste ( ani_mal now-a-days, improver of feed, are of foods, 2 flavouring agent and ditterent laboratory reagents besides their nutritional importance (Table 1) . Table 1. Amino acid application ( Plachy, 1989) Amino acid Area of application Application Lysine, Methionine, Feedstuffs Fortification of Threonine. Feedstuffs. Lysine, Methionine, Food Food additives, Threonine, Glutamic Industry seasioning acid, Glycine, Alanine, Sweeteners. Asparagine, Pheny !alanine. Lysine, Methionine, Medical Therapeutic 1hreonine, Arginine, field application Tryptophan, Other inf'usion essential and non solutions, synthesis essential amino acids. ot peptides and vitamins. Poly- L-alanine, Chemical Surface active poly- {?> -glutamic Industry agents, detergents acid etc. polymers, synthetic fibres and leather. Mixture ot essential amino acids are used in treatment of nephrosis, ulcers, early stages ot liver cirrhosis, drug toxicity, anaemia, during gastric and duodenal convalescence from 3 infectious diseases, before and a Her surgery (Dulaney, 1967). Japanese people add amino acids as condiments tor improvement ot' taste ot tood. 1he organoleptic property of L-glutamic acid is used widely for improving the taste of "Kombu' a traditional preparation made from Laminaria. L-glutamate is marketed for its characteristic flavour in the trade name "Ajinomoto". Lysine derivative have been made to improve the flavour in alcoholic and non-alcoholic beverages. Lysine adipate and lysine succinate enhance the food taste (Hause and Todd, 1962). The characteristic taste of Emmentaler cheese is said to be imparted by certain amino acids like threonine, ci.. -aminobutyric acid, glycine, alanine and proline. In United States primary dietary use of DL ·methionine is used for supplementation of chick feed. Mono hydrochloride of lysine, arginine, glutamic acid and histidine are used {Miyake et al, 1971) as meat improving agent. Lysine is used as a supplement for bread and other food stuffs. It is the limiting essential amino acid required lysine is in being vaccine (Dorval the diet used by ~ ~ of pol:1.HTJ {David, industry to 1987). stabilize the Recently, poliovirus 1990). 1hus it is obvious that if the amino acids are to be used as nutritional supplements or as organoleptic agents these are to be produced commercially and to be marketed at a cheaper rate. For this, several methods have been adopted to produce essential amino acids in a large scale. These include chemical synthesis, extraction, enzymatic synthesis and fermentation ; of which fermentative method is popular because it is cheaper, easier and the amino acids obtained from microbial fermentation are exclusively in the biologically active 1-form. Several methods are used to produce amino acids (Table 2 ) • Table 2. Methods of amino acid preparation (Plachy. 1989). Method Amino acids Extraction from protein Cysteine, leucine, tyrosine, DL-alanine, hydrolyzates asparagine, phenylalanine, glycine, 4 Method Amino acids methi onine, threonine, tryptophan, valine. Microbiological Lysine, L-alanine. methods Enzymic method Aspartic acid, tryptophan Precursor method Lysine, arginine. Fermentation Phenylalanine, histidine, glutamic acid, isoleucine, glutamine, leucine, proline, threonine, valine. An individual liquors extensive ami.no of acids food literature from exists simple processing on the production precursors or industries, of from waste supplemented with appropriate nitrogen sources and cofactors. Recently, amino acids are isolated from farm wastes ( Tsuruoka, 1987). Production of Lamino acids by fermentation is now being used in industrial scale and this potentiality of microbes have been exploited commercially in countries like Japan and USA (Dulaney, 1967 J • Japan is a main producer of amino acids ( lable 3). Among other countries amino acids Union. are produced commercially in Czechoslovakia and Soviet 5 Table 3 • Amino acid market in Japan (Plachy, 1989). Amino acid 1 Annual production p nee . 2 Producer Glutamic acid 10000 - 100000 5-10 Ajinomoto, Kyowa Methionine 10000 - 100000 5-10 Degussa, Ajinomoto, Rhone - Poulene Lysine 5-10 10000 - 100000 Ajinomoto, Kyowa, Toray. Isoleucine 100- 1000 Ajinomoto, Tanabe, 100-500 Kyowa. 100- 1000 Threonine 50-100 Ajinomoto, Kyowa Aspartic acid 10D- 1000 5-10 Ajinomoto, Tanabe Glycine 100- 1000 5-10 Ajinomoto, Kyowa. Pheny !alanine 100-1000 100-500 Ajinomoto, Kyowa. Tryptophan 100-1000 ·lOD-500 Ajinomoto, 1anabe Valine 100-1000 10-50 Ajinomoto, Tanabe 50 10-50 Ajinomoto, Kyowa, Tyrosine 1anabe. 1. Annual production in tons; 2. Price in $/Kg. In Japan, Kinoshita et ( 1957) for the first time showed the ~ cheap way of amino acid production by microbial fermentation. In microbial fermentation microorganism excrete large quantities of amino acid in the fermentation medium from which they could be isolated, purified and marketed at a product obtained through reasonably low cost. The fermentation needs no resolution before biological consumption because amino acids are produced exclusively in 1 - torms. microorganism Kinoshita ( 1959) isolated a glutamic acid producing Micrococcus Corynebacterium glutamicum then Kinoshita ( 1959) glutamicus later on reidentified (Nakayama and Kinoshita, made an extensive study on as 1966). Since amino acid 6 production by screening thousands of micro-organisms and reported that amino acid production is not restricted to any particular group of microorganism. Production of important amino acids through genetically modified bacterial strain and to improve the yield is one ot the aims of modern biotechnological research. It is known that the vegetable proteins are deficient in essential amino acid content. It can be thought, therefore, that in the coming years a large scale microbial production of amino acids will be necessary as food supplement to avoid malnutrition in developing countries (Albanese, 1967). The objective of the present study was to isolate mutant strains that would produce threonine, to optimise the cultural conditions for threonine production and to improve the threonine yield further by isolation of mutants resistant to threonine analogues. The problem was investigated through the following steps (..'"' 1. Revival of lyophilized culture of the threonine producing strain\ S{':--"" M56 (L-methionine auxotroph). 2. Confirmation of the nature of amino acid selected strain by produced by the paper chromatography and microbiological assay. 3. Taxonomic identification of the strain. 4. Mutagenic treatment of the strain with N-methyl-N •-nitroN-nitrosoguanidine and selection of double auzotrophs in order to improve the threonine yield. and triple , 7 5. Selection ot' potent threonine producing auxotrophic mutants. 6. Standardization of optimal conditions for threonine production by the selected single, double and triple auxotrophic mutants. 7. Improvement of the threonine yield by isolation of mutants resistant to threonine analogue and lysine analogue. 8. Study ol the activity and regulation of aspartokinase, the key enzyme pertaining to threonine accumulation. 9. Standardization of method for the product recovery and study of the homogeneity of the isolated product. A review of literature pertaining to threonine and acid following pages perspective. some to other analyse amjno the microbial is production presented programme in the in of the proper