User:Nandini Datta/sandbox

= Amino Acid Production = Amino acids are molecules containing an amino and carboxyl group and are the building blocks of proteins. There are standard 20 amino acids which are classified as essential and non-essential amino acids. These molecules play important roles in providing nutrition, in enhancing flavor and also act as parts of medicinal products and cosmetics. The proteins made from these amino acids are structurally important for muscles, ligaments and skin. Some amino acids trigger hormone secretion. Due to their immense importance, there is now a high demand for the production of amino acids.

= History of amino acid production = The history of amino acid production and usage can be traced back to the 1900s in Japan. Japanese food involves the usage of a lot of sea weeds and flavoring agents. In 1907, Kikunae Ikeda conducted his experiments at Tokyo University to isolate the flavoring molecule from seaweed Laminaria japonica. The isolated substance was in the form of crystals of monosodium glutamate (MSG). After this discovery, the extraction of MSG by Ajinomoto Co. from acid hydrolyzed wheat gluten or defatted soyabean began and it started being sold as a flavoring agent. The use of fermentation for MSG production started soon after World War II.

= Production Process = There are mainly three known procedures for the production of amino acids. These are namely:
 * Extraction from Protein Hydrolysates
 * Chemical Synthesis
 * Microbial or biotechnological methods including enzyme catalysis and fermentation

Extraction from Protein Hydrolysates
The extraction method is one of the oldest methods of amino acid manufacture. It is based on the physicochemical property differences of amino acids which are used to separate them. This method involves acid hydrolysis of residues from humans and animals like human hair, bird feathers etc. This is followed by addition of active charcoal for discolouration and neutralization. Electrodialysis is then used for the extraction of amino acids from the hydrolysates into separate fractions. There are a lot of amino acids produced this way till date.The extraction of L-cysteine is carried out from chemically hydrolyzed keratin present in feather,hair, bristles etc. L-alanine, L- serine are obtained from proteinaceous waste materials from animals. Recent studies focus on extraction of amino acids from fish protein.

Chemical Synthesis
Chemical Synthesis of amino acids utilizes the Strecker reaction. This involves formation of amino acid from an aldehyde or ketone. In this reaction, an aldehyde is treated with ammonium chloride in the presence of potassium cyanide. This results in the formation of α- aminonitrile. This is followed by hydrolysis producing the desired amino acid. This method yields racemic mixtures of amino acids and has been traditionally used for obtaining a racemic mixture of D,L-methionine and D,L-alanine. Nowadays, a variant of Strecker synthesis known as Bucherer-Bergs method is also used for industrial chemical synthesis of amino acids. In this method, Ammonium carbonate and sodium cyanide is used for the reaction with aldehydes or ketones to form hydantoions. These are then hydrolysed in a basic medium to obtain racemic amino acid mixtures. Aminocylase enzyme obtained from Aspergillus niger is used to get active L-forms. The drawbacks of this method are that it uses hazardous chemicals as well as expensive catalysts. It also produces a mixture of D and L forms of amino acids which need to be separated later. Moreover the reactions are quite time consuming and hence not very commercially viable. However, this method is employed even today for production of valine, threonine, methionone, glycine, alanine, phenylalanine and tryptophan.

Enzyme Catalysis
This method involves utilisation of enzymes obtained from various microbes for the production of amino acids. These enzymes are mostly obtained from organisms like Psudomonas dacunhae, Escherichia coli, Saccharomyces cerevisiae, Cryptococcus lurendii etc. Enzymes used are ammonia lyases, hydrolytic enzymes, NAD+ - dependent L-amino acid dehydrogenase etc. Precursors of amino acids are converted to amino acids by using pure enzymes and hence no microbial growth is seen here. Eg: Enzymes Alcalase and Neutrase are used for amino acid extraction from dried fishes. L-Methionine is produced widely using this technology. Acylase obtained from Apergillus niger is used in the enzyme membrane reactor. For production of L-cysteine, enzymes L-ATC hydrolase, S-carbamoyl-L-cysteine hydrolase and ATC racemase are used on thiazoline derivative DL-2-amino-2-thiazoline-4-carboxylic acid obtained from Pseudomonas thiazolinophilum.

Fermentation
The idea behind the fermentation method is the ability of microbes to convert nutrients into components they require. Raw materials like syrups are supplied to the media for fermentation and microorganisms are allowed to produce amino acids. Usage of fermentation technology for amino acid production began after glutamic acid producing Corynebacterium glutamicum was discovered. Auxotrophic bacteria are used in the production of some amino acids like L-lysine and L-glutamic acid. Since the amount of amino acid produced is less than the desired quantities, mutations are introduced in the auxotrophs. The mutation causes inactivation of the repressor leading to accumulation of amino acid. This is then used further for commercially for fermentation.



Amino Acid producing bacteria
Corynebacterium glutamicum and E. coli are mostly used for amino acid production by fermentation. C. glutamacium is used to produce L-glutamate, L-lysine, L-phenylalanine, L-threonine, L-serine, L-tryptophan, L-arginine, L-glutamine, L-proline and L-isoleucine''. In case of C. glutamicum'' the preferred carbon source is glucose but usage of fructose, ribose, mannose, maltose etc is also in practice. The bacterium utilizes glycolysis, pentose phosphate pathway and TCA cycle for the carbon metabolism for amino acid biosynthesis. The optimum temperature is 30°C and pH 7 for its growth. Various techniques are employed for strain improvement. Mutations are induced in genes for target amino acids resulting in use of larger carbon source range like lactose, xylose etc and feedstock like glycerol. L-Lysine and L-isoleucine are produced in large quantities in the presence of high NADPH in the bacteria.

Escherichia coli is used in the production of L-lysine, L-methionine, L-threonine, L-tryptophan, L-phenylalanine, L-tyrosine. Mutant strains have the ability of production of L-valine, L-leucine, L-isoleucine. E. coli uses glucose, xylose, sucrose etc as the substrates. 37°C temperature and pH 7 act as optimum growth conditions. E. coli metabolises carbon via glycolysis, PPP and TCA cycle. The main contributor for biosynthesis of amino acid is the Pentose Phosphate Pathway which also produces NADPH required for the biosynthesis. The strain improvement in E. coli allows usage of various other substrates for production and also increases the yield.

Process design
Fermentation technique requires constant inspection of parameters like inoculum quality, feed rate, aeration, temperature, pH etc. Proper inoculum preparation is key to get maximum yield and productivity. Amino acid production is also influenced by the oxygen transfer rate (OTR). L-Phenyalanine shows higher yield with an increase in OTR. Temperature is also a crucial criteria. Thermotolerant bacteria like Bacillus methanolicus produces amino acids like L-lysine and L-glutamate at around 50°C.

Fed-batch operation mode is most commonly used for production although continuous production provides about 2.5 times higher output than fed-batch. In the fed batch method a small amount of inoculum and medium is introduced followed by the addition of the carbon source. Necessary nutrients like ammonium sulfate, biotin, vitamins etc are supplied with the inoculum. Adequate oxygen concentration is maintained throughout the process satisfying the oxygen demand. This also prevents undesirable by product formation. Hence, there is a better control of concentration of nutrients and good productivity.

Downstream Processing and purification
Centrifugation or filtration techniques are used to separate amino acids from the fermentation broth. After this chromatographic separation is done on the basis of isoelectric point, adsorbent affinity etc for purification. Due to the high cost and the loss of products during the steps, membrane based methods like ion exchange chromatography are gaining momentum. Nanofiltration technique is also employed nowadays where the membranes are integrated with the fermentors making purification and production in the same unit.

= Some amino acids and their production =

= Current Scenario = Fermentation is the most preferred method for industrial scale production of amino acids. This is due to the fact that it is economically and environmentally more feasible. Strain improvement done by inducing mutations forms an important part of enhancing the productivity of amino acids. Downstream processing also is optimized for cost reduction. However for non proteinogenic amino acids, enzyme catalysis is a preferred method.

= References =