Production of L Amino Acids

They may also be used as drugs that are not degraded as quickly as natural amino acids by protease enzymes. There are 4 methods to obtain amino acids for commercial use which include; extraction from natural sources, chemical synthesis, fermentation and enzymatic catalysis (Ault, 2004). Extraction from natural sources engages hydrolysis with aqueous acid as the standard procedure, followed by passage of the hydrolysate over a strongly acidic ion exchange resin to capture amino acids. After the resin is washed with water, elution with aqueous ammonia frees the amino acids collected in fractions (Ault, 2004).

Chemical synthesis can be carried out on a very large scale, the significant disadvantage however is that it typically gives a racemic mixture of the enantiomeric forms of the amino acid. Therefore the product of chemical synthesis must be resolved into the R and S forms and recovered. (Ault, 2004), this increases the cost of production substantially. However, chemical synthesis is ideal for the preparation of (R,S) methionine as both isomers are metabolised by poultry and swine in contrast to the majority of amino acids, chemical synthesis is thus the predominant method for the industrial production of methionine. Ault, 2004) All amino acids can be prepared via fermentation, whether they will or not depends on the costs of competing technologies (Ikeda, 2009). Bacterial strains that produce amino acids are mainly derived from Corynebacterium sp. , Baccilus sp. or E. coli. Strains used in production are wild-type natural overproducers, auxotrophic or regulatory mutants that have altered feedback inhibition pathways, or derepressed enzyme synthesis, and/or genetically engineered organisms that have multiple copies of genes encoding rate-limiting enzymes (Ikeda, 2009).

In enzymatic synthesis of amino acids pure enzymes are used rather than the enzyme systems of living organisms, as is the case with fermentation (Ault, 2004). The amino acid market has undergone rapid development since the 1980’s, due to cost effective production and isolation of amino acid products, specifically fermentation and enzymatic catalysis with their economic and ecological advantages responsible for the spectacular growth (Leuchtenberger 2005).

Glutamine is the most abundant free amino acid in the human body and may be the main physiological nitrogen vehicle between different mammalian tissues (Mates, 2009); it is one of the few amino acids to cross the blood brain barrier. Glutamine is utilised by cells in a variety of ways including oxidation by the Krebs cycle to produce ATP, providing nitrogen for nucleotide synthesis and is a precursor for glutathione, the major non enzymatic cellular antioxidant (Yuneva, 2007).

Although it is made by cells and thus classified non-essential there is a body of research suggesting that glutamine is ‘conditionally’ essential under stressful situations which causes rapid depletion of plasma glutamine levels (Mates 2009). This is significant in clinical trauma such as major surgery or intense exercise (Mates, 2009). Intravenous glutamine supplementation is standard care when parenteral nutrition is given for critical illness and there is research supporting reduced mortality following administration to critically ill patients (Werneman, 2011).

These findings support the hypothesis by Wakisaka et al. that plasma glutamine levels are an independent risk factor for mortality in critical illness (Wakisaka. , 1998). In addition to its use in clinical trauma, it is currently manufactured for use as a therapeutic agent against gastroenterological disorders, improvement of liver and brain functions, immunoenhancement agent and against gastric ulcer and alcoholism. It has recently been observed in human tissue samples that a small piece of RNA was associated with reduced activity of glutamine synthase, (EC 6. 3. 1. ), the research findings in this article suggest that glutamine deficiency is connected to increased intestinal permeability significantly increasing the likelihood of irritable bowel disease symptoms that follow (Zhou, 2010). Clinical studies have also demonstrated that glutamine supplements strengthen the immune system and reduce infection, particularly those associated with surgery and glutamine supplements may also aid in the recovery of severe burns (Singh, 2011). When the body is stressed, it releases the hormone cortisol into the bloodstream and high levels of cortisol may lower the body’s store of glutamine (Singh, 2011).

Thus, it is not surprising that there is general interest and speculation into the effect of glutamine as possible treatment for stress related illnesses including depression. Furthermore it is applied in cosmetics and as a food additive. However, research such as that carried out by Yuneva et al. reveals the possibility that metabolism of glutamine does not merely support accelerated proliferation or an increased requirement for ATP but that glutamine may be used for unrecognised pathways that are required for the survival of cell and cancer cells in particular (Yuneva, 2007).

This is out of the scope of this paper; however their findings raise the concern that the requirement of normal cells for glucose and intraspecies difference in glucose metabolism need to be considered in evaluating depletion of glutamine for therapeutic needs and also suggest that further research into the mechanism by which glutamine is required for cells to remain viable may provide a new approach to killing cancer cells selectively (Yuneva, 2007. Industrial production of L-glutamine started in the late 1960s In 2001, worldwide annual production using bioprocesses with coryneform bacteria was approximately 2000 metric tonnes (Kusumoto, 2001). L-glutamine is manufactured by several manufacturers worldwide all utilising fermentation (Kusumoto, 2001). The manufacturing process of an amino acid by fermentation comprises fermentation, crude isolation and purification processes (Kusumoto, 2001). In the fermentation process the desired amino acid is produced by the specific microorganism. The crude isolation involves the extraction of contaminants contained in the broth.

Finally purification is performed to ensure the required quality for the intended use (Kusumoto, 2001). In order to produce L-glutamine it is imperative to maintain a sterile fermentation tank as the Lglutamine producing bacterial strains are weaker than wild-type strains and are thus compromised in a contaminated environment , L-glutamine is easily degraded by other microorganisms thus proper control of the process and process design is imperative(Kusumoto, 2001). This may be achieved by maintaining the tank under positive pressure by aeration during fermentation to prevent contamination by other microorganisms and external aterials (Kusumoto, 2001). The medium is composed of glucose, ammonia and minerals and vitamins as growth factors. Control factors include pH, temperature and dissolved oxygen, the amount of fed glucose is decreased with proliferation of the fermentation bacteria and when the initial concentration of glucose falls, additional glucose is added to improve productivity (Kusumoto, 2001). To isolate crude L-glutamine, the broth is centrifuged or filtered through a membrane filter to separate cells and debris and the crude crystals through direct crystallisation of the supernatant or filtrate respectively.

However, it is difficult to harvest crude crystals with adequate purity and preparatory steps may be required involving repeated ion exchange resin treatment, chromatographic treatment and crystallisation (Kusumoto, 2001). The chemical and physical and biological properties of the amino acid are all taken into account to obtain the pure amino acid. Lglutamine is stable around its isoelectric point of pH 5. 65, (this pH is optimal for glutamine synthetase activity) if the pH shifts to acid or alkaline conditions L-glutamine is easily hydrolysed to glutamate and ammonia thus it is imperative that pH is controlled (Kusumoto , 2001).

The solubility of L-glutamine is minimally affected by temperature; therefore crystallisation by cooling is not applicable to L-glutamine (Kusumoto, 2001). To use crystallisation for purification simple repetitive crystallisation is employed for L-glutamine for its one crystalline form; however this purification step is relatively inefficient (Kusumoto, 2001). Compared with other amino acids, L-glutamine is difficult to design, thus the purity of the fermentation broth is vitally important to obtain a yield with high purity and productivity (Kusumoto, 2001).