Bio processing Requirements to manufacture Amino Acids
One of the first organic molecule appeared on this earth are Amino acids. Amino acids are biologically important compounds that are made from the functional group of amine and carboxylic acid. All peptides and polypeptides are polymers of α-amino acids. In our nature all the proteins that are available to us are actually made up from a set of blocks that contains a group of 20 amino acids. There are also some other amino acids are found freely or in combined state. The amino acids that are found in proteins also provides for several functions other than the formation of proteins and peptides. For instance tyrosine in the formation of thyroid hormones could be the best suited example. (King, 2012)
General form of amino acids:
(Amino Acid General Structure)
Aside from the synthesis of proteins, amino acids serve other biological functions like energy metabolites, and many of them are essential nutrients. During 1953, conditions of primordial Earth were recreated by Miller and Urey. A combination of hydrogen, ammonia, methane, and water vapor and provided some electrical sparks in the flask (Miller/Urey Experiment) . They found the formation of some molecules and identified them in 11 standard amino acids. Nitrogen is the key atom in the composition of Amino Acids. The inert gas Nitrogen in the environment is the main source for the biosynthesis of the amino acids. Though nitrogen is prominent in the composition of amino acids but it is almost unreactive due to the triple bond that is extremely difficult to break. Glutamine is also considered an essential metabolite for ammonia storage. Moreover glutamine is one of the universal donors of amine groups that helps in the formation several other amino acids as well as other biosynthetic products.
The method that is used for the production of amino acids is known as “Amino acid fermentation method”. Microorganisms help in conversion of nutrients to several vital necessary components to themselves that is the main focus in this procedure. (Healthcare, 2010)
Raw material such as syrups are so added in the culture media of microorganism during the fermentation method and the proliferating to produce amino acids in microorganisms are also allowed.” (Ajinomoto, 2003) Moreover the enzymes also play a prominent role here, enzymes are actually the proteins that catalyze the chemical reactions in the living body that is hardly dispensable to degrade and formulate substances. Amino acids are used as essential components of the microbial cells and thus it is necessary to regulate and maintain an optimal level of its biosynthesis. They are properly synthesized in particular amounts and are subject to negative feedback control. Mutants and a kind of semi-fermentation process called precursor addition fermentation is also used to reduce the negative impact of the production of amino acids. “Two types of mutants have been used for amino acid overproduction: autotrophic and regulatory mutants. In some cases, mutant strains have been further improved through DNA-recombination” (Industrial production of amino acids by fermentation and chemo-enzymatic methods, 2007).
A pathway is a series of all the biochemical reactions that are used for compound synthesis in metabolism. There is variety of ways for Amino acids synthesis. Precursor molecules can help in the synthesis of amino acids that too in simple steps. Alanine, aspartate, and glutamate are synthesized after a transamination reaction step in the form of pyruvate, oxaloacetate, and alpha-ketoglutarate, respectively from keto acids. In the similar manner, asparagine and glutamine by an amidation reaction step, are synthesized from aspartate and glutamate, respectively. equires more steps; Between one and thirteen biochemical reactions are necessary to produce the different amino acids from their precursors of the central metabolism that are required for the synthesis of other amino acids. (The Chemistry of Amino Acids, 2003)
There are different ways of purification of Amino acids. (Healthcare, 2010)
One-step purification of a GST-tagged protein and on-column tag removal:
The GST (Glutathione S-transferase) provides for the purification of Glutathione Sepharose columns. The usage of this method helps to purify the amino acids in one single step. The GST tag increases the solubility of the desired protein but being large in size, it is often removed after purification by using a sequence-specific protease, for example, PreScission Protease.
Two-step purification of an MBP-tagged protein: in this, a two step purification is done using a combination of AC for capture and GF for polishing. It is considered as the most useful of achieving high-purity tagged proteins. Most of the task of purification task can be performed with no or a minimum of optimization, by simply following the instructions for the different products. (Healthcare, 2010)
Three-step purification of a protein without an affinity tag: this is the most common strategies of purification for untagged proteins, IEX for capture, HIC for intermediate purification, and GF for the polishing step. Here obtaining highly purified Deacetoxycephalosporin C synthase (DAOCS), an oxygen-sensitive enzyme for crystallization and structure determination is the main task.
There are several other methods for purification of Amino acids:
- Unattended multistep purification and tag removal
- Two-step purification of membrane proteins
- Screening of conditions for membrane protein purification
- Matrix-assisted refolding of proteins expressed as inclusion bodies
- Screening and scaling up matrix-assisted protein refolding (Nature.com, 2010)
Synthesis of amino acids is not possible in all organisms. Even the synthesis of most of the amino acids is usually done by pathways that are found in certain types of plants and bacterias. It is also reported that mammals for instance requires eight of twenty amino acids from their diets. Thus it was necessary to classify amino in the categories of essentials and non-essentials. “The loss of the ability to synthesize essential amino acids likely emerged very early in evolution, for the source of amino acids they are dependent on other organisms that is common among all eukaryotes, not only those of mammals” (Nature.com)
In short it would not be wrong to say that the study of the evolution of amino acid metabolism has helped us understand the evolution of metabolism in general. The primary application of amino acids is to function as a monomer unit during the synthesis of proteins, using them as substrates for deriving nucleotide bases, biosynthetic reactions; and several other hormones and neurotransmitters from amino acids.