Microbial production of recombinant chymosin: Difference between revisions
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==Chymosin mechanism of action== | ==Chymosin mechanism of action== | ||
Like other acidic proteases from the gastric juice, chymosin is secreted as an inactive precursor, prochymosin. The precursor is stable at weakly alkaline pH whereas the active enzyme is rapidly denatured at pH values above 7. Below | Milk consists of water, fat, protein, phosphate, lactose, citric acid and inorganics such as calcium phosphate. The protein component of milk can be divided into two groups, the casein fraction (αs1-casein, αs2-casein, β-casein and κ-casein)and the whey proteins (β-lactoglobulin, β-lactoalbumin, immunoglobulins and serum albumin). The casein proteins are the ones that will form the curd during cheese making, specifically κ-casein. | ||
Like other acidic proteases from the gastric juice, chymosin is secreted as an inactive precursor, prochymosin. The precursor is stable at weakly alkaline pH whereas the active enzyme is rapidly denatured at pH values above 7. Below pH 5 prochymosin is converted into chymosin by a limited proteolysis during which a peptide segment is cleaved from the N-terninus. The proteolytic activity of chymosin has optimum pH about pH 3-5. | |||
The milk-clotting activity of chymosin is due to a limited proteolysis of the casein. Casein consists of two parts: one hydrophobic and one hydrophilic. During milk-clotting, a Phe-Met bond is hydrolysed, the hydrophilic part of the casein is liberated and aggregation occurs(ref). | The milk-clotting activity of chymosin is due to a limited proteolysis of the κ-casein. κ-Casein consists of two parts: one hydrophobic and one hydrophilic. During milk-clotting, a Phe-Met bond is hydrolysed, the hydrophilic part of the κ-casein is liberated and aggregation occurs(ref). Chymosin specifically recognises the sequence from His 98 to Lys 111 and cleaves the peptide bond between Phe 105 and Met 106 in the κ-casein chain. | ||
==Production of recombinant chymosin== | ==Production of recombinant chymosin== | ||
Revision as of 03:56, 26 March 2013
Introduction
Cheese production requires the use of a 381 aminoacids protein called chymosin (also known as rennin). Chymosin is a proteolitic enzyme which is usually obtained from calf stomachs. The role of this enzyme is to coagulate the milk, which is very important for digestion of milk in young animals. Initially, chymosin is secreted as inactive prochymosin and then activated at low pH. Once it is active, it breaks milk protein k-casein in a specific point and effects clotting which is the first step in cheese production. By clotting the milk, a solid product called curd is obtained. Curd is then processed to make cheese.
The increasing world production of cheese, coupled with a decline in the number of slaughtered calves, has stimulated a search for alternative sources of chymosin. One of these alternative sources is the use of recombinant chymosin produced by microorganisms.
Recombinant chymosin is mainly produced by funghi, but it can also be produced using bacteria.
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Chymosin mechanism of action
Milk consists of water, fat, protein, phosphate, lactose, citric acid and inorganics such as calcium phosphate. The protein component of milk can be divided into two groups, the casein fraction (αs1-casein, αs2-casein, β-casein and κ-casein)and the whey proteins (β-lactoglobulin, β-lactoalbumin, immunoglobulins and serum albumin). The casein proteins are the ones that will form the curd during cheese making, specifically κ-casein.
Like other acidic proteases from the gastric juice, chymosin is secreted as an inactive precursor, prochymosin. The precursor is stable at weakly alkaline pH whereas the active enzyme is rapidly denatured at pH values above 7. Below pH 5 prochymosin is converted into chymosin by a limited proteolysis during which a peptide segment is cleaved from the N-terninus. The proteolytic activity of chymosin has optimum pH about pH 3-5.
The milk-clotting activity of chymosin is due to a limited proteolysis of the κ-casein. κ-Casein consists of two parts: one hydrophobic and one hydrophilic. During milk-clotting, a Phe-Met bond is hydrolysed, the hydrophilic part of the κ-casein is liberated and aggregation occurs(ref). Chymosin specifically recognises the sequence from His 98 to Lys 111 and cleaves the peptide bond between Phe 105 and Met 106 in the κ-casein chain.
Production of recombinant chymosin
Recombination is the process by which genetic material is broken and then joined to a new genetic material. In this case, the chymosin gene from the cow is taken out of the genome and then introduced into a plasmid. The plasmid is then introduced into a microorganism which will start producing the chymosin by transcribing and translating the gene from the plasmid.
The first step in this process is to obtain a piece of tissue from the calf stomach and isolate the DNA from the cells.
Section 3
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Conclusion
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References
Edited by (your name here), a student of Nora Sullivan in BIOL187S (Microbial Life) in The Keck Science Department of the Claremont Colleges Spring 2013.
