Enzymatic hydrolysis of proteins occurs under the action of proteolytic enzymes (proteases). They are classified into endo- and exopeptidases. Enzymes do not have strict substrate specificity and act on all denatured and many native proteins, cleaving in them peptide bonds-CO-NH-.

Endopeptidases (proteinases) - directly hydrolyze the protein through internal peptide bonds. As a result, a large number of polypeptides and few free amino acids are formed.

Optimal conditions for the action of acidic proteinases: pH 4.5-5.0, temperature 45-50 ° C.

Exopeptidases (peptidases) act mainly on polypeptides and peptides, breaking the peptide bond from the end. The main products of hydrolysis are amino acids. This group of enzymes is divided into amino, carboxy, dipeptidases.

Aminopeptidases catalyze the hydrolysis of the peptide bond adjacent to the free amino group.

H2N - CH - C - - NH - CH - C ....

Carboxypeptidases hydrolyze the peptide bond adjacent to the free carboxyl group.

CO -NH- C - H

Dipeptides catalyze the hydrolytic cleavage of dipeptides into free amino acids. Dipeptidases cleave only those peptide bonds adjacent to which are simultaneously free carboxyl and amine groups.

dipeptidase

NH2CH2CONHCH2COOH + H2O 2CH2NH2COOH

Glycine-Glycine Glycocol

Optimal conditions for action: pH 7-8, temperature 40-50 ° C. An exception is carboxypeptidase, which exhibits maximum activity at a temperature of 50 ° C and pH 5.2.

The hydrolysis of protein substances in the canning industry is necessary in the production of clarified juices.

Advantages of an enzymatic method for producing protein hydrolysates

In the production of biologically active substances from protein-containing raw materials, the most important is its deep processing, which provides for the splitting of protein molecules into constituent monomers. Hydrolysis of protein raw materials with the aim of producing protein hydrolysates - products containing valuable biologically active compounds: polypeptides and free amino acids - is promising in this regard. As a raw material for the production of protein hydrolysates, any natural proteins of complete amino acid composition can be used, the sources of which are blood and its constituent components; tissues and organs of animals and plants; waste dairy and Food Industry; veterinary confiscations; food and low-value food products obtained during processing different types animals, birds, fish; wastes from meat processing plants and glue factories, etc. When obtaining protein hydrolysates for medical and veterinary purposes, proteins of animal origin are mainly used: blood, muscle tissue and internal organs, protein membranes, as well as whey proteins.

The problem of protein hydrolysis and its practical implementation have attracted the attention of researchers for a long time. On the basis of protein hydrolysis, various preparations are obtained that are widely used in practice: as blood substitutes and for parenteral nutrition in medicine; to compensate for protein deficiency, increase resistance and improve the development of young animals in veterinary medicine; as a source of amino acids and peptides for bacterial and cultural nutrient media in biotechnology; in the food industry, perfumery. The quality and properties of protein hydrolysates intended for various applications are determined by the feedstock, the hydrolysis method and subsequent processing of the resulting product.

Varying the methods of obtaining protein hydrolysates allows obtaining products with desired properties. Depending on the amino acid content and the presence of polypeptides in the range of the corresponding molecular weight, the area of ​​the most efficient use of hydrolysates can be determined. Protein hydrolysates obtained for various purposes have different requirements, depending primarily on the composition of the hydrolyzate. So, in medicine, it is desirable to use hydrolysates containing 15 ... 20% of free amino acids; in veterinary practice, to increase the natural resistance of young animals, the content of peptides in hydrolysates is predominant (70 ... 80%); for food purposes, the organoleptic properties of the resulting products are important. But the main requirement when using protein hydrolysates in various fields is balance in amino acid composition.

Protein hydrolysis can be carried out in three ways: by the action of alkalis, acids, and proteolytic enzymes. During alkaline hydrolysis of proteins, lanthionine and lysinoalanine residues are formed, which are toxic to humans and animals. With this hydrolysis, arginine, lysine and cystine are destroyed; therefore, it is practically not used to obtain hydrolysates. Acidic protein hydrolysis is a widespread method. Most often, protein is hydrolyzed with sulfuric or hydrochloric acid. Depending on the concentration of the acid used and the hydrolysis temperature, the process time can vary from 3 to 24 hours. Hydrolysis with sulfuric acid is carried out for 3 ... 5 hours at a temperature of 100 ... 130 ° C and a pressure of 2 ... 3 atmospheres; hydrochloric - for 5 ... 24 hours at the boiling point of the solution under low pressure.

With acid hydrolysis, a large depth of protein breakdown is achieved and the possibility of bacterial contamination of the hydrolyzate is excluded. This is especially important in medicine, where hydrolysates are used mainly parenterally and it is necessary to exclude anaphylactogenicity, pyrogenicity and other undesirable consequences. V medical practice acid hydrolysates are widely used: aminocrovin, hydrolysin L-103, TsOLIPK, infusamin, gemmos and others.

The disadvantage of acid hydrolysis is complete destruction tryptophan, partial hydroxyamino acids (serine and threonine), deamination of amide bonds of asparagine and glutamine with the formation of ammoniacal nitrogen, destruction of vitamins, as well as the formation of humic substances, the separation of which is difficult. In addition, during the neutralization of acid hydrolysates, a large amount of salts are formed: chlorides or sulfates. The latter are especially toxic to the body. Therefore, acid hydrolysates require subsequent purification, for which ion exchange chromatography is usually used in production.

In order to avoid the destruction of labile amino acids during the preparation of acid hydrolysates, some researchers used mild hydrolysis regimes in an inert gas atmosphere, and also added antioxidants, thioalcohols, or indole derivatives to the reaction mixture. Acid and alkaline hydrolysis have, in addition to these, still significant limitations associated with the reactivity of the medium, which leads to rapid corrosion of equipment and necessitates compliance with stringent safety requirements for operators. Thus, the acid hydrolysis technology is rather laborious and requires the use of complex equipment (ion-exchange columns, ultramembranes, etc.) and additional stages of purification of the resulting preparations.

Research has been carried out on the development of an electrochemical enzymatic technology for obtaining hydrolysates. The use of this technology makes it possible to exclude the use of acids and alkalis from the process, since the pH of the medium is provided as a result of electrolysis of the processed medium containing a small amount of salt. This, in turn, makes it possible to automate the process and provide a finer and more efficient control of technological parameters.

As you know, in the body, protein is broken down by digestive enzymes into peptides and amino acids. A similar cleavage can be carried out outside the body. For this, the tissue of the pancreas, the mucous membrane of the stomach or intestines, pure enzymes (pepsin, trypsin, chymotrypsin) or enzyme preparations of microbial synthesis are added to the protein substance (substrate). This method of protein breakdown is called enzymatic, and the resulting hydrolyzate is called enzymatic hydrolyzate. The enzymatic method of hydrolysis is more preferable in comparison with chemical methods, since it is carried out under "mild" conditions (at a temperature of 35 ... 50 ° C and atmospheric pressure). The advantage of enzymatic hydrolysis is the fact that during its implementation, amino acids are practically not destroyed and do not enter into additional reactions (racemization and others). In this case, a complex mixture of degradation products of proteins with different molecular weights is formed, the ratio of which depends on the properties of the enzyme used, the raw materials used and the process conditions. The resulting hydrolysates contain 10 ... 15% of total nitrogen and 3.0 ... 6.0% of amine nitrogen. Its technology is relatively simple.

Thus, in comparison with chemical technologies, the enzymatic method for obtaining hydrolysates has significant advantages, the main of which are: availability and ease of implementation, low energy consumption and environmental safety.

Proteins can undergo a variety of transformations during the preparation and cooking process of food.

Melanoidin reaction

Soluble amino acids (glycine, alanine, asparagine, etc.) react vigorously with sugars that have a free carbonyl group (xylose, fructose, glucose, maltose). The melanoidin reaction proceeds most easily when the molar ratio between amino acids and sugars is 1: 2.

The amino acid reacts with sugar as follows:

CH 2 OH- (CHOH) 4 -COH + H 2 N-CH 2 -COOH -------- 

glucose glycine

----------  CH 2 OH- (CHOH) 4 -C-NH-CH 2 -COOH

Poorly soluble acids (cystine, tyrosine) are less active. The melanoidin reaction is accompanied by the formation of intermediate compounds: aldehydes, cyclic groups of furfural and then pyrrole character. Melanoidin reactions are activated at elevated temperatures, especially in the case of repeated heating.

As a result of this reaction, a darkening of the crust of white bread occurs: when baking, amino acids on the surface of the bread react with sugars formed during the fermentation of the dough.

Melanoidins can also be formed during the storage of canned food.

Protein hydrolysis

It can be influenced by enzymes, acids or alkalis. In this way, you can get any of the amino acids that make up proteins. Of practical importance is the hydrolysis of the biomass of yeast grown on hydrocarbon-containing raw materials and containing up to 40% proteins. Carbon dioxide, alcohol, oil paraffins, natural gas, waste from the wood processing industry can also serve as raw materials for obtaining biomass by microbiological means. Amino acids obtained from protein hydrolysates are separated by ion exchange chromatography, electrophoresis, and gas-liquid chromatography.

Protein hydration

Proteins bind water, i.e. exhibit hydrophilic properties. At the same time, they swell, their mass and volume increase. The swelling of the protein is accompanied by its partial dissolution. The hydrophilicity of individual proteins depends on their structure. Hydrophilic -CO-NH- (peptide bond), amino groups -NH2, carboxyl -COOH- groups, which are present in their composition and located on the surface of the protein macromolecule, attract water molecules, strictly orienting them on the surface of the molecule.

The hydration (water) shell surrounding protein globules prevents aggregation, and therefore contributes to the stability of protein solutions and prevents its precipitation.

H 3 N + - (CH 2) n-COOH + NH 3 - (CH 2) n-COO - NH 2 - (CH 2) n-COO -

isoelectric point

pH = 1.0 pH = 7.0 pH = 11.0

At the isoelectric point (see diagram) proteins have the least ability to bind water, the hydration shell around the protein molecules is destroyed, so they combine to form large aggregates. When the pH of the medium changes, the protein molecule becomes charged and its hydration capacity changes. With limited swelling, concentrated protein solutions form complex systems called studios. Globular proteins can be completely hydrated by dissolving in water (for example, milk proteins), forming solutions with a low concentration.

Hydrophilic properties of proteins, i.e. their ability to form studios, stabilize suspensions, emulsions and foams are of great importance in the food industry. The different hydrophilicity of gluten proteins is one of the features that characterize the quality of wheat grain and flour obtained from it (the so-called strong and weak wheat). The hydrophilicity of proteins in grain and flour plays an important role in the storage and processing of grain, in baking. The dough, which is obtained in the bakery industry, in the manufacture of flour confectionery products, is a protein swollen in water, a concentrated jelly containing starch grains.

Protein- natural polypeptides with a huge molecular weight. They are part of all living organisms and perform various biological functions.

Protein structure.

Proteins have 4 levels of structure:

• primary protein structure- a linear sequence of amino acids in a polypeptide chain, folded in space:

• secondary protein structure- the conformation of the polypeptide chain, because twisting in space due to hydrogen bonds between NH and CO in groups. There are 2 ways of styling: α -spiral and β - structure.

• tertiary protein structure is a three-dimensional representation of a swirling α -spiral or β -structures in space:

This structure is formed by disulfide bridges -S-S- between cysteine ​​residues. Oppositely charged ions participate in the formation of such a structure.

• quaternary protein structure formed due to the interaction between different polypeptide chains:

Protein synthesis.

The synthesis is based on the solid-phase method, in which the first amino acid is fixed on a polymer carrier, and new amino acids are sequentially sewn to it. The polymer is then separated from the polypeptide chain.

Physical properties of protein.

The physical properties of a protein are determined by its structure, therefore proteins are divided into globular(water soluble) and fibrillar(insoluble in water).

Chemical properties of proteins.

1. Protein denaturation(destruction of the secondary and tertiary structure with the preservation of the primary). An example of denaturation is the curdling of egg whites when boiling eggs.

2. Protein hydrolysis- irreversible destruction of the primary structure in an acidic or alkaline solution with the formation of amino acids. So you can establish the quantitative composition of proteins.

3. Qualitative reactions:

Biuret reaction- interaction of peptide bonds and copper (II) salts in an alkaline solution. At the end of the reaction, the solution turns purple.

Xanthoprotein reaction- when reacting with nitric acid a yellow color is observed.

The biological significance of protein.

1. Proteins - a building material, muscles, bones, tissues are built from it.

2. Proteins are receptors. They transmit and receive a signal from neighboring cells from the environment.

3. Squirrels play important role in the body's immune system.

4. Proteins perform transport functions and carry molecules or ions to the place of synthesis or accumulation. (Hemoglobin carries oxygen to tissues.)

5. Proteins - catalysts - enzymes. These are very powerful selective catalysts that accelerate reactions millions of times over.

There are a number of amino acids that cannot be synthesized in the body - irreplaceable, they are obtained only with food: tizine, phenylalanine, methinine, valine, leucine, tryptophan, isoleucine, threonine.

Like other chemical reactions, protein hydrolysis is accompanied by the exchange of electrons between specific atoms of the reacting molecules. Without a catalyst, this exchange is so slow that it cannot be measured. The process can be accelerated by adding acids or bases; the former give H-ions upon dissociation, the latter - OH-ions. Acids and bases play the role of true catalysts: they are not consumed during the reaction.

When boiling protein with concentrated acid its complete decomposition into free amino acids occurs. If such a decay took place in a living cell, this would naturally lead to its death. Proteins also break down under the action of proterlytic enzymes, and even faster, but without the slightest harm to the body. And while H-ions act indiscriminately on all proteins and on all peptide bonds in any protein, proteolytic enzymes are specific and break only certain bonds.

Proteolytic enzymes are proteins themselves. How does a proteolytic enzyme differ from a substrate protein (a compound is called a substrate, which is the object of the enzyme's action)? How does a proteolytic enzyme manifest its catalytic activity without destroying itself or the cell? Answering these basic questions would help to understand the mechanism of action of all enzymes. Since 30 years ago M. Kunitz first isolated trypsin in crystalline form, proteolytic enzymes have served as models for studying the relationship between protein structure and enzymatic function.

Proteolytic enzymes of the digestive tract are associated with one of the most important functions human body- assimilation of nutrients. This is why these enzymes have long been the subject of research; in this regard, ahead of them, perhaps, are only yeast enzymes involved in alcoholic fermentation... The best studied digestive enzymes are trypsin, chymotrypsin and carboxy-peptidases (these enzymes are secreted by the pancreas). Namely, by their example, we will consider all that is now known about the specificity, structure and nature of the action of proteolytic enzymes.

Proteolytic enzymes of the pancreas are synthesized in the form of precursors - zymogens - and are stored in intracellular bodies, the so-called zymogen granules. Zymogens are devoid of enzymatic activity and, therefore, cannot act destructively on the protein components of the tissue in which they were formed. Entering the small intestine, zymogens are activated by another enzyme; at the same time, small, but very important changes occur in the structure of their molecules. We will dwell on these changes in more detail later.

"Molecules and Cells", ed. G.M. Frank

Chemistry, like most exact sciences, requiring a lot of attention and solid knowledge, has never been the favorite discipline of schoolchildren. And in vain, because with its help it is possible to understand the many processes taking place around and inside a person. Take, for example, the hydrolysis reaction: at first glance, it seems that it matters only for chemical scientists, but in fact, without it, no organism could function fully. Let's find out about the features of this process, as well as about its practical significance for humanity.

Hydrolysis reaction: what is it?

This phrase is a specific reaction of exchange decomposition between water and a substance dissolved in it with the formation of new compounds. Hydrolysis can also be called solvolysis in water.

This chemical term is derived from 2 Greek words: water and decomposition.

Hydrolysis products

The reaction under consideration can occur during the interaction of H 2 O with both organic and non-organic organic matter... Its result directly depends on what the water was in contact with, as well as whether additional catalyst substances were used, whether the temperature and pressure were changed.

For example, the salt hydrolysis reaction promotes the formation of acids and alkalis. And if we are talking about organic substances, other products are obtained. Water solvolysis of fats promotes the formation of glycerol and higher fatty acids. If the process takes place with proteins, various amino acids are formed as a result. Carbohydrates (polysaccharides) are decomposed into monosaccharides.

In the human body, which is unable to fully assimilate proteins and carbohydrates, the hydrolysis reaction "simplifies" them to substances that the body is able to digest. So solvolysis in water plays an important role in the normal functioning of every biological individual.

Hydrolysis of salts

Having learned hydrolysis, it is worth familiarizing yourself with its course in substances of inorganic origin, namely, salts.

The peculiarities of this process is that when these compounds interact with water, the ions of a weak electrolyte in the composition of the salt are detached from it and form new substances with H 2 O. It can be either acid or both. As a result of all this, there is a shift in the equilibrium of dissociation of water.

Reversible and irreversible hydrolysis

In the above example, in the latter, instead of one arrow, you can see two, and both are directed to different sides... What does it mean? This sign signals that the hydrolysis reaction is reversible. In practice, this means that, interacting with water, the taken substance simultaneously not only decomposes into components (which allow new compounds to arise), but also forms again.

However, not every hydrolysis is reversible, otherwise it would not make sense, since new substances would be unstable.

There are a number of factors that can contribute to the fact that such a reaction becomes irreversible:

• Temperature. Whether it rises or falls depends on the direction in which the equilibrium shifts in the ongoing reaction. If it gets higher, there is a shift towards an endothermic reaction. If, on the contrary, the temperature decreases, the advantage is on the side of the exothermic reaction.
• Pressure. This is another thermodynamic quantity that actively influences ionic hydrolysis. If it rises, the chemical equilibrium is shifted towards the reaction, which is accompanied by a decrease in the total amount of gases. If it goes down, vice versa.
• High or low concentration of substances participating in the reaction, as well as the presence of additional catalysts.

Types of hydrolysis reactions in saline solutions

• By anion (ion with negative charge). Solvolysis in water of salts of weak acids and strong foundations... This reaction is reversible due to the properties of the interacting substances.

Hydrolysis degree

Studying the features of hydrolysis in salts, it is worth paying attention to such a phenomenon as its degree. This word means the ratio of salts (which have already entered into a decomposition reaction with H 2 O) to the total amount of the contained of this substance in solution.

The weaker the acid or base involved in hydrolysis, the higher its degree. It is measured in the range of 0-100% and is determined by the formula below.

N is the number of molecules of the substance that have undergone hydrolysis, and N 0 is their total number in solution.

In most cases, the degree of aqueous solvolysis in salts is low. For example, in a 1% sodium acetate solution, it is only 0.01% (at a temperature of 20 degrees).

Hydrolysis in organic substances

The process under study can also occur in organic chemical compounds.

In almost all living organisms, hydrolysis occurs as part of energy metabolism (catabolism). With its help, proteins, fats and carbohydrates are broken down into easily digestible substances. At the same time, water itself is often rarely able to start the process of solvolysis, so organisms have to use various enzymes as catalysts.

If we are talking about chemical reaction with organic substances, aimed at obtaining new substances in a laboratory or production, then to accelerate and improve it, strong acids or alkalis are added to the solution.

Hydrolysis in triglycerides (triacylglycerols)

This difficult to pronounce term refers to fatty acids, which most of us know as fats.

They are of both animal and plant origin. However, everyone knows that water is not able to dissolve such substances, how does the hydrolysis of fats occur?

The reaction in question is called fat saponification. It is an aqueous solvolysis of triacylglycerols under the influence of enzymes in an alkaline or acidic environment. Depending on it, alkaline hydrolysis and acidic hydrolysis are released.

In the first case, as a result of the reaction, salts of higher fatty acids (better known to everyone as soaps) are formed. Thus, from NaOH, ordinary solid soap is obtained, and from KOH, liquid soap. So alkaline hydrolysis in triglycerides is the process of forming detergents. It should be noted that it can be freely carried out in fats of both vegetable and animal origin.

The reaction in question is the reason that soap is rather difficult to wash in hard water and does not wash at all in salt water. The fact is that H 2 O is called hard, which contains an excess of calcium and magnesium ions. Soap, once it gets into water, undergoes hydrolysis again, breaking down into sodium ions and a hydrocarbon residue. As a result of the interaction of these substances in water, insoluble salts are formed, which look like white flakes. To prevent this from happening, sodium bicarbonate NaHCO 3, better known as baking soda, is added to the water. This substance increases the alkalinity of the solution and thereby helps the soap to perform its functions. By the way, in order to avoid such troubles, in modern industry they make synthetic detergents from other substances, for example from salts esters higher alcohols and sulfuric acid. Their molecules contain from twelve to fourteen carbon atoms, so that they do not lose their properties in salt or hard water.

If the environment in which the reaction takes place is acidic, this process is called acid hydrolysis of triacylglycerols. In this case, under the action of a certain acid, substances evolve to glycerol and carboxylic acids.

The hydrolysis of fats has another option - the hydrogenation of triacylglycerols. This process it is used in some types of cleaning, for example, when removing traces of acetylene from ethylene or oxygen impurities from various systems.

Hydrolysis of carbohydrates

The substances under consideration are among the most important constituents of human and animal food. However, the body is not able to assimilate sucrose, lactose, maltose, starch and glycogen in their pure form. Therefore, just as in the case of fats, these carbohydrates are broken down into digestible elements using a hydrolysis reaction.

Also, water solvolysis of carbon is actively used in industry. From starch, due to the considered reaction with H 2 O, glucose and molasses are extracted, which are part of almost all sweets.

Another polysaccharide that is actively used in industry for the manufacture of many useful substances and products is cellulose. Technical glycerin, ethylene glycol, sorbitol and ethyl alcohol, well-known to all, are extracted from it.

Cellulose hydrolysis occurs with prolonged exposure to high temperatures and the presence of mineral acids. The end product of this reaction is, as is the case with starch, glucose. It should be borne in mind that the hydrolysis of cellulose is more difficult than that of starch, since this polysaccharide is more resistant to the effects of mineral acids. However, since cellulose is the main component of the cell walls of all higher plants, the raw materials containing it are cheaper than for starch. At the same time, cellulose glucose is more used for technical needs, while the starch hydrolysis product is considered better suitable for nutrition.

Protein hydrolysis

Proteins are the basic building blocks for the cells of all living organisms. They are composed of numerous amino acids and are very important product for the normal functioning of the body. However, being high molecular weight compounds, they can be poorly absorbed. To simplify this task, they are hydrolyzed.

As with other organic substances, this reaction breaks down proteins into low molecular weight products that are easily absorbed by the body.