This article is based on the lecture given by Dr. Sara Borrell, from Regulatory Affairs Department, at the XXXI Biovet International Symposium in Tarragona, during the fourth session dedicated to enzymes and mycotoxin binders, which took place on the 29th May 2018. With the title, “Enzymes proteases. Review and economic importance” the lecture deepened on the study of proteases describing the different types, the mechanism of action, the methods of analysis and uses, etc. The veterinarian of the team of Biovet S.A, Dr. Maria Soriano, has made the adaptation of the paper.

ENZYMES: Definition, units and classification

In every chemical reaction there are different reactants (A + BC) transformed later into different products (AB + C) that have an intermediate step called transition state (A-B-C), in which the reactant bonds are weakened so they can be modified. This state of transition has a higher energy than the reactants or the products, so it is necessary an energy contribution called activation energy (Ea) to make the reaction possible.

Enzymes are efficient and specific biocatalysts that boost these biological reactions, increasing the reaction rate by decreasing the activation energy (Picture 1). These catalysts allow, at low amount and without being consumed, to reach the equilibrium of the chemical reaction more easily, without altering the rest of the energetic parameters of the reaction.

Enzymatic reactions are adjustable, and most enzymes are proteins.

Picture 1: Chemical reaction and activation energy differences whether

using an enzyme (red colour) or not (orange colour).

Enzymatic activity is measured according to the speed of disappearance of the substrate or the speed of appearance of the product. The enzymatic activity can be expressed in:

• International Units of enzymatic activity (UI): refers to the amount of enzyme needed under optimal conditions of temperature, pH and ionic strength to transform 1 μmol of substrate per minute.
• Katal: in relation to the amount of enzyme that is required under optimum conditions to transform 1 mol of substrate per second. The equivalence between both units is: 6×10⁷ IU = 1 katal.

These units are not usually used industrially, for example, in the case of proteases, there are several units accepted in practice, such as: King-Armstrong units, Bodansky units, Somogyi units or Anson units. These last units, the Anson units for peptide activity, describe the amount of enzyme needed to hydrolyze 1 mmol of tyrosine with hemoglobin as a substrate in 10 minutes, where 1 Anson unit corresponds to 550 IU.

Enzymes are classified according to the Enzyme Committee with the nomenclature “EC a.b.c.d“, where:

• a: Class. There are 6 main divisions related to the reaction that is catalyzed (Chart 1).
• b: Subclass.
• c: Sub-subclass.
• d: Serial number of the sub-subclass.

Chart 1: Main enzyme division depending on the reaction that they were catalyzing.

PROTEASES: Types, mechanism of action, methods of analysis and uses

In order to obtain the essential amino acids presents in the diet, usually included in more complex proteins, it is necessary to break them down by using specific enzymes, called proteases.

According to the Enzyme Committee, proteases are included in group 3 hydrolases because their mechanism of action is the breakdown of protein peptide bonds using a water molecule and obtaining as a product simpler peptides or amino acids (Picture 2).

Picture 2: Hydrolysis performed by a protease

Proteases are classified according to various criteria such as:

• Origin: animal, vegetable, bacterial or fungal.
• Mode of catalytic action: endogenous or exogenous activity, depending on whether they break the peptide bonds located at the ends or inside the peptide chain.
• According to the amino acid present in the active center, responsible for catalyzing the reaction:
  • Serine proteases
  • Cysteine proteases
  • Aspartate proteases
  • Metalloproteases

The active center of the enzyme is the place where the substrate joins and where the catalysis of the reaction takes place (Picture 3). The active site is very small in size in comparison to the total size of the enzyme and is composed of a side chain of amino acids that bind to the product and a side chain of amino acids that carry out the catalysis.

Picture 3: Active center of an enzyme.

In order to have a more detailed explanation of the protease hydrolysis process, the mechanism of action of a serine protease is shown as an example (Pictures 4, 5, 6, 7 and 8).

MECHANISM OF ACTION OF A SERINE PROTEASE

This mechanism consists of an acylation phase in which a covalent acyl-enzyme intermediate is formed with the release of the first product, and a deacylation phase in which a water molecule breaks the intermediate and releases the second product.

Black spaces present in the pictures below are the representation of the enzyme active site, where initially there are (Picture 4):

• The triad of amino acids: serine (Ser 195), histidine (His 57) and aspartate (Asp 102), common in all active sites of serine proteases.
• The protein that is going to be hydrolyzed, present in the lower left part of the image.

Picture 4: Mechanism of action of a serine protease (Initially).

The catalytic action begins when His 57 removes a proton from the alcoholic group of serine, leaving the histidine with positive charge and the serine with negative charge. This serine is the one that is initially related to the protein, attacking the carbonyl group (C = O) of the cleavable bond, resulting in the transition state 1 (acylation phase, picture 5).

Image 5: Mechanism of action of a serine protease (Acylation phase).

Next, histidine 57 gives its proton to this complex, which separates the first product (NH2-R) and the complex remains as Acyl-enzyme (Picture 6), ready to begin the second phase of the reaction.

Image 6: Mechanism of action of a serine protease (Acyl-enzyme).

The second phase of the reaction takes place because of the entrance of a water molecule (the second substrate) to the active site. Then, Histidine 57 removes a proton from the water molecule, which is converted into a hydroxyl ion (OH-) linked to the acyl-enzyme and serine (Picture 7).

Picture 7: Mechanism of action of a serine protease (Acyl-enzime + Hydroxyl).

The hydroxyl group attacks the carbonyl bond again, producing the second transition state, which resolves with the output of product 2. Finally, the histidine yields its proton to the serine, which results in a return to the initial state of the active site (Picture 8).

Picture 8: Mechanism of action of a serine protease (End of the process).

METHODS OF ANALYSIS

There are three methods for the quantification of enzymes:

• ELISA (Enzyme-Linked Immunosorbent Assay): Based on the ability of the enzyme to bind to a substrate, in this case antibodies. This union will result in a coloured product, so the colour intensity is proportional to the amount of enzyme.

Picture 9: Types of ELISA: Direct and indirect.

• ELECTROPHORESIS: Technique based on the separation of molecules depending on their size and electrical charge. It consists of a gel (agar or polyacrylamide) placed in a chamber subjected to a potential difference caused by a cathode (terminal with negative charge) and an anode (terminal with positive charge) on opposite sides of the chamber. The electric field that is created causes the migration of molecules: large and heavy molecules will migrate more slowly than small and light molecules.

Picture 10: Results of an electrophoresis test.

• SPECTROFOTOMETRY: It is based on the formation of a coloured complex when enzyme and substrate establish contact in the presence of appropriate reactants. The absorbance emitted by this complex is measured and allows to perform a calibration line with the patterns of the enzyme.

Picture 11: Optical cuvettes for evaluation in spectrophotometer.

Chart 2: Concentration results regarding the obtained absorbances.

APPLICATIONS

Protein of animal origin has become an element of the diet with a high cost and with many fluctuations in the supply. Therefore, vegetable protein has become the alternative source, especially legumes, composed of high quality protein, vitamins, phosphorus and iron, with the disadvantage of containing antinutritional factors such as protease inhibitors, which interfere in the digestive and metabolic use of proteins in the diet, or glycosidase inhibitors, especially soy α-amylase.

By increasing the use of protein of plant origin, the inclusion of proteases in the diet has been extended. The use of an efficient protease allows to:

• Improve the digestibility of proteins and amino acids in the diet, which is reflected in a better conversion rate, in a decrease in the excretion of nitrogen to the environment and a reduction of intestinal clostridiosis.
• Compensate anti-nutritional factors.

The selection of protease in the diet should have as considerations:

• The species of destiny.
• The specificity for a substrate with a greater range of action.
• Physiological conditions: for example, the pH in the different digestive tracts.
• The encapsulation of vegetable seeds.
• The stability during manufacturing, and its resistance to heat during processing.
• The synergy with other enzymes used.

ECONOMIC IMPORTANCE

Proteases generated sales for 150 million dollars in 2016, representing 15% of the total enzyme market. The Organization for Economic Cooperation and Development (OECD) foresees a growth of the market for this type of enzymes for the coming years, globally around 1280 million USD in 2019 and 2000 million USD for 2024.

Chart 1: Projected evolution for global protease sells in the coming years (Source: OECD).

CONCLUSIONS

The growing demand for proteins and amino acids needed in the diet of production animals to achieve the conversions that the industry needs is a key factor to support the penetration of proteases in the enzyme markets.

Improving the efficiency of food while maintaining intestinal health is the most beneficial property of proteases.