Proteolytic Enzymes

Proteolytic Enzymes: Enzymes are proteins that catalyze biochemical functions. They are required for various physiological processes. Proteolytic enzymes are also known as proteases that digest proteins, i.e. breakdown of a long chain of protein molecules into shorter peptides and their components such as amino acids. They act as digestive aids, blood cleansers, rebalance the immune system and reduce oedema in inflamed regions.

Papain

Biological Source: It is obtained from green fruits of papaya, Carica papaya.

Family: Caricaceae

Characters:

  • Commercial papain is buff to light brown powder.
  • It is soluble in water and glycerol, but insoluble in most organic solvents.
  • Commercial papain contains chymopapain, lysozyme, lipase, peptidase-A.
  • It activates at pH 3-11.
  • Its activity will retain at 70o C at pH 7.0.
  • The optimum pH for papain activity is 5-7.
  • The optimum temperature range is 60-70o C.

Chemical Composition:

  • It is a sulfhydryl protease enzyme.
  • It contains 212 amino acid residues with cysteine 25 bearing the essential active thiol groups.
  • The isoelectric point at pH 8.75.
  • Its molecular weight is 21,000 to 23,700 dalton.
  • Its 3-D structure is identified by X-ray crystallography which shows the presence of two parts, each part containing about 100 amino acid residues. It hydrolyses into proteins, peptides, amides, and esters.

Activators: Glutathione, Cysteine, Bisulphite, Sulphide, Ammonium sulphate.

Inhibitors: Thiol reagents, Heavy metals ions like Zn+2, Fe+2, Ascorbic acid etc.

Uses: It is used as anti-inflammatory, anti-ulcer, wound healing, digestive aids, oedema, stabilizer etc.

Method of isolation: It has two steps: (a) Extraction of latex followed by (b) Latex to papain.

The method is as follows:

Method-I (Fig.1):

Papain isolation from papaya fruit
Fig.1: Papain isolation from papaya fruit

Methods-II:

Papain isolation method
Fig.2: Papain isolation method

Storage: Lyophilized powder or crystalline suspension of papain is stored in NaCl solution at near-neutral pH at 4-5°C for 6 months.

Bromelain

Biological Source: It is obtained from the stem and fruits of the pineapple of Ananus comosus.

Family: Bromeliaceae.

Characters:

  • It is a sulphydryl proteolytic enzyme.
  • It contains a mixture of proteases and non-proteolytic enzymes like acid phosphatase, peroxidase etc.
  • It is colourless.
  • It is slightly soluble in water and glycerol, but insoluble in organic solvents.
  • The optimum pH required is 5-8.
  • Optimum temperature: 50-60°C.
  • Molecular weight is 15000 to 18000 dalton.

Extraction (Fig.3):

Bromelain isolation method
Fig.3: Bromelain isolation method

Uses: It is used as a digestive agent, in CVS diseases, Joint inflammation, arthritis etc.

Serratiopeptidase

Serratiopeptidase is a proteolytic enzyme. Biological Source: Non-pathogenic enterobacterium Serratia marcescens E-15. This microorganism was originally isolated in the late 1960s from the silkworm Bombyx mori. Family: Enterobacteriaceae.

Properties:

  • It has a high degree of substrate specificity.
  • The molecular weight of Serrapeptase ranges from about 45 kDa – 60 kDa.
  • It is a metalloprotease and contains three zinc atoms as ligands and one active site. The presence of zinc atom is essential and also enhances the proteolytic activity of Serrapeptase.
  • The gene encoding Serrapeptase reveals that it is made up of 470 amino acids. The amino acid sequence is free of Sulphur containing amino acids, cysteine and methionine.
  • The maximum enzyme activity of Serrapeptase is observed at pH 9.0 and a temperature of 40°C.
  • It is degraded or inactivated completely at a temperature of 55°C.
  • It is an active enzyme that binds to the alpha-2 macroglobulin in biological fluids and blood, it binds in the ratio of 1 : 1 and this binding helps to mask its antigenicity.
  • The doses usually range from 10 mg to 60 mg per day.

Production: The production of Serrapeptase depends upon a secretory protein on the membrane of the host cell and it is secreted by the N-terminal signal peptide-independent pathway.

Soil and contaminated water are rich sources of a diverse variety of microorganisms. Isolated pure cultures of the bacterial strains are maintained on nutrient agar plates and stored at 4°C. Serratia marcescens is a Gram-negative bacterium. It grows in a wide range of temperatures (5-40°C) and pH (5.0-9.0) and secretes a variety of enzymes such as serine and thiol proteases, metalloproteases, lipases, chitinases etc.

Medium Preparation (Fig.4): Serratia marcescens are usually cultured in trypticase soy broth. A medium containing carbon source – maltose, organic nitrogen source – peptone, inorganic nitrogen source – ammonium sulphate, dihydrogen phosphate, sodium bicarbonate, inorganic salt source – sodium acetate, glycerin and ascorbic acid is used as a production medium.

Another medium reported for the production of Serrapeptase contained maltose 45 g/lit, soybean meal 65 g/lit, KH2PO4 8.0 g/lit, and NaCl 5.0 g/lit at a pH 7.0 which gives maximum yield. Casein medium is also used, but trypticase soy is a preferred substrate over casein as the specific activity is higher when trypticase soy is used as the substrate in the production medium.

Industrial production of Serratiopeptidase
Fig.4: Industrial production of Serratiopeptidase

Uses: It is used to reduce pain and swelling associated with conditions like back pain, arthritis, tension headaches and migraine headaches. It also has anti-atherosclerotic effects due to its fibrinolytic and caseinolytic properties.

Urokinase

Biological Source: It is obtained from human urine.

Characters: They are plasminogen activators and are obtained from human renal cells. They are serine protease enzymes. Urokinase is a 411-residue protein, consisting of the domains: the serine protease domain, the kringle domain and the growth factor domain. Urokinase is synthesized as a zymogen form and is activated by proteolytic cleavage between Lys 158 and Ile 159. The two resulting chains are kept together by a disulphide bond.

Isolation (Fig.5):

Urokinase isolation
Fig.5: Urokinase isolation

Uses: It is a thrombolytic agent, used in the treatment of severe or massive deep venous thrombosis, pulmonary embolism, myocardial infarction and dialysis cannulas. It is used intrapleural to improve the drainage of complicated pleural effusions and empyemas.

Streptokinase

Biological Source: It is obtained from the bacteria Beta haemolytic streptococci.

Family: Streptococcaceae.

Characters: It is a white powder, soluble in water. The optimum pH range is 7-8. It is an extracellular enzyme-containing single-chain polypeptide. Molar mass is 47 kDa. It is made up of 414 amino acid residues. The protein exhibits its maximum activity at a pH of 7.5 and its isoelectric pH is 4.7.

Isolation (Fig.6):

Medium Composition: Casein solution, potassium dihydrogen phosphate, lysine, dextrose, uracil, adenine sulphate, nicotinic acid, pyridoxine, tryptophan, calcium pantothenate, thiamine-HCl, riboflavin, thioglycolic acid and some salts of trace elements.

Procedure:

Extraction of streptokinase
Fig.6: Extraction of streptokinase

Uses: It is used as thrombolytic medication, in case of a blood clot in myocardial infarction, treatment of burns, respiratory problems etc.

Storage: The enzyme is supplied as a lyophilized white powder in 50 ml infusion bottles or 6.5 ml vials with a coloured label and is stored at room temperature of 15-30°C.

Pepsin

Biological Source: Pepsin is the principal proteolytic enzyme of vertebrate gastric juice.

Properties

  • It is an endopeptidase enzyme.
  • It is buff coloured or white coloured amorphous powder.
  • It has a little acidic or saline taste with a slight meaty odour.
  • It is soluble in water but insoluble in alcohol, ether and chloroform.
  • It breaks down proteins into peptones and proteases.
  • The molecular weight of Pepsin: 34.5 kDa.
  • Pepsin is a monomeric, two-domain, mainly beta protein with a high percentage of acidic residues.
  • It is produced in the stomach and is one of the main digestive enzymes in the digestive system.
  • It has a three-dimensional structure, of which one or more polypeptide chains twist and fold, bringing together a small number of amino acids to form the active site.
  • It is an aspartic protease, using a catalytic aspartate in its active site.
  • It is efficient in cleaving peptide bonds between hydrophobic and aromatic amino acids such as phenylalanine, tryptophan and tyrosine.
  • Pepsinogen is the proenzyme of pepsin.
  • Pepsin is most active in acidic environments between 37 °C and 42 °C.
  • Its primary site of synthesis and activity is in the stomach (pH 1.5 to 2).
  • Pepsin exhibits maximal activity at pH 2.0 and is inactive at pH 6.5.
  • Optimal pH: 1.0-4.0
  • Isoelectric Point: 1.0
  • There are four reported pepsin proteins: pepsin A, pepsin B (parapepsin I), pepsin C (gastricsin), and pepsin D (an unphosphorylated version of pepsin A).

Preparation: It is prepared by using stomach linings. The mucous membrane is separated from the stomach by the stripping process. Then minced stomach linings are digested with hydrochloric acid for autolysis at 37°C for 2 hours. The liquid sample contains pepsin and peptone. Then the sample is subjected to dialysis and concentrated through vacuum evaporation. The spongy pepsin is obtained.

Activator: Pepsinogen.

Inhibitor: Pepstatin is a low molecular weight compound and potent inhibitor specific for acid proteases with a Ki value of about 10−10 M for pepsin. Sucralfate also inhibits pepsin activity.

Specificity: Pepsin has broad specificity for peptides containing linkages with aromatic or carboxylic L-amino acids. It preferentially cleaves C-terminal to Phe and Leu and a lesser extent Glu linkages. The enzyme does not cleave at Val, Ala, or Gly.

Uses:

  • Digestion of antibodies.
  • Preparation of collagen for cosmeceutical purposes.
  • Assessment of digestibility of proteins in food chemistry.
  • Subculture of viable mammary epithelial cells.

Storage: Pepsins should be stored at very low temperatures (between −80°C and −20°C) to prevent autolysis.

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