The catalytic site of proteases is flanked on one or both sides by specificity subsites, each able to accommodate the side chain of a single amino acid residue from the substrate. These sites are numbered from the catalytic site S1 through Sn toward the N terminus of the structure and Sl′ through Sn′ toward the C terminus. The residues which they accommodate from the substrate are numbered Pl through Pn and P1′ through Pn′, respectively (Fig. (Fig.2).2)
Catalysis Catalysis is achieved by one of two mechanisms: Aspartic, glutamic and metallo proteases activate a water molecule which performs a nucleophilic attack on the peptide bond to hydrolyse it. Serine, threonine and cysteine proteases use a nucleophilic residue in a (usually in a catalytic triad). That residue performs a nucleophilc attack to covalently link the protease to the substrate protein, releasing the first half of the product. This covalent acyl-enzyme intermediate is then hydrolysed by activated water to complete catalysis by releasing the second half of the product and regenerating the free enzyme.
In addition, proteases have largely re‐ placed bisulfite, which was previously used to control consistency through reduction of gluten protein disulfide bonds, while proteolysis breaks down peptide bonds. In both cases, the final effect is a similar weakening of the gluten network
Treatment of soy proteins with alcalase at pH 8 results in soluble hydrolysates with high solubility, good protein yield, and low bitterness.
Protease Enzyme Application in Food Processing
BY : MOHAN NAIK . G
M.Tech (Food Processing
College of FPT & BE,
Proteases are abundantly and widely distributed in biological
world including plant, animal and microbes .
Protease is group of enzyme that performs proteolysis known
as hydrolysis of the peptide bonds that link amino acids
together in the polypeptide chain forming the protein .
Proteases constitute more than 70% of industrial enzyme alone
and microbial sources (bacterial and fungal) are leading
supplier of these enzyme.
These enzymes possess catalytic activity in broad range of
temperature and pH (temperature ranges 5 -100 °C and in pH
range 0-14 ).
Protease widely used as detergent, in food, pharmaceutical and
leather tanning industries.
The vast variety of proteases, with their specificity of their
action and application has attracted worldwide attention to
exploit their physiological as well as biotechnological
The specificity of protease binding is not defined solely by the
nature of the catalytic centre. On either side of the catalytic
centre there is a series of binding sites (S) that favour particular
amino acids (P).
By source organism:
1. Animal : chymosin, trypsin, pepsin
2. Plant : bromelain, papain, ficin
3. Bacterial: subtilisin(Bacillus subtilis), bacillopeptidases
4. Fungal : Aspergillopepsin
By proteolytic mechanism:
a. Serine proteases
b. Threonine proteases
c. Cysteine proteases
d. Aspartic proteases
e. Metallo proteases
f. Glutamic acid proteases
By active pH range:
1. Acid proteases
2. Neutral proteases
3. Alkaline proteases
4. High-alkaline proteases
Major fungi producing alkaline proteases
Aspergillus candidus , A. flavus , A. fumigatus , A. melleus ,
A. niger , A. oryzae , A. soja, Cephalosporium sp.
Major bacteria producing alkaline proteases
Streptomyces microflavus , Streptomyces moderatus ,
Streptomyces rectus, Pseudomonas aeruginosa , Pseudomonas
maltophilia , Pseudomonas sp. SJ320
The serine proteases contribute major industrial and
therapeutic protease where serine serves as the nucleophilic
In this class of enzyme chymotrypsin/trypsin and subtilisin
are commercially available serine protease .
With their tremendous scope in industry and medicine several
recombinant serine protease have been produced and are in
Threonine protease is another industrially significant protease
where threonine (Thr) residue lies on catalytic site.
These proteases have much significance in physiology and
proteasome and acyl transferases are classical example
Cysteine proteases also known as thiol protease possess great
importance in industrial applications.
These proteases perform catalysis associated with nucleophilic
thiol in a catalytic triad or dyad.
These proteases are primarily present in all the fruits including
papaya, pineapple, fig and kiwi fruit.
Cysteine proteases had shown their potential in poultry
industry and are key competent of meat tenderizers.
Aspartic acid proteases are quite different from other proteases
as their contribution in maintaining physiological functions.
The classical examples are pepsins, cathepsins, and renins key
enzyme which maintain physiology .
Glutamic acid proteases (EC 3.4.19)
Most important glutamic acid proteases which are widely
present in fungal species are key enzyme for food processing
and modern therapeutics such as antitumor and anticancer .
A novel protease group called metalloprotease which
enormously used in drug development involve metal ion for
Due to wide range of substrate affinity and diverse sources of
proteases the applications of proteases are unique and widely
applicable in different industries .
Used on a large commercial scale in the production of
bread, baked goods, crackers and waffles .
To reduce mixing time,
To decrease dough consistency,
To assure dough uniformity,
To regulate gluten strength in bread,
To control bread texture and to improve flavour .
Endo and exo-proteinases from Aspergillus oryzae have
been used to modify wheat gluten by limited proteolysis
The major application of proteases in the dairy industry is
in the manufacture of cheese.
The milk-coagulating enzymes fall into three main
(i) animal rennets,
(ii) microbial milk coagulants, and
(iii) genetically engineered chymosin.
In cheese making, the primary function of proteases is to
hydrolyze the specific peptide bond to generate para-κ-casein
and macro peptides.
Chymosin is preferred due to its high specificity for
casein, which is responsible for its excellent
performance in cheese making.
The microbial enzymes exhibited two major drawbacks,
(i) the presence of high levels of nonspecific and heat-
stable proteases, which led to the development of
bitterness in cheese after storage; and
(ii) a poor yield.
Proteases have been used from ancient times to prepare
soy sauce and other soy products.
The alkaline and neutral proteases of fungal origin play
an important role in the processing of soy sauce.
Proteolytic modification of soy proteins helps to
improve their functional properties.
The hydrolysate is used in protein-fortified soft drinks
and in the formulation of dietetic feeds.
The medicinal application of protease for diagnostic and
therapeutic is widely accepted and several enzymes are in use
since many years.
clot dissolving agents
The most common types of enzymatic tenderizers are
Bromelain, which is made from pineapples.
Papain, which is made from papayas
Actinidin, which is made from kiwis
Ficin, which is made from figs.
Proteases of Aspergillus find application as digestive aids in
gastro-intestinal disorders such as dyspepsia.
Papain and bromelain have been used to improve the
nutritional value of feeds. Papain has been used to
manufacture yeast extract and SCP.
It has also been used in the extraction of flavour and colour
compounds from plants.
Other applications, which exploit the hydrolytic property of
proteases, include soy protein hydrolysis, soy sauce
production, gelatine hydrolysis, casein and whey protein
hydrolysis, meat protein recovery, fish protein hydrolysis, and
Proteases from Bacillus subtilis have been used to deproteinize
crustacean waste to produce chitin.
Neutrase is a bacterial protease which is used in alcohol
production for improving yeast growth In brewing.
Pharmacology & drug manufacture
Laundry & dishwashing detergents
Hard surface cleaning formulations
Contact lens cleaning formulations
Fermentation (fuel EtOH, etc.)
Chondroitin & heparin production
J. Srilakshmi, ;J. Madhavi, Lavanya S and Ammani (2014)
Commercial Potential of Fungal Protease: Past, Present and
Future Prospects , Journal of Pharmaceutical, Chemical and
Biological Sciences ,2(4)p.218-234.
Sumantha (2006).Food-Grade Proteases, journal of Food
Technology & Biotechnology. 44 (2), p.211–220
Riddhi Sawant and Saraswathy Nagendran (2014),protease:
an enzyme with multiple industrial application,