The document discusses the use of proteases, especially subtilisin proteases, for industrial applications. It notes that over 60% of the global enzyme market is for proteases. Subtilisin proteases from Bacillus species are commonly used because they are stable and have broad substrate specificity. They are used in food processing, leather production, detergents, waste management and more. The mechanism of subtilisin proteases involves cleavage of peptide bonds via a catalytic triad of amino acids and stabilization of intermediates in the active site.

1. Engineering Proteases for
Industrial Applications

2. There are more than 3000 different enzymes known but
only 5% are commercially used . Over 500 commercial
products are made using enzymes
In regard to the total enzyme market, its global figures
depend on the consulted source. In one case, the Global
enzyme market reached $12.1 billion in 2021 and is
predicted to grasp $16.9 billion in 2027.
The indian enzyme market reached a valuation of USD
0.25 billion in 2023 and is projected to grow at a CAGR
of 5.95% through 2029.
Over 60% of the worldwide enzyme market is devoted
to proteases.

3. Definition: Proteases are enzymes that hydrolyze peptide
bonds and are an essential constituent of all
forms of life.
Classification: Proteases can be classified into different
groups based on their optimal pH activity:
acidic, neutral, or alkaline.
The second criterion for protease classification is by the
catalytic residues responsible for peptide bond cleavage,
for example,
metallo-, aspartic-, cysteine-, or sulfydryl-, or serine-type

4. Role of Proteases
Physiological processes
maturation of proenzymes and hormones,
protein hydrolysis in the extracellular environment
blood clotting,
processing and transport of secretory proteins across
membranes, and as pathogenic factors
In Food Industry:
Proteases are used in the preparation of protein
hydrolyzates of high nutritional value and in the production
of meat extract powder.
Furthermore, proteases are employed in fish, seafood, and
animal protein processing in order to enhance oil recovery,
improve digestibility, reduce allergenicity, and improve
flavor.

5. In dairy products, proteases are used to accelerate
cheese ripening and to reduce the allergenic
properties of milk proteins, for example, in the
production of infant milk formulas from cow milk.
Flavor in dairy, meat, and fish products is enhanced
using proteolytic processes or in combination with
fermentation
leather-processing industry:
Alkaline proteases with elastolytic and keratinolytic
activities are used.
They are used in soaking, dehairing, and bating stages of
the preparation of skins and hides

6. In wool industry:
to hydrolyze the overlapping scales that are covering
the wool fibers contributing to a silky luster
In waste management:
Proteases find their use in products from various food-
processing industries and household activities by
solubilizing proteinaceous residues and thus help
lowering the biological oxygen demand of aquatic
systems

7. In biopharmaceutical products such as contact lens
cleaning agents, proteases are used as active agents, as
well as in wound debridement, removal of dead tissue,
and in the treatment of osteoarthritis with a benefit
comparable to that of anti-inflammatory drugs

8. Serine Proteases and Subtilisins:
Serine proteases refer to proteolytic enzymes
containing an essential serine, which initiates the
nucleophilic attack on the peptide bond with an
electronic environment provided by a neighboring
histidine and aspartic acid. Serine proteases are
divided into subclasses depending on substrate
specificity and structural homology to well-established
proteases
The main subclasses are
chymotrypsin-like, subtilisin-like, wheat serine
carboxypeptidase-II-like, prolyl-oligopeptidase-like,
myxobacter α-lytic, and staphylococcal proteases

9. Subtilisin-like proteases can be clearly distinguished from
other serine proteases by their amino acid sequence and
three-dimensional structure, which are common among
bacterial subtilisins.
Subtilisins have a molecular weight ranging from 15 to 90
kDa, whereas subtilisins employed in detergent industry
have a size of appro. 27 kDa.
The main properties of subtilisins, their inherent high
stability and broad substrate specificity are key to their
success.
Subtilisins are naturally produced as extracellular
enzymes by various Bacillus spp. such as Bacillus
amyloliquefaciens, Bacillus licheniformis, and Bacillus
subtilis , which simplifies their separation from biomass,
facilitating other downstream processing steps.

10. Furthermore, the wide availability of X-ray and NMR
structures together with extensive experimental data
enabled numerous structure– function studies in
subtilisins, therefore, becoming a model system for
protein engineering.
All subtilisin proteases have the same reaction
mechanism and catalytic triad consisting of an aspartic
acid, a histidine, and a serine. Once the substrate is
bound, a hydroxyl group of the nucleophilic serine
attacks the sessile peptide carbonyl group.
A covalent bond forms between the serine and the
carbonyl group of the substrate to yield the complex
known as tetrahedral intermediate

11. The tetrahedral intermediate is stabilized by two amide
hydrogens coordinating the anionic oxygen. This region
of the active site is known as oxyanion hole since it is
occupied by the intermediate’s oxyanion group.
The tetrahedral intermediate decomposes back to an
acyl-enzyme intermediate by breaking the peptide bond
and releasing the N-terminal portion of the substrate.
The remaining substrate is temporarily covalently linked
to the enzyme.
The ester bond is broken by a nucleophilic attack of a
water molecule present in the active site. Histidine is
protonated while forming a covalent bond to the
carbonyl carbon.

12. The result is a second tetrahedral intermediate
stabilized by the amide groups in the oxyanion hole. In
the last step, the second tetrahedral intermediate
decomposes by breaking the bond with the hydroxyl
group of the serine. The carboxylic acid product is
released and the enzyme is restored to its initial state .

14. Scheme of the active site of a typical subtilisin
protease. The catalytic triad is shown in red and the
oxyanion hole in yellow. The nonspecific peptide-
binding pocket is shown in green, whereas the
specificity pocket is in blue. The enzyme and the
relevant residues are represented in white; the
substrate peptide is represented in green.