Catalysis Letters

https://doi.org/10.1007/s10562-020-03316-7

PERSPECTIVE

Protease—A Versatile and Ecofriendly Biocatalyst with Multi‑Industrial

Applications: An Updated Review
Muhammad Naveed1 · Fareeha Nadeem2 · Tahir Mehmood3 · Muhammad Bilal4   · Zahid Anwar2 · Fazeeha Amjad5

Received: 31 May 2020 / Accepted: 5 July 2020

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Abstract 
Proteases are important industrial biocatalysts that constitute the largest group of enzymes acting as proteinases, peptidases,
and amidases with a broad range of industrial applications. In this review, particular attention has been given to compre-
hensively scrutinize the proteases. After the succinct introduction, classification of proteases as exopeptidases (amino and
carboxy proteases) and endopeptidases (serine, aspartic, cysteine, and metalloproteases), sources of alkaline, acidic and
neutral protease like animal, plant and microbial sources along with the multi-industrial applications have been dissertated.
Now a day’s, mostly proteases, which are present in the market, are produced from microbial sources because of the fast
production rate and the limited requirement of cultivation. In addition to this, a critique on the applications of proteases in
food, detergent, leather, pharmaceutical, cosmetics, silk degumming, silver recovery, chemical industry, and wastewater
treatment industries is also concisely addressed. Finally, protein engineering and immobilization strategies to improve the
catalytic properties of protease are thoroughly vetted. The quest for novel sources of protease enzyme has been encouraged
to fulfill their ever-increasing demands for industrial exploitation.

* Muhammad Naveed
naveed.quaidian@gmail.com
* Muhammad Bilal
bilaluaf@hotmail.com
1
Department of Biotechnology, Faculty of Life Sciences,
University of Central Punjab, Lahore, Pakistan
2
Department of Biochemistry & Biotechnology, University
of Gujrat, Punjab 50700, Pakistan
3
Institute of Biochemistry and Biotechnology, University
of Veterinary and Animal Sciences-UVAS, Lahore 54000,
Pakistan
4
School of Life Science and Food Engineering, Huaiyin
Institute of Technology, Huaian 223003, China
5
Department of Biotechnology, Quaid-I-Azam University,
Islamabad 15320, Pakistan

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Vol.:(0123456789)
 M. Naveed et al.

Graphic Abstract

Food industry Silver recovery

Detergent Exo-peptidase Silk degumming

industry
Digestion

Endo-peptidase Protease Waste treatment

Leather industry

Cosmecs
Chemical and
pharmaceucal industry

Improving catalyc
acvity

Protein
Immobilizaon
engineering

Keywords  Proteases · Protein engineering · Immobilization · Catalytic properties · Industrial applications

1 Introduction
Enzymes are the biological catalysts that exist in all living
organisms. In green chemistry, enzymes have emerged as
preferred tools and are being used increasingly in indus-
trial processes due to their capacity to perform reactions
with high specificity and efficiency. The enzymes have got
a special interest in many eco-friendly industrial and chemi-
cal processes [1–3]. Proteases are an important group of
industrial enzymes and reported for almost 60% of the whole
enzyme marketplace and also account for 40% of the entire
global enzyme sale [4–6]. Proteases consist of not only a

13
Protease—A Versatile and Ecofriendly Biocatalyst with Multi-Industrial Applications: An…
single enzyme but also contain a consortium of enzymes
that includes (proteinases, peptidases, and amidases). Pro-
teinases are involved in the hydrolysis of whole protein mol-
ecules releasing peptones and amino acids. Peptidases cause
peptones hydrolysis and liberate amino acids, whereas the
amidases catalyze the hydrolysis of amino acids and release
ammonia [7, 8]. Based on pH, proteases can be classified
as acidic (pH 2.0–6.0), neutral (pH 6.0–8.0), and alkaline
(pH 8.0–13.0) enzymes [8, 9]. Proteases have a molecular
weight of 18 to 90 kDa [4]. Different types of proteases have
different properties like catalytic activity, the specificity of
the substrate, pH, temperature, active site-specificity, and
stability profiles [10].
Proteases are fundamental industrial enzymes having
wide applications in chemical and biochemical processes
because they can break peptide bonds present in proteins.
The major advantages of using proteases are stereo-specific-
ity, specificity, biodegradability, the opportunity of produc-
ing natural products and activity under mild reaction condi-
tions [11]. Proteases are present among all living organisms
and vital for the process of cell differentiation, the period
of growth, food proteins digestion, cell division, protein
turnover, and cascades involved in blood clotting, apoptosis,
signal transduction, and numerous infection-causing organ-
isms’ life-cycle including the retroviruses replication [4, 9].
It is interesting to know that protease is the largest family of
enzymes that accounts for 2% of the human genome. Due to
structural and functional diversity, proteases have a range of
intracellular protein recycling as well as a cascade of nutri-
ent digestion and amplification of the immune system [12].
Proteases contribute 60% of the total enzyme market
because of their numerous applications in many industries
like in detergent industry, leather industry, food industry,
processing of meat, cheese making, paper and pulp, recov-
ery of silver from photographic films, and bioremediation
processes. These enzymes can also be used as a therapeutic
treatment for inflammation and harmful lesions [4, 6, 7, 13].
Enzymes used in different industries like detergent, textile,
and paper and pulp encompass the leading segment through
52% of market stock among the leading share of the enzymes
market place is detained by alkaline proteases [11].
The production of life products daily, for example, tex-
tile, paper, feed, food, pharmaceuticals, and chemicals use
raw materials and energy in bulk amount and release a large
number of waste materials that have a negative impact on
the environment and quality of life. The rising worldwide
population and improved economy in various countries raise
global consumption and pressure on the environment. There-
fore, it is an urgent need to sustain human desires without
depleting natural resources. Industries looking for some
alternative technologies around the world that can convey a
large number of products to fulfill every year’s demand with
less consumption of natural resources. Enzymatic processing
is one of the sustainable and promising alternatives to con-
ventional processing [14].
Currently, the worth of the universal sale of industrial-
ized enzymes has been estimated above 3 billion US$ [15].
The worldwide protease enzyme market is growing at a
compound annual growth rate (CAGR) of 6.1% by 2024.
The overall protease enzyme market income is expected
to be valued as 3 billion US Dollars by 2024 [16]. Now a
day, most of the enzymes used in industries are hydrolytic
and used for the degradation of different natural materials.
Because of the wide use in dairy and detergent industries,
proteases remain the dominant type of enzyme [17]. More-
over, they are eco-friendly and typically tend to decrease
the level of toxicity in the atmosphere after their usage in
industries [14]. This review provides an updated overview
of proteases and its classification, sources of protease, indus-
trial applications, protein engineering, and immobilization
strategies to improve the catalytic activity of the protease.
2 Classification of Proteases
The diversity and specificity of these indigenous enzymes
owe their broad characterization. Based on the active
site of proteases, they are classified as exopeptidases and
endopeptidases.
2.1 Exopeptidases
Exopeptidases catalyze the splitting of distinctive protein
peptide bonds adjacent to the carboxyl or amino terminals
embodied in the substrate. Depending upon their precision
regarding the site of action either C or N terminal, they are
additionally characterized as two major classes carboxy-
peptidases or aminopeptidases [15, 18].
2.1.1 Aminopeptidases
Aminopeptidases are biased towards the free N-terminal
of the polypeptide chain releasing a tripeptide, dipeptide,
and single amino acid residue. After the recognition, they
endeavor to remove the N-terminal methionine that might
be present in proteins, which are heterologous in their
expression but not present in many natural (mature) pro-
teins. Aminopeptidases occur in a wide range of microbial
strains including fungi and bacteria. Overall, aminopepti-
dases behave as intracellular enzymes, however, one report
highlighted that aminopeptidases originated from Aspergil-
lus oryzae are extracellular enzymes [19].
13

2.1.2 Carboxypeptidases
The carboxypeptidases exhibit their catalytic activities at a
free C-terminal of the polymer of amino acids with a release
of dipeptide and single amino acids. They are not primar-
ily treated as endopeptidases because they leave the amino
acid at a target protein. Rather, they can be labored for the
removal of tags added at the C-terminal of the target protein.
Amongst metallocarboxyprotease, type A carboxypeptidase
primarily removes the branch or aromatic side chain com-
prising enzyme. While type, B is brought into action for
basic amino acids [20].
2.2 Endopeptidases
Endopeptidases cleave the peptide bond at a distant site of
the substrate [15, 21]. Influenced by the presence of a par-
ticular functional group present on active site, endopepti-
dases are further cataloged as serine, aspartic, cysteine, and
metalloproteases, which are summarized as follows.
2.2.1 Serine Proteases
Serine proteases enjoy wide diversity in nature occurring
not only in the whole kingdom of cellular organisms but
also present in many viral genomes. From the known proteo-
lytic enzymes, one-third of them are serine proteases. These
are generally endopeptidases in which the cleavage of the
bond takes place in the central portion of the chain. Some of
them are exopeptidases, which detach amino acids from the
ending of the polypeptide sequence. Their name originates
from the Ser residue, which is nucleophilic and resides at
the active site of the enzyme. An acyl-enzyme intermediate
is formed by the attack of the serine residue at the carbonyl
end of the upcoming substrate peptide bond. Its nucleophilic
activity is due to the triad complex of ‘Asp’, ‘His’ and ‘Ser’
residues [22, 23].
2.2.2 Cysteine/Thiol Proteases
The active site of this class of proteases is comprised of
cysteine residues. They have natural origin both in prokar-
yotic and eukaryotic organisms. Optimum pH for their
proteolytic activity ranges from 6 to 8 and the optimum
temperature is 50–70 °C. Hydrogen cyanide portrays the
consequential role in the activation of this enzyme, owing to
the regeneration of the SH group. Proteases can be inhibited
by oxidizing agents and display sensitivity to the sulfhydryl
agents e.g. p-CMB, however, they remain unchanged by
metal-chelating agents [24].
13
M. Naveed et al.
2.2.3 Metalloproteases
Metalloproteases are mostly zinc-containing endopeptidases.
Innumerable metal ions, for instance, cobalt, calcium, and
zinc are involved in their reactivation, present in fungi. Most
of the fungal and bacterial metalloproteases possess zinc-
containing enzymes. Zinc is required for their enzymatic
activity and calcium is essential for the stability of protein
structure. These enzymes have optimum pH ranging from
5 to 9, and are sensitive to metal chelating agents such as
EDTA but are insensitive to cysteine inhibitors or sulfhydryl
[25].
2.2.4 Aspartic Proteases
A comparatively nominal class of endopeptidases is aspartic
proteases. Structurally, these proteases are bi-lobed having a
central catalytic site, which is composed of a pair of aspar-
tates. These proteases function on acidic pH and have shown
their occurrence in animals, plants, and microorganisms. A
range of microbes secretes them as their virulence secretions
that can also be mutualistic in breaking the proteins yield-
ing the nitrogen in urea and thus illustrating the duality in
their nature. Aspartic proteases are predominantly inclined
towards the hydrophobic amino acids adjacent to the dipep-
tides bond [26–28]. The former two endoproteases employ
residues residing in the active site having a nucleophilic
attribute for proteolysis. While the latter two operate those
residues that are significant in activating the molecules of
water to undergo the nucleophilic attacks [29]. Some diverse
proteases do not lie in the above-mentioned classification,
for example, ATP-dependent proteases, which require ATP
for their activation [19].
3 Sources of Proteases
In living organisms, proteases play a vital role so their pro-
duction takes place from different sources like plants, ani-
mals, and microbes [4, 30, 31]. The proteases produced from
different sources have been represented in Fig. 1.
3.1 Plants
Plants are used as a source of proteases that depends upon
numerous factors like the accessibility of land intended
for cultivation and the suitability of climate circumstances
for growth. Furthermore, the production of proteases is a
time taking process from plants. Some of the well-known
proteases such as papain, bromelain, and keratinases are
produced from plant sources [15]. Papain is produced
from Papaya fruit (Carica papaya). It has the properties of
milk clotting and protein-digesting with a wide pH range.
Protease—A Versatile and Ecofriendly Biocatalyst with Multi-Industrial Applications: An…

Fig. 1  Proteases produced from

different sources Sources

Animal Plant Microbial

Trypsin Bromelain Endoproteinase

Chymotrypsin Papain Subtilism

Enterokinase Ficin Proteinase K

Elastase WNV Protease

Thrombin Rhino virus 3C

Factor Xa TEV protease

Cathepsin D TVMV protease

Pepsin Endoproteinase

DAPase Thermolysin

Cathepsin C Collagenase

Carboxypeptidase Dipase

Carboxypeptidase Carboxypeptidase

Bromelain is a plant protease that is obtained from the leaf,
juice, stem, and peel of pineapples. It can efficiently control
the growth of tumor cells [32]. Keratinases are produced
from botanical groups of plants, which are used for dehair-
ing. Wool and hair digestion is significant for the formation
of crucial amino acid-like lysine used for the avoidance of
blockage of sewage systems [19]. In Pakistan, cysteine pro-
tease has been purified and characterized by maize leaves
[33].
with high-quality flavor [34]. Pepsin enzyme has an acidic
nature that is present in the stomach of approximately all
vertebrates. As early as 1913, the use of pepsin was only in
detergents, which are now replaced using a combination of
microbial proteases (metal) and serine, which become resist-
ant to degradation through alkaline conditions, detergents
and elevated temperatures [35].
3.3 Microbes

3.2 Animals Proteases that are available nowadays in the market are

Trypsin, chymotrypsin, pepsin, and rennin are the most
well-known proteases, which are originated from animals.
The digestive enzyme, trypsin is found in the intestine
and responsible for the food protein hydrolysis. Chymot-
rypsin is found in the excretory products of the pancreas
(animals). Purified chymotrypsin is a costly enzyme, and is
used in the analytical and diagnostic applications. Protease
(pepsin) such as rennet is produced as an inactive precur-
sor in the stomach of all mammals. By the action of pepsin
enzyme, it is converted into its activated form. In the dairy
industry, it is used widely for the formation of stable curd
obtained from microorganisms. This is due to several reasons
such as high rate of production, the limited requirement of
cultivation space, wide biochemical diversity, easy genetic
manipulation, and attractive characteristics that make them
appropriate for biotechnological applications [36–38]. How-
ever, due to the incapability of animal and plant proteases to
fulfill the recent demands of the world industrial sector, sci-
entists found an alternative solution in the form of microbial
sources [13]. Microbial-derived proteases account for about
40% of entire global enzymes sale [39]. Microorganisms
are responsible for the production of both intracellular and
extracellular proteases. Intracellular proteases are vital for

13

numerous metabolic end-products of cellular processes like
differentiation, turnover of protein, sporulation, processing of
hormones and proteins, removal of unnecessary protein, while
the extracellular proteases are important for the consump-
tion and hydrolysis of proteinaceous nutrients. Extracellular
proteases have a significant role and multiple applications in
different industries [40]. Proteases are isolated from different
microbes like fungus, bacteria, and yeast.
3.3.1 Fungal
Proteases isolated from fungus are the curiosity of researchers
due to their wide range of substrate specificity, stability under
the unfavorable conditions, high diversity, and mycelium sepa-
ration by the process of simple filtration. Fungal proteases are
used in the modification of food proteins [34]. Proteases pro-
duced from fungal sources have superior advantages over the
proteases produced from bacterial sources and they are gener-
ally recognized as genetically regard as safe (GRAS) strains
[41, 42]. Protease producing fungi are Aspergillus niger,
Aspergillus clavatus ES1, Aspergillus flavus, Aspergillus
fumigates, Aspergillus melleus, Aspergillus nidulans HA-10,
Aspergillus sojae and sydomi, Aspergillus terreus, Aspergil-
lus oryzae, Cephalosporium, Fusarium species, Rhizopus and
Penicillium italicum [31, 42–44]. Among these fungal strains,
Aspergillus is the famous group for protease production and
microbial strains such as Myxococcus, Neurospora, Penicil-
lium, Ophiostoma and Rhizopus are also frequent protease
producers [45]. Cheotomuim globusum is a novel indigenous
strain, which produces good alkaline protease [46].
3.3.2 Bacterial
Bacterial proteases, which are alkaline in nature, have eco-
nomic significance in different industries such as leather,
food, laundry, silk, and detergent industry due to their higher
catalytic activities and production capacities. Bacterial pro-
teases (alkaline) are distinguished via their higher activ-
ity at pH (alkaline) 8 to 12 with a range of temperatures
between 50 and 70 °C [34]. Some of the protease producing
bacteria are Bacillus subtilis [47, 48]. Bacillus alkaloophi-
lus, Bacillus amyloliquefaciens, Bacillus clausii, Bacillus
halodurans, Bacillus lentus and Bacillus licheniformis [40],
Bacillus pumilus [49], Bacillus circulans [50], and Bacillus
safensis [3].
4 Applications of Proteases in Different
Industrial Sectors
Today, enzymes are widely used in many biotechnological
industries [51]. The usage of enzymes produced opportuni-
ties for the development of green, modern, and sustainable
13
M. Naveed et al.
industrial chemistry due to exceptional specificity, mild
reaction conditions, being economic, simplicity, and energy-
saving process. The worldwide enzyme market increased
from 1.0 billion dollars in 1995 to 1.5 billion dollars in 2000
and then reached 2.2 billion dollars in 2006 [52]. About
75% of the enzyme industrial market was fulfilled by hydro-
lytic enzymes, for example, lipase, protease, and amylase
[53]. It has been reported that 75% of industrially produced
enzymes are used by major industries including detergents
(37%), textiles (12%), starch (11%), baking (8%), and animal
feed (6%) [54].
4.1 Food Industry
For more than forty years, proteases are commonly used for
protein hydrolysates production that can be used as addi-
tives for improving the nutritional quality of food and mixed
feed. It can be administered to patients who have digestive
disorders and food allergies. It can be obtained from differ-
ent substrates such as casein, whey, meat, and soy. Commer-
cially available proteases “SEB tender 70” which are widely
used in animal protein tenderization for the breakdown of
collagen in meat to make it more edible for eating [36]. In
food processing, major applications of proteases are cereal
mashing, brewing, clarification of beer haze, production of
protein hydrolysates, cheese making (coagulation step), and
baking changing the viscoelastic characteristics of dough
[9]. Proteases are used in the manufacturing of cheese within
the dairy industry, whereas the essential function of enzymes
is the hydrolysis of the peptide linkage to produce macro
peptides along with casein [8, 19]. In the baking industry,
it is used in protein degradation for the manufacturing of
baked wafers, cookies, and biscuits from flour [13]. In the
brewing industry, proteases are used for the extraction of
enough proteins from barley as well as malt and also used
for getting the preferred number of nutrients such as nitrogen
[21]. In the dairy industry, it plays a role in the modification
of flavor and lactose reduction [48, 55].
4.2 Detergent Industry
In detergent industries, a total of 30% of global enzyme pro-
duction is done by proteases and play as one of the leading
roles in many applications of the recent industrial biotech-
nology [13]. In the U.S., 25% of detergents (powder), 50%
of detergents (liquid) and approximately every powered
bleach contain enzymes that are helpful for the removal of
stains, which are difficult to remove with usual surfactants
alone. Proteases play an important role in hydrolyzing the
large protein molecule linked with hard strains. During the
hydrolysis, the peptide bonds are broken down that hold the
protein molecule together, releasing smaller polypeptides
and amino acids. They act like scissors and cut off strain
Protease—A Versatile and Ecofriendly Biocatalyst with Multi-Industrial Applications: An…
in pieces from the surface of cloth [23, 40]. The protease
produced from Aspergillus niger [56], Tritirachium album
[57], Bacillus cereus [58] , Aspergillus flavus [59], Bacillus
firmus MTCC 7728 [60], Penicillium chrysogenium [61] are
used in laundry detergent. Protease produced from novel
indigenous strain Cheotomium globusum is also used in the
detergent industry [46].
4.3 Leather Industry
Different steps are carried out in the leather industry such as
soaking, liming, hair removal, deliming, bating, degreasing,
and pickling to obtain processed leather. All steps are exe-
cuted by quite poisonous chemicals like lime, salt, solvents,
and sodium sulfide, leading to environmental pollution [5].
Protein like collagen is the major protein in the leather mak-
ing, which is present in skins and hides in contact with vari-
ous other globular proteins and fibrous proteins. Removal of
non-collagenous material is required in leather processing;
the extent to which these constituents remove decides the
final leather softness and durability. Environmental pollution
caused due to leather processing is controlled by the substi-
tution of enzymes like proteases in the place of chemicals.
The removal of unwanted protein by the eco-friendly and
inexpensive method is the essential requirement in leather
industries [6, 40]. Figure 2 represents the protease used in
different steps of the leather industry. The enzyme produced
from Bacillus subtilis [12], Aspergillus tamarii [62], Strep-
tomyces avermectinus NRRL B-8165 [63], Bacillus sp. JB
99 [64], and Bacillus halodurans JB 99 [65] are useful for
dehairing.
4.4 Pharmaceutics
Proteases are ever-present in all living organisms that take
part inside the lifecycle of numerous organisms responsible
for causing infections, which made them become a possi-
ble mark for increasing curative agents against severe dis-
eases like a tumor, acquired immunodeficiency syndrome
(AIDS), malaria and bacterial infection. Proteases produced
from microbial sources are used for the treatment of diseases
such as dermal ulcers, cancer, cystic fibrosis, cardiac prob-
lems, digestive disorders, and inflammation [32]. Proteases
are considered as the amplifying category of drugs. Several
Fig. 2  Use of protease in dif-
ferent steps of leather industry
[120]
protease drugs are approved by the FDA being successfully
trialed at preclinical and clinical phases, some of them are
shown in Fig. 3. Protease engineering and advanced delivery
systems have a strong impact on the therapeutic properties
of these efficacious protease drugs [66]. Such proteases have
increased specificity for physiological substrates for curative
purposes [67]. The proteolytic enzyme is used for cleaned
and disinfected contact lenses in a single step by heating at
the temperature of between 60 and 100 °C [68].
In cosmetics and pharmaceutics, keratinases are used in
the eradication of keratin in spots (acne) or itching depila-
tion, elimination of human being callus, degeneration of
keratin covered skin, vaccine preparation for dermatophy-
tosis, and enhance the ungula delivery of drug [37, 69].
Elastoterase enzyme produced by Bacillus subtilis 316M is
utilized for the healing of purulent wounds, deep abscesses,
furuncles, burns, and carbuncles [70]. Oral administration
of proteolytic enzymes produced from Aspergillus oryzae
has been utilized as a digestive relief for the treatment of
deficiency syndromes of various lytic enzymes [19]. In a
study, it was reported that protease (alkaline) from Bacillus
CK 11-4 strain having fibrinolytic activity has been used as
a thrombolytic agent [24]. In addition, studies have also been
demonstrated the use of alkaline proteases from Aspergillus
nigerare in the medicinal industry [71].
4.5 Cosmetic Industry
In the cosmetic industry, proteases catalyze the hydrolysis of
peptide bonds that join keratin, elastin, and collagen found
in the skin. Proteases like bromelain, papain, and many oth-
ers have been used in softening and peeling of the skin. The
function of these proteases is associated with cell renewal,
promoting the deceased cells removal from the epidermis
and re-establish the similar cells, and exercising kerati-
nolytic activity [72]. Keratinases obtained from Bacillus
licheniformis ZJUEL31410 have the potential to be used in
the cosmetics industry [73].
4.6 Silk Degumming
Unrefined threads of silk should be degummed to get rid of
a proteinaceous material such as sericin, which forms the
covering of silk fiber. It is performed usually in a solution
13

Fig. 3  FDA approved proco-
agulants, sepsis, thrombolysis,
digestion and neuromuscular
drugs
having soap, which is alkaline in nature. Fiber attacked itself
so it is harsh treatment. It has other disadvantages such as
time consumption, high energy utilization, and loss in the
luster of silk due to the high amount of water. On the other
hand, the use of ‘proteolytic’ enzymes is a superior tech-
nique because they play a role in removing the proteinaceous
substance such as sericin without attacking fiber [23]. Tests
are performed with higher enzyme concentrations and results
showed that no damage in fiber is practically observed and
threads of silk are firm than conventional methods [74]. The
process of enzymatic action improved the properties of silk
thread like elongation and potency [75]. In a study, it was
reported that the degumming of silk was optimized by pro-
tease produced from Bacillus subtilis C4 through statisti-
cal methods via an RSM and Plackett–Burman design [47].
Proteases isolated from Bacillus firmus MTCC 7728 [60] are
used in silk degumming.
4.7 Silver Recovery
The waste photographic films of X-rays that contain metal-
lic black silver is a very good source of silver revival. Silver
is present in the gelatin layer of X-ray film, which is about
1.5%–2% (by weight). The silver recovered through the burn-
ing of X-ray films generates a foul smell that is responsible
for causing environmental pollution. Proteolytic enzymes
produced from alkali hydroxides and different microorgan-
isms can be used by the stripping methods for the recovery
of silver [6]. A proteolytic enzyme such as protease is used
to breakdown the suspension layer consists of silver, which
13
M. Naveed et al.
contains the protein gelatin. Protease extract from Bacillus
subtilis ATCC 6633 [76] and Conidiobolus coronatus [77]
have been used in silver recovery. Protease isolated from
Cheotomium globusum is also used for the removal of the
gelatin layer from used X-ray films [46].
4.8 Chemical Industry
Proteases have a unique capability to produce esters and pep-
tides in the solvent medium. In organic solvents, the rate
of synthesis of the peptide is low because of the inactiva-
tion and denaturation of enzymes in such an environment.
The use of organic solvent tolerant microbes for protease
production can solve the problem of low product output
in organic media; and inactivation and denaturation of the
enzyme [78]. Highly stable alkaline proteases have a huge
requirement in organic solvents for their burgeoning applica-
tions in organic synthesis. Proteases from Aspergillus flavus
[59], and Pseudomonas aeruginosa [78] are used for the
synthesis of the peptide in a non-aqueous medium. Highly
stable serine alkaline proteases SAPB and KERAB isolated
from Bacillus pumilus CBS and Streptomyces. AB1 strains
have a great prospect for utilization within the synthesis
of the peptide in below level ­H2O systems [79]. Alkaline
protease like alcalase is used for the synthesis of Bz-Arg-
Gly-NH2 (N-benzoylargininylglycinamide) [a dipeptide
originator of (Arginine-Glycine-Aspartic-Serine)] in the
acetonitrile/sodium carbonate/sodium bicarbonate buffer
system [80]. It has been reported that the production of
Protease—A Versatile and Ecofriendly Biocatalyst with Multi-Industrial Applications: An…
2H-1-benzopyran-2-one derivatives by protease (alkaline)
from Bacillus licheniformis [81].
4.9 Waste Management
Protease causes solubilization of waste (proteinaceous) and
also helps in decreasing the biological demand of oxygen
by marine organisms. Currently, a new period opened the
usage of proteases for waste management from different
household activities and food-processing industries [5]. In
a commercial poultry processing, a waste of feathers, which
is generated in huge quantity as a by-product contains a large
amount of keratin protein and amino acids that could be
effectively utilized as an animal feed and foodstuff. On the
other hand, its poor digestibility restricted its application.
Using the process of thermal degradation, feather digestibil-
ity can be improved, which requires a high cost of thermo-
energy and generate some toxic by-products, restricting its
practical use [73]. Microbial proteases such as (keratinases)
might have a significant role in the degradation of wastes
containing keratin from various industries like domestic
poultry and leather by developing nonpolluting processes
[82]. After the process of feather degradation, feather hydro-
lysates can be used as an additive for films, glues, fertilizers,
and feedstuffs or used for the formation of the amino acid
such as proline, cysteine along with serine [36]. Proteases
from Bacillus licheniformis ZJUEL31410 [73], Bacillus
pseudofirmus FA30-01 [83], Streptomyces [82], Bacillus
RV. B2.90 [84], Bacillus licheniformis RG1 [85], Bacillus
amyloliquefaciens [86], Pseudomomanas sp., MS21 [87],
Aspergillus oryzae NRRL-447 [88], and Bacillus JB 99 [64]
are being used for feather degradation. Proteases produced
from Chetomium globusum also have a keratinase activity
useful for feathers degradation [46]. The applications of pro-
teases isolated from different microbial sources in different
industries are represented in Table 1.
5 Improving Catalytic Properties
of Proteases
5.1 Protein Engineering Approaches—Improving
Catalytic Activity and Thermostability
High catalytic performance and improved thermal stability
are highly desired characteristics for industrial-scale exploi-
tation of proteases. In the industrial bio-process, the use of
native biocatalysts is often hindered due to their inherent
constraints under aggressive industrial environments. In par-
ticular, the temperature is considered as a critical factor that
determines the catalytic efficiency of an enzyme [89, 90].
Thermo-tolerant enzymes can retain their catalytic activi-
ties in unfavorable conditions [91], and thus promote the
development of cost-efficient strategies for large-scale appli-
cations of enzymes [92]. In this avenue, protein engineering
offers a promising solution to overcome the above-mentioned
drawbacks and boost up the stability attributes of proteases
to realize target achievements [93]. Table 2 demonstrated the
list of improved catalytic properties of proteases by protein
engineering approaches. Three major types of protein engi-
neering strategies explicitly, rational design, directed evo-
lution, and de novo design are commonly used to improve
the catalytic features of the proteases. Among these, the uti-
lization of rational design remains the superior choice for
modifying enzyme properties due to smaller variant librar-
ies and hastened processing time. A vast number of reports
have demonstrated the efficacy of this engineering strategy
for improving enzymatic catalytic performance and thermal
steadiness via in-depth structural analysis [94, 95]. Many
studies have proven the rational design efficiency to generate
effective and cost-efficient enzyme mutants for large-scale
applications [96, 97]. For example, the rational modification
of serine alkaline protease of Bacillus pumilus led to aug-
mented biocatalytic activity and thermostability by creating
a single N99Y mutant [98]. Likewise, increased catalytic
activity and substrate specificity of Bacillus pumilus serine
alkaline protease has been observed by the replacement of an
uncharged amino acid (L31 and T33). The resultant modified
variants were suggested as promising biocatalysts for use
in the leather industry [99]. Recently, Ashraf et al. [100]
reported the protein engineering of a serine peptidase, iso-
lated from Pseudomonas aeruginosa rational design of non-
catalytic residues to improve its catalytic potentiality and
thermal stability features. Amongst the eight variants, two
mutants namely V336I and A29G produced desired impacts.
In contrast to the wild-type, the newly identified mutants
A29G and V336I showed a 1.4-fold increase in catalytic
activity. The Tm of both mutants was increased by 5 °C, they
presented a pronounced improvement in residual enzyme
activities at higher temperatures. Identical findings were also
achieved using a similar approach to modify alkaline pro-
teases obtained from Bacillus pumilus [101]. Rational engi-
neering of transformed amino acids at the sites with maxi-
mum solvent exposure enhanced the resistance of trypsin
to Aflatoxin-detoxifizyme and β-mannanase MAN47 [102,
103]. The catalytic steadiness of the trypsin and pepsin in
resisting β-mannanase MAN47 was substantially improved
following the introduction of N-glycosylation sequences by
rational designing of molecules orientation [104].
Directed evolution is an emerging biotechnological tool
not only in enzymes engineering for industrial biocataly-
sis but also to elucidate the association between sequence,
structures, and functions of proteins [105, 106]. A library of
mutants is constructed in directed evolution and the variants
exhibiting the desired traits are recognized by using a suit-
able screening and selection method. The cycle is continued
13
 M. Naveed et al.

Table 1  Applications of proteases isolated from microbial strains

Microbial strains Applications References

Aspergillus niger Food, pharmaceutical, cosmetic [121]

Bacillus subtilis Leather industry [120, 122]
Aspergillus niger Detergents and leather industry [123]
Aspergillus niger Detergent [124]
Aspergillus niger Detergent [125]
Aspergillus tamarii Leather processing [62]
Bacillus subtilis Industrial purposes [126]
Aspergillus niger Peptide production [127]
Bacillus sp. No. AH-101 Degradation of human hairs [128]
Conidiobolus coronatus Silver recovery [77]
Aspergillus Oryzae NRRL-447 Feather degradation [88]
Aspergillus flavus MTCC 277 Leather treatment, detergents [120,129]
Bacillus amyloliquefaciens Feather degradation [86]
Aspergillus sojae ATCC20235 Peptides synthesis [130]
Bacillus sp. SSR1 Laundry detergent [131]
Pseudomonas fluorescens Detergent and textile industries [132]
Aspergillus oryzae U1521 Animal feed and food processing [133]
Aspergillus flavus Leather processing [134]
Bacillus cereus Laundry detergent [58]
Bacillus subtilis PE-11 Detergent industry [135]
Bacillus horikoshii Industrial, medical applications [136]
Bacillus subtilis Deproteinize crustacean wastes [137]
Bacillus subtilis Industrial applications [138]
Bacillus sp. KG5 Food industry [139]
Engyodontium album BTMFS10 Detergent industry [140]
Bacillus licheniformis RP1 Chitin extraction, chicken feather degradation, dehairing agent [141]
Stenotrophomonasmaltophilia (MTCC 7528) Detergent and environmental bioremediation in cold regions [142]
Mutant Bacillus licheniformis Bl8 Collagen replacement therapy, detergent stability, waste treatment [143]
Streptomyces Leather, keratin waste treatment, animal feeding industry, cosmetic industry [82]
Cheotomium globusum Detergent industry, Feathers degradation, removal of gelatin layer from X-ray film [46]
Penicillium chrysogenium Textile industry [61]
Bacillus safensis Detergent industry [3]

until the achievement of a variant with target attributes. It
holds notable benefits over the use of the rational design in a
way that it does not necessitate any meticulous prior knowl-
edge of the protein regarding mutation regions. Though sim-
ple, significant efforts are usually required in the directed
evolution approach to creating a library accompanied by
high-throughput screening/selection methods. It should be
emphasized that evolutionary and rational techniques are
not irreconcilable and integrated approaches have recently
been frequently employed [107]. This approach is called as
targeted or semi-rational design mutagenesis that entails the
utilization of randomized or site-saturation mutagenesis over
a selected portion of the protein molecules rather than the
whole enzyme [108, 109]. In a recent report, [110] applied
a directed evolution approach to enhance the resistance of
metalloprotease PT121 in organic solvents. The modified
mutants (H224 F, H224Y, and T46Y) presented exceptional
solvent tolerant ability in the presence of acetone and ace-
tonitrile. In comparison to the wild-type, the half-lives of
modified enzyme variants were improved by 1.2–3.5-folds
after directed evolution. Remarkably, mutual variants T46Y/
H224 F and T46Y/H224Y possessed tremendous catalytic
stability and caseinolytic performance.
6 Immobilization of Proteases—A Key
to Optimal Catalytic Performance
In batch reactions, the extensive utilization of the free state
of the enzyme is challenged by stability, recovery, and
recyclability. Among the many strategies, enzyme immo-
bilization has been proposed as a promising approach to

13
Protease—A Versatile and Ecofriendly Biocatalyst with Multi-Industrial Applications: An…
Table 2  List of improved catalytic properties of proteases by protein engineering approaches
Type of protease Strategies employed
Metalloprotease Directed evolution
Salinovibrio proteolyticus protease Site-directed mutagenesis
Serine protease Site-directed mutagenesis
Neutral protease Site-directed mutagenesis
Alkaline serine proteases Directed evolution
Keratinase Site-directed mutagenesis
Alkaline protease Single-site substitutions
Keratinase Domain exchange with KerSMF More than two-fold increase in catalytic activity towards
KerSMD
Tobacco etch virus protease Directed evolution, error-prone Mutant, TEVSH, in which three amino acid replacements
PCR and gene shuffling
convenient and cost-effective employment of enzymes in
industrial biocatalysis [111, 112]. Immobilization, attach-
ment of biomolecule or enzyme molecule onto a solid
support, does not only integrate enzymes enabling reus-
ability but also modify their catalytic and stability to facili-
tate industrial exploitability. Process control, repeatabil-
ity, reduction in costs, and easy enzyme separation after
enzyme-catalyzed transformation are the notable benefits
of enzyme immobilization [113]. Although there are many
techniques, but generally four methods including adsorption,
co-polymerization, entrapment, and covalent attachment
are commonly employed for immobilizing enzymes. The
choice of a suitable method for enzyme coupling depends
on the highest preservation of enzyme activity, durability,
and stability. Physical adsorption and covalent attachment
are the frequently adopted methods for enzyme immobi-
lization among others. The covalent attachment furnishes
Improved properties References
The modified enzyme presented exceptional solvent tolerant [110]
ability in acetone and acetonitrile. Half-lives of modified
enzyme variants were improved by 1.2–3.5-folds
Combined variants T46Y/H224 F and T46Y/H224Y pos-
sessed tremendous catalytic stability and caseinolytic
performance
The catalytic efficiency of Y23V and N248G mutants [144]
increased about 2.6 and 1.8 folds in methanol and DMF
About 5.0 and 3.8 folds in n-propanol and isopropanol
In contrast to the wild-type, the newly identified variants [100]
A29G and V336I showed 1.4-fold increase in catalytic
activity. The Tm of both mutants was increased by 5 °C,
they presented a pronounced improvement in residual
enzyme activities at higher temperatures
Improved thermostabilities of the Lys11Arg and Lys211Arg [145]
mutants compared to the wild-type
Increase in catalytic efficiency and thermostability than the [146]
wild-type enzyme
Increased proteolytic activity at
15 °C towards synthetic peptide substrates and casein
Two variants (Y94F and Y215F) exhibited higher specific [147]
activity and keratinolytic activity
The A218S and A218G possessed more than 30% improve-
ment of relative activity at 70 °C
As compared with the wild-type, the double-site variant [148]
(W106K/V149I) possessed an enhancement in hydrolytic
activity by 2.3-fold t at 15 °C toward casein
Improved thermal stability with the half-life at 60 and 70 °C
by 2.7- and 5.0-fold, respectively than that to the wild-type
[147]
casein
Substitution of the C-terminal domain increased keratino-
lytic activity
[149]
result in a fivefold improvement in the yield of purified
protease with retained activity
the strongest connection, therefore establishing the most
stabilized enzyme-support bio-conjugates without leach-
ing enzyme into the mixture. Nevertheless, this approach
sometimes results in drastic alterations in the catalytic and
conformational properties of the coupled enzyme presum-
ably due to the aggressive conditions. Adsorption may be
a suitable technique for immobilizing enzymes since it is
simple requiring little time and effort. The catalytic supports
could also be recycled after the enzyme desorption and thus
the final cost of the process can be substantially reduced
[114, 115]. Table 3 represents the types of different support-
ing materials and immobilization techniques for improving
the catalytic properties of proteases.
Recently, Poonsin et  al. [116] immobilized purified
trypsin onto octyl sepharose CL-4B, 50-4,40-dimeth-
yltryptamine-thymidine-succinyl controlled pore glass
and glutaraldehyde functionalized silica to improve their
13


Table 3  A recent summary of improved catalytic properties of proteases by immobilization on different support materials
Type of protease Support material Immobilization technique Improved catalytic properties References

13
Bromelain Cellulose triacetate nanofiber membranes Cross-linking Immobilized biocatalyst showed a high recycling ability [117]
maintaining about 60% of its original activity after four
consecutive cycles. It also showed an elevated activity
recovery of about 675%
Trypsin Octyl sepharose CL-4B, glutaraldehyde activated silica Covalent immobilization Immobilized trypsin displayed high tolerance in organic [117]
and 50-4,40-dimethyltryptamine-thymidine-succinyl solvents and thermal inactivation
controlled pore glass Less vulnerability to inhibition by the trypsin inhibitor
than that to the non-immobilized form of the enzyme
The as-engineered biocatalytic system showed a high
recycling ability maintaining about 60% of its original
activity after four consecutive cycles
Protease Surface modified κ-carrageenan gel beads Covalent immobilization Enhanced thermal stability at elevated temperatures along [118]
with an increment in the half-life time (from 24.06 to
79.95 min)
Immobilized protease retained more than 80% of its
residual activity after storage for 8 weeks at 4 °C
Promising silver removal activity from X-ray films for six
consecutive cycles
Trypsin Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) matrix - Improved and sustained enzyme activity of immobilized [150]
trypsin
Rapid enzyme biosensor for the detection of azocasein
Trypsin Polyvinyl alcohol-coated magnetic nanoparticles activated Covalent immobilization Immobilized enzyme was more stable than the free [151]
with glutaraldehyde enzyme at elevated temperature
It preserved 50% of its original activity after 12-days at
4 °C
Immobilized trypsin showed effective proteolytic activity
in a shorter time (15 min) than free trypsin (24 h)
Endopeptidase Chitosan beads Covalent immobilization The chitosan-immobilized enzyme exhibited improved [119]
thermal stability and presented an identical catalytic
performance at 20 °C or 50 °C. The newly developed
system in a fluidized bed reactor efficiently catalyzed the
reduction of gluten content in the commercial beer from
barley malt
Alkaline protease Chitin, chitosan, alumina, ceramic, PVC, silica gel and Adsorption Enhanced pH and temperature stability [152]
polystyrene The immobilized enzyme had greater stability in various
detergents and metal ions
Alkaline protease Chitosan Covalent binding Improved pH and temperature stability features [152]
Greater stability in various detergents and metal ions
Alkaline protease Cation exchange resin (Amberlite IR-120) and anion Ionic bonding Superior pH and temperature stability [152]
exchange resins (Amberlite IRA-35, DEAE Sephadex Immobilized enzymes had greater stability in various
A50) detergents and metal ions
M. Naveed et al.
 Protease—A Versatile and Ecofriendly Biocatalyst with Multi-Industrial Applications: An…

catalytic features. As compared to the free form of the

References enzyme, the efficiently immobilized biocatalyst displayed
high tolerance in organic solvents and thermal inactivation.
It was also less vulnerable to inhibition by the trypsin inhibi-
[152]

[153]
[154]
tor than that to the non-immobilized form of the enzyme.
The as-engineered biocatalytic system showed a high recy-
cling ability maintaining about 60% of its original activ-
The immobilized enzyme had greater stability in various

ity after four consecutive cycles. Immobilized bromelain

onto glutaraldehyde cross-linked nanofiber membranes
showed an elevated activity recovery of about 675% [117].
In another study, protease enzyme was successfully immo-
A marked increase in activity and stability

bilized onto surface modified κ-carrageenan gel beads and

Increased pH and temperature stability

exhibited enhanced thermal stability at elevated tempera-

tures along with an increment in the half-life time (from
Immobilization technique Improved catalytic properties

24.06 to 79.95 min). The κ-carrageenan gel beads encapsu-

detergents and metal ions
Increase thermal stability

lated protease retained more than 80% of its residual activity

after storage for 8 weeks at 4 °C. Additionally, it showed
promising silver removal activity from X-ray films for 6
consecutive cycles [118]. Benucci et al. [119] developed a
sustainable and innovative biocatalytic system by the immo-
bilization of Aspergillus niger endopeptidase chitosan beads
and employed to produce gluten-reduced beer for the first
time. The chitosan-immobilized enzyme exhibited improved
thermal stability and presented an identical catalytic per-
formance at 20 °C or 50 °C. The newly developed system
α-Amylase and protease Non-woven electrospun poly(styrene-co-maleic anhydride) Covalent binding

in a fluidized bed reactor efficiently catalyzed the reduction

of gluten content in the commercial beer from barley malt.
Entrapment

7 Conclusions and Perspectives
–

In this review, the decisive role of proteases in a vast array

of industrial fields is summarized. Proteases are a signifi-
cant group of multifunctional enzymes. The unique char-
acteristics of proteases like fast action, working under mild
operating conditions, high specificity, biodegradability, and
reduced generation of waste makes it a particularly interest-
ing enzyme for diverse biotechnological applications. The
worldwide protease market is growing 3 billion US Dollars
at a compound annual growth rate (CAGR) of 6.1% by 2024.
Proteases have far-reaching applications in food, detergents,
chemical, pharmaceutical, leather, and wastewater treatment
Support material

Oligopeptidases
Polyacrylamide

industries. Due to the depletion of natural resources and the

nanofiber

increasing population around the world, proteases provide

enormous potential at the industrial level to fulfill the chal-
lenges that will be faced in the coming years. Also, there
is a dire need to identify new sources for efficient, stable,
and reusable catalysts for diverse industrial applications
Table 3  (continued)

that require enzymes with specific properties. Engineering

Alkaline protease
Type of protease

Oligopeptidases

of proteases for novel function and stability seems to be an

addressed area in the future. Industries based on proteases
look onward for getting novel engineered fusion proteases
with numerous activities united in one. Proteases market that

13

is rapidly growing always demands fast-acting and active
proteases at low prices.
Compliance with Ethical Standards  Biol Rev 62(3):597–635
Conflict of interest  The authors declare that they have no competing
interests.
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