biomolecules

Review
Serratiopeptidase: An integrated View of Multifaceted
Therapeutic Enzyme
Sreelakshmi R. Nair and Subathra Devi. C *

Department of Biotechnology, School of Bio Sciences and Technology, Vellore Institute of Technology,
Vellore 632014, India
* Correspondence: [email protected]

Abstract: Microbial products have been used for the treatment of different diseases for many centuries.
The serratiopeptidase enzyme provides a new hope for COVID-19-infected patients. Nowadays, anti-
inflammatory drugs are easy to obtain at minimal expenditure from microbial sources. Serratia sp. is
identified as one of the most efficient bacteria produced from serratiopeptidase. Screening for new and
efficient bacterial strains from different sources has been of interest in recent years. Serratiopeptidase
remains the most well-known anti-inflammatory drug of choice. Serratiopeptidase is a cheaper and
safer anti-inflammatory drug alternative to NSAIDs. The multifaceted properties of serratiopeptidase
may lead towards arthritis, diabetes, cancer and thrombolytic treatments. Existing serratiopeptidase
treatments in combination with antibiotics are popular in the treatment of postoperative swelling.
Although an exclusive number of serratiopeptidase-producing strains have been derived, there is an
urge for new recombinant strains to enhance the production of the enzyme. This review explores
the properties of serratiopeptidase, different therapeutic aspects, industrial production, and various
analytical techniques used in enzyme recovery. In addition, the review highlights the therapeutic and
clinical aspects of the serratiopeptidase enzyme to combat COVID-19-induced respiratory syndrome.

Keywords: Serratia sp.; serratiopeptidase; anti-inflammatory; COVID-19; mucolytic; anti-biofilm;

Citation: Nair, S.R.; C, S.D.
fibrinolytic
Serratiopeptidase: An integrated
View of Multifaceted Therapeutic
Enzyme. Biomolecules 2022, 12, 1468.
https://doi.org/10.3390/biom12101468 1. Introduction
Academic Editor: Vladimir
Nowadays, enzymes are used as an alternative drug of choice to treat many ailments.
N. Uversky De Duve [1] was the first to suggest that enzymes can be an alternative treatment for
hereditary diseases. Trypsin, α-chymotrypsin, prozyme, and bromelain are the most com-
Received: 1 September 2022 monly administered oral anti-inflammatory enzymes [2]. Serratiopeptidase is one of the
Accepted: 29 September 2022
most dominant anti-inflammatory drugs, with numerous therapeutic applications. The
Published: 13 October 2022
enzyme has anti-inflammatory, anti-biofilm, mucolytic, fibrinolytic, and wound-healing
Publisher’s Note: MDPI stays neutral properties. The enzyme has a molecular size of 52kDa, and has the ability to bind with
with regard to jurisdictional claims in alpha-2-macroglobulin in blood at a ratio of 1:1 [3]. It is widely used in treating carpal tunnel
published maps and institutional affil- syndrome, arthritis, fibrocystic breast disease, bronchitis, and sinusitis [4]. Serratiopepti-
iations. dase has a strong affinity for cyclooxygenase (COX) I and II, which are crucially linked
with interleukin (IL), prostaglandin (PGs), and thromboxane (TXs) production [5]. Drugs
such as NSAIDs (nonsteroidal anti-inflammatory drugs), either alone or in combination
with other medicines, are the most often prescribed treatment for acute inflammation [5].
Copyright: © 2022 by the authors.
They act on bonds between Arg and Gly, CysSO3 H and Gly, Asn and Gln, Tyr and Tyr,
Licensee MDPI, Basel, Switzerland.
His and Leu, Gly and Ala, Ala and Leu, Tyr and Leu, Gly and Gly, Phen and Tyr, and Tyr
This article is an open access article
and Thr. This helps with reducing inflammation and controls the release of interleukins,
distributed under the terms and
thromboxanes, and prostaglandins. Serratiopeptidase has a long history of use as a ther-
conditions of the Creative Commons
apeutic enzyme, and its demand in industries has been satisfied by wild, recombinant,
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
and mutated strains. Serratiopeptidase has also been used in the treatment of Alzheimer’s
4.0/).
disease. The enzyme has the ability to degrade amyloid plaques. In vivo studies on rat

Biomolecules 2022, 12, 1468. https://doi.org/10.3390/biom12101468 https://www.mdpi.com/journal/biomolecules

Biomolecules 2022, 12, 1468 2 of 12

models have shown that the enzyme is capable of fighting against Alzheimer’s disease,
as it helped in amyloid fibrin degradation [6]. This review is an attempt to examine and
understand the available evidence regarding clinical, productive, and therapeutic aspects
of serratiopeptidase. In addition, this review emphasizes the various bacterial strains used
in the production of serratiopeptidase.

2. The Enzyme and Its Properties

Japanese researchers were the first to report and introduce the anti-inflammatory
drug serratiopeptidase to the world. Enzyme formulations were created, and were widely
used as medicines. After 1970, these enzyme formulations were eventually successfully
marketed worldwide. The clinical studies carried out by researchers in Europe and Japan
suggested serratiopeptidase as a potent anti-inflammatory drug [7,8]. Hence, the demand
for enzyme began increasing worldwide. Serratiopeptidase is a metalloprotease enzyme
with a molecular weight of 45–60 kDa. The enzyme contains zinc at the active site. Ser-
ratiopeptidase belongs to the group serralysin and has an EC number of 3.4.24.40. [7].
The enzyme consists of 470 amino acids which are important for its proteolytic activity.
The enzyme is devoid of sulfur-containing amino acids such as cysteine and methionine.
Serratiopeptidase showed maximum activity at pH 9 and 40 ◦ C, and can be inactivated at
55 ◦ C for 15 min [8,9].

2.1. Anti-Inflammatory Action of Serratiopeptidase

Inflammation is an innate immune response that causes redness, swelling, and pain
in the human body. It is regarded as a response of the human body against any irritant,
and can be caused by many reasons, such as pathogens, injuries, and damage of cells [3].
Hence, inflammation can be regarded as a healing mechanism of our bodies to main-
tain homeostasis [10]. It has been observed that NSAIDs are the most commonly used
drugs for inflammation [7,10]. Anti-inflammatory drugs can interact with (cyclooxyge-
nase) COX-I and COX-II molecules. Among these enzymes, COX-I is responsible for the
breakdown of arachidonic acid, which is responsible for the production of interleukins
and prostaglandins [9,11]. Serine proteases are known to have a great affinity for these
molecules, and can act as anti-inflammatory agents [3]. The enzymes regulated inflamma-
tory cytokines, modified cell adhesion molecules, and acted at the site of inflammation [4,9].
In the absence of this enzyme, pain and swelling occurred at the area of injury and initi-
ated the release of prostaglandins. (Figure 1a). This led to the onset of cascade reactions.
Serratiopeptidase has the ability to bind with cyclooxygenase and suppress the release of
interleukins and prostaglandins. (Figure 1b). The oral administration of serratiopeptidase
tablets reduce pain and inflammation. The enzyme has its mode of action on arachidonic
acid pathway (COX I and COX II), and acts on the cyclooxygenase pathway, but not on
the lipooxygenase pathway (LOX). The lipooxygenase pathway (LOX) is involved in the
regulation of inflammation by mediating the catalysis of SPM (specialized pro-resolving
mediators) biosynthesis, and non-specific NSAID inhibition [11].

2.2. Wound-Healing Activity of Serratiopeptidase

In addition to the anti-inflammatory property, the enzyme also helps in wound heal-
ing. The enzyme acts by dissolving the dead tissue around the wound and hydrolyses
bradykinin, serotonin, and histamine. This improves the microcirculation at the site of
injury and results in wound healing [12]. There are four phases in a typical wound healing
mechanism. These include the hemostasis phase, the inflammatory phase, the proliferative
phase, and the maturation phase [12,13]. This enzyme can enhance microcirculation and
help to maintain hemostasis [14]. Serratiopeptidase is known to reduce the capillary per-
meability induced by histamine, bradykinin, and serotonin, and has the ability to break
the abnormal exudates and proteins as well as to improve the absorption of decomposed
products through blood and lymphatics [13]. Serratiopeptidase, along with metronidazole,
was found to be effective in improving wound healing in rabbits [15]. Another finding
Biomolecules 2022, 12, 1468 3 of 12

regarding serratiopeptidase was related to the tissue repair mechanism. At the site of an
inflamed wound, the enzyme assisted in reducing the amount of fluids drained to the
wound and facilitated microcirculation, hence improving tissue repair [13]. In a recent
comparative study, the effectiveness of an enteric-coated tablet comprising fixed-dose
combination (FDC) of trypsin 48 mg, bromelain 90 mg, and rutoside trihydrate 100 mg
with serratiopeptidase 10 mg was observed. The results showed that serratiopeptidase
was less effective than trypsin, bromelain, and rutoside trihydrate [16]. One reason for the
Biomolecules 2022, 12, x
lower efficiency may be a low dosage. A higher concentration of the drug may be more
stable at gastric pH, and can facilitate the healing process.

Figureacid
Figure 1. Arachidonic 1. Arachidonic
pathway: (a) acid pathway:
release (a) releaseand
of interleukins of interleukins and induce
prostaglandins prostaglandins
the pain induce th
and swelling. (b)and swelling.
Mode (b). serratiopeptidase
of action: Mode of action: serratiopeptidase acts on the enzyme
acts on the cyclooxygenase cyclooxygenase enzyme (COX
(COX I and
COX II) the
COX II) and suppresses andrelease
suppresses the releaseand
of interleukins of interleukins and prostaglandins.
prostaglandins.

2.3. Antibiofilm2.2.
Activity of Serratiopeptidase
Wound-Healing Activity of Serratiopeptidase
In biofilms, serratiopeptidase
In addition to the cananti-inflammatory
alter the pathogenic phenotype
property, of a bacterium.
the enzyme Thein wound
also helps
use of dispersion agents may improve the effectiveness of current therapeutics.
ing. The enzyme acts by dissolving the dead tissue around the wound and hydr The enzy-
matic agents dispersin
bradykinin,B, lysostaphin,
serotonin, and alpha amylase, This
histamine. V8 protease,
improves andtheserratiopeptidase
microcirculation at the
were tested against
injurymethicillin-resistant
and results in wound and susceptible
healing strains
[12]. There S. aureus
areoffour phases biofilms, bothwound h
in a typical
individually and in combination with vancomycin and rifampicin. When
mechanism. These include the hemostasis phase, the inflammatory phase, the pro coupled with
any of the dispersal agents,and
tive phase, thethe
effectiveness
maturationofphasethe antibiotics
[12,13]. This wasenzyme
increased.canLysostaphin
enhance microcircu
and serratiopeptidase were found to be the most effective dispersion
and help to maintain hemostasis [14]. Serratiopeptidase is known agents against all of the cap
to reduce
the tested strains [14]. Serratiopeptidase, a proteolytic enzyme, was originally
permeability induced by histamine, bradykinin, and serotonin, and has the ability tosuggested
by Selan et al. the
[17]abnormal
for the treatment
exudatesofand biofilm-related
proteins as wellillnesses nearly twenty
as to improve years ago.
the absorption of decom
an S. epidermidis
Most recently, products (a high-slime-producing strain) infected rat
through blood and lymphatics [13]. Serratiopeptidase, along with metromodel was
treated with anzole,
intramuscular
was foundinjection of serratiopeptidase.
to be effective in improving wound It was noted
healingthatin 94.4%
rabbitsof[15].
the Anothe
infected mice were recovered when compared to 62.5% in the group treated
ing regarding serratiopeptidase was related to the tissue repair mechanism. At the with antibi-
otics [18]. In the
anininflamed
vivo animal models,
wound, serratiopeptidase
the enzyme assisted ineffectively
reducing acted againstof
the amount bacteria
fluids drained
that produced biofilms. The antibiofilm function of enzyme may enhance the effectiveness
wound and facilitated microcirculation, hence improving tissue repair [13]. In a
of antibiotics in reducing Staphylococcal infections [18].
comparative study, the effectiveness of an enteric-coated tablet comprising fixed
Another observation regarding the serratiopeptidase enzyme based on its anti-biofilm
combination (FDC) of trypsin 48 mg, bromelain 90 mg, and rutoside trihydrate 1
activity was against a fully matured Staphylococcus aureus biofilm [19]. The researchers
with serratiopeptidase 10 mg was observed. The results showed that serratiopep
constructed an Spep mutant by replacing the glutamic acid in the catalytic site with another
was less effective than trypsin, bromelain, and rutoside trihydrate [16]. One reason f
amino acid (alanine), and evaluated the anti-biofilm activity of the Spep mutant. The
lower efficiency may be a low dosage. A higher concentration of the drug may be
research reports revealed that there was no proteolytic activity for the mutant strain;
stable at gastric pH, and can facilitate the healing process.
nevertheless, it was able to retain its anti-biofilm activity [19]. Serratiopeptidase is known
to exhibit the property of modifying the adhesion molecules and thereby reducing the
2.3. Antibiofilm Activity of Serratiopeptidase
cell surface proteins [19]. Selan et al. [20] reported that the enzyme could alter the biofilm
association of virulentIn biofilms, serratiopeptidase
strains, and that it showedcan alteragainst
activity the pathogenic phenotype
a completely of a bacterium
developed
use of dispersion agents may improve the effectiveness of
biofilm. Biofilms are normally difficult to destroy. Serratiopeptidase, in combination with current therapeutics. The
matic agents dispersin B, lysostaphin, alpha amylase,
other antibiotics, exhibited potent anti-bioflim activity. The serratiopeptidase enzymeV8 protease, and serratiopep
has reduced the were tested against
expression methicillin-resistant
of Listeria monocytogens cell and susceptible
surface proteinsstrains
suchofasS.Ami4b,
aureus biofilms
individually and in combination with vancomycin and rifampicin. When coupled
any of the dispersal agents, the effectiveness of the antibiotics was increased. Lysost
and serratiopeptidase were found to be the most effective dispersion agents agains
the tested strains [14]. Serratiopeptidase, a proteolytic enzyme, was originally sugg
by Selan et al. [17] for the treatment of biofilm-related illnesses nearly twenty year
Biomolecules 2022, 12, 1468 4 of 12

internalin B, Act A, and autolysin. The enzyme significantly precluded the adhesion of
Listeria monocytogens in the human digestive tract [21]. According to previous reports,
interestingly, it was found that the enzyme has the ability to interact only with the cell
adhesion molecules that formed the biofilm. No cytotoxic activity was recorded [19,20].
The enzyme showed its effect on discrete surface proteins such as At1. It can act on these
surface proteins by altering adhesins and autolysins. In a study reported by Artini et al. [22],
it was stated that serratiopeptidase and carboxypeptidase showed activity against biofilm
formation of different strains of Staphylococcus aureus and Staphylococcus epidermidis. The
test results of the previous studies showed that only serratiopeptidase inhibited the activity
of all strains. The enzyme has the ability to modify the phenotype of virulent bacteria and
enhance anti-bacterial properties [22]. Another interesting fact was reported regarding the
enzyme: it regulates the recruitment of immune cells to the site of inflammation [23]. The
efficacy of serratiopeptidase against biofilm-forming bacteria was proven in experimental
animal models. The enzyme serratiopeptidase increased the effectiveness of antibiotics
in the treatment of Staphylococcal infections [18]. The enzyme can be supplemented with
antibiotics for more effective medication.

2.4. Mucolytic Activity of Serratiopeptidase

Sputum production, nasal congestion, and cough are observed as some of the preva-
lent symptoms in COVID-19 patients. Mucolytics can increase bronchial mucus output
or decrease mucus viscosity and make it easier to cough up the mucus. Serratiopeptidase
may be helpful due to its caseinolytic and mucolytic effects on sputum. In patients with
respiratory disorders, serratiopeptidase has improved mucociliary transportability and
mucociliary clearance by lowering neutrophils and modifying the viscoelasticity of spu-
tum [24]. Research has revealed a new combination therapy for COVID-19. A combination
of vitamin D and serratiopeptidase acts as a strong mucolytic agent, and has the ability to
fight against the severe effects of COVID-19 syndrome [25]. Kim et al. [26] has detailed the
occurrence of other symptoms such as rhinorrhoea, hypogeusia, and nasal congestion in a
large number of patients. Treatment methods such as administration of bronchodilators
and mucolytic agents, along with tracheal suction, were the remedial measures for such
patients [26]. Several proteolytic enzymes are known to act in a synchronized manner in the
control and coordination mechanism of viral entry, viral propagation, and establishment in
host cells [27]. The serratiopeptidase enzyme plays a vital role in the treatment of COVID-19
infection. Sharma et al. [28] has conferred the possibility of serratiopeptidase being used as
a mucolytic drug in COVID-19 patients. It was found that serratiopeptidase can inhibit the
cytokine storm in COVID-19 patients. The elevated expression of transforming growth fac-
tor (TGF-α), IL-6, and other chemokines may lead to cytokine storms in COVID-19 patients.
Increased levels of IL-6 may cause acute lung disorders. This condition can be treated with
medicines. Serratiopeptidase has been suggested as an effective medicine to treat the severe
complications of COVID-19 [28]. Another post-COVID syndrome is cardiovascular disor-
der due to increased levels of D-dimers, as well as fibrin or fibrinogen products [26,29]. The
cytokine storm may increase the risk of atherosclerosis and cardiac arrest. The fibrinolytic
activity of serratiopeptidase, along with its proteolytic and anti-inflammatory activity,
increased its potential for reducing the severity of vascular complications in COVID-19
patients [27]. Kase et al. [30] has detailed the importance of serratiopeptidase as a mucolytic
agent, and compared the mucolytic activity of serratiopeptidase with seaprose. Seaprose is
a proteolytic enzyme commonly used in the treatment of bronchitis. Both enzymes showed
considerable mucolytic activity in the in vivo animal models.
Biomolecules 2022, 12, 1468 5 of 12

2.5. Hemolytic Activity of Serratiopeptidase

The formation of blood clots in blood vessels is a major cause for cardiovascular
disorders. Serine proteases are a group of enzymes that includes fibrinolytic enzymes.
Serratiopeptidase, which is a serine protease, has high substrate specificity and fibrinolytic
activity [31]. The enzyme serratiopeptidase has been shown to contain the property of blood
clot lysis, and is able to remove arterial blocks and cysts [31]. The serine metalloprotease
extracted from marine Serratia marcescens subsp. sakuensis showed efficient fibrinolytic
activity [32]. Shank et al. [33] compared the hemolytic activity of both mutant and wild type
Serratia marcescens. Mutant strains of Serratia marcescens exhibited hyper hemolysis. The
compound serratamolide, a small cyclic amino-lipid produced by Serratia marcescens, was
reported as an effective hemolytic and anti-microbial agent [34]. It has been observed that
the swrW gene played an important role in the biosynthesis of serratamolide, also known as
serrawettin [33]. Serratamolide was previously reported as a broad-spectrum antibiotic [33].
The swrW gene is responsible for the production of serratamolide. Wasserman et al. [35]
reported that mutations in swrW gene expression or the hexS transcription factor gene (an
inhibitor of the swrW gene) enhance the production of serratamolide. In vitro cytotoxic
activity of serratamolide was reported against corneal limbal epithelial cells, as well as
sheep and mouse red blood cells [35]. The compound serratamolide extracted from Serratia
marcescens will be an effective anti-microbial and anti-cancer agent in the future.

2.6. Synergistic Property of Serratiopeptidase

Maheshwari et al. [36] found that the enzyme was capable of displaying a vast
synergistic antimicrobial property with penicillins, fluoroquinolones, tetracycline, and
cephalosporins. In combination with antibiotics, the enzyme can exhibit more intense
synergistic activity in preventing biofilms [17]. Bacteria have the potential to colonise on
any surface and orchestrate a coordinated response. According to reports, COX inhibitors
prevent the growth of biofilms effeciently [17,37]. Previous findings have suggested that
cyclooxygenase-dependent synthesis of prostaglandins is necessary for biofilm develop-
ment. COX inhibitors effectively inhibited the biofilm formation when combined with
aspirin, etodolac, diclofenac, celecoxib, nimesulide, ibuprofen, and meloxicam [37]. After
48 h of incubation with aspirin, etodolac along with diclofenac, which were COX-II inhibitors,
showed the greatest effect, while aspirin showed 95% inhibition against biofilms [37].
Presently, researchers are focusing more intently on combination therapy to enhance the
anti-inflammatory activity of serratiopeptidase. Vancomycin and rifampicin, combined
with enzymatic agents such as serratiopeptidase, dispersin B, alpha-amylase, V8 pro-
tease, and lysostaphin, showed an ample amount of action against biofilms formed by
methicillin-resistant susceptible strains of S. aureus [14] (Table 1). The efficiency and syn-
ergistic action of antibiofilms and serratiopeptidase was improved when combined with
dispersal agents [14]. Serratiopeptidase is the most effective dispersion agent against most
biofilm-forming bacterial strains. Table 1 represents the synergistic action of dispersal
agents with different antibiotics on biofilm formation [38,39].

Table 1. Synergistic property of enzyme with different antibiotics.

Sl No Antibiotics Effect of Enzymes References

Enhanced the activity of ofloxacin and
1. Ofloxacin [17]
inhibited biofilm formation.
Effective against different strains of biofilm
2. Azithromycin [38]
forming Staphylococcus sp.
3. Levofloxacin Eradicated > 90% of the preformed biofilm. [39]
Vancomycin and Effective in dispersing most of the biofilm
4. [14]
rifampicin forming bacteria
Biomolecules 2022, 12, 1468 6 of 12

3. Enzyme Production
3.1. Serratia Marcescens
Serratia marcescens E-15 was identified as one of the potent producers of serratiopepti-
dase. However, the pathogenic nature of the organism has made it difficult to adapt using
common production methods [40]. Serratia was first identified as an opportunistic human
pathogen in 1959, and belongs to the Enterobacteriaceae family. It has similar character-
istics to the Klebsiella and Enterobacter groups of bacteria [41]. Much more sophisticated
and safe production strategies could make large-scale production in industries easier and
more cost effective [42]. The maximum yield of enzyme production was obtained from
mutant strains of Serratia marcescens [43]. Enhanced production of serratiopeptidase was
effectively achieved by physical and chemical mutagenesis. Multiple exposures of Serratia
marcescens to UV radiation and chemical mutagens (ethyl sulfonate) enhanced the yield
and activity of the enzyme [43]. It was noted that mutant Serratia marcescens showed higher
serratiopeptidase activity when exposed to UV and chemical mutagens. Industries have
always given priority to such stable organisms. The study on thermoactive serratiopep-
tidase [44] from the soil isolate Serratia marcescens AD-W2 from India’s North-Western
Himalayan area showed a specific activity of 20,492 units/mg protein with 5.28-fold pu-
rification. The molecular weight of the metalloprotease was approximately 51 kDa. At
pH 9.0 and 50 ◦ C, the purified serratiopeptidase showed maximum activity, in addition
to stability over a wide range of pH values and temperatures [44]. The thermostability of
the enzyme was considered to be one of the most significant properties for the large-scale
industrial production.

3.2. Alternative Species for Production

A genetically engineered non-pathogen could be an effective replacement for much
higher production of the enzyme [42]. A study conducted by Srivastava et al. [42] on recom-
binant expression of mature serratiopeptidase in E. coli resulted in failure of transformation.
According to previous reports, transformed E. coli C43 (DE3 ) cells expressed proteins with
lesser yield. It was also inferred that the number of transformants in pET23b(+) (without
gene) and pMSrp (with mature gene) in E. coli DH5 was similar. There was a significant dif-
ference in the DE3 variant. Srivastava et al. [42] indicated that the gene had some negative
effects on cells. Optimization of parameters such as nutrient composition, post induction
duration, inducer concentration, and point of induction resulted in an increased expression
of mature serratiopeptidase [42].
From silkworm gut, five different protease-producing Serratia strains were isolated [45].
The isolated strains are S. indica, S. marcescens, S. piscatorum, S. plymuthica, and S. marcescens
E-15. According to reports, the E-15 strain produced the maximum amount of the enzyme
compared to other strains [45]. From different species of Serratia, different molecular
sizes of serratiopeptidase were identified (Figure 2). Koul et al. [46] identified two potent
producers of serratiopeptidase. Serratia marcescens MES-4, an endophyte, showed 95 U/mL,
and Serratia marcescens MRS-11, a soil isolate, showed 156 U/mL of activity [46]. Recently,
recombinant expression of serratiopeptidase genes in E. coli was reported by Doshi et al. [47].
Fed-batch fermentation was used for the mass production of recombinant serratiopeptidase
protein fusion constructs. The optimized bioreactor parameters revealed a high yield of
protein and cell mass. The downstream solubilization and purification methods were
also improved for the enhanced production of functional serratiopeptidase. In addition,
the enzyme exhibited a novel, unanticipated self-proteolytic activity that cleaved the
propeptide’s N-terminal His-SUMO fusion tag [47].
 recombinant serratiopeptidase protein fusion constructs. The optimized bioreactor pa-
rameters revealed a high yield of protein and cell mass. The downstream solubilization
and purification methods were also improved for the enhanced production of functional
serratiopeptidase. In addition, the enzyme exhibited a novel, unanticipated self-proteo-
Biomolecules 2022, 12, 1468 lytic activity that cleaved the propeptide’s N-terminal His-SUMO fusion tag [47]. 7 of 12

Figure
Figure 2. Molecular
2. Molecular weight
weight of serratiopeptidase
of serratiopeptidase produced
produced fromfrom different
different strains
strains of of Serratia.
Serratia. Serratia
Serratia
marcescens E-15 [40], Serratia marcescens VITSD2
marcescens E-15 [40], Serratia marcescens VITSD2 [48], Serratia marcescens VITAP [4], Serratia mar-
Serratia marcescens VITAP [4], Serratia marcescens
cescens AD-W2 [44], Serratia marcescens subsp. sakuensis [32] Serratia marcescens MES 4[46], Serratia
AD-W2 [44], Serratia marcescens subsp. sakuensis [32] Serratia marcescens MES 4 [46], Serratia marcescens
marcescens MR 11 [46].
MR 11 [46].

4. Analytical
4. Analytical Approaches
Approaches of of Enzyme
Enzyme
Both
Both qualitative
qualitative andand quantitative
quantitative analysis
analysis were were widely
widely carried
carried outthe
out for forpurpose
the purpose
of
of detailed study of the serratiopeptidase enzyme, ranging from simple
detailed study of the serratiopeptidase enzyme, ranging from simple chemical techniques chemical tech-
to niques to more sophisticated
more sophisticated chromatography
chromatography methodsmethods [48]. Thin-layer
[48]. Thin-layer chromatography
chromatography was
was known to be a routine method for separation, and
known to be a routine method for separation, and Rf values were comparedRf values were compared
with the with
the standards.
standards. TheseThese
were were the most
the most simple,
simple, quick,quick,
and and
easyeasy methods
methods used used
forfor compound
compound
identification
identification [7,48].High-performance
[7,48]. High-performanceliquidliquid chromatography
chromatography (HPLC)
(HPLC)data dataonon serratiopep-
serrati-
tidase produced by Serratia marcescens VITSD2 showed a chromatogram
opeptidase produced by Serratia marcescens VITSD2 showed a chromatogram peak of peak of 3.45 min
3.45
retention time. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
min retention time. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS— (SDS—PAGE)
becomes
PAGE) a more
becomes relevant
a more analytical
relevant tool in
analytical identifying
tool the molecular
in identifying the molecularsize size
of serratiopep-
of serra-
tiopeptidase. Quantitative analysis and molecular weight determination werecarried
tidase. Quantitative analysis and molecular weight determination were carriedoutoutby
by Ananthakrishnan et al. [49]. X-ray powder diffractometry has been applied by manyre-
Ananthakrishnan et al. [49]. X-ray powder diffractometry has been applied by many
searchers in
researchers inorder
ordertotocrystallize
crystallizethethemolecule
moleculeand andtotounderstand
understanditsitsstructure.
structure.This Thishashas
relevance
relevance in in identifying
identifying thethe molecular
molecular weight
weight of of
thethe compound.
compound. OneOne
suchsuch dataset
dataset showed
showed
that the molecular size of serratiopeptidase was between 45,000 and
that the molecular size of serratiopeptidase was between 45,000 and 48,000 Da [7]. From48,000 Da [7]. From
the details regarding the analytical study of this enzyme, a cost-effective method may be
the details regarding the analytical study of this enzyme, a cost-effective method may be
more practical for researchers. Replacing commercially available nutrient sources with
more practical for researchers. Replacing commercially available nutrient sources with
cheaper sources, as well as optimization of parameters, can increase the production rate,
cheaper sources, as well as optimization of parameters, can increase the production rate,
which, in turn, can benefit the industries. Caseinolytic assay was one of the most common
which, in turn, can benefit the industries. Caseinolytic assay was one of the most common
methods used by most of the researchers for quantitative analysis of serratiopeptidase.
methods used by most of the researchers for quantitative analysis of serratiopeptidase.
Hawa et al. [50] purified and characterized serratiopeptidase from Pseudomonas sp., and
Hawa et al. [50] purified and characterized serratiopeptidase from Pseudomonas sp., and
Salamone et al. [51] quantified serratiopeptidase from Serratia marcescens using casein and
Salamone et al. [51] quantified serratiopeptidase from Serratia marcescens using casein and
bovine serum albumin as a substrate.
bovine serum albumin as a substrate.
Like every pharmacological analysis, spectroscopy has been used as a valid tool in
Like every pharmacological analysis, spectroscopy has been used as a valid tool in
the qualitative analysis of the serratiopeptidase enzyme. Researchers have compared serra-
the qualitative analysis of the serratiopeptidase enzyme. Researchers have compared
tiopeptidase with other drugs and standardized the dosages for oral administration. ELISA
(Enzyme linked immuno sorbent assay) is the most common assay used in the quantifi-
cation of serratiopeptidase. Universally, ELISA is accepted as one of the most accurate
methods for quantification. Many researchers have applied ELISA to determine the activity
and concentration of serratiopeptidase [52]. Louis et al. [52] quantified serratiopeptidase
produced by Pseudomonas sp. using ELISA. Radio immunoassays and UV microplate assays
are the most sensitive methods for determining the concentration of enzymes. Even very
Biomolecules 2022, 12, 1468 8 of 12

low concentrations of the enzyme produced by different microbial strains could be detected
using UV micro plate assays. When compared to other assay methods, the UV microplate
method is novel, simple, fast, and specific. Sandhya et al. [53] used the UV microplate assay
method for quantification of serratiopeptidase. In order to purify the enzyme, an effective,
robust, and simple methodology is always needed. Pakhale et al. [54] have explained a
novel strategy for purification of serratiopeptidase from Serratia marcescens NRRL B 23112,
using an ultrasound-assisted, three-phase partitioning system [54]. According to reports,
the maximum purity and recovery rate of the enzyme was obtained by the ultrasound-
assisted, three-phase partitioning system when compared to three-phase partitioning (TPP).
The time taken for the purification of the enzyme was dramatically reduced from 1 h to
5 min in the ultrasound-assisted, three-phase partitioning system [54]. Fuchs et al. [55]
emphasized the remarkable purification efficiency of the chitin affinity chromatography
method for multiple chitinolytic proteins produced from Serratia marcescens.

5. Therapeutic Aspects of Serratiopeptidase

The anti-inflammatory effects of serratiopeptidase, aspirin, trypsin, and chymotrypsin
in Albino rats against carrageenan-induced paw edoema were compared by Viswanatha,
Swamy, and Patil [56]. In both acute and subacute types of inflammation in rats, serra-
tiopeptidase had superior anti-inflammatory action both on its own and in combination
with aspirin. Along with a histological analysis, several inflammatory indicators, such
as C-reactive protein, glutathione, myeloperoxidase, and nitric oxide, were found. When
compared to the control group, serratiopeptidase decreased the disease activity index
and stopped the formation of nitric oxide, as well as colonic shortening, glutathione de-
pletion, spleen enlargement, and lipid peroxidation. Serratiopeptidase-treated mice had
significantly lower C-reactive protein levels than the control mice. Moreover, the use of
serratiopeptidase decreased myeloperoxidase, a significant enzyme marker of inflamma-
tion. These findings support serratiopeptidase’s ability to reduce inflammation, and thus it
has been recognized as a multi-channel enzyme in terms of its wide application in treat-
ments [57]. The enzyme has been successfully applied in atherosclerosis, in which plaques
in arteries were dissolved by the proteolytic action of the enzyme. When compared with
other enzymes, serratiopeptidase has been successfully used in ortholaryngiology [58]. Re-
searchers have reported the fibrinolytic activity of serratiopeptidase and successfully used
it in fibrinolytic therapy [56]. Another known application of serratiopeptidase is in dental
implantation, where soft and hard gums developed inflammation upon peri implants, and
anti-inflammatory enzymes were used as a treatment [58]. Serine proteases, along with
other drugs, are commonly used in orthopedic medicines to treat chronic inflammation,
pain, and swelling. The enzyme has great affinity with COX I and COX II, which are
pain mediators [1]. An appropriate study on dosage of the enzyme must be conducted
in order to control levels of the enzyme concentration in plasma, as it was found that the
amount of enzymes in blood varies with body mass [59]. In 2022, it was reported that the
enzyme was not able to bind with LOX or to block lipoxygenase-catalyzed biosynthesis of
specialized pro-resolving mediators [60]. A pre-clinical study reported by Jadav et al. [61]
indicated that serratiopeptidase was orally effective, and had anti-inflammatory activity
which was equivalent to diclofenac sodium in both chronic and acute phases of inflamma-
tion. Serratiopeptidase can be used to treat osteoarthritis in combination with metformin.
Ateia et al. [62] reported the impact of metformin and serratiopeptidase on knee osteoarthri-
tis in obese patients. Metformin and serratiopeptidase combination tablets were efficient in
the treatment of knee osteoarthritis. Ai-Khateeb and Nusair’s [59]. clinical study reports
revealed the effect of serratiopeptidase in pain reduction, trismus, and post-operative
swelling after molar surgery. Small studies in the field of dentistry, otorhinolaryngology,
and orthopaedics have revealed reductions in pain and inflammation for ailments such as
carpal tunnel syndrome, arthritis, and tooth extraction. Serratiopeptidase tablets have also
been used in the treatment of pneumonitis, joint pain, and dermatitis. According to clinical
case reports, serratiopeptidase did not show many adverse effects in treated patients [8].
Biomolecules 2022, 12, 1468 9 of 12

Very few studies have been reported on the anti-cancer activity of serratiopeptidase. The
in vitro cytotoxic activity of serratiopeptidase against colon cancer cell lines (Caco-2) was
reported by Araghi et al. [63]. The findings of previous reports suggested that the enzyme
has anti-cancer potential, but further in vitro and in vivo mechanistic pathway studies are
needed in order to confirm the biological activity of the enzyme.

6. Clinical Significance
Enteric coated tablets are the most commonly available form of serratiopeptidase.
Panthi et al. [64] formulated enteric coated tablets for serratiopeptidase, which exhibited
persistent, stable, and significantly high drug release in the intestine. In general, glyceryl
monooleate-based systems give protection to metallo-enzymes in the gastric environment.
In addition, they enhanced the sustained release of the enzyme after oral administration [64].
Shah and Paradkar [65] suggested that a microenvironment-controlled, in situ, cubic phase
transforming glyceryl monooleate system may give protection to serratiopeptidase as well
as meticulous release. Serratiopeptidase has been used in traumatic and postoperative
inflammation, laryngitis, bronchitis, expectoration of sputum in bronchial asthma, gyne-
cology, venous inflammatory disease, cystitis, epididymitis, traumatic swelling, carpal
tunnel syndrome, osteoarticular infection, sinusitis, rhinitis, and dentistry [3]. This has
caused an increase in demand for the production of the enzyme, and various combinational
drugs have been developed [27]. The tablets were taken orally on an empty stomach or
30 min before food. According to clinical studies, when compared to methylprednisolone,
serratiopeptidase showed low analgesic action and efficient management of edema and
trismus [66]. The oral administration of this enzyme can reduce inflammation and pain
in AIDS, as well as in hepatitis B & C infections [67]. This led to an increase in the use of
serratiopeptidase in the field of medicine. Cancer nanomedicine has created a revolution
in the field of medicine [68]. Serratiopeptidase in combination with nanodrug delivery
systems has been an emerging technology in cancer therapy. Anti-inflammatory agents
such as serratiopeptidase may help in overcoming the adverse effects of anti-cancer agents.
Jaiswal and Mishra [69] reported that the co-delivery of curcumin and serratiopeptidase
along with nanoparticles showed enhanced anti-cancer activity against HeLa and MCF-7
cells. Serratiopeptidase can be viewed as a viable competitor in contemporary medicine.
Hence, the synergistic activity of serratiopeptidase has a vital role in emphasizing its clinical
importance [17,38,39].

7. Conclusions
The proteolytic enzyme serratiopeptidase has an enormous number of therapeutic
applications and significant analytical importance. Anti-inflammatory, anti-biofilm, mu-
colytic, and synergistic action are the potential targets of drug therapy. Being a mucolytic
agent, serratiopeptidase has been used in treatment of the COVID-19 infection. Among
these studies, the serratiopeptidase was known to exhibit significant synergistic activity
with various antibiotics in resolving infection and inflammation. Serratiopeptidase has
fibrinolytic and anti-cancer activity. In the future, there will be a great demand for the en-
zyme due to its multifaceted properties. Researchers are also focusing on serratiopeptidase
nanoparticles for the purpose of targeted delivery and restricted action on the selected sites.
Most researchers emphasize the synergistic action of serratiopeptidase in the treatment
of arthritis, diabetes, and Alzheimer’s disease. The current review explores the different
agents involved in the industrial production of serratiopeptidase. So far, very few studies
have been conducted on serratiopeptidase coding genes. Hence, a more detailed study on
the genome of Serratia species will lead to the development of new and potent strains for
large-scale production. Gene cloning and vector therapy will be helpful for industrialists
to enhance the production rate of the enzyme. Nowadays, pharmaceutical companies are
targeting serratiopeptidase production due to its multifaceted properties. Pharmaceutical
companies are focused on the construction of recombinant strains to enhance the yield
and purity of the enzyme. Serratiopeptidase is a miracle enzyme which has the potential
Biomolecules 2022, 12, 1468 10 of 12

to replace NSAIDs. With this multifaceted value, this enzyme could be a pioneer in the
treatment of COVID-19 and other infectious diseases.

Author Contributions: S.R.N.—Conceptualization, Writing, original draft preparation; S.D.C.—

Conceptualization, Critically revised, editing, supervision and funding acquisition. All authors have
read and agreed to the published version of the manuscript.
Funding: The APC was funded by Vellore Institute of Technology, Vellore, Tamil Nadu, India.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: The authors are thankful to Vellore Institute of Technology, Vellore, for the
constant encouragement, help and support for extending necessary facilities.
Conflicts of Interest: Authors confirm that this article content has no conflict of interest.

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