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REVIEW
Production, purification, characterization, immobilization, and
application of Serrapeptase: a review
Selvarajan Ethiraj (✉), Shreya Gopinath
Department of Genetic Engineering, School of Bioengineering, SRM University, Kattankulathur, Tamil Nadu, India
© Higher Education Press and Springer-Verlag Berlin Heidelberg 2017
BACKGROUND: Serrapeptase is a proteolytic enzyme with many favorable biological properties like anti-inflammatory,
analgesic, anti-bacterial, fibrinolytic properties and hence, is widely used in clinical practice for the treatment of many diseases.
Although Serrapeptase is widely used, there are very few published papers and the information available about the enzyme is
very meagre. Hence this review article compiles all the information about this important enzyme Serrapeptase.
METHODS: A literature search against various databases and search engines like PubMed, SpringerLink, Scopus etc. was
performed.
RESULTS: We gathered and highlight all the published information regarding the molecular aspects, properties, sources,
production, purification, detection, optimizing yield, immobilization, clinical studies, pharmacology, interaction studies,
formulation, dosage and safety of the enzyme Serrapeptase.
CONCLUSION: Serrapeptase is used in many clinical studies against various diseases for its anti-inflammatory, fibrinolytic
and analgesic effects. There is insufficient data regarding the safety of the enzyme as a health supplement. Data about the anti-
atherosclerotic activity, safety, tolerability, efficacy and mechanism of action of the Serrapeptase are still required.
Keywords Serrapeptase, proteolytic enzyme, anti-inflammatory, fibrinolytic
Introduction
Enzymes and enzyme pathways are an integral part of the
human body. Enzymes are proteins in nature and act as
biocatalysts for all the chemical reactions and hence act as
therapeutic agents for most of the metabolic disorders (Cech
and Bass, 1986). Enzymes are highly specific and increase the
rate of a chemical reaction by lowering the activation energy
without any alterations to the enzyme (Aldridge, 2013). Until
the late 19th century, enzymes were used in treating only a
handful of disorders like gastrointestinal disorders and as a
digestive aid but the potential use of enzymes in infections,
cancer and other diverse diseases have slowly emerged (Sabu,
2003). Enzymes are classified into six classes by the Enzyme
Commission namely oxidoreductases, transferase, ligase,
lyases, isomerases and hydrolases based on the type of
reaction they catalyze (Singh et al., 2016).
Microorganisms are the most favorable sources of enzymes
in contrast to plant or animal origin due to ease of availability
and remarkable growth rate stability, ease of modification and
production (Singh et al., 2016). Gene manipulations and
genetic engineering of microorganisms can be easily
performed using recombinant DNA technology to increase
the rate of enzyme production (Illanes et al., 2012). Microbial
enzymes have widespread applications in food, pharmaceu-
tical, textile, paper, leather, medical and other industries and
their demand is rapidly increasing over other conventional
methods due to its greater efficiency, high-quality products
and eco-friendly nature (Jordon, 1929; Kamini et al., 1999;
Gurung et al., 2013).
When abundant protein particles aggregate in the body,
protein lumps are formed, that obstruct arteries and organs
causing multiple organ failure, which results in fatalities. Due
to excess protein accumulation in diseased conditions, an
overwhelming stress is created, affecting the tissues and thus
the body works slower than usual to keep up with the protein
decomposition. Hence, these proteins have to be eliminated
from the body. Proteolytic enzymes thus aid in degrading
protein masses accumulated in the body (Jickling et al.,
2010). Proteolytic enzymes represent one of the three largest
classes of enzymes, the hydrolases that can catalyze the
Received April 11, 2017; accepted July 20, 2017
Correspondence: E. Selvarajan
E-mail: selvarajan.e@ktr.srmuniv.ac.in
Front. Biol. 2017, 12(5): 333–348
DOI 10.1007/s11515-017-1461-3
hydrolysis of peptide bonds in proteins and peptides (Bach et
al., 2012; Fadl et al., 2013). Proteolytic enzymes accounts for
about 60%of total sale in the worldwide market (Anil and
Kashinath, 2013).
Serrapeptase (EC number 3.4.24.40) is an effective
proteolytic enzyme belonging to serine protease family that
first came into interest to the Japanese biochemist 25 years
ago, since when it has been used widely in health care in
Asian and European countries. Serrapeptase made its debut in
the United States in 1977. Serrapeptase is isolated initially
from the Enterobacteria Serratia marcescens strain E-15
found in the gut of the Japanese silkworm Bombyx mori.Itis
also called as Serratiopeptidase or Serratia-peptidase as a
reason of its origin from Serratia marcescens (Anil and
Kashinath, 2013). Serrapeptase has an affinity to the dead
proteins in the end of silkworm threads and dissolves the
proteins that make up the cocoon and it selectively dissolves
the proteins involved in non-living tissues found in the
cocoon and not the living tissue (Sellman, 2003). Serrapep-
tase does not affect healthy tissues in the body because the
chemical structure of Serrapeptase inhibits attachment to
proteins in healthy tissues (Robert, 2009). The production of
Serrapeptase depends upon a secretory protein on the
membrane of the host cell and it is secreted by the N-terminal
signal peptide-independent pathway (Kaviyarasi et al., 2016).
Serrapeptase has been analyzed to have a high degree of
substrate specificity (Miyata et al., 1970b; Aiyappa and
Harris, 1976). It is an immunologically active enzyme and it
is anti-oedemic, analgesic, anti-inflammatory, solubilizes
non-living tissues such as mucous, plaques and blood clots
hence it is named as fibrinolytic/thrombolytic enzymes since
it has the ability to degrade insoluble proteins like fibrin and
other mediators of inflammation (Klein and Kullich, 2000).
Serrapeptase is taken as a supplement that can boost the
cardiovascular system and greatly augment overall health
(Robert, 2009). The serine family proteases play important
roles in not only obtaining nutrients but also in pathogenesis
(Miyagawa et al., 1991). Serrapeptase can affect mammalian
cells by degrading various protease inhibitors in the immune
system and can also be the main factor behind infections in
human epithelial cells (Shanks et al., 2015).
Serrapeptase has a potential to cure and treat disorders like
atherosclerosis, arthritis, bronchitis, carpal tunnel syndrome,
fibrocystic breast disease, Crohn’s disease, leg ulcers,
traumatic swelling, fibromyalgia, breast engorgement,
migraine, Alzheimer’s disease, sinusitis, hepatitis, lung
disorders, arthritis, diabetes, carotid artery blockage, throm-
bosis, uterine fibroids (Klein and Kullich, 2000). Serrapeptase
may also be used as a remedy for women suffering from
endometriosis. Serrapeptase is also considered a healing
enzyme as it heals sprained muscles, leg ulcers, traumatic
injuries, torn ligaments, post-operative inflammation, drains
mucus, reduces elasticity and viscosity of nasal mucus. It also
drains pooling of fluids in mastectomies and dissolves lumps
in the breasts (Sellman, 2003). Serrapeptase acts as an
amyloid dissociating agent with a potential to degrade insulin
amyloids and hence can be considered a potential drug for
different amyloid associated diseases (Metkar et al., 2016).
Serrapeptase has been found to thin the extracellular matrix
thereby reducing the incidence of cancer metastasis (Robert,
2009). Serrapeptase is called the “miracle enzyme”or “super
enzyme”due to its wide range of applications and actions on
the human body (Anil and Kashinath, 2013). Research is still
in progress to find the applications of Serrapeptase in treating
other chronic disorders.
Molecular aspects of Serrapeptase
The molecular weight of Serrapeptase ranges about 45 kDa –
60 kDa (Fig.1). It is a metalloprotease and contains three zinc
atoms as ligands and one active site (Hamada et al., 1996;
Bhagat et al., 2013). The presence of zinc atom is essential
and also enhances the proteolytic activity of Serrapeptase.
The structure of Serrapeptase as containing three zinc ligands
was predicted and confirmed by comparing the structure of
Serrapeptase with thermolysin and Bacillus subtilis neutral
protease (Anil and Kashinath, 2013). The gene encoding
Serrapeptase reveals that it is made up of 470 amino acids.
The amino acid sequence is free of Sulfur containing amino
acids, cysteine and methionine (Matsumoto et al., 1984). The
G+C content of the coding region for the mature protein is
58%(Nakahama et al., 1986). The maximum enzyme activity
of Serrapeptase is observed at pH 9.0 and at a temperature of
40°C. Serrapeptase is degraded or inactivated completely at a
temperature of 55°C (Kaviyarasi et al., 2015).It possesses an
isoelectric point of 5.3 (Matsumoto et al., 1984). It is an active
enzyme that binds to the α-2 macroglobulin in biological
fluids and in blood, it binds in the ratio of 1:1 and this binding
helps mask its antigenicity, retaining the enzymatic activity
(Anil and Kashinath, 2013; Juhi et al., 2015).
The gene for Serrapeptase has been cloned and sequenced
(Nakahama et al., 1986; Braunagel and Benedik, 1990) and
Figure 1 Crystal structure of Serrapeptase generated by
PyMOL software.
334 Production, purification, characterization, immobilization, and application of Serrapeptase
its crystal structure has been determined (Baumann, 1994).
Docking studies reveal that inhibitors of Serrapeptase like
EDTA and Lisinopril show favorable interaction and binding
at the zinc binding site of Serrapeptase with minimal free
energy (Kaviyarasi et al., 2016). Genetic characterization of
Serratia marcescens can be done indirectly by 16s rRNA
sequence analysis, neighbor joining method, phylogenetic
analysis and peptide mass fingerprinting of Serrapeptase.
Peptide mass fingerprinting is used to elucidate the amino
acid sequence of Serrapeptase and can be confirmed by
performing a pairwise alignment (Mohankumar and Raj,
2011). Homogenous purification for the large-scale produc-
tion of Serrapeptase by conventional methods from the
isolated organism is intricate but homogenous preparation is
required for various applications and characterization. Thus,
the versatile rDNA approach is ideal to achieve homogeneous
commercial grade Serrapeptase (Kaviyarasi et al., 2015).
The Serrapeptase gene from Serratia marcescens E-15 was
originally cloned into pTSP26 and expressed in E. coli-JM
103 but the expressed Serrapeptase was found inside the E.
coli cells and not in the culture medium (Nakahama et al.,
1986). In another study, observations showed that the
Serrapeptase gene from Serratia marcescens strain SM6
expressed in E.coli using a lac promoter was secreted into the
medium but as an inactive protein with a marginally higher
molecular weight (Braunagel and Benedik, 1990). In
addition, Serrapeptase gene from Serratia marcescens HR-3
was expressed in E.coli (DE3)/pLysS strain using the
expression vector pET32a (+) and reported that the enzyme
was highly expressed as inclusion bodies and the purified
Serrapeptase was found to be dormant (Tao et al., 2007).
Hence, we can conclude that the problem of secreting
Serrapeptase into the medium using E. coli may be due to the
fact that Serratia marcescens secretion genes are not being
clustered near the Serrapeptase structural gene or due to the
incorrect processing of the protease zymogen, which is
dysfunctional in E. coli (Letoffe et al., 1991).
Recombinant Serrapeptase was produced by cloning
Serrapeptase gene into a pET28b vector and expressing it in
E. coli BL21. This resulted in the absence of inclusion bodies
in the cytoplasm, with the recombinant proteins secreted
properly in the extracellular medium despite a small amount
of it in the intracellular sample (Selan et al., 2015). The
Serrapeptase gene was also cloned into pJET 1.2 cloning
vector and expressed in E. coli DH5-αand proper cloning of
Serrapeptase was observed by sequencing. The sequence
analysis reported the presence of a single Open reading frame
(ORF) comprising of 1464 nucleotides. The sequence
obtained showed 100%homology with serralysin metallo-
protease from Serratia marcescens strain 2170 (Kaviyarasi et
al., 2015). Hence pET28 and pJET series vectors are most
suitable for expression of Serrapeptase in E. coli without
forming inclusion bodies. Serrapeptase gene was also cloned
into pPICZαA Pichia expression vector and electro-
transformed into Pichia pastoris GS115 and maximum
expression was found to be at 72 h Apart from E. coli
expression system, Yeast can also be a favorable alternative
host for the expression of proteins (Kaviyarasi et al., 2016).
Therapeutic properties of Serrapeptase
Anti-inflammatory
Chronic inflammation is an epidemic of the 21st century.
Inappropriate diet, high glucose level, food intolerance, aging
are certain factors influencing inflammation and pain (Sell-
man, 2003). All diseased conditions generate a certain
amount of inflammation which is proportional to the
aggressiveness of the disease. Inflammation provokes the
immune system into exempting and activating the white
blood cells that travel through the circulatory system
annihilating pathogenic bacteria, foreign substances and
cancer cells that it encounters. White blood cells can escape
into organs and tissues during their repair mechanisms and
this accelerated activity of the white blood cells results in
tissue damage (Robert, 2009). Serrapeptase thus helps to
abate the inflammation in arteries that promotes accumulation
of cholesterol and narrowing of the arteries and hence is used
to treat atherosclerosis, artery blocks and other cardiovascular
diseases (Fig.2) (Liver doctor, 2013). Serrapeptase reduces
inflammation in 3 ways: 1. by breaking down insoluble
protein by-products like fibrin, 2. By thinning the fluids
formed during injury which in turn speeds up tissue repair
process, 3. Reducing pain by inhibiting the release of pain-
inducing substances like amines (Sellman, 2003). It can also
modify the cell adhesion molecules that are involved in
guiding inflammatory cells to the site of infection (Klein and
Kullich, 2000). It is orally effective in treating inflammation
caused by laryngitis, catarrhal rhino-pharyngitis, sinusitis,
Figure 2 Properties of Serrapeptase.
Selvarajan Ethiraj and Shreya Gopinath 335
breast engorgement, carpal tunnel syndrome, inflammation in
prostate gland, acute and chronic ear-nose-throat disease, and
chronic emphysema (Tachibana et al., 1984; Vicari et al.,
2005; UmaMaheswari et al., 2016).
Analgesic
Serrapeptase reduces pain by restricting the inflamed tissues
from releasing pain-inducing amines such as bradykinin
(Mazzone et al., 1990). It can also hydrolyze bradykinin,
histamine and serotonin, which are responsible for oedemic
responses (Malshe, 2000). Docking studies have revealed that
the substrate bradykinin that binds near the zinc binding site
of Serrapeptase can be effectively inhibited by cleaving the
peptide bonds of bradykinin (Kaviyarasi et al., 2016). It is
used in treating various diseases as an alternative to
salicylates, ibuprofen and other NSAIDs (Non-steroidal
anti-inflammatory drugs) (Aso et al., 1981).
Fibrinolytic
Fibrin belongs to a category of proteins that are naturally
adept in repairing damage occurring from trauma, surgery and
injuries by replacing the old cells with new cells, tissues, and
muscles (Jickling et al., 2010). When a tissue is damaged, the
blood vessels secrete a compound called thromboplastin and
simultaneously the platelets adhere to the broken blood
vessels releasing platelet factor. Now both thromboplastin
and platelet factor react with calcium ions and other factors to
form prothrombin activator that is converted into insoluble
fibrin which accumulates as a clot in the body obstructing the
blood flow, oxygen supply to tissues, causing myocardial
infarction, strokes in the brain, pulmonary emboli and
thrombi in veins (Guyton, 1974). Hence dissolving the clot
is necessary to avoid significant risk of damage to the body
and this is where these fibrinolytic enzymes play an important
role.
Serrapeptase has the ability to digest non-living tissues
such as mucous, plaques and blood clots. Since it has the
ability to degrade insoluble protein like fibrin without
harming other living tissue, it is designated as a fibrinolytic
enzyme (Klein and Kullich, 2000). Serrapeptase possesses
the ability to dissolve and reduce arterial plaques, fatty
cholesterol, calcium and other foreign protein substances
from sticking to arterial walls. Serrapeptase is used as an
alternative to chelation therapy as it is more effective than
EDTA-mediated chelation for removing arterial plaques by
dislodging the excess fatty deposits in the arteries allowing
optimum blood flow which lowers the blood pressure and
arterial resistance (Nieper, 2010). Serrapeptase, therefore,
helps people with limited mobility such as those affected by
orthostatic hypotension, which is characterized by a drop-in
blood pressure brought about when a person changes their
body position (Liver Doctor, 2013).
Anti-pathogenic agent
Bacteria create a biofilm within the microbiome beneath
which they thrive. Staphylococcus aureus possesses a number
of virulence factors and has the ability to invade eukaryotic
cells and forms surface biofilms causing staphylococcal
infections. Blocking S. aureus colonization may reduce the
incidence of invasive infectious diseases. The anti-infective
properties of Serrapeptase can be used in impairing
staphylococcal properties like attachment to inert surfaces
and invasion on eukaryotic cells. But the exact mechanism of
action is yet to be elucidated (Selan et al., 2015). Thus, the
anti-biofilm efficacy of Serrapeptase may enhance the
antibacterial effects on staphylococcal infections. Serrapep-
tase has antibacterial activity on Escherichia coli and
Pseudomonas aeruginosa with a zone of clearance of
15mm and 12mm respectively and the maximum antibacterial
effect seen in dialysis based partial purification of Serrapep-
tase (Devi et al., 2013).
Sources of Serrapeptase
Serrapeptase is produced by a variety of microorganisms
isolated from different sources (Table 1). Soil and contami-
Table 1 Bacterial strains that produce Serrapeptase
Sl. No. Name of the bacteria Strain Source References
1Serratia marcescens E 15 Intestine of silkworm Anil et al., 2013
ASerratia marcescens VITSD2 Soil Robert et al., 2009
3Serratia marcescens NRRL B-23112 Soil Salamone et al., 1997
4Serratia sp. ZF03 Hot springs Salarizadeh et al., 2014
5Serratia marcescens NCIM 2919 Not mentioned Wagdarikar et al., 2015
6Bacillus licheniformis NCIM 2042 Not mentioned Wagdarikar et al., 2015
7Serratia marcescens SRM Flowers of summer squash Kaviyarasi et al., 2016
8Streptomyces hydrogenans var. Not mentioned Mangrove soil Nageswara et al., 2016
9Streptomyces hydrogenans MGS1 Soil Vanama et al., 2014
10 Serratia marcescens P3 Soil Bach et al., 2012
336 Production, purification, characterization, immobilization, and application of Serrapeptase
nated water are a rich source of a diverse variety of
microorganisms. Isolated pure cultures of the bacterial strains
that produce Serrapeptase are maintained on nutrient agar
plates and stored at 4°C (Devi et al., 2013). Serrapeptase was
first isolated from Serratia marcescens strain E-15 found in
the gut of the silkworm Bombyxmori (Sellman, 2003).
Serratia marcescens is a Gram-negative bacterium that
belongs to Enterobacteriaceae family that can grow in a wide
range of temperatures (5–40°C) and pH (5.0–9.0) and secretes
a variety of enzymes such as serine and thiol proteases,
metalloproteases, lipases, chitinases, hemolysin, and
nucleases (Jayaratne, 1996). Serratia marcescens can be
differentiated from other Gram-negative bacteria by its ability
to hydrolyze casein (Stancu, 2016). It is well known for
producing a cell-associated, red pigment called prodigiosin,
which resembles human blood. Factors such as medium
composition and oxygen supply, affect the production of
prodigiosin and incubation at 37°C inhibit the pigmentation
which makes it tough to identify it in a pool of bacteria
(Gerber, 1975). It is pathogenic to both humans and plants,
involved in food spoilage (Abdou, 2003) and acts as a
powerful insecticide (Salamone and Wodzinski, 1997). It
promotes plant growth by inducing resistance against plant
pathogens (Kloepper et al., 1993), producing antagonistic
substances (Queiroz and Melo, 2006) and solubilizing
phosphate molecules (Tripura et al., 2007).
Production of Serrapeptase
Strains producing Serrapeptase especially Serratia marces-
cens are usually cultured in trypticase soy broth (Fig.3). A
medium containing carbon source- maltose, organic nitrogen
source- peptone, inorganic nitrogen source- ammonium
sulfate, dihydrogen phosphate, sodium bicarbonate, inorganic
salt source- sodium acetate, glycerin and ascorbic acid can be
used as a production medium and this medium yielded about
27.36 U/ml (Badhe et al., 2009). Another medium reported
for production of Serrapeptase contained maltose 45 g/l,
soybean meal 65 g/l, KH
2
PO
4
8.0 g/l, and NaCl 5.0 g/l at a pH
7.0 which gave a maximum yield of 32575EU/mg. This
maximum yield was due to presence of maltose as carbon
source (Pansuriya and Singhal, 2010). A combination of
tryptic soy broth (30 g/l) and skim milk (5%w/v) (TSB-SM)
medium can also be used which is equal to Serrapeptase
production using glucose minimal medium for 48 h with
subsequent addition of 10%(w/v) skim milk at intervals of 12
h (Salamone and Wodzinski, 1997). Casein medium can also
be used but trypticase soy is a preferred substrate over casein
as the specific activity is higher when trypticase soy is used as
the substrate in the production medium (Devi et al., 2013).
Another medium containing tryptone, yeast extract, glycine,
sodium chloride, skim milk 1%(w/v) and 0.5%(w/v) glucose
is used for Serrapeptase production. Induction of the
Serrapeptase enzyme was due to the presence of Skim milk
and glucose (Romero et al., 2001). Feather meal broth can
also be used for enzyme production that contains feather
meal, sodium chloride, KH
2
PO
4
and K
2
HPO
4
(Bach et al.,
2012). The enzyme produced can be filtered using filter paper
and stored at 4°C for further use (Mohankumar and Raj,
2011).
Growth curve analysis of Serrapeptase shows that
Serrapeptase production is observed at 12 h of growth time
and maximum production can be observed at 48 h of growth
time (Devi et al., 2013). The medium used for production of
Serrapeptase by Streptomyces hydrogenans contains soya
bean meal, glucose, glycerol, CaCO
3
, tryptone and KH
2
PO
4
(Vanama et al., 2014). Horse gram (Microtylona uniflorum)is
one of a few low-cost substrates for the production of
Serrapeptase by Streptomyces hydrogenans var.under solid
state fermentation conditions (Nageswara et al., 2016). The
enzyme produced is expressed in terms of units/ml using a
special standard curve (Ammar et al., 1998; Mohankumar and
Raj, 2011). The medium used for production of Serrapeptase
by Bacillus licheniformis contains glycerin, glucose, tryp-
tone, ammonium oxalate, sodium acetate, disodium
hydrogen phosphate and ammonium sulfate at a composition
Figure 3 Industrial processing of Serrapeptase.
Selvarajan Ethiraj and Shreya Gopinath 337
of 10 g/l each maintained at a pH of 7.5 to get a yield of
22.85 IU/ml (Wagdarikar et al., 2015). The growth medium
used for production of Serrapeptase in yeast is Minimal
medium with Histidine (MMH) and the production medium
used is minimal medium with Glycerol and Histidine
(MGYH) and a maximum yield of 0.6 mg/ml was obtained
using these medium (Kaviyarasi et al., 2016).
Purification of Serrapeptase
Partial purification of the enzyme can be performed by
ammonium sulfate precipitation, dialysis, ultra-filtration,
aqueous two-phase systems, High-Performance Liquid Chro-
matography (HPLC) etc. (Bach et al., 2002; Devi et al.,
2013). Serrapeptase can also be purified using ultrasound
assisted three phase partitioning method, which not only
purifies the enzyme but also concentrates it. This method has
several advantages like single step purification, easy scale-up,
economical and accounts for about 96%recovery of
Serrapeptase with a 9.4-fold degree of purification in 5 min
of process time under optimal conditions, 30%w/v
ammonium sulfate concentration, 1:1 t-butanol to crude
ratio, pH 7.0, 0.05 W/cm
2
ultrasonic intensity, 25 kHz
frequency and 20%duty cycle (Pakhale and Bhagwat, 2016).
The activity of Serrapeptase, estimated by gelatin clearing
zone increased from 24mm zone of clearance to 36mm zone
of clearance after purification of the enzyme by ammonium
sulfate precipitation (Anil and Kashinath, 2013). Casein assay
showed that specific activity of Serrapeptase in the crude
enzyme, precipitated and dialyzed samples to be 12.00 U/ml,
21.33 U/ml, and 25.7 U/ml respectively, with a maximum
purification fold of 2.1 in dialyzed samples followed by a 1.8-
fold purification in precipitated samples and a 1-fold
purification in the crude samples and hence proves that
partial purification by ammonium sulfate precipitation and
dialysis gives better enzyme activity than the crude enzyme
(Devi et al., 2013). Another study showed a maximum
purification fold of 5.7 for dialysis followed by 3.8 for
acetone fractionation, 1.8 for ammonium sulfate precipitation
and a 1-fold purification for crude samples (Salamone and
Wodzinski, 1997). A 2013 study reported a maximum
specific activity of ammonium sulfate precipitated Serrapep-
tase to be 63623 EU/mg and dialysis purified Serrapeptase to
be 190451 EU/mg (Ayswarya et al., 2013). Recombinant
Serrapeptase was purified in a single step using Nickel-NTA
based affinity chromatography in which His
6
tag helps in
purification and the yield of the purified Serrapeptase was
found to be 0.6mg/ml (Kaviyarasi et al., 2016). Hence, we can
conclude that dialysis is the best method for partial
purification of Serrapeptase. Complete purification of the
enzyme can be achieved by Chromatographic methods
among which reverse phase HPLC is so far used. Reverse
phase HPLC can be used for purification and is regarded as a
versatile, accurate, robust and the precision values lay under
the ICH guidelines of validation. This method resulted in a
100.23%- 100.71%recovery of the Serrapeptase (Patel et al.,
2015). Another study of Reverse phase HPLC purification
showed a percentage recovery of 98.9%to 99.5%(Reddy
et al., 2015). Hence chromatographic techniques can be used
for high rate of purification of enzymes.
Detection and determination of activity of
Serrapeptase
Serrapeptase can be estimated by widely used methods such
as reverse phase HPLC, UV based methods, radioimmunoas-
say, Lowry’s assay (Lowry et al., 1951), Bradford assay etc
(Miyata et al., 1970). Proteolytic activity of Serrapeptase can
be detected by skim milk agar plate method where a clear
zone formation indicates the enzyme has proteolytic activity
(Salarizadeh et al., 2014). Proteolytic activity of Serrapeptase
can also be distinguished by well diffusion method in which
culture filtrates will be added in wells in the agar medium and
stained and de-stained to visualize a clear zone around the
well (Devi et al., 2013). Serrapeptase has absorption maxima
at 275-280 nm (Anil and Kashinath, 2013). The enzyme
activity of Serrapeptase can be determined by casein protease
assay or by gelatin clearing zone assay (Salamone and
Wodzinski, 1997). The enzyme’s kinetic parameters, K
m
and
V
max
values can be determined by Lineweaver-Burk plot
according to Michaelis-Menten kinetics and were found to be
0.00105 mg/ml and 0.0531 mM/min, respectively (Salariza-
deh et al., 2014). HPLC can also be used to detect the
presence of Serrapeptase. The enzyme extract with a
maximum retention time of 3.45 min was observed by this
method (Devi et al., 2013). Reverse phase HPLC was used for
estimation of enzyme and a correlation co-efficient of 0.998
was obtained in this study which is closely equal to 1 and
hence suggests good concentration of purified enzyme (Patel
et al., 2015). Another study evaluating the activity of
Serrapeptase by Reverse phase HPLC showed a regression
co-efficient of 0.998, limit of detection at a concentration of
3.33 µ/ml, limit of quantitation of 10.9 µ/ml (Reddy et al.,
2015).When trypticase soy broth is used as the substrate, the
maximum specific activity of Serrapeptase is found to be
60.7U/mg with a clearance zone of 23mm on a skim milk agar
plate (Devi et al., 2013) but when casein is used as the
substrate, specific activity of Serrapeptase is found to be
0.65U/ml (Subbaiya et al., 2011). Serrapeptase was estimated
in formulations by using microplate readers which use the
principle of vertical photometry. A linear relationship was
observed between Serrapeptase concentration and absorbance
at 230 nm with the co-efficient of regression being 0.9911,
percentage recovery was found to be 97%-98%, abiding the
standard limits, low standard deviation of ±0.020 to ±0.044
which confirms the method to be precise, accurate and free
from any positive or negative interference of the excipient
(Sandhya et al., 2008). The presence of Serrapeptase was
338 Production, purification, characterization, immobilization, and application of Serrapeptase
detected using a zymogram which produced a clearance zone.
The recombinant Serrapeptase had an enzyme activity of 30
U/ml and specific activity of 50 U/mg (Kaviyarasi et al.,
2016).
Optimization studies on production of
Serrapeptase
The media composition, temperature, pH, and other condi-
tions can be optimized for increased yield of Serrapeptase
with better enzymatic activity.
Effect of media composition
Maximum production of Serrapeptase can be obtained when
both tryptone and yeast extract are added to the medium, in
the absence of glucose is absent in the medium (Anil and
Kashinath, 2013). The richest source of carbon is glucose for
Serratia marcescens, both Glycerin and Maltose for Bacillus
licheniformis. The best nitrogen source is tryptone for both
Serratia marcescens and Bacillus licheniformis.After
optimizing the media for Bacillus licheniformis the concen-
tration of Serrapeptase increased from 16.52 IU/ml to 22.85
IU/ml (Wagdarikar et al., 2015). In optimizing batch process
study, the production medium containing tryptone, together
with maltose as carbon source gave a maximum activity of
36,415 EU/mg at the 68th hour (Ayswarya et al., 2013). The
suggested amino acid for maximum yield of Serrapeptase is
valine. The addition of any acids would lead to inhibition of
Serrapeptase production (Mohankumar and Raj, 2011).
A surface response method of media components was
considered to enhance Serrapeptase yield by Streptomyces
hydrogenans MGS13. Response surface method is an
empirical statistical modeling technique used for multiple
regression analysis to solve multi-variable equations simulta-
neously. Here, the medium for maximum production was
optimized by ‘one-variable-at-a-time approach by studying
effects of dextrose, soybean meal as substrate variables, pH,
inoculum level. The coefficient of determination (R
2
) was
estimated to be 0.9559 for Serrapeptase production which is
statistically significant as R
2
lies from 0 to 1 and 0.9559 is
nearly equal to 1 which implies that the model is valid.
Maximum Serrapeptase production of 254.65 IU/ml was
observed with dextrose and soybean meal concentrations of
2.04 (%w/v) and 2.09 (%w/v) respectively (Vanama et al.,
2014). Another study used 1%soybean meal as optimal
nitrogen source to obtain a maximum yield of 85U/gds from
Streptomyces hydrogenans (Nageswara et al., 2016). The
Gelatin Clearing Zone (GCZ) exhibited by Serrapeptase
produced from Serratia marcescens shows maximum Serra-
peptase production with a clearing zone of 36mm at gelatin
concentration of 0.5%w/v and higher or lower concentrations
of gelatin results in decrease in Serrapeptase production
(Mohankumar and Raj, 2011). 5 g of horse gram is the
optimal substrate concentration for maximum Serrapeptase
yield of 85 U/gds from Streptomyces hydrogenans (Nages-
wara et al., 2016).
Effect of agitation and aeration
Aeration and agitation rates are both key parameters in the
fermentative production of Serrapeptase. The maximum
specific productivity, 78.8 EU/g maltose/hour, has been
obtained at the optimum fermentation conditions of 400
rpm agitation and 0.075 vvm aeration with the maximum
yield of 11580 EU/ ml (Ruchir et al., 2011) which is the
highest yield to date (Decedue et al., 1979; Miyazaki et al.,
1990).
Effect of temperature
Temperatures between 32°C (Mohankumar and Raj, 2011)
and 37°C (Anil and Kashinath, 2013) show maximum
Serrapeptase production from Serratia marcescens. Hence
32°C –37°C is generally the favorable temperature for
maximum Serrapeptase production. Temperatures below or
above this range decreases the yield (Anil and Kashinath,
2013). The optimum temperature for maximum Serrapeptase
production from Bacillus licheniformis is about 35°C
(Wagdarikar et al., 2015). Serrapeptase is stable up to 42°C
and activity of the enzyme rapidly decreases above 42°C
(Salamone and Wodzinski, 1997). Serrapeptase is optimally
active in the range of 50°C-55°C, and at 45°C it retained 85%
of its enzyme activity and declined to 25%at 60°C
(Salarizadeh et al., 2014). In a comparative study of exposing
Serratia marcescens to different temperatures of 28°C, 32°C,
37°C, and 40°C, it was observed that activity of enzyme
retained till a maximum temperature of 32°C (Manal, 2015).
Effect of pH
Optimum pH for maximum Serrapeptase production from
Serratia marcescens is 5.0 to 9.0 with phosphate buffer as the
best buffer and a notable decline in productivity can be seen at
both higher and lower pH values from the optimum pH value
(Mohankumar and Raj, 2011). Even at pH 9.0, the enzyme is
in its active form and is stable (Salarizadeh et al., 2014).
Another study found that the optimum pH for maximum
Serrapeptase production from Serratia marcescens was 7.3
(Anil and Kashinath, 2013). Optimum pH for maximum
Serrapeptase production from Bacillus licheniformis was 6.5
(Wagdarikar et al., 2015). Optimal pH for maximum
Serrapeptase yield of 85 U/gds from Streptomyces hydro-
genans was found to be 6.5-7.0 (Nageswara et al., 2016).
Effect of incubation period
Incubation time is very important in determining the yield and
activity of any enzyme. The optimum incubation period for
Serrapeptase production from Serratia marcescens varies
Selvarajan Ethiraj and Shreya Gopinath 339
from 24 h (Mohankumar and Raj, 2011) to 25 h (Anil and
Kashinath, 2013). The optimal incubation duration for
maximal Serrapeptase production from Bacillus Lichenifor-
mis is 24 h (Wagdarikar et al., 2015).
Effect of mutations
Mutations can be induced by different means and the easiest
way is by exposure to UV light. In a study, Serratia
marcescens isolates were exposed to UV light for different
time intervals of 20, 40, 60seconds and was observed that the
maximum hydrolysis of casein was at 20 s of UV exposure at
32°C (Manal, 2015). Another study confirmed that the
maximum Serrapeptase activity of 1575.3 EU/ml was
observed at 20 s of UV exposure (Ayswarya et al., 2013).
Streptomyces also are regarded as an efficient producer of
Serrapeptase. Streptomyces isolates were subjected to nitrous
acid treated chemical mutation and observed a maximum
activity of 60.1%higher than wild-type strain. When the same
strain was subjected to UV mutation with a UV lamp of 220
V, 40 W, 50 Hz with the exposure time of 0, 30, 60, 90, 120,
150, 180, 240 and 360 s, it was observed that UV mutant
exhibited 33.9%higher activity than the wild-type strain
(Vanama et al., 2014).
Immobilization of Serrapeptase
Immobilization of drugs and other biological agents these
days are used for targeted drug delivery. Immobilized
enzymes have several advantages like the fact that they can
be recycled easily, removed easily from the reaction mixtures,
minimal amount of enzyme is lost in the reaction mixture,
have greater thermal stability (Yu et al., 2012). The idea of
immobilization emerged from the advent of nanotechnology.
Magnetic nanoparticles are widely used for immobilization
of enzymes. Magnetic nanoparticles have several advantages
over other materials including the fact that they are easy to
prepare, come in a wide range of sizes, chemically
modifiable, superparamagnetic in nature, possess large sur-
face area, low mass transfer, highly active, inert,and highly
stable (Kumar et al., 2010; Krukemeyer et al., 2012; Verma
et al., 2013).
MNPs can be removed easily from the body through a
process called opsonization (Chen et al., 2011). Some
magnetic nanoparticles (MNPs) used for immobilization of
Serrapeptase are Fe
3
O
4
nanoparticles, carboxyl-functiona-
lized magnetic nanoparticles, Amino-functionalized magnetic
nanoparticles (Namdeo and Bajpai, 2009), chitosan/glutar-
aldehyde MNPs, 3-amino propyltriethoxysilane (APTES)
magnetic nanoparticles etc. (Kumar et al., 2014). Other
materials used for enzyme immobilization include Gold, ionic
fluids, albumin, streptavidin, polymer-coating cellulose,
dextran, silica (Bi et al., 2009; Ziv-Polat et al., 2010; Yu et
al., 2012). Alginate based microspheres encapsulated with
Serrapeptase have also proved to be very efficient in wound
healing therapies (Rath et al., 2010).
When Serrapeptase was immobilized using ethyl cellulose
microparticles, the entrapment efficiency was found to be
85%and the yield, calculated using the weight of the raw
materials and the microparticles obtained was found to be
96%. Serrapeptase undergoes metabolism causing gastro-
intestinal disturbance and systemic toxicity which can be
overcome by using a transdermal drug delivery system. In this
system, lipid-based transferosomes are used to delivery
Serrapeptase with greater encapsulation of about 90%due
to the presence of more cholesterol. It showed a greater tensile
strength of 2.95±0.71 to 2.98 ±0.89 kg/cm
2
and hence
showed no risk of cracking.The drug release was slow and
controlled when compared to percentage release from an
aqueous solution of Serrapeptase (Pravin et al., 2015). When
Serrapeptase was immobilized with carboxyl- functionalized
magnetic nanoparticles, the kinetic parameters K
m
increased
from 0.096 mg/ml to 0.121 mg/ml, V
max
decreased from
0.061 to 0.045 µmol/min. Serrapeptase immobilized on
carboxyl functionalized magnetic nanoparticles was found to
be better than amino-functionalized magnetic nanoparticles
with a yield of 115.78 mg protein/g (Kumar et al., 2013).
When Serrapeptase was immobilized on Fe
3
O
4
magnetic
nanoparticles, the kinetic parameter, Km of free and
immobilized enzyme was found to be 0.078mg/ml and 0.1
mg/ml respectively showing a significant increase in Km of
Serrapeptase after immobilization due to factors like change
in structure of enzyme, steric hindrance, effects of diffusion
etc., (Zhu et al., 2009). In vivo studies suggest that magnetic
targeting has the potential to increase the permeation of
Serrapeptase through the membrane and also enhance the
anti-inflammatory effects in carrageenan induced paw edema
in rats even at very less dose ofimmobilized enzyme than
required. V
max
for immobilized enzyme also decreased from
0.064 µmol/min to 0.055 µmol/min respectively and relative
activity for immobilized enzyme was found to be 67.875.
Studies have also found that immobilization of Serrapeptase
does not affect the crystallinity and has very less effect on the
size and magnetic properties of nanoparticles used (Kumar
et al., 2013).
Pharmacokinetic studies
Serrapeptase is usually administered orally. Serrapeptase is
considered a systemic enzyme in humans as they are absorbed
by the intestines and then is directly released into the
bloodstream (Moriya et al., 1994). Absorption studies on
proteolytic enzymes have confirmed that they are absorbed
intact into the bloodstream in its active form and are actively
transported through the gut wall (Ambrus et al., 1967).
Serrapeptase, when used therapeutically may lead to low
bioavailability due to low membrane permeability and
enzymatic degradation (Woodley, 1994). At an oral dose of
340 Production, purification, characterization, immobilization, and application of Serrapeptase
100 mg/kg, Serrapeptase can be detected in the serum and
lymph of rats at a concentration of 0.87 +/-0.41ng/mL and
43 +/-42ng/mL respectively. This was measured by an
enzyme immunoassay technique with a maximum time
interval of 15-30 min.This study reveals that Serrapeptase is
absorbed by the intestinal tract and transferred to the
inflammatory site via blood or lymph in an enzymatically
active state (Moriya et al., 1994). To improve the oral
absorption, a liposomal formulation of Serrapeptase can be
used as liposomal mediated delivery shows a 50%increase in
Serrapeptase entrapment efficiency but the permeation value
in terms of a log was found to be -7.72cm/s which is lower
than the Biopharmaceutics Classification System (BCS)
classification (Ruell et al., 2002; Sandhya et al., 2008).
Clinical studies of Serrapeptase
The potential effects of Serrapeptase in different disease
conditions have been tested on both animals and humans
(Fig.4, Tables 2 and 3).
Contraindications
Since Serrapeptase is very effective at degrading fibrin, there
may be increased bleeding when taken with natural agents
like turmeric, garlic, certain oils like fish oil and chemical
agents like Aspirin and Warfarin. Serrapeptase cross reacts
with statins, NSAIDs, anticoagulants, anti-platelet agents,
heparin and heparinoids (Bhagat et al., 2013).
Formulation of Serrapeptase
Serrapeptase, being an enzyme, can be degraded easily by
other digestive enzymes in the stomach and gastrointestinal
tract. Therefore, Serrapeptase is manufactured as enteric
coated tablets. The enteric coating is basically a polymer
Figure 4 Pre-clinical and clinical studies of Serrapeptase.
Selvarajan Ethiraj and Shreya Gopinath 341
Table 2 Pre-Clinical studies
Sl. no Disorder Dosage Time(d) Study animal Outcome Reference
1 Gingival infection STP (20mg/kg) +4
antimicrobial
(100mg/kg orally)
Not mentioned Rat Serrapeptase enhanced the activity and concentration of antimicrobials used Aratani et al., 1980
Sub-acute bronchitis 20mg/kg Not mentioned Rabbit Reduced viscosity of sputum Kase et al., 1982
Pleuritis and pneumonitis STP +Cefotiam 30min after
injection
Rabbit Serrapeptase increases plasma concentration of cefotiam Ishihara et al., 1983
Peri-prosthetic infection STP +antibiotic 14 days Rat Serrapeptase eradicated infection caused by biofilm- forming bacteria and
increased antibiotic efficacy. In 5.6%cases, the infection persisted
Mecikoglu et al.,
2006
Hind paw oedema and
granuloma
STP 3 hours Albino rats Serrapeptase showed enhanced anti-inflammatory activity Viswanatha et al.,
2008
paw oedema 3mg/kg 3 hours Rats Serrapeptase reduced inflammation and reduced oedema by 44%Mundhava et al.,
2016
Chronic paw oedema 20mg/kg 8 hours Albino rats Serrapeptase significantly inhibited inflammation induced by formalin by 68%Jadav et al., 2010
Insulin amyloid 0.55mg/ml 2-32 hours Zebrafish Serrapeptase degraded the amyloid fibrils formed by insulin Metkar et al., 2016
10 ulcerative colitis 1.3mg/kg Not mentioned Albino female
mice
Serrapeptase showed significant anti-inflammatory activity Rajinikanth et al.,
2014
11 Chronic respiratory disease 2 g/litre 3days Broiler chicks Serrapeptase significantly decreased the total cholesterol, serum LDH and the
inflammatory markers CRP and ESR, better immunological response to NDV
vaccination and decreased the shedding of Mycoplasma gallisepticum and E. coli
El-Hamid et al.,
2014
12 Alzheimer’s Disease 10.800 U/ kg b.wt and
21.600 U/ kg b. wt
45 days Albino rats Histopathological investigation of Alzheimer’s induced rat brain tissue showed
the disappearance of most of the amyloid plaques after Serrapeptase treatment
and also decreased cholinesterase activity, TGF- β, IL-6 and P53 levels
accompanied with a significant increase in Bcl-2 level
Ahmed et al., 2014
13 Alzheimer’s disease 17 mg/kg b.wt 45 days Albino rats Serrapeptase decreased brain AchE activity, TGF-b, Fas and IL-6 levels and
increased the expression levels of ADAM9 and ADAM10 genes in the brain
tissue of the treated rats, which are factors of Alzheimer’s disease
Fadl et al., 2013
342 Production, purification, characterization, immobilization, and application of Serrapeptase
Table 3 Clinical Studies of Serrapeptase
Sl.no. Disorder Dosage Time (days) No. of patient Study design Outcome Reference
1 Osteo-articular infection STP 30mg/d +
sulbenicillin
6 8 Open Reduced the inflammation by transferring sulbenicillin to cell
exudates
Okumura et al., 1977
2 Chronic respiratory
disease
10mg, thrice daily 14 376 (128 on STP)Random, double-blind No significant improvement Nagaoka et al., 1979
3 Post-operative buccal
swelling
30mg/d 7 174 (88 on STP) Random, double-blind Significant reduction in swelling of the buccal cavity Tachibana et al., 1984
4 Secretory otitis media 0.5mg/d 10 75 Random, double-blind Tests unclear Bellussi 1984
5 Chronic respiratory
disease
30mg/d 7 40 Random Relaxation of sputum elasticity was observed Shimura et al., 1983
6 Chronic sinusitis 30mg/d 30 Not mentioned Open Reduction in the viscosity of mucus was seen Majima et al., 1988
7 Breast engorgement 30mg/d 3 70 Random, double-blind Improvement in breast pain, swelling Kee et al., 1989
8 Lung cancer patients
undergoing thoracotomy
20mg Thrice daily Not mentioned 35 (18 on STP) Random, open STP group patients had more amounts of antibiotics in their
tissues
Koyama et al., 1986
9 Post-operative swelling 5mg thrice daily 5 40 Not given No significant improvement Surachai et al., 1981
10 Post-operative and
traumatic swelling
Not given Not clear 66 Random The pain disappeared on the 10
th
day of treatment and swelling
reduced by 50%
Esch et al., 1989
11 Post-operative and
traumatic swelling
Not given Not clear 98 Random, double-blind Reduced swelling Tsuyama et al., 1977
12 Superficial thrombo-
phlebitis
30mg/d 14 40 Random 65%patients taking STP showed a reduction in pain and
improvement in other symptoms like erythema
Bracale et al., 1996
13 Carpal tunnel syndrome 10mg twice daily 44 20 Prospective trial 65%of patients showed improvement in symptoms Panagariya et al., 1999
14 Acute and chronic ear,
nose, and throat disorder
30mg/d 7-8 193 Random, double-blind Reduced pain, amount of secretions, difficulty in swallowing and
nasal obstruction
Mazzone et al., 1990
15 Chronic airway disease 30mg/d 30 29 (15 on STP) Random, open Reduced solid component, viscosity, the elasticity of sputum and
sputum neutrophil count
Nakamura et al., 2003
16 Perennial rhinitis, chronic
rhinitis
STP +Cephalexin Not mentioned 93 Random, double-blind rhinorrhea, nasal stuffiness were reduced due to STP intake Brewer science., 1999
17 Removal of mandibular
third molars
5mg STP +1000mg
paracetamol
7 24 Individual, random,
double-blind
Reduction in cheek swelling and pain Khateeb et al., 2008
18 Post-operative swelling
after removal of molars
5mg thrice daily 5 40 Not given No improvements in symptoms Chopra et al., 2009
19 Bronchitis,
pneumonia,
bronchial
Asthma
30mg/d 14 140 (69 on STP) Random, double-blind. Serrapeptase was found to be effective and safe expectorant in
controlling the sticky sputum formation
Kai-sheng et al., 2009
20 Upper and lower limb, soft
tissue trauma
30mg/d 14 100 (50 on STP) Prospective trail Serratiopeptidase showed a significant anti-inflammatory effect
and mild analgesic effect
Garg et al., 2012
Selvarajan Ethiraj and Shreya Gopinath 343
which is sensitive to pH i.e., these tablets remain intact in
acidic pH of the stomach and the gastrointestinal tract and
gets dissolved in the alkaline pH of the small intestine
(Bodhankar et al., 2011). Liposomal formulations of
Serrapeptase are used as effective oral drug delivery systems
as it has increased permeability and hence can increase oral
absorption of Serrapeptase (Sandhya et al., 2008). Lipid-
based transferosomes are also one mode of enzymatic carriers
of drugs that are studied recently (Pravin et al., 2015).
Dosage of Serrapeptase
Serrapeptase doses usually ranges from 10 mg to 60 mg per
day. Most pharmaceutical firms formulate the drug dosage to
be 10mg, taken 2-3 times a day. 10mg corresponds to about
20000 enzyme units of Serrapeptase. Serrapeptase must be
taken on an empty stomach. Also, the person should not
consume any food up to half an hour after taking Serrapeptase
(Bhagat et al., 2013). If it is consumed with a meal, then the
body will utilize it to digest the food (Liver Doctor, 2013). It
is to be ensured that Serrapeptase should be double dosed.
Safety of Serrapeptase
There are limited adverse drug reactions reported so far for
Serrapeptase. They include skin conditions like dermatosis,
dermatitis, erythema, muscle and joint aches, coagulation
abnormalities (Mazzone et al., 1990). There may also be
certain gastric related issues like nausea, anorexia, stomach
upset, cough, pneumonitis (Sasaki et al., 2000). Serrapeptase
may also cause granulomatous hepatitis (only 1 case reported
so far), acute eosinophilic pneumonia (Dohmen et al., 1998;
Sasaki et al., 2000). Serrapeptase may induce hemorrhage and
hence while consuming this drug or any proteolytic drugs to
prevent thrombosis, bleeding risk should be taken into
consideration (Celik et al., 2013). It has no inhibitory effects
on prostaglandins and is free from serious effects like
stomach ulceration, joint destruction, kidney problems,
stomach upset, psychiatric problems (Sellman, 200). A 69-
year-old man developed acute renal failure following
treatment with diclofenac/Serrapeptase which also led to leg
swelling and tests revealed pedal edema with concentration of
urea 99 mg/dL, plasma creatinine 9.4 mg/dL, plasma sodium
133 mEq/L and plasma potassium 4.9 mEq/l, reduced urinary
sodium and osmolality and hence Serrapeptase was with-
drawn (Dhanvijay et al., 2013).
Conclusion and future perspectives
Enzymes from microbial sources play a vital role in pharma
and health care. Serrapeptase is being used in many clinical
studies against various diseases for its anti-inflammatory,
fibrinolytic, anti-bacterial and analgesic effects. It is also
recommended for preventing cardiovascular blocks due to its
fibrinolytic activity. Although many bacteria produce Serra-
peptase, Serratia marcescens is the best producer of
Serrapeptase. Many studies on production strategies, purifi-
cation, optimization studies and immobilization of the
enzyme have been studied and reported to be successful
and efficient. Preclinical studies on various animal models
and clinical studies have shown significant effects for various
diseases. Absorption study confirms that the Serrapeptase is
absorbed through the intestines and released into the blood-
stream. Serrapeptase may have side effects if taken along with
anti-fibrinolytic agents and hence care must to take when
consuming Serrapeptase. Serrapeptase is manufactured as
enteric coated tablets to avoid getting degraded by other
digestive enzymes. When Serrapeptase is used to treat any
disorder, it should never be consumed along with meals, as it
will be utilized to digest food. Data regarding the safety and
drug interaction studies of the enzyme is insufficient to use it
as a health supplement. Data and research about the anti-
atherosclerotic activity, safety, tolerability, efficacy and
mechanism of action of the Serrapeptase are still required to
be widely accepted for treating various diseases.
Acknowledgments
The authors would like to thank SRM University, Chennai, India for
supporting and performing the study. SG is obliged to the management
of SRM for partial completion of masters in Genetic Engineering.
Compliance with ethics guidelines
E. Selvarajan and Shreya Gopinath declare that they have no conflicts of
interest. This manuscript is a review article and does not involve a
research protocol requiring approval by the relevant institutional review
board or ethics committee.
References
Abdou A M (2003). Purification and partial characterization of
psychrotrophic Serratia marcescens lipase. J Dairy Sci, 86(1): 127–
132
Ahmed H H, Fadl N N, El- Shamy K A, Hamza H A (2014). Miracle
enzymes serrapeptase and nattokinase mitigate neuroinflammation
and apoptosis associated with Alzheimer’s disease in experimental
model. World J Pharm Pharm Sci, 3(2): 876–891
Aiyappa P S, Harris J O (1976). The extracellular metalloprotease of
Serratia marcescens: I. Purification and characterization. Mol Cell
Biochem, 13(2): 95–100
Al-Khateeb T H, Nusair Y (2008). Effect of the proteolytic enzyme
serrapeptase on swelling, pain and trismus after surgical extraction of
mandibular third molars. Int J Oral Maxillofac Surg, 37(3): 264–268
Aldridge S (2013). Industry backs biocatalysis for greener manufactur-
ing. Nat Biotechnol, 31(2): 95–96
Ambrus J L, Lassman H B, DeMarchi J J (1967). Absorption of
344 Production, purification, characterization, immobilization, and application of Serrapeptase
exogenous and endogenous proteolytic enzymes. Clin Pharmacol
Ther, 8(3): 362–368
Ammar M S, El-Ssaway M, Yassin M, Sherif Y M (1998). Hydrolytic
Enzymes of fungi isolated from Certain Egyptian Antiquities objects
while utilizing the Industrial Wastes of Sugar and Integrated
Industries Company (SIIC). Egypt J Biotechnol, (3): 60–90
Anil C S, Kashinath M A (2013). Production, Characterization and
optimization of potent protease (Serratiopeptidase) from Serratia-
marcescensE 15. Int Res J Pharm App Sci, 3(4): 95–98
Aratani H, Tateishi H, Negita S (1980). Studies on the distributions of
antibiotics in the oral tissues: Experimental staphylococcal infection
in rats, and effect of serratiopeptidase on the distributions of
antibiotics (author’s transl). Jpn J Antibiot, 33(5): 623–635
Aso T, Noda Y, Matsuura S, Nishimura T (1981). Breast engorgement
and its treatment: Clinical effects of Danzen an anti-inflammatory
enzyme preparation. The world of Obsttrics and Gynecology
(Japanese), 33: 371–379
Ayswarya A, Ramesh B, Muthuraman M S (2013). Optimisation studies
in the production and purification of serratiopeptidase from
Serratiamarscecens UV mutant SM3. International Journal of
Pharmacy and Pharmaceutical Sciences, 5(3): 0975–1491
Bach E, Anna S V, Daroit D J, Correa A P F, Segalin J, Brandelli A
(2012). Production, one-step purification, and characterization of a
keratinolytic protease from Serratia marcescens P3. Process
Biochem, 47(12): 2455–2462
Badhe R V, Nanda R K, Kulkarni M B, Bhujabal M N, Patil P S, Badhe
S R (2009). Medium optimization studies for Serratiopeptidase
production from Serratia marcescens ATCC 13880. Hindustan
Antibiotics Bulletin, 51(1–4): 17–23
Baumann U (1994). Crystal structure of the 50 kDa metallo protease
from Serratia marcescens. J Mol Biol, 242(3): 244–251
Bellussi L, Ciferri G, De S E, Passali D (1984). Effect of 2-(alpha-
thenoylthio)propionyl-glycine in treatment of secretory otitis media.
CurrTher Res, 36(3): 596–605
Bhagat S, Agarwal M, Roy V (2013). Serratiopeptidase: a systematic
review of the existing evidence. Int J Surg, 11(3): 209–217
Bi F, Zhang J, Su Y, Tang Y C, Liu J N (2009). Chemical conjugation of
urokinase to magnetic nanoparticles for targeted thrombolysis.
Biomaterials, 30(28): 5125–5130
Bodhankar M M, Agnihotri V V, Bhushan S B (2011). Feasibility,
formulation and characterization of innovative microparticles for oral
delivery of peptide drug. Int J Res Pharm Chem, 1(3): 630–636
Bracale G, Selvetella L (1996). Clinical study of the efficacy of and
tolerance to seaprose S in inflammatory venous disease. Controlled
study versus serratio-peptidase. Minerva Cardioangiol, 44(10): 515–
524
Braunagel S C, Benedik M J (1990). The metalloprotease gene of
Serratia marcescens strain SM6. Mol Gen Genet, 222(2-3): 446–451
Cech T R, Bass B L (1986). Biological catalysis by RNA. Annu Rev
Biochem, 55(1): 599–629
Celik M M, Kavvasoglu G, Celik E, Bulgurcu M (2013). Serratiopepti-
dase Induced Hemorrhage in a Patient with Behçet’s Disease.
International Journal of Basic and Clinical Studies, 1(1): 190–196
Chen Y, Zhang Y (2011). Fluorescent quantification of amino groups on
silica nanoparticle surfaces. Anal Bioanal Chem, 399(7): 2503–2509
Chopra D, Rehan H S, Mehra P, Kakkar A K (2009). A randomized,
double-blind, placebo-controlled study comparing the efficacy and
safety of paracetamol, serratiopeptidase, ibuprofen and betametha-
sone using the dental impaction pain model. Int J Oral Maxillofac
Surg, 38(4): 350–355
Decedue C J, Broussard E A 2nd, Larson A D, Braymer H D (1979).
Purification and characterization of the extracellular proteinase of
Serratia marcescens. Biochim Biophys Acta, 569(2): 293–301
Devi C S, Joseph R E, Saravanan H, Naine S J, Srinivansan V M (2013).
Screening and molecular characterization of Serratia marcescens
VITSD2: A strain producing optimum serratiopeptidase. Front Biol,
8(6): 632–639
Dhanvijay P, Misra A K, Varma S K (2013). Diclofenac induced acute
renal failure in a decompensated elderly patient. J Pharmacol
Pharmacother, 4(2): 155–157
Dohmen K, Matsunaga K, Irie K, Shirahama M, Onohara S, Miyamoto
Y, Takeshita T, Ishibashi H (1998). Granulomatous hepatitis induced
by saridon and serrapeptase. Hepatol Res, 11(2): 133–137
El-Hamid H S, Ahmed H A, Sadek K M, El-Bestawy A R, Ellakany H F
(2014). Impact of Serratiopeptidase treatment on performance and
health parameters in broiler chicken. Int J Pharm Sci Rev Res, 26(2):
84–90
Esch P M, Gerngross H, Fabian A (1989). Reduction of postoperative
swelling. Objective measurement of swelling of the upper ankle joint
in treatment with serrapeptase–a prospective study. Fortschr Med,
107(4): 67–68, 71–72
Fadl N N, Ahmed H H, Booles H F, Sayed A H (2013). Serrapeptase and
nattokinase intervention for relieving Alzheimer’s disease pathophy-
siology in rat model. Hum Exp Toxicol, 32(7): 721–735
Garg R, Aslam A, Garg A, Walia R (2012). A prospective comparative
study of serratiopeptidase and aceclofenac in upper and lower limb
soft tissue trauma cases. Int J PharmacolPhamaceutTechnol, 1(2):
11–16
Gerber N N (1975). Prodigiosin-like pigments. CRC Crit Rev Microbiol,
3(4): 469–485
Gurung N, Ray S, Bose S, Rai V (2013). A broader view: microbial
enzymes and their relevance in industries, medicine, and beyond.
BioMed Res Int, 2013: 329121
Guyton A (1974). Function of the Human Body. WB Saunders, 4:8384
Hamada K, Hata Y, Katsuya Y, Hiramatsu H, Fujiwara T, Katsube Y
(1996). Crystal structure of Serratia protease, a zinc-dependent
proteinase from Serratia sp. E-15, containing a beta-sheet coil motif
at 2.0 A resolution. J Biochem, 119(5): 844–851
Illanes A, Cauerhff A, Wilson L, Castro G R (2012). Recent trends in
biocatalysis engineering. Bioresour Technol, 115(1): 48–57
Ishihara Y, Kitamura S, Takaku F (1983). Experimental studies on
distribution of cefotiam, a new beta-lactam antibiotic, in the lung and
trachea of rabbits. II. Combined effects with serratiopeptidase. Jpn J
Antibiot, 36(10): 2665–2670
Jadav S P, Patel N H, Shah T G, Gajera M V, Trivedi H R, Shah B K
(2010). Comparison of anti-inflammatory activity of serratiopepti-
dase and diclofenac in albino rats. J Pharmacol Pharmacother, 1(2):
116–117
Jayaratne P (1996). Major metalloprotease gene of Serratia marcescens
is conserved and provides a molecular typing method to differentiate
Selvarajan Ethiraj and Shreya Gopinath 345
clinical isolates. J Microbiol Methods, 26(3): 261–269
Jickling G C, Zhan X, Ander B P, Turner R J, Stamova B, Xu H, Tian Y,
Liu D, Davis R R, Lapchak P A, Sharp F R (2010). Genome response
to tissue plasminogen activator in experimental ischemic stroke.
BMC Genomics, 11(1): 254
Jordon D L (1929). Red heat in salted hides. J IntSoc Leather Trade
Chem, 13: 538–569
Juhi K P, Divya T, Mandev B P (2015). Development and Validation of
Analytical Method for Estimation of Paracetamol, Lornoxicam and
Serratiopeptidase in their Combined Dosage Form. J Pharm Sci
Bioscientific Res, 5(5): 425–433
Kaisheng Y I N, Wanzhen F U, Lixian H E, Shanqun L I, Yi S H I,
Yongying X I A O, Hao Y U, Zhenjie H A N (2000). A multicenter
randomized double blind and controlled clinical trial of serrapeptase
enteric coated tablets as an expectorant. Chinese journal of new
Drugs and Clinical Remedies, 15(10): 737–740
Kamini N R, Hemchander C, Geraldine J (1999). Microbial enzyme
technology as an alternative to conventional chemical in leather
industry. Curr Sci, 76: 101
Kasé Y, Seo H, Oyama Y, Sakata M, Tomoda K, Takahama K, Hitoshi
T, Okano Y, Miyata T (1982). A new method for evaluating
mucolytic expectorant activity and its application. I. Methodology.
Arzneimittelforschung, 32(4): 368–373
Kaviyarasi N S, Prashantha C N, Suryanarayan V V S (2016). In Silico
Analysis Of inhibitor and substrate binding site of serrapeptidase
from SerratiamarcescensMTCC 8708. Int J Pharm Pharm Sci, 8(4):
975–1491
Kaviyarasi N S, Sarkar S, Suryanarayan V V S (2015). Characterization
of a Gene Encoding Serrapeptidase from SerratiamarcescensStrain
(SRM) MTCC 8708, a Plant Isolate. Int J Curr Microbiol Appl Sci, 4
(8): 206–214
Kaviyarasi N S, Suryanarayan V V S (2016). Serrapeptidase gene of
Serratia marcescens from plant origin expressed by Pichia pastoris
has protease activity. Indian Journal of Medical Research and
Pharmaceutical Sciences, 3(6): 2349–5340
Kee W H, Tan S L, Lee V, Salmon Y M (1989). The treatment of breast
engorgement with Serrapeptase (Danzen): a randomised double-blind
controlled trial. Singapore Med J, 30(1): 48–54
Klein G, Kullich W (2000). Short-term treatment of painful osteoarthritis
of the knee with oral enzymes: randomised, double blind study versus
Diclofenac. Clin Drug Investig, 19(1): 15–23
Kloepper J W, Tuzun S, Liu L, Wei G (1993). Plant growth-promoting
rhizobacteria as inducers of systemic disease resistance. In: Lumsden
RD, Waughn J (eds) Pest management: biologically based technol-
ogies, American Chemical Society Books,156–165
Koyama A, Mori J, Tokuda H, Waku M, Anno H, Katayama T,
Murakami K, Komatsu H, Hirata M, Arai T (1986). [Augmentation
by serrapeptase of tissue permeation by cefotiam. Jpn J Antibiot, 39
(3): 761–771
Krukemeyer M G, Krenn V, Jakobs M, Wagner W (2012). Magnetic
drug targeting in a rhabdomyosarcoma rat model using magnetite-
dextran composite nanoparticle-bound mitoxantrone and 0.6 tesla
extracorporeal magnets- sarcoma treatment in progress. J Drug
Target, 20(2): 185–193
Kumar S, Jana A K, Dhamija I, Singla Y, Maiti M (2013). Preparation,
characterization and targeted delivery of serratiopeptidase immobi-
lized on amino-functionalized magnetic nanoparticles. Eur J Pharm
Biopharm, 85(3 Pt A 3A): 413–426
Kumar S, Jana A K, Maiti M, Dhamija I (2014). Carbodiimide-mediated
immobilization of serratiopeptidase on amino-, carboxyl-functiona-
lized magnetic nanoparticles and characterization for target delivery.
J Nanopart Res, 16(2): 2233
Kumar S, Mohan U, Kamble A L, Pawar S, Banerjee U C (2010). Cross-
linked enzyme aggregates of recombinant Pseudomonas putida
nitrilase for enantioselective nitrile hydrolysis. Bioresour Technol,
101(17): 6856–6858
Létoffé S, Delepelaire P, Wandersman C (1991). Cloning and expression
in Escherichia coli of the Serratia marcescens metalloprotease gene:
secretion of the protease from E. coli in the presence of the Erwinia
chrysanthemi protease secretion functions. J Bacteriol, 173(7): 2160–
2166
Liver Doctor (2013). Serrapeptase Health guide
Lowry O H, Rosebrough N J, Farr A L, Randall R J (1951). Protein
measurement with the Folin phenol reagent. J Biol Chem, 193(1):
265–275
Majima Y, Inagaki M, Hirata K, Takeuchi K, Morishita A, Sakakura Y
(1988). The effect of an orally administered proteolytic enzyme on
the elasticity and viscosity of nasal mucus. Arch Otorhinolaryngol,
244(6): 355–359
Malshe P C (2000). A preliminary trial of serratiopeptidase in patients
with carpal tunnel Syndrome. J Assoc Physicians India, 48(11): 1130
Manal K M (2015). Effect of temperature and mutation on serratio-
peptidase secreted from Serratiamarcesence. Journal of Genetic and
Environmental Resources Conservation, 3(1): 35–37
Matsumoto K, Maeda H, Takata K, Kamata R, Okamura R (1984).
Purification and characterization of four proteases from a clinical
isolate of Serratia marcescens kums 3958. J Bacteriol, 157(1): 225–
232
Mazzone A, Catalani M, Costanzo M, Drusian A, Mandoli A, Russo S,
Guarini E, Vesperini G (1990). Evaluation of Serratia peptidase in
acute or chronic inflammation of otorhinolaryngology pathology: a
multicentre, double-blind, randomized trial versus placebo. J Int Med
Res, 18(5): 379–388
Mecikoglu M, Saygi B, Yildirim Y, Karadag-Saygi E, Ramadan S S,
Esemenli T (2006). The effect of proteolytic enzyme serratiopepti-
dase in the treatment of experimental implant-related infection. J
Bone Joint Surg Am, 88(6): 1208–1214
Metkar S K, Girigoswami A, Murugesan R, Girigoswami K (2016). In
vitro and in vivo insulin amyloid degradation mediated by
Serratiopeptidase. Materials Science & Engineering C, 70(2017):
728–735
Miyagawa S, Matsumoto K, Kamata R, Okamura R, Maeda H (1991).
Spreading of Serratia marcescens in experimental keratitis and
growth suppression by chicken egg white ovomacroglobulin. Jpn J
Ophthalmol, 35(4): 402–410
Miyata K, Maejima K, Tomoda K, Isono M (1970). Serratia protease part
1. Purification and general properties of the enzyme. AgrBiolChem,
34(2): 310–318
Miyata K, Tomoda K, Isono M (1970b). Serratia protease. Part II.
Substrate specificity of the enzyme. Agric Biol Chem, 34(10): 1452–
346 Production, purification, characterization, immobilization, and application of Serrapeptase
1457
Miyazaki H, Yanagida N, Horinouchi S, Beppu T (1990). Specific
excretion into the medium of a serine protease from Serratia
marcescens. Agric Biol Chem, 54(10): 2763–2765
Mohankumar A, Raj R H K (2011). Production and characterization of
Serratiopeptidase enzyme from Serratia marcescens. Int J Biol, 3(3):
39–44
Moriya N, Nakata M, Nakamura M, Takaoka M, Iwasa S, Kato K,
Kakinuma A (1994). Intestinal absorption of serrapeptase (TSP) in
rats. Biotechnol Appl Biochem, 20(Pt 1): 101–108
Mundhava S G, Mehta D S, Thaker S J (2016). A Comparative Study to
Evaluate Anti-Inflammatory and analgesic activity of commonly
used Proteolytic Enzymes and their Combination with Diclofenac in
Rats. Int J Pharm Sci Res, 7(6): 2615–2619
Nagaoka S, Tanaka M, Tsubura E (1979). A double blind clinical study
on the expectorant effect of Seaprose-S. RinshoHyoka, 7: 205–232
(ClinEval)
Nageswara S, Singam R, Guntuku G (2016). Design of a low cost
fermentation medium for the production of Serratiopeptidase enzyme
by a novel Streptomyces sp. World J Pharm Pharm Sci, 5(10): 829–
842
Nakahama K, Yoshimura K, Marumoto R, Kikuchi M, Lee I S, Hase T,
Matsubara H (1986). Cloning and sequencing of Serratia protease
gene. Nucleic Acids Res, 14(14): 5843–5855
Nakamura S, Hashimoto Y, Mikami M, Yamanaka E, Soma T, Hino M,
Azuma A, Kudoh S (2003). Effect of the proteolytic enzyme
serrapeptase in patients with chronic airway disease. Respirology, 8
(3): 316–320
Namdeo M, Bajpai S K (2009). Immobilization of a-amylase onto
cellulose-coated magnetite (CCM) nanoparticles and preliminary
starch degradation study. J MolCatalEnzym, 59: 134–139
Nieper H A (2010). “The Curious Man: The Life and Works of Dr. Hans
Nieper”. AuthorHouse.
Okumura H, Watanabe R, Kotoura Y, Nakane Y, Tangiku O (1977).
Effects of a proteolytic-enzyme preparation used concomitantly with
an antibiotic in osteoarticular infections (author’s transl). Jpn J
Antibiot, 30(3): 223–227
Pakhale S V, Bhagwat S S (2016). Purification of serratiopeptidase from
Serratiamarcescens NRRL B 23112 using ultrasound assisted three
phase partitioning. Ultrasonics Sonochemistry, 31: 532–538
Panagariya A, Sharma A K (1999). A preliminary trial of serratiopepti-
dase in patients with carpal tunnel syndrome. J Assoc Physicians
India, 47(12): 1170–1172
Pansuriya R, Singhal R (2011). Effects of dissolved oxygen and agitation
on production of serratiopeptidase by Serratia marcescens NRRL B-
23112 in stirred tank bioreactor and its kinetic modeling. J Microbiol
Biotechnol, 21(4): 430–437
Pansuriya R C, Singhal R S (2010). Evolutionary operation (EVOP) to
optimize whey independent serratiopeptidase production from
Serratia marcescens NRRL B-23112. J Microbiol Biotechnol, 20
(5): 950–957
Patel J K, Thakkar D, Patel M B (2015). Development and Validation of
Analytical Method for Estimation of Paracetamol, Lornoxicam and
Serratiopeptidase in their Combined Dosage Form. J Pharm Sci
BioscientificRes, 5(5): 425–433
Pravin K S, Ravindra L B, Gaudb R S, Kiran N B, Madhugandha S K
(2015). Modulation of serratiopeptidase transdermal patch by lipid-
based transfersomes. J Adhes Sci Technol, 29(23): 2622–2633
Queiroz B P V, Melo I S (2006). Antagonism of Serratiamarcescens
towards Phytophthoraparasitica and its effects in promoting the
growth of citrus. Braz J Microbiol, 37(4): 448–450
Rajinikanth B, Venkatachalam V V, Manavalan R (2014). Investigations
on the potential of serratiopeptidase –A proteolytic enzyme, on
acetic acid induced ulcerative colitis in mice. Int J Pharm Pharm Sci,
6(5): 525–531
Rath G, Johal E S, Goyal A K (2011). Development of serratiopeptidase
and metronidazole based alginate microspheres for wound healing.
Artif Cells Blood Substit Immobil Biotechnol, 39(1): 44–50
Reddy A R, Rajamani K, Mounika A, Reddy G S (2015). Development
and validation of RP-HPLC Method for simulations estimation of
paracetamol, aceclofenac and serratiopeptidase in combined tablet
dosage form. World J Pharm Pharm Sci, 4(3): 1008–1023
Robert S R (2009). The ‘Miracle’Enzyme is Serrapeptase, the 2nd Gift
from Silkworms Giving the answer to Pain, Inflammation and Clear
Arteries. Naturally Healthy Publications, 3(4): 12–19
Romero F J, Garcia L A, Salas J A, Diaz M, Quiros L M (2001).
Production, purification and partial characterization of two extra-
cellular proteases from Serratia marcescens grown in whey. Process
Biochem, 36(6): 507–515
Ruell J A, Avdeef A, Du C, Tsinman K (2002). A Simple PAMPA Filter
for Passively Absorbed Compounds. 224th National Meeting of the
American Chemical Society.
Sabu A (2003). Sources, Properties and Applications of Microbial
Therapeutics Enzymes. Indian J Biotechnol, 2(3): 334–341
Salamone P R, Wodzinski R J (1997). Production, purification and
characterization of a 50-kDa extracellular metalloprotease from
Serratia marcescens. Appl Microbiol Biotechnol, 48(3): 317–324
Salarizadeh N, Hasannia S, Noghabi K A, Sajedi R H (2014).
Purification and Characterization of 50 kDa Extracellular Metallo-
protease from Serratia sp. ZF03. Iranian J Biotechnol, 12(3): 18–27
Sandhya K V, Devi S G, Mathew S T (2008). Quantitation of
serrapeptase in formulations by UV method in the microplate format.
Curr Drug Deliv, 5(4): 303–305
Sasaki S, Kawanami R, Motizuki Y, Nakahara Y, Kawamura T, Tanaka
A, Watanabe S (2000). Serrapeptase-induced lung injury manifesting
as acute eosiniphilic pneumonia. Nihon Kokyuki Gakkai Zasshi, 38
(7): 540–544
Selan L, Papa R, Tilotta M, Vrenna G, Carpentieri A, Amoresano A,
Pucci P, Artini M (2015). Serratiopeptidase: a well-known
metalloprotease with a new non-proteolytic activity against S. aureus
biofilm. BMC Microbiol, 15(1): 207
Sellman S (2003). Serrapeptase, An Amazing Gift from The Silk Worm.
World rights reserved.
Shanks R M, Stella N A, Hunt K M, Brothers K M, Zhang L, Thibodeau
P H (2015). Identification of SlpB, a Cytotoxic Protease from Serratia
marcescens. Infect Immun, 83(7): 2907–2916
Shimura S, Okubo T, Maeda S, Aoki T, Tomioka M, Shindo Y,
Takishima T, Umeya K (1983). Effect of expectorants on relaxation
behavior of sputum viscoelasticity in vivo. Biorheology, 20(5): 677–
683
Selvarajan Ethiraj and Shreya Gopinath 347
Singh R, Kumar M, Mittal A, Mehta P K (2016). Microbial enzymes:
industrial progress in 21st century. 3 Biotech, 6(2): 174
Stancu M M (2016). Response Mechanisms in Serratia marcescens
IBBPo15 During Organic Solvents Exposure. Curr Microbiol, 73(6):
755–765
Subbaiya R, Ayyappadasan G, Mythili S V, Dhivya E, Ponmurugan P
(2011). Pilot scale microbial production and optimization of Serratia
peptidase from Serratia marcescens. J Ecobiotechnol, 3(12): 10–13
Tachibana M, Mizukoshi O, Harada Y, Kawamoto K, Nakai Y (1984). A
multi-centre, double-blind study of serrapeptase versus placebo in
post-antrotomy buccal swelling. Pharmatherapeutica, 3(8): 526–530
Tao K, Yu X, Liu Y, Shi G, Liu S, Hou T (2007). Cloning, expression,
and purification of insecticidal protein Pr596 from locust pathogen
Serratia marcescens HR-3. Curr Microbiol, 55(3): 228–233
Tripura C, Sashidhar B, Podile A R (2007). Ethyl methanesulfonate
mutagenesis-enhanced mineral phosphate solubilization by ground-
nut-associated Serratia marcescens GPS-5. Curr Microbiol, 54(2):
79–84
Tsuyama N, Yoshio O, Makoto F (1977). Clinical evaluation on
antiswelling drug-A-4700 in orthopaedic field. A comprehensive
double blind controlled trial compared with serratiopeptidase and
placebo in 20 orthopedic clinics. RinshoHyoka, 5: 535–575
(ClinEval)
UmaMaheswari T, Hemalatha T, Sankaranarayanan P, Puvanakrishnan
R (2016). Enzyme therapy: current perspectives. Indian J Exp Biol,
54(1): 7–16
Vanama J, Chintada H, Guntuku G, Tadimalla P (2014). Application of
response surface methodology in medium components optimization
to enhance serratiopeptidase production by Streptomyces hydroge-
nans MGS13. Eur Sci J, 10(12): 1857–7431
Verma M L, Barrow C J, Puri M (2013). Nanobiotechnology as a novel
paradigm for enzyme immobilisation and stabilisation with potential
applications in biodiesel production. Appl Microbiol Biotechnol, 97
(1): 23–39
Vicari E, La Vignera S, Battiato C, Arancio A (2005). Treatment with
non-steroidal anti-inflammatory drugs in patients with amicrobial
chronic prostato-vesiculitis: transrectal ultrasound and seminal
findings. Minerva Urol Nefrol, 57(1): 53–59
Viswanatha Swamy A H, Patil P A (2008). Effect of some clinically used
proteolytic enzymes on inflammation in rats. Indian J Pharm Sci, 70
(1): 114–117
Wagdarikar J M, Joshi A M, Shaikh A A (2015). Media Optimization
Studies for Enhanced Production of Serratiopeptidase from Bacillus
Licheniformis (NCIM 2042). Asian Journal of Biomedical and
Pharmacutical Sciences, 5(42): 19–23
Woodley J F (1994). Enzymatic barriers for GI peptide and protein
delivery. Crit Rev Ther Drug Carrier Syst, 11(2-3): 61–95
Yu C C, Kuo Y Y, Liang C F, Chien W T, Wu H T, Chang T C, Jan F D,
Lin C C (2012). Site-specific immobilization of enzymes on magnetic
nanoparticles and their use in organic synthesis. Bioconjug Chem, 23
(4): 714–724
Zhu H, Pan J, Hu B, Yu H L, Xu J H (2009). Immobilization of glycolate
oxidase from Medicago falcata on magnetic nanoparticles for
application in biosynthesis of glyoxylic acid. J Mol Catal, B
Enzym, 61(3–4): 174–179
Ziv-Polat O, Topaz M, Brosh T, Margel S (2010). Enhancement of
incisional wound healing by thrombin conjugated iron oxide
nanoparticles. Biomaterials, 31(4): 741–747
348 Production, purification, characterization, immobilization, and application of Serrapeptase
