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, pharmaceutical, 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 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. It is 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). Serrapeptase 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, thrombosis, 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 a-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 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 production 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-a and 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 metalloprotease 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 pPICZaA 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 (
Sellman, 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, 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. Serrapeptase 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 Serrapeptase (
Devi et al., 2013).
Sources of Serrapeptase
Serrapeptase is produced by a variety of microorganisms isolated from different sources (Table 1). Soil and contaminated 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 marcescens 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
2PO
4 8.0 g/l, and NaCl 5.0 g/l at a pH 7.0 which gave a maximum yield of 32,575EU/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
2PO
4 and K
2HPO
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
2PO
4 (
Vanama et al., 2014). Horse gram (
Microtylona uniflorum) is one of a few low-cost substrates for the production of Serrapeptase by
Streptomyces hydrogenansvar
. 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 licheniformiscontains glycerin, glucose, tryptone, ammonium oxalate, sodium acetate, disodium hydrogen phosphate and ammonium sulfate at a composition 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, 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 Serrapeptase 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, radioimmunoassay, 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 (
Salarizadeh et al., 2014). High-Performance Liquid Chromatography 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 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 conditions 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 concentration 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 simultaneously. Here, the medium for maximum production was optimized by ‘one-variable-at-a-timeʼ 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 Serrapeptase 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(
Nageswara 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 hydrogenans 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 from 24 h (Mohankumar and Raj, 2011) to 25 h (Anil and Kashinath, 2013). The optimal incubation duration for maximal Serrapeptase production from
Bacillus Licheniformis 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 surface 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
3O
4 nanoparticles, carboxyl-functionalized magnetic nanoparticles, Amino-functionalized magnetic nanoparticles (Namdeo and Bajpai, 2009), chitosan/glutaraldehyde 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 gastrointestinal 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
3O
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 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 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 withdrawn (
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 Serrapeptase,Serratia marcescens is the best producer of Serrapeptase. Many studies on production strategies, purification, 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 bloodstream. 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.
Higher Education Press and Springer-Verlag Berlin Heidelberg
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