Microbiological Studies on The Production of Serratia-peptidase As an Anti-inflammatory from Serratia Species

M.Sc. Thesis Submitted in Partial Fulfillment of the Requirements for The master’s degree in Pharmaceutical Sciences (Microbiology & Immunology)

Presented by

Ahmed Abd El Monaem Ammar B.Sc. Pharmaceutical Science, October 6 university, 2005 Microbiological Quality Control Specialist – NODCAR

Under the supervision of Prof. Dr. Magdy Ali Amin Professor of Microbiology & Immunology Faculty of Pharmacy, Cairo University

Dr. Reham Samir Dr. Wael Mohamed Abu El Assistant professor of Wafa Microbiology & Immunology Assistant Professor of Microbiology Faculty of Pharmacy, National Organization for Drug Control Cairo University and Research

Microbiology & Immunology Department Faculty of Pharmacy Cairo University 2019

Abstract

Proteases constitute one of the most important groups of industrial , accounting for more than 65% of the industrial market. Microbial produced from microbes belonging to bacteria, fungi, yeast and actinomycete, account for approximately 40% of the total worldwide sales of enzymes. Serratiopeptidase is a proteolytic enzyme that has been used as anti-inflammatory agent in sinusitis, bronchitis and other inflammatory disorders. The present study aimed to isolate serratia species producing enzyme and increase the enzyme production by optimization of some nutrition demands and environmental conditions, studying the physicochemical properties of the purified protease and in-vivo evaluation of the anti-inflammatory effect of serratiapeptidase by using animal model (rat). Results revealed that out of 170 bacterial isolates retrieved from soil samples collected from different geographical regions, Egypt, only 20 (11.8%) isolates were primarily identified as Serratia species. Serratia S6 was the most potent Serratia isolate in protease production, which preliminary identified by both cultural and morpho-chemical characteristics and finally confirmed by sequencing of 16SrRNA gene and phylogenetic tree. To maximize the production of protease from Serratia S6, some nutritional supplements (casein concentrations, K2HPO4 concentrations and type of sugars), and some environmental conditions (initial pH level, inoculum size and incubation temperature) should be adapted. The maximum protease production (327.32 U/ml) by Serratia S6 were obtained by adding 10 g/L casein, 1.0 g/L K2HPO4 and 1% (w/v) fructose, 2.0 mM of ZnSO4, KCl and NaCl, at initial pH level 9.0, inoculum size (1%) and incubation temperature at 37oC for 48h. For precipitation of protease enzyme, 80% ammonium sulphate saturation salt had been added. The molecular weight of the proteins by SDS-PAGE was found to be 79,60 and 65 KDa. Characterization of proteases from Serratia S6 were investigated and the results showed that serratiapeptidase exhibited some improvements in its physiochemical properties. The optimum temperature for maximum protease activity (435 U/ml) was 40oC and stability (422 U/ml) was obtained at 30⁰ C - 40oC for 120 min. Also, the optimum pH level for maximum protease activity (440 U/ml) and stability (438 U/ml) was at pH 9.0. The activity of protease was gradually decreased by increasing of some inhibitor concentrations including EDTA, Tween 20 and PMSF. Regarding in-vivo evaluation Serratiopeptidase in rat, obtained results revealed that different molecular weights protease significantly inhibited acute inflammation in lung of rat after intranasal infection with strain of Acinetobacter baumannii bacteria which was comparable with non-treated group. Treated groups revealed focal few inflammatory cells infiltration in the peribronchiolar tissue and mild congestion in the interalveolar and peribronchiolar blood vessels when compared with non-treated group which showed sever congestion in the peribronchiolar and interalveolar blood vessels associated with focal aggregation as well as infiltration of leucocytes cells in the peribronchiolar tissue. Key words: Serratia, Serratiapeptidase, environmental conditions, nutritional supplements, anti-inflammatory effect, 16S rRNA, rat.

INTRODUCTION

The genus Serratia a member of the Enterobacteriaceae is comprised of a group of bacteria that are related both phenotypically and by DNA sequence. The type species of the genus is Serratia marcescens. Some species and biotypes of Serratia produce a non-diffusible red pigment, prodigiosin, or 2-methyl-3-amyl-6- methoxyprodigiosene.

At the start of this century, more than 76 known species had been described with red or pink pigmentation, and 23 Serratia species were listed in the first edition of Bergey’s Manual. This number progressively decreased to five in the fifth edition of Bergey’s Manual and later to one species: S. marcescens. The only Serratia species recognized in the eighth edition of Bergey’s Manual was S. marcescens. Ten species are presently known to belong in the genus Serratia.

Serrapeptase or serratiopeptidase is a proteolytic enzyme isolated from the nonpathogenic Serratia a member of the Enterobacteriaceae. Proteases perform highly specific and selective modifications of proteins such as activation of zymogenic forms of enzymes by limited proteolysis, blood clotting and lysis of fibrin clots, processing and transport of secretory proteins across the membranes, and so on. Proteases are ubiquitous in nature.

Protease is of commercial value and various industrial applications. They are widely used as detergent, in food, pharmaceutical and leather tanning industries. The vast variety of proteases, with their specificity of their action and application has attracted worldwide attention to exploit their physiological as well as biotechnological applications. It has been considered as eco-friendly because the appropriate producers of these enzymes for commercial exploitation are non-toxic and non- pathogenic that are designated a safe. Serratiopeptidase, has been found useful in patients suffering from acute or chronic inflammatory disorders of ear, nose or throat, such as laryngitis, catarrhal rhino-pharyngitis and sinusitis.

Aim of work:

The present study aims to produce the anti-inflammatory agent (serratiapeptidase) from Serratia species as an alternative treatment for people severing from inflammatory disorders.

REVIEW OF LITERATURE

Protease enzymes

Proteolytic enzymes catalyze the hydrolytic cleavage of bonds. They are also called proteinases or proteases; these enzymes are present in all living organisms and are essential for cell growth and differentiation. Proteases are classified according to their structure or the properties of the . Microorganisms produce a variety of intracellular and/or extracellular proteases such as serine-, metallo-, carboxyl-, acidic-, neutral-, and alkaline proteases.

Highly specified and selective modifications of proteins performed by proteases such as activation of zymogenic forms of enzymes by limited proteolysis, blood clotting and lysis of fibrin clots, processing and transport of secretory proteins across the membranes, correspondingly Proteases represents one of the three large groups of industrial enzymes and find application in detergents, leather, food, pharmaceutical industries and bioremediation processes. The largest application of proteases particularly the alkaline proteases has probably been in the laundry detergent where they enhance the removal of protein-based stains from clothing.

Enzyme Nomenclature

Enzymes are identified by a common nomenclature system based on the description of what function it performs in the cell and ends with a common phrase. The International Union of Biochemistry and Molecular Biology and the International Union of Pure and Applied Chemistry developed a nomenclature system wherein each enzyme is given an called as the EC number. Accordingly, the top-level classes based on the mechanism of operation of an enzyme are (, , , , and .

Classification of proteases

Proteases are grossly subdivided into two major groups, i.e., exopeptidases and endopeptidases, depending on their site of action. Based on the functional group present at the active site, proteases are further classified into four prominent groups, i.e., serine proteases, aspartic proteases, cysteine proteases, and metalloproteases. Currently, proteases are classified based on three major criteria (type of reaction catalyzed, chemical nature of the catalytic site and relationship to the structure).

The physiological function of proteases is essential for all living organism, from viruses to humans and the enzymes can be classified based on their origin: microbial (bacterial, fungal and viral), plant, animal and human enzymes can be distinguished. Based on the site of action on protein substrates, proteases are broadly classified as endopeptidases or exopeptidases enzymes. Exopeptidases cleave the peptide bond proximal to the amino or carboxy termini of the substrate. Based on the site of action at the N or C terminus, they are classified as aminopeptidases and carboxypeptidases. Endopeptidases cleave peptide bonds distant from the termini of the substrate. Based on the functional group present at the active site, endo-peptidases are further classified into four prominent groups, i.e., serine proteases, aspartic proteases, cysteine proteases and metallo-proteases also Based on the pH optima, they are referred to as acidic, neutral, or alkaline proteases.

Importance of serratiapeptidase and its applications

Serratiopeptidase enzyme (SRP) or serrapeptase is a 50-kDa metalloprotease produced by Serratia marcescens species. SRP has been gaining wide acceptance in Europe and Asia as a potent anti-inflammatory, anti-oedemic and fibrinolytic activity and acts rapidly on localized inflammation.

Sources of proteases

Proteases are ubiquitous and distributed widely in all living organisms: in plants (, ), in animal and mainly in microbes (bacteria, fungi and viruses). Microbial proteases account for approximately 4000 of the total Worldwide sales of enzymes. Due to possessing almost all the characteristics desired for their biotechnological applications, microbial proteases are preferred to the plant and animal enzymes.

Plant Proteases

Proteases of plant origin perform many vital functions, ranging from the mobilization of storage proteins during germination to the initiation of cell death and senescence. Plant enzymes (such as , and ficin) have been extensively investigated as meat tenderizers. New plant proteases (actinidin and zingibain) and microbial enzyme preparations have been of recent interest due to controlled meat tenderization and other advantages.

Animal Proteases

Animal proteases are less used to tenderize meat. In a recent study, injection of pancreatin on beef semitendinosus tended to improve overall tenderness, and the results showed that taste was not affected.

The most familiar proteases of animal origin are pancreatic trypsin, chymotrypsin, pepsin, and rennin. These are prepared in pure form in bulk quantities. However, their production depends on the availability of livestock for slaughter, which in turn is governed by political and agricultural policies.

Microbial Proteases Proteases are widely distributed in microbial population viz. bacteria, actinomycetes, viruses and fungi. Although proteases are widespread in nature, microbes serve as a preferred source of these enzymes and account for around two- thirds of commercial production worldwide. Alkaline serine proteases are the most important group of commercial enzymes.

Taxonomy and Nomenclature of the genus Serratia

(Martinec, 1961) mentioned that the oldest known species of the genus Serratia is Serratia marcescens. It was named as early as 1823 and since this time both its nomenclature and its taxonomy have undergone many changes. Recognition of this species as the type species of the genus Serratia was proposed by (Buchanan et al., 1966) It is so recognized in all seven editions of the Bergey' s Manual.

Isolation, characterization, identification

Isolation of Serratia in Water and Soil:

It is well known that Serratia marcescens has a propensity for water and moist environments, which explains, in part, the ease with which it disseminates within hospitals. However, contaminated faucets have not been previously reported as possible point sources in outbreaks of Serratia marcescens infection and colonization. Moreover, administration of drugs orally or via a nasogastric tube with tap water as vehicle has not been previously considered as a possible mechanism of infection acquisition.

(Tasić et al., 2013) isolated Serratia Fonticola strain ST2 from oligo- mineral bottled water. The strain showed the ability to survive in specific conditions that characterize this bottled water (especially the very low mineralization <50 mg/L, which is an unfavorable medium for most bacterial species. Serratia plymuthica is a Gram-negative bacterium (Enterobacteriaceae family) isolated from a raw vegetable-processing line in an industrial kitchen. Prevalent in soil, air, and water, Serratia species are commonly associated with raw food materials and are often implicated in the spoilage of various foods.

Isolation of Serratia from Plants

Cucurbit yellow vine disease (CYVD) attacks important agricultural crops such as watermelon, pumpkin, cantaloupe and squash. The causal agent of CYVD is Serratia marcescens, which resides and multiplies within the phloem, eventually killing the plant by blocking its vascular system.

Isolation of Serratia from animals and Humans

Serratia marcescens was first reported to cause infection in humans in 1951 when it was associated with bacterial endocarditis. Since then, Serratia marcescens has been identified as the cause of various infections such as bacteremia, pneumonia, keratitis, endocarditis, urinary tract infection, meningitis and necrotizing fasciitis.

Effect of different nutritional and environmental conditions on protease production

The bacterium Serratia marcescens has been widely reported as a good producer of extracellular metalloprotease. Research on the purification and characterization of Serratiopeptidase enzyme from different strains of Serratia marcescens isolates, such as Serratia marcescens E-15, Serratia marcescens ATCC 25419, and Serratia marcescens NRRL B-23112, has shown micro-heterogeneity among the metalloproteases produced.

Effect of different substrate sources on protease production

The optimal conditions for serratiopeptidase enzyme production by Serratia marcescens were 0.5 % substrate (Gelatin) concentration after 24 hour of incubation period.

Effect of different carbon sources on protease production

The optimum carbon source for serratiopeptidase enzyme production was glucose. On the other hand, (Sonnleitner, 1983; Sen and Satyanarayana, 1993) reported that glucose suppress protease production.

Effect of different nitrogen sources on protease production

(Mohankumar and Hari Krishna Raj, 2011) stated that the optimum nitrogen source for serratiopeptidase enzyme production was tryptone. While, (Makino et al., 1981) reported that Marine Pseudomonas strain 1452 have the ability to produce extracellular protease using casein as a nitrogen source. Also, (Nigam et al., 1981) reported that a strain of Pseudomonas aeruginosa from soil produced large quantities of extracellular neutral proteinase. However, (Henriette et al., 1993) revealed that the active effect of protease peptone was significantly affected by the addition of NH4Cl at increasing concentrations to a culture of Serratia marcescens 532S. Moreover, (Femi-Ola et al., 2014) declared that the best nitrogen source for protease production by Serratia marcescens was casein. Also, (Ananthakrishnan et al., 2013) mentioned that the best nitrogen source for protease production was tryptone by Serratia marcescens UV mutant SM3.

Effect of different phosphorus sources on protease production

(Mohankumar and Hari Krishna Raj, 2011) stated that the best buffer for production of Serratiopeptidase enzyme was phosphate buffer. Additionally,

(Pansuriya and Singhal, 2010) declared that the best concentration of K2HPO4 for the protease production is 1.2 (%w/v)

Effect of different initial pH levels on protease production (Mohankumar and Hari Krishna Raj, 2011) stated that the maximum proteolytic Yield was achieved at 6.0 pH. Also, (Venil and Lakshmanaperumalsamy, 2009) mentioned that optimum initial pH for protease production by Serratia marcescens SB08 was pH 6. Additionally, (Pansuriya and Singhal, 2010) declared that initial pH of 6.0 of the fermentation medium supported maximum Serratiopeptidase enzyme production (950±80 EU/ml).

Effect of different incubation temperatures on protease production

(Mohankumar and Hari Krishna Raj, 2011) stated that the maximum proteolytic Yield was achieved at 32ºC. While, (Joseph and Palaniyandi, 2011) declared that maximum protease production was attained at 40 ⁰ C by all the production mediums using Serratia marcescens Sp7. Also, (Femi-Ola et al., 2014) stated that The optimum temperature for protease production by Serratia marcescens isolate was found to be 40°C.

(Doddapaneni et al., 2007) reported that protease production was seen by Serratia rubidaea strain in the medium incubated at 4⁰ C, indicating the organism as a psychrophile. Also, the optimum temperature for the growth of Serratia rubidaea strain was observed at 10-30⁰ C and protease production was detected in mid logarithmic phase (after 10 hrs.), increased until its optimum production after 48 h incubation. 65% protease activity was observed after 72 hrs. Moreover, (Salamone and Wodzinski, 1997) reported that though protease production noticed all most all studied environments higher production was noticed at 33⁰ C (5400U/ml). A very low enzyme activity of 2385U/ml observed at 40⁰ C. Additionally, (Bach et al., 2012) mentioned that Maximal protease production by Serratia marcescens P3 was (12.5 U mL−1) was observed at 18–30 ◦C. Furthermore, (Sumathi et al., 2012) stated that the optimum temperature for protease production from Serratia marcescens NPLR1 was 40⁰ C. Chemo-physical properties of the protease and effect of metal ions and inhibitors

(Salamone and Wodzinski, 1997) reported that the extracellular metalloprotease (SMP 6.1) produced by a soil isolate of Serratia marcescens NRRLB-23112 was resistant to inhibition by all compounds tested with the exception of EDTA which indicate that extracellular protease enzyme is a metalloprotease enzyme.

(Romero et al., 2001) reported that the activity was totally inhibited by PMSF, but not by 1,10-phenanthroline and only slightly by EDTA, indicating that it was a serine protease. In addition, the major protease purified from the whey supernatant was analyzed in a similar way to that described for the serine protease. This enzyme activity was not inhibited by PMSF, but both 1,10- phenanthroline and EDTA produced strong inhibition of the protease indicating that it should be classified as a metalloprotease. The effects of some divalent cations on the activity of this metallo-protease were also analyzed, by the addition of the respective cation to the assay mixture. The presence of Ca2+ at 2 mM slightly increased the protease activity, while 2 mM Mg2+ produced a partial inhibitory effect, but the most important effect was produced by the presence of Zn2+ which was able to increase the velocity of the reaction very strongly.

Effect of temperature on protease activity

Femi-Ola et al. (2014) stated that optimum protease activity was obtained at 50°C. At temperatures above and below these points, there was reduction in protease activity. While, Tariq et al. (2011) mentioned that the maximum temperature for the cold active protease of Serratia marcescens TS1 was 20°C in 100 mM Tris HCl buffer, The activity declined rapidly above 25°C and was negligible above 50°C. The enzyme retained its 82% activity at 25°C when temperature increased the enzyme activity decreases rapidly and lost at 50°C.

Effect of temperature on protease stability

(Doddapaneni et al., 2007) mentioned that both the proteases showed considerable thermal stability at 30–60 ⁰ C. Also, (Singh et al., 2012) stated that the optimal temperature for protease activity was 67 ⁰ C by Serratia marcescens isolate. Furthermore, (Salamone and Wodzinski, 1997) reported that the temperature stability of the protease was examined at 40, 50, and 55 °C. At 40 °C the enzyme retained 80% activity after a 1-hr incubation. The enzyme had no activity after a 15-min incubation at 55 °C. The enzyme exhibited 50% maximal activity at 51 °C and 26 °C.

(Nam et al., 2013) stated that in light of heat stability, the alkaline protease produced by Serratia marcescens S3-R1 was examined at 30, 40, 50, and 60⁰ C. At 30 and 40⁰ C, the enzyme retained close to 100% activity at 60 min incubation. Additionally, (Sumathi et al., 2012) declared that the thermos- stability of alkaline protease enzyme produced by Serratia marcescens NPLR1 increased from 55% to 85% at 70⁰ C in the presence of Ca2+ which supports the fact that calcium plays a key role in the enzyme stability at high temperatures.

Effect of pH on protease activity

Femi-Ola et al. (2014) stated that protease was active in pH range of 6 with optimum activity at pH 8. No protease activity was obtained below pH 5 and at pH above 12. While, Tariq et al. (2011) mentioned that The hydrogen ion concentration of cold active protease Serratia marcescens strain TS1 was 8.5 with a sharp decrease in activity above pH 9.0. The protease had half- maximal activity near pH 7.5 and exhibited a little activity below pH 3.5. The protease retained its maximum activity from pH 6.5 to 9.0.

Effect of pH on protease stability

Doddapaneni et al. (2007) mentioned that protease CP-1 was very stable in a broad pH range, maintaining over 90% of its original activity between pH 6 and pH 11, and more than 50% of its activity was retained at pH 12. Protease CP-2 was stable between pH 5 and 9, and much lower activity beyond this pH range. while, Salamone and Wodzinski (1997) reported that the pH stability of for the extracellular metalloprotease (SMP 6.1) produced by a soil isolate of Serratia marcescens NRRLB-23112 was examined by incubating the protease in the pH range 4.5 - 11.5 for 60 min at 30 °C, with residual activity. The protease retained more than 50% of its activity from pH 6.5 to 10.5. Also, Nam et al. (2013) stated that the protease enzyme produced by Serratia marcescens S3-R1 exhibited good pH stability with greater than 90% of the maximal activity in alkaline pH ranging from 7 to 11. The stability of the enzyme was significantly decreased below pH 6. Moreover, Sumathi et al. (2012) stated that the optimum relative pH stability for protease enzyme produced by Serratia marcescens NPLR1 was retained about 90% of stability at pH 12.

Metalloprotease Gene

The gene of metalloprotease enzyme (SRP) from various S. marcescens species has been isolated, cloned, and sequenced (Nakahama et al., 1986; Braunagel and Benedik, 1990) and the crystal structure determined (Baumann, 1994). Summary

Serratiopeptidase is a proteolytic enzyme that has been used as anti- inflammatory agent in sinusitis, bronchitis and other inflammatory disorders. Out of 170 bacterial isolates retrieved from soil samples, Serratia marcescens S6, identified by 16s rDNA, was the most potent isolate in protease production that carried an allele of metallo-protease gene in its genome. The maximum protease production (534 U/ml) was obtained by 10 g/l casein, 1.0 g/l K2HPO4 and 1% (w/v) fructose, 2mM of ZnSO4, KCl and NaCl, at initial pH 9, with inoculum size 1% (1×108 CFU/ml) incubated at 37⁰ C for 48 h. The extracted enzyme was active and stable at 20-50oC. The optimum pH for its activity were 7- 9. The activity was highly affected by metal ions and some inhibitors like EDTA and Tween 20. After purification and fractionation, S6 protease significantly inhibited acute inflammation in mice lungs after intranasal infection with Acinetobacter baumannii compared to non-treated group. Serratia marcescens - S6 produces stable and active metallo-protease that can be used as a co-treatment in A. baumanii pneumonia infections.

Conclusion it could be concluded that, serratiopeptidase has a specific, anti-inflammatory effect, superior to that of other proteolytic enzymes. It is also used as an anti- inflammatory agent against sinusitis, by thinning the mucous secretion. Serratiopeptidase may be particularly effective for those who have lung problems, as it clears out the inflammation, mucus and dead scar tissues, enabling the body's own natural healing system to replace the bad tissue with healthy tissue resulting in better lung function.