Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 19  |  Issue : 3  |  Page : 238-243

Production and one-step purification of serratiopeptidase enzyme from Serratia marcescens with potent anti-inflammatory and antioxidant power


1 Department of Chemistry of Natural and Microbial Products, National Research Centre, Dokki, Giza, Egypt
2 Department of Plant Biochemistry, National Research Centre, Dokki, Giza, Egypt

Date of Submission27-Dec-2019
Date of Decision05-Feb-2020
Date of Acceptance04-May-2020
Date of Web Publication24-Aug-2020

Correspondence Address:
Mostafa A El-Abd
Department of Chemistry of Natural and Microbial Products, Lab 174, National Research Centre, Dokki 12311, Giza
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/epj.epj_65_19

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  Abstract 

Background and objective Inflammation is a huge problem facing the world. NSAIDs are the most popular and widespread drugs used for lowering the impaired symptoms accompanied with inflammation. This group has plentiful disorders to the gastrointestinal tract reaching to gastric ulcer. The objective of this study was to produce a natural product having an anti-inflammatory power without the side effects concomitant with the used drugs. The serratiopeptidase enzyme has a potent anti-inflammatory action without any abnormal disorders.
Materials and methods Different media and cultural conditions were evaluated for the optimal production of the serratiopeptidase enzyme from a local bacterial strain. Partial purification was employed using the ammonium sulfate precipitation technique.
Results and conclusion Serratiopeptidase enzyme was produced by the bacterial strain Serratia marcescens and its anti-inflammatory and antioxidant power were declared. The best medium was chosen for the optimal productivity and the optimum pH was 6 that recorded the highest anti-inflammatory and anti-oxidant activity at 67.5 and 71.8%, respectively, which is powerful than any chemical drugs used. Serratiopeptidase enzyme produced by this strain was partially purified by ammonium sulfate precipitation revealed an increase in specific activity by two times than the crude one with 14.7 recovery. The authors conclude the use of serratiopeptidase enzyme for treating the inflammation instead of the traditional ways.

Keywords: anti-inflammatory, antioxidant, Serratia marcescens, serratiopeptidase


How to cite this article:
El-Abd MA, Ibrahim EA. Production and one-step purification of serratiopeptidase enzyme from Serratia marcescens with potent anti-inflammatory and antioxidant power. Egypt Pharmaceut J 2020;19:238-43

How to cite this URL:
El-Abd MA, Ibrahim EA. Production and one-step purification of serratiopeptidase enzyme from Serratia marcescens with potent anti-inflammatory and antioxidant power. Egypt Pharmaceut J [serial online] 2020 [cited 2020 Nov 26];19:238-43. Available from: http://www.epj.eg.net/text.asp?2020/19/3/238/293319


  Introduction Top


Nonhealed inflammation is one of the most predominant problems facing the medical circles worldwide. Inflammation is a self-defense response of the body’s immune system against most infectious and noninfectious disorders such as sinusitis, arthritis, lung diseases, cardiovascular disorders, etc. It is characterized by heat, redness, and pain. There are many groups of chemically synthesized anti-inflammatory agents that were conventionally used in the market. NSAIDs are the most potent and widely prescribed group especially for inflammatory joint problems. Their effects are mediated through inhibition of the biosynthesis of prostaglandins, a hormone responsible for mucosal protection of the stomach, leading to gastric ulcers.

Unlike these drugs, serratiopeptidase is a naturally occurring enzyme with no inhibitory effects on prostaglandins and with no effect on the gastrointestinal tract. Serratiopeptidase is a proteolytic enzyme available for clinical use from many years. Serratiopeptidase binds to α-2-macroglobulin in the blood which obscured its antigenicity but retains its enzymatic activity and is slowly, transferred to the site of inflammation. Serratiopeptidase hydrolyzes bradykinin, histamine, and serotonin responsible for the edematous status. Serratiopeptidase reduces swelling, improves microcirculation and expectoration of sputum, etc. [1]. Also, serratiopeptidase is widely used to reduce pain and inflammation associated with some diseases such as arthritis, chronic bronchitis, and atherosclerosis [2].


  Materials and methods Top


Microorganism

Serratia marcescens bacterial strain was obtained from the cultural collection of El-Diwany Laboratory, Natural and Microbial Products Division, National Research Center. The experimental research that is reported in this manuscript has been performed with the approval of an appropriate ethics committee. No experiments had done on humans.

Production media

Nutrient broth was prepared and inoculates with S. marcescens mother culture. Culture broth was maintained in 150 rpm for 24 h at 27°C for the preparation of preproduction media. 2% of the preproduction media are taken for the main production media with different compositions as follows (g/l):
  1. Medium 1: KH2PO4 7, K2HPO4 2, MgSO4 0.1, yeast extract 1.0, casein 2.
  2. Medium 2: Beef extract 5, peptone 8, NaCl 2, dextrose 5, casein 7.5.
  3. Medium 3: Luria Bertani (LB) medium.
  4. Medium 4: Nutrient broth (NB) composed of glucose 5, beef extract 5, peptone 5, NaCl 5.
  5. Medium 5: It composed of 3% ground sesame in tap water.
  6. Medium 6: It composed of 3% ground peanuts in tap water.
  7. Medium7: It composed of 3% rice bran in tap water.


pH was adjusted to 7.

Enzyme assay

Qualitative estimation of serratiopeptidase activity

This was done using 10% gelatin medium poured in equal volumes in test tubes. One tube was inoculated with 0.5 ml culture filtrate of pregrown S. marcescens and another one was inoculated with water and used as control. Both tubes were incubated for 2 h at 37°C, and then kept at 4°C for 30 min. The positive one shows the liquefaction of gelatin.

Quantitative estimation of serratiopeptidase activity

Serratiopeptidase activity was assayed by the modified method of Tsuchida et al. [3]. In brief, 0.5 ml of the enzyme was mixed thoroughly with 2.5 ml of 0.75% of casein solution (using Tris buffer pH 8.5). The mixture was incubated at 37°C for 30 min. Then the reaction was terminated by the addition of 2 ml of 15% trichloroacetic acid. The incubated solution was filtered through Whatman No. 1 filter paper. Then 1 ml from the filtrate was estimated using the Lowry method. The final solution was measured at 750 nm. One unit of serratiopeptidase activity was defined as the amount of enzyme required to liberate 1 µmol (181 μg) of tyrosine in 30 min at 37°C.

Protein assay

Protein concentration was measured by the method of Lowry et al. [4] using bovine serum albumin (Sigma Scientific Services Co., St. Louis, Missouri, USA) as the standard. The specific activity was expressed as the enzyme.

In-vitro anti-inflammatory assay

Anti-inflammatory activity was tested using the method of Rahman et al. [5]. The samples or standard drug diclofenac sodium were mixed with 0.45 ml bovine albumin serum. Samples were incubated at 37°C for 20 min, heated at 57°C for 3 min, after cooling the samples, 2.5 ml phosphate buffer at 6.3 pH was added to the samples. The absorbance was measured using a ultraviolet-visible spectrophotometer at 255 nm.

In vitro total antioxidant capacity assay

Phosphomolybdic acid-free radial scavenging was assayed according to Prieto et al. [6]. A measure of 1 ml of samples and standard ascorbic acid was mixed with 3 ml of reagent solution. Tubes were incubated at 95°C for 90 min. After cooling, the absorbance of each sample was measured at 695 nm.

Partial purification of serratiopeptidase

Sequential ammonium sulfate precipitation

The enzyme was first precipitated from the cell-free supernatant by the addition of ammonium sulfate at different saturation levels ranging from 20 to 80% and kept for 2 h at 4°C and the precipitated protein was collected by centrifugation at 10 000 rpm for 20 min and at 4°C and then resuspended in a minimal volume of 0.1 M phosphate buffer, pH 8.0 to obtain the concentrated enzyme suspension. The enzyme solution was finally dialyzed against the same buffer and this was repeated till the buffer outside the bag was free from sulfate ions; then the enzyme solution was lyophilized and weighed.


  Results and discussion Top


S. marcescens was tested for the serratiopeptidase enzyme production qualitatively as shown in [Figure 1]. The left tube showed positive result, where the protease enzyme liquefied the substrate by hydrolyzing the gelatin.
Figure 1 Qualitative estimation of serratiopeptidase production by Serratia marcescens.

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As shown in [Figure 2], S. marcescens can successfully grow and produce serratiopeptidase enzyme in a wide range of unselective media with even simple compositions. Medium no. 2 was selected because of its higher enzyme productivity and activity. The highest productivity obtained by medium no. 2 related to the presence of casein in its composition which acts as a protein source that was hydrolyzed by proteolytic enzymes produced by S. marcescens. These results are in agreement with that of Subbaiya et al. [7], who produce serratiopeptidase enzyme in a pilot scale using casein as the sole carbon source. Also, medium no.2 showed the highest anti-inflammatory activity by inhibiting the denaturation of heat-induced albumin as shown in [Table 1] with maximum inhibition (74.27%) at a concentration of 25 µg/ml compared either with different media or with diclofenac sodium as the most potent drug used in the market.
Figure 2 Serratiopeptidase activities produced by Serratia marcescens at different shaken culture media.

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Table 1 Anti-inflammatory and antioxidant activities of different shaken culture media

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On the other hand, the anti-oxidant capacity of serratiopeptidase was evaluated by scavenging of phosphomolybdic acid as shown in [Table 1]. Most of the media possessed the ability of scavenging of phosphomolybdic acid radical; the inhibition % ranged from 7.83 to 85.03% at a concentration of 50 μg/ml. The highest antioxidant effect was observed in media 4 (85.03%) at a concentration of 50 μg/ml. These results were superior than that of Wang et al. [8] who produced the proteolytic enzyme along with the antioxidant with 72% capacity from Serratia ureilytica.

By the same, medium no. 2 was the most favored for serratiopeptidase productivity and anti-inflammatory activity when growing S. marcescens on the prior media under static conditions but with lower yield than the shaken one. While medium no.6 enhanced the antioxidant capacity than other media that are related to the presence of peanuts in the medium enriched with vitamin E ([Figure 3] and [Table 2]). This is referred to the need of high aeration for growth and metabolism.
Figure 3 Serratiopeptidase activities produced by Serratia marcescens at different static culture media.

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Table 2 Anti-inflammatory and antioxidant activities of different static culture media

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Panchanathan et al. [9] showed that protease from marine actinobacterium Streptomyces spp. MAB18 has antioxidant activity against free radicals of DPPH, O, NO, Fe2+ chelating and reducing power.

The effect of different initial pH values on the productivity of S. marcescens was studied as shown in [Figure 4] and [Table 3]. The activity of serratiopeptidase enzyme was nearly the same, while at pH 6 the yield increased obviously. pH 7.5 was the best for anti-inflammatory and antioxidant activities. These results match with that of Mohankumar [1], who declared that pH 6 was the optimal for serratiopeptidase production by S. marcescens using phosphate buffer.
Figure 4 Effect of initial pH on serratiopeptidase activity.

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Table 3 Anti-inflammatory and antioxidant activities at different pHs in shaken culture

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From the results summarized in [Table 4] and [Table 5], an extracellular serratiopeptidase was precipitated from the culture filtrate of S. marcescens using different saturation levels of ammonium sulfate ranging from 25 to 100%. The results showed that the 40% ammonium sulfate saturation fraction possessed the highest enzyme activity (3905 Ul/ml) with an increase of purification fold to two times and recovered activity 14.7%. These results are in agreement with that of El-Khonezy et al. [10], who illustrated that 40–-60% saturation of ammonium sulfate was the best for protease enzyme recovery (4.28) with 3.5 purification fold. Also, Nageswara et al. [11] illustrated the partial purification of serratiopeptidase produced by S. hydrogenans var. MGS13 at 50% saturation of ammonium sulfate yielded a maximum recovery of 52%.
Table 4 Summary of the results of the partial purification steps of serratiapeptidase enzyme produced by Serratia marcescens

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Table 5 Anti-inflammatory and antioxidant activities of different partial purification steps of culture filtrate produced by Serratia marcescens

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There were no effects of partial purification on anti-inflammatory and antioxidant activities. Serratiopeptidase enzyme purification by 40, 65, and 80 of ammonium sulfate saturation declared the highest anti-inflammatory activity at 25 μg/ml (70.33, 70.32, and 70.18%, respectively). While a low percentage of inhibition was demonstrated at 100% ammonium sulfate saturation compared with the drug. Also, the highest scavenging ability was possessed by serratiopeptidase purification at ammonium sulfate saturation of 65% (70.33%), followed by 40% (70.32%) and 80% (70.18%).The highest anti-inflammatory property of serratiopeptidase from S. marcescens was reported by Umesh et al. [12], who declared that serine protease inhibits cyclooxygenase (COX-I and COX-II). As this cyclooxygenase produce different inflammatory mediators including interleukins (IL), prostaglandins (PGs), and thromboxane (TXs). Rajinikanth et al. [13] found that the anti-inflammatory activity of serratiopeptidase is exhibited by reducing reactive oxygen species, lipid peroxidation, neutrophil accumulation and nitric oxide in acetic acid induced ulcer colitis in mice.


  Conclusion Top


Serratiopeptidase enzyme is a promising substitute for the ordinary anti-inflammatory drugs present in the market without the dangerous and harmful side effects accompanied with their use for long term with an additional antioxidant benefit. Shaken culture having a casein in its constituents at 27°C incubation temperature is the optimal for the production of serratiopeptidase enzyme by S. marcescens.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Mohankumar A. Production and characterization of serratiopeptidase enzyme from Serratia marcescens. Int J Biol 2011;3:3.  Back to cited text no. 1
    
2.
Tiwari M. The role of serratiopeptidase in the resolution of inflammation. Asian J Pharm Sci 2017;12:209–215.  Back to cited text no. 2
    
3.
Tsuchida OY, Yamagata T, Ishizuka J, Arai M, Yamada T, Ichishima E. An alkaline protease of an alkalophilic Bacillus spp., Curr Microbiol 1986;14:7–12.  Back to cited text no. 3
    
4.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265–275.  Back to cited text no. 4
    
5.
Rahman H, Eswaraiah MC, Dutta AM. In-vitro anti-inflammatory and anti-arthritic activity of Oryza sativa Var.Joha Rice (an aromatic indigenous rice of Assam). American-Eurasian J Agric Environ Sci 2015;15:115–121.  Back to cited text no. 5
    
6.
Prieto P, Pineda M, Aguilar M. Spectrophotometric quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: specific application to the determination of vitamin E. Anal Biochem 1999;269:337–341.  Back to cited text no. 6
    
7.
Subbaiya R, Ayyappadasan G, Mythili SV, Dhivya E, Ponmurugan P. Pilot scale microbial production and optimization of Serratia peptidase from Serratia marcescens. J Ecobiotechnol 2011; 3:10–13.  Back to cited text no. 7
    
8.
Wang SL, Lin CL, Liang TW, Liu KC, Kuo YH. Conversion of squid pen by Serratia ureilytica for the production of enzymes and antioxidants. Bioresour Technol 2009;100:316–323.  Back to cited text no. 8
    
9.
Panchanathan M, Venkatesan J, Sivakumar K, Kim SK. Production, characterization and antioxidant potential of protease from Streptomyces spp. MAB18 Using Poultry Wastes. Biomed Res Int 2013;2013:1–12.  Back to cited text no. 9
    
10.
El-Khonezy MI, El-Gammal EW, Atwa NA, El-Abd MA. Partial purification and characterization of an alkaline serine protease produced by Streptomyces griseus NCRRT and its antifungal effect on Fusarium solani. World Appl Sci J 2015;33:831–842.  Back to cited text no. 10
    
11.
Swathi Nageswara S, Guntuku G, Yakkali BL. Purification, characterization, and structural elucidation of serralysin-like alkaline metalloprotease from a novel source. J Genet Eng Biotechnol 2019;17:1.  Back to cited text no. 11
    
12.
Umesh L, Nishtha KS, Vrushali B, Vandana G, Patil RR. Media optimization for serratiopeptidase by statistical approach followed by isolation and product purification. Int J Sci Res Manage 2014;2:1608–1614.  Back to cited text no. 12
    
13.
Rajinikanth B, Venkatachalam VV, Manavalan R. Investigations on the potential of serratiopeptidase on acetic acid induced ulcerative colitisin mice. Int JPharm Pharm Sci 2014; 5:525–531.  Back to cited text no. 13
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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