Synthesis and Characterization of Thrombolytic Serratiopeptidase from Environmental Isolates of Serratia marcescens

 

Natarajan K1, Subashkumar R2*

1Department of Biomedical Engineering, Vinayaka Mission’s Kirupananda Variyar Engineering College, Vinayaka Mission’s Research Foundation (Deemed to be University), Salem – 636308, Tamil Nadu, India.

2Department of Biotechnology, Sri Ramakrishna College of Arts and Science,

Coimbatore - 641006, Tamil Nadu, India.

*Corresponding Author E-mail: rsubashkumar@gmail.com.

 

ABSTRACT:

Serratiopeptidase is an extracellular enzyme produced by Serratia marcescens and possesses anti-inflammatory, analgesic, anti-atherosclerotic, anti-edemic, and thrombolytic properties. The objective of this study was to produce and characterize the serratiopeptidase from environmental isolates of Serratia marcescens. Serratia marcescens strains were isolated from soil samples by spread plate technique and identified by 16S rRNA-PCR assay. Trypticase soy broth was used as the production medium and the crude enzymes were prepared from the culture by centrifugation. The protein in the crude extract was measured by Lowry’s method. Further, the enzyme was purified by column chromatography, and the fractions were concentrated using ice-cold acetone. Serratiopeptidase in the fraction was confirmed by SDS PAGE and characterized for thrombolytic activity on human blood sample. 150 soil samples were processed and 21 isolates of Serratia marcescens were identified. The protein content in the crude enzyme preparation was estimated in the range of 9 to 12.4mg/mL. SDS PAGE of the purified serratiopeptidase produced a distinct band with a molecular weight of 52 kDa. The significant thrombolytic activity produced by 15 isolates and the isolate, SM24 shows the maximum thrombolytic activity of 95%. In this study, serratiopeptidase produced from the isolates of Serratia marcescens shows significant thrombolytic activity on the human blood clot.

 

KEYWORDS: Serratiopeptidase, Serratia marcescens, Thrombolytic enzymes, extracellular enzymes, therapeutic enzymes.

 

 


INTRODUCTION: 

Enzymes are promising therapeutic tools in medicine today for numerous medical conditions. These enzymes are acting on certain molecules to restore the normal physiological condition of the body1. Many manufacturers targeting enzyme-based drugs and the industrial market for the enzyme is expected to increase sharply. Many enzymes are investigated and proteolytic enzymes are one among them, which have been used for the treatment of many illnesses.

 

Serratiopeptidase (EC number 3.4.24.40) is a metalloprotease (zinc-containing enzyme) with molecular weight ranges from 45 to 60 kDa2. This enzyme is being used commonly by healthcare professionals in various therapeutic applications like surgery, orthopedics, otolaryngology, gynecology, and dentistry because of its anti-inflammatory, analgesic, anti-atherosclerotic and anti-edemic properties3. Serratiopeptidase is conventionally produced from Serratia marcescens, a Gram-negative bacterium belonging to the family Enterobacteriaceae4. Environmental isolates of S. marcescens characteristically produce a red pigment, prodigiosin5,6. Serratia spp. normally inhabit in gut of the silkworm and plays a vital role in morphological transformation of silkworms by proteolysis of cocoons7. The pigmented bacterium is found in various ecological niches, including soil, water, plants, and animals.

 

Cardiovascular diseases were the leading cause of death and caused 10.8 million deaths in Asia in 20198. The development of a blood clot within a blood vessel (intravascular thrombosis) is one of the main causes of cardiovascular diseases. Thrombolytic enzymes rapidly dissolute thrombi within the cardiovascular system. So, several investigations are being pursued to enhance the efficacy and specificity of fibrinolytic therapy, and microbial thrombolytic enzymes have attracted much more medical interest in recent decades9,10. Serratiopeptidase is also found to break down atherosclerotic plaque in cardiovascular diseases. This enzyme target non-living tissue and effectively removes the deposits of fatty substances, cholesterol, cellular waste products, calcium, and fibrin on the inside of the arteries11.

 

The commercial production, purification, and characterization of serratiopeptidase by employing various methodologies are very essential for improving the activity of enzymes and reducing the cost of the final product. The major focus of many investigators is to make their production economically competitive by modifying microorganisms to improve product yields and increase substrate ranges. The present investigation is an attempt to reveal the production of serratiopeptidase in Serratia marcescens, isolated from the environmental sources which might be the better choice for commercial production when compared to the previously reported strains.

 

MATERIALS AND METHODS:

Isolation of S. marcescens:

Serratia marcescens strains were isolated from the soil sample. The soil samples (n=150) were collected near the slaughterhouse located around Erode, Tamilnadu, India. The samples were transported in a sterile test tube and processed immediately. The soil samples were serially diluted (tenfold dilution) and 0.1mL of each dilution was plated on Nutrient agar pales by spread plate technique. The plates were incubated under the aerobic condition at 32°C for 24 to 48 h. The purified isolates were preserved and maintained at 4°C in Nutrient agar slants. Reference strain, Serratia marcescens MTCC 4301, procured from Microbial Type Culture Collection, Institute of Microbial Technology (IMTECH), Chandigarh was used for comparative study.

 

Identification of S. marcescens by 16S rRNA-PCR assay:

Total DNA isolation and agarose gel electrophoresis were carried out under standard conditions12. Unique primers were designed for the amplification of the genes coding for the 16S rRNA of Serratia marcescens. The primers were searched for their uniqueness and specificity to the respective 16S rRNA gene from different strains of Serratia marcescens using the basic local alignment search tool (BLAST). A 1500 bp fragment from the Serratia marcescens encoding the 16S rRNA gene was identified by using a primer set. The sequence of forward primer and the reverse primer is 5’ - CGGACGGGTGAGTAATGT - 3’ and 5’ - GCAGGTTCCCCTACGGTT - 3’ respectively.

 

The PCR was performed in a 0.2mL microcentrifuge tube by adding 1µL of template DNA, 1µL of forward primer, 1µL of reverse primer, 5µL of PCR master mix (Ampliqon-III 2X),  and 2µL of nuclease free water to make 10µL of the reaction mixture and homogenized by quick centrifugation. The PCR was performed with an initial denaturation at 95°C for 5 min, 34 cycles of denaturation at 94°C for 1min, annealing at 51°C for 1 min, extension at 72°C for 1min followed by a final extension at 72°C for 8 min. The reaction tubes were cooled at 4°C. 

 

The amplified PCR product (10µL) was electrophoresed using 1.5% agarose gel along with the DNA molecular weight marker for 2h at 50 V in Tris-acetate-EDTA buffer to analyze the amplicon. The sizes of the amplification products were determined by comparing with 500 bp DNA ladder (Helini biomolecules, Chennai).

 

Synthesis and preparation of crude enzyme (Serratiopeptidase):

The isolated colony of each strain was inoculated into appropriately labeled 10mL of sterile trypticase soy broth (Pancreatic digest of casein - 15.0gm, Peptic digest of soybean meal - 5.0 gm, Sodium chloride - 5.0 gm, Distilled water - 1000mL, Final pH - 7.3 +/- 0.2 at 25°C) and incubated at 32°C for 24 h. The 10 mL of the 24h incubated inoculum was transferred to 90mL sterile trypticase soy broth and incubated at 32°C for 24hours in a shaker at 80rpm/min. The broth was centrifuged at 10,000rpm for 20 min at 4°C to obtain a cell-free filtrate. The cell-free filtrate was filtered through a membrane filter and the filtrate was stored in the refrigerator at 4°C. The concentration of protein in the filtrate was measured by Lowry’s method13. The protein content in the serially diluted sample was measured using bovine serum albumin as a standard.

 

Preparation of purified sample:

Column chromatography was performed by packing with Sephadex G-50 (Sigma-Aldrich) in 5mL of the column. One gram of the Sephadex was dissolved in 10 mL of the 1M phosphate buffer (pH 7) and incubated overnight at 4°C in the column for self-packing.

 

The crude enzyme (2.5mL) was mixed with 0.5mL of 0.1M phosphate buffer. The prepared sample was allowed to run on the prepared column. Six fractions with a volume of 0.5mL were collected in sterile Eppendorf tubes.

 

The obtained fractions were mixed with an equal volume of ice-cold acetone and incubated in a freezer for 15min. After the incubation period, the tubes were centrifuged at 5000rpm for 10min, the protein precipitates as a pellet was collected in a sterile tube and the supernatant was discarded. The pellet was dissolved in 1M phosphate buffer and stored in a refrigerator14.

 

Profiling of Serratiopeptidase by SDS PAGE:

The SDS PAGE was done to determine the presence of the desired enzyme, serratiopeptidase with a molecular weight of 52 kDa. The acetone purified samples were mixed with sample buffer in the ratio of 1:1 and denatured in a boiling water bath for 10 sec.

 

Electrophoresis was performed with 4% stacking gel and 12% resolving gel. The gel was stained with Coomassie brilliant blue R-250 staining solution for 4 to 6h with uniform shaking and treated with a destining solution (Methanol-Acetic acid-Water) until the background of the gel appear clear. The molecular weight of fractioned proteins was determined by a standard molecular weight marker with a range of 14 to 97 kDa.

 

Thrombolytic activity:

Venous blood from healthy individuals was collected and immediately transferred (0.5mL) to sterile microcentrifuge tubes. The serum was pipetted out carefully after clotting and the weight of the tube with clotted blood was determined. 0.1mL of purified serratiopeptidase enzyme from each isolate was added to the labeled tube and distilled water was added to the control tube. The tubes were incubated at 37°C for 90 min. The fluid observed after the incubation period, on the top of the blood clot was carefully removed and the weight of the tube with remaining clotted blood was determined. The percentage of clot lysis was calculated based on the weight difference obtained before and after clot lysis15.

 

RESULTS:

Isolation and identification of S. marcescens:

The presumptive identification of Serratia marcescens was carried out based on cultural characteristics. Colony morphology was observed from the 24h old cultures on Nutrient agar. 21 well-isolated colonies with red, smooth, convex, entire, and round characteristics were selected and subjected to the 16S rRNA identification method. All the 21 isolates were identified as Serratia marcescens by the 16S rRNA identification method. Amplification of conserved region in S. marcescens using specific PCR primer revealed the presence of 1500bp on agarose gel documentation. Results of the 16s rRNA-PCR assay are shown in figure 1.

 

 

Fig.1: Identification of S. marcescens by 16S rRNA–PCR method. Lane (from left to right) – DNA Ladder, MTCC 4301, SM01,SM02, SM05, SM06, SM07, SM08, SM09, SM10, SM13, SM15, SM16, SM17, SM19, SM23, SM24, SM25, SM26, SM27, SM29, SM30, SM31.

 

Synthesis and preparation of crude enzyme (Serratiopeptidase):

The crude enzymes were prepared as cell-free filtrate and the concentration of protein in the filtrate obtained from all the isolates was measured by Lowry’s method. The concentration of proteins in the crude enzyme preparation was determined in the range between 9 and 12.4mg/mL (Table 1). The highest protein concentration of 12.4mg/mL was found in the cell-free filtrate of SM24 and the lowest concentration of 9mg/mL was found in the cell-free filtrate of SM08.

 

Table 1: Determination of protein content by Lowry’s method.

S. No.

Isolates

Protein Concentration (mg/mL) Mean ± SD

1.

SM01

10.2 ± 1.2

2.

SM02

9.6 ± 0.5

3.

SM05

9.7 ± 1

4.

SM06

11 ± 1.2

5.

SM07

11.8 ± 0.8

6.

SM08

9 ± 0.8

7.

SM09

10.5 ± 0.6

8.

SM10

9.8 ± 1.3

9.

SM13

12.2 ± 1

10.

SM15

9.4 ± 1.1

11.

SM16

9.7 ± 0.6

12.

SM17

10.3 ± 0.8

13.

SM19

9.3 ± 0.6

14.

SM23

12 ± 1

15.

SM24

12.4 ± 0.9

16.

SM25

9 ± 0.6

17.

SM26

11.3 ± 1.2

18.

SM27

9.2 ± 0.6

19.

SM29

10.4 ± 0.4

20.

SM30

11.8 ± 0.6

21.

SM31

9.6 ± 0.6

22.

MTCC 4301

9.7 ± 0.8

Values are the mean of triplicate determination (n =3) ± SD

Preparation of Purified Sample:

The enzymatic proteins in the cell-free filtrate were purified by column chromatography packed with Sephadex G-50. Six fractions with a volume of 0.5mL were collected in sterile Eppendorf tubes. The protein content in the fractions was precipitated by mixed with an equal volume of ice-cold acetone and the protein precipitates as a pellet. The pellet was collected and dissolved in 1M phosphate buffer and stored in a refrigerator for further characterization.

 

Profiling of Serratiopeptidase by SDS PAGE:

SDS PAGE was performed for all the collected fractions to identify the 52 kDa protein with reference to the standard protein marker (14 to 97 kDa). Fraction 6 of the sample was found to contain 52 kDa molecular weight protein, serratiopeptidase (Figure 2).

 

 

Fig.2: SDS PAGE of Serratiopeptidase. Lane M – Protein marker (14-97 kDa); Lane 1 – SM13; Lane 2 – SM23; Lane 3 – SM30.

 

Thrombolytic Activity:

Of the 21 isolates, 15 isolated strains have substantial thrombolytic activity. The clot lysis percentage of serratiopeptidase produced from the isolate was found to differ significantly. The highest thrombolytic activity of 95% was observed with SM24. The isolates SM07, SM09, SM10, SM16, SM17, and SM19 fail to produce significant thrombolytic activity (Figures 3 and 4).

 

 

 

Fig. 3: Thrombolytic activity of the isolates. Tube (from left to right) – SM01, SM02, SM05, SM06, SM07, SM08, SM09, SM10, SM13, SM15, SM16, SM17, SM19, Control, SM23, SM24, SM25, SM26, SM27, SM29, SM30, SM31, MTCC 4301.

 

 

Fig.4: Clot lysis percentage of the isolates

 

DISCUSSION:

Samples to isolate S. marcescens were collected near the slaughterhouse. Normally, the microbial flora colonized in this location may derive their nutrition from the blood and blood products due to the production of thrombolytic enzymes. The colony morphology of all the isolates of S. marcescens showed red, convex, entire, and round. All the isolates were shown a distinct band with a molecular weight of 1.5kb on optimized PCR conditions. Salarizadeh et al., also identified the metalloprotease gene on Serratia, using a specific primer, and the DNA fragment of 1.5kbp in size was isolated from the genome of Serratia16.

 

Mohankumar and Krishna Raj also reported that the incubation temperature of 32ºC was generally more favorable for the enzyme production in trypticase soy broth17. The acetone method offers more advantages than any other method to concentrate the soluble proteins in the cell-free extract because ice-cold acetone removes most of the salts and lipid-soluble contaminants. The molecular weight of Serratiopeptidase ranges from 45 to 60 kDa, The SDS-PAGE analysis of Serratiopeptidase showed that 52 kDa metalloprotease and Mohankumar also reported a similar result17. The vigorous thrombolytic activity of SM24 may be associated with increased enzyme production. Now a day’s rapid and precise spectrophotometric method for estimation of serratiopeptidase was available which can be utilised for the screening of effective wild stains18,19.

 

CONCLUSION:

Strain improvement using induced mutation techniques, optimization of incubation temperature, time, pH, carbon source, and production media may increase the yield of Serratiopeptidase in already reported isolates of S. marcescens, but still, the searching for effective wild stain is one of the old and golden methods of choice in many investigators. The present investigation reported the isolate, SM24 as a good candidate for the production of serratiopeptidase.

 

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

 

REFERENCES:

1.      De la Fuente M, et al. Enzyme therapy: Current challenges and future perspectives. International Journal of Molecular Sciences. 2021; 22(17):9181.doi:10.3390/ijms22179181

2.      Jadhav SB, et al. Serratiopeptidase: Insights into the therapeutic applications. Biotechnology Reports. 2020; 28(e00544): e00544.doi:10.1016/j.btre.2020.e00544

3.      Bhagat S, et al. Serratiopeptidase: a systematic review of the existing evidence. International Journal of Surgery. 2013;11(3): 209-217.doi:10.1016/j.ijsu.2013.01.010

4.      Archana L, et al. Extraction of Serralysin: A Fibrinolytic enzyme from Serratia sp. isolated from soil. Research J. Pharm. and Tech. 2018;11(7):2911-2913. doi:10.5958/0974-360X.2018.00536.X

5.      Dhanalakshmi S, et al. Development and Optimization of Natural Colourant from Microbial Pigment. Research J. Pharm. and Tech. 2019;12(7):3475-3478.doi:10.5958/0974-360X.2019.00589.4

6.      Namratha K, et al. A study on extraction and characterization of natural red pigment from a soil isolate Serratia marcescens CN14. Research Journal of Pharmacy and Technology. 2022; 15(1):148-2.doi:10.52711/0974-360X.2022.00024

7.      Rawat M, et al. An Overview of Miracle Enzyme- Serratiopeptidase. Research J. Pharm. and Tech. 2008;1(3):124-131.

8.      Zhao D. Epidemiological features of cardiovascular disease in Asia. JACC: Asia. 2021;1(1),1-13. doi:10.1016/j.jacasi.2021.04.007

9.      Tough J. Thrombolytic therapy in acute myocardial infarction. Nurs Stand. 2005;19(37):55-64; quiz 66.doi:10.7748/ns2005.05.19.37.55.c3878

10.   Monisha V, et al. Screening for Streptokinase producing Streptococcus sp. from Food and Soil samples. Research J. Pharm. and Tech, 2018;11(7):2845-2847.doi:10.5958/0974-360X.2018.00524.3.

11.   Tiwari M. The Role of Serratiopeptidase in the Resolution of Inflammation. Asian Journal of Pharmaceutical Sciences. 2017; 12(3):209-215.doi:10.1016/j.ajps.2017.01.003.

12.   Sambrook J, et al.  Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, New York. 2001

13.   Lowry OH, et al.  Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry. 1951;193(1):265-75

14.   Morita Y, et al. Purification and characterization of a cold-active protease from psychrotrophic Serratia marcescens AP3801. J Amer Oil Chem Soc. 1997;74:1377-1383.doi:10.1007/s11746-997-0240-8

15.   Gopinath S, et al. Enhancement of Serrapeptase Hyper Producing Mutant by Combined Chemical and UV Mutagenesis and its Potential for Fibrinolytic Activity. J Pure Appl Microbiol. 2020;14(2):1295-1303. doi:10.22207/JPAM.14.2.25

16.   Salarizadeh N, et al. Purification and characterization of 50 kda extracellular metalloprotease from Serratia sp. ZF03. Iran J Biotechnol. 2014;12:18-27.doi:10.15171/ijb.1009.

17.   Mohankumar A, KrishnaRaj RH. Production and Characterization of Serratiopeptidase Enzyme from Serratia Marcescens. International Journal of Biology. 2011;3(3): 3951.doi:10.5539/ijb.v3n3p39.

18.   Patel P, Rabadiya B. New Visible Spectrophotometric Method for Estimation of Serratiopeptidase from Tablet Formulations. Asian J. Research Chem. 2010; 3(3): 631-633

19.   Alpesh BL, et al. Development and Validation of Analytical Method for Simultaneous Estimation of Diclofenac potassium and Serratiopeptidase in Pharmaceutical Formulation. Research J. Pharm. and Tech. 2014;7(6): 655-659.

 

 

 

 

 

Received on 27.07.2022            Modified on 23.12.2022

Accepted on 07.04.2023           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(10):4698-4702.

DOI: 10.52711/0974-360X.2023.00763