Serratia peptidase is active proteolytic enzyme which has the potential of cleaving peptide bond.Â  Present investigation deals about the Microbial production of serratia peptidase using Serratia marcescens in small scale fermentor. Batch fermentor has been run continuously throughout the night to analyze the production of protein as well as kinetics. Culture broth was maintained at 150rpm for 72 hrs. Protein sample was isolated by centrifuging at 3000rpm for 10mints. The result revealed that Serratia marcescens showed the enormous production of protein in fed batch fermentor compared to the small scale level.Â  Different substrates were been used for the production of enzyme. Among all cysteine showed the better activity as 2 units/ml of enzyme. Enzymatic assay of Serratia peptidase was done at different time interval of crude broth. Enzyme activity showed that maximum at 40ÂºC for 72hrs. It was observed that 0.65 units/ml of enzyme. Fed batch pilot scale production of Serratia peptidase was done at 0.5%cystein and 700rpm for 48hrs of run time.Â

Ayyappadasan, G

Dhivya, E

Mythili, S V

Ponmurugan, P

Subbaiya, R

Journal of Ecobiotechnology

English

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Journal of Ecobiotechnology 2011, 3(12): 10-13 ISSN: 2077-0464 Available Online: http://journal-ecobiotechnology.com/    Pilot scale microbial production and optimization of Serratia peptidase from Serratia marcescens  R. Subbaiya, G.Ayyappadasan, S.V.Mythili, E.Dhivya and P.Ponmurugan  Department of Biotechnology, K.S.Rangasamy College of Technology, Tiruchengode, Namakkal-637 215, Tamil Nadu, India.  Abstract  Serratia peptidase is active proteolytic enzyme which has the potential of cleaving peptide bond.  Present investigation deals about the Microbial production of serratia peptidase using Serratia marcescens in small scale fermentor. Batch fermentor has been run continuously throughout the night to analyze the production of protein as well as kinetics. Culture broth was maintained at 150rpm for 72 hrs. Protein sample was isolated by centrifuging at 3000rpm for 10mints. The result revealed that Serratia marcescens showed the enormous production of protein in fed batch fermentor compared to the small scale level.  Different substrates were been used for the production of enzyme. Among all cysteine showed the better activity as 2 units/ml of enzyme. Enzymatic assay of Serratia peptidase was done at different time interval of crude broth. Enzyme activity showed that maximum at 40ºC for 72hrs. It was observed that 0.65 units/ml of enzyme. Fed batch pilot scale production of Serratia peptidase was done at 0.5%cystein and 700rpm for 48hrs of run time.  Keywords: Serratia marcescens, Serratia peptidase, casein.  INTRODUCTION       The natural Chelation-Anti-Inflammatory Serratia peptidase has had wide clinical use - spanning over twenty-five years throughout Europe and Asia - as a viable alternative to salicylates, ibuprofen and the more potent NSAIDs. Unlike these drugs, Serratia peptidase is a naturally occurring, physiological agent with no inhibitory effects on prostaglandins and is devoid of gastrointestinal side effects. Uses: 1. Cardiovascular Disease 2. Arthritis 3.Rheumatoid Arthritis 4.Lung Problems 5.Eye Problems 6.Running Nose and sinusitis problems 7.Sports Injuries 8. Inflammation of any kind Serratia peptidase is a proteolytic enzyme isolated from the micro-organism Serratia marcescens. This enzyme is naturally processed commercially today through fermentation and was discovered in the silkworm intestine. This immunologically active enzyme is completely bound to the alpha 2 macroglobulin in biological fluids. Histological studies reveal powerful anti-inflamma-tory effects of this naturally occurring enzyme. Serratia peptidase digests non-living tissue, blood clots, cysts, and arterial plaque and inflammation in all forms.      NSAIDs are among the most widely prescribed drugs for rheumatoid arthritis and other inflammatory joint conditions. Their effects are mediated through inhibition of the biosynthesis of prostaglandins. They work by irreversibly blocking cyclooxygenase, the enzyme which catalyses the reactions of arachidonic acid to endoperoxide compounds. The neurological and gastrointestinal side effects of these agents have been reviewed in considerable detail. All of the NSAIDs, with the exception of Cytotec, inhibit prostaglandin El, a local hormone responsible for gastric mucosa cyto protection. A common side effect from these medications is gastric ulcers. More serious adverse reactions such as blood dyscrasias, kidney damage and cardiovascular effects have been noted. Most physicians rotate among the ten most widely prescribed NSAIDs, as soon as one causes side effects or stops working.      The search for a physiologic agent that offers anti-inflammatory properties without causing side effects may have ended with the discovery of the Serratia peptidase (SP) enzyme. SP is an anti-inflammatory, proteolytic enzyme isolated from the microorganism, SerratiaEl5. This enzyme is naturally present in the silkworm intestine and is processed commercially today through fermentation. The silkworm has a symbiotic relationship with the Serratia microorganisms in its intestines. The enzymes secreted by the bacteria in silkworm intestines have a specific affinity to a vital tissue and have no detrimental effect on the host’s living cells. By dissolving a small hole in the silkworm’s protective cocoon (avital tissue), the winged creature is able to emerge and fly away. The discovery of this unique biological phenomenon led researchers to study clinical applications of the SP enzyme in man.  MATERIALS AND METHODS Screening For Serratia peptidase Enzyme Production Casein Hydrolysis       Skimmed milk agar was prepared as follows: Dissolve the ingredients, adjust the pH to 7.2, distribute in 200ml portions into 250ml conical flasks and sterilize by autoclaving at 15lb pressure for 15minutes. Don’t autoclave skimmed milk powder. Pasteurize the skimmed milk at 70oC for 15-20 min. Cool and mix with the autoclaved and cooled medium containing peptone and agar. Pour the autoclaved and cooled (45-500C) medium into sterile Petri plates (20ml each) and allow these to solidify. Label 3 skim milk agar plates with name of bacterial organism to be inoculated and the fourth plate as control. Make a single line streak inoculation from each culture into its labeled Petri plate across the surface of the medium.   Received: Oct 12, 2011; Revised: Nov 15, 2011; Accepted: Dec 08, 2011.  *Corresponding Author R. Subbaiya  Department of Biotechnology, K.S.Rangasamy College of Technology,  Tiruchengode, Namakkal-637 215, Tamil Nadu, India. Tel: +91-4288-274741; Fax: +91-4288-274860 Email: ramsubbubio@gmail.com       Journal of Ecobiotechnology 2011, 3(12): 10-13  11Incubate the plates (inoculated & uninoculated) for 24-48 hours at 300C.   Production Media       Nutrient broth was prepared and inoculates the 1% of serratia marcescens mother culture.  Culture broth was maintained in 150rpm for 24hrs for preparation of preproduction media. 2% of the preproduction media are taken for the main production media with different substrate concentration.   Composition of Different Production Media: Casein as Substrate Tryptone   - 3g Yeast extract  - 5g Glucose   - 5g Casein    - 5g Glycerol   - 1g Distilled water  - 1000ml pH        - 7  Cysteine as Substrate Tryptone         - 15g Yeast extract     - 5g L-cysteine      - 0.2g NaCl       - 1g Disodium phosphate anhydrous- 0.3g Sodium bicarbonate   - 2g Sodium acetate anhydrous  - 12g Sucrose      - 5g Distilled water     - 1000ml pH        - 7  Enzyme Assay       Serratia peptidase activity was assayed by the modified method of Keay et al[2]. In brief, 0.2 ml of the enzyme was mixed thoroughly with 0.5 ml of 0.5% of casein solution. The mixture was incubated at 37°C for 10 min. Then the reaction was terminated by the addition of 2 ml of 110 mM Trichloroacetic acid and the mixture was again incubated for 30 min at 37°C. The incubated solution was filtered through Whatman No. 1 filter paper. Then 2 ml from the filtrate with 5 ml of 500 mM Na2CO3 and 0.5 ml of Folin’s phenol reagent was added and mixed thoroughly. Again the mixture was incubated at 37°C for 30 min and the final solution was measured at 660 nm. One unit of protease activity was defined as the amount of enzyme required to liberate 1 µmol of tyrosine in 30 min at 37°C.  Protein Assay       Protein concentration was measured by the method of Lowry et al[6] using bovine serum albumin (Sigma, USA) as the standard. In column chromatography elution, the amount of protein was measured in terms of the absorbance at 660 nm. The specific activity was expressed as the enzyme activity per mg of protein.  Unit Definition       One unit will hydrolyze casein to produce color equivalent to 1.0 µmole (181 µg) of tyrosine per minute at pH 7.5 at 37°C (color by Folin & Ciocalteu’s reagent).  Final Assay Concentration       In a 6.00 ml reaction mix, the final concentrations are 42 mM potassium phosphate, 0.54% (w/v)casein, 1.7 mM sodium acetate, 0.8 mM calcium acetate, and 0.1 - 0.2 unit protease.  RESULTS       In small scale we have got higher results when compare to large scale. If we continue further to scale up process, the production will be comparatively low. To overcome this we have to do alternative methods such as Media Optimization, changing the PH, changing various carbon source, nitrogen source & percentage of the inoculum. We can also perform immobilization process for improving the enzyme efficiency. Table 1. Estimation of Protein By Lowry’s Method  Sample Vol of BSA(ml) Vol of Sample (ml) Vol of Water (ml) Reagents (ml)  FC (ml)  OD Concentration ( Ug/ml) STD 1 0.2  0.8 5  0.5  0.103 20 STD 2 0.4  0.6 5 10 0.5 30 0.256 40 STD 3 0.6  0.4 5 min 0.5 min 0.344 60 STD 4 0.8  0.2 5  0.5  0.423 80 STD 5 1  0 5  0.5  0.561 100 T1 2 0.1 0.8 5  0.5  1.269 Un1 T2 2 0.1 0.8 5  0.5  1.405 Un2    R.Subbaiya et al.,   12Table 2. Estimation of Protein By Lowry’s Method  Sample Vol of BSA (ml) Vol of Sample (ml) Vol of Water (ml) Reagents (ml)  FC (ml)  OD Concentration  ( Ug/ml) STD 1 0.2  0.8 5  0.5  0.103 20 STD 2 0.4  0.6 5 10 0.5 30 0.256 40 STD 3 0.6  0.4 5 Min 0.5 min 0.344 60 STD 4 0.8  0.2 5  0.5  0.423 80 STD 5 1  0 5  0.5  0.561 100 T1 2 0.2 0.8 5  0.5  1.152 Un1 T2 2 0.2 0.8 5  0.5  1.255 Un2 Pdn media 2 0.2 0.8 5  0.5   1.066 Un3  Table 3. Enzyme Activity  Fermentor Reading Enzyme Activity(µg/ml) Specific Enzyme Activity(µg/ml)  Filterate (mg/ml) Non-Filterate (mg/ml) Filterate (mg/ml) Non-Filterate (mg/ml) T1(5.30 pm) 0 0 0 0 T2(7.30 pm) 0.16 0.14 0.28 0.68 T3(9.30 pm) 0.32 0.25 0.37 0.91 T4(11.30 pm) 0.47 0.35 0.43 1.12s T5(7.00 am) 0.61 0.44 0.78 1.89 T6(9.00 am) 0.61 0.44 0.78 1.89 T7(11.00 am) 0.42 0.38 0.99 2.38 T8(1.00 pm) 0.34 0.23 1.89 4.54              Fig1. Production Media For Casein                    Fig 2. Casein                 Fig 3. Estimation of Protein By Lowry’s Method Fig                  4. Large Scale Level                        Fig 5. Enzymatic Assay                          Fig 6. Growth Curve                                                        Fig 7. Enzyme Activity Journal of Ecobiotechnology 2011, 3(12): 10-13  13 DISCUSSION       Present work reveals the Casein as substrate for the production of serratia peptidase enzyme. Casein acts as protein source which leads to the production of proteolytic enzyme. It has clearly confirmed that Serratia marcescens can able to produce proteolytic enzyme by casein hydrolysis. Large scale production of enzyme is much useful for biomedical application in commercial aspects. In present result enzyme activity of both filtrate and non filtrate has been taken in different time interval. Fermentor conditions were optimized by setting the parameter in different ranges. Growth curve of Serratia marcescens in large scale in relatively low compared to the small scale level. It has been observed that 0.33 and 0.65.  It was observed that twice times lower than the normal scale production. The result revealed that maximum activity of enzyme at temperature of 40ºC for two days. Maximum production was observed in the exponential phase of the microorganism during the growth. Casein assay shows the filtrate sample has the better enzyme activity and has the lower specific enzyme activity. It was noticed that maximum activity of 0.61Units/ml enzyme in filtrate solution. But in case of non-filtrate has shown better specific enzyme activity as 1.89 units/mg of solid. Enzyme activity was checked for the commercial available serratia peptidase.  Compared to the previous result we had observed the less activity due to the environmental factors.  Hereby it needs to optimize the concentration of media for large scale level.  CONCLUSION       Serratia peptidase is one of the important enzyme in medicinal field.  It used to cure the different diseases like cardiovascular attack and heart diseases. Current study reveals that effective production of Serratia peptidase from Serratia marcescens. It has been observed the highest activity than previous result in large scale level. Characterization of enzyme was done and it was effective as 40ºC and pH 7.0.  The activity of enzyme was low in large scale level than small scale level of production. Further it needs to optimize the substrate to increase the production of enzyme in large scale level.   ACKNOWLEDGEMENT       We are thankful to the management of KSR College of Technology, Tiruchengode for providing the necessary facility and our beloved Head of the Biotechnology Department Dr.P.Ponmurugan.  REFERENCES  [1] Andrew, T. and Michael, G. 2005. Adrienne, Production of chitinolytic enzymes by Serratia marcescens QMB1466 using various chitinous substrates, J. of chem tech and biotech, vol. 80, no1, pp. 28-34 [7 page(s) (article)] (45 ref.) [2] Longo, M. A. Novella, I. S. Garcia, L. A. and Diaz, M. 1999. Comparison of Bacillus Subtilis and Serratia marcescens as protease producers under different operating conditions, J of bioscience and bioengg.   vol. 88, no1, pp. 35-40 (32 ref.) [3] Hyun-Soo Kim, Peter, N. Golyshin Kenneth, N. 2007. TimmisCharacterization and role of a metalloprotease induced by chitin in Serratia sp. KCK, J Ind Microbiol Biotechnol 34:715–721DOI 10.1007/s10295-007-0245-1 [4] Jens Frankowski, MatteoLorito, Felice Scala·Roland, Schmid, Gabriele Berg, Hubert Bahl, 2001. Purification and properties of two chitinolytic enzymes of Serratia plymuthica HRO-C48 Arch Microbiol 176 421–426 DOI 10.1007/s002030100347 [5] KiSeok Kim,1. Tae Un Kim,1. In Jung Kim,2. SiMyung Byun,2. and YongChul Shin, 1995. Characterization of a Metalloprotease Inhibitor Protein(SmaPI) of Serratiamarcescens. App And Environmental Microbiol, p. 3035–3041 Vol. 61, No. 80099-2240/95/$04.0010 [6] Esch, P.M. Gerngross, H. Fabian, A. 1989. Reduction of postoperative swelling. Objective measurement of swelling of the upper ankle joint in treatment with Serratia peptidase-a prospective study (German). Fortschr Med.;107(4):67-8, 71-2 [7] Kee, W.H. Tan, S.L. Lee, V. Salmon, Y.M. 1989. The treatment of breast engorgement with Serratia peptidase (Danzen): a randomized double-blind controlled trial. Singapore Med J.;30(1):48-54. [8] Le`toffee, S. Delepelaire, P. Wandersman, C. Cloning and Expression in Escherichia coli of the Serratia marcescensMetalloprotease Gene: Secretion of the Protease from E.coli in the presence of the Erwiniachrysanthemi Protease Secretion functions, J Bacteriol. Vol. 173, No, p.2160-2166 [9] Aso T et al. 1981 Breast engorgement and its treatment: Clinical effects of Danzen an anti-inflammatory enzyme preparation. The world of Obstetrics and Gynecology (Japanese).; 33:371-9. [10] Majima, Y. Inagaki, M. Hirata, K. Takeuchi, K. Morishita, A. Sakakura, Y. 1988. The effect of an orally administered proteolytic enzyme on the elasticity and viscosity of nasal mucus. .Arch Otorhinolaryngology.;244(6):355-9 [11] Mazzone A, et al. 1990. Evaluation of Serratia peptidase in acute or chronic inflammation of otorhinolaryngology pathology: a multicenter, double-blind, randomized trial versus placebo. J Int Med Res.; 18(5):379-88 [12] Mete Mecikoglu, BaranselSaygi, YakupYildirim, EvrimKaradag-Saygi, SaimeSezgin     [13] Ramadan and TanilEsemenli. 2006. The Effect of Proteolytic Enzyme Serratia peptidase in the treatment of experimental Implant-Related Infection, The Journal of Bone and Joint  Surgery (American).;88:1208-1214.doi:10.2106/JBJS.E.00007.   

Pilot scale microbial production and optimization of Serratia peptidase from Serratia marcescens

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Pilot scale microbial production and optimization of Serratia peptidase from Serratia marcescens

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