OPTIMIZATION ON THE PRODUCTION OF SERRAPEPTIDASEFROM SERRATIA MARCESCENS

PROTOCOL FOR

M.PHARM DISSERTATION

SUBMITTED TO

RAJIVGANDHIUNIVERSITY OF HEALTH SCIENCES

4TH ‘T’ BLOCK, JAYANAGAR, BANGALORE.

KARNATAKA

BY

MOHAMMED SHAMSEER

I M. PHARM PART I ,

DEPARTMENT OF PHARMACEUTICAL BIOTECHNOLOGY,

BHARATHICOLLEGE OF PHARMACY,

BHARATHI NAGARA.

UNDER THE GUIDANCE OF

Dr. K. N. SHANTI,Ph.D.

PROFESSOR,

DEPARTMENT OF PHARMACEUTICAL BIOTECHNOLOGY,

BHARATHICOLLEGE OF PHARMACY,

BHARATHI NAGARA,

KARNATAKA-571422.

RAJIVGANDHIUNIVERSITY OF HEALTH SCIENCES, KARNATAKA.

ANNEXURE-II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1. / Name of the Candidate and Address (In Block Letters) / MOHAMMED SHAMSEER,
S/O HAMZA P. V,
PUTHUKAYIL VALAPPIL(H),
BP ANGADI(PO),
TIRUR-2,
MALAPPURAM,
KERALA.
2. / Name of the Institution / BHARATHICOLLEGE OF PHARMACY,
BHARATHI NAGARA,
MANDYA- 571422.
3. / Course of Study and Subject / MASTER OF PHARMACY IN PHARMACEUTICAL BIOTECHNOLOGY.
4. / Date of Admission of Course / 30-06-2008
5. / Title of Topic / “OPTIMIZATION ON THE PRODUCTION OF SERRAPEPTIDASE FROM SERRATIA MARCESCENS”
6. / Brief Resume of the Intended Work
6.1 Need for the study
6.2 Review of the literature
6.3 Objectives of the study / Enclosure-I
7 /

Materials and Methods

7.1 Source of data

7.2 Method of collection of data

7.3 Does study require any investigations or interventions to conducted on patients or other human or animal? If so, please describe briefly.

7.4 Has ethical clearance been obtained from your institution in case of 7.3?

/ Enclosure-II
8 / List of References / Enclosure-III

enclosure-i

6. BRIEF RESUME OF THE INTENDED WORK

6.1 NEED FOR STUDY:

Serrapeptidase (E.C. 3.4.24.40) is an extra cellular alkaline metalloprotease isolated from the non pathogenic Enterobacteria serratia E15, present in the gut of the Japanese silk worm. The enzyme in its natural form is utilized by the silk worms to dissolve the cocoons and emerge as moths outsides. This enzyme is discovered by German physician Dr, Hans Niper, who described it as a “Miracle Enzyme” for its ability to treat arterial blockage in patients suffering form coronary artery diseases1.Physicians throughout Europe and Asia have recognized the anti-inflammatory and pain-blocking benefits of this naturally occurring substance and are using it in treatment as an alternative to salicylates, ibuprofen, and other NSAIDs2. It is also been used in the successful treatment of fibrocystic breast disease and it is a safe and effective method for the treatment of breast engorgement3.

Serrapeptidase induces fibrinolytic, anti-inflammatory and anti-edemic (prevents swelling and fluid retention) activity in a number of tissues, and that its anti-inflammatory effects are superior to other proteolytic enzymes. Besides reducing inflammation, it is one of the most profound benefits is reduction of pain, due to its ability to block the release of pain-inducing amines from inflamed tissues4. . Serrapeptidase has wide clinical use for the treatment of assorted inflammatory disorders5. It also works by modifying cell-surface adhesion molecules, which guide inflammatory cells to their targets. These adhesion molecules are known to play an important role in the development of arthritis and other auto immune disease6Serrapeptidase in this study was found to exert a beneficial effect on mucous clearance by reducing neutrophil numbers and altering the viscoelasticity of sputum in patients with chronic disease7.

The research programme planned to isolate the serrapeptidase from microorganism like Serratia marcescens to study the importance and to optimize the production.

6.2 REVIEW OF LITERATURE:

1. Aiyappa,et al., worked on an extracellular protease of Serratia marcescens produced during growth on skim milk medium was isolated by ethanol precipitation. The protease was purified by salt fractionation, DEAE-cellulose ion exchange chromatography and gel filtration chromatography on Agarose P-100. It has a broad optimum from pH 6.0 to 9.0 and a temperature optimum of 45º C for proteolytic activity on casein. It was classified as a metallo-protease by virtue of its inactivation by metal-ion chelators and reactivation by ferrous ions. Proteolytic activity was not affected by diso-propylfluorophosphate, p-chloromercuribenzoate and dithiothreitol.8

2.Le toffee,et al.,studied the Serratia marcescens extracellular protease SM is secreted by a signal peptide-independent pathway. When the prtSM gene was cloned and expressed in Escherichia coli, the cell did not secrete protease SM. The lack of secretion could be very efficiently complemented by the Erwinia chrysanthemi protease B secretion apparatus constituted by the PrtD, PrtE, and PrtF proteins. As with protease B and alpha-hemolysin, the secretion signal was located within the last 80 amino acids of the protease. These results indicate that the mechanism of S.marchescens protease SM secretion is analogous to the mechanisms of protease B and hemolysin secretion.10

3.Perna,etal., evaluated the effects of administering the antibiotic cephalexin in conjunction with serrapeptidase or a placebo to 93 patients suffering from either perennial rhinitis, chronic rhinitis with sinusitis or chronic relapsing bronchitis. The serrapeptidase-treated group experienced significant improvement in rhinorrhea, nasal stuffiness, coryza and improvement of the para-nasal sinus shadows.11

4. Esch, et al., analysed in Germany and other European countries, serrapeptidase is a common treatment for inflammatory and traumatic swellings and much of the research that exists on this substance is of European origin. One double-blind study was conducted by German researchers to determine the effect of serrapeptidase on post-operative day, the group receiving serrapeptidase exhibited a 50 percent reduction of swelling, compared to the controls. The patients receiving serrapeptidase also became more rapidly pain-free than the controls, and by the tenth day, the pain had disappeared completely.9

5.Aso, et al.,worked on serrapeptidase has also been used in the successful treatment of fibrocystic breast disease. In a double-blind study, 70 patients complaining of breast engorgement randomly were divided into a treatment group and a placebo group. Serrapeptidase was superior to the placebo for improvement of breast pain, breast swelling and induration (firmness). Nearly eighty-six percent of the patients receiving serrapeptidase reported moderate to marked improvement. No adverse reactions to serrapeptidase were reported and the researchers concluded that serrapeptidase is a safe and effective method for the treatment of breast engorgement.2

6.Mete Mecikoglu,et al.,To evaluate the effectiveness of serrapeptidase, in the eradication of a periprosthetic infection in an in vivo animal model was conducted and it was seen that serrapeptidase was effective for eradicating infection caused by biofilm-forming bacteria in this experimental animal model and the antibiofilm property of the enzyme could enhance the antibiotic efficacy in the treatment of staphylococcal infections.12

6.3 OBJECTIVES OF THE STUDY:

  • To isolate, identify and screen the Serratia marcescens for the production serrapeptidase enzyme.
  • To optimize the production by using different parameters like pH, temperature, media supplements.
  • To evaluate the kinetic parameters of the purified enzymes.

ENCLOSURE-II

7.METHODS AND MATERIALS:

1.To isolate, identify and screen the Serratia marcescens for the production serrapeptidase enzyme

1.1) Collection of soil samples from different places

1.2) Isolation of the organisms by serial dilution agar plating method

1.3) Identifying the organisms by using Bergeys manual of Bacteriology

1.4) Screening the organism by casien hydrolysis method

2. To optimize the production by using different parameters like pH, temperature, media supplements

2.1) Optimization of production and purification by using standard method reffered the protocol.8

  1. To evaluate the kinetic parameters of the purified enzymes

3.1) To obtain the maximum activity by using Temperature, pH, V max, Km, Activators, and Inhibitors by using standard methods

7.1 SOURCE OF DATA:

1. Bharathi College of Pharmacy library,Bharathi Nagara.

2. E-Library from BharathiCollege of Pharmacy.

3.IISC Library,Bangalore

4. Relevant Compact Discs regarding informations on Enzymology.

5. Relevant Compact Discs regarding informations on Biotechnology.

7.2 METHOD OF COLLECTION OF DATA:

The preliminary data required for the experimental study were obtained from

1.Internet

2.Scientific Abstracts

3.Scientific Journals

4.Relevant Books on applications of Biotechnology

5.Relevant Books on Biotechnology

6.Relevant Books on Enzymology

7.Relevant Compact Discs on Enzymology and Biotechnology

7.3 Does the study require any investigations or intervensions to conduct on patients or other human or animal?If so,please describe briefly.

-NO-

7.4 Has ethical clearance been obtained from your institution in case of 7.3?

-NOT APPLICABLE-

ENCLOSURE-III

8.LIST OF REFERENCES:

1. Nieper, HA., Basic Cardiovascular Packet, Richland Centre, Wise: Brewer Science Library. Available from the A. Keith Brewer International Science Library at US 1999; 608:647-6513.

2. Aso. Breast engorgement and its treatment: Clinical effects of Danzen an anti-inflammatory enzyme preparation. The world of Obstetrics and Gynecology (Japanese). 1981; 33:371-9.

3. Kee, Tan SL, Lee, Salmon. The treatment of breast engorgement with Serrapeptase (Danzen): A randomized double-blind controlled trial. Singapore Med J.1989;30(1):48-54

4. Mazzone A, Catalani M, Costanzo M, Drusian A, Mandoli A,et al., Evaluation of Serratia peptidase in acute or chronic inflammation of otorhinolaryngology pathology: a multicentre, double-blind, randomized trial versus placebo. J Int Med Res. 1990; 18(5):379-88.

5. Rothschild, J. "Clinical Use of Serrapeptase: An Alternative To Non-Steroidal Anti-inflammatory Agents."The American Chiropractor. 1991;58:17.

6.Kleine, M.W. "The Intestinal Absorption of Orally Administered Hydrolytic Enzymes and Their Effects in the Treatment of Acute Herpes Zoster as compared with those of oral Acylclovir Therapy."Phytomedicine, 1995;2(1): 7-15.

7. Nakamura S, Hashimoto Y, Mikami M, Yamanaka E, Soma T, et al., Effect of the proteolytic enzyme serrapeptase in patients with chronic airway disease Respirologu 2003; 8: 316-320.

8. Aiyappaand Harris.The extracellular metalloprotease of. Serratia marcescens. Purification and characterization. Mol. Cell Biochem 1976;13:95-100.

9. Esch PM, Gerngross H, Fabian A. Reduction of postoperative swelling. Objective measurement of swelling of the upper ankle joint in treatment with serrapeptase-a prospective study (German). Fortschr Med. 1989;107(4):8-67.

10.Letoffee S , Delepelaire P, Wandersman C. Cloning and Expression in Escherichiacoliof the Serratia marcescensmetalloprotease Gene: Secretion of the Protease from E.coli in the presence of the Erwinia chrysanthemiProtease Secretion functions, J Bacteriol. 1990;173:2160-2166.

11. Perna L,Majima Y, InagakiM, HirataK, Takeuchi K,et al., The effect of an orally administered proteolytic enzyme on the elasticity and viscosity of nasal mucus. Arch Otorhinolaryngol. 1988;244(6):355-9.

12. Mete Mecikogu, Baransel Saygi, Yakup Yildirim, Evrim Karadag-Saygi, Saime Sezgin,et al., The Effect of Proteolytic Enzyme Serratiopeptidase in the Treatment of experimental Implant-Related Infection, Istanbul, TurkeyJBJSAmerican). 2006;88:1208-1214.

13. Tachibana M, Mizukoshi O, Harada Y, Kawamoto K, Nakai Y. A multi-centre, double-blink study of Serrapeptase versus placebo in post-antrotomy buccal swelling. Pharmatherapeutica 1984; 3(8):526-30.

9.
10.
11.
12. / Signature of the candidate:
(Mohammed Shamseer)

The study involves the serrapeptidase
Remarks of the guide: optimization from serratia marcescens
found to be a advance study in the field of
biotechnology

Name and Designation of:
Dr. K.N. SHANTI, Ph.D
11.1 Guide Professor
Department of Pharmaceutical
Biotechnology,
BharathiCollege of Pharmacy,
Bharathinagara,
Karnataka-571422.
11.2 Signature

11.3 Co-Guide Dr.GURUKAR MATHEW.S, Ph.D
11.4 Signature

11.5 Head of the Department Dr.GURUKAR MATHEW.S, Ph.D
Professor
Department of Pharmaceutical
Biotechnology,
Bharathi college of pharmacy,
Bharathinagara,
Karnataka-571422.
11.6 Signature

12.1 Remarks of the Chairman and Principal:Recommended for approval.
12.2 Signature Prof. Dr. Tamizh Mani. T
Principal,
BharathiCollege of pharmacy,
Bharathinagara,
Karnataka-571422.