ISOLATION, PRODUCTION AND PURIFICATION OF PEROXIDASE FROM FUNGI BY CHANGING MEDIA COMPOSITION (CARBON SOURCE)
PROTOCOL FOR
M.PHARM DISSERTATION
SUBMITTED TO
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
4TH T BLOCK, JAYANAGAR
BANGALORE, KARNATAKA
BY
SALEEM C.P
M. PHARM PART-I
DEPARTMENT OF PHARMACEUTICAL BIOTECHNOLOGY,
BHARATHI COLLEGE OF PHARMACY,
BHARATHI NAGARA.
UNDER THE GUIDANCE OF
Dr.TAMIZH MANI .T, Ph.D.
PROFFESSOR,
DEPARTMENT OF PHARMACEUTICAL BIOTECHNOLOGY,
BHARATHI COLLEGE OF PHARMACY,
BHARATHI NAGARA,
KARNATAKA-571422.
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA.
ANNEXURE-II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
1. / Name of the Candidate and Address (In Block Letters) / SALEEM C.PS/O ABOOBACKER T.M,
SHAHINA MANZIL,
ANJARAKKANDY,
KANNUR,
KERALA-670 612.
2. / Name of the Institution / BHARATHI COLLEGE 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 JUNE 2008
5. / Title of Topic / “ISOLATION, PRODUCTION AND PURIFICATION OF PEROXIDASE FROM FUNGI BY CHANGING MEDIA COMPOSITION (CARBON SOURCE)”
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-II8 / List of References / ENCLOSURE-III
ENCLOSURE-I
6. BRIEF RESUME OF THE INTENDED WORK:
6.1 NEED FOR STUDY
Peroxidase (E.C.1.11.1.7) an iron porphyrin organic catalyst belongs to class oxidoreductase, which occurs naturally in plants, animals and microorganisms1. It exerts bactericidal activity in vitro, and the participation of the enzyme in the antibacterial actions of intact leukocytes has been proposed2. Peroxidase, functionally linked to hydrogen peroxide-generating systems, could provide phagocytic cells with the ability to kill many fungal species3.
Peroxidase is widely used as an indicator. It has been found that the enzyme is well-suited for the preparation of enzyme conjugated anti- bodies (labeling of anti-bodies) since it is able to produce chromogenic products. Immuno-globins that are peroxidase- labeled, for example, have been used as immuno-histological probes for the demonstration of tissue antigens4. Peroxidase can also replace a number of harsh chemicals, eliminating harsh reaction conditions5. Studies have shown that it could be a potential replacement for carcinogenic formaldehyde in glues and varnishes. In Europe, peroxidase has started to replace potassium bromate in bread-making and dough conditioning6. Companies are even investigating the use of peroxidase in manufacturing adhesives, computer chips, car parts, and linings of drums and cans. Horseradish peroxidase is a widely used label for immunoglobulins in many different immunochemistry applications including ELISA, immunoblotting, and immunohistochemistry4. Peroxidase is also utilized for the determination of glucose and peroxides in solution7.
Our aim of current research is to focus on obtaining peroxidase enzyme of higher concentration and optimal activity. Since such an enzyme is widely used in degrading environmental pollutants and xenobiotics.
6.2 REVIEW OF LITERATURE:
1. Kent, et al., Reported the Iignin-degrading enzyme system of white-rot fungi, which are mostly basidiomycetes, has been studied intensively in recent years. The extracellular component of the system is comprised of Iignin peroxidase, manganese peroxidase, glyoxal oxidase and certain metabolites. Lignin is fragmented by this system, and the plethora of degradation products taken up by the hyphae and further metabolized by the intracellular system. The intracellular system has received very little research attention. The structural complexity and heterogeneity of Iignin show in fact that this enzyme system is so nonspecific that it also degrades a variety of hazardous compounds, including polycyclic aromatics, some polychlorinated biphenyls and dioxins, DDT, and many chlorinated phenols. Using pentachlorophenol (PCP) as a model substrate, we have studied the possibility of using white-rot fungi to remediate soils contaminated with hazardous compounds. Successful laboratory results led to a field study in the summer of 1989. Results showed that the laboratory findings could be duplicated in the field.9
2. Ziai, et al., Found peroxidase used in oxidation of a wide range of aromatic chemicals. We used a domestic source of peroxidase (garden radish, Raphanous sativus L.) to treat the following hazardous chemicals: phenol, aniline, benzidine, alachlor, butachlor, acid red 88 and acid blue 62. Crude enzyme removed the above-mentiond chemicals more efficiently in 24 hr treatment. Changes in pH cause a removal efficacy change, for example lowering pH from7 to 4.0 for phenol treatment increased removal from 0.5 to 100% for crude enzyme. In conclusion, Garden radish is a good substitute for horseradish in order to treat aromatic wastewaters.13
3. Urra, et al., Reported the production of manganese dependent peroxidase (MnP) by Phanerochaete chrysosporium and the level of decolorization of 13 dyes were evaluated using static and agitated batch cultures and continuous cultures. A screening carried out under static conditions showed that the oxidative system has a certain affinity for azoic structures. For concentrations of 100 mg/l of Acid Black 1, Reactive Black 5, Reactive Orange 16 and Acid Red 27, decolorization percentages higher than 90% were obtained. In batch cultures with Acid Black 1 and Reactive Black 5 a significant increment in primary post-metabolism biomass was observed. For these last two dyes, it was possible to explore the response of the continuous system during 32 to 47 days, with concentrations between 25 to 400 mg /l, obtaining decolorization percentages greater than 70% for 400 mg/l.8
4. Hilhorst, et al., Selected bread doughs supplemented with xylanase and xylanase plus peroxidase were fractionated into 4 insoluble and 3 soluble fractions. Chemical analysis and high-performance size-exclusion chromatography analysis of apparent molecular weight distribution indicated that xylanase acts on both cold-water-extractable arabinoxylans and on those that can be solubilized from cell wall fragments by hot water extraction. Peroxidase action increased the amount of insoluble small cell wall fragments, notably the amount of protein and arabinoxylan. Arabinoxylans were retained in the small cell wall fragments because cross-linking of arabinoxylans through ferulic acid residues to other arabinoxylans rendered them insoluble. Peroxidase did not affect the composition of gluten, nor was evidence obtained for peroxidase-catalyzed cross-linking of arabinoxylans to protein in the gluten and other fractions.6
5. Dan Wang, et al., Reported the activity of lignin peroxidase (LiP) in reversed micelles of polyoxyethylene laurylether (Brij30) changed with the molar ratio of water to the surfactant and the denaturant concentration of guanidinium chloride. At low water contents the activity of LiP could be enhanced by the denaturant at moderate concentration. This phenomenon, together with the spectral characteristics of the intrinsic fluorescence of LiP, suggested that the conformation of the active center of LiP was flexible.10
6.3 OBJECTIVES OF THE STUDY
· To isolate, identify and screening of fungal species for the production of Peroxidase
· Optimum Production of peroxidase using different media composition (changing carbon source) and purification.
· . To characterize the enzyme for its maximum activity
ENCLOSURE-II
7. MATERIAL AND METHODS
1. To isolate, identify and screening of fungal species for the production of
Peroxidase
· Collection of soil samples from forest areas
· Isolation of the organisms by serial dilution agar plating method
· Identifying the organisms by physical appearance and microscopic characteristic
· Screening the organism for production of peroxidase
2. Optimum Production of peroxidase using different media composition
(Changing carbon source) and purification.
· To purify peroxidase using standard method
3. To characterize the enzyme for its maximum activity.
· To study enzyme characteristics in different parameters by using Temperature, PH, Substrate Concentration etc.
7.1 SOURCE OF DATA:
1. Bharathi college of pharmacy library, Bharathinagara.
2. E-Library from Bharathi College of pharmacy.
3.IISC Library,Bangalore
7.2 METHOD OF COLLECTION OF DATA:
The priliminary data required for the experimental study were obtained from
1. Internet
2. Scientific Abstracts
3. Scientific Journals
4. Relevent Books
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
- Farzana Habib, Khalil-ur-Rehman, Anjum Zia M, Khalid Saeed M Peroxidase: purification from soybean Seeds, Pakistan Journal of Biological sciences,2003; 6 (2): 130-132
- Agner K, Studies on peroxidative detoxification of purified diphtheria toxin. J. Exp. Med.1950; 92: 337-347.
- Lehrer R.I, Antifungal Effects of Peroxidase Systems, Journal of bacteriology, 1969; 99: 361-365.
- Deshpande SS, Enzyme Immunoassays, From Concept to Product Development, Chapman and Hall, 1996; 169-171.
5. Daniel D, Lefebvre, Peter Chenaux, and Maureen Edwards, Dye Degradation by Fungi: An Exercise in Applied Science for Biology Students Bioscene,2005; 31(3): 13-15.
6. Hilhrost R, Gruppen H, Orsel R, Laane C, Scholas HA, Voragen AGJ, Effects of Xylanase and Peroxidase on Soluble and Insoluble Arabinoxylans in Wheat Bread Dough Journal of Food Science, 2006; 67(2):497–506 .
7. Bergmeyer, H.U, Methods of Enzymatic Analysis (Bergmeyer, H.U., ed.), 1974; 1205-1227.
8. Urra .J, Sepulveda .L, Contreras .E and Palma .C screening of static culture and comparison of batch and continuous culture for the textile dye biological decolorization by phanerochaete chrysosporium Brazilian Journal of Chemical Engineering. 2006; 23: 281 –290.
9. Kent Kirk T, and Richard T, Lamar and John A, Glaser the potential of white-rot fungi in bioremediation Biotechnology and environmental science–molecular approaches. Proceedings of an international conference on biotechnology and environmental science: molecular approaches: Plenum Press; 1992; 131-138.
- Dan Wang, Xi Rong Huang, Cai Xia Liu, Yue Zhong Li, Yin Bo Qu, Pei Ji Gao Activity Regulation of Lignin Peroxidase from Phanerochaetechrysosporium in Nonionic Reversed Micellar Medium, Chinese Chemical Letters, 2005;
16(6): 819-822.
11. Shannon, Baltimore, "Peroxidase Isozymes from Horseradish Roots." The
Journal of Biological Chemistry, 1966; 241(9): 2166-2172.
12. Delincee H. and Radola, BJ. Determination of isoelectric points in thin-layer
Isoelectric focusing: The importance of attaining the steady state and the role of
CO2 interference, Eur. J. Biochemistry, 1975; 52: 321–330.
13. Ziai SA, Eshraghi SS, Taghizadeh M, Vahabzadeh F, Gerden radish (Raphanus
Sativus L.) Peroxidase in the detoxication of hazardous aromatic wastes J. of
Medicinal Plants. 1998; 241: 2166–2172.
Signature of the candidate:
(SALEEM C.P)
Remarks of the guide: The research found to locate the source of
peroxidase and their applications
Name And Designation of:
Dr. TAMIZH MANI.T, Ph.D.
11.1 Guide Professor,
Department of Pharmaceutical Biotechnology,
Bharathi College 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, Ph.D.
Principal
Bharathi College of pharmacy
Bharathinagara
Karnataka-571422