Production, Purification and Characterization of Anticancerous Enzymes from Different

PRODUCTION, PURIFICATION AND CHARACTERIZATION OF ANTICANCEROUS ENZYMES FROM DIFFERENT MICROORGANISMS

PROTOCOL FOR M.PHARM DISSERTATION

SUBMITTED TO RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, BANGALORE, KARNATAKA.

BY RASHID.S M.PHARM PART-1 DEPARTMENT OF PHARMACEUTICAL BIOTECHNOLOGY 2008-2009

UNDER THE GUIDANCE OF Dr.GURUKAR MATHEW.S. Ph.D. PROFESOR, DEPARTMENT OF PHARMACEUTICAL BIOTECHNOLOGY, BHARATHI COLLEGE OF PHARMACY, BHARATHI NAGARA, KARNATAKA-571422. RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA, BANGALORE.

ANNEXURE-II PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1. Name of the Candidate and Address RASHID.S, S/o YAHU.S (In Block Letters) SATHIYAKKAL HOUSE PADINJARAKKARA (PO) MALAPPURAM KERALA-676 562. 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 – 06 - 2008 5. Title of Topic PRODUCTION, PURIFICATION AND CHARACTERIZATION OF ANTICANCEROUS ENZYMES FROM DIFFERENT MICROORGANISMS 6. Brief Resume of the Intended Work 6.1 Need for the study 6.2 Review of the literature ENCLOSURE-I 6.3 Objectives of the study 7 Materials and Methods 7.1 Source of data 7.2 Method of collection of Data. 7.3 Does study require any investigations ENCLOSURE-II 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. 8 List of References ENCLOSURE-III

ENCLOSURE-I

6. BRIEF RESUME OF THE INTENDED WORK 6.1 Need For The Study: Asparaginase (EC 3.5.1.1) is an enzyme that catalyzes the hydrolysis of asparagine to aspartic acid. The rationale behind asparaginase is that it takes advantage of the fact that Acute Lymphoblastic Leukemia (ALL) leukemic cells are unable to synthesize the non-essential amino acid asparagine whereas normal cells are able to make their own asparagine. leukemic cells require high amount of asparagine. These leukemic cells depend on circulating asparagine. Asparaginase however catalyzes the conversion of L-asparagine to aspartic acid and ammonia. This deprives the leukemic cell of circulating asparagine.The main side effect is an allergic or hypersensitivity reaction. Asparaginase has also been associated with pancreatitis. Additionally, it can also be associated with a coagulopathy as it decreases protein synthesis, including synthesis of coagulation factors (examples progressive isolated decrease of fibrinogen) and anticoagulant factor (generally antithrombin III; sometimes protein C & S as well), leading to bleeding or thrombotic events such as stroke. 2

Application: To treat Acute Lymphoblastic Leukemia (ALL) and is also used.3 In some mast cell tumor protocols Unlike other chemotherapy agents, It can be given as an intramuscular, subcutaneous, or intravenous injection without fear of tissue irritation

Production of L-asparaginase in yields of sufficient quantity and purity for more extensive clinical evaluation the nutritional requirements for optimal production of biologically active L- asparaginase by a strain of different fungi has been ascertained. The highest yields of enzyme were obtained when cells were grown aerobically in a corn steep medium.Good enzyme production was associated with media containing L-glutamicacid, L-methionine, and lactic acid. The addition of glucose to the medium, however, resulted in depressed production of L-asparaginase. Sodium ion appeared to suppress L-asparaginase production. With the procedure described for isolation of biologically active L-asparaginase from fungi stable L-asparaginase preparations with a specific activity of 1200 IU per mg of protein were obtained.11 Recent investigation showed that, production of enzymes, from an isolated organism, and its effect on animal studies, L-Asparaginase is now known to be a potent antineoplastic agent in animals and has given complete remission in some human leukemia’s. Extensive clinical trials of this enzyme, however, were not possible in the past because of inadequate production of this substance Along with strain improvement of an organisms we can enhance the production of enzymes, and increase the activity of the enzymes, can also be produced by using solid as well as submerged fermentation technique to cheque the yield of enzyme activity

6.2 Review Of The Literature:

1. Abha Mishra, Found the production of high levels of L-asparaginase from a new isolate of Aspergillus niger in solid state fermentation using agro wastes from three leguminous crops (bran of Cajanus cajan, Phaseolus mungo, and Glycine max). When used as the sole source for growth in SSF, bran of G. max showed maximum enzyme production followed by that of P. mungo and C. cajan.. Developing an asparaginase production process based on bran of G. max as a substrate in SSF is economically attractive as it is a cheap and readily available raw material in agriculture- based countries.1 2. Ashok Shenoy, et al., Reported the hyperglycemia may occur as a complication in patients with leukemia during induction of remission with Lasparaginase and steroids. The reported incidence is about 10%. The presentation of this complication may vary from mild glucose intolerance to severe, or even fatal, diabetic ketoacidosis. We report a case of a 15-yearold girl who developed transient diabetes mellitus following L-asparaginase therapy with ketoacidosis as the mode of presentation.2 3. Thomas, et al., Reported the relapse rate in childhood acute lymphoblastic leukemia is approximately 30% but few reinduction regimens have investigated the intensive use of polyethylene glycol E.coli asparaginase (PEG-Asp). Asparaginase levels and antibody to both E coli asparaginase and PEG-asp were measured weekly just before each PEG-asp dose. Low asparaginase levels were associated with high antibody titers to either native (P 5 .024) or PEG asp (P 5 .0013).14 4. Kudgi1, et al., Reported the L-asparaginase is commonly used in combination with doxorubicin, vincristine, methotrexate and p rednisolone for the treatment of acute lymphoblastic leukemia (ALL). Leukemic cells are highly sensitive to chemotherapy. In over 90% of patients, a complete remission in 4-6 weeks can be achieved with prednisolone, vincristine, Lasparaginase and doxorubicin combination chemotherapy. We report a case of severe hyperglycemia and ketoacidosis following administration of L asparaginase in an adolescent girl.2 5. Maria, et al., Found the L-asparaginase production was investigated in the filamentous fungi Aspergillus tamarii and Aspergillus terreus. The fungi were cultivated in medium containing different nitrogen sources. A. terreus showed the highest L-asparaginase (activity) production level (58 U/L) when cultivated in a 2% proline medium. Both fungi presented the lowest level of L-asparaginase production in the presence of glutamine and urea as nitrogen sources. These results suggest that L-asparaginase production by of filamentous fungi is under nitrogen regulation.11 6. Mijoon Lee, et al, Found the Metastasis, the dissemination of tumor cells to distant organs, is often associated with fatal outcome in cancer patients. Formation of metastasis requires degradation of extracellular matrices and several families of proteases have been implicated in this process, including matrix metalloproteinases (MMPs), serine and cysteine proteases. Inhibition of these enzymes in animal models of metastasis has shown impressive therapeutic effects. This report discusses the various approaches used for enzyme inhibition and describes new developments in drug design for inhibition of proteases in metastatic disease.10 7. Gulati, et al, Reported in the present investigation, a novel and semi-quantitative plate assay for screening L-Aspaginase producing micro organism is reported. It is generally observed that L- Asparginase production is accompanied by an increase in pH of the culture filtrates. The plate assay was devised using this principle by incorporating the pH indicator phenol red in medium containing asparaginase. Phenol red at acidic pH is yellow and alkaline pH turns pink, thus a pin zone is formed around the microbials colonies producing L-Aspaginase.5 8. Sukumaran.C.P, et al, Reported the production of L-Asparaginase by two mutants of Serratia marcescens grown on 14 different media was studied. The enzyme content increased from trace levels to 2·4 international units per ml when the organisms were grown in glycerol-peptone yeast extract medium. Glucose was the best carbon source under aerobic conditions. The enzyme content increased when L-asparagine was present in the growth medium.13 9. John, et al, Studied the homogeneous L-asparaginase with anti-lymphoma activity was prepared from Vibrio succinogenes, an anaerobic bacterium from the bovine rumen. An overall yield of pure L-asparaginase of 40 to 45% and a specific activity of 200 + 2 IU per mg of protein were obtained. The pure enzyme can be stored at ~ - 20” for at least 3 months with no loss of activity. The isoelectric point of the L-asparaginase is 8.74. No carbohydrate, phosphorus, tryptophan, disulfide, or sulfhydryl groups were detected. The enzyme has a molecular weight of 146,000 and a subunit molecular weight of approximately 37,000.9 10. Liu, et al, Reported the maximum L-asparaginase activity was obtained when 1.0% lactose and 1.5% yeast extract were supplied as carbon and nitrogen sources, respectively. Glucose inhibited the enzyme formation. The diauxie phenomenon was observed with Erwinia aroideae NRRL B- 138 grown in a medium containing glucose and lactose.9 11. George, et al., Come across significant production of more growth inhibition without a marked increase in host toxicity than did treatment with L-asparaginase administered alone. In the 3-day- old tumor treated with the combination of L-asparaginase and 6-diazo-5-oxo-L-norleucine, there was a significant enhancement of growth inhibition as compared with the effect of either drug used alone. A less significant increase of growth inhibition was obtained with combination of L- asparaginase and L-glutamyl-'y-hydrazide as compared with the effect of this chemical administered alone. Combinations of L-asparaginase and 6- diazo-5-oxo-L-nor leucine produced increased antitumor effects in mouse melanoma Bl 6 and to a lesser degree in the solid form of Ehrlich tumor and Walker carcinosarcoma 256 of the rat.4 12. Joseph, et al, Reported the L-Asparaginase is now known to be a potent antineoplastic agent in animals and has given complete remission in some human leukemias. Extensive clinical trials of this enzyme, the highest yields of enzyme were obtained when cells were grown aerobically in a corn steep medium. Good enzyme production was associated with media containing L-glutamic acid, L-methionine, and lactic acid. The addition of glucose to the medium, however, resulted in depressed production of L-asparaginase. Sodium ion appeared to suppress L-asparaginase production. With the procedure described for isolation of biologically active L-asparaginase from E. coli, stable L-asparaginase preparations with a specific activity of 620 IU per mg of protein (1,240-fold purification with 40% total recovery) were obtained.6

3 6.3 Objectives Of The Study: The Overall aim of the proposed study is to production of anticancerous enzymes from microorganisms.  Isolation, identification and Screening of microorganisms for production of anticancerous enzymes  Strain improvement of an organism for production of anticancerous enzymes  Production, purification and characterization of an anticancerous enzymes  Study anticancerous activity on cell lines

ENCLOSURE-II

7. MATERIALS AND METHODS:  Isolation, identification and Screening of microorganisms for production of anticancerous enzymes 5 o Isolation of microorganisms from natural sources o Identification of unknown organisms o Screening for the production of anticancerous enzymes from identified microorganisms  Strain improvement of an organism for enhanced production of enzyme o Strain improvement of an organisms by chemical mutagens o Strain improvement of an organism by exposing it physical mutagens  Production, purification and characterization of an Anticancerous enzymes 1 o Production of organisms in a production media o Crude extraction of anticancerous enzymes o Purification of Anticancerous enzyme  Protein precipitation technique  Dialysis  Ion exchange chromatography

o Characterization of a purified enzyme  Optimization of purified enzyme (pH, Temp, Activators, Inhibitors)  Enzyme kinetics  SDS PAGE  Study of an enzyme activity on cell lines

. 7.1 Source Of Data:

 Bharahti College of pharmacy library, Bharathi nagara.  E-library from Bharahti College of pharmacy library, Bharathi nagara.  IISC library, Bangalore.  RGUHS library, Bangalore

7.2 Method Of Collection Of Data . Data Collected From: Journals:.  Indian Journal of Biotechnology.  Indian Journal of Pharmaceutical Education and Research.  Applied Biochemistry and Biotechnology  Kuwait Medical Journal  Applied microbiology  Journal of bacteriology Related Links:  http://www.google.com  http://www.Rxlist.com  http://www.sciencedirect.com  http://www.pubmed.com  http://www.medline.com  http://www.wileyinterscience.com  http://www.drugbank.com

7.3 7.3 Does the study require any investigation or interventions to be conducted on patients or other humans or animals?

-NOT APPLICABLE-

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

-NOT APPLICABLE- ENCLOSURE-III

8. LIST OF REFERENCES:

1) Abha mishra, Production of L-asparaginase, an anticancer agent, from Aspergillus niger using agricultural waste in solid state fermentation, Applied Biochemistry and Biotechnology 2006;135(1):33-42. 2) Ashok Shenoy Kudgi1, Mukta Nithyananda Chowta1, Rajeev Aravindakshan, Transient Diabetes Mellitus following L- Asparaginase Therapy, Kuwait Medical Journal 2007,39 (2):188-189. 3) Appel IM, van Kessel-Bakvis C, Stigter R, Pieters R, "Influence of two different regimens of concomitant treatment with asparaginase and dexamethasone on hemostasis in childhood acute lymphoblastic leukemia",Leukemia 2007, 21:2377. 4) George S. Tarnowski, Isabel M, Mountain, ChesterStock.C, Combination Therapy of Animal Tumors with L-Asparaginase and Antagonists of Glutamine or Glutamic Acid', [CANCER RESEARCH] 1970,30,1118-1122. 5) Gulati.R, Saxena R.K, Gupta R, A rapid plate assay for screening L-Asparginase producing micro organisms, Applied microbiology 1997, 24, 23-26. 6) Joseph roberts, Gene burson, Joseph M. Hill, New Procedures for Purification of L- Asparaginase with High Yield from Escherichia coli, Journal of bacteriology 1968,95(6) 2117-2123. 7) John A. Distasio, Robert A. Niederman, Malvin l. Stern, Purification and Characterization of L-Asparaginase with Anti-lymphoma Activity from Vibrio succinogenes.The journal of biological 1976,251(22),6929-6933. 8) John W. Boyd, Arthur W. Phillips, Purification and Properties of L-Asparaginase from Serratia marcescens, Journal of bacteriology, American Society for Microbiology, 1971, 106(2),578-587. 9) Liu. F.S, J. E. Zajic, L-Asparaginase Synthesis by Erwinia aroideae, Applied microbiology,1972,23(3)667-668. 10) Mijoon Lee,a Rafael Fridmanb and Shahriar Mobashery, Extracellular proteases as targets for treatment of cancer metastases, Chem Soc Rev , 2004 ,33 ,401–409 11) Maria Inez de Moura Sarquis, Edna Maria Morais Oliveira, Alberdan Silva Santos, Gisela Lara da Costa, Production of L-asparaginase by Filamentous Fungi Rio de Janeiro,2004 99(5): 489-492. 12) Peterson.R.E., Ciegler.A, L-Asparaginase Production by Various Bacteria, Applied microbiology,1969, 17(6)929-930. 13) Sukumaran,C.P., Singh.D.V., Mahadevan,P.R., Synthesis of L-asparaginase by Serratia marcescens (Nima) J.Biosci.,1979,1(3),263–269. 14) Thomas C. Abshire, Brad H. Pollock, Amy L. Billett, Patricia Bradley,George R. Buchanan Weekly polyethylene glycol conjugated L-asparaginase compared with biweekly dosing produces superior induction remission rates in childhood relapsed acute lymphoblastic leukemia: a pediatric oncology group study, Blood, 2008, 96: 1709-1715.

9 Signature of Candidate

(RASHID.S)

10 Remarks of Guide Production of anticancerous enzymes from different microorganisms found to be note the source and their application may enhance the knowledge of production source

11 Name and Designation of 11.1 Guide DrGURUKAR MATHEW.S.Ph.D. Profesor,Department of Pharmaceutical Biotechnology, Bharathi college of pharmacy, Bharathi nagara. 11.2 Signature

11.3 Co- Guide (if any) NOT APPLICABLE. 11.4 Signature

11.5 Head of Department Dr.GURUKAR MATHEW.S.Ph.D. Profesor,Department of Pharmaceutical Biotechnology, Bharathi college of pharmacy, Bharathi nagara.

11.6 Signature

12.1 Remarks of the Chairman and Forwarded for approval 12 Principal: Prof. Dr. T. Tamizh Mani.Ph.D. Principal Bharathi College of pharmacy Bharathinagara Karnataka-571422 Signature