Production of fungal L-asparaginase
enzyme as anticancer agent.
Mohammed Mahmoud Shawky
Kassab ,specialist of
pharmacology,toxicology,microbiology,
immunology and molecular biology,faculty of
pharmacy,Cairo university,Egypt.
Abstract
Aspergillus Sp is a fungal source of L-asparaginase enzyme from different soil environments.It is similar to human L-asparaginase enzyme. Bacterial L-asparaginase enzyme usually produced from Erwinia chrysanthem or Escherichia coli is used in the treatment of acute lymphoblastic leukaemia and decreasing acrylamide production which is carcinogenic during food preparation.Fungal L-asparaginase is superior to bacterial L-asparaginase due to less glutaminase like activity and less hypersensitivity reactions such as anaphylactic reactions and drug neutralization.In our study,fungal L-asparaginase was produced on mineral asparagine agar(MAM) selective medium(only fungi which utilize asparagine as sole source for carbon and nitrogen can grow on it) at PH 6.5 , temperature 25c and incubation for 3 days .Malt agar medium was used for sub-culturing fungal L-asparaginase producing strains.Fungal L-asparaginase showed high efficacy and bio-availability as anticancer agent.
According to the morphological ,biochemical tests and DNA blotting hybridization ,the main fungal isolate which produced this enzyme was Aspergillus Sp.We recommend in the following studies to determine and optimize the effective dose of fungal L-asparaginase as anticancer agent and to design new primer for better expression of it by recombinant DNA technology.
Key wards: Fungal,L-asparaginase,production,anticancer agent.
Introduction
A-structure and growth of fungi:
Fungi(yeasts and molds) are eukaryotic organisms (1).There are two types of fungi:yeasts and molds(2).Molds grow as long filaments (hyphae)and form a mat(mycelium)(3).Some hyphae form transverse walls (septate hyphae) where as others do not form these transverse
1
walls(nonseptate hyphae)(4).The growth of hyphae does not occur by the cell division but occurs by the extension of the tip of the hyphae (5).The nonseptate hyphae are multicellular(coenocytic)(6).Most fungi are obligate aerobes ,some are faculatative aerobes, but none are obligate anaerobes(7).Several medically important fungi are thermally dimorphic((8).They exist as yeasts in human tissues at body temperature and as molds in the environment at ambient temperature (9).
B-Role of microbial L-asparaginase as anticancer agent:
Bacterial L-asparaginase enzyme usually produced from Erwinia chrysanthem or Escherichia coli was utilized in the treatment of acute lymphoblastic leukaemia and decreasing acrylamide production which is carcinogenic during food preparation(10).L-asparaginase converts L- asparagine amino acid( which is essential for growth of cancer cells which can not synthesize it and obtain it from external environments such as blood ,lymph and extracellular fluids) into aspartic acid and ammonia (11) Both Escherichia coli and Erwinia chrysanthem are prokaryotes(12).Bacterial L-asparaginase causes many side effects such as hypersensitivity reactions(anaphylactic reactions and drug neutralization) and glutaminase like activity(13). Fungal enzyme is similar human L-asparaginase because both are from eukaryotic cells(14).Our study could overcome these drawbacks by production of this enzyme from new fungal source such as aspergillus Sp.This new enzyme was devoid of these side effects and has better efficacy and higher yield than bacterial enzyme.
Collection of the samples
Soil samples were collected from different environments in Egypt.
Methodology
(A) Screening of positive fungal L-asparaginase producing isolates: (1) Mineral asparaginine medium (MAM):
selective medium was used for screening of fungal L-asparaginase producing isolates.It was composed of the following components: KH2PO4(1.2),MgSO4(0.6),FeSO4(0.005),KCL(0.4),D- glucose(7),Agar(13),L-asparaginine(15),Thiamphenicol antibiotic(0.15).Thiamphenicol was added to prevent bacterial growth.The PH of medium was 6.5 and incubation was carried out for 3 days.Only the colonies which were able to utilize L-asparaginine as nitrogen and carbon source were grown in this selective medium.
The positive isolates were stored at 4c for later studies.
(2) Malt extract agar medium :
It is a general purpose growth acidic medium for cultivation and isolation of fungi.PH was adjusted at 6.5 using NaOH.Subcultures of the positive
2
isolates (grown on MAM)were grown and isolated on malt extract agar medium at temperature 20c,PH 6.5 after 3 days incubation period.
(3) Microscopic examination of positive fungal isolates after dissolving in 10%KOH.
(4) Molecular detection of positive fungal isolates using DNA probe hybridization.
(B) Determination of fungal L-asparaginase production and activity:
(1) Direct nesselerization test:
It was used to characterize the enzyme production and activity from positive fungal isolates grown in MAM.L-asparaginase catalyzes the hydrolysis of L-asparagine amino acid to L-aspatric acid and ammonium.The released ammonium was identified and assayed spectrophotometerically at wave length 425 nm.The intensity of the light was directly proportional to ammonium concentration .The amount of released ammonium was directly proportional to the enzyme activity.
(2) Salicylate method :
This method is a variation of the well known phenate method.It is free from mercury salts and phenol.it is useful for low range ammonium nitrogen determination.
L-asparaginase catalyzes the hydrolysis of L-asparagine amino acid to L- aspatric acid and ammonium.The released ammonium was identified and assayed spectrophotometerically at UV wavelength 425 nm.The intensity of the light was directly proportional to ammonium concentration .The amount of released ammonium was directly proportional to the enzyme activity.
(3) In vitro cell viability assay:
CCL-120 cancer cell line was used for assessment of the
physiologic ,pharmacologic and toxicological effects of the enzyme on the cancer cells.
Vero cell line was used for assessment of physiologic ,pharmacologic and toxicological effects of the L-asparaginase enzyme on the mammalian cells.
MTT((dimethylthiazol-2-yl)diphenyl tetrazonium)method was used for in vitro cell viability assay of fungal L-asparaginase.
Results an discussion
41 soil samples from different environments in Egypt revealed that the fungal Aspergillus SP was the main L-asparaginase producing isolate grown on MAM selective medium and malt extract agar.It was characterized by mold with septate hyphae seen by light
microscopy .Molds with green spores and conidia in radiating channels
3
were grown on MAM selective medium and malt extract agar.This was confirmed by molecular technique hybridization with DNA probes . L-asparaginase catalyzes the hydrolysis of L-asparagine amino acid into L-aspatric acid and ammonia.The concentration of ammonia was determined by direct nesslerization and salicylate tests.The amount of released ammonium was directly proportional to the enzyme activity.Optimal conditions for the production and biological activity of enzyme were:PH 6.5 at temperature 25c.The activators of enzyme production were KH2PO4(1.2 g),MgSO4(0.6 g),FeSO4(0.005g),KCL (0.4 g).In comparison with bacterial enzyme,this fungal enzyme showed higher efficacy as antileukaemic and anticancer agent than bacterial L- asparginase produced from Escherichia coli or Erwinia chrysanthem. Also, this fungal enzyme could overcome the drawbacks of bacterial L- asparaginase .It showed less glutaminase activity and less hypersensitivity reactions (such as drug neutralization and anaphylactic reactions)than bacterial L- asparaginase .This was revealed by in vitro cell viability MTT assay test on cell lines:
1-CCL-120 cancer cell line which was used for assessment of the biological,physiologic ,pharmacologic and toxicological effects of the enzyme on the cancer cells.
2- Vero cell line which was used for assessment of biological, physiologic ,pharmacologic and toxicological effects of the L- asparaginase enzyme on the mammalian cells.
Conclusion
Fungal L-asparaginase was produced from aspergillus SP grown on MAM selective medium.This enzyme showed superior antileukaemic and anticancer activity to bacterial L-asparaginase produced from Escherichia coli or Erwinia chrysanthem.It is also showed less glutaminase and less hypersensitivity reactions than bacterial L-asparaginase.We recommend future studies to determine the optimal dose of this fungal enzyme as anticancer agent and also, to design new primer for its expression by recombinant DNA technology to get higher yield,efficacy and purity.
References
1-Parveen Kumar(2017).Kumarm,Clarks clinical medicine.Ninth edition,Elsevier Edinburgh London.
2-Caroline S,Zeind Michael G(2018).Applied therapeutics,the clinical use of drugs.Eleventh edition,Wolters Kluwer, London
3-Parveen Kumar(2017).Kumarm,Clarks clinical medicine.Ninth edition,Elsevier Edinburgh London.
4
4-Parveen Kumar(2017).Kumarm,Clarks clinical medicine.Ninth edition,Elsevier Edinburgh London.
5-Caroline S,Zeind Michael G(2018).Applied therapeutics,the clinical use of drugs.Eleventh edition,Wolters Kluwer, London
6-Parveen Kumar(2017).Kumarm,Clarks clinical medicine.Ninth edition,Elsevier Edinburgh London.
7-Parveen Kumar(2017).Kumarm,Clarks clinical medicine.Ninth edition,Elsevier Edinburgh London.
8-Caroline S,Zeind Michael G(2018).Applied therapeutics,the clinical use of drugs.Eleventh edition,Wolters Kluwer, London
9-Parveen Kumar(2017).Kumarm,Clarks clinical medicine.Ninth edition,Elsevier Edinburgh London.
10Parveen Kumar(2017).Kumarm,Clarks clinical medicine.Ninth edition,Elsevier Edinburgh London.
11-Caroline S,Zeind Michael G(2018).Applied therapeutics,the clinical use of drugs.Eleventh edition,Wolters Kluwer, London
12-Parveen Kumar(2017).Kumarm,Clarks clinical medicine.Ninth edition,Elsevier Edinburgh London.
13-Caroline S,Zeind Michael G(2018).Applied therapeutics,the clinical use of drugs.Eleventh edition,Wolters Kluwer, London
14-Parveen Kumar(2017).Kumarm,Clarks clinical medicine.Ninth edition,Elsevier Edinburgh London.