Bioprospection of Microorganisms from North Western Himalayas for Asparaginase Enzyme

Abstract

L-asparaginase, an essential biopharmaceutical, has been an aid to cancer patients, particularly for the therapeutic treatment of acute lymphoblastic leukemia. However, current asparaginase formulations derived from Escherichia coli and Erwinia species are the only available sources for enzyme production. Moreover, long term administration of asparaginase in general, produces corresponding antibodies in the tissues and is associated with possible side effects causing hindrances towards a successful therapeutic treatment. Therefore, there is a dire need for exploring novel microbial sources for asparaginase production to obtain a serologically different L-asparaginase possessing similar therapeutic properties. Optimization and production of asparaginase from varied microbial sources has been the aim of several studies to overcome the hypersensitive and toxicological responses associated with presently used drug formulations. In this study, L-asparaginase producing bacterial culture R16C1 was isolated from the black gram soil sample of Rajouri, Jammu. The primary screening using rapid plate assay showed that out of 44 isolates, 2 strains were found asparaginase positive. A quantitative estimation of enzyme activity using nessler s method showed that culture R16C1 has highest enzyme activity of 9.14 U/ml as compared to second isolate RA8C1, which displayed 4.22 U/ml activity after 72 h of incubation. This culture was characterized as Enterobacter aesburiae, strain R16C1/MT93543. An attempt was made to optimize different parameters for the production of enzyme using submerged fermentation. Box Behnken design was used to optimize and to study interactive effect of rpm, inoculum size (%) and temperature for asparaginase activity. Comparable values for enzyme activity were obtained from experimental results and software predicted vL-asparaginase, an essential biopharmaceutical, has been an aid to cancer patients, particularly for the therapeutic treatment of acute lymphoblastic leukemia. However, current asparaginase formulations derived from Escherichia coli and Erwinia species are the only available sources for enzyme production. Moreover, long term administration of asparaginase in general, produces corresponding antibodies in the tissues and is associated with possible side effects causing hindrances towards a successful therapeutic treatment. Therefore, there is a dire need for exploring novel microbial sources for asparaginase production to obtain a serologically different L-asparaginase possessing similar therapeutic properties. Optimization and production of asparaginase from varied microbial sources has been the aim of several studies to overcome the hypersensitive and toxicological responses associated with presently used drug formulations. In this study, L-asparaginase producing bacterial culture R16C1 was isolated from the black gram soil sample of Rajouri, Jammu. The primary screening using rapid plate assay showed that out of 44 isolates, 2 strains were found asparaginase positive. A quantitative estimation of enzyme activity using nessler s method showed that culture R16C1 has highest enzyme activity of 9.14 U/ml as compared to second isolate RA8C1, which displayed 4.22 U/ml activity after 72 h of incubation. This culture was characterized as Enterobacter aesburiae, strain R16C1/MT93543. An attempt was made to optimize different parameters for the production of enzyme using submerged fermentation. Box Behnken design was used to optimize and to study interactive effect of rpm, inoculum size (%) and temperature for asparaginase activity. Comparable values for enzyme activity were obtained from experimental results and software predicted values. As per interaction data obtained for the selected fermentation parameters, rpm, size of inoculum and temperature showed significant effects at interactive levels, thus, showing increase in production of enzyme. Higher enzyme activity of 40.36 U/ml was observed in M9 medium which was 4.4 fold higher than the initial activity of enzyme. After optimization, production of asparaginase was carried out in a 6 L fermenter under optimized conditions. L-asparaginase activity of 44.23 U/ml was obtained in 72 h showing specific activity of 6.5 U/mg and a protein content of 6.8 mg/ml. L-asparaginase produced was purified using ammonium sulphate precipation, dialysis and anion exchange chromatrography on Mono-Q column 5/50GL. Maximum activity of enzyme i.e., 41.61 U/ml was obtained in 60 - 90% fraction, yielding 3.7 mg/ml of protein with specific activity of 11.25 U/mg. Dialysate of this fraction was loaded on pre-equilliberated Mono-Q column for anion exchange chromatography using fast protein liquid chromatography. FPLC purified enzyme gave a specific activity of 20.7 U/mg. A 2.85 fold purification with a yield of 70.1% was achieved after sequential purification of enzyme. Purified enzyme was tested against 8 different human cancer cell lines originated from seven different tissues using SRB assay and the enzyme showed 58%, 55% and 50% growth inhibition of breast (MCF-7, MDA-MB-231) and pancreatic (MIAPACA) cancer cells respectively. The results demonstrated that the bacterial culture Enterobacter aesburiae isolated from black gram soil sample of Rajouri is a potential producer of L-asparaginase with efficacy against breast and pancreatic cancer cell lines that can be further modified to enhance its drug potential to treat cancer patients.alues. As per interaction data obtained for the selected fermentation parameters, rpm, size of inoculum and temperature showed significant effects at interactive levels, thus, showing increase