PRODUCTION, PURIFICATION AND CHARACTERISATION OF LASPARAGINASE FROM ACTINOMYCETES AND EVALUATION OF ITS ANTI-CANCER AND ACRYLAMIDE REDUCTION ACTIVITIES

Abstract

The potential of L-asparaginase to inhibit acrylamide formation (a carcinogenic compound from fried starchy foods) and the growth of cancer cells have attracted scientific attention in biomedical fields. Utilisation of L-asparaginase from several sources has been limited due to high rate of allergic reactions, after a prolonged use because of the presence of glutaminase activity which deamidates glutamine to glutamic acid and ammonia, thereby increasing the enzyme toxicity. Hence, it is necessary to search for other L-asparaginase sources with no Lglutaminase activity as alternatives. Production trials with Actinomycetes and their characterisation have been limited. The work aim was to produce, purify and characterize Lasparaginase with anticancer and acrylamide reduction potential from Actinomycetes. Actinomycetes were isolated from rhizospheric soil of Moringa plants in the botanical garden, University of Ibadan, and were screened for L-asparaginase activity. The selected Lasparaginase producers were screened for L-glutaminase activity using plate assay method. The L-asparaginase producers with lower glutaminase activity were identified using molecular methods. Effects of temperature, pH, metal ions, incubation time, carbon and nitrogen sources, agitation and medium composition on the growth and L-asparaginase production by identified isolates were determined. The L-asparaginase produced was purified by ammonium sulphate precipitation, dialysis and column chromatography using Sephadex G-50. The molecular weight of the enzyme produced was determined using SDS-PAGE. Effect of pH, temperature, metal ions, inducers and inhibitors and substrate concentration on activity and stability of the partially purified enzyme was determined. The in-vitro anticancer activity of the partially purified L-asparaginase on colon cancer cell line at different concentrations using 3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was done. The acrylamide reduction activity of L-asparaginaseinpotato after heat treatment (above 120oC) was determined using standard methods. Data were analysed using descriptive statistics. One hundred and forty-five bacteria were isolated, 46% of them were L-asparaginase producers. The best six L-asparaginase producers with lower glutaminase activity were identified as Amycolatopsis japonica, Sphingobium yanoikuyae, Sphingobacterium caenis, Stenotrophomonas pavani, Paenibacillus cineris and Actinomycetal bacterium. Amycolatopsis japonica showed highest L-asparaginase activity (0.2772±0.001 U/mL) and no glutaminase activity after 25 days of screening. The highest L-asparaginase was produced at pH 7.0, 35oC, 7 days incubation time, Mg2+, 150rpm, M9 medium, maltose (0.2% w/v) and yeast extract (0.2% w/v). Partially purified L-asparaginase from Amycolatopsis japonica had total activity of 1968.98 U/mL, total protein (26.69 mg), specific activity (73.75 U/mg), purification fold (6.42), recovery yield (42.86%) and molecular weight of 37.5 kDa. Ethylenediamine Triacetic Acid, Mg2+, pH 8, 45oC supported optimum activity and stability of the enzyme. The Michaelis constant (Km) and maximum velocity (Vmax) were 7.874 mM and 2.57 U/mL, respectively. The enzyme showed high anticancer activity against colon cancer cell line with half Maximum Inhibitory Concentration (IC50) of 36 μg/mL. It also had 11% reduction of acrylamide formation in potato which is a carcinogenic compound in starchy food. Amycolatopsis japonica L-asparaginase exhibited anticancer potential and reduced formation of acrylamide in fried starchy food. This enzyme has the potential to be used as drug to complement the ones currently in use.