Optimization and characterization of l-asparaginase production by a novel isolated streptomyces spp. strain

Abstract

Introduction and purpose Microbial l-asparaginase (l-asparagine amino hydrolase, E.C.3.5.1.1) has been applied as the most important chemotherapeutic agent in the treatment of certain human cancers, especially in acute lymphoblastic leukemia. Actinomycetes are recognized as a comparatively less explored source for l-asparaginase and therefore act as candidates for the production of l-asparaginase. The possibility of using novel actinomycete as a source of production of some industrially important microbial l-asparaginase was investigated in this study. Materials and methods Genomic DNA of the actinomycetes isolate from soil samples was extracted using the Gene JET Genomic DNA purification kit (Thermo scientific #k0721). The actinomycete isolate was identified by 16S rDNA. The identified actinomycete isolate was inoculated on starch casein agar slants and incubated for 7–10 days at 28°C, and then it was maintained at 4°C until further use. Inoculum was prepared from a 7-day old culture of the strain. Production of l-asparaginase was initially tested in four different media. The actinomycete strain was used further for the optimization of cultural conditions, namely, l-asparagine substrate concentrations, pH, temperature, and incubation conditions. The pH of the medium was varied from pH 3.0 to pH 9.0; the incubation temperature was varied from 30 to 50°C. The effect of carbon source and nitrogen source on l-asparaginase production was studied. Modified Czapex Dox broth was supplemented with different carbon and nitrogen sources such as starch, mannitol, mannose, sucrose, cellulose, and fructose at a concentration of 1% (w/v) and ammonium sulfate, beef extract, yeast extract, and peptone at a concentration of 0.2% (w/v), respectively, keeping other components constant. The properties of Streptomyces spp. l-asparaginase were also studied such as pH, assay temperature, and thermal stability. Results and discussion Genotypic characterization of the most promising unknown actinomycete isolate showing the maximum production was identified by 16S rDNA. PCR amplified the 16 s rDNA region using primers. Genotypic characterization of the most promising unknown actinomycete isolate showing the maximum production was identified by16S rDNA. The 16 s rDNA region was amplified by PCR (about 1000 bp) using primers. According to sequencing similarities and multiple alignments, the isolate was found to be closely related to Streptomyces griseoplanus strain NRRL-ISP 5009 16 s ribosomal RNA gene with 85% identity. Higher enzyme activity was observed in modified Czapex Dox broth as compared with other media used. Modified Czapex Dox broth was supplemented with different concentrations of l-asparagine; the enzyme production was maximum at 1.5% l-asparagine (126.20 U/ml). Analysis of the culture supernatant showed that the enzyme activity rise from 126.20 U/ml on the fifth day to 141.11 U/ml on the 10th day which its peak enzyme production activity. Different carbon sources such as starch, mannitol, lactose, sucrose, and glucose were amended in asparagine-modified Czapek Dox broth to determine their impact on l-asparaginase production. Biosynthesis of l-asparaginase by S. griseoplanus strain has been reported to be higher when the basal medium was supplemented with starch. For the production of l-asparaginase by S. griseoplanus strain, yeast extract has been reported as a good nitrogen source. According to the properties of the enzyme, the maximum activity was achieved at 45°C. The half-lives of the free enzyme were calculated to be 521 min (8.5 h) at 50°C, 312.6 min (5.2 h) at 55°C, and 195.2 min (3.25 h), at 60°C.