In silico analysis, molecular cloning, expression and characterization of l-asparaginase gene from Lactobacillus reuteri DSM 20016

Abstract

l-Asparaginase is employed in leukaemic treatment and in processing starchy foods. The in silico analysis of Lactobacillus reuteri DSM 20016 reveals the presence of an l-asparaginase gene with theoretical pI value of 4.99. 3D structure prediction was carried out and one model was selected based on the validation scores of 86.293 for ERRAT, 92.10% for VERIFY 3D and Ramachandran plot. Multiple sequence alignment of the protein sequences of l-asparaginases I and II of Escherichia coli, Erwinia chrysanthemum and Homo sapiens shows their sequence similarity. The ORF LREU_RS09880 from L. reuteri DSM 20016 genome was cloned and expressed in E. coli. The recombinant protein was purified to homogeneity using Ni–NTA chromatography and showed higher substrate specificity for l-asparagine. Kinetic parameters like Km and Vmax of recombinant l-asparaginase were calculated as 0.3332 mM, 14.06 mM/min, respectively. Temperature and pH profile of recombinant l-asparaginase were analysed and maximum activity was found between 30 and 40 °C and at pH 6. The recombinant enzyme was thermally stable up to 24 h at 28 °C. Recombinant l-asparaginase has a recovery percentage of 92 and 10.5 fold purification. HPLC–MS–MS and SDS-PAGE analysis of the purified protein indicated a molecular weight of 35 kDa as a monomer.