DefinePK

Abstract

ABSTRACT L-asparaginase (E.C 3.5.1.1) is a highly valuable enzyme owing to its ability to function as a chemotherapeutic agent against certain lymphomas such as acute lymphoblastic leukemia. This enzyme also mitigates the level of acrylamide production in processed food items. L-asparaginase performs the two-step hydrolytic reaction that results in the deamination of amino acid asparagine into aspartic acid and ammonia. L-asparaginases are widely distributed in three domains of life but their microbial source is getting much attention in the field of pharmaceutical industry due to cost effectiveness and ease of production. The focus of current research work is the utilization of various in silico tools for structural and functional analysis of an asparaginase-like domain of ansA gene from Methanocaldococcus sp. The DNA fragment coding for the asparaginase-like domain was PCR amplified, cloned, and expressed using recombinant DNA technology. Specifically designed primers were used for PCR amplification of ansA(T) gene from the genomic DNA of M. jannaschii DSM 2661. After PCR amplification, the gene was first cloned into the cloning vector (pJET1.2/blunt) and thereafter cloned in expression vector pET-28a (+). E. coli BL21 CodonPlus (DE3)-RIL was used as an expression host to produce an asparaginase-like protein, AnsA(T). The recombinant protein AnsA(T) was expressed in soluble form in E. coli. Moreover, the thermostability of the enzyme was determined by heating the supernatant at different temperatures ranging from 60-90 °C for different durations of time. Additionally, the AnsA(T) enzyme was partially purified by heat treatment at 80 °C for 15 min followed by Ni-NTA chromatography. The highly thermostable asparaginase AnsA(T) of Methanocaldococcus sp. can make it a potential candidate for its applications in the food industry.