Overexpression of type I L-asparaginase ofBacillus subtilis inEscherichia coli, rapid purification and characterisation of recombinant type I L-asparaginase

Abstract

The present study focused on cloning and expression of the ansA gene, encoding putative asparaginase I of Bacillus subtilis in Escherichia coli , and investigation of the basic properties of the recombinant enzyme for application in food processing industry. The ansA gene of B. subtilis was cloned by polymerase chain reaction and expressed in E. coli Rosseta Gami B. It consisted of an open reading frame of 987 nucleotides encoding a protein of 329 amino acids with a calculated molecular mass of 36441 Da. The recombinant enzyme showed increased expression (21.7 U mg −1 ) and was purified to homogeneity by a two-step procedure that included L-asparagine affinity column chromatography. Based on the kinetic properties and primary structure of the native enzyme, the product of the ansA gene was classified as L-asparaginase I. The enzyme showed a high specific activity compared with that of L-asparaginase II from E. coli . Results of this study will allow us to apply the enzyme to food industry.