Molecular Cloning And Expression Of An Amidase Gene From Nocardia Sp. In Escherichia Coli

Acrylamide and acrylic acid are two kinds of important chemicals widely used in many industrial fields. Amidase is one of the key enzymes received considerable recognition as biocatalysts for microbial-production of acrylic acid and byproduct inhibition of acrylamide. Consequently, the cloning and expression of the gene encoding the amidase was focused and emphasized in this thesis.At first, three different strategies were designed to obtain the amidase gene from Nocardia YS-2002 based on sequence analyses. A novel amidase gene, gene bank accession number of DQ342287, was successfully cloned by PCR. Multiple sequence alignment, phylogenetic tree plotting, catalysis stability prediction, rare codon and hydrophobicity profile analyses, in-cell position determination and secondary structure prediction of the new amidase were further carried out thereafter.Secondly, the amidase gene was inserted into both the plasmid pET-28a and pMAL-p2x. Some recombinant strains were successfully constructed by transforming the plasmids into Escherichia coli BL21 (DE3) and TB1, respectively. Cell culture and amidase induction were further performed under different conditions, and the highest amidase...