Affinity purification and characterization of recombinant Bacillus sphaericus phenylalanine dehydrogenase produced by pET expression vector system.

ABSTRACT

Cloning and expression of the L-phenylalanine dehydrogenase gene, from B. sphaericus in E. coli were done. The gene was cloned in the vector pET16b and transformed into E. coli BL21 [DE3]. The functional form of the L-phenylalanine dehydrogenase enzyme was purified by affinity purification techniques, taking advantage of the ability of this enzyme to bind to the nucleotide site affinity dye, Reactive Blue 4. Approximately 3 mg of highly purified recombinant enzyme was obtained from 950 mg cell pellet [wet weight]. The Relative molecular mass of the L-phenylalanine subunits was about 41 kDa by 10% SDS-PAGE. Using this method, the enzyme was obtained with a yield of 28%, and had a specific activity of 577.3 U/mg proteins, which is purified 88 times. This method was provided a facile and effective way for preparing the enzyme with a good yield that suitable for analytical purposes: