Nucleotide sequencing and cloning of alanine dehydrogenase gene from Aeromonas hydrophila

Abstract

Alanine dehydrogenase [EC 1.4.1.1] catalyses the reversible deamination of L-alanine to pyruvate and ammonia. Nowadays, the enzyme is used for quantitating L-alanine and also used for synthesis of L-amino acid and its derivatives, which are used in drug and food industries. Aeromonas hydrophila, an isolate from soil in Bangkok, is one of the bacteria, which have high activity of alanine dehydrogenase. Previous work presented that the enzyme had molecular mass of about 230 kDa and consisted of six identical subunits. Amino acid sequence at N-terminus was known as MIIGVPKEIKN HEYRVGMVPASVRELTARNHTVFVQSGAGN and one of the internal amino acid sequences of this enzyme was LAEQGYRNLLSDPHLRHGLNVMAGKK. From the obtained data, the degenerated primers were designed for amplification of one part of alanine dehydrogenase gene by PCR technique using EcoRI or HindIII digested chromosomal DNA as a template. These PCR products were sequenced and the obtained data was used to design primers for amplification of other parts of the gene. The alanine dehydrogenase gene from Aeromonas hydrophila encodes for 371 amino acids residues. The whole gene was amplified by using primers designed from N-terminal and C-terminal nucleotide sequences and then cloned into E.coli JM109. The specific activity of the crude extracts from recombonant clones were about 0 to 10 units/ mg protein. The specific activity of the clone that has highest activity was about 50 times more than that of Aeromonas hydrophila.