Nucleotide Sequencing and Cloning of the Phenylalanine Dehydrogenase Gene from Thermotolerant Bacillus badius BC1

Abstract

Phenylalanine dehydrogenase (EC 1.4.1.20) catalyzes the NAD-dependent reversible oxidative deamination of L-phenylalanine to form ammonia, phenylpyruvate, and NADH. The enzyme is important as a catalyst for the synthesis of L-phenylalanine used in the artificial sweetener, aspartame, production. Moreover, it is therefore applicable to diagonosis of neonatal hyperphenylalaninaemia and phenylketonuria. Thermotolerant bacterial strain BC1, which had high phenylalanine dehydrogenase activity, was identified by morphological and biochemical properties as well as 16S rRNA gene sequence. The results indicated that this strain was Bacillus badius. Amino acid sequence at the N-terminus was TSIIKDFTLFEKMSEHEQV VFANDPATGLR and 6 internal sequences of this enzyme were GMTYKXAASDVDFGGGKAVIIGDPQKDKSPELFRAFG QFVDSLGGRFYTGTDMGTNMEDFIHAMK, ATNK, DDLGGVTYAIQGLGKVGYKVAEGLLEEGAHLFVT, AIAGSANNQLLTEDHGRHLADK, ERVLAK, and WDIRN. The amino acid sequence was used to design degenerated primers which were used to amplify an internal part of phenylalanine dehydrogenase gene by PCR technique using restriction enzyme digested chromosomal DNA as templates. These PCR products were sequenced and the data was used for design next primers for amplification of 5’ and 3’ regions of the gene by using cassette-ligation mediated PCR. The whole nucleotide sequence of the structural gene consisted of 1140 nucleotides. The open reading frame encoding a polypeptide of 380 amino acids. The phenylalanine dehydrogenase gene was successfully cloned and expressed in E. coli JM109 cells. The enzyme total activities were found in crude extract of the clones in the range of 0 to 360 units/100 ml culture. The highest activity was 60 fold higher than that of the enzyme from Bacillus badius BC1. The optimum time to induce phenylalanine dehydrogenase gene expression was 120 minutes before cell harvesting. Stability of phenylalanine dehydrogenase gene in E. coli JM109 was studied. After daily subculturing recombinant clones that showed high phenylalanine dehydrogenase activity for 15 days, five from six recombinant clones still gave the plasmid pattern and enzyme activity similar to their original clones, however, the inserted gene from one of them was removed by host cell deletion process. Retransformation of recombinant plasmids into the host cell E. coli JM109 showed that all of them gave the same pattern with their original recombinant plasmids and phenylalanine dehydrogenase activity of all retransformants was still constantly remained.