Cloning and expression of Bacillus sphaericus phenylalanine dehydrogenase gene in Bacillus subtilis cells: purification and enzyme properties

Cloning and expression of the L-phenylalanine dehydrogenase (PheDH) gene from Bacillus sphaericus in B. subtilis was performed. It was ligated into the pHY300PLK shuttle vector and the resulting plasmid, pHYDH encoding polypeptide with molecular weight of 340 kDa, then transformed in B. subtilis ISW1214 and Escherichia coli JM109 competent cells for expression. Bacillus subtilis ISW1214/pHYDH only produced PheDH enzyme (4700 U/l). The recombinant PheDH was purified to near homogeneity as judged by SDS–polyacrylamide gel electrophoresis (Mr 41000 Da) and the result was 40-fold with a yield of about 54%. Apparent Km values for L-phenylalanine (Phe), L-tyrosine and NAD+ were 0.24, 0.48 and 0.19 mM respectively. The optimum pH of the recombinant enzyme was 11 for the oxidative deamination, 10.2 for the reductive amination. The features of recombinant PheDH enzyme were comparable with the wild type PheDH protein.