MOLECULAR CLONING AND EVALUATION OF WILD PROMOTER IN EXPRESSION OF BACILLUS SPHAERICUS PHENYLALANINE DEHYDROGENASE GENE IN BACILLUS SUBTILIS CELLS

Abstract

To evaluate the role of wild promoter of L-phenylalanine dehydrogenase (PheDH) gene, referred to as pdh, from Bacillus sphaericus in expression, cloning of pdh gene in Bacillus subtilis was performed. The whole pdh gene was cloned in pHY300PLK shuttle vector and amplified, construct (pHYDH) then transformed in B. subtilis ISW1214 and E. coli JM109. The pdh endogenous promoter presented no effect on transcription of the target gene in E. coli JM 109. But B. subtilis ISW1214/pHYDH produced PheDH enzyme (4700 U/L). The level of PheDH protein expression with native promoter was about 12%. It was purified to near homogeneity as judged by SDS-polyacrylamide gel electrophoresis (Mr 41 kDa) and the result was 18 fold with a yield of about 31%. The Mr of PheDH was estimated to be approximately 340 kDa (octamer) by a gel filtration on a G-200 sephadex column. Apparent Km values for L-phenylalanine, L-tyrosine and NAD+ were 0.24 mM, 0.48 mM 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 compatible with the wild type PheDH protein.