Medium Optimization for Improved Production of Dihydrolipohyl Dehydrogenase from Bacillus sphaericus PAD-91 in Escherichia coli

• Published in: Molecular biotechnology 2017-07, Vol.59 (7), p.260-270
• Main Authors: Shahbazmohammadi, Hamid, Omidinia, Eskandar
• Format: Article
• Language: English
• Subjects: Bacillus - classification; Bacillus - enzymology; Bacillus - genetics; Bacillus - isolation & purification; Bacteria; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Batch Cell Culture Techniques; Biochemistry; Biological Techniques; Bioreactors; Bioreactors - microbiology; Biotechnology; Cell Biology; Chemistry; Chemistry and Materials Science; Dehydrogenase; Dehydrogenases; Dihydrolipoamide Dehydrogenase - genetics; Dihydrolipoamide Dehydrogenase - metabolism; E coli; Enzymatic activity; Enzyme activity; Enzymes; Escherichia coli - genetics; Escherichia coli - growth & development; Fermentation; Flavin-adenine dinucleotide; Human Genetics; Hydrogen-Ion Concentration; Mathematical analysis; Molecular Weight; NAD; NADH; Nicotinamide adenine dinucleotide; Optimization; Original Paper; Oxidation; Phylogeny; Protein Engineering; Protein Science; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Response surface methodology; Scale (corrosion); Scaling; Scaling up; Sodium chloride; Soil Microbiology; Sucrose; Sugar; Temperature; Temperature effects; Yeast; Yeasts
• ISSN: 1073-6085; 1559-0305
• DOI: 10.1007/s12033-017-0013-z

Dihydrolipohyl dehydrogenase (DLD) is a FAD-dependent enzyme that catalyzes the reversible oxidation of dihydrolipoamide. Herein, we report medium optimization for the production of a recombinant DLD with NADH-dependent diaphorase activity from a strain of Bacillus sphaericus PAD-91. The DLD gene that consisted of 1413 bp was expressed in Escherichia coli BL21 (DE3), and its enzymatic properties were studied. The composition of production medium was optimized using one-variable-at-a-time method followed by response surface methodology (RSM). B. sphaericus DLD catalyzed the reduction of lipoamide by NAD + and exhibited diaphorase activity. The molecular weight of enzyme was about 50 kDa and determined to be a monomeric protein. Recombinant diaphorase showed its optimal activity at temperature of 30 °C and pH 8.5. K m and V max values with NADH were estimated to be 0.025 mM and 275.8 U/mL, respectively. Recombinant enzyme was optimally produced in fermentation medium containing 10 g/L sucrose, 25 g/L yeast extract, 5 g/L NaCl and 0.25 g/L MgSO 4 . At these concentrations, the actual diaphorase activity was calculated to be 345.0 ± 4.1 U/mL. By scaling up fermentation from flask to bioreactor, enzyme activity was increased to 486.3 ± 5.5 U/mL. Briefly, a DLD with diaphorase activity from a newly isolated B. sphaericus PAD-91 was characterized and the production of recombinant enzyme was optimized using RSM technique.