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

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Abstract

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 MgSO4. 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.

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Acknowledgments

Research finding was from Department of Biochemistry.

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Authors and Affiliations

  1. Enzyme Technology Laboratory, Department of Biochemistry, Genetic and Metabolism Research Group, Pasteur Institute of Iran, Tehran, Iran

    Hamid Shahbazmohammadi & Eskandar Omidinia

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  1. Hamid Shahbazmohammadi
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  2. Eskandar Omidinia
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Correspondence to Eskandar Omidinia.

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This article does not contain any studies with human participants performed by any of the authors.

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Shahbazmohammadi, H., Omidinia, E. Medium Optimization for Improved Production of Dihydrolipohyl Dehydrogenase from Bacillus sphaericus PAD-91 in Escherichia coli . Mol Biotechnol 59, 260–270 (2017). https://doi.org/10.1007/s12033-017-0013-z

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