Shahbazmohammadi Hamid, Omidinia Eskandar
Enzyme Technology Laboratory, Department of Biochemistry, Genetic and Metabolism Research Group, Pasteur Institute of Iran, Tehran, Iran.
Mol Biotechnol. 2017 Jul;59(7):260-270. 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 and V 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. 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.
二氢硫辛酰胺脱氢酶（DLD）是一种依赖黄素腺嘌呤二核苷酸（FAD）的酶，可催化二氢硫辛酰胺的可逆氧化反应。在此，我们报道了从球形芽孢杆菌PAD - 91菌株生产具有NADH依赖型黄递酶活性的重组DLD的培养基优化过程。由1413 bp组成的DLD基因在大肠杆菌BL21（DE3）中表达，并对其酶学性质进行了研究。采用一次一个变量法，随后结合响应面法（RSM）对生产培养基的组成进行了优化。球形芽孢杆菌DLD催化NAD还原硫辛酰胺，并表现出黄递酶活性。该酶的分子量约为50 kDa，经测定为单体蛋白。重组黄递酶在30℃和pH 8.5时表现出最佳活性。其对NADH的米氏常数（Km）和最大反应速度（Vmax）值分别估计为0.025 mM和275.8 U/mL。重组酶在含有10 g/L蔗糖、25 g/L酵母提取物、5 g/L氯化钠和0.25 g/L硫酸镁的发酵培养基中产量最佳。在这些浓度下，实际黄递酶活性经计算为345.0±4.1 U/mL。通过从摇瓶发酵扩大到生物反应器发酵，酶活性提高到486.3±5.5 U/mL。简而言之，对新分离的球形芽孢杆菌PAD - 91中具有黄递酶活性的DLD进行了表征，并使用RSM技术优化了重组酶的生产。
