Ding Haitao, Gao Fen, Yu Yong, Chen Bo
Key Laboratory for Polar Science of State Oceanic Administration, Polar Research Institute of China, Shanghai 200136, China.
East China Sea Fisheries Research Institute, Shanghai 200090, China.
Int J Mol Sci. 2017 Jun 5;18(6):1198. doi: 10.3390/ijms18061198.
Due to the dual cofactor specificity, glucose 1-dehydrogenase (GDH) has been considered as a promising alternative for coenzyme regeneration in biocatalysis. To mine for potential GDHs for practical applications, several genes encoding for GDH had been heterogeneously expressed in BL21 (DE3) for primary screening. Of all the candidates, GDH from sp. ZJ (BzGDH) was one of the most robust enzymes. BzGDH was then purified to homogeneity by immobilized metal affinity chromatography and characterized biochemically. It displayed maximum activity at 45 °C and pH 9.0, and was stable at temperatures below 50 °C. BzGDH also exhibited a broad pH stability, especially in the acidic region, which could maintain around 80% of its initial activity at the pH range of 4.0-8.5 after incubating for 1 hour. Molecular dynamics simulation was conducted for better understanding the stability feature of BzGDH against the structural context. The in-silico simulation shows that BzGDH is stable and can maintain its overall structure against heat during the simulation at 323 K, which is consistent with the biochemical studies. In brief, the robust stability of BzGDH made it an attractive participant for cofactor regeneration on practical applications, especially for the catalysis implemented in acidic pH and high temperature.
由于具有双辅因子特异性，葡萄糖1-脱氢酶（GDH）被认为是生物催化中辅酶再生的一种有前景的替代物。为了挖掘用于实际应用的潜在GDH，几个编码GDH的基因已在BL21（DE3）中进行异源表达以进行初步筛选。在所有候选物中，来自sp. ZJ的GDH（BzGDH）是最具活力的酶之一。然后通过固定化金属亲和色谱将BzGDH纯化至同质，并进行生化表征。它在45°C和pH 9.0时表现出最大活性，并且在低于50°C的温度下稳定。BzGDH还表现出较宽的pH稳定性，特别是在酸性区域，在4.0-8.5的pH范围内孵育1小时后，其初始活性可维持在80%左右。进行分子动力学模拟以更好地理解BzGDH在结构背景下的稳定性特征。计算机模拟表明，BzGDH是稳定的，并且在323 K的模拟过程中能够抵抗热保持其整体结构，这与生化研究一致。简而言之，BzGDH强大的稳定性使其成为实际应用中辅酶再生的有吸引力的参与者，特别是对于在酸性pH和高温下进行的催化反应。
