Abstract：
A broad application spectrum ranging from clinical diagnostics to biosensors in a variety of sectors, makes the enzyme Lactate dehydrogenase (LDH) highly interesting for recombinant protein production. Expression of recombinant LDH is currently mainly carried out in uncontrolled shake-flask cultivations leading to protein that is mostly produced in its soluble form, however in rather low yields. Inclusion body (IB) processes have gathered a lot of attention due to several benefits like increased space-time yields and high purity of the target product. Thus, to investigate the suitability of this processing strategy for ldhL1 production, a fed-batch fermentation steering the production of IBs rather than soluble product formation was developed. It was shown that the space-time-yield of the fermentation could be increased almost 3-fold by increasing qs to 0.25 g g-1 h-1 which corresponds to 21% of qs,max, and keeping the temperature at 37°C after induction. Solubilization and refolding unit operations were developed to regain full bioactivity of the ldhL1. The systematic approach in screening for solubilization and refolding conditions revealed buffer compositions and processing strategies that ultimately resulted in 50% product recovery in the refolding step, revealing major optimization potential in the downstream processing chain. The recovered ldhL1 showed an optimal activity at pH 5.5 and 30∘C with a high catalytic activity and KM values of 0.46 mM and 0.18 mM for pyruvate and NADH, respectively. These features, show that the here produced LDH is a valuable source for various commercial applications, especially considering low pH-environments.
摘要：
广泛的应用范围，从临床诊断到各种行业的生物传感器，使乳酸脱氢酶（LDH）对重组蛋白生产非常有趣。重组LDH的表达目前主要在不受控制的摇瓶培养中进行，导致蛋白质主要以其可溶形式产生。然而，收益率相当低。由于诸如增加的时空产率和目标产物的高纯度的若干益处,包涵体(IB)方法已经引起了很多关注。因此，为了调查这种处理策略对ldhL1生产的适用性，开发了一种指导IBs生产而不是可溶性产物形成的分批补料发酵。结果表明，通过将qs增加到0.25gg-1h-1，相当于qs的21％，发酵的时空产量可以增加近3倍，max,诱导后将温度保持在37°C。开发了增溶和重折叠单元操作以恢复ldhL1的全部生物活性。筛选溶解和重折叠条件的系统方法揭示了缓冲液组成和处理策略，最终在重折叠步骤中产生50%的产物回收率。揭示了下游加工链中的主要优化潜力。回收的ldhL1在pH5.5和30°C下表现出最佳活性，具有高催化活性，丙酮酸和NADH的KM值为0.46mM和0.18mM，分别。这些特点,表明这里生产的LDH是各种商业应用的有价值的来源，特别是考虑低pH环境。
