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首页> 外文期刊>BMC Biotechnology >Optimization of production, purification and lyophilisation of cellobiose dehydrogenase by Sclerotium rolfsii
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Optimization of production, purification and lyophilisation of cellobiose dehydrogenase by Sclerotium rolfsii

机译：罗氏菌的纤维素二糖脱氢酶的生产，纯化和冻干的优化

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Background The enzyme cellobiose dehydrogenase (CDH) can be used to oxidize lactose to lactobionic acid. As Sclerotium rolfsii is known to be a good producer of CDH, the aim of this paper was to simplify its production and secondly to systematically study its purification aiming for a high yield. Two preservation methods (freezing and freeze-drying) and the influence of several protectants were investigated. Results Production of cellobiose dehydrogenase was optimized leading to a more simplified medium composition. Purification of the enzyme was evaluated by determining breakthrough profiles on different ion exchange (IEX) and hydrophobic interaction (HIC) materials with regard to buffer composition. Highest purification with an acceptable loss during the capture step using IEX was obtained with a Q Sepharose XL medium and a 100 mM sodium acetate buffer at pH 4.5. Subsequent purification using hydrophobic interaction chromatography was done at 1.1 M ammonium sulfate concentration. Purification was moderate, yielding a specific activity of 11.9 U/mg (56% yield). However, as could be shown in a preliminary experiment, purity of the obtained enzyme solution was sufficient for its intended use to oxidize lactose to lactobionic acid. Various sugars and sugar alcohols were investigated to study their protective effect during lyophilisation and freezing at -20°C. Glucose and lactulose could be identified to have a high lyoprotective effect while loss of enzyme activity was high (77%) when using no additives. Conclusion By simplifying the cultivation medium of Sclerotium rolfsii , the costs of cellobiose dehydrogenase production could be reduced. Simultaneously, CDH production was increased by 21%. The production of lactobionic acid from lactose is possible using partially purified and unpurified enzyme. Storage at -20°C using 50% (w/v) glycerol was considered to be most suited for preservation of the enzyme.
机译：背景技术纤维二糖脱氢酶（CDH）可用于将乳糖氧化为乳糖酸。由于众所周知，Sclerotium rolfsii是CDH的良好生产者，因此本文的目的是简化其生产，其次是系统地研究其纯化以实现高收率。研究了两种保存方法（冷冻和冷冻干燥）以及几种保护剂的影响。结果优化了纤维二糖脱氢酶的生产，从而简化了培养基组成。通过确定关于缓冲液组成的不同离子交换（IEX）和疏水相互作用（HIC）材料的突破特性来评估酶的纯化。使用Q Sepharose XL培养基和pH 4.5的100 mM乙酸钠缓冲液，在使用IEX的捕获步骤中获得了最高纯度且可接受的损失。随后使用疏水相互作用色谱法在1.1 M硫酸铵浓度下进行纯化。纯化是中等的，产生的比活为11.9 U / mg（产率56％）。但是，如初步实验所示，所获得的酶溶液的纯度足以使其用于将乳糖氧化为乳糖酸的预期用途。研究了各种糖和糖醇，以研究其在冻干和-20°C冷冻期间的保护作用。当不使用添加剂时，可以确定葡萄糖和乳果糖具有很高的冻干保护作用，而酶活性的损失则很高（77％）。结论通过简化菌核菌的培养基，可以降低纤维二糖脱氢酶的生产成本。同时，CDH产量增加了21％。使用部分纯化的和未纯化的酶可以从乳糖生产乳糖酸。使用50％（w / v）甘油在-20°C下保存被认为最适合保存该酶。

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