Three‐Enzyme Phosphorylase Cascade Immobilized on Solid Support for Biocatalytic Synthesis of Cello−oligosaccharides,ChemCatChem

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Three‐Enzyme Phosphorylase Cascade Immobilized on Solid Support for Biocatalytic Synthesis of Cello−oligosaccharides
ChemCatChem ( IF 3.8 ) Pub Date : 2020-01-22 , DOI: 10.1002/cctc.201901964
Chao Zhong ₁ , Božidar Duić ₁ , Juan M. Bolivar ₁ , Bernd Nidetzky _{1,

2}

Affiliation

Enzyme cascades are promising for multistep biocatalytic synthesis, but their effective use beyond the proof‐of‐concept stage is challenging. Strategies to recycle the individual enzymes are critical for the applicability of such cascades. Immobilization on solid support is well developed for single enzymes but remains difficult for enzyme ensembles. Here, we show a controlled co‐immobilization of three glycoside phosphorylases to establish a highly active and recyclable biocatalyst for the conversion of sucrose and glucose into soluble (short‐chain) cello−oligosaccharides. We use protein fusion with the binding module Z_basic2 to enable non‐covalent surface tethering of all enzymes according to a uniform principle and in a programmable fashion. We thus achieve loading of the phosphorylases in an activity ratio optimal for the overall conversion and for controlling the cello−oligosaccharide chain length (≤6), hence the solubility, in the reaction. We demonstrate efficient production of ∼12 g/L cello−oligosaccharides with integrated enzyme re‐use, retaining ∼85 % of the overall initial activity after five reaction cycles. This study presents a major advance toward the practical use of systems bio‐catalysis on solid support.

中文翻译：

固定在固体支持物上的三酶磷酸化酶级联用于生物催化合成纤维寡糖

酶级联技术有望用于多步生物催化合成，但是在概念验证阶段之外如何有效使用酶仍具有挑战性。回收单个酶的策略对于此类级联的适用性至关重要。固相支持物对单一酶的固定作用已得到很好的发展，但对于酶的组合仍然很困难。在这里，我们显示了三种糖苷磷酸化酶的受控共固定化，从而建立了一个高活性和可回收的生物催化剂，用于将蔗糖和葡萄糖转化为可溶性（短链）纤维寡糖。我们使用结合模块Z _basic2的蛋白质融合以统一的原则和可编程的方式实现所有酶的非共价表面束缚。因此，我们以对整个转化和控制纤维素-寡糖链长（≤6）（因此在反应中的溶解度）最佳的活性比实现了磷酸化酶的负载。我们证明了通过整合的酶重复使用可有效生产约12 g / L的纤维寡糖，在五个反应循环后，可保留约85％的总体初始活性。这项研究显示了在固体载体上系统生物催化的实际应用方面的重大进展。

更新日期：2020-01-23

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