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首页> 外文期刊>Biotechnology Progress >Design and Characterization of a Prototype Enzyme Microreactor: Quantification of Immobilized Transketolase Kinetics
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Design and Characterization of a Prototype Enzyme Microreactor: Quantification of Immobilized Transketolase Kinetics

机译：原型酶微反应器的设计和表征：固定化转酮醇酶动力学的定量。

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In this work, we describe the design of an immobilized enzyme microreactor (IEMR)for use in transketolase (TK) bioconversion process characterization. The prototype microreactor is based on a 200-μm ID fused silica capillary for quantitative kinetic analysis. The concept is based on the reversible immobilization of His6-tagged enzymes via Ni-NTA linkage to surface derivatized silica. For the initial microreactor design, the mode of operation is a stop-flow analysis which promotes higher degrees of conversion. Kinetics for the immobilized TK-cata-lysed synthesis of L-erythrulose from substrates glycolaldehyde (GA) and hydroxypyruvate (HPA) were evaluated based on a Michaelis-Menten model. Results show that the TK kinetic parameters in the IEMR (V_(max(app)) = 0.1 ± 0.02 mmol min~(-1), K_(m(app)) = 26 ±4 mM) are comparable with those measured in free solution. Furthermore, the k_(cat) for the microreactor of 4.1 × 10~5 s~(-1) was close to the value for the bioconversion in free solution. This is attributed to the controlled orientation and monolayer surface coverage of the His6-immobilized TK. Furthermore, we show quantitative elution of the immobilized TK and the regeneration and reuse of the derivatized capillary over five cycles. The ability to quantify kinetic parameters of engineered enzymes at this scale has benefits for the rapid and parallel evaluation of evolved enzyme libraries for synthetic biology applications and for the generation of kinetic models to aid bioconversion process design and bioreactor selection as a more efficient alternative to previously established microwell-based systems for TK bioprocess characterization.
机译：在这项工作中，我们描述了用于转酮醇酶（TK）生物转化过程表征的固定化酶微反应器（IEMR）的设计。微型反应器原型基于200-μm内径的熔融石英毛细管，用于定量动力学分析。该概念基于通过Ni-NTA键可逆地将His6标记的酶固定在表面衍生的二氧化硅上。对于最初的微反应器设计，操作模式是停止流分析，可促进更高的转化度。基于Michaelis-Menten模型评估了固定化TK催化由底物乙醇醛（GA）和羟基丙酮酸（HPA）合成L-赤藓糖的动力学。结果表明IEMR中的TK动力学参数（V_（max（app））= 0.1±0.02 mmol min〜（-1），K_（m（app））= 26±4 mM）与游离条件下的可比性解。此外，微反应器的4.1×10〜5 s〜（-1）的k_（cat）接近游离溶液中生物转化的值。这归因于His6固定的TK的受控取向和单层表面覆盖。此外，我们显示了固定的TK的定量洗脱以及衍生的毛细管在五个周期内的再生和重复使用。在这种规模上量化工程化酶动力学参数的能力有利于快速并行评估合成酶应用中进化的酶文库，并有助于动力学模型的生成，以帮助生物转化工艺设计和生物反应器选择，这是以前的更有效替代方案建立了基于微孔的传统知识生物过程表征系统。

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