Investigation of micro-immobilised enzyme reactors containing endoglucanases for efficient hydrolysis of cellodextrins and cellulose derivatives
Investigation of micro-immobilised enzyme reactors containing endoglucanases for efficient hydrolysis of cellodextrins and cellulose derivatives
复制标题
研究含有内切葡聚糖酶的微固定化酶反应器，用于有效水解纤维糊精和纤维素衍生物
DOI：
10.1016/j.aca.2005.06.070
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发表时间：
2005
影响因子：
6.2
通讯作者：
L. Gorton
中科院分区：
文献类型：
--
作者：
C. Melander;M. Bengtsson;H. Schagerlöf;F. Tjerneld;T. Laurell;L. Gorton
将水解纤维素内部部分的纤维素酶（也称为内切葡聚糖酶）固定在由多孔硅制成的微固定化酶反应器（μ MER）中，目的是研究此类μ MER用于水解纤维糊精和可溶性纤维素衍生物的用途。内切葡聚糖酶里氏木霉Cel 12A（TrCel 12A）和粘琼脂芽孢杆菌Cel 5A（BaCel 5A）通过席夫碱形成共价偶联到硅微芯片的表面。为了表征，使用纤维六糖作为固定化酶的底物。通过研究底物溶液浓度、流速、温度和pH值对产物形成的影响，考察了μIMER的特性。在与色谱系统在线连接的μIMER中进行水解，其中使用高效阴离子交换色谱（HPAEC）偶联脉冲安培检测（PAD）分离和检测产物。BaCel 5A和TrCel 12A之间的水解模式进行了比较，结果表明，这两种研究的内切葡聚糖酶在所形成的产物中给出了特定的水解模式，提供了有关酶的重要信息。μ MER是耐用的，并且可以在至少几天的时间内连续使用。此外，在适当的储存条件下，60天后未见活性损失。还使用羧甲基纤维素（CMC）作为底物研究了含μIMER的BaCel 5A水解衍生纤维素的能力。使用具有折射率检测（RI）的尺寸排阻色谱（SEC）进行分离和检测。结果表明，μIMERS是耐用的，可以用于在线水解的纤维糊精和高分子量的衍生纤维素。
Cellulases hydrolysing the interior parts of cellulose, also called endoglucanases, were immobilised in micro-immobilised enzyme reactors (μIMER) made of porous silicon with the purpose of investigating the use of such μIMERs for hydrolysis of cellodextrins and soluble cellulose derivatives. The endoglucanases Trichoderma reesei Cel 12A (TrCel 12A) and Bacillus agaradhaerens Cel 5A (BaCel 5A) were covalently coupled to the surface of a silicon microchip through Schiff base formation. For characterisation cellohexaose was used as substrate for the immobilised enzymes. The characteristics of the μIMER were investigated by studying the product formation when varying the concentration, flow-rate, temperature and pH of the substrate solution. Hydrolysis was performed in the μIMER connected on-line to a chromatographic system, where the products were separated and detected using high-performance anion exchange chromatography (HPAEC) coupled to pulsed amperometric detection (PAD). A comparison of the hydrolytic pattern between BaCel 5A and TrCel 12A was carried out and the results show that the two investigated endoglucanases give specific hydrolytic patterns in the products formed that provide important information about the enzymes. The μIMERs are robust and can be employed continuously over a period of at least several days. Moreover, on appropriate storage, no activity loss is seen after 60 days. The ability of the BaCel 5A containing μIMER to perform hydrolysis of derivatised cellulose was also investigated using carboxymethyl cellulose (CMC) as substrate. Separation and detection were carried out using size exclusion chromatography (SEC) with refractive index detection (RI). The results show that the μIMERs are robust and can be employed for on-line hydrolysis of both cellodextrins and derivatised cellulose of high molecular weight.