Forest Products Biotechnology/Bioenergy Group, University of British Columbia, 2424 Main Mall, Vancouver, British Columbia, V6T1Z4, Canada.
Biotechnol Biofuels. 2013 May 20;6(1):80. doi: 10.1186/1754-6834-6-80.
It is widely recognised that fast, effective hydrolysis of pretreated lignocellulosic substrates requires the synergistic action of multiple types of hydrolytic and some non-hydrolytic proteins. However, due to the complexity of the enzyme mixture, the enzymes interaction with and interference from the substrate and a lack of specific methods to follow the distribution of individual enzymes during hydrolysis, most of enzyme-substrate interaction studies have used purified enzymes and pure cellulose model substrates. As the enzymes present in a typical "cellulase mixture" need to work cooperatively to achieve effective hydrolysis, the action of one enzyme is likely to influence the behaviour of others. The action of the enzymes will be further influenced by the nature of the lignocellulosic substrate. Therefore, it would be beneficial if a method could be developed that allowed us to follow some of the individual enzymes present in a cellulase mixture during hydrolysis of more commercially realistic biomass substrates.
A high throughput immunoassay that could quantitatively and specifically follow individual cellulase enzymes during hydrolysis was developed. Using monoclonal and polyclonal antibodies (MAb and PAb, respectively), a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed to specifically quantify cellulase enzymes from Trichoderma reesei: cellobiohydrolase I (Cel7A), cellobiohydrolase II (Cel6A), and endoglucanase I (Cel7B). The interference from substrate materials present in lignocellulosic supernatants could be minimized by dilution.
A double-antibody sandwich ELISA was able to detect and quantify individual enzymes when present in cellulase mixtures. The assay was sensitive over a range of relatively low enzyme concentration (0 - 1 μg/ml), provided the enzymes were first pH adjusted and heat treated to increase their antigenicity. The immunoassay was employed to quantitatively monitor the adsorption of cellulase monocomponents, Cel7A, Cel6A, and Cel7B, that were present in both Celluclast and Accellerase 1000, during the hydrolysis of steam-pretreated corn stover (SPCS). All three enzymes exhibited different individual adsorption profiles. The specific and quantitative adsorption profiles observed with the ELISA method were in agreement with earlier work where more labour intensive enzyme assay techniques were used.
人们普遍认识到，要快速有效地水解预处理木质纤维素底物，需要多种水解酶和一些非水解酶的协同作用。然而，由于酶混合物的复杂性，酶与底物的相互作用以及缺乏特定的方法来跟踪水解过程中单个酶的分布，大多数酶-底物相互作用的研究都使用了纯化酶和纯纤维素模型底物。由于典型“纤维素酶混合物”中的酶需要协同作用才能实现有效水解，一种酶的作用可能会影响其他酶的行为。酶的作用还将受到木质纤维素底物性质的影响。因此，如果能够开发出一种方法，使我们能够在水解更具商业现实意义的生物质底物时跟踪纤维素酶混合物中存在的一些单个酶，那将是有益的。
开发了一种高通量免疫测定法，可在水解过程中定量和特异性地跟踪单个纤维素酶。使用单克隆和多克隆抗体（MAb 和 PAb），开发了双抗体夹心酶联免疫吸附测定法（ELISA），以特异性定量来自里氏木霉的纤维素酶：纤维二糖水解酶 I（Cel7A）、纤维二糖水解酶 II（Cel6A）和内切葡聚糖酶 I（Cel7B）。通过稀释，可以最大限度地减少存在于木质纤维素上清液中的底物材料的干扰。
双抗体夹心 ELISA 能够在纤维素酶混合物中检测和定量存在的单个酶。该测定法在相对较低的酶浓度（0-1μg/ml）范围内具有敏感性，前提是酶首先进行 pH 调节和热处理以提高其抗原性。该免疫测定法用于定量监测存在于 Celluclast 和 Accellerase 1000 中的纤维素酶单一组分 Cel7A、Cel6A 和 Cel7B 在蒸汽预处理玉米秸秆（SPCS）水解过程中的吸附。这三种酶都表现出不同的个体吸附曲线。ELISA 方法观察到的特异性和定量吸附曲线与早期使用更费力的酶测定技术的工作结果一致。
