Automated assay for screening the enzymatic release of reducing sugars from micronized biomass.
Automated assay for screening the enzymatic release of reducing sugars from micronized biomass.
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DOI：
10.1186/1475-2859-9-58
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发表时间：
2010-07-16
影响因子：
6.4
通讯作者：
Bignon C
中科院分区：
文献类型：
--
作者：
Navarro D;Couturier M;da Silva GG;Berrin JG;Rouau X;Asther M;Bignon C
为了降低木质纤维生物质(即第二代生物乙醇)降解所产生的糖类发酵所获得的生物乙醇的生产成本，有必要筛选出具有更有效的生物质降解特性的新的酶。这就要求建立高通量的筛选方法。已经设计了几种方法，都使用工业SBS格式的微型板。尽管这种尺寸缩小和标准化极大地改进了筛选过程，但已公布的方法包括一个或多个严重降低产量的手动步骤。因此，我们致力于设计一种不需要任何人工步骤的筛查方法。我们描述了一种全自动的测定小麦秸秆和云杉生物质降解酶释放还原糖的方法。该方法包括两个独立的自动化步骤。第一步是“基板”的制作。它包括用微粉化底物的浆液悬浮液填充96孔微孔板，然后冷冻储存直到使用。第二步是酶活性测定。解冻后，机器人在必要时在底物板上补充细胞壁降解酶，从添加酶到定量释放的糖的整个过程由机器人自主执行。我们描述了如何选择关键参数(底物数量、酶的数量、孵化时间和温度)来适应我们的特定用途。使用一组商业酶对这种自动化的小规模分析区分不同酶活性的能力进行了验证。使用自动微孔板封口机解决了在建立测量植物细胞壁降解酶释放糖活性的方法时通常遇到的三个主要问题：吞吐量、自动化和蒸发损失。在目前的设置下，该机器人每天可以自主处理120个三份小麦-秸秆样本。如果孵育时间从24小时减少到4小时(例如，用于初始速率测量)，则产量可以翻一番。这种方法可能适用于任何可微粉化的不溶性底物。说明该方法的视频可在以下网址上看到：http://www.youtube.com/watch?v=NFg6TxjuMWU
To reduce the production cost of bioethanol obtained from fermentation of the sugars provided by degradation of lignocellulosic biomass (i.e., second generation bioethanol), it is necessary to screen for new enzymes endowed with more efficient biomass degrading properties. This demands the set-up of high-throughput screening methods. Several methods have been devised all using microplates in the industrial SBS format. Although this size reduction and standardization has greatly improved the screening process, the published methods comprise one or more manual steps that seriously decrease throughput. Therefore, we worked to devise a screening method devoid of any manual steps. We describe a fully automated assay for measuring the amount of reducing sugars released by biomass-degrading enzymes from wheat-straw and spruce. The method comprises two independent and automated steps. The first step is the making of "substrate plates". It consists of filling 96-well microplates with slurry suspensions of micronized substrate which are then stored frozen until use. The second step is an enzymatic activity assay. After thawing, the substrate plates are supplemented by the robot with cell-wall degrading enzymes where necessary, and the whole process from addition of enzymes to quantification of released sugars is autonomously performed by the robot. We describe how critical parameters (amount of substrate, amount of enzyme, incubation duration and temperature) were selected to fit with our specific use. The ability of this automated small-scale assay to discriminate among different enzymatic activities was validated using a set of commercial enzymes. Using an automatic microplate sealer solved three main problems generally encountered during the set-up of methods for measuring the sugar-releasing activity of plant cell wall-degrading enzymes: throughput, automation, and evaporation losses. In its present set-up, the robot can autonomously process 120 triplicate wheat-straw samples per day. This throughput can be doubled if the incubation time is reduced from 24 h to 4 h (for initial rates measurements, for instance). This method can potentially be used with any insoluble substrate that is micronizable. A video illustrating the method can be seen at the following URL: http://www.youtube.com/watch?v=NFg6TxjuMWU
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影响因子：
3.8
作者：
Chundawat, Shishir P. S.;Venkatesh, Balan;Dale, Bruce E.
通讯作者：
Dale, Bruce E.
影响因子：
6.3
作者：
Inoue H;Yano S;Endo T;Sakaki T;Sawayama S
通讯作者：
Sawayama S
影响因子：
2.4
作者：
Alberto, F.;Navarro, D.;Record, E.
通讯作者：
Record, E.
影响因子：
3.1
作者：
BLAKENEY, AB;HARRIS, PJ;STONE, BA
通讯作者：
STONE, BA
影响因子：
3.8
作者：
King, Brian C.;Donnelly, Marie K.;Gibson, Donna M.
通讯作者：
Gibson, Donna M.