Automated assay for screening the enzymatic release of reducing sugars from micronized biomass.
BACKGROUND
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.


RESULTS
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.


CONCLUSIONS
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.
Navarro
David
D
Architecture et Fonction des Macromolécules Biologiques, UMR 6098, CNRS et Université d'Aix-Marseille I et II, 163 Avenue de Luminy CP 925, 13288 Marseille Cedex 09, France.
Couturier
Marie
M
da Silva
Gabriela Ghizzi Damasceno
GG
Berrin
Jean-Guy
JG
Rouau
Xavier
X
Asther
Marcel
M
Bignon
Christophe
C
D018533:Biomass; D050260:Carbohydrate Metabolism; D002241:Carbohydrates / Q000737:chemistry; D057075:Enzyme Assays / Q000295:instrumentation* / Q000379:methods; D004798:Enzymes / Q000378:metabolism; D057166:High-Throughput Screening Assays; D010084:Oxidation-Reduction; D012371:Robotics; D013379:Substrate Specificity
D002241:Carbohydrates; D004798:Enzymes