How to better measure the hydrolytic potential of biomass deconstruction enzyme mixtures

The filter paper assay (FPA) has been used for decades as the standardised method for measuring the hydrolytic potential of enzyme mixtures. This method has a number of shortcomings such as only measuring the initial rate of hydrolysis over a short period of time (1 hour) with this typical 4% conversion supposedly representing the extent and rate of more complete hydrolysis.  It is also recognised that addition of β-glucosidase to the enzymes can significantly influence the outcome of the assay by diminishing the accumulation of cellobiose and its effect on end product inhibition.  Current studies with hydrolytic enzyme mixtures have raised a number of additional concerns regarding this assay.  The vast majority of more realistic deconstruction studies have been carried out using complex lignocellulose substrates that have been modified through a pretreatment step. These substrates consist not only of cellulose, but also hemicellulose and lignin, depending on the type of substrate and pretreatment.  Therefore, it is questionable how suitable the FPA might be in predicting the hydrolytic potential of an enzyme mixture on a pretreated substrate.  A number of recent studies have demonstrated the importance of accessory enzymes, such as xylanases and lytic polysaccharide monooxygenases in achieving effective hydrolysis of the cellulose within “real-life” lignocellulose substrates and the contribution of these enzymes is difficult to assess using the FPA.  The presentation will review the strengths and weaknesses of the FPA, while suggesting improvements to this method to obtain a more representative method for assessing the hydrolytic potential of enzyme mixtures.