A modified, rapid ninhydrin assay for the quantification of protein/enzymes during the hydrolysis of pretreated biomass

Accurate protein quantification is necessary in many of the steps during the enzymatic hydrolysis of biomass, from the determination of enzyme kinetics to improving enzyme recycling strategies. However, many of the commonly used protein assays such as the Bicinchoninic, Bradford and Lowry assay demonstrate limited compatibility with carbohydrates, protein glycosylation and phenolic derivatives which are commonly encountered during biomass processing. Thus, the selection of protein assay used can have a significant impact on the protein concentration measured, leading to difficulties when comparative studies are made based on literature values. The ninhydrin assay has been a leading method of protein quantification due to its specificity for protein and its compatibility with most compounds present during biomass processing. The method is a simple two-step process where the protein is hydrolyzed in strong mineral acid followed by the addition of the ninhydrin reagent. The presentation will show how acidic hydrolysis is preferred over alkaline hydrolysis as it does not destroy the serine and threonine components, which account for approximately 20% of the amino acids present in cellulases. As higher solids and lower enzymes loading are becoming more commonplace and the dilution of samples can no longer overcome interfering effects, a modified acidic ninhydrin method was developed and evaluated for its compatibility with conditions encountered during biomass processing and hydrolysis. The presentation will also describe ways to decrease the time required for the assay in comparison to traditional methods.