1. Technical Field
The present disclosure relates to methods of analyzing, screening and/or evaluating enzyme(s), polypeptide(s), mixtures of enzymes, mixtures of polypeptides, and/or mixtures of enzymes and polypeptides of interest. The methods are useful for, inter alia, detecting residual cellulose and evaluating hydrolysis activity of enzymes, polypeptides, or mixtures of interest. The present disclosure further relates to high-throughput assay(s), for example, a high-throughput assay for quantifying cellulolytic activity and/or cellulase activity of enzyme(s) or polypeptide(s) of interest in a pretreated corn stover (PCS) hydrolysis is disclosed.
2. Description of the Related Art
Current methods of analyzing, evaluating, or screening the hydrolysis activity of an enzyme, polypeptide, or mixtures of interest are problematic and not well designed for high-throughput analysis. For example, one method of analyzing pretreated corn stover (hereinafter referred to as “PCS”) hydrolysis requires a lengthy high pressure liquid chromatography (hereinafter referred to as “HPLC”) analysis for determining the quantity of sugars. Here, cellulose, in the PCS is enzymatically hydrolyzed to glucose, cellobiose, and higher beta-glucans. HPLC is used to measure the glucose and cellobiose. One of skill in the art, knowing the cellulose content of the substrate, can then use this information to calculate the percent conversion of cellulose into sugars. Accordingly, cellulolytic activity of the enzymes in the hydrolysis can be measured. However, the HPLC step is time consuming and laborious. Assays using HPLC are not suitable for high-throughput analysis, and/or a quick analysis of multiple enzymes of interests in a single assay.
Attempts have been made to improve the HPLC assay using a pipettable substrate loaded in deep well plates; however post-hydrolysis HPLC sugar analysis is still required. The HPLC assay is time-consuming, for example, a 96 well plate takes approximately 19 hours of HPLC time. The attempted improvements are problematic in that they include time-consuming filtration, pipetting, and dilution steps prior to HPLC.
Problems with the known assays lead to higher research costs, tedious assay formatting, as well as time-consuming enzyme activity evaluation. Accordingly, there is a continuing need for assays and analysis methods having improved accuracy and/or reduced performance time, having excellent accuracy, an excellent reaction rate, and/or excellent cellulose conversion, especially where high-throughput analysis is desirable.