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Microplate-based filter paper assay to measure total cellulase activity
Zhizhuang Xiao1, Reginald Storms, Adrian Tsang
1Centre for Structural and Functional Genomics, Concordia University, 7141 Sherbrooke Street West, Montreal H4B 1R6 Quebec, Canada.
Biotechnology and Bioengineering
|October 2, 2004
Summary
A new microplate method offers a faster way to measure total cellulase activity using a reduced filter paper assay (FPA) volume. This high-throughput approach is accurate and reproducible for analyzing large sample numbers.
Area of Science:
- Biochemistry
- Enzymology
- Analytical Chemistry
Background:
- The standard filter paper assay (FPA) by the International Union of Pure and Applied Chemistry (IUPAC) is a common method for determining total cellulase activity.
- The conventional IUPAC FPA is not amenable to high-throughput screening due to its large reaction volume and manual processing.
- There is a need for a more efficient and scalable method for cellulase activity determination.
Purpose of the Study:
- To develop and validate a microplate-based filter paper assay (FPA) for high-throughput determination of total cellulase activity.
- To adapt the standard FPA for reduced reaction volumes suitable for 96-well assay plates.
- To assess the accuracy, reproducibility, and sensitivity of the modified microplate FPA.
Main Methods:
- A modified filter paper assay (FPA) was developed using a reduced enzymatic reaction volume of 60 microliters in 96-well microplates.
- The modified FPA was validated by comparing results with the standard IUPAC FPA using commercial cellulases from Trichoderma reesei and Aspergillus species.
- The effect of adding excess beta-glucosidase on assay sensitivity was investigated.
Main Results:
- The modified 60-microliter FPA yielded cellulase activity measurements that were not significantly different from the standard IUPAC FPA.
- The microplate-based FPA demonstrated quantitative performance and high reproducibility across multiple analyses.
- The addition of excess beta-glucosidase enhanced the sensitivity of the assay by up to 60%.
Conclusions:
- The developed 60-microliter microplate FPA is a reliable and efficient alternative to the standard IUPAC method for high-throughput analysis of total cellulase activity.
- This method facilitates the parallel analysis of a large number of samples, significantly improving workflow efficiency in enzyme activity studies.
- The enhanced sensitivity with beta-glucosidase addition allows for more precise measurements, particularly with low enzyme concentrations.