A Low-Volume, Parallel Copper-Bicinchoninic Acid (BCA) Assay for Glycoside Hydrolases
The quantitation of liberated reducing sugars by the copper-bicinchoninic acid (BCA) assay provides a highly sensitive method for the measurement of glycoside hydrolase (GH) activity, particularly on soluble polysaccharide substrates. Here, we describe a straightforward method adapted to low-volume polymerase chain reaction (PCR) tubes which enables the rapid, parallel determination of GH kinetics in applications ranging from initial activity screening and assay optimization, to precise Michaelis–Menten analysis.
Key wordsGlycoside hydrolase (GH) Glycosidase Carbohydrate-active enzymes (CAZymes) Copper-bicinchoninic acid (BCA) Polysaccharide Enzymology Reducing sugar
Funding from the Natural Sciences and Engineering Research Council of Canada (NSERC) via the Strategic Partnership Grants for Networks (for the Industrial Biocatalysis Network) and Discovery Grant programs is gratefully acknowledged. Equipment infrastructure was funded by the Canada Foundation for Innovation and the British Columbia Knowledge Development Fund. We thank Sean McDonald (Brumer group, UBC) for comments on an early version of this chapter.
- 1.Sinnott M (2013) Carbohydrate chemistry and biochemistry: structure and mechanism, 2nd edn. RSC Publishing, LondonGoogle Scholar
- 12.Stoll VS, Blanchard JS (1990) Buffers: principles and practice. In: Deutscher MP (ed) Methods in enzymology: guide to protein purification. Academic Press, Cambridge, MA, pp 24–38.Google Scholar
- 14.Cornish-Bowden A (2012) Practical aspects of kinetics. In: Fundamentals of enzyme kinetics, 4th edn. Wiley-Blackwell, Hoboken, NJ, pp 85–106Google Scholar
- 18.Skoog DA, Holler FJ Crouch SR (2006) Appendix I. In: Douglas A et al (eds) Principles of instrumental analysis, 6th edn. Brooks Cole, Pacific Grove, CA.Google Scholar