Esterase hydrolyzes esters of small faty acids, and a colorimetric substrate p-nitrophenyl acetate can be utilized to assay the esterase activity by determining the release of p-nitrophenol. This is easy, quantitative and highly reproducible method!

Dear Sir. Concerning your issue about the performance colorimetric assay for acetyl xylan esterase . A bromothymol blue-based colorimetric assay has been devised to screen for acetyl xylan esterase or cephalosporin C (CPC) deacetylase activities using 7-amino cephalosporanic acid (7-ACA), CPC, or acetylated xylan as substrate. These enzymes are not screened with their natural substrates because of the tedious procedures available previously. Acetyl xylan esterase from Bacillus pumilus CECT 5072 was cloned, expressed in Escherichia coli Rosetta (DE3), and characterized using this assay. Similar K(M) values for 7-ACA and CPC were obtained when compared with those described using HPLC methods. The assay is easy to perform and can be carried out in robotic high-throughput colorimetric devices normally used in directed evolution experiments. The assay allowed us to detect improvements in activity at a minimum of twofold with a very low coefficient of variance in 96-well plates. This method is significantly faster and more convenient to use than are known HPLC and pH-stat procedures. I think the following below links may help you in your analysis:

https://www.ncbi.nlm.nih.gov/pubmed/17651681

https://www.ncbi.nlm.nih.gov/pubmed/28417360

https://link.springer.com/protocol/10.1007/978-1-4939-6899-2_5

https://www.pubfacts.com/detail/28417360/Colorimetric-Detection-of-Acetyl-Xylan-Esterase-Activities

Thanks

Esterase hydrolyzes esters of small faty acids, and a colorimetric substrate p-nitrophenyl acetate can be utilized to assay the esterase activity by determining the release of p-nitrophenol. This is easy, quantitative and highly reproducible method!