A colorimetric assay for the determination of acetyl xylan esterase or cephalosporin C acetyl esterase activities using 7-amino cephalosporanic acid, cephalosporin C, or acetylated xylan as substrate.
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.
Martínez-Martínez
Irene
I
Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Murcia, E-30071 Murcia, Spain.
Montoro-García
Silvia
S
Lozada-Ramírez
José Daniel
JD
Sánchez-Ferrer
Alvaro
A
García-Carmona
Francisco
F
D000107:Acetylation; D000115:Acetylesterase / Q000032:analysis / Q000378:metabolism*; D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D001681:Biological Assay / Q000379:methods*; D002511:Cephalosporins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003124:Colorimetry / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D014990:Xylans / Q000737:chemistry* / Q000378:metabolism
D002511:Cephalosporins; D014990:Xylans; C025163:cephalosporin C; C030735:7-aminocephalosporanic acid; D000115:Acetylesterase; C081851:acetylxylan esterase