TY - JOUR
T1 - One-step zymogram method for the simultaneous detection of cellulase/xylanase activity and molecular weight estimation of the enzyme
AU - Cano-Ramírez, Claudia
AU - Santiago-Hernández, Alejandro
AU - Rivera-Orduña, Flor Nohemí
AU - Pineda-Mendoza, Rosa María
AU - Zúñiga, Gerardo
AU - Hidalgo-Lara, María Eugenia
N1 - Publisher Copyright:
© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
PY - 2017/2/1
Y1 - 2017/2/1
N2 - Here, we describe a zymographic method for the simultaneous detection of enzymatic activity and molecular weight (MW) estimation, following a single electrophoresis step. This involved separating cellulase and xylanase activities from bacteria and fungi, obtained from different sources, such as commercial extracts, crude extract and purified proteins, under denaturing conditions, by 10% polyacrylamide gel electrophoresis, using polyacrylamide gels copolymerized with 1% (w/v) carboxymethylcellulose or beechwood xylan as substrates. Then, enzymes were refolded by treatment with 2.5% Triton X-100 in an appropriate buffer for each enzymatic activity, and visualized by Coomassie blue staining for MW estimation. Finally, Congo red staining revealed bio-active cellulase and xylanase bands after electrophoretic separation of the proteins in the preparations. This method may provide a useful additional tool for screening of particular cellulase and xylanase producers, identification and MW estimation of polypeptides that manifest these activities, and for monitoring and control of fungal and bacterial cellulase and xylanase production.
AB - Here, we describe a zymographic method for the simultaneous detection of enzymatic activity and molecular weight (MW) estimation, following a single electrophoresis step. This involved separating cellulase and xylanase activities from bacteria and fungi, obtained from different sources, such as commercial extracts, crude extract and purified proteins, under denaturing conditions, by 10% polyacrylamide gel electrophoresis, using polyacrylamide gels copolymerized with 1% (w/v) carboxymethylcellulose or beechwood xylan as substrates. Then, enzymes were refolded by treatment with 2.5% Triton X-100 in an appropriate buffer for each enzymatic activity, and visualized by Coomassie blue staining for MW estimation. Finally, Congo red staining revealed bio-active cellulase and xylanase bands after electrophoretic separation of the proteins in the preparations. This method may provide a useful additional tool for screening of particular cellulase and xylanase producers, identification and MW estimation of polypeptides that manifest these activities, and for monitoring and control of fungal and bacterial cellulase and xylanase production.
KW - Cellulase
KW - Enzyme activity
KW - Molecular weight
KW - Xylanase
KW - Zymogram method
UR - http://www.scopus.com/inward/record.url?scp=85005917733&partnerID=8YFLogxK
U2 - 10.1002/elps.201600347
DO - 10.1002/elps.201600347
M3 - Artículo
C2 - 27873329
SN - 0173-0835
VL - 38
SP - 447
EP - 451
JO - Electrophoresis
JF - Electrophoresis
IS - 3-4
ER -