Turbidity-based assay for polygalacturonic acid depolymerase activity.
A technically simple and inexpensive discontinuous turbidity assay for qualitative and/or quantitative assessments of polygalacturonic acid depolymerase activity is presented. The enzyme reaction is initiated by the addition of enzyme preparation to a reaction mixture containing 0.02% polygalacturonic acid (PGA) in acetate buffer. The progress of the reaction is monitored by terminating aliquots of the reaction mixture (via heat treatment at appropriate times), subsequently adding poly(diallyldimethylammonium chloride) (PDADMAC) for turbidity development (approximately 30 min), and then measuring the turbidity (typically at 420 nm) of the resulting PGA-PDADMAC complex-containing solution. PGA depolymerase activity causes a decrease in the observed turbidity of PGA-PDADMAC-containing solutions because the stability of the interpolyelectrolyte PGA-PDADMAC complex is a function of the degree of polymerization of the PGA. The rate of turbidity change is herein shown to be proportional to a relatively wide range of enzyme concentrations. The assay is demonstrated using a commercial pectinase preparation and tomato fruit extracts.
Chen
Chi-Shen
CS
Department of Food Science and Technology, Oregon State Univeristy, Corvallis, Oregon 97331-6602, USA.
Penner
Michael H
MH
D005638:Fruit / Q000201:enzymology; D007700:Kinetics; D018551:Solanum lycopersicum / Q000201:enzymology; D009391:Nephelometry and Turbidimetry / Q000379:methods*; D010368:Pectins / Q000032:analysis / Q000378:metabolism; D011095:Polyethylenes / Q000032:analysis / Q000378:metabolism; D011096:Polygalacturonase / Q000032:analysis* / Q000378:metabolism*; D000644:Quaternary Ammonium Compounds / Q000032:analysis / Q000378:metabolism
D011095:Polyethylenes; D000644:Quaternary Ammonium Compounds; C041004:poly-N,N-dimethyl-N,N-diallylammonium chloride; D010368:Pectins; D011096:Polygalacturonase; C003181:polygalacturonic acid