Activation and stabilization of 10 starch-degrading enzymes by Triton X-100, polyethylene glycols, and polyvinyl alcohols

Abstract

A dilute solution (0.01–0.1 unit/mL) of porcine pancreatic α-amylase (PPA) at pH 6.5, 24 °C, and 1 mM CaCl 2 was found to lose 98% of its activity on standing for 2 h. Addition of 0.02% (w/v) Triton X-100 gave 41% activation and stabilization for 3 h. Because Triton X-100 has a polyethylene glycol side chain, seven polyethylene glycols (PEGs), ranging in MWs of 400–8000 Da and two polyvinyl alcohols (PVAs) of MWs of 10,000 and 50,000 Da were tested as activators and stabilizers of 10 starch-degrading enzymes: PPA, human salivary α-amylase (HSA), Aspergillus oryzae α-amylase (AOA), Bacillus amyloliquefaciens α-amylase (BAA), Bacillus licheniformis α-amylase (BLA), Aspergillus niger glucoamylase (GA), barley β-amylase (β-A), Pseudomonas amylodermosa isoamylase (IA), Bacillus acidopullulyticus pullulanase (PUL), and Bacillus macerans cyclomaltodextrin glucanyltransferase (CGTase). Although nearly all of the additives gave activation and stabilization of the 10 enzymes, there was one specific additive and concentration for each enzyme that gave a maximum degree of activation: 0.02% (w/v) PEG 1500 Da gave 77% activation for β-A; 0.04% PEG 1500 Da gave 70% for PPA; 0.04% PEG 2000 Da gave 58% for IA; 0.04% PEG 1500 Da gave 53% for AOA; 0.04% PVA 50,000 Da gave 48% for GA; 0.04% PEG 4500 Da gave 46% for BLA; 0.02% Triton X-100 gave 45% for HSA; 0.04% PEG 1000 Da gave 42% for BAA; 0.02% Triton X-100 gave 27% for PUL; and 0.02% PEG 1500 Da gave 20% for CGTase. The mechanism of activation and stabilization is postulated to be the binding of the additives to the enzyme-proteins to give a single tertiary structure that results in a maximum enzyme activity, whereas without the additive the enzymes have several tertiary structures in equilibrium, each with a specific activity that results in an average lower activity.