ABSTRACT
Enzymatic browning is a major quality issue in fruit and vegetable processing and can be counteracted by different natural inhibitors. Often, model systems containing a single polyphenol oxidase (PPO) are used to screen for new inhibitors. To investigate the impact of the source of PPO on the outcome of such screening, this study compared the effect of 60 plant extracts on the activity of PPO from mushroom ( Agaricus bisporus , AbPPO) and PPO from potato ( Solanum tuberosum , StPPO). Some plant extracts had different effects on the two PPOs: an extract that inhibited one PPO could be an activator for the other. As an example of this, the mate ( Ilex paraguariensis ) extract was investigated in more detail. In the presence of mate extract, oxygen consumption by AbPPO was found to be reduced >5-fold compared to a control reaction, whereas that of StPPO was increased >9-fold. RP-UHPLC-MS analysis showed that the mate extract contained a mixture of phenolic compounds and saponins. Upon incubation of mate extract with StPPO, phenolic compounds disappeared completely and saponins remained. Flash chromatography was used to separate saponins and phenolic compounds. It was found that the phenolic fraction was mainly responsible for inhibition of AbPPO and activation of StPPO. Activation of StPPO was probably caused by activation of latent StPPO by chlorogenic acid quinones.
Subject(s)
Agaricus/enzymology , Catechol Oxidase/antagonists & inhibitors , Catechol Oxidase/metabolism , Enzyme Inhibitors/chemistry , Fungal Proteins/antagonists & inhibitors , Fungal Proteins/metabolism , Ilex paraguariensis/chemistry , Plant Extracts/chemistry , Plant Proteins/antagonists & inhibitors , Plant Proteins/metabolism , Solanum tuberosum/enzymology , Phenols/chemistry , Saponins/chemistryABSTRACT
To enhance the phosphorylating activity of the bacterial nonspecific acid phosphatase from Salmonella enterica ser. typhimurium LT2 towards dihydroxyacetone (DHA), a mutant library was generated from the native enzyme. Three different variants that showed enhanced activity were identified after one round of epPCR. The single mutant V78L was the most active and showed an increase in the maximal DHAP concentration to 25 % higher than that of the wild-type enzyme at pH 6.0. This variant is 17 times more active than the wild-type acid phosphatase from Salmonella enterica ser. typhimurium LT2 in the acid phosphatase/aldolase cascade reaction at pH 6.0 and is also six times more active than the phosphatase from Shigella flexneri that we previously used.
Subject(s)
Acid Phosphatase/metabolism , Dihydroxyacetone/metabolism , Directed Molecular Evolution , Aldehyde-Lyases/metabolism , Amino Acid Sequence , Dihydroxyacetone/chemistry , Hydrogen-Ion Concentration , Mutagenesis, Site-Directed , Phosphorylation , Protein Conformation , Salmonella enterica/enzymologyABSTRACT
A new enzymatic method for the generation of dihydroxyacetone phosphate (DHAP) using the acid phosphatase from Shigella flexneri (PhoN-Sf) and the cheap phosphate donor pyrophosphate (PPi) is described. The utility of this method was demonstrated in an aldolase-catalyzed condensation carried out in one pot in which DHAP was generated and coupled to propionaldehyde to give a yield of 53% of the isolated dephosphorylated end product.
Subject(s)
Carbohydrate Metabolism , Carbohydrates/chemical synthesis , Dihydroxyacetone Phosphate/chemistry , Dihydroxyacetone Phosphate/metabolism , Phosphates/chemistry , Aldehydes/chemistry , Carbohydrates/chemistry , Magnetic Resonance Spectroscopy , Molecular Structure , Shigella flexneri/enzymologyABSTRACT
Nonspecific acid phosphatases share a conserved active site with mammalian glucose-6-phosphatases (G6Pase). In this work we examined the kinetics of the phosphorylation of glucose and dephosphorylation of glucose-6-phosphate (G6P) catalysed by the acid phosphatases from Shigella flexneri (PhoN-Sf) and Salmonella enterica (PhoN-Se). PhoN-Sf is able to phosphorylate glucose regiospecifically to G6P, glucose-1-phosphate is not formed. The K(m) for glucose using pyrophosphate (PPi) as a phosphate donor is 5.3 mM at pH 6.0. This value is not significantly affected by pH in the pH region 4-6. The K(m) value for G6P by contrast is much lower (0.02 mM). Our experiments show these bacterial acid phosphatases form a good model for G6Pase. We also studied the phosphorylation of inosine to inosine monophosphate (IMP) using PPi as the phosphate donor. PhoN-Sf regiospecifically phosphorylates inosine to inosine-5'-monophosphate whereas PhoN-Se produces both 5'IMP and 3'IMP. The data show that during catalysis an activated phospho-enzyme intermediate is formed that is able to transfer its phosphate group to water, glucose or inosine. A general mechanism is presented of the phosphorylation and dephosphorylation reaction catalysed by the acid phosphatases. Considering the nature of the substrates that are phosphorylated it is likely that this class of enzyme is able to phosphorylate a wide range of hydroxy compounds.
