ABSTRACT
Glucitol, also known as sorbitol, is a major photosynthetic product in plants from the Rosaceae family. This sugar alcohol is synthesized from glucose-6-phosphate by the combined activities of aldose-6-phosphate reductase (Ald6PRase) and glucitol-6-phosphatase. In this work we show the purification and characterization of recombinant Ald6PRase from peach leaves. The recombinant enzyme was inhibited by glucose-1-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate and orthophosphate. Oxidizing agents irreversibly inhibited the enzyme and produced protein precipitation. Enzyme thiolation with oxidized glutathione protected the enzyme from insolubilization caused by diamide, while incubation with NADP+ (one of the substrates) completely prevented enzyme precipitation. Our results suggest that Ald6PRase is finely regulated to control carbon partitioning in peach leaves.
Subject(s)
Aldehyde Reductase/metabolism , Plant Leaves/enzymology , Plant Proteins/metabolism , Prunus domestica/enzymology , Aldehyde Reductase/antagonists & inhibitors , Aldehyde Reductase/genetics , Fructosediphosphates/metabolism , Fructosediphosphates/pharmacology , Fructosephosphates/metabolism , Fructosephosphates/pharmacology , Glucosephosphates/metabolism , Glucosephosphates/pharmacology , Glutathione Disulfide/metabolism , Hexosephosphates/metabolism , Hexosephosphates/pharmacology , Immunoblotting , Kinetics , Models, Biological , NADP/metabolism , Oxidants/metabolism , Oxidants/pharmacology , Phosphates/metabolism , Phosphates/pharmacology , Phylogeny , Plant Leaves/genetics , Plant Proteins/classification , Plant Proteins/genetics , Prunus domestica/genetics , Recombinant Proteins/metabolism , Substrate Specificity , Sulfhydryl Compounds/metabolismABSTRACT
Derivatized-agarose supports are suitable for enzyme immobilization by different methods, taking advantage of different physical, chemical and biological conditions of the protein and the support. In this study, agarose particles were modified with MANAE, PEI and glyoxyl groups and evaluated to stabilize polygalacturonase from Streptomyces halstedii ATCC 10897. A new immobilized biocatalyst was developed using glyoxyl-agarose as support; it exhibited high performance in degrading polygalacturonic acid and releasing oligogalacturonides. Maximal enzyme activity was detected at 5h of reaction using 0.05g/mL of immobilized biocatalyst, which released 3mg/mL of reducing sugars and allowed the highest product yield conversion and increased stability. These results are very favorable for pectin degradation with reusability up to 18 successive reactions (90h) and application in juice clarification. Plum (4.7°Bx) and grape (10.6°Bx) juices were successfully clarified, increasing reducing sugars content and markedly decreasing turbidity and viscosity.