Purification and characterization of alpha-amylase from poplar leaves

Endoamylase (EC 3.2.1.1) from mature poplar leaves (Populus x canadensis Moench ''robusta') was purified 17 700-fold over the crude protein extract to a specific activity of 166 mu mol min(-1) mg(-1) by (NH4)(2)SO4-precipitation, treatment with anion exchange resins, affinity chromatography on a starch grain column, and gel filtration. The purified enzyme was classified as alpha-amylase by its substrate specificity and by comparison with pop]ar wood alpha-amylase. It migrates on SDS-PAGE gels as a single band (M(r) 44000), but it shows electrophoretic polymorphism as detected by activity staining on native PAGE gels containing amylopectin. The purified cr-amylase is reversibly inactivated by oxidation in the absence of reducing agents and by chelation of divalent cations. The activity was restored by reductants and metal ions, a combination of Ca2+, DTT, and thioredoxin being most effective. The amylase was stable at 300 over several hr in the presence of DTT and thioredoxin whereas 2-mercaptoethanol could stabilize the activity only at 4 degrees. The treatment with divalent cations and reductants also changed the native PAGE banding pattern and resulted in a shift of the pH optimum to a higher value. It is concluded that the electrophoretic polymorphism is caused by the different affinity to the immobilized amylopectin of the enzyme forms associated with the individual bands. Some of these effects were also observed with the main endoamylase from poplar wood.