PURIFICATION AND CHARACTERIZATION OF ALPHA-AMYLASE FROM POPLAR LEAVES

Endoamylase (EC 3.2.1.1) from mature poplar leaves (Populus x canadens is 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 chr omatography on a starch grain column, and gel filtration. The purified enzyme was classified as alpha-amylase by its substrate specificity a nd by comparison with pop]ar wood alpha-amylase. It migrates on SDS-PA GE gels as a single band (M(r) 44000), but it shows electrophoretic po lymorphism as detected by activity staining on native PAGE gels contai ning amylopectin. The purified cr-amylase is reversibly inactivated by oxidation in the absence of reducing agents and by chelation of dival ent cations. The activity was restored by reductants and metal ions, a combination of Ca2+, DTT, and thioredoxin being most effective. The a mylase was stable at 300 over several hr in the presence of DTT and th ioredoxin 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 electrophoreti c 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 fro m poplar wood.