Purification and characterization of an endoamylase from tulip (Tulipa gesneriana) bulbs

Amylase activity extracted from tulip (Tulipa gesneriana L. cv, Apeldoorn) bulbs that had been stored for 6 weeks at 4 degrees C was resolved to 3 pea ks by anion-exchange chromatography on diethylaminoethyl-Sephacel. These 3 amylases exhibited different relative mobilities during non-denaturing poly acrylamide gel electrophoresis (PAGE), The most abundant amylase form (amyl ase I) was purified to apparent homogeneity using hydrophobic interaction c hromatography, gel filtration and chromatofocusing. The apparent molecular mass of the purified amylase was estimated to be 51 kDa by sodium dodecyl s ulfate-PAGE and 45 kDa by gel filtration chromatography, The purified amyla se was determined to be an endoamylase (EC 3.2.1.1) based on substrate spec ificity and end-product analysis. The enzyme had a pH optimum of 6.0 and a temperature optimum of 55 degrees C. The apparent K-m value with soluble st arch (potato) was 1.28 mK ml(-1). The presence of Ca2+ increased the activi ty and thermal stability of the enzyme. The presence of dithiothreitol enha nced the activity, while beta-mercaptoethanol and reduced glutathione had n o significant effect. When pre-incubated in the absence of the substrate, N -ethylmaleimide and 5,5'-dithiobis-(2-nitrobenzoic acid) partially inhibite d the enzyme, alpha-cyclodestrins or beta-cyclodextrins had no effect on en zyme activity up to 10 mM. In addition to CaCl2, CoCl2 slightly enhanced ac tivity, while MgCl2 and MnCl2 had no significant effect at a concentration of 2 mM. ZnCl2, CuSO4, AgNO3 and EDTA partially inhibited enzyme activity, while AgNO3 and HgCl2 completely inhibited it at 2.0 mM.