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 peaks by anion-exchange chromatography on diethylaminoethyl-Sephacel. These 3 amylases exhibited different relative mobilities during non-denaturing polyacrylamide gel electrophoresis (PAGE), The most abundant amylase form (amylase I) was purified to apparent homogeneity using hydrophobic interaction chromatography, gel filtration and chromatofocusing. The apparent molecular mass of the purified amylase was estimated to be 51 kDa by sodium dodecyl sulfate-PAGE and 45 kDa by gel filtration chromatography, The purified amylase was determined to be an endoamylase (EC 3.2.1.1) based on substrate specificity 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 starch (potato) was 1.28 mK ml(-1). The presence of Ca2+ increased the activity and thermal stability of the enzyme. The presence of dithiothreitol enhanced the activity, while beta-mercaptoethanol and reduced glutathione had no significant effect. When pre-incubated in the absence of the substrate, N-ethylmaleimide and 5,5'-dithiobis-(2-nitrobenzoic acid) partially inhibited the enzyme, alpha-cyclodestrins or beta-cyclodextrins had no effect on enzyme activity up to 10 mM. In addition to CaCl2, CoCl2 slightly enhanced activity, 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.