CHARACTERIZATION OF THREE SOLUBLE INVERTASES FROM LILIUM LONGIFLORUM FLOWER BUDS

Three soluble invertases (EC 3.2.1.26) previously identified in developing flower buds of Lilium longiflorum Thunb (HortSci. 25:1076, 1990) have been further investigated. These enzymes are fully separable on DEAE-Sephacel and differ substantially in enzymological properties. Each enzyme was further purified by consecutive use of Sephacryl S-200 gel filtration, Con A Sepharose affinity chromatography and Phenyl-Agarose hydrophobic interaction chromatography. This produced 135, 189 and 202 fold purification of invertase I, II and III, respectively. Each was an acid invertase showing pH optima between 4.0 and 5.0. The molecular weight of each invertase was estimated to be 75,000 Da by gel filtration. Invertase I, II and III showed temperature optimum at 40, 50 and 45°C, respectively. A temperature stability study revealed that Invertase III was the most stable followed by II and I. Invertase I, II and III had Km values of 1.0, 6.4 and 6.6 mM for sucrose, respectively. Invertase II and III had lower affinity to raffinose and stachyose than Invertase I. All three invertases were completely inhibited by Hg 2+ and Ag + ions at 1.7 mM concentration. At this concentration Cu 2+ inhibited 45% of activity of Invertase I, but only 30% of activity of Invertase II and III.