Purification and characterization of an alkaline invertase from shoots of etiolated rice seedlings

Abstract

One alkaline invertase and two acid invertase activities were detected in the shoots of etiolated rice (Oryza sativa) seedlings. The alkaline invertase (AIT) was purified to homogeneity through steps of ammonium sulphate fractionation, concanavalin A-Sepharose affinity chromatography (non-retained), DEAE-Sephacel chromatography and preparative electrophoresis. The pH optimum of AIT was 7.0 and the molecular mass, determined by gel filtration, was 240 kDa. It is apparently a homotetrameric enzyme (subunit molecular mass 60 kDa). The isoelectric point was 4.4 by isoelectric focusing. The best substrate of the enzyme was sucrose, with a K m of 2.53 mM. The enzyme also hydrolysed raffinose, but not maltose or lactose, so it is a β-D-fructofuranosidase. It gave negative glycoprotein staining. Of the hydrolysis products, fructose was a competitive inhibitor and glucose was a non-competitive inhibitor. Treatment with an alkaline phosphatase could activate AIT, whereas other proteins such as BSA, concanavalin A and urease had no effect on the enzyme activity. The enzyme activity was inhibited by Tris, thiol reagents and heavy metal ions.

HbNIN2, a cytosolic alkaline/neutral‐invertase, is responsible for sucrose catabolism in rubber‐producing laticifers of Hevea brasiliensis (para rubber tree)

“…pH 7.0–7.2, but had no detectable enzyme activity at pH 5.0 or lower (Fig. c), this behavior being similar to those of the native or recombinant A/N‐Invs of other plant species (Chen & Black, ; Lee & Sturm, ; Lin et al ., ; Sturm, ; Vargas et al ., ). Activity of recombinant HbNIN2 was temperature‐dependent, peaking at 45°C (Fig.…”

“…Another unexpected finding is that the enzyme activity of HbNIN2 was markedly stimulated by pyridoxal or pyridoxine (Table ). In other studies, the activities of A/N‐Invs were found to be completely or severely inhibited by pyridoxal or pyridoxine (Chen & Black, ; Van den Ende & Van Laere, ; Lin et al ., ). The new character of enzymatic activation by pyridoxal was further confirmed by assaying the soluble extracts of E. coli expressing the HbNIN2 gene (Table S3).…”

“…Our findings contribute to understanding the enzymatic characteristics of A/N‐Invs in plants. All previous studies have shown that A/N‐Invs cannot use maltose as a substrate (Chen & Black, ; Van den Ende & Van Laere, ; Lee & Sturm, ; Gallagher & Pollock, ; Lin et al ., ; Qi et al ., ). In this study, the semipurified HbNIN2 enzyme hydrolyzed maltose with an efficiency of c .…”

Biochemical Characterization of Soluble Acid and Alkaline Invertases from Shoots of Etiolated Pea Seedlings

“…Invertases are widely distributed in the plant world and numerous studies describing their presence have been published (Isla et al 1995; Vorster and Botha 1998; Lin et al 1999; Hashizume et al 2003; Bruskova et al 2004; Liu et al 2006; Huang et al 2008). Traditionally, invertases have been classified as ‘soluble’, readily extractable from plant tissues in buffer solutions, and ‘insoluble’, extractable only in buffer solutions containing high concentrations of salt.…”

Characterization of Neutral Invertase from Fast Growing Pea (Pisum sativum L.) Seedlings after Gibberellic Acid (GA) Treatment

“…Invertases (INV) are widely distributed in the plant world and numerous studies describing them have been published [11,14,18,19]. Plant invertases can be classified into different subgroups based on solubility, optimum pH, isoelectric point, and subcellular localization [23,29].…”