Purification and Characterization of Three Soluble Invertases from Barley (Hordeum vulgare L.) Leaves

Abstract

Three soluble isoforms of invertase (8-fructofuranosidase; EC 3.2.1.26) were purified from 7-d-old primary leaves of barley (Hordeum vurgare 1.). lnvertase I, a monomeric protein of 64 kD, was purified to apparent homogeneity as shown by sodium dodecylsulfate-polyacrylamide gel electrophoresis. lnvertases IIA and IIB, multimeric proteins with molecular masses of 116 and 155 kD, were purified 780-and 1370-fold, respectively, but were not yet homogeneous. Extracts of epidermal strips of leaves contained only invertase IlB. l h e specific activity of invertase was more than 100-fold higher in the epidermis than in the mesophyll. All three isoforms were acidic invertases, with p H optima of around 5.0 and little activity in the alkaline range. lnvertase I had a K, for sucrose of 8.1 mM, and invertases IIA and IIB had much lower values of 1.0 and 1.7 mM, respectively. lnvertase I was more than 2-fold more resistant than the other two invertases to the inhibitors HgClz and pyridoxal. AI1 three constitutive invertases were found t o act also as sucrose-sucrose fructosyltransferases when supplied with high concentrations of sucrose, forming 1-kestose as principal product. However, the fructosyltransferase activity of all three enzymes was inhibited by pyridoxal in the same way as their invertase activity. This characteristic clearly differentiates them from the inducible sucrose-sucrose fructosyltransferase of barley leaves, the activity responsible for the initial steps of fructan biosynthesis, which has previously been shown to be insensitive to pyridoxal.

Fructan biosynthesis in excised leaves of Lolium temulentum VII. Sucrose and fructan hydrolysis by a fructan‐polymerizing enzyme preparation

“…The M r of 57 kDa is identical to that of the FEH of Dactylis glomerata (Yamomoto & Mino, 1985) and the results closely parallel those reported for L. rigidum where an invertase of M r 132 kDa was separated from an FEH of M r 68 kDa using SEC . The values for L. temulentum fructan hydrolase and invertase fall within the range of 57-68 kDa and 40-155 kDa reported for FEH and acid invertases, respectively, from grasses Obenland et al, 1993 ;Henson & Livingston, 1996).…”

Purification and Recovery of Invertase from Potato Tubers (Solanum tuberosum) by Three Phase Partitioning and Determination of Kinetic Properties of Purified Enzyme

“…Optimum pH for the same enzyme was detected as 4.6 by the Matsushita and Uritani [5]. For the bamboo invertase optimum pH was reported as 4.5 [2], and for barley invertase 5.0 [4]. When the enzyme was incubated in pH 2-9 buffer solution at 37°C for 30 min, the enzyme showed good stability in the pH range from 4.0 to 6.0 and nearly 60% of the activity remained at pH 7.0 (Fig.…”

“…3.2.1.26) is a glycoenzyme that hydrolysis the terminal non-reducing β-fructofuronoside residues in β-D-fructofuronoside, like sucrose [1]. Invertase is found in many plants and it is a key enzyme for the carbohydrate mobilization as sucrose is catalyzed irreversibly by invertase into glucose and fructose residues which are main carbon source in plant metabolism [2][3][4][5]. This enzyme has many applications in cosmetics, pharmaceutical, paper but especially food and beverage industries [6][7][8].…”

“…which are led to low yields. They were also unscalable, time consuming, expensive multistep protocols [2][3][4][5][9][10][11][12]. Three phase partitioning has relatively recent use for bioseperation of enzymes and inhibitors [13][14][15][16][17].…”

Patterns of solute in individual mesophyll, bundle sheath and epidermal cells of barley leaves induced to accumulate carbohydrate

“…The synthesis of fructans can occur in mesophyll and bundle sheath cells; it requires high sucrose concentrations (Housley & Pollock, 1985). The paradoxical situation is that the activity of acid invertase in barley leaves is also quite high (Obenland et al, 1993). The reason could be spatial separation of sucrose from acid invertase.…”