Purification and Partial Characterization of Potato (Solanum tuberosum) Invertase and Its Endogenous Proteinaceous Inhibitor

Abstract

Invertase plays an important role in the hydrolysis of sucrose in higher plants, especially in the storage organs. In potato (Solanum tuberosum) tubers, and in some other plant tissues, the enzyme seems to be controlled by interaction with an endogenous proteinaceous inhibitor. An acid invertase from potato tubers (variety russet) was purified 1560-fold to electrophoretic homogeneity by consecutive use of concanvalin A-Sepharose 4B affinity chromatography, DEAE-Sephadex A-50-120 chromatography, Sephadex G-150 chromatography, and DEAE-Sephadex A-50-120 chromatography. The enzyme contained 10.9% carbohydrate, had an apparent molecular weight of 60,000 by gel filtration, and was composed of two identical molecular weight subunits (M, 30,000). The enzyme had a Km for sucrose of 16 millimolar at pH 4.70 and was most stable and had maximum activity around pH 5. The endogenous inhibitor was purified 610-fold to homogeneity by consecutive treatment at pH 1 to 1.5 at 370C for 1 hour, (NH4)2SO4 fractionation, Sephadex G-100 chromatography, DEAESephadex G-50-120 chromatography, and hydroxylapatite chromatography. The inhibitor appears to be a single polypeptide (M, 17,000) without glyco groups. The purified inhibitor was stable over the pH range of 2 to 7 when incubated at 370C for 1 hour.Sucrose is the most abundant transportable free carbohydrate in the plant kingdom. Sucrose serves as an important reserve carbohydrate in plants, especially in such storage organs as tuber, root, and seed. During germination, sucrose is a readily degradable source of energy. In storage organs, invertase (fl-fructofuranosidase; EC 3.2.1.26) functions in the hydrolytic degradation of sucrose to glucose and fructose.Invertase was one of the first enzymes discovered; it was first isolated from yeast more than a century ago (3). The enzyme occurs widely in microbial, plant, and animal sources.

Sucrose protects cell wall invertase but not vacuolar invertase against proteinaceous inhibitors

“…Apart from control of gene expression, enzyme activity may be affected by modulation of vacuolar (VI) or apoplasmic (CWI) pH. In addition, plant invertases may be regulated by proteinaceous inhibitors found in different species [11][12][13][14][15][16][17][18]. However, it has been questioned whether inhibitors and invertases are localized in vivo in the same cellular compartment [19].…”

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

“…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].…”

“…This enzyme has many applications in cosmetics, pharmaceutical, paper but especially food and beverage industries [6][7][8]. Invertase was firstly isolated from yeast more than a century ago [3]. From the date until now researchers reported several purification methods that involve salt precipitation, different chromatographic techniques and ultrafiltration etc.…”

“…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].…”

“…Purification and recovery of invertase from potato tubers (Solanum tuberosum) by three phase partitioning and determination of kinetic properties of purified enzyme [Üçlü faz ayırma yöntemi ile patates (Solanum tuberosum) yumrularından invertazın saflaştırılması ve geri kazanımı ve saflaştırılmış enzimin kinetik özelliklerinin belirlenmesi] Determination of invertase activity 800 µL substrate (50 mM sucrose in 50 mM pH 4.7 acetate buffer) and 200 µL suitably diluted enzyme solution were taken in a test tube (extinction coefficient of o-sucrose equals to 2.568x10 3 M -1 cm -1 ). The reaction was incubated at 37 ºC for 30 min then was stopped by adding 0.1 ml of DNS (3,5-Dinitrosalicylic acid) reagent.…”