Original Articles
Purification and Characterization of Two β-d-Glucosidases from an Aspergillus Niger Enzyme Preparation: Affinity and Specificity Toward Glucosylated Compounds Characteristic of the Processing of Fruits

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Abstract

Two β-d-glucosidases were purified to homogeneity from an Aspergillus niger enzyme preparation. Their respective molecular weights (118 kDa for β-d-glucosidase A and 109 kDa for β-d-glucosidase B) and pH and temperature properties were in the range of values reported for fungal β-d-glucosidases. They were competitively inhibited by glucose, gluconolactone, and deoxynojirimycin. Their substrate specificities (specific activities, affinity and catalytic constants) were determined on three glucosylated compounds corresponding to the technological applications of β-d-glucosidases in the processing of fruits; malvidin-3-glucoside (decolorization), geranyl-β-d-glucoside (aroma enhancing), and cellobiose (cellulolysis); in comparison with the activity on p-nitrophenyl-β-d-glucoside. β-d-Glucosidase A presented similar affinities toward the four substrates (Km in the range 0.43–0.5 mm) but displayed very different activities. Cellobiose and geranyl-β-d-glucoside were degraded with respective catalytic constants of 200 μmol min−1 mg−1 and 28 μmol min−1 mg−1, whereas the degradation of malvidin-3-glucoside was very slow (kcat ∼ 1 μmol min−1 mg−1). On the contrary, β-d-glucosidase B displayed its maximum activity toward malvidin-3-glucoside (Km ∼ 0.11 mm, kcat ∼ 50 μmol min−1 mg−1) and had no significant activity on geranyl-β-d-glucoside and cellobiose (Km ∼ 0.25 mm, kcat ∼ 0.4 μmol min−1 mg−1); thus, the two enzymes presented different potential applications to the fruit juice industry, aroma enhancing and liquefaction/maceration with β-d-glucosidase A, and decolorization with β-d-glucosidase B.

Introduction

Commercial enzyme preparations are complex mixtures of pectinases, cellulases, hemicellulases, and glycosidases.1, 2, 3 They are used by the fruit- and vegetable-processing industry to improve the technological properties of juices (Brix level, juice yield, flavor and color extraction, filterability, clarification, and juice viscosity).1, 3, 4, 5 These enzymes can also degrade fruit cell walls to increase juice yields and improve color and flavor extraction.6, 7, 8

β-d-glucosidases (EC 3.2.1.21) present in commercial enzyme preparations participate in cellulolysis through the hydrolysis of cellobiose and contribute to aroma enhancing (particularly in wine) by hydrolyzing terpenyl-β-d-glucosides into terpenols.9, 10, 11 In red fruit juices and wines, they can induce a loss of color.5, 12 Anthocyanins, responsible for the color of red fruits,13, 14 are formed by the addition of a mono- or disaccharide to anthocyanidins. The main sugar residue of anthocyanins is glucose. It is present in fruits such as blueberry, cranberry, blackcurrant, plum, and grape.15, 16 The decolorization is due to the breaking of the linkage between the sugar residue and the anthocyanidin which degrades spontaneously into colorless compounds.17, 18 The discolorating effect of fungal preparations from Aspergillus niger on raspberry,[19] strawberry,[20] blackberry,[21] cranberry,[5] and grape21, 22 anthocyanins has been reported. In grapes juices and wines, since the main anthocyanins are anthocyanidin-β-d-glucosides the decolorization is due to β-d-glucosidases.15, 23

Fungal β-d-glucosidases have been isolated from Humicola grisea,[24] Fusarium oxysporum,[25] Gliocladium virens,[26] Neocallimatix frontalis,[27] Botrytis cinerea,28, 29 Talaromyces emersonii,[30] Trichoderma reesei,[31] Aspergilli aculeatus,[32] foetidus,[33] roseus,[34] terreus,[35] and niger36, 37, 38, 39, 40 All β-d-glucosidases from A. niger reported so far have molecular weights in the range 90–130 kDa, optimum pHs of about 4, optimum temperatures between 50–70°C, and an acidic isoelectric point in the pH range 3.5–4.5.

Some glycosidases active on anthocyanins have been partially purified from crude preparations of A. niger. Their activity on strawberries20, 41 and hawthorn extracts[22] has been reported. An endo-β-d-glucosidase from an A. niger preparation exhibited anthocyanase activity together with side activities on tannins and aroma precursors.[42] Most A. niger glucosidases have been studied for their activity on p-NP-β-d-glucoside and cellobiose but very few authors20, 43 paid attention to the degradation of anthocyanidin-β-d-glucosides. As far as we are aware, no study reported the effect of purified β-d-glucosidases on several substrates representing the different technological targets of β-d-glucosidases: cellobiose (cellulolysis), terpenyl-β-d-glucosides (flavor enhancing), and anthocyanidin-β-d-glucosides (decolorization) in comparison with the “reference” activity on p-nitrophenyl-β-d-glucoside.

One challenge in improving the enzyme preparations used for red-fruit processing, particularly for red wine production, is to obtain enzyme preparations containing glucosidases active on target substrates such as cellobiose and terpenyl-β-d-glucoside but with a low decolorization effect. In this paper, we described the purification and characterization of two β-d-glucosidases of A. niger which differ in their respective affinities and activities toward cellobiose, geranyl-glucoside, and anthocyanidin-β-d-glucosides.

Section snippets

Enzyme Preparation

An experimental enzyme preparation from A. niger (Gist-Brocades, Seclin, France) was used as source of β-d-glucosidases.

Substrates for Enzyme Assays

Cellobiose, p-NP-β-d-glucopyranoside (p-NP-β-d-glucoside), p-NP-α-l-glucopyranoside, p-NP-β-d-galactopyranoside, p-NP-α-l-arabinofuranoside, p-NP-α-l-rhamnopyranoside, and p-NP-β-d-cellobioside were purchased from Sigma (St. Louis, MO).

Geranyl-β-d-glucoside was a generous gift from Dr. Z. Günata (Unité de Recherches des Arômes et des Substances Naturelles-IPV-INRA,

Purification of Two β-d-Glucosidases from A. niger

The purification of the two β-d-glucosidases was followed by measuring in parallel β-d-glucosidase activity on p-NP-β-d-glucoside and the decolorization activity of an anthocyanin extract from V. vinifera var. cabernet franc. The first steps of purification (ammonium sulfate precipitation and hydrophobic interaction) were required to eliminate brown pigments from the crude preparation and induced a 50% loss of total and specific glucosidase activity. The β-d-glucosidase specific activity

Discussion

β-d-glucosidases are used in the fruit juice industry to improve the maceration or the liquefaction processes through their participation to cellulolysis and increase the aroma by hydrolyzing their glucosylated precursors; however, their presence in fungal enzyme preparations can induce a loss of color of red fruit juices when anthocyanidin-3-β-d-glucosides are hydrolyzed into unstable anthocyanidins. The aim of this study was to determine if all A. niger β-d-glucosidases are involved in these

List of Symbols

    .

    p-NP-,

    p-nitrophenyl-;

    .

    HPAEC,

    high performance anion-exchange chromatography

    .

    Enzymes:

    β-glucosidase (EC 3.2.1.21).

Acknowledgements

The authors wish to thank Gist-Brocades France SA (Seclin, France) for financial support. We also thank P. Sarni-Manchado, J. M. Souquet (Unité de Recherche des Polymères et Techniques Physico-Chimiques-Institut des Produits de la Vigne-INRA, Montpellier), and Z. Günata (Unité de Recherches des Arômes et des Substances Naturelles-IPV-INRA, Montpellier) for their respective assistance during the malvidin-3-glucoside purification, HPLC use, and for the gift of pure geranyl-glucoside.

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