Abstract
A bacterium Ensifer adhaerens FERM P-19486 with the ability of alliinase production was isolated from a soil sample. The enzyme was purified for characterization of its general properties and evaluation of its application in on-site production of allicin-dependent fungicidal activity. The bacterial alliinase was purified 300-fold from a cell-free extract, giving rise to a homogenous protein band on polyacrylamide gel electrophoresis. The bacterial alliinase (96 kDa) consisted of two identical subunits (48 kDa), and was most active at 60°C and at pH 8.0. The enzyme stoichiometrically converted (-)-alliin ((-)-S-allyl-L-cysteine sulfoxide) to form allicin, pyruvic acid, and ammonia more selectively than (+)-alliin, a naturally occurring substrate for plant alliinase ever known. The C-S lyase activity was also detected with this bacterial enzyme when S-alkyl-L-cysteine was used as a substrate, though such a lyase activity is absolutely absent in alliinase of plant origin. The enzyme generated a fungicidal activity against Saccharomyces cerevisiae in a time- and a dose-dependent fashion using alliin as a stable precursor. Alliinase of Ensifer adhaerens FERM P-19486 is the enzyme with a novel type of substrate specificity, and thus considered to be beneficial when used in combination with garlic enzyme with respect to absolute conversion of (±)-alliin to allicin.
Highlights
Allicin is the best-known active compound of freshly crushed garlic extract, and is known to possess a vast variety of biological effects: antimicrobial, anti-inflammatory, antithrombotic, anticancer, and antiatherosclerotic activities (Stoll and Seeback 1951; Block 1985; Tsai et al 1985; Agarwal 1996; Ankri and Mirelman 1999; Siegel et al 1999)
We evaluate a combination of the enzyme and alliin with respect to on-site generation of allicin-dependent fungicidal activity using the yeast Saccharomyces cerevisiae as a model of fungal cells
Identification of strain FERM P-19486 As summarized in Table 1, strain FERM P-19486 is a rod-shaped bacterium with a size of 1.2-2.0 μm in length and 0.6-0.7 μm in diameter
Summary
Allicin (diallyl thiosulfinate, Figure 1a) is the best-known active compound of freshly crushed garlic extract, and is known to possess a vast variety of biological effects: antimicrobial, anti-inflammatory, antithrombotic, anticancer, and antiatherosclerotic activities (Stoll and Seeback 1951; Block 1985; Tsai et al 1985; Agarwal 1996; Ankri and Mirelman 1999; Siegel et al 1999) This allyl-sulfur compound is synthesized as a result of condensation of allyl sulfenate, which is produced depending on the C-S lyase activity of alliinase (EC 4.4.1.4) on (+)-alliin ((+)-S-allyl-L-cysteine sulfoxide), a naturally occurring diastereomer, as illustrated in Figure 1a (Siegel et al 1999; Jones et al 2004). We evaluate a combination of the enzyme and alliin with respect to on-site generation of allicin-dependent fungicidal activity using the yeast Saccharomyces cerevisiae as a model of fungal cells
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