Characterization of a novel steviol-producing β-glucosidase from Penicillium decumbens and optimal production of the steviol

Abstract

This study aimed to develop an economically viable enzyme for the optimal production of steviol (S) from stevioside (ST). Of 9 commercially available glycosidases tested, S-producing β-glucosidase (SPGase) was selected and purified 74-fold from Penicillium decumbens naringinase by a three-step column chromatography procedure. The 121-kDa protein was stable at pH 2.3-6.0 and at 40-60 °C. Hydrolysis of ST by SPGase produced rubusoside (R), steviolbioside (SteB), steviol mono-glucoside (SMG), and S, as determined by HPLC, HPLC-MS, and (1)H- and (13)C-nuclear magnetic resonance. SPGase showed higher activity toward steviol mono-glucosyl ester, ST, R, and SMG than other β-linked glucobioses. The optimal conditions for S production (30 mM, 64 % yield) were 47 mM ST and 43 μl of SPGase at pH 4.0 and 55 °C. This is the first report detailing the production of S from ST hydrolysis by a novel β-glucosidase, which may be useful for the pharmaceutical and agricultural areas.

Selective production of rubusoside from stevioside by using the sophorose activity of β-glucosidase from Streptomyces sp. GXT6

“…(Wan et al 2012), Sulfolobus solfataricus (Chen et al 2014), and Thermus thermophilus (Nguyen et al 2014) focused on the optimization of steviol or rubusoside production, but they provided limited information about their specificity for stevioside. The remaining four enzymes were all β-glucosidases: Clavibacter michiganense β-glucosidase (CMGase) (Nakano et al 1998), Flavobacterium johnsoniae β-glucosidase (FJGase) (Okamoto et al 2000), Aspergillus aculeatus β-glucosidase (SSGase) (Ko et al 2012), and Penicillium decumbens β-glucosidase (SPGase) (Ko et al 2013). Each has been described in detail with respect to hydrolysis of steviol glucoside substrate.…”

“…SPGase hydrolyzed all the glucosidic bonds and glucosyl ester linkages between the glucosidic moiety and steviol, such as in stevioside, rubusoside, steviolmono-glucoside, and steviol mono-glucosyl ester. However, it did not hydrolyze rebaudioside A. SPGase was the only one of these β-glucosidases that could produce steviol from stevioside in a single-enzyme system (Ko et al 2013). SSGase did not hydrolyze the glucosyl ester linkages at the 19-carboxyl group of rebaudioside A, stevioside, rubusoside, and steviol mono-glucosyl ester or the glucosidic bonds in the saccharide chain at C-13 of rebaudioside A.…”

“…Selective cleavage of β-1,2-glucosidic linkage of sophorosyl moiety at C-13 of stevioside produces rubusoside. Enzymatic preparation of stevioside from rubusoside has specific advantages over the chemical preparation, including higher selectivity, milder reaction conditions, convenient workup, and minimal damage to the environment (Ko et al 2013). However, previous studies have shown that few of the 30 enzymes tested can convert stevioside to rubusoside as a primary product (Nguyen et al 2014).…”

“…However, previous studies have shown that few of the 30 enzymes tested can convert stevioside to rubusoside as a primary product (Nguyen et al 2014). Many β-linkage-hydrolyzing enzymes were screened for stevioside hydrolysis (Chen et al 2014;Ko et al 2012;Ko et al 2013;Nakano et al 1998;Nguyen et al 2014;Okamoto et al 2000;Wan et al 2012). However, only three enzymes have been identified that can produce rubusoside (Ko et al 2012;Nguyen et al 2014;Wan et al 2012).…”

Heterologous Expression and Characterization of a GH3 β-Glucosidase from Thermophilic Fungi Myceliophthora thermophila in Pichia pastoris

“…β-Glucosidases have been widely applied in various industrial fields such as food, feed, textile, detergents, pharmaceutical, and bio-ethanol production [3][4][5][6]. In addition, some β-glucosidases with transglycosylation activity can be used to produce the high-value-added rare oligosaccharides [7,8].…”