Identification, purification and characterization of a novel glycosidase (BgLm1) from Leuconostoc mesenteroides

Highlights

• LEUM_0847 gene is identified as a functional β-glycosidase enzyme.

• Recombinant L. mesenteroides BgLm1 has been high-yield purified and characterized.

• BgLm1 showed β-galactosidase, β-glucosidase and β-fucosidase activities.

• This study describes BgLm1 as a β-galactosidase since greatly hydrolyzed lactose.

Abstract

This study describes the identification and characterization of a novel recombinant Leuconostoc mesenteroides glycosidase (BgLm1). Since the protein encoded by LEUM_0847 gene was annotated as putative β-glucosidase, characterization procedures were done using p-nitrophenyl-β-d-glucopyranoside as substrate. A high yield of purified recombinant BgLm1 was obtained (12 mg/L). The enzyme showed an optimal activity at pH 6.0 and 40 °C and preserved 65% of residual activity after 48 h of incubation at 25 °C. Ca²⁺ and Mn²⁺ ions greatly increased the β-glucosidase activity. Moreover, BgLm1 demonstrated β-galactosidase and β-fucosidase activities. Kinetic parameters of BgLm1 revealed its low affinity to p-nitrophenyl-β-d-glucopyranoside (K_m of 9.93 mmol/L).

Then, although LEUM_0847 gene was annotated as a β-glucosidase, our results suggest that BgLm1can be indeed considered as a β-galactosidase since high hydrolysis using skimmed milk (lactose) as natural substrate and high affinity (K_m of 0.56 mmol/L) and specific constant (2254 mmol/L⁻¹ s⁻¹) to p-nitrophenyl-β-d-galactopyranoside were observed. In conclusion, the enzymatic properties observed in this study, support the interest of BgLm1 for food industrial applications.

Introduction

Glycosidases (E.C.3.2.1) catalyze the hydrolysis of glycosidic bonds in complex sugars (Speciale, Thompson, Davies, & Williams, 2014). Among the glycosidases, β-glucosidases are the enzymes that catalyze the hydrolysis of the glycosidic bonds of a part of carbohydrates to release nonreducing terminal glycosyl residues, glycosides and oligosaccharides (Li et al., 2013). Because of their enzymatic properties, the food industry uses β-glucosidases to decrease bitterness by the hydrolysis of specific compounds and to release flavored compounds from glycosylated precursors present in fruit juices, wine tea and fermented products (Acebrón, Curiel, de las Rivas, Muñoz, & Mancheño, 2009; Olguín, Oriol-Alegret, Bordons, & Reguant, 2011). Besides, β-glucosidases are used as cheap biocatalysts in the synthesis of oligosaccharides and alkyl-glycosides synthesis (Bruins, Strubela, van Lieshoutb, Janssena, & Boom, 2003). In addition to applications in the of food industry, β-glucosidases are used for the production of biofuel and ethanol from biomass (Li et al., 2013), in the pharmaceutical sector, for the synthesis of phytoestrogen precursors from daizin and genistin (Otieno, Ashton, & Shah, 2005), and also in chemical, cosmetic, and detergent industries (Bankova, Bakalova, Petrova, & Kolev, D. 2006).

β-glucosidase activity is widespread among Lactic Acid Bacteria (LAB) mainly involved in carbohydrate metabolism. Considering the industrial intereset of β-glucosidases and the fact that LAB are classified as Generally Regarded as Safe (GRAS), several β-glucosidases from LAB have been identified and their biochemical properties characterized, as for example those isolated from Lactobacillus brevis (Michlmayr, Schumann, Barreira Braz Da Silva, Kulbe, & Del Hierro, 2010a), Oenococcus oeni (Dong et al., 2014; Michlmayr et al., 2010b), Lactobacillus plantarum (Sestelo, Poza, & Villa, 2004), Lactobacillus casei (Coulon, Chemardin, Gueguen, Arnaud, & Galzy, 1998), Weissella cibaria (Lee, Han, & Kim, 2012), and Lactococcus sp. (Fang et al., 2014).

Leuconostoc mesenteroides is a LAB species widely used in food industry as starter leading kimchi and sauerkraut fermentations (Zabat, Sano, Wurster, Cabral, & Belenky, 2018). Moreover, L. mesenteroides is mainly characterized for the synthesis of exopolysaccharides such as dextran, with broad medical and chemical applications (Han et al., 2014; Lule, Singh, Pophaly, Pooan, & Tomar, 2016) and bacteriocins as well (Okuda, Tulini, Winkëlstroter, & De Martinis, 2017). As for others LAB, β-glucosidase activity in L. mesenteroides has been previously described (Eom, Hwang, Kim, Kim, & Paik, 2018; Lee et al., 2016; Zhu, Wang, & Zhang, 2019), and an enzyme has been partially-purified and characterized (Gueguen, Chemardin, Labrot, Arnaud, & Galzy, 1997). However, to our knowledge no β-glucosidase from L. mesenteroides has been previously genetically identified and characterized for its physicochemical properties.

Thus, because of i) the potential industrial applications of β-glucosidases from LAB; and ii) although β-glucosidase activity has been described in L. mesenteroides, the enzyme identification and physicochemical properties still remain unknown; this study describes for the first time the genetic identification and biochemical characterization of a recombinant glycosidase from L. mesenteroides.