Glycosylation of Caffeic Acid and Structural Analogues Catalyzed by Novel Glucansucrases from Leuconostoc and Weissella Species
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Biocatalysis and Agricultural Biotechnology 19 (2019) 101114 Contents lists available at ScienceDirect Biocatalysis and Agricultural Biotechnology journal homepage: www.elsevier.com/locate/bab Glycosylation of caffeic acid and structural analogues catalyzed by novel T glucansucrases from Leuconostoc and Weissella species ∗ Johannes Nolte, Alexander Kempa, Arne Schlockermann, Matthias Hochgürtel, Ulrich Schörken Faculty of Applied Natural Sciences, TH Köln-Campus Leverkusen, Chempark Leverkusen E39, Kaiser-Wilhelm-Allee, 51368, Leverkusen, Germany ARTICLE INFO ABSTRACT Keywords: Twelve Leuconostoc and seven Weissella strains with extracellular glucansucrase activity were obtained from an Caffeic acid analysis of 41 lactic acid bacteria. Culture supernatants of all glucansucrase positive strains catalyzed the gly- Glucansucrase cosylation of caffeic acid with sucrose as donor substrate. Eighteen enzymes produced one major peak, which Leuconostoc was identified as caffeic acid-4′-O-α-D-monoglucoside by LC-MS and NMR spectroscopy. Only W. beninensis DSM Weissella 22752 formed significant amounts of the corresponding 3´-O-α-D-monoglucoside. The Weissella strain and five Acceptor reaction Leuconostoc strains with high glycosylation activity were selected for further studies. All glucansucrases cata- Transglycosylation lyzed the glycosylation of the catechol protocatechuic acid, a side-chain truncated analogue of caffeic acid. The Leuconostoc enzymes displayed a preference for the 4′-O-α-D isomer, while the DSM 22752 glucansucrase also produced the protocatechuic acid-3′-O-α-D-monoglucoside. Lower activities with non-catecholic caffeic acid derivatives and no activity with mono-methylated caffeic acid were observed with all glucansucrases. Time- course analyses confirmed that glucansucrase from L. citreum DSM 5577 was the most efficient biocatalyst for catechol glucosylation with yields of up to 74% caffeic acid glucosides after 24 h. The enzyme displayed ahigh regio-preference for the 4′-O-α-D-isomer and formed less than 10% oligoglucosides. Gel electrophoretic analysis and activity staining of the PEG-enriched enzyme showed a single protein band with a molecular mass of 171 kDa. The DSM 5577 glucansucrase was tolerant against the co-solvents dimethyl sulfoxide and ethanol. −1 Kinetic analysis revealed a KM of 27.6 mM for caffeic acid and 31 mM for sucrosecat withk values of 131 s and 438 s−1. 1. Introduction sufficient process stability in the presence of solvents (Andre et al., 2010; Girard and Legoy, 1999; Meng et al., 2016). Natural polyphenols are widely distributed secondary plant meta- Glucansucrase catalyzed glycosylation of non-physiological poly- bolites with attributed health effects and applications in dietary sup- phenols was originally shown with the flavanol (+)-catechin by plements, cosmetics and pharmaceuticals (Nadim et al., 2014; Quideau Nakahara et al. (1995). Other successfully glycosylated catechols in- et al., 2011; Sauer and Plauth, 2017). Covalent attachment of a gly- clude epigallocatechin gallate (Kim et al., 2016), L-DOPA (Yoon et al., cosyl-residue protects polyphenols from oxidation, increases water so- 2010) and caffeic acid (Auriol et al., 2012). The glucoside of caffeic acid lubility and can lead to enhanced bioavailability (Desmet et al., 2012; is marketed as a cosmetic bioactive with antioxidant properties under ® Křen, 2008; Nadim et al., 2014; Thuan and Sohng, 2013; Xu et al., the brand name Inoveol CAFA (Induchem). Also a few non-catecholic 2016). Chemical glycosylation of polyphenols, requiring extensive phenols like resveratrol (Shim et al., 2003), hydroquinone (Seo et al., protective group chemistry, needs greener alternatives (Desmet et al., 2009) and salicyl alcohol (Seo et al., 2005) were glycosylated by glu- 2012; Roode et al., 2003). Glucansucrases of glycoside hydrolase family cansucrases, though with comparably lower activity. 70 (Lombard et al., 2014), naturally catalyzing extracellular poly- The two aerotolerant genera Leuconostoc and Weissella were shown saccharide formation in lactic acid bacteria, are promising glycosyla- to be rich sources of glucansucrases (Bounaix et al., 2009), however; tion catalysts. They are secreted to the outer medium (Meng et al., studies concerning polyphenol glycosylation are currently limited to 2016; van Hijum et al., 2006), from which they can be isolated and enzymes from a few Streptococcus species, Leuconostoc mesenteroides and purified (Majumder et al., 2007). Glucansucrases use sucrose as cost Lactobacillus reuteri. Glucansucrases from the species Weissella have not efficient donor substrate in a one-step transglycosylation and possess been analyzed for polyphenol acceptor specificity, so far. This study ∗ Corresponding author. E-mail address: [email protected] (U. Schörken). https://doi.org/10.1016/j.bcab.2019.101114 Received 7 May 2018; Received in revised form 19 January 2019; Accepted 24 March 2019 Available online 26 March 2019 1878-8181/ © 2019 Elsevier Ltd. All rights reserved. J. Nolte, et al. Biocatalysis and Agricultural Biotechnology 19 (2019) 101114 presents the isolation and characterization of novel glucansucrases from 2.3. Isolation and concentration of glucansucrases Leuconostoc and Weissella and the evaluation of their glycosylation po- tential with caffeic acid and structurally related polyphenol acceptors. Pre-cultures of selected strains were grown in a volume of 15 mL and main cultures in a volume of 200 mL in shake flasks at 100 rpm ® essentially according to the Biolector micro-scale method described in 2. Materials and methods 2.2. After harvesting of the main cultures glucansucrases were isolated according to the method of Paul et al. (1986). An aqueous (50% w/v) 2.1. Bacterial strains and chemicals polyethylene glycol 4000 solution was added under continuous stirring to the supernatants to reach a final concentration of 25% (w/v). The Forty-one Leuconostoc and Weissella strains obtained from DSMZ, mixtures were centrifuged at 2800×g, 4 °C for 15 min and the pre- USDA, Agroscope, Sacco S.r.l., Ernst Böcker GmbH and Moguntia Food cipitates containing glucan and glucansucrase were suspended in Group AG were used in this study (Table 1). All strains were maintained 20 mM sodium acetate, pH 5.4. The PEG precipitation was repeated at −80 °C as 20% (v/v) glycerol stocks obtained from single colonies twice and the final precipitates were dissolved in 20 mM sodium and propagated in De Man, Rogosa and Sharpe (MRS) medium con- acetate, 0.45 mM CaCl2, pH 5.4 containing protease inhibitor cocktail taining 2% (w/v) glucose at 30 °C. Caffeic acid, 4-coumaric acid and (EDTA-free, Roche) and stored at 4 °C. umbellic acid were purchased from Sigma Aldrich. Protocatechuic acid was from Abcr Gmbh, ferulic acid from Acros organics and resveratrol 2.4. Biocatalytical glycosylation and glycoside purification was obtained from Carl Roth. Glycosylation mixtures contained 200 mM sucrose, 10 or 40 mM 2.2. Strain cultivation and selection polyphenol in 20 mM sodium acetate, pH 5.4 supplemented with 0.45 mM CaCl2 and 15% (v/v) dimethyl sulfoxide (DMSO). Reactions Bacterial cultures from glycerol stocks were grown for 15 h in 2 mL were started by addition of glucansucrase-containing supernatants or −1 pre-culture containing MRS media with 2% (w/v) glucose at 30 °C, concentrated glucansucrases (to a final activity of 0.33 UmL or 1.0 U −1 288 rpm in 48 well round plates covered with an air permeable sealing mL ) and incubated statically for up to 24 h at 30 °C. Samples were ® tape in an 80% humidified atmosphere using a BioLector micro bior- taken periodically and the reaction was stopped by addition of 9 vol of eactor system. For cultivation of Weissella strains, the medium was ethanol (−20 °C). Samples were vortexed for 20 s and precipitated supplemented with 0.1% (w/v) cysteine hydrochloride. Cells from the glucans were removed by centrifugation at 3300×g for 20 min at 4 °C pre-cultures were centrifuged (4 °C, 15 min at 2800×g), washed once according to the method of Overwin et al. (2015). with sodium acetate buffer, pH 5.4, and suspended in 2 mL main culture Ethanol was evaporated from the glycoside containing samples under media (MRS) supplemented with 2% (w/v) sucrose to a final (600OD nm) reduced pressure. The samples were lyophilized and dissolved in 20% (v/ ® of 0.25. Main cultures were incubated in the Biolector system at 25 °C v) acetonitrile in water. Preparative HPLC was carried out with an and 288 rpm for 48 h. Samples of 250 μL were taken periodically, the Interchim puriflash 4250/250 system equipped with a Kromasil 100C18 cells were removed by centrifugation and the supernatants were used column (20 × 250 mm, 5 μm) and a PDA detector. The eluents were for the determination of glucansucrase activity and caffeic acid glyco- water (eluent A) and acetonitrile (eluent B) each supplemented with −1 sylation. 0.2% (v/v) formic acid. The flow-rate was 8 mL min . Caffeic acid and umbellic acid glycosides were purified in a water/acetonitrile gradient Table 1 and protocatechuic acid glycosides were purified isocratically with 90% List of strains and analysis of glucansucrase activities in supernatants. eluent A. Glycoside containing fractions were pooled and lyophilized. Microorganism Designation, supplier and glucansucrase activity 2.5. Determination of glucansucrase activity L. citreum 1DSM 20188 (++), 1DSM 5577 (++), Glucansucrase activity was determined spectrophotometrically in 6 50018 (−) microtiter plate format with 3,5-dinitrosalicylic