Accepted Manuscript Title: Weissella confusa Cab3 Dextransucrase: Properties and in Vitro Synthesis of Dextran and Glucooligosaccharides Author: Shraddha Shukla Qiao Shi Ndegwa H. Maina Minna Juvonen MaijaTenkanen Arun Goyal PII: DOI: Reference:
S0144-8617(13)00988-0 http://dx.doi.org/doi:10.1016/j.carbpol.2013.09.087 CARP 8176
To appear in: Received date: Revised date: Accepted date:
7-8-2013 20-9-2013 25-9-2013
Please cite this article as: Shukla, S., Shi, Q., Maina, N. H., Juvonen, M., & Goyal, A., Weissella confusa Cab3 Dextransucrase: Properties and in Vitro Synthesis of Dextran and Glucooligosaccharides, Carbohydrate Polymers (2013), http://dx.doi.org/10.1016/j.carbpol.2013.09.087 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Weissella confusa Cab3 Dextransucrase: Properties and in Vitro Synthesis of Dextran and
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Glucooligosaccharides
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Shraddha Shukla1, Qiao Shi2, Ndegwa H. Maina2, Minna Juvonen2, MaijaTenkanen2.and
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Arun Goyal1*
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Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati 781 039, Assam, India 2
Department of Food and Environmental Sciences, P.O. Box 27, FI-00014 University of Helsinki, Finland
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*Corresponding author and address for correspondence:
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Dr. Arun Goyal
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Professor
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Department of Biotechnology,
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Indian Institute of Technology Guwahati Guwahati 781 039, Assam Email: [email protected] Tel. 361-258 2208 Fax: 361-258 2249
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Abstract Food-derived Weissella spp. have gained attention during recent years as efficient dextran
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producers. Weissella confusa Cab3 dextransucrase (WcCab3-DSR) was isolated applying PEG
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fractionation and used for in vitro synthesis of dextran and glucooligosaccharides. WcCab3-DSR
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had a molar mass of 178 kDa and was activated by Co2+ and Ca2+ ions. Glycerol and Tween 80
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enhanced enzyme stability, and its half-life at 30°C increased from 10 h to 74 h and 59 h,
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respectively. The 1H and
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presence of main chain α-(1→6) linkages with only 3.0% of α-(1→3) branching, of which some
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were elongated. An HPSEC analysis in DMSO revealed a high molecular weight of 1.8x107 g/mol.
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Glucooligosaccarides produced through the acceptor reaction with maltose, were analyzed with
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HPAEC-PAD and ESI-MS/MS. They were a homologous series of isomaltooligosaccharides with
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reducing end maltose units. To our knowledge, this is a first report on native W. confusa
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dextransucrase.
C NMR spectral analysis of the produced dextran confirmed the
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Keywords: Weissella confusa; dextransucrase; dextran; glucooligosaccharides
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Introduction Various lactic acid bacteria (LAB) among genera Leuconostoc, Lactobacillus,
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Streptococcus, Pediococcus, and Weissella produce extracellular α-glucooligosaccharides (GLOS)
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and polysaccharides catalyzed by glucansucrases (often also called glucosyltransferases, EC
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2.4.1.5) (Patel, Kothari, & Goyal, 2011; Leemhuis et al., 2013). The α-glucans differ in their
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glycosidic linkage composition and organization and are classified into dextrans, mutans, alternans,
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and reuterans (Leemhuis et al., 2013). The glucansucrases are named accordingly, based on the α-
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glucans they synthesize, such as dextransucrases (DSR) and alternansucrases (ASR). Because of
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the hydrocolloid properties and the potential health benefits of α-glucans, they are of high interest
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in food applications (Hernández, Livings, Aguilera, & Chiralt, 2011; Patel, Majumder, & Goyal,
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2012).
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Dextrans are α-glucans that contain consecutive α-(1→6) linkages in the main chain and α-
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(1→2), α-(1→3), or α-(1→4) branch linkages (Bounaix et al., 2009; Naessens, Cerdobbel,
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Soetaert, & Vandamme, 2005). The type and percentage of branch linkages depend on the
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dextransucrases elaborated by individual LAB. Dextransucrases catalyze dextran synthesis and are
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classified as part of the glycoside hydrolase (GH) family 70, which contains solely glucansucrases /
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glucosyltransferases. The GH70 enzymes are mechanistically, structurally, and evolutionary closely
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related to GH13 and GH77 enzymes (Stam, Danchin, Rancurel, Coutinho, & Henrissat, 2006).
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Although glucansucrases are classified as transferases based on their activity, structurally, they are
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close to glycoside hydrolases. Indeed, the first step in the synthesis is the hydrolysis of sucrose,
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from which the glucosyl residue is transferred to the growing α-glucan chain. The three-
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dimensional structure of truncated glucansucrases has recently become available. Based on the
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findings, it has been proposed that α-glucans are synthesized through the step-wise addition of
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glucose moieties to the non-reducing end of growing chains (Brison et al., 2012; Ito et al., 2011;
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Leemhuis et al., 2013; Vujicic-Zagar et al., 2010. When exogenous acceptor molecules are present
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beside sucrose, glucose moieties can also be transferred to them, and the resulting molecules may
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in turn act as acceptors, giving rise to acceptor products in addition to α-glucans. The product
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pattern of glucansucrase for a specific acceptor depends on enzyme specificity. Leuconostoc
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mesenteroides NRRL B-512F dextransucrase predominantly synthesizes α-(1→6) linkages. It
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produces homologous GLOS in the presence of maltose, with additional glucose moieties all α-
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(1→6) linked (Robyt & Eklund, 1983). Because of its α-(1→2)-branch synthesizing property, Lc.
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mesenteroides NRRL B-1299 dextransucrase synthesizes three homologous GLOS families with
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maltose, the major family, constituting linear α-(1→6) elongated GLOS. The second and third
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families have α-(1→6)-linked main chains, with a single unit α-(1→2) branch respectively at the
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terminal and the penultimate unit of the non-reducing end (Dols, Remaud-Simeon, Willemot,
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Vignon, & Monsan, 1997).
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The dextran-producing Weissella spp. have received significant attention in the recent years
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due to their relatively higher dextran production ability, as compared to other lactic acid bacteria,
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especially in sourdough bread applications (Di Cagno et al., 2006; Galle, Schwab, Arendt, &
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Gänzle, 2010; Galle et al., 2012; Katina et al., 2009; Schwab, Mastrangelo, Corsetti, & Gänzle,
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2008). The genus Weissella was proposed in 1993 after reclassification of some Leuconostoc-like
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bacteria (Collins, Samelis, Metaxopoulos, & Wallbanks, 1993). They are rod-shaped
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heterofermentative LAB from the family Leuconostocaceae. Dextran production is a phenotypic
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criterion for the identification of bacteria under this genus. Weissella strains have been isolated
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from various sources, such as sourdough breads and vegetables (Ahmed, Siddiquia, Armanb, &
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Ahmeda, 2012; Bounaix et al., 2010; Shukla & Goyal, 2011). Dextrans produced by Weissella
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strains have been reported to possess highly similar structures, consisting of predominantly α-
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(1→6) linkages and only a few α-(1→3) branch linkages (2.4–4%), with some of the branches
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elongated (Ahmed at al., 2012; Bejar et al., 2013; Bounaix et al., 2009; Maina, Tenkanen,
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Maaheimo, Juvonen, & Virkki, 2008; Maina, Virkki, Pynnönen, Maaheimo, & Tenkanen, 2011).
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They contain less branch linkages than the most commercially used Lc. mesenteroides NRRL B-
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512F dextran. In wheat sourdough, Weissella confusa VTT E-90392 synthesized a high yield of
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dextran, while Lc. mesenteroides NRRL B-512F produced almost none under the same conditions.
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In situ-produced W. confusa dextran improved the shelf life, volume, and softness of sourdough
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wheat bread. These positive effects were attributed to the low degree of branching, the high
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molecular weight of W. confusa dextran, and possibly to the GLOS produced concomitantly
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through acceptor reaction with maltose (Katina et al., 2009). In situ-produced exopolysaccharides
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by LAB are regarded as natural food thickeners from fermentation and could replace other added
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hydrocolloid additives, leading to clean label foods.
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Dextransucrases from Weissella spp., especially W. confusa, had not been studied until
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recently. Several dextran-producing W. cibaria and W. confusa strains exhibited constitutive
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dextransucrase activity, assigned to a 180 kDa protein (Bounaix et al., 2010). The effect of
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temperature, pH, and ionic strength on the activity of crude W. confusa Cab3 dextransucrase
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(WcCab3-DSR) has been reported (Shukla & Goyal, 2011). In 2012, the first complete gene
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sequence encoding dextransucrase from W. confusa strain (LBAE K39) became available, followed
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by the expression of the gene in E. coli and the characterization of the recombinant dextransucrase
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(Amari et al., 2012). The crude dextransucrase from pear-derived Weissella sp. TN610 has also
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been characterized. It induced the solidification of sucrose-supplemented milk and was suggested
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as a promising, safe food additive (Bejar et al., 2013).
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In the present study, the dextransucrase from the highly efficient dextran producer W.
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confusa Cab3, WcCab3-DSR, originating from fermented cabbage, was partially purified using
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polyethylene glycol partitioning and functionally characterized. The dextran produced by in vitro
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enzyme synthesis was characterized using FT-IR spectroscopy, NMR spectroscopy, and HPSEC.
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The GLOS produced by acceptor reaction with maltose were purified and analyzed by tandem mass
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spectrometry. To our knowledge, this was the first study characterizing a native W. confusa
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dextransucrase. Knowing the properties of W. confusa dextransucrase and its dextran and GLOS
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products enhances the usability of this potential dextran producer.
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2. Materials and methods
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2.1. Microorganism and culture conditions The bacterial strain Weissella confusa Cab3 (Genbank Accession Number JX649223)
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isolated from fermented cabbage (Shukla & Goyal, 2011) was used for the production of WcCab3-
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DSR. The microorganism was maintained in a modified MRS medium (Goyal & Katiyar, 1996) as
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stabs incubated at 25°C, stored at 4°C, and sub-cultured every two weeks. For the preparation of
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the inoculum of W. confusa Cab3, a loopful of culture from a modified MRS agar stab (Goyal &
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Katiyar, 1996) was transferred to a 5 ml medium as described by Tsuchiya et al., 1952. W. confusa
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Cab3 was incubated at 25C at 180 rpm for 12 h; 1% of the culture was again inoculated in a 2x100
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ml enzyme production medium in a 250 ml Erlenmeyer flask and incubated at 25°C for 12 h. The
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broth was centrifuged at 8000g at 4°C for 10 min and the cell-free supernatant containing WcCab3-
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DSR was subjected to purification.
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2.2. Enzyme purification
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The cell-free supernatant of W. confusa Cab3 was fractionated by polyethylene glycol
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according to the method established by Purama & Goyal, 2008. The ice-cold polyethylene glycols
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of two different molecular weights, i.e., PEG-400 (Qualigens Pvt. Ltd., India) and PEG-1500
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(BDH, U.K), were added to 30 ml of the cell-free supernatant. The PEG-400 concentration varied
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from 10 to 50% (v/v); 40% (w/v) of the PEG-1500 solution was prepared in distilled water and
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added to the 30 ml cell-free supernatant to get final concentrations from 5 to 25% (v/v). The
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mixtures were incubated at 4°C for 12 h to allow the dextransucrase (WcCab3-DSR) to fractionate,
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after which they were centrifuged at 12,000g for 30 min at 4°C to separate the WcCab3-DSR. The
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enzyme pellets were dissolved in a 20 mM sodium acetate buffer pH 5.4 and subjected to dialysis
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against the same buffer using a 14 kDa cut off membrane. The samples were analyzed for
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dextransucrase activity and protein content.
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2.3. Dextransucrase activity assay The activity assay of WcCab3-DSR was conducted in 1 ml of a reaction mixture containing
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20 mM sodium acetate buffer pH 5.4, 5% sucrose, and a 20 μl enzyme sample. The reaction
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mixture was incubated at 35C for 15 min. Aliquots (100 l) from the reaction mixture were
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analyzed for reducing sugars by the method desctibed by Nelson, 1944 and Somogyi, 1945. The
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absorbance was recorded at 500 nm in a UV-visible spectrometer (Varian, model Cary 100) against
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a blank. D-fructose (Hi-Media Pvt. Ltd., India) was used as a standard.
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2.4. Protein determination
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The protein content of the cell-free extract containing WcCab3-DSR and partially purified
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WcCab3-DSR were estimated by the method established by Lowry, Rosebrough, Farr, & Randall,
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1951, using bovine serum albumin as a standard.
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2.5. SDS-PAGE analysis
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SDS-polyacrylamide gel electrophoresis was performed with a vertical slab mini-gel unit
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(BioRad) using 1.5 mm thick gel, following the method established by Laemmli, 1970; 7.0% (w/v)
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acrylamide for resolving gel and 4% (w/v) for stacking gel were prepared. For running the enzyme
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samples (WcCab3-DSR) under non-denaturing conditions, the sample buffer (5x) used did not
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contain β-mercaptoethanol and the enzyme sample mixed with the sample buffer (62.5 mM Tris-
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HCl buffer pH 6.8, containing 2.0%, w/v sodium dodecyl sulfate, 20%, w/v glycerol, and 0.025%,
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w/v bromophenol blue), was not subjected to heat denaturation. The samples were loaded in
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duplicate on 7.0% acrylamide gel for non-denaturing SDS-PAGE. The electrophoresis was carried
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out using a running buffer (200 mM glycine, 0.1% SDS, 50 mM Tris-HCl pH 8.3) with a current of
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2 mA per lane. Following the run, the gel was cut into two parts. One part of the gel was subjected
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to silver staining and the other part of was analyzed for in situ activity detection by periodic acid
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(PA)-Schiff staining protocol (Miller, & Robyt, 1986a).
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The WcCab3-DSR isolated by 30%, w/v PEG-400 was also run under native condition on a
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7.0% gel and subsequently analyzed by periodic acid (PA)-Schiff staining protocol (Miller, & 7 Page 7 of 37
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Robyt, 1986a). For this the enzyme sample was prepared in 62.5 mM Tris-HCl buffer (pH 6.8),
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containing 20% (w/v) glycerol, and 0.025% (w/v) bromophenol blue. The electrophoresis was
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carried out using running buffer (200 mM glycine, 50 mM Tris-HCl pH 8.3) with a current of 2 mA
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per lane.
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2.6. Effects of divalent metals, additives and temperature on WcCab3-DSR The effects of different divalent cations using MgCl2, CaCl2, CoCl2, NiSO4, ZnCl2, MnCl2
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salt solutions (0 - 10 mM), and EDTA (0 to 7 mM) on WcCab3-DSR activity was evaluated. The
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assays were carried out in a 1.0 ml reaction mixture containing salt, WcCab3-DSR preparation
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(10.5 U/mg, 0.84 mg/ml), and a substrate as described earlier.
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The effects of various additives on the stability of WcCab3-DSR at 30°C were tested.
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Aqueous solutions of dextran (500 kDa) (Sigma Aldrich Pvt. Ltd., USA), PEG-8000 (Hi-Media
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Pvt. Ltd., India), glutaraldehyde (Qualigens Pvt. Ltd., India), glycerol (Qualigens Pvt. Ltd., India)
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and Tween 80 (Merck, India) were added to 1.0 ml WcCab3-DSR (10.5 U/mg, 0.84 mg/ml) to
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obtain final concentrations of 2 μg/ml (w/v) dextran (500 kDa), 10 μg/ml (w/v) PEG-8000, 0.1%
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(v/v) glutaraldehyde, 0.5% (v/v), glycerol and 0.1% (v/v) Tween 80, respectively. Storage stability
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was also studied by incubating the WcCab3-DSR (10.5 U/mg, 0.84 mg/ml) at various temperatures
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(0°C, 4°C, and –20°C). The aliquots (20 l) were taken periodically for residual enzyme activity
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assays as described earlier. The residual activity and half-life (t1/2) of WcCab3-DSR were measured
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at various temperatures (30°C, 4°C, and -20°C) with respect to time without any additives. The
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half-life (t1/2) of additives-treated WcCab3-DSR was determined only at 30°C. For determining the
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thermostability, the WcCab3-DSR preparation (10.5 U/mg, 0.84 mg/ml) was incubated at different
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temperatures (10–50°C) for 1 h and the aliquots (10–20 l) were assayed for residual enzyme
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activity.
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2.7. In vitro synthesis of dextran
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For WcCab3-DSR dextran synthesis, 24.0 ml of WcCab3-DSR (10.5 U/mg, 0.84 mg/ml) in
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200 ml of 5% (w/v) sucrose in a 20 mM sodium acetate buffer pH 5.4 containing 0.3 mM CaCl2 8 Page 8 of 37
and 15 mM sodium azide was incubated at 35°C for 24 h. The viscous solution (200 ml) containing
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the enzyme-synthesized WcCab3-DSR dextran was treated with three volumes of ethanol (90%,
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v/v) to precipitate the dextran. The solution was centrifuged at 12,000g at room temperature for 20
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min and the pellet was re-suspended in 500 ml of deionized water. The dextran was precipitated
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again and the same process was repeated two more times to ensure removal of sucrose and fructose
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residues. Finally, the precipitated dextran was dissolved in 500 ml of deionized water. The purified
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dextran solution was analyzed for protein content using bovine serum albumin as the standard
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(Lowry et al., 1951). The solution was then freeze-dried using a lyophilizer (Christ GmbH, model
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ALPHA 1-4 LD) at –51°C at a vacuum pressure of 35 x 10-3 mbar for 24 h. The sample was stored
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at room temperature for further characterization.
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2.8. Production of GLOS by acceptor-reaction
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The acceptor products (GLOS) were produced by WcCab3-DSR using maltose as the
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acceptor molecule following the method established by Dols et al., 1997. The reaction mixture (100
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ml) contained 10% (w/v) sucrose, 5% (w/v) maltose, and 12.0 ml of WcCab3-DSR (10.5 U/mg,
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0.84 mg/ml) in a 20 mM sodium acetate buffer pH 5.4. The reaction was allowed to proceed for 24
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h at 35°C. The enzyme action was terminated by keeping the reaction mixture in a boiling water
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bath for 10 min, after which, polymeric dextran was removed by ethanol precipitation with three
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volumes of pre-chilled ethanol (AltiaEtax A, Finland). After being centrifuged at 12,000 g for 10
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min, the supernatant containing fructose, residual sucrose, residual maltose, and GLOS was
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concentrated to 22 ml with a rotary vacuum evaporator (Heidolph LABOROTA digital 4001) and is
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referred to as the acceptor-product solution henceforth.
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GLOS were purified according to the degree of polymerization (DP) by gel permeation
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chromatography (GPC) using a Biogel P2 column (5 x 95 cm; Biorad, Hercules, USA) with water
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as the eluent. A 5 ml of 10-fold diluted acceptor-product solution was filtered with 0.45 µm
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membrane filters (Acrodisc 13, Pall Corporation, Ann Arbor, Michigan, USA) and injected into the
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column. The flow rate was first kept at 1.0 ml/min to elute void volume (600 ml) and adjusted to 9 Page 9 of 37
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0.3 ml/min for the collection of 4 ml fractions. The fractions were analyzed for oligosaccharides by
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HPAEC-PAD (section 2.10), and the selected fractions of isolated GLOS of varying degrees of
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polymerization (DP) were subsequently analyzed by tandem mass spectrometry (section 2.15).
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2.9. Estimation of total carbohydrate content The total carbohydrate content of the purified WcCab3-DSR dextran and acceptor-product
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solution was determined by the phenol-sulphuric acid method in a micro-titre plate (Dubois, Gilles,
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Hamilton, Rebers, & Smith, 1956; Fox & Robyt, 1991). To 25 μl of a sample in a microtitre plate,
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25 μl of 5% (v/v) phenol was added and mixed by shaking the plate on a vortex mixer for 30 s. The
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plate was placed on an ice bath, and 125 μl of concentrated sulphuric acid was added to each well
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containing the mixture. The plate was again shaken for 30 s to ensure proper mixing of the contents
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of the wells. The plate was wrapped in a cling film and incubated in a water bath at 80C for 30
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min. After cooling to room temperature, the absorbance was determined at 490 nm on a multimode
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microplate reader (Tecan, model InfiniteTM 200). Standard graphs using dextran T40 (Sigma
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Aldrich, USA) and glucose (Hi-Media Pvt. Ltd., India) in the concentration range 0.1–1.0 mg/ml
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were plotted for the estimation of the total carbohydrate content of WcCab3-DSR dextran and
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acceptor-product solution, respectively.
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2.10. Mono- and oligosaccharide analysis
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Monosaccharide analysis of WcCab3-DSR dextran was carried out by high performance
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anion exchange chromatography (HPAEC); 50 mg of lyophilized WcCab3-DSR dextran sample
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was hydrolyzed with1 ml of 50 mM sulfuric acid at 100oC for 6 h. The hydrolyzed dextran was
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neutralized by adding a 2 M Na2CO3 (100 µl) solution (filtered with a 0.2 µm membrane). The
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sample was analyzed by HPAEC-PAD using an ion-chrome system (ICS-3000 system, equipped
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with the ICS-3000 detector/chromatographic module, Dionex Corporation, USA) with the
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CarboPac PA-20 analytical column (3 x 150 mm) and the CarboPac PA-20 guard column (3 x 30
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mm). The temperature of the column was adjusted to 30°C and the injection volume was 25 μl. The
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eluent used was 250 mM NaOH at 0.2 ml/min. D-glucose and D-fructose (Hi-Media Pvt. Ltd.,
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India) were used as external standards. WcCab3-DSR dextran hydrolyzed with a mixture of enzymes (section 2.14) and acceptor-
260
product solution were analyzed by HPAEC-PAD-applying equipment and the method described by
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Rantanen, et al., 2007. The samples were filtered using 0.45 μm membrane filters (Acrodisc 13,
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Pall Corporation, Ann Arbor, USA), and the injection volume was 10 μl. D-glucose (Merck,
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Germany), D-fructose (Merck), sucrose (Merck), maltose (Sigma–Aldrich), isomaltose (TCI
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Europe, Belgium), isomaltotriose (TCI Europe), and panose (TCI Europe) were used as external
265
standards.
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2.11. Fourier-transform infrared spectrometric analysis
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Fourier transform infra-red (FT-IR) spectrum of WcCab3-DSR dextran was recorded in the
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frequency range of 4000–400 cm-1 (Perkin-Elmer Instruments, Spectrum One FT-IR Spectrometer).
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The dried sample was ground with potassium bromide powder and pressed into pellet for FT-IR
270
measurement.
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2.12. Nuclear magnetic resonance spectroscopy analysis
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Nuclear magnetic resonance (NMR) spectra of WcCab3-DSR dextran were recorded at 50°C
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with a 600 MHz NMR spectrometer (Bruker Avance III NMR spectrometer). The dextran (10
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mg/ml) was exchanged three times with D2O and placed in NMR tubes (Wilmad NMR tubes, 5
275
mm, ultra-imperial grade, from Aldrich chemical company, Milwaukee, WI, USA). The chemical
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shifts were referenced to internal acetone (1H = 2.225 ppm and 13C = 31.55 ppm).
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2.13. High performance size exclusion chromatography
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For the molar mass analysis, a 2.0 mg/ml solution of the WcCab3-DSR dextran was prepared
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in DMSO with 10 mM LiBr. The high-performance, size-exclusion chromatography (HPSEC)
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equipment comprised an integrated autosampler and pump module (GPCmax, Viscotek Corp.,
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Houston, TX, USA), a combined light-scattering and viscometric detector (270 Dual Detector,
282
Viscotek Corp.), and a refractive-index (RI) detector (VE 3580, Viscotek Corp.). The light11 Page 11 of 37
scattering detector angles were 7 and 90. Two LF-804 columns (8 x 300 mm, exclusion limit 2 x
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106, Showa Denko, Tokyo, Japan) with a LF-G (4.6 x 10 mm) guard column were used. Before
285
injection, the sample was filtered using a 0.45 μm membrane filter. The flow rate was 1 ml/min and
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the injection volume was 100 μl. The HPSEC data were processed with the OmniSEC 4.5 software
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(Viscotek Corp.) to determine the average molecular weight (Mw) of the WcCab3-DSR dextran. A
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d/dc value of 0.072 ml/g for dextran in DMSO-based eluent was used (Basedow & Ebert, 1979).
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2.14. Enzymatic hydrolysis of dextran and GLOS
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The in vitro-synthesized WcCab3-DSR dextran was treated by a mixture of Chaetomium
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erraticum dextranase (Sigma-Aldrich, Germany) and Aspergillus niger α-glucosidase (E-TRNGL,
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Megazyme, Ireland) as previously described by Maina et al., 2011. The acceptor-product solution
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was treated separately with dextranase (3.37 nkat/μl acceptor product solution) and Bacillus
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stearothermophilis α-glucosidase (0.42 nkat/μl acceptor product solution, E-TSAGL, Megazyme,
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Ireland). After boiling to inactivate the enzymes, the residual oligosaccharides were analysed with
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HPAEC-PAD (section 2.10).
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2.15. Mass spectrometry analysis
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Mass spectrometry was used to determine the molar masses of the GLOS obtained by
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WcCab3-DSR in the presence of sucrose and maltose. To check whether the GLOS-product
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mixture
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desorption/ionization with ion trap mass spectrometry (AP-MALDI-ITMS) was conducted using
302
the acceptor-product solution filtrated with 0.45 µm membrane filters and desalted with a strongly
303
acidic cation exchanger (Dowex® 50W, Fluka, Sigma-Aldrich, Switzerland). The sample was
304
diluted 500-fold with MilliQ-water before MS analysis, which was performed on an Agilent 6330
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ion trap mass spectrometer (Agilent Technologies, Waldbronn, Germany) equipped with an AP-
306
MALDI ion source (Mass Tech Inc. Columbia, MD, USA) in the positive ionization mode (sodium
307
adduct ions [M+Na]+) according to the method described by Chong et al., 2011 with modifications.
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The capillary voltage was set at 3000 V. The ion optics voltages were set as follows: the capillary
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exit, 300 V; the skimmer potential, 40 V; and the trap drive value, 70. Furthermore, the purified fractions containing DP 3, 4, and 5 were analyzed by electrospray
311
ionization-tandem mass spectrometry (ESI-MS/MS), as previously described (Maina, et al., 2013).
312
The analysis was carried out in the negative mode (chloride adduct ions [M+Cl]-) with the Agilent
313
XCT Plus Ion Trap mass spectrometer with an electrospray ion source (Agilent Technology, Palo
314
Alto, CA, USA).
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3. Results and Discussion
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3.1. Isolation of WcCab3-DSR
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The cell-free supernatant of Weissella confusa Cab3 (6.0 mg protein /ml) contained 6.0 U/ml
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crude WcCab3-DSR (Table 1). Because polyethylene glycol fractionation is known as a potential
320
method of isolating dextransucrases (Purama & Goyal, 2008), isolation of WcCab3-DSR applying
321
PEG-400 (20% to 45% v/v) and PEG-1500 (5% to 20% v/v) were tested (Table 1). The
322
concentration of 30% (v/v) PEG-400 gave the highest specific activity of 10.5 U/mg with 10-fold
323
purification and 25.5% overall yield (Fig.. 1S, Table 1); 15% (v/v) concentration of PEG-1500
324
resulted in a slightly higher specific activity of 10.9 U/mg, with 11-fold purification and 24%
325
overall yield (Fig. 1S, Table 1). WcCab3-DSR (10.5 U/mg, 0.84 mg/ml) isolated by fractionation
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with 30% (v/v) PEG-400 was selected for further experiments, because PEG-400 is more
327
economical to use than PEG-1500.
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WcCab3-DSR obtained after fractionation using 30% PEG-400 and 15% PEG-1500 were
329
analyzed in a single SDS-PAGE gel in duplicate run under a non-denaturing condition (Fig. 1).
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WcCab3-DSR showed a single band of about 178 kDa molecular mass on the non-denaturing gel
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stained for proteins (Fig. 1, lanes 1 and 2). The other part of the gel was subjected to activity
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staining for in situ activity detection. The presence of magenta color bands after PAS staining (Fig.
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1, lanes 3 and 4) were consistent with the protein staining of the dextransucrase bands. The 13 Page 13 of 37
appearance of some minor faint bands in the activity stained gel (Fig. 1, lanes 3 and 4) might be
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due to the slight degradation of WcCab3-DSR under the non-denaturing SDS condition applied
336
during their run. Furthermore, to confirm this, WcCab3-DSR isolated by PEG-400 (30%, w/v) was
337
also run under native condition and analysed by PAS staining (Fig. 1, lanes 5). The presence of a
338
single activity band on the native-PAGE, confirmed that the WcCab3-DSR exist as a single
339
enzyme.
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3.2. Effect of temperature and additives on WcCab3-DSR
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WcCab3-DSR exhibited a mesophilic nature and showed stability up to 40°C, above which
342
it rapidly lost activity during 1 h incubation (Fig. 2A). The optimum temperature range for the
343
assay of WcCab3-DSR was also in the range of 30–35°C (Shukla & Goyal, 2011). Thus, WcCab3-
344
DSR showed a more thermostable nature as compared with the dextransucrase from Lc.
345
mesenteroides NRRL B-640, which rapidly lost its activity above 30°C after 10 min of incubation
346
(Purama, Agrawal, & Goyal, 2010). Dextranscrase from W. cibaria JAG8 was also stable up to
347
35°C, but further increase in temperature to 40°C caused the enzyme to lose 96% of its activity
348
(Tinirikari & Goyal, 2013).
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Certain metal ions show positive effects on the activity of enzymes. Salts are believed to
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affect the structure of enzymes, thus affecting their solubility and activity (Forman & Kennedy,
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1977). The effects of selected metal ions were studied on WcCab3-DSR activity. The Co2+ and
352
Ca2+ ions increased the enzyme activity of WcCab3-DSR (Table 2). The enzyme activity was
353
enhanced by 20% and 21% in the presence of 1 mM CoCl2 and CaCl2, respectively (Table 2). Ca2+
354
ion has been reported to be associated with the catalytic sites of dextransucrases (Miller & Robyt,
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1986b). CaCl2 (6 mM) increased the activity for Pediococcus pentosaceus dextransucrase by 150%
356
and 4 mM CaCl2 by 108% for Lc. mesenteroides NRRL B-640 dextransucrase (Patel et al., 2011;
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Purama, et al., 2010). The addition of ZnCl2 (1 mM), MgCl2 (2 mM), and NiSO4 (2 mM) resulted in
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9%, 8%, and 7% increase in enzyme activity, respectively (Table 2). On the other hand, MnCl2
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affected enzyme activity negatively. At lower 1 mM concentration, MnCl2 decreased the activity by
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4%, and at 10 mM MnCl2, the enzyme activity was lost by 42% of its initial value (Table 2). In the
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presence of 5 mM EDTA, WcCab3-DSR displayed only 2% residual activity (Table 2). The effects of different additives on the stability of WcCab3-DSR were studied at 30°C.
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The residual activity of WcCab3-DSR after 24 h with glycerol, Tween 80, and PEG 8000 were
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80%, 76%, and 37.0%, respectively (Fig. 2B). The dextran (500 kDa) and glutaraldehyde did not
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stabilize the enzyme, because the residual activity with dextran (500 kDa) and glutaraldehyde after
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24 h was only 25% and 16%, respectively (Fig. 2B). The residual activity of WcCab3-DSR with
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glutaraldehyde at 30°C after 24 h was even lower than that of WcCab3-DSR without any additive,
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which was 20%. Thus, glutaraldehyde negatively affected WcCab3-DSR. The storage stability of
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WcCab3-DSR was also followed at 4°C and –20°C. WcCab3-DSR lost only 1.5% and 4% of the
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activity after 5 days of incubation at 4°C and –20°C, respectively (Fig. 2C).
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The half-life times (t1/2) of WcCab3-DSR and additives-treated WcCab3-DSR were
372
calculated by assuming that the decay followed first-order kinetics (Table 2). Among all the
373
additives used, glycerol and Tween 80 were the most efficient stabilizers, resulting in t1/2 of 74.1 h
374
and 59.3 h at 30°C, respectively (Table 2). A small enhancement in the t1/2 (16.7 h) was observed
375
using PEG 8000 at 30°C as compared with the control (10.3 h). The half-life (t1/2) of the enzyme
376
increased from 10.3 h to 69.4 d by decreasing the storage temperature from 30°C to 4°C. The
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WcCab3-DSR was also quite stable at –20°C with a half-life (t1/2) of 37.9 d.
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3.3 Characterization of synthesized dextran
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The in vitro-synthesized WcCab3-DSR dextran was isolated by ethanol precipitation and
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lyophilized for further structural and functional characterization. The total carbohydrate and protein
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content of the purified WcCab3-dextran was 98.27% (w/w) and 1.73% (w/w) (7.5 mg/ml and 0.13
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mg/ml), respectively. HPAEC-PAD analysis after acid hydrolysis proved that the purified sample
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contained no carbohydrate other than glucose.
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Enzyme-aided structure analysis An enzyme-aided structure analysis of dextrans is a simple and quick method for determining
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the structural variation in dextrans produced by different strains or in different conditions (Katina et
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al., 2009). The method involves specific enzymatic hydrolysis of dextran with a mixture of
390
dextranase and α-glucosidase that converts the dextran to glucose and a set of enzyme-resistant
391
oligosaccharides. The chromatographic profiles of the enzyme-resistant oligosaccharides vary
392
depending on the structural components of dextran, and can therefore be used as structural markers
393
(Maina, 2012). An HPAEC-PAD analysis showed that the enzyme-resistant oligosaccharides from
394
WcCab3-DSR dextran (Fig. 4A) had a similar profile to the oligosaccharides as obtained earlier
395
from W. confusa VTT E-90392 and Lc. mesenteroides NRRL B-512F dextran (Katina et al., 2009),
396
indicating high similarities in their structural features. Dextrans from these strains have few (
S0144-8617(13)00988-0 http://dx.doi.org/doi:10.1016/j.carbpol.2013.09.087 CARP 8176
To appear in: Received date: Revised date: Accepted date:
7-8-2013 20-9-2013 25-9-2013
Please cite this article as: Shukla, S., Shi, Q., Maina, N. H., Juvonen, M., & Goyal, A., Weissella confusa Cab3 Dextransucrase: Properties and in Vitro Synthesis of Dextran and Glucooligosaccharides, Carbohydrate Polymers (2013), http://dx.doi.org/10.1016/j.carbpol.2013.09.087 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Weissella confusa Cab3 Dextransucrase: Properties and in Vitro Synthesis of Dextran and
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Glucooligosaccharides
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Shraddha Shukla1, Qiao Shi2, Ndegwa H. Maina2, Minna Juvonen2, MaijaTenkanen2.and
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Arun Goyal1*
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Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati 781 039, Assam, India 2
Department of Food and Environmental Sciences, P.O. Box 27, FI-00014 University of Helsinki, Finland
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*Corresponding author and address for correspondence:
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Dr. Arun Goyal
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Professor
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Department of Biotechnology,
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Indian Institute of Technology Guwahati Guwahati 781 039, Assam Email: [email protected] Tel. 361-258 2208 Fax: 361-258 2249
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Abstract Food-derived Weissella spp. have gained attention during recent years as efficient dextran
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producers. Weissella confusa Cab3 dextransucrase (WcCab3-DSR) was isolated applying PEG
33
fractionation and used for in vitro synthesis of dextran and glucooligosaccharides. WcCab3-DSR
34
had a molar mass of 178 kDa and was activated by Co2+ and Ca2+ ions. Glycerol and Tween 80
35
enhanced enzyme stability, and its half-life at 30°C increased from 10 h to 74 h and 59 h,
36
respectively. The 1H and
37
presence of main chain α-(1→6) linkages with only 3.0% of α-(1→3) branching, of which some
38
were elongated. An HPSEC analysis in DMSO revealed a high molecular weight of 1.8x107 g/mol.
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Glucooligosaccarides produced through the acceptor reaction with maltose, were analyzed with
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HPAEC-PAD and ESI-MS/MS. They were a homologous series of isomaltooligosaccharides with
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reducing end maltose units. To our knowledge, this is a first report on native W. confusa
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dextransucrase.
C NMR spectral analysis of the produced dextran confirmed the
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Keywords: Weissella confusa; dextransucrase; dextran; glucooligosaccharides
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Introduction Various lactic acid bacteria (LAB) among genera Leuconostoc, Lactobacillus,
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Streptococcus, Pediococcus, and Weissella produce extracellular α-glucooligosaccharides (GLOS)
53
and polysaccharides catalyzed by glucansucrases (often also called glucosyltransferases, EC
54
2.4.1.5) (Patel, Kothari, & Goyal, 2011; Leemhuis et al., 2013). The α-glucans differ in their
55
glycosidic linkage composition and organization and are classified into dextrans, mutans, alternans,
56
and reuterans (Leemhuis et al., 2013). The glucansucrases are named accordingly, based on the α-
57
glucans they synthesize, such as dextransucrases (DSR) and alternansucrases (ASR). Because of
58
the hydrocolloid properties and the potential health benefits of α-glucans, they are of high interest
59
in food applications (Hernández, Livings, Aguilera, & Chiralt, 2011; Patel, Majumder, & Goyal,
60
2012).
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Dextrans are α-glucans that contain consecutive α-(1→6) linkages in the main chain and α-
62
(1→2), α-(1→3), or α-(1→4) branch linkages (Bounaix et al., 2009; Naessens, Cerdobbel,
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Soetaert, & Vandamme, 2005). The type and percentage of branch linkages depend on the
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dextransucrases elaborated by individual LAB. Dextransucrases catalyze dextran synthesis and are
65
classified as part of the glycoside hydrolase (GH) family 70, which contains solely glucansucrases /
66
glucosyltransferases. The GH70 enzymes are mechanistically, structurally, and evolutionary closely
67
related to GH13 and GH77 enzymes (Stam, Danchin, Rancurel, Coutinho, & Henrissat, 2006).
68
Although glucansucrases are classified as transferases based on their activity, structurally, they are
69
close to glycoside hydrolases. Indeed, the first step in the synthesis is the hydrolysis of sucrose,
70
from which the glucosyl residue is transferred to the growing α-glucan chain. The three-
71
dimensional structure of truncated glucansucrases has recently become available. Based on the
72
findings, it has been proposed that α-glucans are synthesized through the step-wise addition of
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glucose moieties to the non-reducing end of growing chains (Brison et al., 2012; Ito et al., 2011;
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Leemhuis et al., 2013; Vujicic-Zagar et al., 2010. When exogenous acceptor molecules are present
75
beside sucrose, glucose moieties can also be transferred to them, and the resulting molecules may
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in turn act as acceptors, giving rise to acceptor products in addition to α-glucans. The product
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pattern of glucansucrase for a specific acceptor depends on enzyme specificity. Leuconostoc
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mesenteroides NRRL B-512F dextransucrase predominantly synthesizes α-(1→6) linkages. It
79
produces homologous GLOS in the presence of maltose, with additional glucose moieties all α-
80
(1→6) linked (Robyt & Eklund, 1983). Because of its α-(1→2)-branch synthesizing property, Lc.
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mesenteroides NRRL B-1299 dextransucrase synthesizes three homologous GLOS families with
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maltose, the major family, constituting linear α-(1→6) elongated GLOS. The second and third
83
families have α-(1→6)-linked main chains, with a single unit α-(1→2) branch respectively at the
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terminal and the penultimate unit of the non-reducing end (Dols, Remaud-Simeon, Willemot,
85
Vignon, & Monsan, 1997).
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The dextran-producing Weissella spp. have received significant attention in the recent years
87
due to their relatively higher dextran production ability, as compared to other lactic acid bacteria,
88
especially in sourdough bread applications (Di Cagno et al., 2006; Galle, Schwab, Arendt, &
89
Gänzle, 2010; Galle et al., 2012; Katina et al., 2009; Schwab, Mastrangelo, Corsetti, & Gänzle,
90
2008). The genus Weissella was proposed in 1993 after reclassification of some Leuconostoc-like
91
bacteria (Collins, Samelis, Metaxopoulos, & Wallbanks, 1993). They are rod-shaped
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heterofermentative LAB from the family Leuconostocaceae. Dextran production is a phenotypic
93
criterion for the identification of bacteria under this genus. Weissella strains have been isolated
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from various sources, such as sourdough breads and vegetables (Ahmed, Siddiquia, Armanb, &
95
Ahmeda, 2012; Bounaix et al., 2010; Shukla & Goyal, 2011). Dextrans produced by Weissella
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strains have been reported to possess highly similar structures, consisting of predominantly α-
97
(1→6) linkages and only a few α-(1→3) branch linkages (2.4–4%), with some of the branches
98
elongated (Ahmed at al., 2012; Bejar et al., 2013; Bounaix et al., 2009; Maina, Tenkanen,
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Maaheimo, Juvonen, & Virkki, 2008; Maina, Virkki, Pynnönen, Maaheimo, & Tenkanen, 2011).
100
They contain less branch linkages than the most commercially used Lc. mesenteroides NRRL B-
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512F dextran. In wheat sourdough, Weissella confusa VTT E-90392 synthesized a high yield of
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dextran, while Lc. mesenteroides NRRL B-512F produced almost none under the same conditions.
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In situ-produced W. confusa dextran improved the shelf life, volume, and softness of sourdough
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wheat bread. These positive effects were attributed to the low degree of branching, the high
105
molecular weight of W. confusa dextran, and possibly to the GLOS produced concomitantly
106
through acceptor reaction with maltose (Katina et al., 2009). In situ-produced exopolysaccharides
107
by LAB are regarded as natural food thickeners from fermentation and could replace other added
108
hydrocolloid additives, leading to clean label foods.
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Dextransucrases from Weissella spp., especially W. confusa, had not been studied until
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recently. Several dextran-producing W. cibaria and W. confusa strains exhibited constitutive
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dextransucrase activity, assigned to a 180 kDa protein (Bounaix et al., 2010). The effect of
112
temperature, pH, and ionic strength on the activity of crude W. confusa Cab3 dextransucrase
113
(WcCab3-DSR) has been reported (Shukla & Goyal, 2011). In 2012, the first complete gene
114
sequence encoding dextransucrase from W. confusa strain (LBAE K39) became available, followed
115
by the expression of the gene in E. coli and the characterization of the recombinant dextransucrase
116
(Amari et al., 2012). The crude dextransucrase from pear-derived Weissella sp. TN610 has also
117
been characterized. It induced the solidification of sucrose-supplemented milk and was suggested
118
as a promising, safe food additive (Bejar et al., 2013).
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In the present study, the dextransucrase from the highly efficient dextran producer W.
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confusa Cab3, WcCab3-DSR, originating from fermented cabbage, was partially purified using
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polyethylene glycol partitioning and functionally characterized. The dextran produced by in vitro
122
enzyme synthesis was characterized using FT-IR spectroscopy, NMR spectroscopy, and HPSEC.
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The GLOS produced by acceptor reaction with maltose were purified and analyzed by tandem mass
124
spectrometry. To our knowledge, this was the first study characterizing a native W. confusa
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dextransucrase. Knowing the properties of W. confusa dextransucrase and its dextran and GLOS
126
products enhances the usability of this potential dextran producer.
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2. Materials and methods
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2.1. Microorganism and culture conditions The bacterial strain Weissella confusa Cab3 (Genbank Accession Number JX649223)
131
isolated from fermented cabbage (Shukla & Goyal, 2011) was used for the production of WcCab3-
132
DSR. The microorganism was maintained in a modified MRS medium (Goyal & Katiyar, 1996) as
133
stabs incubated at 25°C, stored at 4°C, and sub-cultured every two weeks. For the preparation of
134
the inoculum of W. confusa Cab3, a loopful of culture from a modified MRS agar stab (Goyal &
135
Katiyar, 1996) was transferred to a 5 ml medium as described by Tsuchiya et al., 1952. W. confusa
136
Cab3 was incubated at 25C at 180 rpm for 12 h; 1% of the culture was again inoculated in a 2x100
137
ml enzyme production medium in a 250 ml Erlenmeyer flask and incubated at 25°C for 12 h. The
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broth was centrifuged at 8000g at 4°C for 10 min and the cell-free supernatant containing WcCab3-
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DSR was subjected to purification.
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2.2. Enzyme purification
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The cell-free supernatant of W. confusa Cab3 was fractionated by polyethylene glycol
142
according to the method established by Purama & Goyal, 2008. The ice-cold polyethylene glycols
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of two different molecular weights, i.e., PEG-400 (Qualigens Pvt. Ltd., India) and PEG-1500
144
(BDH, U.K), were added to 30 ml of the cell-free supernatant. The PEG-400 concentration varied
145
from 10 to 50% (v/v); 40% (w/v) of the PEG-1500 solution was prepared in distilled water and
146
added to the 30 ml cell-free supernatant to get final concentrations from 5 to 25% (v/v). The
147
mixtures were incubated at 4°C for 12 h to allow the dextransucrase (WcCab3-DSR) to fractionate,
148
after which they were centrifuged at 12,000g for 30 min at 4°C to separate the WcCab3-DSR. The
149
enzyme pellets were dissolved in a 20 mM sodium acetate buffer pH 5.4 and subjected to dialysis
150
against the same buffer using a 14 kDa cut off membrane. The samples were analyzed for
151
dextransucrase activity and protein content.
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2.3. Dextransucrase activity assay The activity assay of WcCab3-DSR was conducted in 1 ml of a reaction mixture containing
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20 mM sodium acetate buffer pH 5.4, 5% sucrose, and a 20 μl enzyme sample. The reaction
157
mixture was incubated at 35C for 15 min. Aliquots (100 l) from the reaction mixture were
158
analyzed for reducing sugars by the method desctibed by Nelson, 1944 and Somogyi, 1945. The
159
absorbance was recorded at 500 nm in a UV-visible spectrometer (Varian, model Cary 100) against
160
a blank. D-fructose (Hi-Media Pvt. Ltd., India) was used as a standard.
161
2.4. Protein determination
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The protein content of the cell-free extract containing WcCab3-DSR and partially purified
163
WcCab3-DSR were estimated by the method established by Lowry, Rosebrough, Farr, & Randall,
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1951, using bovine serum albumin as a standard.
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2.5. SDS-PAGE analysis
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SDS-polyacrylamide gel electrophoresis was performed with a vertical slab mini-gel unit
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(BioRad) using 1.5 mm thick gel, following the method established by Laemmli, 1970; 7.0% (w/v)
168
acrylamide for resolving gel and 4% (w/v) for stacking gel were prepared. For running the enzyme
169
samples (WcCab3-DSR) under non-denaturing conditions, the sample buffer (5x) used did not
170
contain β-mercaptoethanol and the enzyme sample mixed with the sample buffer (62.5 mM Tris-
171
HCl buffer pH 6.8, containing 2.0%, w/v sodium dodecyl sulfate, 20%, w/v glycerol, and 0.025%,
172
w/v bromophenol blue), was not subjected to heat denaturation. The samples were loaded in
173
duplicate on 7.0% acrylamide gel for non-denaturing SDS-PAGE. The electrophoresis was carried
174
out using a running buffer (200 mM glycine, 0.1% SDS, 50 mM Tris-HCl pH 8.3) with a current of
175
2 mA per lane. Following the run, the gel was cut into two parts. One part of the gel was subjected
176
to silver staining and the other part of was analyzed for in situ activity detection by periodic acid
177
(PA)-Schiff staining protocol (Miller, & Robyt, 1986a).
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The WcCab3-DSR isolated by 30%, w/v PEG-400 was also run under native condition on a
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7.0% gel and subsequently analyzed by periodic acid (PA)-Schiff staining protocol (Miller, & 7 Page 7 of 37
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Robyt, 1986a). For this the enzyme sample was prepared in 62.5 mM Tris-HCl buffer (pH 6.8),
181
containing 20% (w/v) glycerol, and 0.025% (w/v) bromophenol blue. The electrophoresis was
182
carried out using running buffer (200 mM glycine, 50 mM Tris-HCl pH 8.3) with a current of 2 mA
183
per lane.
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2.6. Effects of divalent metals, additives and temperature on WcCab3-DSR The effects of different divalent cations using MgCl2, CaCl2, CoCl2, NiSO4, ZnCl2, MnCl2
186
salt solutions (0 - 10 mM), and EDTA (0 to 7 mM) on WcCab3-DSR activity was evaluated. The
187
assays were carried out in a 1.0 ml reaction mixture containing salt, WcCab3-DSR preparation
188
(10.5 U/mg, 0.84 mg/ml), and a substrate as described earlier.
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The effects of various additives on the stability of WcCab3-DSR at 30°C were tested.
190
Aqueous solutions of dextran (500 kDa) (Sigma Aldrich Pvt. Ltd., USA), PEG-8000 (Hi-Media
191
Pvt. Ltd., India), glutaraldehyde (Qualigens Pvt. Ltd., India), glycerol (Qualigens Pvt. Ltd., India)
192
and Tween 80 (Merck, India) were added to 1.0 ml WcCab3-DSR (10.5 U/mg, 0.84 mg/ml) to
193
obtain final concentrations of 2 μg/ml (w/v) dextran (500 kDa), 10 μg/ml (w/v) PEG-8000, 0.1%
194
(v/v) glutaraldehyde, 0.5% (v/v), glycerol and 0.1% (v/v) Tween 80, respectively. Storage stability
195
was also studied by incubating the WcCab3-DSR (10.5 U/mg, 0.84 mg/ml) at various temperatures
196
(0°C, 4°C, and –20°C). The aliquots (20 l) were taken periodically for residual enzyme activity
197
assays as described earlier. The residual activity and half-life (t1/2) of WcCab3-DSR were measured
198
at various temperatures (30°C, 4°C, and -20°C) with respect to time without any additives. The
199
half-life (t1/2) of additives-treated WcCab3-DSR was determined only at 30°C. For determining the
200
thermostability, the WcCab3-DSR preparation (10.5 U/mg, 0.84 mg/ml) was incubated at different
201
temperatures (10–50°C) for 1 h and the aliquots (10–20 l) were assayed for residual enzyme
202
activity.
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2.7. In vitro synthesis of dextran
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For WcCab3-DSR dextran synthesis, 24.0 ml of WcCab3-DSR (10.5 U/mg, 0.84 mg/ml) in
205
200 ml of 5% (w/v) sucrose in a 20 mM sodium acetate buffer pH 5.4 containing 0.3 mM CaCl2 8 Page 8 of 37
and 15 mM sodium azide was incubated at 35°C for 24 h. The viscous solution (200 ml) containing
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the enzyme-synthesized WcCab3-DSR dextran was treated with three volumes of ethanol (90%,
208
v/v) to precipitate the dextran. The solution was centrifuged at 12,000g at room temperature for 20
209
min and the pellet was re-suspended in 500 ml of deionized water. The dextran was precipitated
210
again and the same process was repeated two more times to ensure removal of sucrose and fructose
211
residues. Finally, the precipitated dextran was dissolved in 500 ml of deionized water. The purified
212
dextran solution was analyzed for protein content using bovine serum albumin as the standard
213
(Lowry et al., 1951). The solution was then freeze-dried using a lyophilizer (Christ GmbH, model
214
ALPHA 1-4 LD) at –51°C at a vacuum pressure of 35 x 10-3 mbar for 24 h. The sample was stored
215
at room temperature for further characterization.
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2.8. Production of GLOS by acceptor-reaction
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The acceptor products (GLOS) were produced by WcCab3-DSR using maltose as the
218
acceptor molecule following the method established by Dols et al., 1997. The reaction mixture (100
219
ml) contained 10% (w/v) sucrose, 5% (w/v) maltose, and 12.0 ml of WcCab3-DSR (10.5 U/mg,
220
0.84 mg/ml) in a 20 mM sodium acetate buffer pH 5.4. The reaction was allowed to proceed for 24
221
h at 35°C. The enzyme action was terminated by keeping the reaction mixture in a boiling water
222
bath for 10 min, after which, polymeric dextran was removed by ethanol precipitation with three
223
volumes of pre-chilled ethanol (AltiaEtax A, Finland). After being centrifuged at 12,000 g for 10
224
min, the supernatant containing fructose, residual sucrose, residual maltose, and GLOS was
225
concentrated to 22 ml with a rotary vacuum evaporator (Heidolph LABOROTA digital 4001) and is
226
referred to as the acceptor-product solution henceforth.
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GLOS were purified according to the degree of polymerization (DP) by gel permeation
228
chromatography (GPC) using a Biogel P2 column (5 x 95 cm; Biorad, Hercules, USA) with water
229
as the eluent. A 5 ml of 10-fold diluted acceptor-product solution was filtered with 0.45 µm
230
membrane filters (Acrodisc 13, Pall Corporation, Ann Arbor, Michigan, USA) and injected into the
231
column. The flow rate was first kept at 1.0 ml/min to elute void volume (600 ml) and adjusted to 9 Page 9 of 37
232
0.3 ml/min for the collection of 4 ml fractions. The fractions were analyzed for oligosaccharides by
233
HPAEC-PAD (section 2.10), and the selected fractions of isolated GLOS of varying degrees of
234
polymerization (DP) were subsequently analyzed by tandem mass spectrometry (section 2.15).
235
2.9. Estimation of total carbohydrate content The total carbohydrate content of the purified WcCab3-DSR dextran and acceptor-product
237
solution was determined by the phenol-sulphuric acid method in a micro-titre plate (Dubois, Gilles,
238
Hamilton, Rebers, & Smith, 1956; Fox & Robyt, 1991). To 25 μl of a sample in a microtitre plate,
239
25 μl of 5% (v/v) phenol was added and mixed by shaking the plate on a vortex mixer for 30 s. The
240
plate was placed on an ice bath, and 125 μl of concentrated sulphuric acid was added to each well
241
containing the mixture. The plate was again shaken for 30 s to ensure proper mixing of the contents
242
of the wells. The plate was wrapped in a cling film and incubated in a water bath at 80C for 30
243
min. After cooling to room temperature, the absorbance was determined at 490 nm on a multimode
244
microplate reader (Tecan, model InfiniteTM 200). Standard graphs using dextran T40 (Sigma
245
Aldrich, USA) and glucose (Hi-Media Pvt. Ltd., India) in the concentration range 0.1–1.0 mg/ml
246
were plotted for the estimation of the total carbohydrate content of WcCab3-DSR dextran and
247
acceptor-product solution, respectively.
248
2.10. Mono- and oligosaccharide analysis
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Monosaccharide analysis of WcCab3-DSR dextran was carried out by high performance
250
anion exchange chromatography (HPAEC); 50 mg of lyophilized WcCab3-DSR dextran sample
251
was hydrolyzed with1 ml of 50 mM sulfuric acid at 100oC for 6 h. The hydrolyzed dextran was
252
neutralized by adding a 2 M Na2CO3 (100 µl) solution (filtered with a 0.2 µm membrane). The
253
sample was analyzed by HPAEC-PAD using an ion-chrome system (ICS-3000 system, equipped
254
with the ICS-3000 detector/chromatographic module, Dionex Corporation, USA) with the
255
CarboPac PA-20 analytical column (3 x 150 mm) and the CarboPac PA-20 guard column (3 x 30
256
mm). The temperature of the column was adjusted to 30°C and the injection volume was 25 μl. The
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eluent used was 250 mM NaOH at 0.2 ml/min. D-glucose and D-fructose (Hi-Media Pvt. Ltd.,
258
India) were used as external standards. WcCab3-DSR dextran hydrolyzed with a mixture of enzymes (section 2.14) and acceptor-
260
product solution were analyzed by HPAEC-PAD-applying equipment and the method described by
261
Rantanen, et al., 2007. The samples were filtered using 0.45 μm membrane filters (Acrodisc 13,
262
Pall Corporation, Ann Arbor, USA), and the injection volume was 10 μl. D-glucose (Merck,
263
Germany), D-fructose (Merck), sucrose (Merck), maltose (Sigma–Aldrich), isomaltose (TCI
264
Europe, Belgium), isomaltotriose (TCI Europe), and panose (TCI Europe) were used as external
265
standards.
266
2.11. Fourier-transform infrared spectrometric analysis
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Fourier transform infra-red (FT-IR) spectrum of WcCab3-DSR dextran was recorded in the
268
frequency range of 4000–400 cm-1 (Perkin-Elmer Instruments, Spectrum One FT-IR Spectrometer).
269
The dried sample was ground with potassium bromide powder and pressed into pellet for FT-IR
270
measurement.
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2.12. Nuclear magnetic resonance spectroscopy analysis
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Nuclear magnetic resonance (NMR) spectra of WcCab3-DSR dextran were recorded at 50°C
273
with a 600 MHz NMR spectrometer (Bruker Avance III NMR spectrometer). The dextran (10
274
mg/ml) was exchanged three times with D2O and placed in NMR tubes (Wilmad NMR tubes, 5
275
mm, ultra-imperial grade, from Aldrich chemical company, Milwaukee, WI, USA). The chemical
276
shifts were referenced to internal acetone (1H = 2.225 ppm and 13C = 31.55 ppm).
277
2.13. High performance size exclusion chromatography
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For the molar mass analysis, a 2.0 mg/ml solution of the WcCab3-DSR dextran was prepared
279
in DMSO with 10 mM LiBr. The high-performance, size-exclusion chromatography (HPSEC)
280
equipment comprised an integrated autosampler and pump module (GPCmax, Viscotek Corp.,
281
Houston, TX, USA), a combined light-scattering and viscometric detector (270 Dual Detector,
282
Viscotek Corp.), and a refractive-index (RI) detector (VE 3580, Viscotek Corp.). The light11 Page 11 of 37
scattering detector angles were 7 and 90. Two LF-804 columns (8 x 300 mm, exclusion limit 2 x
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106, Showa Denko, Tokyo, Japan) with a LF-G (4.6 x 10 mm) guard column were used. Before
285
injection, the sample was filtered using a 0.45 μm membrane filter. The flow rate was 1 ml/min and
286
the injection volume was 100 μl. The HPSEC data were processed with the OmniSEC 4.5 software
287
(Viscotek Corp.) to determine the average molecular weight (Mw) of the WcCab3-DSR dextran. A
288
d/dc value of 0.072 ml/g for dextran in DMSO-based eluent was used (Basedow & Ebert, 1979).
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2.14. Enzymatic hydrolysis of dextran and GLOS
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The in vitro-synthesized WcCab3-DSR dextran was treated by a mixture of Chaetomium
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erraticum dextranase (Sigma-Aldrich, Germany) and Aspergillus niger α-glucosidase (E-TRNGL,
292
Megazyme, Ireland) as previously described by Maina et al., 2011. The acceptor-product solution
293
was treated separately with dextranase (3.37 nkat/μl acceptor product solution) and Bacillus
294
stearothermophilis α-glucosidase (0.42 nkat/μl acceptor product solution, E-TSAGL, Megazyme,
295
Ireland). After boiling to inactivate the enzymes, the residual oligosaccharides were analysed with
296
HPAEC-PAD (section 2.10).
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2.15. Mass spectrometry analysis
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Mass spectrometry was used to determine the molar masses of the GLOS obtained by
299
WcCab3-DSR in the presence of sucrose and maltose. To check whether the GLOS-product
300
mixture
301
desorption/ionization with ion trap mass spectrometry (AP-MALDI-ITMS) was conducted using
302
the acceptor-product solution filtrated with 0.45 µm membrane filters and desalted with a strongly
303
acidic cation exchanger (Dowex® 50W, Fluka, Sigma-Aldrich, Switzerland). The sample was
304
diluted 500-fold with MilliQ-water before MS analysis, which was performed on an Agilent 6330
305
ion trap mass spectrometer (Agilent Technologies, Waldbronn, Germany) equipped with an AP-
306
MALDI ion source (Mass Tech Inc. Columbia, MD, USA) in the positive ionization mode (sodium
307
adduct ions [M+Na]+) according to the method described by Chong et al., 2011 with modifications.
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was
homologous
in
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atmospheric
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matrix-assisted
laser
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The capillary voltage was set at 3000 V. The ion optics voltages were set as follows: the capillary
309
exit, 300 V; the skimmer potential, 40 V; and the trap drive value, 70. Furthermore, the purified fractions containing DP 3, 4, and 5 were analyzed by electrospray
311
ionization-tandem mass spectrometry (ESI-MS/MS), as previously described (Maina, et al., 2013).
312
The analysis was carried out in the negative mode (chloride adduct ions [M+Cl]-) with the Agilent
313
XCT Plus Ion Trap mass spectrometer with an electrospray ion source (Agilent Technology, Palo
314
Alto, CA, USA).
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3. Results and Discussion
317
3.1. Isolation of WcCab3-DSR
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The cell-free supernatant of Weissella confusa Cab3 (6.0 mg protein /ml) contained 6.0 U/ml
319
crude WcCab3-DSR (Table 1). Because polyethylene glycol fractionation is known as a potential
320
method of isolating dextransucrases (Purama & Goyal, 2008), isolation of WcCab3-DSR applying
321
PEG-400 (20% to 45% v/v) and PEG-1500 (5% to 20% v/v) were tested (Table 1). The
322
concentration of 30% (v/v) PEG-400 gave the highest specific activity of 10.5 U/mg with 10-fold
323
purification and 25.5% overall yield (Fig.. 1S, Table 1); 15% (v/v) concentration of PEG-1500
324
resulted in a slightly higher specific activity of 10.9 U/mg, with 11-fold purification and 24%
325
overall yield (Fig. 1S, Table 1). WcCab3-DSR (10.5 U/mg, 0.84 mg/ml) isolated by fractionation
326
with 30% (v/v) PEG-400 was selected for further experiments, because PEG-400 is more
327
economical to use than PEG-1500.
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WcCab3-DSR obtained after fractionation using 30% PEG-400 and 15% PEG-1500 were
329
analyzed in a single SDS-PAGE gel in duplicate run under a non-denaturing condition (Fig. 1).
330
WcCab3-DSR showed a single band of about 178 kDa molecular mass on the non-denaturing gel
331
stained for proteins (Fig. 1, lanes 1 and 2). The other part of the gel was subjected to activity
332
staining for in situ activity detection. The presence of magenta color bands after PAS staining (Fig.
333
1, lanes 3 and 4) were consistent with the protein staining of the dextransucrase bands. The 13 Page 13 of 37
appearance of some minor faint bands in the activity stained gel (Fig. 1, lanes 3 and 4) might be
335
due to the slight degradation of WcCab3-DSR under the non-denaturing SDS condition applied
336
during their run. Furthermore, to confirm this, WcCab3-DSR isolated by PEG-400 (30%, w/v) was
337
also run under native condition and analysed by PAS staining (Fig. 1, lanes 5). The presence of a
338
single activity band on the native-PAGE, confirmed that the WcCab3-DSR exist as a single
339
enzyme.
340
3.2. Effect of temperature and additives on WcCab3-DSR
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WcCab3-DSR exhibited a mesophilic nature and showed stability up to 40°C, above which
342
it rapidly lost activity during 1 h incubation (Fig. 2A). The optimum temperature range for the
343
assay of WcCab3-DSR was also in the range of 30–35°C (Shukla & Goyal, 2011). Thus, WcCab3-
344
DSR showed a more thermostable nature as compared with the dextransucrase from Lc.
345
mesenteroides NRRL B-640, which rapidly lost its activity above 30°C after 10 min of incubation
346
(Purama, Agrawal, & Goyal, 2010). Dextranscrase from W. cibaria JAG8 was also stable up to
347
35°C, but further increase in temperature to 40°C caused the enzyme to lose 96% of its activity
348
(Tinirikari & Goyal, 2013).
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Certain metal ions show positive effects on the activity of enzymes. Salts are believed to
350
affect the structure of enzymes, thus affecting their solubility and activity (Forman & Kennedy,
351
1977). The effects of selected metal ions were studied on WcCab3-DSR activity. The Co2+ and
352
Ca2+ ions increased the enzyme activity of WcCab3-DSR (Table 2). The enzyme activity was
353
enhanced by 20% and 21% in the presence of 1 mM CoCl2 and CaCl2, respectively (Table 2). Ca2+
354
ion has been reported to be associated with the catalytic sites of dextransucrases (Miller & Robyt,
355
1986b). CaCl2 (6 mM) increased the activity for Pediococcus pentosaceus dextransucrase by 150%
356
and 4 mM CaCl2 by 108% for Lc. mesenteroides NRRL B-640 dextransucrase (Patel et al., 2011;
357
Purama, et al., 2010). The addition of ZnCl2 (1 mM), MgCl2 (2 mM), and NiSO4 (2 mM) resulted in
358
9%, 8%, and 7% increase in enzyme activity, respectively (Table 2). On the other hand, MnCl2
359
affected enzyme activity negatively. At lower 1 mM concentration, MnCl2 decreased the activity by
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14 Page 14 of 37
360
4%, and at 10 mM MnCl2, the enzyme activity was lost by 42% of its initial value (Table 2). In the
361
presence of 5 mM EDTA, WcCab3-DSR displayed only 2% residual activity (Table 2). The effects of different additives on the stability of WcCab3-DSR were studied at 30°C.
363
The residual activity of WcCab3-DSR after 24 h with glycerol, Tween 80, and PEG 8000 were
364
80%, 76%, and 37.0%, respectively (Fig. 2B). The dextran (500 kDa) and glutaraldehyde did not
365
stabilize the enzyme, because the residual activity with dextran (500 kDa) and glutaraldehyde after
366
24 h was only 25% and 16%, respectively (Fig. 2B). The residual activity of WcCab3-DSR with
367
glutaraldehyde at 30°C after 24 h was even lower than that of WcCab3-DSR without any additive,
368
which was 20%. Thus, glutaraldehyde negatively affected WcCab3-DSR. The storage stability of
369
WcCab3-DSR was also followed at 4°C and –20°C. WcCab3-DSR lost only 1.5% and 4% of the
370
activity after 5 days of incubation at 4°C and –20°C, respectively (Fig. 2C).
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The half-life times (t1/2) of WcCab3-DSR and additives-treated WcCab3-DSR were
372
calculated by assuming that the decay followed first-order kinetics (Table 2). Among all the
373
additives used, glycerol and Tween 80 were the most efficient stabilizers, resulting in t1/2 of 74.1 h
374
and 59.3 h at 30°C, respectively (Table 2). A small enhancement in the t1/2 (16.7 h) was observed
375
using PEG 8000 at 30°C as compared with the control (10.3 h). The half-life (t1/2) of the enzyme
376
increased from 10.3 h to 69.4 d by decreasing the storage temperature from 30°C to 4°C. The
377
WcCab3-DSR was also quite stable at –20°C with a half-life (t1/2) of 37.9 d.
378
3.3 Characterization of synthesized dextran
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The in vitro-synthesized WcCab3-DSR dextran was isolated by ethanol precipitation and
380
lyophilized for further structural and functional characterization. The total carbohydrate and protein
381
content of the purified WcCab3-dextran was 98.27% (w/w) and 1.73% (w/w) (7.5 mg/ml and 0.13
382
mg/ml), respectively. HPAEC-PAD analysis after acid hydrolysis proved that the purified sample
383
contained no carbohydrate other than glucose.
384 385 15 Page 15 of 37
386
Enzyme-aided structure analysis An enzyme-aided structure analysis of dextrans is a simple and quick method for determining
388
the structural variation in dextrans produced by different strains or in different conditions (Katina et
389
al., 2009). The method involves specific enzymatic hydrolysis of dextran with a mixture of
390
dextranase and α-glucosidase that converts the dextran to glucose and a set of enzyme-resistant
391
oligosaccharides. The chromatographic profiles of the enzyme-resistant oligosaccharides vary
392
depending on the structural components of dextran, and can therefore be used as structural markers
393
(Maina, 2012). An HPAEC-PAD analysis showed that the enzyme-resistant oligosaccharides from
394
WcCab3-DSR dextran (Fig. 4A) had a similar profile to the oligosaccharides as obtained earlier
395
from W. confusa VTT E-90392 and Lc. mesenteroides NRRL B-512F dextran (Katina et al., 2009),
396
indicating high similarities in their structural features. Dextrans from these strains have few (
