Weissella confusa Cab3 dextransucrase: Properties and in vitro synthesis of dextran and glucooligosaccharides

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 25C 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 35C 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 80C 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-

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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

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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

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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

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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,

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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

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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

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.

Ac ce

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was

homologous

in

structure,

atmospheric

pressure

matrix-assisted

laser

12 Page 12 of 37

308

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).

cr

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310

316

3. Results and Discussion

317

3.1. Isolation of WcCab3-DSR

us

315

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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328

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

cr

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334

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).

ed

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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).

an

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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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379

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 (<5%)

397

α-(1→3)-linked branches that are either single unit or elongated by two or more α-(1→6)-linked

398

glucosyl units (Maina et al., 2011). Characterization of the resistant oligosaccharides from W.

399

confusa VTT E-90392 and Lc. mesenteroides NRRL B-512F dextrans showed that they included

400

oligosaccharides with terminal α-(1→3)-linked glucosyl units originating from single-unit branches

401

and α-(1→3)-linked isomaltosyl units that originate from branches that are elongated with α-(1→6)

402

linkages (Maina et al., 2011).

403

FT-IR analysis

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404

An FT-IR spectrum of the in vitro synthesized WcCab3-DSR dextran is presented in Fig.

405

3A. It shows bands in the 950–1200 cm-1 region, which is a typical fingerprinting region for all

406

polysaccharides (Cerna et al., 2003). The bands at 3394 cm-1and 2930 cm-1 represent hydroxyl-

407

stretching vibration and C–H stretching vibration of the polysaccharide, respectively. Similar

408

results have been reported earlier (Cao et al., 2006; Shingel, 2002). The transmission peak at 908

409

cm-1 is a characteristic peak for a α-glycosidic bond. The band at 1154 cm-1 is assigned to valent

410

vibrations of C–O–C bond and a glycosidic bridge. The peak, at 1022 cm-1, indicates great chain

16 Page 16 of 37

411

flexibility around the α-(1→6) glycosidic bonds in the dextran . Similar findings were observed by

412

Shingel, 2002.

413

NMR spectroscopy analysis The 1H spectrum of the WcCab3-DSR dextran (Fig. 3B) showed typical main-chain α-

415

(1→6)-linked glucopyranosyl residues with an anomeric signal centered at 4.97 ppm as also

416

reported earlier (Ahmed et al., 2012; Maina et al., 2008). An additional low intensity anomeric

417

proton signal at 5.32 ppm (Fig. 3B) was also observed in the spectrum, which indicated the

418

presence of α-(1→3)-linked branches (Maina et al., 2008). The remaining five resonances in the 1H

419

spectrum, at 3.97, 3.91, 3.77, 3.73, 3.58, and 3.52 ppm (Fig. 3B), corresponded to H-6b, H-5, H-6a,

420

H-3, H-2, and H-4, respectively of the α-(1→6)-linked glucopyranosyl residues in the main chain

421

(Table 1S).

cr

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13

C NMR spectroscopy has been used to study the structure of a series of dextrans. Dextrans 13

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422

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414

have typical

C anomeric signals (C-1), downfield at about 90 ppm, characteristic of anomeric

424

carbon C-1 involved in a glycosidic linkage. The chemical shifts for C-2, C-3, C-4, and C-5 appear

425

in the 70–75 ppm region, and C-6 is normally up-field at about 60 ppm (Seymour, Knapp, &

426

Bishop, 1976). 13C chemical shifts of WcCab3-DSR dextran were determined by 2D heteronuclear

427

single quantum correlation spectroscopy (HSQC). The edited HSQC spectrum of the WcCab3-DSR

428

dextran (Fig. 3C) showed two anomeric

429

corresponding to the 1H anomeric signals at 4.97 ppm of the α-(1→6)-linked residues, and 5.32

430

ppm for the α-(1→3)-linked residues, respectively. Apart from the anomeric signals, five 13C NMR

431

resonances at 73.0, 75.0, 71.3, 71.8, and 67.3 ppm were also observed (Fig. 3C), which were

432

assigned to the α-(1→6)-linked residues in the main chains (Table 1S). A typical downfield shift

433

for a linked C-3 of the 3,6-O-disubstituted glucopyranosyl units at 82.2 ppm was also observed in

434

the HSQC spectra of the WcCab3-DSR dextran, which further established the presence of α-

435

(1→3)-linked branch residues (Maina et al., 2008).

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C chemical shifts at 99.3 ppm and 100.5 ppm

17 Page 17 of 37

NMR spectroscopy data also confirmed the presence of elongated branches in WcCab3-

437

DSR dextran. As described previously, the signal at 5.32 ppm represents both α-(1→3)-linked

438

single unit and elongated branches (Maina et al., 2011). The elongated branches can be determined

439

by the occurrence of a signal at ~4.19 ppm that has been assigned to the H-5 signal of the elongated

440

branch point glucopyranosyl unit (→6)-α-D-Glcp-(1→3)-) (Maina et al., 2011). In the dextran

441

synthesized by WcCab3-DSR, a peak of 4.18 ppm for the elongated branches was observed in the

442

1

443

in the HSQC spectrum (Fig. 3C). The terminal units in the main chain, single unit, or elongated

444

branches had a C-6 signal at 61.8 ppm (Fig. 3C).

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436

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cr

H spectrum (Fig. 3B), and, furthermore, a cross peak (H-5→C-5) of 4.18→71.6 ppm was observed

The relative intensities of the anomeric signals at 4.97 and 5.32 ppm were 97% and 3%,

446

respectively, indicating the relatively low branching degrees in the in vitro synthesized WcCab3-

447

DSR dextran. Previous studies have also shown that dextran from Weissella strains generally have

448

a lower degree of branching compared with dextran from other strains such as Lc. mesenteroides

449

(Bounaix et al., 2009; Maina et al., 2008). This may be a typical feature of Weissella strains, which

450

in combination with their high dextran-production efficiency, makes them highly potential for

451

production of commercial dextran with few branch linkages.

452

HPSEC analysis

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445

An HPSEC analysis of WcCab3-DSR dextran was done in DMSO containing 10 mM LiBr.

454

Although dextrans are commonly analyzed in aqueous solutions, DMSO was preferred in this

455

study, because in aqueous solutions, dextrans are prone to aggregation, which can significantly

456

affect the results (Maina, 2012). Only one major peak for both refractive index (RI) and light

457

scattering at 90° (LS) signals was observed in the HPSEC chromatogram of WcCab3-DSR dextran

458

(results not shown), and the corresponding molar mass was 1.8 x 107 g/mol. Interestingly, when

459

dextran was extracted from the cell mass of W. confusa VTT E-90392, HPSEC analysis in DMSO

460

showed a clearly lower molar mass (1.5 x 106 g/mol) (Maina, 2012), compared with WcCab3-DSR

461

dextran obtained in vitro in the present study. Irague et al., 2012 recently also reported significantly

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18 Page 18 of 37

462

high molar mass values (108 g/mol) for dextran synthesized in vitro with Lc. mesenteroides NRRL

463

B-512F dextransucrase mutants, as analyzed in an aqueous solution. Based on the characterization,

464

WcCab3-DSR dextran seems well suitable for food applications such as sourdough in which high

465

molar mass dextrans with few branches are desirable (Lacaze, Wick, & Cappelle, 2007).

466

3.4. Production and characterization of GLOS The GLOS produced by WcCab3-DSR through an acceptor reaction with maltose were

468

characterized by HPAEC-PAD, enzyme-aided structural analysis and mass spectrometry.

469

According to HPAEC-PAD analysis, sucrose was depleted during the reaction, while 19% of the

470

added maltose was left unutilized (Fig. 4B). A series of GLOS were produced as shown in the

471

HPAEC-PAD chromatogram (Fig. 4B), with some peaks being predominant, while minor peaks

472

were also present.

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467

Selective enzymatic hydrolysis of the GLOS shed light on their structure. Endo-acting

474

dextranase degraded the GLOS of higher DP into their building blocks, i.e., glucose, isomaltose,

475

isomaltotriose, and panose (based on retention time) (Fig. 4C and E). On the other hand, the α-

476

glucosidase exo-acting on α-(1→4) linkages from the non-reducing end of GLOS, converted only

477

maltose into glucose, leaving the acceptor products intact (Fig. 4D). This indicated that GLOS have

478

consecutive α-(1→6) linked glucopyranosyl chains of varying lengths attached to the non-reducing

479

end of the maltose. Because the GLOS were expected to be homologous to panose, their yield was

480

estimated to be 6.8 g by quantifying GLOS peaks using panose as a standard. This GLOS yield was

481

in accordance with the estimate based on the total carbohydrate content (600 mg/ml as determined

482

by phenol sulphuric acid method), in the GLOS solution. Because 81% of added maltose (4.1 g)

483

was utilized in the GLOS production, the weight gain of GLOS (2.7 g) was derived from the

484

glucosyl moiety in sucrose (2.7/162=16.7 mmol). Because sucrose (10/342= 29.2 mmol) was

485

depleted, the acceptor reaction toward GLOS consumed 57.2% (16.7/29.2) sucrose, leaving 42.8%

486

sucrose for dextran production.

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19 Page 19 of 37

The AP-MALDI-ITMS analysis of the GLOS-mixture components confirmed that they

488

belong to a homologous hexose series. The peaks at m/z 527.3, 689.3, 851.4, 1013.5, 1175.5,

489

1337.5, and 1499.5 corresponded to the [M+Na]+ ions of GLOS from DP3 up to DP9, respectively

490

(Fig. 5A). Nonetheless, oligosaccharides most likely of DP10-DP12 in HPAEC-PAD analysis (Fig.

491

4B) were not detected in the AP-MALDI-ITMS analysis under the conditions used. Products up to

492

DP10 were isolated by gel filtration with a Biogel P2 column (Fig. 2S). Interestingly, the HPAEC-

493

PAD analysis of the selected fractions showed that minor products particularly co-eluted in the

494

fractions for DP5, DP6, and DP7, possibly representing another homologous series.

cr

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487

Tandem MS in negative mode is useful in the structural analysis of linear mixed-linkage

496

GLOS (Maina et al., 2013). Here, isolated DP3, DP4, and DP5 ([M+Cl]- ions of m/z at 539.0,

497

701.1, and 863.2, respectively), were further analyzed by ESI-MS/MS to prove the proposed

498

structures. Cross-ring cleavage fragment ions were observed at m/z 424.8 and 382.8 (loss of 78 and

499

120 Da, respectively) and at m/z 280.8, 250.6, and 220.6 (loss of 60, 90 and 120 Da, respectively)

500

in the MS2 spectrum of DP3 (panose) (results not shown). These diagnostic fragment ions (Maina

501

et al., 2013) confirmed the α-(1→4) linkage at its reducing end and the α-(1→6) linkage at the non-

502

reducing end. Similarly, in the MS2 spectrum of the [M+Cl]- ion of DP4 (Fig. 5B), fragment ions at

503

m/z 587.1 and 545.1 (loss of 78 Da and 120 Da, respectively) indicated the presence of a α-(1→4)

504

linkage at the reducing end. The ions at m/z 443.1, 413.0, and 383.1 (loss of 60, 90, and 120 Da,

505

respectively) in the MS3 spectrum, starting with the ion at m/z 503.0, indicated the subsequent α-

506

(1→6) linkage (Fig. 5C). The α-(1→6) linkage at the non-reducing end was confirmed with the

507

MS3 spectrum of the ion at m/z 340.9 (Fig. 5D), which contained fragment ions at m/z 280.8,

508

250.7, and 220.8 (loss of 60, 90, and 120 Da, respectively). The MS/MS of DP5 showed a similar

509

trend (Fig. 3S); a α-(1→4) linkage at its reducing end and the remaining linkages being α-(1→6).

510

The findings confirmed that the principal acceptor products (GLOS) were a homologous series of

511

isomaltooligosaccharides that contain maltose at the reducing end. Similar maltose acceptor

512

products have been reported for dextransucrases from Lc. mesenteroides NRRL B-512F, and B-

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20 Page 20 of 37

513

1299; however, the latter produced another two series of products bearing a α-(1→2) single unit

514

branch (Brison et al., 2012; Robyt & Eklund, 1983). The minor acceptor product series has not been previously reported for dextransucrases

516

producing typical dextrans, which are predominantly α-(1→6)-linked α-glucan with a low degree of

517

branching. Cote & Leathers (2005) analyzed the maltose acceptor products of various Leuconostoc

518

spp. glucansucrases by thin-layer chromatography, and found that strains producing typical

519

dextrans produced only one series of isomaltooligosaccharides, while strains producing highly

520

branched dextrans produced another series of acceptor products. However, the characterization of

521

the minor acceptor product series synthesized by WcCab3-DSR needs further studies.

522

4. Conclusions

us

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515

Recently, Weissella spp. has drawn attention for its high dextransucrase and dextran

524

production capacity and the presence of only a few branches in its dextran. Dextransucrase from W.

525

confusa Cab3 was isolated in this study by fractionation, using 30% PEG-400, resulting in a

526

specific activity of 10.5 U/mg with 10 fold purification. Activity of WcCab3-DSR was enhanced by

527

Co2+ and Ca2+ ions (21% and 20%, respectively). Glycerol and Tween 80 significantly enhanced

528

their stability at 30°C, increasing the half-life (t1/2) to 74 h and 59 h, respectively, compared with

529

the same process without any additive (10 h). WcCab3-DSR dextran had a high molar mass of

530

1.8x107 g/mol according to an HPSEC analysis in DMSO. A 1H and

531

confirmed the presence of main chain α-(1→6) linkages with few (3.0%) α-(1→3) branch linkages.

532

WcCab3-DSR catalyzed acceptor reaction when maltose was present, giving rise to a homologous

533

series of GLOS (DP3 – DP12), which carried maltose at the reducing end and additional glucose

534

units all α-(1→6) linked to the non-reducing end.

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13

C NMR spectral analysis

535 536 537 538 21 Page 21 of 37

539

Acknowledgements

540

The researchers would like to thank Leena Pitkänen for help in the HPSEC analysis. This

541

study was supported by the Department of Biotechnology; the Ministry of Science and Technology,

542

New Delhi, India (AG); the Academy of Finland (contract number 255755, MT) via the joint

543

WISEDextran project; and the ABS Graduate School in Finland (QS).

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555 556 557 558 559

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References

566

Ahmed, R. Z., Siddiquia, K., Armanb, M., & Ahmeda, N. (2012). Characterization of high

567

molecular weight dextran produced by Weissella cibaria CMGDEX3. Carbohydrate Polymers,

568

90(1), 441-446. Amari, M., Arango, L. F. G., Gabriel, V., Robert, H., Morel, S., Moulis, C., Gabriel, B., Remaud-

570

Siméon, M., & Fontagné-Faucher, C. (2012). Characterization of a novel dextransucrase from

571

Weissella confusa isolated from sourdough. Applied Microbiology and Biotechnology, 97,

572

5413-5422.

cr

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569

Basedow, A. M., & Ebert, K. H. (1979). Production, characterization and solution properties of

574

dextran fractions of narrow molecular distributions. Journal of Polymer Sciences: Polymer

575

Symposia, 66(1), 101-115.

an

us

573

Bejar, W., Gabriel, V., Amari, M., Morel, S., Mezghani, M., Maguin, E., Fontagné-Faucher, C.,

577

Bejar, S., & Chouayekh, H. (2013). Characterization of glucansucrase and dextran from

578

Weissella sp. TN610 with potential as safe food additives. International Journal of Biological

579

Macromolecules, 52, 125-132.

ed

M

576

Bounaix, M.-S., Gabriel, V., Morel, S., Robert, H., Rabier, P., Remaud-Siméon, M., Gabriel, B., &

581

Fontagné-Faucher, C. (2009). Biodiversity of exopolysaccharides produced from sucrose by

582

sourdough lactic acid bacteria. Journal of Agricultural and Food Chemistry, 57, 10889-10897.

Ac ce

pt

580

583

Bounaix, M.-S., Robert, H., Gabriel, V., Morel, S., Remaud-Siméon, M., Gabriel, B., & Fontagné-

584

Faucher, C. (2010). Characterization of dextran-producing Weissella strains isolated from

585

sourdoughs and evidence of constitutive dextransucrase expression. FEMS Microbiology

586

Letters, 311(1), 18-26.

587

Brison, Y., Pijning, T., Malbert, Y., Fabre, E., Mourey, L., Morel, S., Potocki-Véronèse, G.,

588

Monsan, P., Tranier, S., Remaud-Siméon, M., & Dijkstra, B. W. (2012). Functional and

589

structural characterization of an α-(1→2) branching sucrase derived from DSR-E

590

glucansucrase. The Journal of Biological Chemistery, 287, 7915-7924. 23 Page 23 of 37

591

Cao, W., Li, X.-Q., Liu, L., Yang, T. H., Li, C., Fan, H. T., Jia, M., Lu, Z. G., & Mei, Q-B. (2006).

592

Structure of antitumor polysaccharide from Angelica sinensis (Oliv.) Diels. Carbohydrate

593

Polymers, 66(2), 149-159. Cerna, M., Barros, A., Nunes, A., Rocha, S. M., Delgadillo, I., Copikova, J., & Coimbra, M. A.

595

(2003). Use of FT-IR spectroscopy as a tool for the analysis of polysaccharide food additives.

596

Carbohydrate Polymers, 51(4), 383-389.

ip t

594

Chong, S. L., Nissilä, T., Ketola, R. A., Koutaniemi, S., Derba-Maceluch, M., Mellerowicz, E. J,

598

Tenkanen, M., & Tuomainen, P. (2011). Feasibility of using atmospheric pressure matrix-

599

assisted laser desorption/ionization with ion trap mass spectrometry in the analysis of acetylated

600

xylooligosaccharides derived from hardwoods and Arabidopsis thaliana. Analytical and

601

Bioanalytical Chemistry, 401, 2995-3009.

an

us

cr

597

Collins, M. D., Samelis, J., Metaxopoulos, J., & Wallbanks, S. (1993). Taxonomic studies on some

603

Leuconostoc-like organisms from fermented sausages: description of a new genus Weissella for

604

the Leuconostoc paramesenteroides group of species. Journal of Applied Microbiology, 75(6),

605

595-603.

ed

M

602

Cote, G. L., & Leathers, T. D. (2005). A method for surveying and classifying Leuconostoc spp.

607

glucansucrases according to strain-dependent acceptor product patterns. Journal of Industrial

608

Microbiology and Biotechnology. 32(2), 53-60.

Ac ce

pt

606

609

Di Cagno, R., De Angelis, M., Limitone, A., Minervini, F., Carnevali, P., Corsetti, A., Gaenzle, M.,

610

Ciati, R., & Gobbetti, M. (2006). Glucan and fructan production by sourdough Weissella

611

cibaria and Lactobacillus plantarum. Journal of Agricultural and Food Chemistry, 54, 9873-

612

9881.

613

Dols, M., Remaud-Simeon, M., Willemot, R. M., Vignon, M. R., & Monsan, P. F. (1997).

614

Structural characterization of the maltose acceptor-products synthesized by Leuconostoc

615

mesenteroides NRRL B-1299 dextransucrase. Carbohydrate Reserch, 305(3-4), 549-559.

24 Page 24 of 37

617 618 619 620 621

Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., & Smith, F. (1956). Colorimetric method for determination of sugars and related substances. Analytical Chemistry, 28(3), 350-356. Forman, H. J., & Kennedy, J. (1977). Effects of chaotropic agents versus detergents on dihydroorotatedehydrogenase. The Journal of Biological Chemistry, 25, 3379-3381. Fox, J. D., & Robyt, J. F. (1991). Miniaturization of three carbohydrate analyses using a microsample plate reader. Analytical Chemistry, 195(1), 93-96.

ip t

616

Galle, S., Schwab, C., Arendt, E., & Gänzle, M. (2010). Exopolysaccharide-forming Weissella

623

strains as starter cultures for sorghum and wheat sourdoughs. Journal of Agricultural and Food

624

Chemistry, 58, 5834-5841.

us

cr

622

Galle, S., Schwab, C., Dal Bello, F., Coffey, A., Gänzle, M. G., & Arendt, E. K. (2012). Influence

626

of in situ synthesized exopolysaccharides on the quality of gluten-free sorghum sourdough

627

bread. International Journal of Food Microbiology, 155(3), 105-112.

M

an

625

Goyal, A., & Katiyar, S. S. (1996). Regulation of dextransucrase productivity of Leuconostoc

629

mesenteroides B-512F by the maintenance media. The Journal of General and Applied

630

Microbiology, 42, 81-85.

ed

628

Hernández, H. G., Livings, S., Aguilera, J. M., & Chiralt, A. (2011). Phase transitions of dairy

632

proteins, dextrans and their mixtures as a function of water interactions. Food

633

Hydrocolloids, 25(5), 1311-1318.

Ac ce

pt

631

634

Irague, R., Rolland-Sabaté, A., Tarquis, L., Doublier, J. L., Moulis, C., Monsan, P., Remaud-

635

Siméon, M., Potocki-Vronèse, G., & Buleon, A. (2012). Structure and property engineering of

636

α-D-glucans synthesized by dextransucrase mutants. Biomacromolecules, 13(1), 187-195.

637

Ito, K., Ito, S., Shimamura, T., Weyand, S., Kawarasaki, Y., Misaka, T., Abe, K., Kobayashi, T.,

638

Cameron, A. D., & Iwata, S. (2011). Crystal structure of glucansucrase from the dental caries

639

pathogen Streptococcus mutans. Journal of Molecular Biology, 408(2), 177-186.

25 Page 25 of 37

640

Katina, K., Maina, N. H., Juvonen, R., Flander, L., Johansson, L., Virkki, L., Tenkanen, M., &

641

Laitila, A. (2009). In situ production and analysis of Weissella confusa dextran in wheat

642

sourdough. Food Microbiology, 26(7), 734-743.

645 646

novel sourdoughs. Food Microbiology, 24(2), 155-160. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of

ip t

644

Lacaze, G., Wick, M., & Cappelle, S. (2007). Emerging fermentation technologies: development of

bacteriophage T4. Nature, 227, 680-685.

cr

643

Leemhuis, H., Pijning, T., Dobruchowska, J. M., van Leeuwen, S. S., Kralj, S., Dijkstra, B. W., &

648

Dijkhuizen, L. (2013). Glucansucrases: Three-dimensional structures, reactions, mechanism, α-

649

glucan analysis and their implications in biotechnology and food applications. Journal of

650

Biotechnology,163(2), 250-272.

652

an

Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with

M

651

us

647

the Folin phenol reagent. The Journal of Biological Chemistry, 193(1), 265-275. Maina, N. H. (2012). Structure and macromolecular properties of Weissella confusa and

654

Leuconostoc citreum dextrans with a potential application in sourdough. Doctoral Thesis, EKT-

655

series 1553, University of Helsinki, Finland.

pt

ed

653

Maina, N. H., Juvonen, M., Domingues, R. M., Virkki, L., Jokela, J., & Tenkanen, M. (2013).

657

Structural analysis of linear mixed-linkage glucooligosaccharides by tandem mass

658

spectrometry. Food Chemistry, 136, 1496-1507.

Ac ce

656

659

Maina, N. H., Tenkanen, M., Maaheimo, H., Juvonen, R., & Virkki, L. (2008). NMR spectroscopic

660

analyses of exopolysaccharides produced by Leuconostoc citreum and Weissella confusa.

661

Carbohydrate Research, 343, 1446-1455.

662

Maina, N. H., Virkki, L., Pynnönen, H., Maaheimo, H., & Tenkanen, M. (2011). Structural analysis

663

of enzyme-resistant isomaltooligosaccharides reveals the elongation of α-(1→3)-linked

664

branches in Weissella confusa dextran. Biomacromolecules, 12(2), 409-418.

26 Page 26 of 37

665

Miller, A. W., & Robyt, J. (1986a). Detection of dextransucrase and levansucrase on

666

polyacrylamide gels by the periodic acid-Schiff stain: Staining artifacts and their

667

prevention. Analytical Biochemistry, 156, 357-363.

668 669

Miller, A. W., & Robyt, J. F. (1986b). Activation and inhibition of dextransucrase by calcium. Biochimica et Biophysica Acta, 880(1), 32-39. Naessens, M., Cerdobbel, A., Soetaert, W., & Vandamme, E. J. (2005). Leuconostoc

671

dextransucrase and dextran: Production, properties and applications. Journal of Chemical

672

Technology and Biotechnology, 80, 845-860.

674

cr

Nelson, N. A. (1944). photometric adaptation of the Somogyi method for the determination of

us

673

ip t

670

glucose. The Journal of Biological Chemistry, 153(2), 375-380.

Patel, S., Kothari, D., & Goyal, A. (2011). Purification and characterization of an extracellular

676

dextransucrase from Pediococcus pentosaceus isolated from the soil of north east India. Food

677

Technology and Biotechnology, 49(3), 297-303.

679

M

Patel, S., Majumder, A., & Goyal, A. (2012). Potentials of exopolysaccharides from lactic acid

ed

678

an

675

bacteria. Indian Journal of Microbiology, 52(1), 3-12. Purama, R. K., & Goyal, A. (2008). Identification, effective purification and functional

681

characterization of dextransucrase from Leuconostoc mesenteroides NRRL B-640. Bioresource

682

Technology, 99, 3635-3542.

684

Ac ce

683

pt

680

Purama, R. K., Agrawal, M., & Goyal, A. (2010). Stabilization of dextransucrase from Leuconostoc mesenteroides NRRL B-640. Indian Journal of Microbiology, 50(1), S57-S61.

685

Rantanen, H., Virkki, L., Tuomainen, P., Kabel, M. A., Schols, H. A., & Tenkanen, M. (2007).

686

Preparation of arabinoxylobiose from rye xylan using family 10 Aspergillus aculeatus endo-

687

1,4-ß-d-xylanase. Carbohydrate Polymers, 68(2), 350-359.

688 689

Robyt, J. F., & Eklund, S. H. (1983). Relative, quantitative effects of acceptors in the reaction of Leuconostoc mesenteroides B-512F dextransucrase. Carbohydrate Reserch, 121, 279-286.

27 Page 27 of 37

690

Schwab, C., Mastrangelo, M., Corsetti, A., & Gänzle, M. (2008). Formation of oligosaccharides

691

and polysaccharides by Lactobacillus reuteri LTH5448 and Weissella cibaria 10M in sorghum

692

sourdoughs. Cereal Chemistry, 85(5), 679-684.

697 698 699 700

Shingel, K. I. (2002). Determination of structural peculiarities of dextran, pullulan and c-irradiated

ip t

696

C-nuclear magnetic resonance spectroscopy. Carbohydrate Research, 51(2), 179-194.

pullulan by Fourier-transform IR spectroscopy. Carbohydrate Research, 337, 1445-1451.

cr

695

13

Shukla, S., & Goyal, A. (2011). 16S rRNA based identification of a glucan hyper-producing Weissella confusa. Enzyme Research, ID 250842, doi:10.4061/2011/250842.

us

694

Seymour, F. R., Knapp, R. D., & Bishop, S. H. (1976). Determination of the structure of dextran by

Somogyi, M. (1945). A new reagent for determination of sugars. The Journal of Biological Chemistry, 160(1), 61-68.

an

693

Stam, M. R., Danchin, E. G. J., Rancurel, C., Coutinho, P. M., & Henrissat, B. (2006). Dividing the

702

large glycoside hydrolase family 13 into subfamilies: towards improved functional annotations

703

of α-amylase-related proteins. Protein Engineering Design and Selection, 19, 555-562.

ed

M

701

Tinirikari, J. M. R., & Goyal, A. (2013). Purification, optimization of assay, and stability studies of

705

dextransucrase isolated from Weissella cibaria JAG8. Preparative Biochemistry and

706

Biotechnology, 43(4), 329-341.

pt

704

Tsuchiya, H. M., Koepsell, H. J., Corman, J., Bryant, M. O., Feger, V. H., & Jackson, R. W.

708

(1952). The effect of certain cultural factors on production of dextransucrase by Leuconostoc

709

mesenteroides. Journal of Bacteriology, 64(4), 521-526.

Ac ce

707

710

Vujicic-Zagar, A., Pijning, T., Kralj, S., López, C. A., Eeuwema, W., Dijkhuizen, L., & Dijk-stra,

711

B. W. (2010). Crystal structure of a 117 kDa glucansucrase fragment provides insight into

712

evolution and product specificity of GH70 enzymes. Procedings of the Natlional Academy of

713

Sciences USA, 107, 21406-21411.

714 715 28 Page 28 of 37

716 717

Legends to Figures Figure 1

Non-denaturing SDS-PAGE analyses on 7% acrylamide gel of WcCab3-DSR isolated with PEG-400 (30%, v/v) fractionation (lanes 1 and 3) and PEG-1500 (15%, w/v)

719

fractionation (lanes 2 and 4). Identification of WcCab3-DSR by protein staining (lanes

720

1 and 2) and staining of dextran formed from sucrose (lanes 3 and 4). PEG-400 (30%,

721

v/v) fraction (lane 5) run on native-PAGE (7% acrylamide gel) and stained with PAS

722

staining using sucrose as substrate. Lane M: Protein molecular weight marker (30-200

723

kDa).

cr

Figure 2

(A) Effect of temperature on stability of WcCab3-DSR. WcCab3-DSR 10.5 U/mg and

us

724

ip t

718

0.84 mg protein/ml) was pre-incubated at different temperatures for 1 h and assayed for

726

residual activity. (B) Effects of various stabilizers on WcCab3-DSR activity. WcCab3-

727

DSR (10.5 U/mg and 0.84 mg protein/ml) in a 20 mM sodium acetate buffer (pH 5.4)

728

was pre-incubated with additives at 30°C and assayed for activity. (C) Effect of

729

temperatures at 30°C, 4°C, and –20°C on the storage stability of dextransucrase.

730

WcCab3-DSR (10.5 U/mg, 0.84 mg/ml) in a 20 mM sodium acetate buffer (pH 5.4)

731

was pre-incubated at 30°C, 4°C, and –20°C and aliquots were assayed for

732

dextransucrase activity at 35°C at 1 day intervals.

M

ed

An FT-IR and NMR spectroscopic analysis of WcCab3-DSR dextran. (A) FT-IR spectrum. (B) The 1D 1H spectrum recorded at 600 MHz in D2O at 50°C. (C) The

734

edited HSQC spectrum recorded at 600 MHz in D2O at 50°C. Peaks in the NMR

735

spectra are referenced to internal acetone (1H = 2.225 ppm and 13C = 31.55 ppm).

736 737

pt

Figure 3

Ac ce

733

an

725

Figure 4

HPAEC-PAD chromatograms showing (A) resistant oligosaccharides from WcCab3-

738

DSR dextran after dextranase and α-glucosidase treatment, with the structure of the

739

shortest four oligosaccharides illustrated; (B) acceptor-reaction products after WcCab3-

740

DSR incubated with 5% (w/v) maltose and 10% (w/v) sucrose; (C) acceptor products

741

after treatment with dextranase; (D) acceptor products after treatment with Bacillus 29 Page 29 of 37

742

stearothermophilis α-glucosidase; (E) standards, from left to right G: Glucose, F:

743

Fructose, IM2: Isomaltose, S: Sucrose, IM3: Isomaltotriose, M: Maltose, and P:

744

Panose.

745

Figure 5

Mass spectra of WcCab3-DSR maltose acceptor products. (A) The positive mode ([M+Na]+) AP-MALDI-ITMS spectrum of the GLOS-acceptor product mixture. (B)

747

The negative mode ([M+Cl]-) MS2 and (C and D) MS3 spectra of the isolated DP4

748

acceptor product. The MS3 spectra in C and D are for the fragment ions at m/z 503 and

749

341, respectively.

751

cr

Figure 1S Isolation of dextransucrase from Weissella confusa Cab3 by applying different concentrations of PEG-400 and PEG-1500.

us

750

ip t

746

Figure 2S HPAEC-PAD chromatograms of GLOS fractions from a Biogel P2 column

753

purification. From bottom to top: the acceptor reaction solution and isolated products of

754

DP3–DP10. The determined m/z of purified DP3-DP5 products as [M+Cl]- indicated.

M

an

752

Figure 3S The negative mode MSn spectra of the isolated DP5 acceptor product of WcCab3-DSR.

756

(A) MS2 spectrum ([M+Cl]-). (B and C) MS3 and MS4 spectra of the fragment ions at

757

m/z 503 and 341, respectively.

pt Ac ce

758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777

ed

755

30 Page 30 of 37

Highlights

778

Dextransucrase from W. confusa Cab3 was biochemically characterized.

779

Dextransucrase was used for in vitro dextran & oligosaccharides synthesis.

780

NMR spectroscopy showed dextran having α(1→6) linkages with 3.0% of α(1→3) branching.

781

HPAEC-PAD analysis revealed glucooligosaccharides from degree of polymerization 3-12.

782

Structural characterization of isolated DP3-5 oligosaccharies was done by ESI-MS/MS.

ip t

777

783

Ac ce

pt

ed

M

an

us

cr

784

31 Page 31 of 37

789 791 793 795 797 799 801 803 805 807 809 811 813 815 817 819 821 823 825 827 829 831 833 835

ip t

Fig. 1

pt Ac ce

836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859

ed

M

an

us

cr

784 785 786 787

32 Page 32 of 37

pt

ed

M

an

us

cr

ip t

Fig. 2

Ac ce

860 862 864

33 Page 33 of 37

B

us

cr

ip t

Fig. 3

Bulk region protons

an

Anomeric protons

ed

M

α-(1→6)-linked residues

pt

α-(1→3)-linked residues

Ac ce

865 866 868 870

C

Terminal glucosyl units

H-5→C-5 (4.18→71.6 ppm) for elongated branches

Linked C3 (82.2 ppm)

34 Page 34 of 37

pt

ed

M

an

us

cr

ip t

Fig. 4

Ac ce

871 872 874 876

35 Page 35 of 37

pt

ed

M

an

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cr

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Fig. 5

OH O OH OH

Ac ce

877 878 879 880

O OH

CH 2

221 251

O

341

OH OH

O

281

OH

CH 2

383

O

O

443

Cl-

O

OH

413

665 OH

OH

OH

545

OH

503

587

OH

36 Page 36 of 37

882 883 884 885

Table 1. Isolation of WcCab3-DSR by Fractionation with Polyethylene Glycols.

Sample

Enzyme Activity (U/ml)

Crude 6.01 PEG-400 (30%) 8.53 PEG-1500 (15%) 4.90

Vol. (ml) 30.0 5.4 9.0

Total Units 180.4 46.1 44.1

Overall Activity Protein Yield (mg/ml) (%) 6.0 25.5 0.84 24.4 0.45

1.0 10.5 10.9

1.0 10.0 11.0

Table 2. Effects of Additives on Purified WcCab3-DSR.

us

cr

886 887 888 889 890 891

Specific PurificActivity ation (U/mg) Fold

ip t

880 881

892 893

Ac ce

pt

ed

M

an

Effect of metal ions Sample Residual enzyme activity (%) Control 100 CoCl2 (1 mM) 121 CaCl2 (1 mM) 120 109 ZnCl2 (1 mM) MgCl2 (2 mM) 108 NiSO4 (2 mM) 107 MnSO4 (10 mM) 58 EDTA (5 mM) 2 Half life (t1/2) of WcCab3-DSR treated with various additives and temperatures Additive 30°C 4°C -20°C Control 10.33h 69.4d 37.9d Glycerol (0.5%) 74.12h nd nd Tween 80 (1.0%) 59.26h nd nd PEG-8000 (10 µg/ml) 16.72h nd nd Dextran T-500 (2 µg/ml) 12.0h nd nd Glutaraldehyde (0.1%) 9.07h nd nd nd: not determined

894 895 896 897

37 Page 37 of 37