Characterization of Dextransucrase from Leuconostoc Mesenteroides T3, Water Kefir Grains Isolate

Characterization of dextransucrase from Leuconostoc mesenteroides T3, water kefir grains isolate Miona G. Miljković1, Slađana Z. Davidović1, Slavko Kralj2, Slavica S. Šiler-Marinković1, Mirjana D. Rajilić-Stojanović1, Suzana I. Dimitrijević-Branković1 1University of Belgrade, Faculty of Technology and Metallurgy, Department for Biochemical Engineering and Biotechnology, Belgrade, Serbia 2Department for Materials Synthesis, Jožef Stefan Institute, Ljubljana, Slovenia Abstract The production of dextransucrase (DS) by Leuconostoc mesenteroides T3, novel isolate SCIENTIFIC PAPER from water kefir grain, was studied and optimized. Bacterial supernatant reached activity of 3.1 U/ml when the culture was grown at 23 °C and under static culture condition using UDC 63(497.113):632.954:66.061:543 classical Tsuchiya medium for DS production. The increase of sucrose concentration to 7% led to an increase of DS activity by 52% compared to the control. Medium with 2% beef extract and 1% yeast extract resulted in 4.52 U/ml, which was 47% higher than in the Hem. Ind. 71 (4) 351–360 (2017) control (with 2% yeast extract). Finally, the increase of K2HPO4 concentration from 2 to 3% resulted in the increased enzyme activity by 28%. Enzyme purified by polyethylene glycol 400 fractionation displayed maximum activity at 30 °C and pH 5.4. Zymogram analysis confirmed the presence of DS of approximately 180 kDa. The addition of divalent cations Ca2+, Mg2+, Fe2+ and Co2+ led to a minor increase of DS activity, while the addition of Mn2+ was the most prominent with 73% increase. These findings classify dextransucrase from Leuconostoc mesenteroides T3 as promising candidate for production of dextran, which has numerous applications in various industries. Keywords: lactic acid bacteria, Leuconostoc mesenteroides, dextransucrase, dextran. Available online at the Journal website: http://www.ache.org.rs/HI/ Lactic acid bacteria (LAB) are one of the main The growing importance of DS stems from the wide classes of microorganisms that are known to produce application of dextran [7–10]. Dextran is a high-mole- several industrially important biomolecules among cular-mass homopolysaccharide made of glucose. The which exopolysaccharides (EPS) have the widest range glucose molecules in the main chain of dextran are of uses [1]. Fermentation processes for EPS production linked by linear alpha 1-6 glycosidic bonds and by alpha are quite expensive due to purification costs, giving an 1-2, alpha 1-3 and/or alpha 1-4 branching linkages [11]. advantage to the use of enzymes. Current challenges in The proportion of branching and the chain length the LAB enzymes production include both strain imp- influenced the rheological properties of dextran [12]. rovement and enhancement of enzyme production. Depending on its solubility and molecular mass, Dextransucrase (DS) is an extracellular enzyme that dextran has had successful industrial application so far, belongs to the class of glucosyltransferases [2] and it is such as in medical, pharmaceutical, food, textile and mainly produced by microorganisms belonging to the chemical industries [7–10]. Dextrans of low molecular families Lactobacillaceae and Streptococcaceae, espe- weight have been used as a blood plasma substitute for cially by the genera Lactobacillus, Leuconostoc and the last 60 years while the high molecular weight dex- Streptococcus [3]. Enzyme DS catalyzes the transfer trans can be used as viscosifying, stabilizing, gelling, reaction of glucosyl residues from sucrose to dextran sweetening and emulsifying agents in food production polymer chain and releases fructose [4,5]. In the pre- [9,13,14]. There are also approved patents related to sence of appropriate acceptor (glucose, maltose, iso- the use of dextran in the preparation of bakery pro- maltose, etc.) this enzyme can also produce oligosac- ducts [15,16]. charides [6]. In the present study, Leuconostoc mesenteroides strain T3, water kefir grains’ isolate described earlier [17] has been used for the production of DS. The growth conditions and culture medium composition for Correspondence: M.G. Miljković, University of Belgrade, Faculty of the highest DS production from the aforementioned Technology and Metallurgy, Department for Biochemical Engineering and Biotechnology, Karnegijeva 4, Belgrade, Serbia. strain were determined. The DS was purified by poly- E-mail: [email protected] ethylene glycol fractionation and characterized in terms Paper received: 21 April, 2016 of temperature, pH value and the influence of ions. Paper accepted: 12 December, 2016 https://doi.org/10.2298/HEMIND160421046M 351 M.G. MILJKOVIĆ et al.: DEXTRANSUCRASE FROM L. mesenteroides Hem. ind. 71 (4) 351–360 (2017) EXPERIMENTAL 1ml of culture was taken out from flask and centrifuged at 6000 rpm and 4 °C for 10 min. The DS activity in cell Microorganism and culture conditions for free supernatant was determined in triplicate. dextransucrase production Effects of culture medium composition on The microorganism used in this study is Leuconostoc dextransucrase production mesenteroides T3 natural isolate from water kefir grain, The effects of various concentrations of medium indentified as L. mesenteroides based on the almost components, i.e., sucrose, nitrogen source (yeast ext- full-length 16S rRNA gene sequence (KT924430), des- ract, meat extract and peptone) and K2HPO4, on DS cribed earlier [17]. production were studied by changing the concentration Production of dextransucrase from L. mesenteroides T3 of one component, and keeping other components constant in Tsuchiya medium [18]. Concentration of For DS production, the organism was grown in 100 sucrose was varied from 1 to 10% with step of 1%, ml enzyme production medium described by Tsuchiya nitrogen sources were varied from 0.5–4.0 % with step et al. [18], with 4% of sucrose. The medium was com- of 0.5%, and K HPO was varied from 1–6% with step of posed of sucrose, 4%; yeast extract, 2%; K HPO , 2%; 2 4 2 4 1%. The isolate was grown in different media at 23 °C MgSO ·7H O, 0.02%; MnSO ·4H O, 0.001%; FeSO ·7H O, 4 2 4 2 4 2 under static condition. Broth samples of 1 ml were peri- 0.001%; CaCl ·2H O, 0.001%; NaCl, 0.001%, while the 2 2 odically withdrawn and analyzed for enzyme activity. pH was adjusted to 6.9. The culture was incubated at 23 °C for 16 h under static condition. The culture broth Dextransucrase purification ° was then centrifuged at 7000 rpm at 4 C for 10 min, DS was precipitated from cell free supernatant by and the cell free supernatant was analyzed for enzyme addition of 25% PEG 400 (Zorka Pharma, Serbia) [21]. activity and protein concentration as described below. After precipitation at 4 °C for 24 h, the mixture was Enzyme assay and protein estimation centrifuged at 10000 rpm for 15 min at 4 °C. The pellet was dissolved in 20 mM sodium acetate buffer (pH 5.4). The enzyme assay was carried out in 450 μl reaction These fractions were subjected to dialysis using 10 kDa mixture containing 10% sucrose in 20 mM sodium ace- cutoff membrane (Thermo scientific, USA). The DS frac- tate buffer (pH 5.4) and 50 μl cell free supernatant at tions were analyzed for enzyme activity and protein 30 °C for 15 min. After that time the reaction was estimation. stopped by adding 500 μl of DNS. The enzyme activity was determined by measuring the concentration of rel- SDS-PAGE analysis and activity staining of eased reducing sugars by DNS method [19], using fruc- dextransucrase tose as a standard. The absorbance was measured at Sodium dodecyl sulfate-polyacrylamide gel electro- 540 nm using spectrophotometer (Ultrospec 3300 pro, phoresis (SDS-PAGE) was performed under denaturing Amersham Biosciences). One unit of DS activity was condition according to the method of Laemmli [22] defined as the amount of enzyme releasing 1 µmol of using 7.5% polyacrylamide running gel. The enzyme reducing sugars per min. was purified by 25% PEG 400 fractionation. The protein The protein concentration of the cell free super- samples were prepared in 0.0625 M Tris-HCl buffer (pH natant was determined using the method of Lowry et 6.8) containing 2% sodium dodecylsulfate, 10% gly- al. [20], using bovine serum albumin as a standard. cerol, 5% β-mercaptoethanol and 0.02% bromphenol Screening for factors affecting dextransucrase blue and boiled at 100 °C for 3 min. The electrophoresis production was carried out using Tris-Glycine buffer (pH 8.3) at room temperature with a current of 12 mA throughout Growth conditions (temperature and aeration) and stacking gel and of 25 mA throughout running gel culture medium composition (sucrose, nitrogen source, (Cleaver Scientific Ltd., OmniPAGE Mini System). Pro- K HPO and Tween 80) for enzyme production were 2 4 teins were stained using Coomassie brilliant blue G-250. screened and optimized. The in situ detection of DS activity in gel was carried Effects of growth conditions on dextransucrase out following the method of Holt et al. [23] with production modification described by Purama and Goyal [24]. The The influence of temperature and shaking on pro- protein samples for non-denaturing SDS-PAGE were duction of DS by L. mesenteroides T3 was tested. The prepared in the same manner as described above, with temperature was varied from 17 to 37 °C under static exclusion of β-mercaptoethanol component and boiling condition, using 100 ml Tsuchiya medium [18]. The step. The gel was washed in 20 mM sodium acetate effect of shaking condition on enzyme production was buffer solution (pH 5.4) with 0.3 mM CaCl2 and 0.1% analyzed at 90, 120, 150 and 180 rpm at fixed tempe- Tween 80 at 30 °C for 30 min to remove SDS. Sub- rature of 23 °C. Every 4 h, during the period of 24 h, sequent incubation in 20 mM sodium acetate buffer 352 M.G. MILJKOVIĆ et al.: DEXTRANSUCRASE FROM L.

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