Ann Microbiol (2014) 64:1715–1724 DOI 10.1007/s13213-014-0815-1

ORIGINAL ARTICLE

Isolation, purification and functional characterization of glucansucrase from probiotic Lactobacillus plantarum DM5

Deeplina Das & Arun Goyal

Received: 10 July 2013 /Accepted: 10 January 2014 /Published online: 28 January 2014 # Springer-Verlag Berlin Heidelberg and the University of Milan 2014

Abstract The probiotic Lactobacillus plantarum DM5 isolat- Introduction ed from fermented beverage Marcha of Sikkim was explored for its ability to produce extracellular glucansucrase during The glucansucrases, commonly called fermentation. The strain L. plantarum DM5 displayed glucosyltransferases (GTFs) (Leemhuis et al. 2013), from glucansucrase activity of 2.71 U/ml (0.48 U/mg) at 27 °C are well known for catalyzing the transfer under static condition. The medium compositions for of glucosyl units from the cleavage of to a growing α- glucansucrase production were optimized and it was found chain (Robyt et al. 2008; Purama and Goyal 2008a). that K2HPO4 (2.5 %, w/v), yeast extract (2.5 %, w/v), Tween Glucansucrase are classified as family 70 80 (0.6 %, v/v), and sucrose (5 %, w/v) enhanced the activity (GH70) according to the CAZy classification system by 20 %, 22 %, 68 % and 230 %, respectively. Glucansucrase (Cantarel et al. 2009). Glucansucrases are also often named was purified using polyethylene glycol 400 and 1500 frac- according to the product they synthesize, e.g. dextransucrase tionation followed by gel filtration that gave specific activity that synthesizes with α-(1 → 6) linkages and of 18.7 U/mg with 40-fold purification. Purified enzyme that synthesizes alternan with alternating α- exhibited maximum activity at 30 °C and pH 5.4. Zymogram (1 → 3) and α-(1 → 6) linkages (Leemhuis et al. 2013). So far, analysis of purified enzyme confirmed the presence of α-glucan formation by extracellular glucansucrase has been glucosyltransferase of approximately 148 kDa. The Vm and reported for the lactic acid bacteria of genera Lactobacillus, Km of purified glucansucrase for sucrose as substrate was 19.6 Leuconostoc, Streptococcus, Pediococcus and Weissella μmoles/mg/min and 4.5 mM. Divalent cations Mg2+,Ca2+ and (Shukla and Goyal 2011a;Leemhuisetal.2013). The pro- Co2+ enhanced glucansucrase activity by 16 %, 18 % and duction of glucan by the help of glucansucrase from lactic acid 19 %, respectively, whereas Hg2+ and Mn2+ decreased the bacteria has numerous potential applications as a viscosifier enzyme activity by 81 % and 79 %, respectively, when assayed and water-binding agent in both food and non-food industries in presence of sucrose. Among stabilizers, dextran T-40, PEG (Purama and Goyal 2008a; Badel et al. 2011). 6000, PEG 8000, glutaraldehyde, glycerol, Tween 80 and Among all species of Lactobacillus genera, Lactobacillus acetonitrile; Tween 80 provided maximum stabilization with reuteri 121, Lactobacillus sakei, Lactobacillus fermentum and half-life of 86 days at –20 °C. The overall biochemical char- Lactobacillus parabuchneri are known to produce acterization reveals a promising novel glucansucrase that can glucansucrase (van Hijum et al. 2006; van Leeuwen et al. compensate for the increasing demand of glucan as viscosifier 2009). Apart from these four Lactobacillus species, recently, and stabilizer in food industry. a novel species Lactobacillus satsumensis isolated from a fermented beverage starter culture produced extracellular Keywords Lactobacillus plantarum . Probiotic . glucansucrase that synthesized dextran (Cote et al. 2013). Glucansucrase . Glucan . Marcha . Tween 80 Several methods, such as fractionation by polyethylene gly- col, ultra-filtration, precipitation by salt, glycerol and alcohol, chromatography and phase-partitioning, are used for purifica- : * D. Das A. Goyal ( ) tion of glucansucrase (Nigam et al. 2006; Purama and Goyal Department of , Indian Institute of Technology Guwahati, Guwahati 781 039, Assam, India 2008a). The presence of associated glucan in glucansucrase e-mail: [email protected] during the purification results in aggregated forms of enzyme, 1716 Ann Microbiol (2014) 64:1715–1724 making the enzyme purification troublesome. However, the 27 °C for 18 h under static condition. The culture broth was purification of glucansucrase by polyethylene glycol fraction- then centrifuged at 10,000 g at 4 °C for 10 min, and the cell ation is a simple, effective and single step purification method, free supernatant was analyzed for enzyme activity and protein as it readily removed by dialysis (Purama and Goyal 2008a; concentration as described below. Majumder et al. 2008). Lactobacillus plantarum is one of the most studied lactic Enzyme assay and protein estimation acid bacteria, due to its ability to reduce and eliminate poten- tially pathogenic micro-organisms by synthesis of antimicro- The enzyme assay was carried out in 1 ml reaction mixture bial agents and by competition with pathogens for receptor containing 5 % (w/v) sucrose, 20 mM sodium acetate buffer sites at the intestinal mucosa (Adlerberth et al. 1996). (pH 5.4) and 20 μl cell free supernatant. The enzymatic Lactobacillus plantarum has been proven effective against reaction was performed at 30 °C in water bath for 15 min. diarrhea, irritable bowel disorder and lactose intolerance Aliquot of 100 μl from the reaction mixture was taken and the (Lonnermark et al. 2010); however, the strain has not thus enzyme activity was determined by estimating the released far been explored for production of glucansucrase. It has been reducing sugar by the Nelson and Somogyi method (Nelson reported that L. plantarum PL916 concomitantly produced 1944;Somogyi1945), using as a standard. The glucansucrase and fructansucrase during sourdough fermen- absorbance was measured at 500 nm using spectrophotometer tation (Cagno et al. 2006), but no report is available on the (Varian, Cary 100). One unit of glucansucrase activity was purification of glucansucrase from L. plantarum. A probiotic defined as the amount of enzyme producing 1 μmol of reduc- lactic acid bacterium, Lactobacillus plantarum DM5 ing sugar per min under the assays conditions of pH 5.4 and (Genbank Accession No: KC020195), was isolated from tra- 30 °C. The protein concentration of the cell free supernatant ditional fermented beverage Marcha of the biodiversity hot was determined using the method of Lowry et al. (1951), spot region Sikkim, India (Das and Goyal 2010), and its using Bovine serum albumin as a standard. ability to produce extracellular glucansucrase was explored, considering its enormous commercial applications in food Effect of temperature and aeration on glucansucrase industry. In the present study, we report the culture and nutri- production ent conditions for higher glucansucrase production from Lactobacillus plantarum DM5. The glucansucrase was puri- The production of enzyme by L. plantarum DM5 was studied fied by polyethylene glycol fractionation and gel filtration, under different physicochemical conditions, such as temper- and its enzymatic properties were also characterized. To the ature and shaking. The effect of temperature on enzyme best of our knowledge, this is the first report on purification of production was studied by varying the temperature from glucansucrase from Lactobacillus plantarum. 20 °C to 35 °C under static condition, using 100 ml of Tsuchiya medium (Tsuchiya et al. 1952). The effect of shak- ing condition on enzyme production was analyzed under Materials and methods different shaking condition of 90, 120, 150 and 180 rpm at 27 °C. At every 4 h until 36 h, culture of 500 μlwas Microorganism and culture medium withdrawn from each flask and centrifuged at 10,000 g and 4 °C for 10 min. The cell free supernatant was used for The strain L. plantarum DM5 was screened from an ethnic enzyme assay and the enzyme activity was calculated by fermented beverage Marcha of Sikkim on the basis of antimi- measuring the released reducing sugar as mentioned earlier. crobial activity against Escherichia coli by agar well diffusion Measurements were carried out in triplicate, and all the data method (Das and Goyal 2013), and maintained in modified expressed were the average of three independent experiments MRS agar medium (Goyal and Katiyar 1996) at 4 °C and with ± standard error. subcultured every 2 weeks. Effect of medium nutrients on glucansucrase production Production of glucansucrase from L. plantarum DM5 The effects of various medium components on glucansucrase The enzyme was produced by inoculating the isolate production were studied by changing the concentration of one L. plantarum DM5 in sterile 50 ml enzyme production medi- variable, while keeping other variables constant in Tsuchiya um as described by Tsuchiya et al. (1952). The composition of medium (Tsuchiya et al. 1952). The isolate was grown in enzyme production medium was (%, w/v) sucrose, 2; yeast different medium at 27 °C under static condition. Broth sam- extract, 2; K2HPO4,2;MgSO4·7H2O, 0.02; MnSO4·4H2O, ples of 5 ml were periodically withdrawn and analyzed for 0.001; FeSO4·7H2O, 0.001; CaCl2·2H2O, 0.001; NaCl, 0.001 enzyme activity by measuring the released reducing sugar. All and the pH was adjusted to 6.9. The culture was incubated at the experiments were carried out in triplicate and the data used Ann Microbiol (2014) 64:1715–1724 1717 was the average of three independent experiments with ± part of the gel was incubated in 20 mM sodium acetate buffer standard error. (pH 5.4) supplemented with 10 % sucrose at 30 °C for 48 h and the other part was incubated under the same conditions Purification of glucansucrase with 10 % raffinose instead of sucrose. After incubation, the gels were washed with 75 % ethanol for 40 min and incubated The purification of enzyme was carried out by fractionation in a periodic acid solution (periodic acid, 0.7 %, w/v and acetic with different concentrations of pre chilled polyethylene gly- acid, 5 %, v/v) for 1 h at 25 °C. After the periodic acid col (PEG) 400 ranging from 25 to 40 % (v/v, final concentra- treatment, the gels were washed with a solution containing tion) and PEG 1500 ranging from 10 to 25 % (w/v, final 0.2 % (w/v) sodium metabisulphite and 5 % (v/v) acetic acid. concentration) in 50 ml cell free supernatant. The mixture Finally the gels were stained with 15 ml Schiff reagent (0.5 %, was incubated overnight at 4 °C to allow the enzyme to w/v Fuchsin basic, 1 %, w/v sodium bisulphite and 0.1 N HCl) precipitate and then centrifuged at 10,000 g at 4 °C for until the discrete magenta colored band within the gel matrix 30 min to separate the fractionated enzyme (Purama and appeared. The gels were washed in distilled water to remove Goyal 2008a). The enzyme pellet was dissolved in 20 mM the excess stain and stored in 10 % (v/v) acetic acid at 25 °C. sodium acetate buffer (pH 5.4) and subjected to dialysis using 14 kDa cut-off membrane (Hi-media Pvt. Ltd., India). The Optimum temperature and thermal stability of glucansucrase dialyzed enzyme was further purified by gel filtration column (1.5 cm × 50 cm) using Sephacryl S-300HR as the matrix. The The optimum assay temperature of glucansucrase was studied column was pre-equilibrated with 20 mM sodium acetate by adding 20 μl of the purified enzyme (18.7 U/mg, buffer (pH 5.4) and was eluted using 20 mM sodium acetate 0.08 mg/ml) to 1 ml enzyme reaction mixture containing buffer (pH 5.4) at a flow rate of 0.3 ml/min, and fractions of 150 mM sucrose in 20 mM sodium acetate buffer (pH 5.4). 3 ml were collected. The purified fractions showing maximum The reaction mixture was incubated at different temperatures specific activity were pooled and analyzed for enzyme activity varying from 15 °C to 55 °C for 15 min. Of the reaction and protein content. mixture, 100 μl was taken for reducing sugar estimation by the Nelson and Somogyi method (Nelson 1944; Somogyi SDS-PAGE analysis and activity staining of glucansucrase 1945). The thermostability of the enzyme was determined by incubating 0.5 ml of enzyme (18.7 U/mg, 0.08 mg/ml) at The molecular weight of glucansucrase was determined by different temperatures ranging from 10 to 50 °C for 30 min, 7.5 % (w/v) SDS-PAGE analysis under denaturing condition and 20 μl aliquots of enzyme were assayed for residual following the method of Laemmli, (1970). The enzymes were enzyme activity as described earlier. Measurements were car- purified by 15 % PEG 1500 fractionation and by gel filtration ried out in triplicate for temperature optima and temperature (Sephacryl S-300HR) were run on the gel along with protein stability experiments. molecular weight marker from Fermentas International Inc. The protein samples were prepared in 0.0625 M Tris–HCl Optimum pH and pH stability of glucansucrase buffer (pH 6.8) containing 2.8 % (w/v) sodium-dodecyl sul- fate, 10 % (w/v) glycerol, 5 % (w/v) β-mercaptoethanol and The optimum pH of purified enzyme was determined by incu- 0.05 % (w/v) bromophenol blue and boiled at 100 °C for bating 20 μl (18.7 U/mg, 0.08 mg/ml) of purified enzyme in 4 min. The electrophoresis was carried out using Tris- 1 ml reaction mixture containing 150 mM concentration of Glycine buffer (pH 8.3) at room temperature with a current sucrose in 20 mM sodium acetate buffer of different pH, rang- of 2 mA per lane. After the migration protein bands were ing from 3 to 7. The reaction mixture was incubated at 30 °C for stained with silver solution (Rabilloud 1992). 15 min and reducing sugar estimation was done as mentioned The in situ glucansucrase activity was determined follow- previously. In order to determine the pH stability, 100 μlof ing the method of Holt et al. (2001) with minor modifications purified enzyme was incubated at different pHs ranging from (Purama and Goyal 2008a). The enzymes samples purified by 3.0 to 6.0 in 20 mM sodium acetate buffer and pH 6.2 to pH 8.4 15 % PEG 1500 fractionation and by gel filtration were loaded in 20 mM sodium phosphate buffer at 30 °C for 30 min, and in duplicate on 7.5 % acrylamide gel and run under non- then the aliquots of 20 μl were assayed for residual enzyme denaturing condition. The protein samples for non- activity as described earlier. Measurements were carried out in denaturing SDS-PAGE were prepared in the same manner as triplicate for pH optima and pH stability experiments. above, but without the addition of β-mercaptoethanol and they were not subjected to boiling. After the run, the gel was Determination of kinetic parameters of glucansucrase divided into two parts and both the parts were washed thrice by 20 mM sodium acetate buffer (pH 5.4) with 0.3 mM CaCl2 The purified glucansucrase (18.7 U/mg, 0.08 mg/ml) was used and 0.1 %, v/v Tween 80, incubating at 30 °C for 30 min. One to study the effect of sucrose concentration on its activity. The 1718 Ann Microbiol (2014) 64:1715–1724 reaction was carried out in 1 ml 20 mM sodium acetate buffer Results and discussion (pH 5.4) containing 20 μl of enzyme and varying concentra- tions of sucrose ranging from 0.05 mM to 400 mM at 30 °C Effect of temperature and aeration on glucansucrase for 15 min. The enzyme activity was determined by estimating production from L. plantarum DM5 the released reducing sugar, as mentioned earlier. The data were used to generate a Lineweaver-Burk plot, and the kinetic The effect of incubation temperature on enzyme production parameters were analyzed from the plot. was studied by varying the temperature from 20 °C to 35 °C, and the maximum activity of 2.71±0.39 U/ml was observed at Effect of salts and denaturing agent on glucansucrase activity 27 °C under static condition (Fig. 1a). The enzyme activity was decreased as the temperature increased above 27 °C, and

The effects of CaCl2,MgCl2, CoCl2,MnSO4 and HgCl2 it decreased by 47 % at 35 °C due to deactivation of the between 0 and 12 mM and EDTA between 0 and 5 mM enzyme at higher temperatures. The enzyme activity was concentrations were studied on the activity of purified lower by 35 % at 20 °C, which might be due to the slower glucansucrase (18.7 U/mg, 0.08 mg/ml). The assays were growth rate of cells consequently resulting in lower enzyme carried out in 1.0 ml reaction mixtures containing the salt or production. The enzyme production was also studied at dif- EDTA, the substrate sucrose (5 %, w/v) in 20 mM sodium ferent shaking conditions of 90 to 180 rpm at 27 °C, and was acetate buffer (pH 5.4), and 20 μlenzyme.Theeffectofurea compared with the static condition (Fig. 1b). The enzyme was studied by prior incubation of enzyme with urea (0–5M, activity of 2.4±0.23 U/ml was observed at 90 rpm at 27 °C, final concentration) at 30 °C for 30 min. Aliquots of 20 μl which was 11 % less than static condition (2.7 U/ml). These were taken and enzyme activity was measured as described results indicated the microaerophilic nature of the bacterium. earlier, and the percent residual activities were calculated with respect to activity in the absence of metal ion and denaturing Effect of medium nutrients on glucansucrase production compound. Measurements were carried out in triplicate and all the data expressed were the averages of three independent Effect of sucrose experiments with ± standard error. The effect of sucrose concentration (1 to 7 %, w/v) on Effect of stabilizer on glucansucrase activity glucansucrase production was studied and compared with the control medium containing 2 % (w/v) sucrose as described To study the effect of different additives on the stability of by Tsuchiya et al. (1952). The maximum enzyme activity of glucansucrase, aqueous solutions of dextran T-40, PEG-6000, 6.23±0.11 U/ml was observed at 5 % (w/v) sucrose PEG-8000, glutaraldehyde, glycerol, Tween-80 and acetonitrile concentration (Fig. 2a). The production of enzyme de- were added to purified glucansucrase (18.7 U/mg, 0.08 mg/ml) creased after 5 % (w/v) sucrose concentration and de- in sodium acetate buffer (pH 5.4) to obtain the final concentra- creasedby21%at7%(w/v)sucroseconcentration tions of 2 μg/ml dextran T-40, 10 μg/ml PEG-6000, 10 μg/ml (Fig. 2a), which might be due to the subsequent utilization of PEG-8000, 0.1 % glutaraldehyde, 0.5 % glycerol, 1 % aceto- available sucrose for the formation of exopolysaccharide by the nitrile and 10 μl/ml Tween 80, respectively, in a final volume of released enzyme. The enhancement of glucansucrase ac- 0.6 ml and stored at 30 °C for 36 h. At different time intervals, tivity by threefold has also been reported in the case of 20 μl of samples were taken and analyzed for residual enzyme Leuconostoc mesenteroides NRRL B-640 (Purama and Goyal activity as mentioned earlier. All the experiments were carried 2008b)andWeissella confusa Cab3 (Shukla and Goyal 2011b) out in triplicate, and the data used were the averages of three in the presence of 7 % and 5 % (w/v) sucrose, respectively. independent experiments with ± standard error. Effect of nitrogen source Effect of storage temperature on stability of glucansucrase The effects of various nitrogen sources like yeast extract, The storage temperature of purified glucansucrase was studied peptone and beef extract on glucansucrase production by by incubating the enzyme at different temperatures (0 °C, 4 °C L. plantarum DM5 were studied. It was found that yeast and −20 °C) with or without additives. The additives used for extract enhanced the production of glucansucrase, and maxi- long-term storage of glucansucrase were dextran T-40 mum enzyme activity of 3.3±0.21 U/ml was achieved at (2 μg/ml) and Tween 80 (10 μl/ml). Samples of 20 μlwere 2.5 % (w/v) yeast extract (Fig. 2b). Beyond 2.5 % yeast taken at different time intervals and analyzed for enzyme extract, the enzyme activity progressively decreased and en- activity as described earlier. The assay was performed in zyme activity of 1.31 U/ml was observed at 4 % (w/v) yeast optimum conditions and the data presented are mean values extract (Fig. 2b), which was 52 % lesser as compared to of three independent experiments with ± standard error. control medium (2.71 U/ml), which contains 2 % (w/v) yeast Ann Microbiol (2014) 64:1715–1724 1719

Fig. 1 Glucansucrase production from Lactobacillus plantarum DM5. a Effect of temperature rangingfrom20to35°Con glucansucrase production under static condition. b Effect of agitation speeds of 90, 120, 150 and 180 rpm on glucansucrase production at 27 °C. The mean value of three independent experiments was presented with ± standard error

extract. The effects of peptone and beef extract as sole nitrogen reports where yeast extract served as a source of vitamin and source on glucansucrase production were studied by varying supplement in the production of glucansucrase their concentration from 0.5 % to 4 % as shown in Fig. 2b.The (Majumder and Goyal 2008; Purama and Goyal 2008b). maximum enzyme activity of 2.4±0.19 U/ml and 1.9±0.20 However, the addition of 1.5 % (w/v) peptone or 2 % (w/v) U/ml was observed by the addition of 1.5 % (w/v) peptone beef extract in control medium (with 2 %, w/v yeast extract) and 2 % (w/v) beef extract, respectively (Fig. 2b), with a enhanced the enzyme production by 29 % and 14 %, respec- reduction of enzyme activity by 11 % and 30 % as compared tively. Similar results were also observed in the case of to control medium, which contained 2 % (w/v) yeast extract. Leuconostoc mesenteroides NRRL B-640 (Purama and Goyal The results indicated that the yeast extract was the most effec- 2008b)andLeuconostoc mesenteroides PCSIR-3 (Ul-Qader tive nitrogen source for production of glucansucrase from et al. 2001), where peptone and beef extract in addition to yeast L. plantarum DM5, and this was in good agreement with other extract also resulted in enhanced enzyme activity.

Fig. 2 Effect of a Sucrose, b Nitrogen source, c Tween 80 and dK2HPO4 on glucansucrase production from Lactobacillus plantarum DM5 at 27 °C.Yeast extract (black shaded box), beef extract (grey shaded box)and peptone (olive green shaded box) were used as sole nitrogen source for production of enzyme. The mean value of three independent experiments was presented with ± standard error 1720 Ann Microbiol (2014) 64:1715–1724

Effect of Tween 80 and K2HPO4 homogeneous fractions were pooled and showed a specific activity of 18.7 U/mg with 40-fold purification (Table 1). The effect of Tween 80 on enzyme production was studied by varying its concentration from 0.1 to 1 % (v/v) in the control SDS-PAGE analysis and activity staining of glucansucrase medium. It was observed that the addition of 0.1 % Tween 80 in the control medium (without Tween 80) stimulated the The molecular weight of the purified enzyme (15 % PEG enzyme production by 8 % (Fig. 2c). Increasing the concentra- 1500) was determined by 7.5 % (w/v) SDS polyacrlymide tion of Tween 80 further increased the enzyme activity, and the gel under denaturing condition and showed two isoforms of maximum enzyme activity of 4.54±0.20 U/ml was observed at approximately 189 kDa and 148 kDa with silver staining 0.6 % (v/v) Tween 80 (Fig. 2c), which was 68 % higher as solution (Fig. 3b, Lane 2). The purified enzyme (15 % PEG compared with the control medium (2.71 U/ml), which did not 1500) was also run on SDS-PAGE gels under nondenaturing contain Tween 80. It has been reported that the use of the condition for in situ activity detection of glucansucrase by PAS surfactant Tween 80 increases the glucansucrase secretion from staining. The PAS staining of the gel showed three activity the cells by altering the fatty acid composition of the cell bands of molecular weights of approximately 189 kDa, membrane (Sato et al. 1989; Majumder and Goyal 2008). 150 kDa and 148 kDa when incubated in 10 % sucrose

The effect of K2HPO4 on enzyme production was also solution. The faint band of 150 kDa observed only in PAS studied by varying its concentration from 1 to 4 % in the staining but not in silver staining might be due to the active enzyme production medium. It was observed that the maxi- form being present in very low amounts (Pg level) that could mum enzyme activity of 3.2±0.48 U/ml was achieved at not be stained by the silver staining. As the active form of

2.5 % (w/v) K2HPO4 concentration (Fig. 2d). The higher enzyme in pg levels also synthesized glucan in the presence of enzyme activity in presence of K2HPO4 might be due to sucrose, it was easily detected by PAS staining. It was observed buffering activity of K2HPO4 in the medium, which lowered that the most active form of glucansucrase was 148 kDa, as it the effect of lactic acid production during the fermentation. showed an intense band with PAS staining and as well as silver

However, the concentration beyond 2.5 % K2HPO4 did not staining (Fig. 3b, Lane 1 and Lane 3). It has been reported that support the enzyme activity and at 3.5 % K2HPO4, it was 1.84 the molecular weight of extracellular glucansucrase was in the ±0.27 U/ml and causing a 44 % decrease (Fig. 2d). range of 120–200 kDa (Leemhuis et al. 2013) and can exist in multiple molecular forms (Purama and Goyal 2008a; Patel Purification of enzyme by polyethylene glycol and gel et al. 2011). No band after activity staining of purified enzyme filtration by 15 % PEG 1500 fractionation was observed upon the incubation of the gel with raffinose, as shown in Fig. 3b, The cell free supernatant containing extracellular Lane 5, which excluded the presence of fructosyltransferase. glucansucrase (0.48 U/mg, 5.7 mg/ml) was subjected to frac- The partially purified enzyme (15 % PEG 1500) was further tionation with various concentrations of PEG 400 and PEG purified by gel filtration, which showed a single distinct band 1500. The maximum specific activity of 6.68 U/mg and 10.1 of a molecular size of approximately 148 kDa on silver staining U/mg was achieved at 36 % PEG 400 and 15 % PEG 1500, (Fig. 3b, Lane 2), as well as on PAS staining (Fig. 3b, Lane 4) respectively (Table 1). The purified enzyme by 15 % PEG after the gel was incubated in 10 % sucrose solution. 1500 fractionation exhibited 21-fold purification with 14 % overall yield in a single step (Table 1). Since the purified Optimum temperature and thermostability of glucansucrase enzyme by PEG 1500 (15 %) showed maximum activity, it was further subjected to gel filtration using Sephacryl The purified glucansucrase showed maximum activity within the S-300HR. The enzyme eluted in the form of a single asym- temperature range of 30–33 °C with a specific activity of ∼18.6 metrical peak (Fig. 3a) and most of the maximum activity was U/mg at pH 5.4 in 20 mM sodium acetate buffer (Fig. 4a). This confined between the eighth and tenth fractions. These result was in accordance with the earlier findings that the

Table 1 Purification of glucansucrase from L. plantarum DM5

Volume (ml) Enzyme activity Total units Overall Protein Total (mg) Specific activity Fold purifi- (U/ml) yield (%) (mg/ml) (U/mg) cation

Crude (cell free supernatant) 50 2.71 136 – 5.70 285 0.48 – Fractionation by PEG-400 (36 %) 5.4 2.54 14 10 0.38 2.1 6.68 7 Fractionation by PEG 1500 (15 %) 5.0 3.84 19 14 0.38 2 10.1 21 Gel filtration by Sephacryl S-300HR 9 1.50 13 9 0.08 0.72 18.7 40 Ann Microbiol (2014) 64:1715–1724 1721

Fig. 3 Purification of glucansucrase from Lactobacillus plantarum purified glucansucrase by 15 % PEG 1500 fractionation under denaturing DM5. a Elution profile of glucansucrase given by gel filtration using condition (silver stained), Lane 2: purified glucansucrase by gel filtration Sephacryl S-300HR. The flow rate was 0.3 ml/min and fractions of 3 ml using Sephacryl S-300 HR under denaturing condition (silver stained), were collected. The fractions were assayed for enzyme activity (−−■–) Lane 3: glucansucrase purified by 15 % PEG 1500 (PAS Staining) and and protein concentration (−−○–). b SDS-PAGE (7.5 %) analysis of Lane 4: purified glucansucrase by glucansucrase using Sephacryl purified glucansucrase. After the electrophoresis, the gels were cut into S-300HR by PAS staining of glucan formed using sucrose as substrate; two parts for silver staining and for activity staining by periodic acid Lane 5: Absence of fructansucrase and confirmation of glucansucrase by schiff (PAS) reagent. Lane M: Protein Molecular Mass marker (10 kDa to PAS staining method using raffinose as substrate 200 kDa) from Fermentas International Inc. (silver stained), Lane 1: optimum temperature for glucansucrase enzyme activity was the strains Leuconostoc mesenteroides NRRL B-640 (Purama within the range of 30 to 35 °C (Majumder and Goyal 2008; and Goyal 2008a), Weissella confusa Cab3 (Shukla and Goyal Pateletal.2011). The loss of activity was observed on either side 2011a)andPediococcus pentosaceus SPA (Patel et al. 2011). of 30–33 °C. The enzyme activity rapidly decreased after 37 °C The enzyme activity decreased sharply below pH 4.5 and above and by 94 % at 55 °C. The thermostability results showed that the pH 5.5. The enzyme lost 38 % and 93 % activity at pH 6.6 and enzyme was stable at lower temperatures (10–30 °C) and rapidly pH 3.4, respectively (Fig. 4b). The enzyme was stable in acidic lost activity at temperatures higher than 35 °C. The enzyme could pH (4.6–5.8) range like other glucansucrases from retain only 6 % of its initial activity at 50 °C (Fig. 4a). Leuconostoc dextranicum NRRL B-1146 (Majumder et al. 2008)andPediococcus pentosaceus SPA (Patel et al. 2011). Optimum pH and pH stability of glucansucrase Determination of kinetic parameters of glucansucrase The maximum glucansucrase activity was observed at pH 5.4 with a specific activity of 18.6 U/mg (Fig. 4b). The optimum pH The effect of sucrose concentration on the enzyme activity was of 5.4 of glucansucrase from L. plantarum DM5 was similar to studied with varying sucrose concentration between 0.05 mM

Fig. 4 Effect of temperature (a) and pH (b) on activity (−−●–)and enzyme was incubated at different temperatures (10–50 °C) and different stability (−−■–) of glucansucrase. The enzyme activity measurements pHs (3–9) for 30 min. The assay was performed in optimum conditions for pH and temperature optima were performed in pH (3.6–6.6) and and the data presented are mean values of three independent experiments temperature (10–55 °C) ranges, respectively. For stability studies, the with ± standard error 1722 Ann Microbiol (2014) 64:1715–1724 to 400 mM. The results showed that it follows the classical 30 h. The enzyme without any stabilizer (control) lost its

Michaelis-Menten kinetics and enzyme saturation was activity rapidly (t1/2=10.3 h) at 30 °C, and only 13.3 % resid- reached at 146 mM (5 %, w/v) sucrose concentration. The ual activity remained after 30 h (Table 3). Glycerol, PEG 6000 purified glucansucrase gave Vm of 19.6±0.83 μmole/mg/min and PEG 8000 acted as stabilizers and displayed stabilizing and Km of 4.5 mM±0.58 mM, which indicated that the en- effects on glucansucrase, as the residual activity was 42 % zyme has a high affinity and specificity for the substrate, and (t1/2=24 h), 38 % (t1/2=21.5 h) and 33 % (t1/2=18.6 h) respec- hence is more effective for production of glucan. The value of tively, whereas glutaraldehyde and acetonitrile (t1/2=5handt1/ Km of glucansucrase from L. plantarum DM5 was comparable 2=7 h, respectively) acted as inhibitsor of enzyme, as activity with the Km value (3.2±0.02 mM) of glucansucrase from was lost by 95 % and 92 %, respectively. Among all the Lactobacillus reuteri 180 (Pijning et al. 2008). stabilizers, Tween 80 displayed the maximum stabilization

of glucansucrase (t1/2 of 64.8 h), followed by dextran T-40 Effect of salts and denaturing agent on glucansucrase activity (t1/2=41 h) at 30 °C. Therefore, Tween 80 and dextran T-40 were used as stabilizers for investigation of long-term storage The Mg2+,Ca2+ and Co2+ salts at low concentrations exhibited a stability at temperatures of −20 °C, 0 °C and 4 °C. The marginal increase in glucansucrase activity, as shown in Table 2. stabilization data of glucansucrase from L. plantarum

The addition of 1 mM MgCl2, 2 mM CaCl2 and3mMCoCl2 to DM5 was in accordance with a previous report, where glucansucrase caused enhancement of enzyme activity by 16 %, glucansucrase from Leuconostoc mesenteroides NRRL 18 % and 19 %, respectively (Table 2). It has been reported that B-512 F showed stabilization in the presence of high these salts stabilize the active site of the enzyme by stabilizing the molecular weight dextran (2 μg/ml) and non-ionic detergent three-dimensional protein structure (Miller and Robyt 1984; Tween 80 (10 μg/ml) (Miller and Robyt 1984).

Majumder et al. 2008). The addition of 0.5 mM MnSO4 and To study suitable storage temperature, glucansucrase was 0.5 mM HgCl2 resulted in 78 % and 71 % inhibition of enzyme incubated with or without Tween 80 or dextran T-40 at three activity, and activity decreased by 79 % and 81 % in the presence different temperatures (−20 °C, 0 °C and 4 °C) and half-lives of 8 mM MnSO4 and8mMHgCl2, respectively. Urea and were calculated (Fig. 5). The enzyme in the presence of EDTA at all concentrations displayed a denaturing effect on dextran T-40 and Tween 80 exhibited enhanced half-life of glucansucrase, and 97 % and 69 % of enzyme inactivation was 80 days and 86 days, respectively, as compared with the observedwith6Mureaand6mMEDTA,respectively(Table2). control (t1/2=65 d) at −20 °C; however, Tween 80 provided Similar results were also found in the case of glucansucrase from 33 %, 35 % and 16 % higher stabilization than dextran T-40 at Leuconostoc dextranicum NRRL B-1146 (Majumder et al. 2008) all the three temperatures of 4 °C, 0 °C and −20 °C. From all and Pediococcus pentosaceus SPA (Patel et al. 2011) for effects above data, it can be summarized that Tween 80 as an additive of chaotropic agents, urea and EDTA. (t1/2=86 days) proved to be best for the stabilization of glucansucrase from L. plantarum DM5 and could be used Effect of stabilizers and storage temperature on glucansucrase for long-term storage. activity Table 3 Effect of stabilizers on glucansucrase activity at 30°C

The residual enzyme activity of glucansucrase was measured Stabilizera Residual Half-life Decay b, d d d at 30 °C with respect to time and with or without stabilizer for Activity (%) (t1/2)(h) constant (λ)

c Table 2 Effect of salts and chaotropic agent on the activity of purified Control 13.3±2.2 10.3±0.1 1.6±1.2 glucansucrase Glycerol (0.5 %, v/v) 42.1±1.2 24.0±1.0 0.8±1.1 PEG 6000 (10 μg/ml, w/v) 38.0±1.9 21.5±1.0 0.8±1.0 Regents Residual activity (%)a PEG 8000 (10 μg/ml, w/v) 33.0±2.0 18.6±0.9 0.9±1.5 Maximum Minimum Dextran T40 (2 μg/ml, w/v) 56.3±1.3 41.0±1.3 0.4±1.1 Glutaraldehyde (0.1 %, v/v) 5.4±2.5 5.0±1.2 3.4±1.2 MgCl 116.2 ± 2.1 (1 mM) 86.1±1.6 (12 mM) 2 Acetonitrile (1 %, v/v) 8.3±1.2 7.0±1.0 2.5±1.5 CaCl 118.3±1.3 (2 mM) 85.2±1.1 (12 mM) 2 Tween 80 (10 μl/ml, v/v) 86.0±1.4 64.8±1.3 0.3±0.8 CoCl2 119.0±1.8 (3 mM) 65.2±1.2 (12 mM) a MnSO4 78.1±2.1 (0.5 mM) 21.4±1.3 (8 mM) The enzyme with or without any stabilizer incubated at 30 °C for 30 h b HgCl2 71.3±1.1 (0.5 mM) 19.2±1.0 (8 mM) Residual enzyme activity was calculated by considering the initial Urea 58.4±1.4 (0.5 M) 3.2±1.3 (6 M) enzyme activity as 100 % c EDTA 70.1±1.01 (1 mM) 31.3±1.2 (6 mM) The enzyme without any stabilizer incubated at 30 °C for 30 h and residual activity was calculated from initial activity as 100 % a The mean values of three independent experiments are presented with ± d The mean value of three independent experiments was presented with ± standard error standard error Ann Microbiol (2014) 64:1715–1724 1723

References

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