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

Abstract

The probiotic Lactobacillus plantarum DM5 isolated from fermented beverage Marcha of Sikkim was explored for its ability to produce extracellular glucansucrase during fermentation. The strain L. plantarum DM5 displayed glucansucrase activity of 2.71 U/ml (0.48 U/mg) at 27 °C under static condition. The medium compositions for glucansucrase production were optimized and it was found that K₂HPO₄ (2.5 %, w/v), yeast extract (2.5 %, w/v), Tween 80 (0.6 %, v/v), and sucrose (5 %, w/v) enhanced the activity by 20 %, 22 %, 68 % and 230 %, respectively. Glucansucrase was purified using polyethylene glycol 400 and 1500 fractionation followed by gel filtration that gave specific activity of 18.7 U/mg with 40-fold purification. Purified enzyme exhibited maximum activity at 30 °C and pH 5.4. Zymogram analysis of purified enzyme confirmed the presence of glucosyltransferase of approximately 148 kDa. The V_m and K_m of purified glucansucrase for sucrose as substrate was 19.6 μmoles/mg/min and 4.5 mM. Divalent cations Mg²⁺, Ca²⁺ and Co²⁺ enhanced glucansucrase activity by 16 %, 18 % and 19 %, respectively, whereas Hg²⁺ and Mn²⁺ decreased the enzyme activity by 81 % and 79 %, respectively, when assayed in presence of sucrose. Among stabilizers, dextran T-40, PEG 6000, PEG 8000, glutaraldehyde, glycerol, Tween 80 and acetonitrile; Tween 80 provided maximum stabilization with half-life of 86 days at –20 °C. The overall biochemical characterization reveals a promising novel glucansucrase that can compensate for the increasing demand of glucan as viscosifier and stabilizer in food industry.

Introduction

The enzyme glucansucrases, commonly called glucosyltransferases (GTFs) (Leemhuis et al. 2013), from lactic acid bacteria are well known for catalyzing the transfer of glucosyl units from the cleavage of sucrose to a growing α-glucan chain (Robyt et al. 2008; Purama and Goyal 2008a). Glucansucrase are classified as glycoside hydrolase family 70 (GH70) enzymes according to the CAZy classification system (Cantarel et al. 2009). Glucansucrases are also often named according to the product they synthesize, e.g. dextransucrase that synthesizes dextran with α-(1 → 6) linkages and alternansucrase that synthesizes alternan with alternating α-(1 → 3) and α-(1 → 6) linkages (Leemhuis et al. 2013). So far, α-glucan formation by extracellular glucansucrase has been reported for the lactic acid bacteria of genera Lactobacillus, Leuconostoc, Streptococcus, Pediococcus and Weissella (Shukla and Goyal 2011a; Leemhuis et al. 2013). The production of glucan by the help of glucansucrase from lactic acid bacteria has numerous potential applications as a viscosifier and water-binding agent in both food and non-food industries (Purama and Goyal 2008a; Badel et al. 2011).

Among all species of Lactobacillus genera, Lactobacillus reuteri 121, Lactobacillus sakei, Lactobacillus fermentum and Lactobacillus parabuchneri are known to produce glucansucrase (van Hijum et al. 2006; van Leeuwen et al. 2009). Apart from these four Lactobacillus species, recently, a novel species Lactobacillus satsumensis isolated from a fermented beverage starter culture produced extracellular glucansucrase that synthesized dextran (Cote et al. 2013). Several methods, such as fractionation by polyethylene glycol, ultra-filtration, precipitation by salt, glycerol and alcohol, chromatography and phase-partitioning, are used for purification of glucansucrase (Nigam et al. 2006; Purama and Goyal 2008a). The presence of associated glucan in glucansucrase during the purification results in aggregated forms of enzyme, making the enzyme purification troublesome. However, the purification of glucansucrase by polyethylene glycol fractionation is a simple, effective and single step purification method, as it readily removed by dialysis (Purama and Goyal 2008a; Majumder et al. 2008).

Lactobacillus plantarum is one of the most studied lactic acid bacteria, due to its ability to reduce and eliminate potentially pathogenic micro-organisms by synthesis of antimicrobial agents and by competition with pathogens for receptor sites at the intestinal mucosa (Adlerberth et al. 1996). Lactobacillus plantarum has been proven effective against diarrhea, irritable bowel disorder and lactose intolerance (Lonnermark et al. 2010); however, the strain has not thus far been explored for production of glucansucrase. It has been reported that L. plantarum PL916 concomitantly produced glucansucrase and fructansucrase during sourdough fermentation (Cagno et al. 2006), but no report is available on the purification of glucansucrase from L. plantarum. A probiotic lactic acid bacterium, Lactobacillus plantarum DM5 (Genbank Accession No: KC020195), was isolated from traditional fermented beverage Marcha of the biodiversity hot spot region Sikkim, India (Das and Goyal 2010), and its ability to produce extracellular glucansucrase was explored, considering its enormous commercial applications in food industry. In the present study, we report the culture and nutrient conditions for higher glucansucrase production from Lactobacillus plantarum DM5. The glucansucrase was purified by polyethylene glycol fractionation and gel filtration, and its enzymatic properties were also characterized. To the best of our knowledge, this is the first report on purification of glucansucrase from Lactobacillus plantarum.

Materials and methods

Microorganism and culture medium

The strain L. plantarum DM5 was screened from an ethnic fermented beverage Marcha of Sikkim on the basis of antimicrobial activity against Escherichia coli by agar well diffusion method (Das and Goyal 2013), and maintained in modified MRS agar medium (Goyal and Katiyar 1996) at 4 °C and subcultured every 2 weeks.

Production of glucansucrase from L. plantarum DM5

The enzyme was produced by inoculating the isolate L. plantarum DM5 in sterile 50 ml enzyme production medium as described by Tsuchiya et al. (1952). The composition of enzyme production medium was (%, w/v) sucrose, 2; yeast extract, 2; K₂HPO₄, 2; MgSO₄ · 7H₂O, 0.02; MnSO₄ · 4H₂O, 0.001; FeSO₄ · 7H₂O, 0.001; CaCl₂ · 2H₂O, 0.001; NaCl, 0.001 and the pH was adjusted to 6.9. The culture was incubated at 27 °C for 18 h under static condition. The culture broth was then centrifuged at 10,000 g at 4 °C for 10 min, and the cell free supernatant was analyzed for enzyme activity and protein concentration as described below.

Enzyme assay and protein estimation

The enzyme assay was carried out in 1 ml reaction mixture containing 5 % (w/v) sucrose, 20 mM sodium acetate buffer (pH 5.4) and 20 μl cell free supernatant. The enzymatic reaction was performed at 30 °C in water bath for 15 min. Aliquot of 100 μl from the reaction mixture was taken and the enzyme activity was determined by estimating the released reducing sugar by the Nelson and Somogyi method (Nelson 1944; Somogyi 1945), using fructose as a standard. The absorbance was measured at 500 nm using spectrophotometer (Varian, Cary 100). One unit of glucansucrase activity was defined as the amount of enzyme producing 1 μmol of reducing sugar per min under the assays conditions of pH 5.4 and 30 °C. The protein concentration of the cell free supernatant was determined using the method of Lowry et al. (1951), using Bovine serum albumin as a standard.

Effect of temperature and aeration on glucansucrase production

The production of enzyme by L. plantarum DM5 was studied under different physicochemical conditions, such as temperature and shaking. The effect of temperature on enzyme production was studied by varying the temperature from 20 °C to 35 °C under static condition, using 100 ml of Tsuchiya medium (Tsuchiya et al. 1952). The effect of shaking condition on enzyme production was analyzed under different shaking condition of 90, 120, 150 and 180 rpm at 27 °C. At every 4 h until 36 h, culture of 500 μl was withdrawn from each flask and centrifuged at 10,000 g and 4 °C for 10 min. The cell free supernatant was used for enzyme assay and the enzyme activity was calculated by measuring the released reducing sugar as mentioned earlier. Measurements were carried out in triplicate, and all the data expressed were the average of three independent experiments with ± standard error.

Effect of medium nutrients on glucansucrase production

The effects of various medium components on glucansucrase production were studied by changing the concentration of one variable, while keeping other variables constant in Tsuchiya medium (Tsuchiya et al. 1952). The isolate was grown in different medium at 27 °C under static condition. Broth samples of 5 ml were periodically withdrawn and analyzed for enzyme activity by measuring the released reducing sugar. All the experiments were carried out in triplicate and the data used was the average of three independent experiments with ± standard error.

Purification of glucansucrase

The purification of enzyme was carried out by fractionation with different concentrations of pre chilled polyethylene glycol (PEG) 400 ranging from 25 to 40 % (v/v, final concentration) and PEG 1500 ranging from 10 to 25 % (w/v, final concentration) in 50 ml cell free supernatant. The mixture was incubated overnight at 4 °C to allow the enzyme to precipitate and then centrifuged at 10,000 g at 4 °C for 30 min to separate the fractionated enzyme (Purama and Goyal 2008a). The enzyme pellet was dissolved in 20 mM sodium acetate buffer (pH 5.4) and subjected to dialysis using 14 kDa cut-off membrane (Hi-media Pvt. Ltd., India). The dialyzed enzyme was further purified by gel filtration column (1.5 cm × 50 cm) using Sephacryl S-300HR as the matrix. The column was pre-equilibrated with 20 mM sodium acetate buffer (pH 5.4) and was eluted using 20 mM sodium acetate buffer (pH 5.4) at a flow rate of 0.3 ml/min, and fractions of 3 ml were collected. The purified fractions showing maximum specific activity were pooled and analyzed for enzyme activity and protein content.

SDS-PAGE analysis and activity staining of glucansucrase

The molecular weight of glucansucrase was determined by 7.5 % (w/v) SDS-PAGE analysis under denaturing condition following the method of Laemmli, (1970). The enzymes were purified by 15 % PEG 1500 fractionation and by gel filtration (Sephacryl S-300HR) were run on the gel along with protein molecular weight marker from Fermentas International Inc. The protein samples were prepared in 0.0625 M Tris–HCl buffer (pH 6.8) containing 2.8 % (w/v) sodium-dodecyl sulfate, 10 % (w/v) glycerol, 5 % (w/v) β-mercaptoethanol and 0.05 % (w/v) bromophenol blue and boiled at 100 °C for 4 min. The electrophoresis was carried out using Tris-Glycine buffer (pH 8.3) at room temperature with a current of 2 mA per lane. After the migration protein bands were stained with silver solution (Rabilloud 1992).

The in situ glucansucrase activity was determined following the method of Holt et al. (2001) with minor modifications (Purama and Goyal 2008a). The enzymes samples purified by 15 % PEG 1500 fractionation and by gel filtration were loaded in duplicate on 7.5 % acrylamide gel and run under non-denaturing condition. The protein samples for non-denaturing SDS-PAGE were prepared in the same manner as above, but without the addition of β-mercaptoethanol and they were not subjected to boiling. After the run, the gel was 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 and 0.1 %, v/v Tween 80, incubating at 30 °C for 30 min. One part of the gel was incubated in 20 mM sodium acetate buffer (pH 5.4) supplemented with 10 % sucrose at 30 °C for 48 h and the other part was incubated under the same conditions with 10 % raffinose instead of sucrose. After incubation, the gels were washed with 75 % ethanol for 40 min and incubated in a periodic acid solution (periodic acid, 0.7 %, w/v and acetic acid, 5 %, v/v) for 1 h at 25 °C. After the periodic acid treatment, the gels were washed with a solution containing 0.2 % (w/v) sodium metabisulphite and 5 % (v/v) acetic acid. Finally the gels were stained with 15 ml Schiff reagent (0.5 %, w/v Fuchsin basic, 1 %, w/v sodium bisulphite and 0.1 N HCl) until the discrete magenta colored band within the gel matrix appeared. The gels were washed in distilled water to remove the excess stain and stored in 10 % (v/v) acetic acid at 25 °C.

Optimum temperature and thermal stability of glucansucrase

The optimum assay temperature of glucansucrase was studied by adding 20 μl of the purified enzyme (18.7 U/mg, 0.08 mg/ml) to 1 ml enzyme reaction mixture containing 150 mM sucrose in 20 mM sodium acetate buffer (pH 5.4). The reaction mixture was incubated at different temperatures varying from 15 °C to 55 °C for 15 min. Of the reaction mixture, 100 μl was taken for reducing sugar estimation by the Nelson and Somogyi method (Nelson 1944; Somogyi 1945). The thermostability of the enzyme was determined by incubating 0.5 ml of enzyme (18.7 U/mg, 0.08 mg/ml) at different temperatures ranging from 10 to 50 °C for 30 min, and 20 μl aliquots of enzyme were assayed for residual enzyme activity as described earlier. Measurements were carried out in triplicate for temperature optima and temperature stability experiments.

Optimum pH and pH stability of glucansucrase

The optimum pH of purified enzyme was determined by incubating 20 μl (18.7 U/mg, 0.08 mg/ml) of purified enzyme in 1 ml reaction mixture containing 150 mM concentration of sucrose in 20 mM sodium acetate buffer of different pH, ranging from 3 to 7. The reaction mixture was incubated at 30 °C for 15 min and reducing sugar estimation was done as mentioned previously. In order to determine the pH stability, 100 μl of purified enzyme was incubated at different pHs ranging from 3.0 to 6.0 in 20 mM sodium acetate buffer and pH 6.2 to pH 8.4 in 20 mM sodium phosphate buffer at 30 °C for 30 min, and then the aliquots of 20 μl were assayed for residual enzyme activity as described earlier. Measurements were carried out in triplicate for pH optima and pH stability experiments.

Determination of kinetic parameters of glucansucrase

The purified glucansucrase (18.7 U/mg, 0.08 mg/ml) was used to study the effect of sucrose concentration on its activity. The reaction was carried out in 1 ml 20 mM sodium acetate buffer (pH 5.4) containing 20 μl of enzyme and varying concentrations of sucrose ranging from 0.05 mM to 400 mM at 30 °C for 15 min. The enzyme activity was determined by estimating the released reducing sugar, as mentioned earlier. The data were used to generate a Lineweaver-Burk plot, and the kinetic parameters were analyzed from the plot.

Effect of salts and denaturing agent on glucansucrase activity

The effects of CaCl₂, MgCl₂, CoCl₂, MnSO₄ and HgCl₂ between 0 and 12 mM and EDTA between 0 and 5 mM concentrations were studied on the activity of purified glucansucrase (18.7 U/mg, 0.08 mg/ml). The assays were carried out in 1.0 ml reaction mixtures containing the salt or EDTA, the substrate sucrose (5 %, w/v) in 20 mM sodium acetate buffer (pH 5.4), and 20 μl enzyme. The effect of urea was studied by prior incubation of enzyme with urea (0–5 M, final concentration) at 30 °C for 30 min. Aliquots of 20 μl were taken and enzyme activity was measured as described earlier, and the percent residual activities were calculated with respect to activity in the absence of metal ion and denaturing compound. Measurements were carried out in triplicate and all the data expressed were the averages of three independent experiments with ± standard error.

Effect of stabilizer on glucansucrase activity

To study the effect of different additives on the stability of glucansucrase, aqueous solutions of dextran T-40, PEG-6000, PEG-8000, glutaraldehyde, glycerol, Tween-80 and acetonitrile were added to purified glucansucrase (18.7 U/mg, 0.08 mg/ml) in sodium acetate buffer (pH 5.4) to obtain the final concentrations of 2 μg/ml dextran T-40, 10 μg/ml PEG-6000, 10 μg/ml PEG-8000, 0.1 % glutaraldehyde, 0.5 % glycerol, 1 % acetonitrile and 10 μl/ml Tween 80, respectively, in a final volume of 0.6 ml and stored at 30 °C for 36 h. At different time intervals, 20 μl of samples were taken and analyzed for residual enzyme activity as mentioned earlier. All the experiments were carried out in triplicate, and the data used were the averages of three independent experiments with ± standard error.

Effect of storage temperature on stability of glucansucrase

The storage temperature of purified glucansucrase was studied by incubating the enzyme at different temperatures (0 °C, 4 °C and −20 °C) with or without additives. The additives used for long-term storage of glucansucrase were dextran T-40 (2 μg/ml) and Tween 80 (10 μl/ml). Samples of 20 μl were taken at different time intervals and analyzed for enzyme activity as described earlier. The assay was performed in optimum conditions and the data presented are mean values of three independent experiments with ± standard error.

Results and discussion

Effect of temperature and aeration on glucansucrase production from L. plantarum DM5

The effect of incubation temperature on enzyme production 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 27 °C under static condition (Fig. 1a). The enzyme activity was decreased as the temperature increased above 27 °C, and it decreased by 47 % at 35 °C due to deactivation of the enzyme at higher temperatures. The enzyme activity was lower by 35 % at 20 °C, which might be due to the slower growth rate of cells consequently resulting in lower enzyme production. The enzyme production was also studied at different shaking conditions of 90 to 180 rpm at 27 °C, and was compared with the static condition (Fig. 1b). The enzyme activity of 2.4 ± 0.23 U/ml was observed at 90 rpm at 27 °C, which was 11 % less than static condition (2.7 U/ml). These results indicated the microaerophilic nature of the bacterium.

Fig. 1

Glucansucrase production from Lactobacillus plantarum DM5. a Effect of temperature ranging from 20 to 35 °C on 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

Effect of medium nutrients on glucansucrase production

Effect of sucrose

The effect of sucrose concentration (1 to 7 %, w/v) on glucansucrase production was studied and compared with the control medium containing 2 % (w/v) sucrose as described by Tsuchiya et al. (1952). The maximum enzyme activity of 6.23 ± 0.11 U/ml was observed at 5 % (w/v) sucrose concentration (Fig. 2a). The production of enzyme decreased after 5 % (w/v) sucrose concentration and decreased by 21 % at 7 % (w/v) sucrose concentration (Fig. 2a), which might be due to the subsequent utilization of available sucrose for the formation of exopolysaccharide by the released enzyme. The enhancement of glucansucrase activity by threefold has also been reported in the case of Leuconostoc mesenteroides NRRL B-640 (Purama and Goyal 2008b) and Weissella confusa Cab3 (Shukla and Goyal 2011b) in the presence of 7 % and 5 % (w/v) sucrose, respectively.

Fig. 2

Effect of a Sucrose, b Nitrogen source, c Tween 80 and d K₂HPO₄ 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

Effect of nitrogen source

The effects of various nitrogen sources like yeast extract, peptone and beef extract on glucansucrase production by L. plantarum DM5 were studied. It was found that yeast extract enhanced the production of glucansucrase, and maximum enzyme activity of 3.3 ± 0.21 U/ml was achieved at 2.5 % (w/v) yeast extract (Fig. 2b). Beyond 2.5 % yeast extract, the enzyme activity progressively decreased and enzyme activity of 1.31 U/ml was observed at 4 % (w/v) yeast extract (Fig. 2b), which was 52 % lesser as compared to control medium (2.71 U/ml), which contains 2 % (w/v) yeast extract. The effects of peptone and beef extract as sole nitrogen source on glucansucrase production were studied by varying their concentration from 0.5 % to 4 % as shown in Fig. 2b. The maximum enzyme activity of 2.4 ± 0.19 U/ml and 1.9 ± 0.20 U/ml was observed by the addition of 1.5 % (w/v) peptone and 2 % (w/v) beef extract, respectively (Fig. 2b), with a reduction of enzyme activity by 11 % and 30 % as compared to control medium, which contained 2 % (w/v) yeast extract. The results indicated that the yeast extract was the most effective nitrogen source for production of glucansucrase from L. plantarum DM5, and this was in good agreement with other reports where yeast extract served as a source of vitamin and amino acid supplement in the production of glucansucrase (Majumder and Goyal 2008; Purama and Goyal 2008b). However, the addition of 1.5 % (w/v) peptone or 2 % (w/v) beef extract in control medium (with 2 %, w/v yeast extract) enhanced the enzyme production by 29 % and 14 %, respectively. Similar results were also observed in the case of Leuconostoc mesenteroides NRRL B-640 (Purama and Goyal 2008b) and Leuconostoc mesenteroides PCSIR-3 (Ul-Qader et al. 2001), where peptone and beef extract in addition to yeast extract also resulted in enhanced enzyme activity.

Effect of Tween 80 and K₂HPO₄

The effect of Tween 80 on enzyme production was studied by varying its concentration from 0.1 to 1 % (v/v) in the control medium. It was observed that the addition of 0.1 % Tween 80 in the control medium (without Tween 80) stimulated the enzyme production by 8 % (Fig. 2c). Increasing the concentration of Tween 80 further increased the enzyme activity, and the maximum enzyme activity of 4.54 ± 0.20 U/ml was observed at 0.6 % (v/v) Tween 80 (Fig. 2c), which was 68 % higher as compared with the control medium (2.71 U/ml), which did not contain Tween 80. It has been reported that the use of the surfactant Tween 80 increases the glucansucrase secretion from the cells by altering the fatty acid composition of the cell membrane (Sato et al. 1989; Majumder and Goyal 2008).

The effect of K₂HPO₄ on enzyme production was also studied by varying its concentration from 1 to 4 % in the enzyme production medium. It was observed that the maximum enzyme activity of 3.2 ± 0.48 U/ml was achieved at 2.5 % (w/v) K₂HPO₄ concentration (Fig. 2d). The higher enzyme activity in presence of K₂HPO₄ might be due to buffering activity of K₂HPO₄ in the medium, which lowered the effect of lactic acid production during the fermentation. However, the concentration beyond 2.5 % K₂HPO₄ did not support the enzyme activity and at 3.5 % K₂HPO₄, it was 1.84 ± 0.27 U/ml and causing a 44 % decrease (Fig. 2d).

Purification of enzyme by polyethylene glycol and gel filtration

The cell free supernatant containing extracellular glucansucrase (0.48 U/mg, 5.7 mg/ml) was subjected to fractionation with various concentrations of PEG 400 and PEG 1500. The maximum specific activity of 6.68 U/mg and 10.1 U/mg was achieved at 36 % PEG 400 and 15 % PEG 1500, respectively (Table 1). The purified enzyme by 15 % PEG 1500 fractionation exhibited 21-fold purification with 14 % overall yield in a single step (Table 1). Since the purified enzyme by PEG 1500 (15 %) showed maximum activity, it was further subjected to gel filtration using Sephacryl S-300HR. The enzyme eluted in the form of a single asymmetrical peak (Fig. 3a) and most of the maximum activity was confined between the eighth and tenth fractions. These homogeneous fractions were pooled and showed a specific activity of 18.7 U/mg with 40-fold purification (Table 1).

Table 1 Purification of glucansucrase from L. plantarum DM5

Fig. 3

Purification of glucansucrase from Lactobacillus plantarum DM5. a Elution profile of glucansucrase given by gel filtration using Sephacryl S-300HR. The flow rate was 0.3 ml/min and fractions of 3 ml were collected. The fractions were assayed for enzyme activity (−−■--) and protein concentration (−−○--). b SDS-PAGE (7.5 %) analysis of purified glucansucrase. After the electrophoresis, the gels were cut into two parts for silver staining and for activity staining by periodic acid schiff (PAS) reagent. Lane M: Protein Molecular Mass marker (10 kDa to 200 kDa) from Fermentas International Inc. (silver stained), Lane 1: purified glucansucrase by 15 % PEG 1500 fractionation under denaturing condition (silver stained), Lane 2: purified glucansucrase by gel filtration using Sephacryl S-300 HR under denaturing condition (silver stained), Lane 3: glucansucrase purified by 15 % PEG 1500 (PAS Staining) and Lane 4: purified glucansucrase by glucansucrase using Sephacryl S-300HR by PAS staining of glucan formed using sucrose as substrate; Lane 5: Absence of fructansucrase and confirmation of glucansucrase by PAS staining method using raffinose as substrate

SDS-PAGE analysis and activity staining of glucansucrase

The molecular weight of the purified enzyme (15 % PEG 1500) was determined by 7.5 % (w/v) SDS polyacrlymide gel under denaturing condition and showed two isoforms of approximately 189 kDa and 148 kDa with silver staining solution (Fig. 3b, Lane 2). The purified enzyme (15 % PEG 1500) was also run on SDS-PAGE gels under nondenaturing condition for in situ activity detection of glucansucrase by PAS staining. The PAS staining of the gel showed three activity bands of molecular weights of approximately 189 kDa, 150 kDa and 148 kDa when incubated in 10 % sucrose solution. The faint band of 150 kDa observed only in PAS staining but not in silver staining might be due to the active form being present in very low amounts (Pg level) that could not be stained by the silver staining. As the active form of enzyme in pg levels also synthesized glucan in the presence of sucrose, it was easily detected by PAS staining. It was observed that the most active form of glucansucrase was 148 kDa, as it showed an intense band with PAS staining and as well as silver staining (Fig. 3b, Lane 1 and Lane 3). It has been reported that the molecular weight of extracellular glucansucrase was in the range of 120–200 kDa (Leemhuis et al. 2013) and can exist in multiple molecular forms (Purama and Goyal 2008a; Patel et al. 2011). No band after activity staining of purified enzyme by 15 % PEG 1500 fractionation was observed upon the incubation of the gel with raffinose, as shown in Fig. 3b, Lane 5, which excluded the presence of fructosyltransferase. The partially purified enzyme (15 % PEG 1500) was further purified by gel filtration, which showed a single distinct band of a molecular size of approximately 148 kDa on silver staining (Fig. 3b, Lane 2), as well as on PAS staining (Fig. 3b, Lane 4) after the gel was incubated in 10 % sucrose solution.

Optimum temperature and thermostability of glucansucrase

The purified glucansucrase showed maximum activity within the temperature range of 30–33 °C with a specific activity of ∼18.6 U/mg at pH 5.4 in 20 mM sodium acetate buffer (Fig. 4a). This result was in accordance with the earlier findings that the optimum temperature for glucansucrase enzyme activity was within the range of 30 to 35 °C (Majumder and Goyal 2008; Patel et al. 2011). The loss of activity was observed on either side of 30–33 °C. The enzyme activity rapidly decreased after 37 °C and by 94 % at 55 °C. The thermostability results showed that the enzyme was stable at lower temperatures (10–30 °C) and rapidly lost activity at temperatures higher than 35 °C. The enzyme could retain only 6 % of its initial activity at 50 °C (Fig. 4a).

Fig. 4

Effect of temperature (a) and pH (b) on activity (−−●--) and stability (−−■--) of glucansucrase. The enzyme activity measurements for pH and temperature optima were performed in pH (3.6–6.6) and temperature (10–55 °C) ranges, respectively. For stability studies, the enzyme was incubated at different temperatures (10–50 °C) and different pHs (3–9) for 30 min. The assay was performed in optimum conditions and the data presented are mean values of three independent experiments with ± standard error

Optimum pH and pH stability 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 of 5.4 of glucansucrase from L. plantarum DM5 was similar to the strains Leuconostoc mesenteroides NRRL B-640 (Purama and Goyal 2008a), Weissella confusa Cab3 (Shukla and Goyal 2011a) and Pediococcus pentosaceus SPA (Patel et al. 2011). The enzyme activity decreased sharply below pH 4.5 and above pH 5.5. The enzyme lost 38 % and 93 % activity at pH 6.6 and pH 3.4, respectively (Fig. 4b). The enzyme was stable in acidic pH (4.6–5.8) range like other glucansucrases from Leuconostoc dextranicum NRRL B-1146 (Majumder et al. 2008) and Pediococcus pentosaceus SPA (Patel et al. 2011).

Determination of kinetic parameters of glucansucrase

The effect of sucrose concentration on the enzyme activity was studied with varying sucrose concentration between 0.05 mM to 400 mM. The results showed that it follows the classical Michaelis-Menten kinetics and enzyme saturation was reached at 146 mM (5 %, w/v) sucrose concentration. The purified glucansucrase gave V_m of 19.6 ± 0.83 μmole/mg/min and K_m of 4.5 mM ± 0.58 mM, which indicated that the enzyme has a high affinity and specificity for the substrate, and hence is more effective for production of glucan. The value of K_m of glucansucrase from L. plantarum DM5 was comparable with the K_m value (3.2 ± 0.02 mM) of glucansucrase from Lactobacillus reuteri 180 (Pijning et al. 2008).

Effect of salts and denaturing agent on glucansucrase activity

The Mg²⁺, Ca²⁺ and Co²⁺ salts at low concentrations exhibited a marginal increase in glucansucrase activity, as shown in Table 2. The addition of 1 mM MgCl₂, 2 mM CaCl₂ and 3 mM CoCl₂ to glucansucrase caused enhancement of enzyme activity by 16 %, 18 % and 19 %, respectively (Table 2). It has been reported that these salts stabilize the active site of the enzyme by stabilizing the three-dimensional protein structure (Miller and Robyt 1984; Majumder et al. 2008). The addition of 0.5 mM MnSO₄ and 0.5 mM HgCl₂ resulted in 78 % and 71 % inhibition of enzyme activity, and activity decreased by 79 % and 81 % in the presence of 8 mM MnSO₄ and 8 mM HgCl₂, respectively. Urea and EDTA at all concentrations displayed a denaturing effect on glucansucrase, and 97 % and 69 % of enzyme inactivation was observed with 6 M urea and 6 mM EDTA, respectively (Table 2). Similar results were also found in the case of glucansucrase from Leuconostoc dextranicum NRRL B-1146 (Majumder et al. 2008) and Pediococcus pentosaceus SPA (Patel et al. 2011) for effects of chaotropic agents, urea and EDTA.

Table 2 Effect of salts and chaotropic agent on the activity of purified glucansucrase

Effect of stabilizers and storage temperature on glucansucrase activity

The residual enzyme activity of glucansucrase was measured at 30 °C with respect to time and with or without stabilizer for 30 h. The enzyme without any stabilizer (control) lost its activity rapidly (t _1/2  = 10.3 h) at 30 °C, and only 13.3 % residual activity remained after 30 h (Table 3). Glycerol, PEG 6000 and PEG 8000 acted as stabilizers and displayed stabilizing effects on glucansucrase, as the residual activity was 42 % (t _1/2  = 24 h), 38 % (t _1/2  = 21.5 h) and 33 % (t _1/2  = 18.6 h) respectively, whereas glutaraldehyde and acetonitrile (t _1/2  = 5 h and t _1/2  = 7 h, respectively) acted as inhibitsor of enzyme, as activity was lost by 95 % and 92 %, respectively. Among all the stabilizers, Tween 80 displayed the maximum stabilization of glucansucrase (t _1/2 of 64.8 h), followed by dextran T-40 (t _1/2  = 41 h) at 30 °C. Therefore, Tween 80 and dextran T-40 were used as stabilizers for investigation of long-term storage stability at temperatures of −20 °C, 0 °C and 4 °C. The stabilization data of glucansucrase from L. plantarum DM5 was in accordance with a previous report, where glucansucrase from Leuconostoc mesenteroides NRRL B-512 F showed stabilization in the presence of high molecular weight dextran (2 μg/ml) and non-ionic detergent Tween 80 (10 μg/ml) (Miller and Robyt 1984).

Table 3 Effect of stabilizers on glucansucrase activity at 30 °C

To study suitable storage temperature, glucansucrase was incubated with or without Tween 80 or dextran T-40 at three different temperatures (−20 °C, 0 °C and 4 °C) and half-lives were calculated (Fig. 5). The enzyme in the presence of dextran T-40 and Tween 80 exhibited enhanced half-life of 80 days and 86 days, respectively, as compared with the control (t _1/2  = 65 d) at −20 °C; however, Tween 80 provided 33 %, 35 % and 16 % higher stabilization than dextran T-40 at all the three temperatures of 4 °C, 0 °C and −20 °C. From all above data, it can be summarized that Tween 80 as an additive (t_1/2 = 86 days) proved to be best for the stabilization of glucansucrase from L. plantarum DM5 and could be used for long-term storage.

Fig. 5

The half-lives of purified glucansucrase from Lactobacillus plantarum DM5 when stored at 4 °C, 0 °C and −20 °C. The purified enzyme was stored with or without dextran T-40 and Tween 80 as stabilizer at 4 °C, 0 °C and −20 °C. The assay was performed in optimum conditions and the data presented are mean values of three independent experiments with ± standard error

Conclusions

An extracellular glucansucrase showing specific activity of 0.48 U/mg from a probiotic L. plantarum DM5 was isolated and biochemically characterized. The medium components such as sucrose, yeast extract, K₂HPO₄ and Tween 80 were imperative nutrients for L. plantarum DM5, as they significantly enhanced the glucansucrase production. The glucansucrase purified by PEG 1500 followed by gel filtration using Sephacryl S-300HR gave a specific activity of 18.7 U/mg with 40-fold purification and showed molecular mass of approximately 148 kDa. The zymogram analysis of purified enzyme confirmed that it was glucansucrase, as it showed a magenta color band only in the presence of sucrose, and not in the presence of raffinose. The purified glucansucrase was maximally active at 30 °C and pH 5.4, and was stable at acidic pH and low temperature. The metal ions Ca²⁺, Mg²⁺ and Co²⁺ stimulated the enzyme activity, whereas Mn²⁺ and Hg²⁺ caused inactivation of enzyme. Urea and EDTA denatured the glucansucrase, and even at lower concentrations, a greater extent of enzyme inactivation was observed. Storage stability studies of the enzyme showed 25 % higher stability in the presence of Tween 80 at -20 °C (t _1/2  = 86 days) as compared to the enzyme (t _1/2  = 65 days) without a stabilizer. Further studies are needed using the pure glucansucrase from L. plantarum DM5 in order to produce glucan to support its strong candidacy for application as a food supplement.