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New dextransucrase purification process of the enzyme produced by Leuconostoc mesenteroides IBUN 91.2.98 based on binding product and dextranase hydrolysis
Journal of Biotechnology 265 (2018) 8–14
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Research Paper
New dextransucrase purification process of the enzyme produced by Leuconostoc mesenteroides IBUN 91.2.98 based on binding product and dextranase hydrolysis
MARK
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Glaehter Yhon Flórez Guzmana, , Gustavo Buitrago Hurtadoa, Sonia Amparo Ospinaa,b a b
Universidad Nacional de Colombia, Institute of Biotechnology, (IBUN) Laboratory of Enzyme Technology, Bogotá, Colombia Director Biopolymers and Biofunctionals Research Group, Bogotá DC, Colombia
A R T I C L E I N F O
A B S T R A C T
Keywords: Dextransucrase Leuconostoc mesenteroides Dextran Glucan Glucosyltransferase Exopolysaccharides
This paper examines a new dextransucrase (DS) purification process of the extracellular enzyme (EC 2.4.1.5) produced by Leuconostoc mesenteroides IBUN 91.2.98. The enzyme was purified using a methodology which combines the immobilization of the enzyme in the produced biopolymer dextran, followed by a concentration step by ultrafiltration, using a membrane with a pore size of 300 kDa and subsequent hydrolysis of dextran by action of a dextranase and finally enzyme purification by anion exchange chromatography. The obtained enzyme has a purification factor of 118 and a yield of 26% from the initial extract. The purified dextransucrase has a specific activity of 335.1 U/mg, electrophoretic analysis shows absence of subunits, and a molecular weight of 170.1 kDa, a Vmax of 28.1 U/ml and a Km of 48 mM. Optimal conditions of pH, temperature and substrate concentration were 5, 30 °C and 584 mM sucrose respectively in a ratio of 0.4 U/mole of substrate. The produced dextran has a molecular size of 800–1000 kDa. Both the hydrolytic and transference activity are inhibited by Fe+3 (96.5%) and Al+3 (99.1%), whereas Mg+2 and K produce activation of 36.7% and 27.2%, respectively.
1. Introduction Dextransucrase (sucrose: 1, 6-α-D-glucan 6-α-glucosyltransferase EC 2.4.1.5) is an enzyme that catalyzes the transfer of α-D-glucopyranose residues from sucrose (S) to low molecular weight acceptors (Edward, 2001) forming oligos and polysaccharides (Vasileva Kirilov et al., 2009). The main DS reaction product is dextran, along with the release of fructose (F) as a waste product (Dols et al., 1998; Shukla et al., 2011). Dextran is a glucose linear polymer joined by α (1 → 6) links (more of 50%) and fewer α (1 → 2), α (1 → 3) and/or α (1 → 4) links (Iliev et al., 2008; Khalikova et al., 2005); the link frequency and type are dependent on the nature of the enzyme and type of microorganism (Jeanes et al., 1954; Vandamme et al., 2001). Dextran has diverse applications in the pharmaceutical, chemical and food industry; as blood plasma substitute chromatographic support, texture improver, viscosifier, fiber source, prebiotic, among others (Freitas et al., 2011; Rehm, 2010). DS is produced mainly by four genera of lactic acid bacteria (LAB) Leuconostoc, Streptococcus, Weissella and Lactobacillus (Parlak et al., 2014; Freitas et al., 2011; Hijum et al., 2006; Kralj et al., 2004). Most of these microorganisms are induced by sucrose for DS production (Marguerite et al., 1997), except Streptococcus where the enzyme is
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usually constitutive. From all DS producer microorganisms, the most studied and used is Leuconostoc. The dextran and enzyme production are concomitants on the fermentation in presence of sucrose, forming an enzyme–dextran complex (Willemot, 1988) leading to high viscosities and polymer aggregates, making more difficult the purification and characterization of the product (Majumder et al., 2007). There have been proposed and carried out several DS purification methods as salting-out and solvents (Hehre, 1946; Gupta and Prabhu, 1995; Rodrigues et al., 2003), phase partition (Otts and Day, 1988; Quirasco et al., 1999; Nigam et al., 2006), polyethylene glycol fractioning (Goyal and Katiyar, 1994; Purama and Goyal, 2008), chromatography column (Miller and Robyt, 1984; Miller et al., 1986; Kobayashi and Matsuda, 1986), ultrafiltration (Sánchez-González et al., 1999; Kitaoka and Robyt, 1998) and combined processes as sugaring and gel permeation chromatography (Monsan and Lopez, 1981; Neubauer, 2003). Only one enzyme purification methodology involving concentration and dialysis of the supernatant and subsequent treatment with dextranase, has been reported by Robyt and Walseth, using a L. mesenteroides B-512F (Robyt and Walseth, 1979) with a specific activity of 53 U/mg. In this study the purification and characterization of a DS
Corresponding author. E-mail address: gyfl[email protected] (G.Y. Flórez Guzman).
http://dx.doi.org/10.1016/j.jbiotec.2017.10.019 Received 10 July 2017; Received in revised form 24 October 2017; Accepted 30 October 2017 Available online 31 October 2017 0168-1656/ © 2017 Elsevier B.V. All rights reserved.
Journal of Biotechnology 265 (2018) 8–14
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Fig. 1. Ultrafiltration process scheme. A: filtered out; B: feeding − retentate; C: washing solution (phosphate buffer50 mM, pH: 5); line a: filter outlet; b: filter inlet; c: washing line; d: retentate line (concentrated); 1, 2, 3 and 4: flow valves. Washing process (diafiltered): valves 1, 2, 3 and 4 enabled, flow c to maintain level constant in B. Concentration process: valves 1, 2 and 3 enabled, valve 4 closed. In both processes temperature B should remain to 10 ± 2 °C.
activity was defined as the amount of enzyme required to transfer 1 μmol of glucose per minute at 30 °C, with 233 mM (8% w/v) sucrose as substrate and 50 mM phosphate buffer (pH: 5).
obtained by a Colombian native strain Leuconostoc mesenteroides IBUN 91.2.98 was achieved using ultrafiltration, dextranase treatment and anion exchange chromatography to obtain an enzyme with an specific activity of 331.6 U/mg, six times higher than reported. The high activity of this enzyme, together with the short time of enzymatic production in the fermentation (4.5 h), makes it a good potential for the production of dextran (Guzman, 2014) The dextran produced simultaneously with the enzyme complex, provides stability and affects the DS activity (Miller and Robyt, 1984; Goyal and Katiyar, 1994), the aggregate enzyme-polymer is used for retaining the enzyme in ultrafiltration processes (Pu et al., 2012; García-Molina et al., 2006) used for purposes of purification and enzymatic characterization. The dextran-enzyme retention is achieved by an ultrafiltration membrane with a pore size of 300 kDa and dextranase treatment allowed to study the effect of the polymer in the enzyme activity, and their characterization.
2.4. Protein determination The total protein content in the samples was determined by Lowry method (Lowry et al., 1951) using bovine serum albumin (BSA) (Merck) as standard. 2.5. Quantification of dextran The biopolymer concentration was determined by high performance liquid chromatography (HPLC), using an spectrophotometer Waters 510 with refractive index detector Waters 2410 in a column Shodex Sugar SC1011, 8 × 300 mm; and water as a mobile phase; 70 °C; flow 0.6 ml/min and a sample volume of 10 μl. Standards: dextran (BioRad) from Leuconostoc mesenteroides B-512, dextran purified by precipitation with ethanol from the fermentation of Leuconostoc mesenteroides strain IBUN 91.2.98, glucose, sucrose and fructose analytical reagents.
2. Materials and methods 2.1. Microorganism The strain used for the production of dextran was the Colombian native Leuconostoc mesenteroides IBUN 91.2.98. Isolated from a sugar cane cultivation, in the city of Moniquirá (Boyacá) (Hurtado et al., 2013; Saenz, 1998). The medium used for this study was a modification of the proposed by Tsuchiya and col. (Tsuchiya et al., 1955) composed by (g/l): Sucrose, 60; Yeast Extract, 10; CaCl2·2H2O, 0.14; MgSO4·7H2O, 0.04; FeSO4·7H2O, 0.04; MnSO4·H2O, 0.02; NaCl, 0.01; H3PO4, 5.7.
2.6. Purification of the enzyme dextransucrose 2.6.1. Concentration and diafiltration by ultrafiltration Fig. 1 shows the process that was performed from 1.8 l of crude cellfree extract stored at 10° C. The DS activity was concentrated 3.6 fold (up to a final volume of 0.5 l) by ultrafiltration using a membrane with a pore size of 300 kDa and 50 cm2 of area (Biomax 300 Millipore Pellicom XL) and a peristaltic pump (Heidolph PD5006 model PUMP drive). The average flow rate was 5.41 ml/min in the filtrate.
2.2. Dextransucrase production Leuconostoc mesenteroides IBUN 91.2.98 was reaching a biomass concentration of 2.29 g/l which is achieved at the end of the exponential phase (4 h). The crude extract whith the dextransucrose activity was obtained by centrifugation at 5875 gravities for 20 min at 4 °C. Total protein and enzymatic activity was determined in cell-free supernatant, stored at 10 °C.
2.6.2. Dextran enzymatic hydrolysis The dextran on ultrafiltration retentate was enzymatically hydrolyzed using commercial enzyme Dextranase Plus L (from Novozymes (EC 3.2.1.11) Chaetomium arraticum), with an enzyme concentration between 12.6–315 U/mg. Reaction was performed in 5 ml, 5 °C; 10 h, and the dextran content quantified by HPLC; separation of both enzymes was achieved by anion exchange chromatography. DS activity was determined in the fractions corresponding to the peaks of interest. One unit (U) of dextranase activity was defined as the amount of enzyme required to produce one micromole (μMol) of glucose per minute at a temperature of 5 °C, pH 5, using dextran as substrate at a concentration of 71.5 mg/ml.
2.3. Enzyme activity assay The enzyme activity assay was performed in 1 ml of reaction using sucrose as substrate 233 mM (8% w/v) in 50 mM phosphate buffer, pH 5 and crude enzyme extract in a ratio 1:1. The reaction mixture was incubated at 30 °C for 40 min and 0.1 ml aliquots were inactivated in boiling water for 5 min at times 0, 5, 10, 15, 20 and 40 min. The enzyme activity was determined estimating the reducing sugars concentration by colorimetric method with 3,5 dinitrosalicylic acid (DNS) at 540 nm (Miller, 1959; Aman et al., 2012; Vettori et al., 2011; Champion et al., 2009) and glucose concentration by the enzymatic method (Kit GOD-POD) at 510 nm. One unit (U) of dextransucrase
2.6.3. Anion exchange chromatography To separate DS and dextranase, a BioRad Biologic Duo flow FPLC chromatograpy was used, with a column 150 × 7 mm of the anionic resin UNOsphere Q BioRad and buffer phosphate 50 mM, pH 5.0 as mobile phase (flow 2 ml/min, 20 °C, sample volume 500 μl) and a NaCl linear gradient of from 0 to 1 M. The fraction volume was 2 ml and the 9
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Fig. 2. Growth kinetics from Leuconostoc mesenteroides IBUN 91.2.98, processes from: activation, inoculum and fermentation. −△−: activation, ( ± DS): 0.048; −◇−: obtaining inoculum, ( ± DS): 0.063; −○−: fermentation, ( ± DS): 0.055; −□−: enzymatic activity, ( ± DS): 0.149; − − −▶: ending and initiation of fermentation and time of inoculation; activation periods and inoculum were performed in flask 100 ml, fermentation was conducted in a fermenter 2000 ml, in all periods was employed 5% the inoculum; the values expressed ( ± DS) are the result of three (3) independent experiments n = 3.
Cholamidopropyl) dimethylammonio] −1-propanesulfonate (CHAPS), 2% (w/v); bromophenol blue, 0.002% (w/v); 1% ampholytes (pH: 3–10); threo-1,4-dimercapto-2,3-butanediol (DTT), 18 mM and 26.4 g sample (150 μl).
total time for each run was 60 min. The analyzed samples were previously filtered and kept at a temperature of 4 °C., fractions were unified and dialyzed using a membrane with a pore size of 10,000 Da in 50 mM phosphate buffer, pH 5.0, by 12 h. They were lyophilized and resuspended in 500 μl of 50 mM phosphate buffer at pH 5.0, to quantify protein and enzyme activity.
2.7. Identification dextransucrase enzyme by peptide mapping
2.6.4. Electrophoresis SDS-PAGE analysis of the purified enzyme Sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS–PAGE) was performed according to the method of Laemmli (U.K. Laemmli, 1970) under not reduced conditions. The concentrations of resolving gel and stacking gel were 8% and 2,4%, respectively. Molecular weight markers from ProSieve™ (225- 5 Kda) were used.
Identifying the DS enzyme by peptide mapping was made on the corresponding point (spot) gel isoelectric focusing (IEF), this was subjected to treatment with trypsin (Promega) overnight at 37 °C.The peptides were extracted from the gel using 60% acetonitrile in trifluoroacetic acid (TFA) 0.2%; They were then concentrated by vacuum drying; salts were removed using a micro- column C18 reverse phase (OMIX Pippetetips, Varian); elution of peptides was performed directly on the sample plate of the mass spectrometer, with 3 μl matrix solution (α-cyano-4-hydroxycinnamic in 60% acetonitrile containing 0.2% TFA). Mass spectra of the digestion mixtures were performed in a spectrometer MALDI-TOF/TOF 4800 (Applied Biosystems), the calibration was performed using a mixture of standard peptides (Applied Biosystems). The proteins were identified in the NCBI database using the values: (m/z) and using the MASCOT program with the following search parameters: monoisotopic mass tolerance of 0.05 Da; tolerance of the mass fractions: 0.25 Da and oxidation of methionine as a possible modification.
2.6.5. Dextransucrase identification by activity gel DS activity was detected by gel electrophoresis under denaturing SDS-PAGE and nonreducing conditions, using a concentration polyacrylamide gel separator and concentrator 8% T, 2.4% C and 5% T, 1.3% C respectively; electrophoresis was run at 80 V constant for 45 min at 10 °C. After the run, SDS is remove using a modification of the protocol described is by Ravi (Purama and Goyal, 2008) which consists in immersing the gel in a 50 mM phosphate buffer (pH 5.0), 0.45 mM CaCl2, and 1% (v/v) Triton X-100, under constant stirring for 10 min at 4 °C and washed with distilled water, this procedure was performed three times in succession. The gel was incubated in a 50 mM phosphate buffer (pH 5.0), 146.1 mM sucrose, for 45 min at 30 °C under constant stirring; then was washed three times with distilled water at 4 °C for 5 min. Identifying sucrose dextran activity was performed using the method PAS (periodic acid-Schiff) as described by Miller and Robyt (1986). The gel was immersed in a 0.5% solution (w/v) of periodic acid for 10 min, then washed three times with distilled water at constant stirring for 5 min. Subsequently the gel is suspended in Schiff's reagent (0.5% (w/v) of fuchsin, 2.5% (w/v), 0.55% NaHSO3 and concentrated HCl) to banding deep pink (Shukla et al., 2011), this confirms the presence of dextran (Miller and Robyt, 1986; Kapitany and Zebrowski, 1973; Bejar et al., 2013).
3. Results and discussion 3.1. Dextransucrase enzyme production Comparing each of the three systematic stages provided for production of the enzyme (activation and fermentation inoculum) a decrease is observed at the time of the growth kinetics of the microorganism strain Leuconostoc mesenteroides IBUN 91.2.98 (See Fig. 2). With the composition of culture medium and fermentation conditions, the microorganism is able to obtain an enzyme extract with maximum DS activity of 6.9 U/ml for 4 h cultivation, coinciding with the end of the exponential phase and maximum biomass of 2.29 g/l. In this work the enzyme productivity was 1533 U/l.h−1, this is one of the highest achieved in a fermentation process; DS reports of some activities achieved in fermentation are: L mesenteroides NRRL B-512F (Fabre et al., 2005), L mesenteroides B512 FMC (Parlak et al., 2014), L. mesenteroides NRRL B-1299 (Moulis et al., 2006) and the alternansucrase of L. citreum NRRL B-1355 (Joucla et al., 2006) with: 5.85 U/ml, 6.33 U/ml, 0.58 U/ml y 0.66 U/ml respectively. The maximum specific growth rate (μmax) was 1.16 h−1 with a doubling time of 0.59 h (35.8 min); the substrate consumption in fermentation were 97.8% and the yield 17.7 g/l dextran (Yp/s: 0.304).
2.6.6. Isoelectric focusing (IEF), two-dimensional gel electrophoresis (2D) Proteomic analysis of crude extract or purified enzyme DS was performed by two-dimensional gel electrophoresis (2D-PAGE). The technique involves both the first dimension (separation based on the isoelectric point within a matrix in a pH gradient containing ampholytes) and second dimension (separation in a matrix based on molecular weight).The first dimension was performed on a system using a computer isoelectric focussing IPG strip ReadyStrip TM IPG Strips (BioRad). Immobilized pH gradient of 7 cm in length, were used with a pH range of 3–10 (Bio-Rad), polyacrylamide containing 4% T and 3% of C. The hydration of the strips IPG, was performed with 150 μl of rehydration solution, whose composition is urea, 8 M; 3 − [(310
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Fig. 3. Anion exchange chromatogram; separation of the sample from the hydrolytic treatment with dextranase ultrafiltration retentate. Peak A: Corresponding fractions 8 to the 12, where dextransucrase activity was detected; paek B: corresponding to fractions: 35 to the 38; dextranase activity was detected; peak C: corresponding to fractions 39 to the 48, no kind of activity was detected.
3.2. Purification of the enzyme dextransucrase The enzyme-dextran complex formed in the fermentation allows the enzyme DS to be stopped in the so-called retained fraction of the ultrafiltration process, achieving a concentration of 13.8 times, relative to the initial feed using a 300 KDa membrane (see Fig. 1). Subsequent to this a 54.9% dextran separation was performed by enzymatic hydrolysis using a dextranase, the separation of these two enzymes together with the residues of the hydrolysis was achieved by anion exchange chromatography (see Fig. 3). It was establish that the minimum dextran concentration is 110.5 mg/ml with an enzyme activity of 72.7%. The enzyme purification steps are shown in Table 1. A purification factor of 118 was achieved along with a yield of 26%, the specific activity obtained was 335.1 U/mg one of the highest reported activities of a purified DS enzyme. Some activities of DS enzymes purified using ultrafiltration are: L. mesenteroides B-512FMC (Kitaoka and Robyt, 1998) C. and L. mesenteroides B-512 (Robyt and Walseth, 1979) are 183 U/mg and 53 U/mg respectively. 3.2.1. Diafiltration and ultrafiltration The first step of purification of DS is concentration and diafiltration by ultrafiltration of fermentation supernatant. Using pore size membrane of 300 kDa it was possible to retain the 100% of DS activity (104 U/ml) in the concentrated fraction of ultrafiltration, the dextran polymer concentration in this fractions is 245 mg/ml. In the fractions of ultrafiltering is not detect DS activity, protein content is 2.56 mg/ml equivalent to 97.6% of the initial. With a calculated molecular weight in 171.5 kDa, the DS was retained by the polymer and remain in the concentrated fraction; this is due to the covalent interaction of the enzyme in the formation of dextran, not allowing this to be released. The concentration limit reached in the ultrafiltration was 13.8 times relative to the initial volume of the filtrate, this is due to increased viscosity by accumulation of dextran.
Fig. 4. Electrophoresis SDS PAGE analysis purification of DS enzyme from the strain Leuconostoc mesenteroides IBUN 91.2.98. 1: Molecular weight markers (225 − 5 kDa), 2: Concentrate from the ultrafiltration membrane 300 kDa (retained), 3: Fermentation supernatant (crude extract), 4: Dextransucrase fraction from the anion exchange column (UNOsphere Q).
established that the enzyme does not have protein subunits and 170.1 kDa molecular size was calculated; isoelectric focusing shows absence of isoproteins and an isoelectric point (pI) of 4.2.
3.3. Electrophoretic analysis Protein separation results are shown in Fig. 4. A single protein band with activity was detected corresponding to dextransucrase; electrophoretic gel showed deformation until rupture by the accumulation of dextran polymer due to the high enzyme activity, see Fig. 5. It was
3.4. Enzymatic properties of dextransucrase IBUN 91.2.98 The effect of sucrose on the activity of the enzyme was determined
Table 1 Purification of dextransucrase of Leuconostoc mesenteroides strain IBUN 91.2.98 by ultrafiltration concentration and subsequent hydrolysis of dextran with dextranase. Purification step
Volume (ml)
Enzime Activity (U/ml)
Total protein (mg)
Specific activity (U/mg)
Yield (%)
Purification (Fold)
Crude extract Ultrafiltration 300 kDa Ion exchange UNO sphere
1800 130 130
6.9 104 24.8
4374 104 9.6
2.8 130 335.1
100 109 26
1 46 118
11
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Fig. 5. Activity gel (zimograma) for dextransucrase and identification of the enzyme produced by the microorganism: Leuconostoc mesenteroides IBUN 91.2.98. 1: Molecular weight markers (220–40 KDa); 2, 3 and 4: crude extract (supernatant from fermentation), bands were stained with Coomassie blue. 5: accumulation of dextran polymer, product of enzyme activity; a: bands exposed treatment Schiff reagent, PAS staining (periodic acid-Schiff), reaction time 2 min; b: reaction time 5 min; c: reaction time 10 min, note accumulated polymer production, which ends at the break of electrophoretic gel.
Fig. 6. Linearization by the method of Hannes Wolf of dextransucrose enzyme produced by the microorganism: Leuconostoc mesenteroides strain: IBUN 91.2.98. S/V = (1/Vmax)S + (Km/Vmax); ( ± DS 0.76), R2 = 0.993. Where: S: substrate (mM) and V: enzymatic activity (U/ml).
using various substrate concentrations in the reaction, between 8.76–1168 mM, adjusting a Michaelis-Menten kinetics with a Vmax of 28.1 U/ml and Km of 48 mM, calculated by the method Hanes-Woolf, see Fig. 6. The enzyme presents substrate inhibition at concentrations over 584 mM sucrose, although at a concentration of 1168 mM their activity decreases only 7.3%, this values are consistent if compared with characteristics kinetics reported by these enzymes (Patel et al., 2011).
Fig. 7. Effect of pH on the activity and enzymatic stability of dextransucrase of Leuconostoc mesenteroides IBUN 91.2.98. A: effect of pH on enzyme activity, ( ± DS): 2.39. B: effect of pH on the stability of the enzyme, −Δ−: time of exposition: 20 min, ( ± DS): 1.88; −□−: time of exposition: 40 min, ( ± DS): 3.19; −◇−: time of exposition: 80 min, ( ± DS): 2.60. The trials were conducted by exposing the dextransucrase to different values pH in times of 20, 40 and 80 min; after this enzymatic activity assays performed at a temperature of 30°C, with 50 mM phosphate buffer, pH: 5, containing 233 mM (8% p/v) sucrose as substrate. The values expressed ( ± DS) are the result of three independent experiments n = 3.
3.4.1. Effect of temperature and pH on the activity and stability of the enzyme The effect of pH and temperature on DS activity is shown in Fig. 7 and Fig. 8, the optimum pH is 5, losing 51.4 and 74.9% of its initial activity at pH 4 and 7 respectively. The enzyme showed high stability against pH values between 4 and 7 at three different exposure times (20, 40 and 80 min). The enzyme activity gradually affected at pH values below 4 decreasing in average 0.48% of its activity per minute exposure. The optimal temperature for DS is 30 °C and enzyme denaturing
occurs at 40 °C where their activity decreases 86.8%. The activation energy (Ea) is 22.6 kJ/mol, this value is lower than that reported for DS of L. mesenteroides NRRL B-1299 and L. mesenteroides NRRL B-512F, 47 12
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act as activators in a 36.7 and 27.2% respectively. Calcium ion (Ca+3) had a slight inhibitory effect of 8% of its initial activity. This differs from other values reported for DS of L. mesenteroides where calcium ions increases enzyme activity and stability (Parlak et al., 2014; Kitaoka and Robyt, 1998). See Table 2. 3.4.3. Identification of the enzyme by peptide mapping A signal is identified (m/z = 2036.8) whose sequence is: −GEFINADGDTFYTSATDGR− which is only present in the protein identified as “dextransucrose DsrD”; the enzyme DS of Leuconostoc mesenteroides strain IBUN 91.2.98. It owns 25% homology with the protein DsrD; similarly a signal detected in the spectrum (m/ z = 1599.8) corresponding to the sequence: −YYFEPGSGNLAILR− which is only present in proteins glucosyl-hydrolases family 70 (GH70) which have repetitive fragments −YG−.Their functioning is related to the accession and stability of the enzyme with the enzymatic reaction product polymer. 4. Conclusions The strain Leuconostoc mesenteroides IBUN 91.2.98 is producing an extracellular DS type enzyme, which synthesizes a dextran polymer of high molecular weight (800–1000 kDa); the fermentation conditions used for growth of the microorganism and production of the enzyme, allowed high enzyme productivity, it is the highest reported so far. DS covalently interacts with dextran polymer product of the enzymatic synthesis, providing stability and allowing its partial purification by ultrafiltration on a filter with a pore size of 1.7 times of DS. The polymer produced in the enzymatic synthesis using DS is associated with enzyme stability, which is confirmed by the complete loss of enzymatic activity in the absence of polymer. Repetitive peptide fractions analyzed by mass spectrometry is associated with enzyme to polymer adhesion and with the enzyme stability. Because of this feature it was not possible for complete hydrolysis of dextran, becoming a limiting factor in the purification process. The enzyme presents low inhibition with calcium ion (Ca+3), differs from other similar enzymes. Dextransucrose was classified as a glycosylhydrolase (GH) belonging to the family 70, showing 25% homology with dextransucrase reported as DsrD.
Fig. 8. Temperature effect on activity and enzymatic stability of dextransucrase of Leuconostoc mesenteroides IBUN 91.2.98. A: Temperature effect on enzyme activity ( ± DS): 2.36. B: Temperature effect on stability activity, −◇−: Time of exposition: 20 min, ( ± DS): 2.61; −□−: Time of exposition: 40 min, ( ± DS): 3.19; −○−: Time of exposition: 80 min, ( ± DS): 2.66. The values expressed ( ± DS) are the result of three independent experiments n = 3.
Table 2 Effect of metal ions on the enzyme activity of dextransucrase (DS) from Leuconostoc mesenteroides strain IBUN 91.2.98. The tests were performed at 30 °C, pH: 5 and a sucrose concentration of 233.7 mM as a substrate. The enzymatic reactions were performed in the absence (control) and presence of metal ions at a final concentration of 2 mM, all as chlorides. The values expressed ( ± DS) are the result of three independent experiments n = 3. Metal ion
Relative activity (%)
Control CaCl2 MnCl2 NaCl KCl AlCl3 ZnCl3 MgCl2 FeCl3
100.0 ± 4.7 91.4 ± 3.4 109.6 ± 4.3 85.9 ± 2.8 127.2 ± 2.0 0.9 ± 0.1 99.9 ± 4.2 136.7 ± 4.0 3.5 ± 1.3
Acknowledgments The authors wish to thank: Biotechnology Institute of the National University of Colombia (IBUN), the Administrative Department of Science, Technology and Innovation (Colciencias), Dr. Jairo Cerón Salamanca, professor at the National University of Colombia, Dr. Jaime Antonio Cardozo Cerquera of Colombian Agricultural Corporation Research (CORPOICA). References Aman, A.I., Siddiqui, N.N., Qader, S.A.U., 2012. Characterization and potential applications of high molecular weight dextran produced by Leuconostoc mesenteroides AA1. Carbohydr. Polym. 87, 910–915. Bejar, W., Gabriel, V., Amari, M., Morel, S., Mezghani, M., Maguin, E., Fontagné-Faucher, C., Bejar, S., Chouayekh, H., 2013. Characterization of glucansucrase and dextran from Weissella sp: TN610 with potential as safe food additives. Int. J. Biol. Macromol. 52, 125–132. Champion, E., André, I., Moulis, C., Boutet, J., Descroix, K., Morel, S., Monsan, P., Mulard, L.A., Remaud-Siméon, M., 2009. Design of α-Transglucosidases of Controlled Specificity for Programmed Chemoenzymatic Synthesis of Antigenic Oligosaccharides. J. Am. Chem. Soc. 131, 7379–7389. Dols, M., Remaud-Simeon, M., Willemot, R.M., Vignon, M., Monsan, P., 1998. Characterization of the different dextransucrase activities excreted in glucose, fructose, or sucrose medium by Leuconostoc mesenteroides NRRL B-1299. Appl. Environ. Microbiol. 64, 1298–1302. Edward, H.J., 2001. Glycosyl transfer: a history of the concept’s development and view of its major contributions to biochemistry. Carbohydr. Res. 331, 347–368. Fabre, E., Bozonnet, S., Arcache, A., Vignon, M., Monsan, P., Remaud-simeon, M., 2005. Role of the two catalytic domains of DSR-E dextransucrase and their involvement in
and 44 kJ/mol respectively (Dols et al., 1998). The enzyme presents low stability with respect to temperature, decreases its activity at 0.73 and 1.52% per minute of exposure to 30 and 40 °C respectively.
3.4.2. Effect of metal ions and agents The effect of some metal ions on the DS produce for Leuconostoc mesenteroides strain IBUN 91.2.98. is shown in Table 2; the enzyme is strongly inhibited by cations Fe+3 and Al+3 in a 96.5 and a 99.1% respectively compared to the initial activity while cations Mg+2 and K 13
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G.Y. Flórez Guzman et al. the formation of highly α −1, 2 branched dextran. J. Bacteriol. 187, 296–303. Freitas, F., Alves, V.D., M.A, Reis, 2011. Advances in bacterial exopolysaccharides: from production to biotechnological applications. Trends Biotechnol. 29, 388–398. García-Molina, V., Esplugas, S., Wintgens, T., Melin, T., 2006. Ultrafiltration of aqueous solutions containing dextran. Desalination 188, 217–227. Goyal, A., Katiyar, S.S., 1994. Fractionation of Leuconostoc mesenteroides NRRL B-512F dextran sucrase by polyethylene glycol: a simple and effective method of purification. J. Microbiol. Methods 20, 225–231. Gupta, A., Prabhu, K.A., 1995. Immobilization and properties of dextransucrase from Leuconostoc mesenteroides culture LM1. J. Gen. Appl. Microbiol. 41, 399–407. Guzman, G.Y.F., 2014. Estudio De La Enzima Dextransacarasa (DS) Producida Por Leuconostoc Mesenteroides Cepa IBUN 91.2.98. Universidad Nacional de Colombia. Hehre, E.J., 1946. Studies on the enzymatic synthesis of dextran from sucrose. J. Biol. Chem. 163, 221–233. Hijum, S.A.F.T., Van, Kralj, S., Ozimek, L.K., Dijkhuizen, L., Geel-schutten, I.G.H. Van, 2006. Structure-Function Relationships of Glucansucrase and Fructansucrase Enzymes from Lactic Acid Bacteria 70, 157–176. Hurtado, G.B., Porras, W.A.V., Sepúlveda, D.J.V., Alvarez, A.O., Guzman, G.Y.F., 2013. Evaluating the effect of the number of generations in IBUN 91.2.98 Leuconostoc mesenteroides cultures on enzyme extract production. Ing. e Investig. 33, 66–70. Iliev, I., Vassileva, T., Ignatova, C., Ivanova, I., Haertlé, T., Monsan, P., Chobert, J.-M., 2008. Gluco-oligosaccharides synthesized by glucosyltransferases from constitutive mutants of Leuconostoc mesenteroides strain Lm 28. J. Appl. Microbiol. 104, 243–250. Jeanes, A., Haynes, W.C., Wilham, C.A., Rankin, J.C., Melvin, E.H., Austin, M.J., Cluskey, J.E., Fisher, B.E., Tsuchiya, H.M., Rist, C.E., 1954. Characterisation and classification of dextrans from ninety six strains of bacteria. J. Am. Chem. Soc. 76, 5041–5052. Joucla, G., Pizzut, S., Monsan, P., Remaud-Simeon, M., 2006. Construction of a fully active truncated alternansucrase partially deleted of its carboxy-terminal domain. FEBS Lett. 580, 763–768. Kapitany, R.A., Zebrowski, E.J., 1973. A high resolution PAS stain for polyacrylamide gel electrophoresis. Anal. Biochem. 56, 361–369. Khalikova, E., Susi, P., Korpela, T., 2005. Microbial dextran-hydrolyzing enzymes: fundamentals and applications. Microbiol. Mol. Biol. Rev. 69, 306–325. Kitaoka, M., Robyt, J.F., 1998. Large-scale preparation of highly purified dextransucrase from a high-producing constitutive mutant of Leuconostoc mesenteroides B-512FMC. Enzyme Microb. Technol. 23, 386–391. Kobayashi, M., Matsuda, K., 1986. Electrophoretic analysis of the multiple forms of dextransucrase from Leuconostoc mesenteroides. J. Biochem. 100, 615–621. Kralj, S., van Geel-Schutten, G.H., Dondorff, M.M.G., Kirsanovs, S., van der Maarel, M.J.E.C., Dijkhuizen, L., 2004. Glucan synthesis in the genus Lactobacillus: isolation and characterization of glucansucrase genes, enzymes and glucan products from six different strains. Microbiology 150, 3681–3690. Laemmli, U.K., 1970. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 277, 680–685. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. Majumder, A., Purama, R., Goyal, A., 2007. An overview of purification methods of glycoside hydrolase family 70 dextransucrase. Indian J. Microbiol. 197–206. Marguerite, D., Magali, R., Monsan, P.F., 1997. Dextransucrase production by leuconostoc mesenteroides NRRL B-1299. comparison with L. mesenteroides NRRL B412F. Enzyme Microb. Technol. 20, 523–530. Miller, A.W., Robyt, J.F., 1984. Stabilization of dextransucrase from Leuconostoc mesenteroides NRRL B-512F by nonionic detergents, poly(ethylene glycol) and highmolecular-weight dextran. Biochim. Biophys. Acta 785, 89–96. Miller, A.W., Robyt, J.F., 1986. Detection of dextransucrase and levansucrase on polyacrylamide gels by the periodic acid-Schiff stain: staining artifacts and their prevention. Anal. Biochem. 156, 357–363.
Miller, A., Eklund, S., Robyt, J., 1986. Milligram to gram scale purifi cation and characterization of dextransucrase from Leuconostoc mesenteroides NRRL B-512F. Carbohydr. Res. 147, 119–133. Miller, G.L., 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31, 426–428. Monsan, P., Lopez, A., 1981. On the production of dextran by free and immobilized dextransucrase. Biotechnol. Bioeng. 23, 2027–2037. Moulis, C., Joucla, G., Harrison, D., Fabre, E., Potocki-Veronese, G., Monsan, P., RemaudSimeon, M., 2006. Understanding the polymerization mechanism of glycoside-hydrolase family 70 glucansucrases. J. Biol. Chem. 281, 31254–31267. Neubauer, H., 2003. Molecular characterization and expression analysis of the dextransucrase DsrD of Leuconostoc mesenteroides Lcc4 in homologous and heterologous Lactococcus lactis cultures. Microbiology 149, 973–982. Nigam, M., Goyal, A., Katiyar, S., 2006. High yield purifi cation of dextransucrase from Leuconostoc mesenteroides NRRL B-512F by phase-partitioning. J. Food Biochem. 30, 12–20. Otts, D.R., Day, D.F., 1988. Dextransucrase secretion in Leuconostoc mesenteroides depends on the presence of a transmembrane proton gradient. J. Bacteriol. 170, 5006–5011. Parlak, M., Ustek, D., Tanriseven, A., 2014. Designing of a novel dextransucrase efficient in acceptor reactions. Carbohydr. Res. 386C, 41–47. Pu, Y., Zou, Q., Liu, L., Han, Z., Wang, X., Wang, Q., Chen, S., 2012. Clinical dextran purified by fractional ultrafiltration coupled with water washing. Carbohydr. Polym. 87, 1257–1260. Purama, R.K., Goyal, A., 2008. Identification, effective purification and functional characterization of dextransucrase from Leuconostoc mesenteroides NRRL B-640. Bioresour. Technol. 99, 3635–3642. Quirasco, M., López-Munguía, A., Remaud-Simeon, M., Monsan, P., Farrés, A., 1999. Induction and transcription studies of the dextransucrase gene in Leuconostoc mesenteroides NRRL B-512F. Appl. Environ. Microbiol. 65, 5504–5509. Rehm, B.H.A., 2010. Bacterial polymers: biosynthesis, modifications and applications. Nat. Rev. Microbiol. 8, 578–592. Robyt, J.F., Walseth, T., 1979. Production, purification and properties of dextransucrase from Leuconostoc mesenteroides NRRL B-512F. Carbohydr. Res. 68, 95–111. Rodrigues, S., Lona, L.M.F., Franco, T.T., 2003. Effect of phosphate concentration on the production of dextransucrase by Leuconostoc mesenteroides NRRL B512F. Bioprocess Biosyst. Eng. 26, 57–62. Sánchez-González, M., Alagón, A., Rodríguez-Sotrés, R., López-Munguía, A., 1999. Proteolytic processing of dextransucrase of Leuconostoc mesenteroides. FEMS Microbiol. Lett. 181, 25–30. Saenz, A.C., M.C.G.P, 1998. Aislamiento E Identificación De Cepas Nativas Con Actividad Levansacarasa. Universidad Nacional de Colombia. Shukla, R., Shukla, S., Bivolarski, V., Iliev, I., Ivanova, I., Goyal, A., 2011. Structural characterization of insoluble Dextran produced by Leuconostoc mesenteroides NRRL B1149 in the presence of maltose. Food Technol. Biotechnol. 49, 291–296. Tsuchiya, H.M., Hellman, N.N., Koepsell, H.J., 1955. Factors affecting molecular weight of enzymatically synthesized dextran. J. Am. Chem. Soc. 77, 2412–2419. E.J. Vandamme, Baets, S., De, Steinbüchel, S.A. (Eds.) 2001. Biopolymers: Polysaccharides I—Polysaccharides from Prokaryotes, Vol. 5. ed. Germany. Vasileva Kirilov, A., Bivolarski, V., Bounaix, M., Gabriel, V., Robert, H., Fontagne-faucher, C., Gabriel, B., Ivanova, I., Iliev, I., 2009. Characterization of glycansucrase activities from leuconostoc mesenteroides Lm17 and ure 13 strains. Biotechnol. Biotechnol. 23, 698–701. Vettori, M.H.P.B., Mukerjea, R., Robyt, J.F., 2011. Comparative study of the efficacies of nine assay methods for the dextransucrase synthesis of dextran. Carbohydr. Res. 346, 1077–1082. Willemot, R., Monsan, P., Durand, G., 1988. Effects of dextran on the activity and stability of dextransucrase from Leuconostoc mesenteroides. Ann. N. Y. Acad. Sci. 542, 169–172.
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Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Research Paper
New dextransucrase purification process of the enzyme produced by Leuconostoc mesenteroides IBUN 91.2.98 based on binding product and dextranase hydrolysis
MARK
⁎
Glaehter Yhon Flórez Guzmana, , Gustavo Buitrago Hurtadoa, Sonia Amparo Ospinaa,b a b
Universidad Nacional de Colombia, Institute of Biotechnology, (IBUN) Laboratory of Enzyme Technology, Bogotá, Colombia Director Biopolymers and Biofunctionals Research Group, Bogotá DC, Colombia
A R T I C L E I N F O
A B S T R A C T
Keywords: Dextransucrase Leuconostoc mesenteroides Dextran Glucan Glucosyltransferase Exopolysaccharides
This paper examines a new dextransucrase (DS) purification process of the extracellular enzyme (EC 2.4.1.5) produced by Leuconostoc mesenteroides IBUN 91.2.98. The enzyme was purified using a methodology which combines the immobilization of the enzyme in the produced biopolymer dextran, followed by a concentration step by ultrafiltration, using a membrane with a pore size of 300 kDa and subsequent hydrolysis of dextran by action of a dextranase and finally enzyme purification by anion exchange chromatography. The obtained enzyme has a purification factor of 118 and a yield of 26% from the initial extract. The purified dextransucrase has a specific activity of 335.1 U/mg, electrophoretic analysis shows absence of subunits, and a molecular weight of 170.1 kDa, a Vmax of 28.1 U/ml and a Km of 48 mM. Optimal conditions of pH, temperature and substrate concentration were 5, 30 °C and 584 mM sucrose respectively in a ratio of 0.4 U/mole of substrate. The produced dextran has a molecular size of 800–1000 kDa. Both the hydrolytic and transference activity are inhibited by Fe+3 (96.5%) and Al+3 (99.1%), whereas Mg+2 and K produce activation of 36.7% and 27.2%, respectively.
1. Introduction Dextransucrase (sucrose: 1, 6-α-D-glucan 6-α-glucosyltransferase EC 2.4.1.5) is an enzyme that catalyzes the transfer of α-D-glucopyranose residues from sucrose (S) to low molecular weight acceptors (Edward, 2001) forming oligos and polysaccharides (Vasileva Kirilov et al., 2009). The main DS reaction product is dextran, along with the release of fructose (F) as a waste product (Dols et al., 1998; Shukla et al., 2011). Dextran is a glucose linear polymer joined by α (1 → 6) links (more of 50%) and fewer α (1 → 2), α (1 → 3) and/or α (1 → 4) links (Iliev et al., 2008; Khalikova et al., 2005); the link frequency and type are dependent on the nature of the enzyme and type of microorganism (Jeanes et al., 1954; Vandamme et al., 2001). Dextran has diverse applications in the pharmaceutical, chemical and food industry; as blood plasma substitute chromatographic support, texture improver, viscosifier, fiber source, prebiotic, among others (Freitas et al., 2011; Rehm, 2010). DS is produced mainly by four genera of lactic acid bacteria (LAB) Leuconostoc, Streptococcus, Weissella and Lactobacillus (Parlak et al., 2014; Freitas et al., 2011; Hijum et al., 2006; Kralj et al., 2004). Most of these microorganisms are induced by sucrose for DS production (Marguerite et al., 1997), except Streptococcus where the enzyme is
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usually constitutive. From all DS producer microorganisms, the most studied and used is Leuconostoc. The dextran and enzyme production are concomitants on the fermentation in presence of sucrose, forming an enzyme–dextran complex (Willemot, 1988) leading to high viscosities and polymer aggregates, making more difficult the purification and characterization of the product (Majumder et al., 2007). There have been proposed and carried out several DS purification methods as salting-out and solvents (Hehre, 1946; Gupta and Prabhu, 1995; Rodrigues et al., 2003), phase partition (Otts and Day, 1988; Quirasco et al., 1999; Nigam et al., 2006), polyethylene glycol fractioning (Goyal and Katiyar, 1994; Purama and Goyal, 2008), chromatography column (Miller and Robyt, 1984; Miller et al., 1986; Kobayashi and Matsuda, 1986), ultrafiltration (Sánchez-González et al., 1999; Kitaoka and Robyt, 1998) and combined processes as sugaring and gel permeation chromatography (Monsan and Lopez, 1981; Neubauer, 2003). Only one enzyme purification methodology involving concentration and dialysis of the supernatant and subsequent treatment with dextranase, has been reported by Robyt and Walseth, using a L. mesenteroides B-512F (Robyt and Walseth, 1979) with a specific activity of 53 U/mg. In this study the purification and characterization of a DS
Corresponding author. E-mail address: gyfl[email protected] (G.Y. Flórez Guzman).
http://dx.doi.org/10.1016/j.jbiotec.2017.10.019 Received 10 July 2017; Received in revised form 24 October 2017; Accepted 30 October 2017 Available online 31 October 2017 0168-1656/ © 2017 Elsevier B.V. All rights reserved.
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Fig. 1. Ultrafiltration process scheme. A: filtered out; B: feeding − retentate; C: washing solution (phosphate buffer50 mM, pH: 5); line a: filter outlet; b: filter inlet; c: washing line; d: retentate line (concentrated); 1, 2, 3 and 4: flow valves. Washing process (diafiltered): valves 1, 2, 3 and 4 enabled, flow c to maintain level constant in B. Concentration process: valves 1, 2 and 3 enabled, valve 4 closed. In both processes temperature B should remain to 10 ± 2 °C.
activity was defined as the amount of enzyme required to transfer 1 μmol of glucose per minute at 30 °C, with 233 mM (8% w/v) sucrose as substrate and 50 mM phosphate buffer (pH: 5).
obtained by a Colombian native strain Leuconostoc mesenteroides IBUN 91.2.98 was achieved using ultrafiltration, dextranase treatment and anion exchange chromatography to obtain an enzyme with an specific activity of 331.6 U/mg, six times higher than reported. The high activity of this enzyme, together with the short time of enzymatic production in the fermentation (4.5 h), makes it a good potential for the production of dextran (Guzman, 2014) The dextran produced simultaneously with the enzyme complex, provides stability and affects the DS activity (Miller and Robyt, 1984; Goyal and Katiyar, 1994), the aggregate enzyme-polymer is used for retaining the enzyme in ultrafiltration processes (Pu et al., 2012; García-Molina et al., 2006) used for purposes of purification and enzymatic characterization. The dextran-enzyme retention is achieved by an ultrafiltration membrane with a pore size of 300 kDa and dextranase treatment allowed to study the effect of the polymer in the enzyme activity, and their characterization.
2.4. Protein determination The total protein content in the samples was determined by Lowry method (Lowry et al., 1951) using bovine serum albumin (BSA) (Merck) as standard. 2.5. Quantification of dextran The biopolymer concentration was determined by high performance liquid chromatography (HPLC), using an spectrophotometer Waters 510 with refractive index detector Waters 2410 in a column Shodex Sugar SC1011, 8 × 300 mm; and water as a mobile phase; 70 °C; flow 0.6 ml/min and a sample volume of 10 μl. Standards: dextran (BioRad) from Leuconostoc mesenteroides B-512, dextran purified by precipitation with ethanol from the fermentation of Leuconostoc mesenteroides strain IBUN 91.2.98, glucose, sucrose and fructose analytical reagents.
2. Materials and methods 2.1. Microorganism The strain used for the production of dextran was the Colombian native Leuconostoc mesenteroides IBUN 91.2.98. Isolated from a sugar cane cultivation, in the city of Moniquirá (Boyacá) (Hurtado et al., 2013; Saenz, 1998). The medium used for this study was a modification of the proposed by Tsuchiya and col. (Tsuchiya et al., 1955) composed by (g/l): Sucrose, 60; Yeast Extract, 10; CaCl2·2H2O, 0.14; MgSO4·7H2O, 0.04; FeSO4·7H2O, 0.04; MnSO4·H2O, 0.02; NaCl, 0.01; H3PO4, 5.7.
2.6. Purification of the enzyme dextransucrose 2.6.1. Concentration and diafiltration by ultrafiltration Fig. 1 shows the process that was performed from 1.8 l of crude cellfree extract stored at 10° C. The DS activity was concentrated 3.6 fold (up to a final volume of 0.5 l) by ultrafiltration using a membrane with a pore size of 300 kDa and 50 cm2 of area (Biomax 300 Millipore Pellicom XL) and a peristaltic pump (Heidolph PD5006 model PUMP drive). The average flow rate was 5.41 ml/min in the filtrate.
2.2. Dextransucrase production Leuconostoc mesenteroides IBUN 91.2.98 was reaching a biomass concentration of 2.29 g/l which is achieved at the end of the exponential phase (4 h). The crude extract whith the dextransucrose activity was obtained by centrifugation at 5875 gravities for 20 min at 4 °C. Total protein and enzymatic activity was determined in cell-free supernatant, stored at 10 °C.
2.6.2. Dextran enzymatic hydrolysis The dextran on ultrafiltration retentate was enzymatically hydrolyzed using commercial enzyme Dextranase Plus L (from Novozymes (EC 3.2.1.11) Chaetomium arraticum), with an enzyme concentration between 12.6–315 U/mg. Reaction was performed in 5 ml, 5 °C; 10 h, and the dextran content quantified by HPLC; separation of both enzymes was achieved by anion exchange chromatography. DS activity was determined in the fractions corresponding to the peaks of interest. One unit (U) of dextranase activity was defined as the amount of enzyme required to produce one micromole (μMol) of glucose per minute at a temperature of 5 °C, pH 5, using dextran as substrate at a concentration of 71.5 mg/ml.
2.3. Enzyme activity assay The enzyme activity assay was performed in 1 ml of reaction using sucrose as substrate 233 mM (8% w/v) in 50 mM phosphate buffer, pH 5 and crude enzyme extract in a ratio 1:1. The reaction mixture was incubated at 30 °C for 40 min and 0.1 ml aliquots were inactivated in boiling water for 5 min at times 0, 5, 10, 15, 20 and 40 min. The enzyme activity was determined estimating the reducing sugars concentration by colorimetric method with 3,5 dinitrosalicylic acid (DNS) at 540 nm (Miller, 1959; Aman et al., 2012; Vettori et al., 2011; Champion et al., 2009) and glucose concentration by the enzymatic method (Kit GOD-POD) at 510 nm. One unit (U) of dextransucrase
2.6.3. Anion exchange chromatography To separate DS and dextranase, a BioRad Biologic Duo flow FPLC chromatograpy was used, with a column 150 × 7 mm of the anionic resin UNOsphere Q BioRad and buffer phosphate 50 mM, pH 5.0 as mobile phase (flow 2 ml/min, 20 °C, sample volume 500 μl) and a NaCl linear gradient of from 0 to 1 M. The fraction volume was 2 ml and the 9
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Fig. 2. Growth kinetics from Leuconostoc mesenteroides IBUN 91.2.98, processes from: activation, inoculum and fermentation. −△−: activation, ( ± DS): 0.048; −◇−: obtaining inoculum, ( ± DS): 0.063; −○−: fermentation, ( ± DS): 0.055; −□−: enzymatic activity, ( ± DS): 0.149; − − −▶: ending and initiation of fermentation and time of inoculation; activation periods and inoculum were performed in flask 100 ml, fermentation was conducted in a fermenter 2000 ml, in all periods was employed 5% the inoculum; the values expressed ( ± DS) are the result of three (3) independent experiments n = 3.
Cholamidopropyl) dimethylammonio] −1-propanesulfonate (CHAPS), 2% (w/v); bromophenol blue, 0.002% (w/v); 1% ampholytes (pH: 3–10); threo-1,4-dimercapto-2,3-butanediol (DTT), 18 mM and 26.4 g sample (150 μl).
total time for each run was 60 min. The analyzed samples were previously filtered and kept at a temperature of 4 °C., fractions were unified and dialyzed using a membrane with a pore size of 10,000 Da in 50 mM phosphate buffer, pH 5.0, by 12 h. They were lyophilized and resuspended in 500 μl of 50 mM phosphate buffer at pH 5.0, to quantify protein and enzyme activity.
2.7. Identification dextransucrase enzyme by peptide mapping
2.6.4. Electrophoresis SDS-PAGE analysis of the purified enzyme Sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS–PAGE) was performed according to the method of Laemmli (U.K. Laemmli, 1970) under not reduced conditions. The concentrations of resolving gel and stacking gel were 8% and 2,4%, respectively. Molecular weight markers from ProSieve™ (225- 5 Kda) were used.
Identifying the DS enzyme by peptide mapping was made on the corresponding point (spot) gel isoelectric focusing (IEF), this was subjected to treatment with trypsin (Promega) overnight at 37 °C.The peptides were extracted from the gel using 60% acetonitrile in trifluoroacetic acid (TFA) 0.2%; They were then concentrated by vacuum drying; salts were removed using a micro- column C18 reverse phase (OMIX Pippetetips, Varian); elution of peptides was performed directly on the sample plate of the mass spectrometer, with 3 μl matrix solution (α-cyano-4-hydroxycinnamic in 60% acetonitrile containing 0.2% TFA). Mass spectra of the digestion mixtures were performed in a spectrometer MALDI-TOF/TOF 4800 (Applied Biosystems), the calibration was performed using a mixture of standard peptides (Applied Biosystems). The proteins were identified in the NCBI database using the values: (m/z) and using the MASCOT program with the following search parameters: monoisotopic mass tolerance of 0.05 Da; tolerance of the mass fractions: 0.25 Da and oxidation of methionine as a possible modification.
2.6.5. Dextransucrase identification by activity gel DS activity was detected by gel electrophoresis under denaturing SDS-PAGE and nonreducing conditions, using a concentration polyacrylamide gel separator and concentrator 8% T, 2.4% C and 5% T, 1.3% C respectively; electrophoresis was run at 80 V constant for 45 min at 10 °C. After the run, SDS is remove using a modification of the protocol described is by Ravi (Purama and Goyal, 2008) which consists in immersing the gel in a 50 mM phosphate buffer (pH 5.0), 0.45 mM CaCl2, and 1% (v/v) Triton X-100, under constant stirring for 10 min at 4 °C and washed with distilled water, this procedure was performed three times in succession. The gel was incubated in a 50 mM phosphate buffer (pH 5.0), 146.1 mM sucrose, for 45 min at 30 °C under constant stirring; then was washed three times with distilled water at 4 °C for 5 min. Identifying sucrose dextran activity was performed using the method PAS (periodic acid-Schiff) as described by Miller and Robyt (1986). The gel was immersed in a 0.5% solution (w/v) of periodic acid for 10 min, then washed three times with distilled water at constant stirring for 5 min. Subsequently the gel is suspended in Schiff's reagent (0.5% (w/v) of fuchsin, 2.5% (w/v), 0.55% NaHSO3 and concentrated HCl) to banding deep pink (Shukla et al., 2011), this confirms the presence of dextran (Miller and Robyt, 1986; Kapitany and Zebrowski, 1973; Bejar et al., 2013).
3. Results and discussion 3.1. Dextransucrase enzyme production Comparing each of the three systematic stages provided for production of the enzyme (activation and fermentation inoculum) a decrease is observed at the time of the growth kinetics of the microorganism strain Leuconostoc mesenteroides IBUN 91.2.98 (See Fig. 2). With the composition of culture medium and fermentation conditions, the microorganism is able to obtain an enzyme extract with maximum DS activity of 6.9 U/ml for 4 h cultivation, coinciding with the end of the exponential phase and maximum biomass of 2.29 g/l. In this work the enzyme productivity was 1533 U/l.h−1, this is one of the highest achieved in a fermentation process; DS reports of some activities achieved in fermentation are: L mesenteroides NRRL B-512F (Fabre et al., 2005), L mesenteroides B512 FMC (Parlak et al., 2014), L. mesenteroides NRRL B-1299 (Moulis et al., 2006) and the alternansucrase of L. citreum NRRL B-1355 (Joucla et al., 2006) with: 5.85 U/ml, 6.33 U/ml, 0.58 U/ml y 0.66 U/ml respectively. The maximum specific growth rate (μmax) was 1.16 h−1 with a doubling time of 0.59 h (35.8 min); the substrate consumption in fermentation were 97.8% and the yield 17.7 g/l dextran (Yp/s: 0.304).
2.6.6. Isoelectric focusing (IEF), two-dimensional gel electrophoresis (2D) Proteomic analysis of crude extract or purified enzyme DS was performed by two-dimensional gel electrophoresis (2D-PAGE). The technique involves both the first dimension (separation based on the isoelectric point within a matrix in a pH gradient containing ampholytes) and second dimension (separation in a matrix based on molecular weight).The first dimension was performed on a system using a computer isoelectric focussing IPG strip ReadyStrip TM IPG Strips (BioRad). Immobilized pH gradient of 7 cm in length, were used with a pH range of 3–10 (Bio-Rad), polyacrylamide containing 4% T and 3% of C. The hydration of the strips IPG, was performed with 150 μl of rehydration solution, whose composition is urea, 8 M; 3 − [(310
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Fig. 3. Anion exchange chromatogram; separation of the sample from the hydrolytic treatment with dextranase ultrafiltration retentate. Peak A: Corresponding fractions 8 to the 12, where dextransucrase activity was detected; paek B: corresponding to fractions: 35 to the 38; dextranase activity was detected; peak C: corresponding to fractions 39 to the 48, no kind of activity was detected.
3.2. Purification of the enzyme dextransucrase The enzyme-dextran complex formed in the fermentation allows the enzyme DS to be stopped in the so-called retained fraction of the ultrafiltration process, achieving a concentration of 13.8 times, relative to the initial feed using a 300 KDa membrane (see Fig. 1). Subsequent to this a 54.9% dextran separation was performed by enzymatic hydrolysis using a dextranase, the separation of these two enzymes together with the residues of the hydrolysis was achieved by anion exchange chromatography (see Fig. 3). It was establish that the minimum dextran concentration is 110.5 mg/ml with an enzyme activity of 72.7%. The enzyme purification steps are shown in Table 1. A purification factor of 118 was achieved along with a yield of 26%, the specific activity obtained was 335.1 U/mg one of the highest reported activities of a purified DS enzyme. Some activities of DS enzymes purified using ultrafiltration are: L. mesenteroides B-512FMC (Kitaoka and Robyt, 1998) C. and L. mesenteroides B-512 (Robyt and Walseth, 1979) are 183 U/mg and 53 U/mg respectively. 3.2.1. Diafiltration and ultrafiltration The first step of purification of DS is concentration and diafiltration by ultrafiltration of fermentation supernatant. Using pore size membrane of 300 kDa it was possible to retain the 100% of DS activity (104 U/ml) in the concentrated fraction of ultrafiltration, the dextran polymer concentration in this fractions is 245 mg/ml. In the fractions of ultrafiltering is not detect DS activity, protein content is 2.56 mg/ml equivalent to 97.6% of the initial. With a calculated molecular weight in 171.5 kDa, the DS was retained by the polymer and remain in the concentrated fraction; this is due to the covalent interaction of the enzyme in the formation of dextran, not allowing this to be released. The concentration limit reached in the ultrafiltration was 13.8 times relative to the initial volume of the filtrate, this is due to increased viscosity by accumulation of dextran.
Fig. 4. Electrophoresis SDS PAGE analysis purification of DS enzyme from the strain Leuconostoc mesenteroides IBUN 91.2.98. 1: Molecular weight markers (225 − 5 kDa), 2: Concentrate from the ultrafiltration membrane 300 kDa (retained), 3: Fermentation supernatant (crude extract), 4: Dextransucrase fraction from the anion exchange column (UNOsphere Q).
established that the enzyme does not have protein subunits and 170.1 kDa molecular size was calculated; isoelectric focusing shows absence of isoproteins and an isoelectric point (pI) of 4.2.
3.3. Electrophoretic analysis Protein separation results are shown in Fig. 4. A single protein band with activity was detected corresponding to dextransucrase; electrophoretic gel showed deformation until rupture by the accumulation of dextran polymer due to the high enzyme activity, see Fig. 5. It was
3.4. Enzymatic properties of dextransucrase IBUN 91.2.98 The effect of sucrose on the activity of the enzyme was determined
Table 1 Purification of dextransucrase of Leuconostoc mesenteroides strain IBUN 91.2.98 by ultrafiltration concentration and subsequent hydrolysis of dextran with dextranase. Purification step
Volume (ml)
Enzime Activity (U/ml)
Total protein (mg)
Specific activity (U/mg)
Yield (%)
Purification (Fold)
Crude extract Ultrafiltration 300 kDa Ion exchange UNO sphere
1800 130 130
6.9 104 24.8
4374 104 9.6
2.8 130 335.1
100 109 26
1 46 118
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Fig. 5. Activity gel (zimograma) for dextransucrase and identification of the enzyme produced by the microorganism: Leuconostoc mesenteroides IBUN 91.2.98. 1: Molecular weight markers (220–40 KDa); 2, 3 and 4: crude extract (supernatant from fermentation), bands were stained with Coomassie blue. 5: accumulation of dextran polymer, product of enzyme activity; a: bands exposed treatment Schiff reagent, PAS staining (periodic acid-Schiff), reaction time 2 min; b: reaction time 5 min; c: reaction time 10 min, note accumulated polymer production, which ends at the break of electrophoretic gel.
Fig. 6. Linearization by the method of Hannes Wolf of dextransucrose enzyme produced by the microorganism: Leuconostoc mesenteroides strain: IBUN 91.2.98. S/V = (1/Vmax)S + (Km/Vmax); ( ± DS 0.76), R2 = 0.993. Where: S: substrate (mM) and V: enzymatic activity (U/ml).
using various substrate concentrations in the reaction, between 8.76–1168 mM, adjusting a Michaelis-Menten kinetics with a Vmax of 28.1 U/ml and Km of 48 mM, calculated by the method Hanes-Woolf, see Fig. 6. The enzyme presents substrate inhibition at concentrations over 584 mM sucrose, although at a concentration of 1168 mM their activity decreases only 7.3%, this values are consistent if compared with characteristics kinetics reported by these enzymes (Patel et al., 2011).
Fig. 7. Effect of pH on the activity and enzymatic stability of dextransucrase of Leuconostoc mesenteroides IBUN 91.2.98. A: effect of pH on enzyme activity, ( ± DS): 2.39. B: effect of pH on the stability of the enzyme, −Δ−: time of exposition: 20 min, ( ± DS): 1.88; −□−: time of exposition: 40 min, ( ± DS): 3.19; −◇−: time of exposition: 80 min, ( ± DS): 2.60. The trials were conducted by exposing the dextransucrase to different values pH in times of 20, 40 and 80 min; after this enzymatic activity assays performed at a temperature of 30°C, with 50 mM phosphate buffer, pH: 5, containing 233 mM (8% p/v) sucrose as substrate. The values expressed ( ± DS) are the result of three independent experiments n = 3.
3.4.1. Effect of temperature and pH on the activity and stability of the enzyme The effect of pH and temperature on DS activity is shown in Fig. 7 and Fig. 8, the optimum pH is 5, losing 51.4 and 74.9% of its initial activity at pH 4 and 7 respectively. The enzyme showed high stability against pH values between 4 and 7 at three different exposure times (20, 40 and 80 min). The enzyme activity gradually affected at pH values below 4 decreasing in average 0.48% of its activity per minute exposure. The optimal temperature for DS is 30 °C and enzyme denaturing
occurs at 40 °C where their activity decreases 86.8%. The activation energy (Ea) is 22.6 kJ/mol, this value is lower than that reported for DS of L. mesenteroides NRRL B-1299 and L. mesenteroides NRRL B-512F, 47 12
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act as activators in a 36.7 and 27.2% respectively. Calcium ion (Ca+3) had a slight inhibitory effect of 8% of its initial activity. This differs from other values reported for DS of L. mesenteroides where calcium ions increases enzyme activity and stability (Parlak et al., 2014; Kitaoka and Robyt, 1998). See Table 2. 3.4.3. Identification of the enzyme by peptide mapping A signal is identified (m/z = 2036.8) whose sequence is: −GEFINADGDTFYTSATDGR− which is only present in the protein identified as “dextransucrose DsrD”; the enzyme DS of Leuconostoc mesenteroides strain IBUN 91.2.98. It owns 25% homology with the protein DsrD; similarly a signal detected in the spectrum (m/ z = 1599.8) corresponding to the sequence: −YYFEPGSGNLAILR− which is only present in proteins glucosyl-hydrolases family 70 (GH70) which have repetitive fragments −YG−.Their functioning is related to the accession and stability of the enzyme with the enzymatic reaction product polymer. 4. Conclusions The strain Leuconostoc mesenteroides IBUN 91.2.98 is producing an extracellular DS type enzyme, which synthesizes a dextran polymer of high molecular weight (800–1000 kDa); the fermentation conditions used for growth of the microorganism and production of the enzyme, allowed high enzyme productivity, it is the highest reported so far. DS covalently interacts with dextran polymer product of the enzymatic synthesis, providing stability and allowing its partial purification by ultrafiltration on a filter with a pore size of 1.7 times of DS. The polymer produced in the enzymatic synthesis using DS is associated with enzyme stability, which is confirmed by the complete loss of enzymatic activity in the absence of polymer. Repetitive peptide fractions analyzed by mass spectrometry is associated with enzyme to polymer adhesion and with the enzyme stability. Because of this feature it was not possible for complete hydrolysis of dextran, becoming a limiting factor in the purification process. The enzyme presents low inhibition with calcium ion (Ca+3), differs from other similar enzymes. Dextransucrose was classified as a glycosylhydrolase (GH) belonging to the family 70, showing 25% homology with dextransucrase reported as DsrD.
Fig. 8. Temperature effect on activity and enzymatic stability of dextransucrase of Leuconostoc mesenteroides IBUN 91.2.98. A: Temperature effect on enzyme activity ( ± DS): 2.36. B: Temperature effect on stability activity, −◇−: Time of exposition: 20 min, ( ± DS): 2.61; −□−: Time of exposition: 40 min, ( ± DS): 3.19; −○−: Time of exposition: 80 min, ( ± DS): 2.66. The values expressed ( ± DS) are the result of three independent experiments n = 3.
Table 2 Effect of metal ions on the enzyme activity of dextransucrase (DS) from Leuconostoc mesenteroides strain IBUN 91.2.98. The tests were performed at 30 °C, pH: 5 and a sucrose concentration of 233.7 mM as a substrate. The enzymatic reactions were performed in the absence (control) and presence of metal ions at a final concentration of 2 mM, all as chlorides. The values expressed ( ± DS) are the result of three independent experiments n = 3. Metal ion
Relative activity (%)
Control CaCl2 MnCl2 NaCl KCl AlCl3 ZnCl3 MgCl2 FeCl3
100.0 ± 4.7 91.4 ± 3.4 109.6 ± 4.3 85.9 ± 2.8 127.2 ± 2.0 0.9 ± 0.1 99.9 ± 4.2 136.7 ± 4.0 3.5 ± 1.3
Acknowledgments The authors wish to thank: Biotechnology Institute of the National University of Colombia (IBUN), the Administrative Department of Science, Technology and Innovation (Colciencias), Dr. Jairo Cerón Salamanca, professor at the National University of Colombia, Dr. Jaime Antonio Cardozo Cerquera of Colombian Agricultural Corporation Research (CORPOICA). References Aman, A.I., Siddiqui, N.N., Qader, S.A.U., 2012. Characterization and potential applications of high molecular weight dextran produced by Leuconostoc mesenteroides AA1. Carbohydr. Polym. 87, 910–915. Bejar, W., Gabriel, V., Amari, M., Morel, S., Mezghani, M., Maguin, E., Fontagné-Faucher, C., Bejar, S., Chouayekh, H., 2013. Characterization of glucansucrase and dextran from Weissella sp: TN610 with potential as safe food additives. Int. J. Biol. Macromol. 52, 125–132. Champion, E., André, I., Moulis, C., Boutet, J., Descroix, K., Morel, S., Monsan, P., Mulard, L.A., Remaud-Siméon, M., 2009. Design of α-Transglucosidases of Controlled Specificity for Programmed Chemoenzymatic Synthesis of Antigenic Oligosaccharides. J. Am. Chem. Soc. 131, 7379–7389. Dols, M., Remaud-Simeon, M., Willemot, R.M., Vignon, M., Monsan, P., 1998. Characterization of the different dextransucrase activities excreted in glucose, fructose, or sucrose medium by Leuconostoc mesenteroides NRRL B-1299. Appl. Environ. Microbiol. 64, 1298–1302. Edward, H.J., 2001. Glycosyl transfer: a history of the concept’s development and view of its major contributions to biochemistry. Carbohydr. Res. 331, 347–368. Fabre, E., Bozonnet, S., Arcache, A., Vignon, M., Monsan, P., Remaud-simeon, M., 2005. Role of the two catalytic domains of DSR-E dextransucrase and their involvement in
and 44 kJ/mol respectively (Dols et al., 1998). The enzyme presents low stability with respect to temperature, decreases its activity at 0.73 and 1.52% per minute of exposure to 30 and 40 °C respectively.
3.4.2. Effect of metal ions and agents The effect of some metal ions on the DS produce for Leuconostoc mesenteroides strain IBUN 91.2.98. is shown in Table 2; the enzyme is strongly inhibited by cations Fe+3 and Al+3 in a 96.5 and a 99.1% respectively compared to the initial activity while cations Mg+2 and K 13
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