Production of Levansucrase from Bacillus subtilis NRC 33a and Enzymic Synthesis of Levan and Fructo-Oligosaccharides

Bacillus subtilis NRC33a was able to produce both inducible and constitutive extracellular levansucrase, respectively, using sucrose and glucose as carbon source. The optimal production of the levansucrase was at 30°C. The effect of different nitrogen sources showed that baker’s yeast with 2% concentration gave the highest levansucrase activity. Addition of 0.15 g/L MgSO₄ was the most favorable for levansucrase production. The enzymic synthesis of levan was studied using 60% acetone fraction. The results indicated that high enzyme concentrations produced increasing amounts of levan, and hence conversion of fructose to levan reached 84% using 1000 μg/ml enzyme protein. Sucrose concentration was the most effective factor controlling the molecular weight of the synthesized levan. The conversion of fructose to levan was maximal at 30°C. The time of reaction clearly affected the conversion of fructose to levan, which reached its maximum productivity at 18 hours (92%). Identification of levan indicated that fructose was the building unit of levan.     

Comparison of characteristics of levan produced by different preparations of levansucrase from Zymomonas mobilis

The characteristics of levan formation by different preparations of levansucrase (free and immobilized enzyme and toluene-permeabilized whole cells), derived from recombinant levansucrase from Zymomonas mobilis expressed in Escherichia coli, were investigated. The maximal yield of levan by the three preparations were similar and were about 70–80% on a fructose-released basis with sucrose as nutrient at 100 g l^–1. Immobilized enzyme and toluene-permeabilized whole cells produced low molecular weight levan (2–3 × 10⁶), as determined by HPLC while high molecular weight levan (>6 × 10⁶) was the major product with the free levansucrase. The size of levan can thus be controlled by immobilized levansucrase and toluene-permeabilized whole cells in high yield.     

Enhanced levan production using chitin-binding domain fused levansucrase immobilized on chitin beads

Levan is a homopolymer of fructose which can be produced by the transfructosylation reaction of levansucrase (EC 2.4.1.10) from sucrose. In particular, levan synthesized by Zymomonas mobilis has found a wide and potential application in the food and pharmaceutical industry. In this study, the immobilization of Z. mobilis levansucrae (encoded by levU) was attempted for repeated production of levan. By fusion levU with the chitin-binding domain (ChBD), the hybrid protein was overproduced in a soluble form in Escherichia coli. After direct absorption of the protein mixture from E. coli onto chitin beads, levansucrase tagged with ChBD was found to specifically attach to the affinity matrix. Subsequent analysis indicated that the linkage between the enzyme and chitin beads was substantially stable. Furthermore, with 20% sucrose, the production of levan was enhanced by 60% to reach 83 g/l using the immobilized levansucrase as compared to that by the free counterpart. This production yield accounts for 41.5% conversion yield (g/g) on the basis of sucrose. After all, a total production of levan with 480 g/l was obtained by recycling of the immobilized enzyme for seven times. It is apparent that this approach offers a promising way for levan production by Z. mobilis levansucrase immobilized on chitin beads.     

Identification and characterisation of the extracellular sucrases of Zymomonas mobilis UQM 2716 (ATCC 39676)

An investigation was conducted to isolate, and characterise the extracellular sucrases of Zymomonas mobilis UQM 2716. Levansucrase (EC 2.4.1.10) was the only extracellular sucrase produced by this organism. This enzyme was responsible for sucrose hydrolysis, levan formation, and oligosaccharide production. It had a molecular mass of 98 kDa, a Michaelis constant (K _m) of 64 mm, and a pH optimum of 5.5. It was inhibited by glucose, but not by fructose, ethanol, sorbitol, NaCl, TRIS or ethylenediaminetetraacetic acid (EDTA). The formation of levan was the principal reaction catalysed by this enzyme at low temperatures. However, levan formation was thermolabile, being irreversibly lost when levansucrase was heated to 35°C. S This did not effect sucrose hydrolysis or oligosaccharide formation, which were optimal at 45°C. Sucrose concentration greatly influenced the type of acceptor molecule used in the transfructosylation reactions catalysed by levansucrase. At low sucrose concentration, the predominant reaction catalysed was the hydrolysis of sucrose to free glucose and fructose. At high sucrose concentrations, oligosaccharide production was the major reaction catalysed.     

Mercuric Ion Stabilizes Levansucrase Secreted by <Emphasis Type="Italic">Acetobacter nitrogenifigens</Emphasis> Strain RG1<Superscript>T</Superscript>

The purification and characterization of an extracellular levansucrase enzyme produced by novel nitrogen-fixer Acetobacter nitrogenifigens strain RG1^T is described. Culture conditions were optimized for maximum levansucrase production. Levansucrase purified to homogeneity by tenfold purification has a molecular weight of 65 kDa, contained four cysteine residues, polymerized raffinose and was stable for 21 days at pH 6.0 when stored at 4 °C or −20 °C but was vulnerable to DTT and β-mercaptoethanol. Interestingly, this enzyme showed enhanced hydrolytic and polymerization activity in the presence of mercuric ion which, to our knowledge, is the first report for any levansucrase enzyme characterized so far. Evidences obtained from Native PAGE, tryptophan fluorescence study and activity measurements at different temperatures and in the presence of thiol modifying agents, show that mercuric ion stabilizes the enzyme. Levan, synthesized by the enzyme, has a molecular weight of 7,080 kDa and was shown to be a homopolymer of fructose.     

Fructan Biosynthesis by Intra‐ and Extracellular Zymomonas mobilis Levansucrase after Simultaneous Production of Ethanol and Levan

The chemical composition of the Zymomonas mobilis biomass and the culture liquid after ethanol and levan synthesis were studied. The activities of intra‐ and extracellular levansucrase produced by the Z. mobilis strain 113 “S” under optimum conditions both for levan and fructooligosaccharide (FOS) synthesis were also determined. It was shown that levan production relates to the reduction of the carbohydrate and lipid content in the biomass by increasing the nucleic acid and protein content. The levan producing activity of cellular levansucrase after ethanol and levan synthesis was approximately 30–40% of the total activity in the second fermentation stage. It was established that the cell free culture liquid, containing ethanol, levan, gluconic acid and sucrose (15%) at 25 °C, did not show any additional levan synthesising activity. At optimum FOS synthesis conditions (45 °C and 70% sucrose), the cell‐free culture liquid exhibited a high FOS synthesising activity (31% from total carbohydrates), with slightly reduced biomass activity. It was concluded that as a result of the simultaneous ethanol and levan production, the remaining biomass as well as the cell‐free culture liquid could be used for FOS production.     

Optimal conditions for levan formation by an overexpressed recombinant levansucrase

Summary A genetically modified levansucrase, which contained His-affinity tag in its C-terminal, was constructed by PCR reaction using two synthetic primers. This modified protein was produced up to 30 % in total cell protein of E. coli, and was purified by a one-step affinity chromatography. The optimum pH for levan production was pH 5 and the optimum temperature was 0 °C. The higher velocity of levan formation within shorter enzyme reaction times was achieved by increasing the levels of enzyme concentration. The optimal sucrose concentration for levan production was around 20 %. Under these conditions, more than 50 g levan/l was produced.     

Levan and fructosyl derivatives formation by a recombinant levansucrase from Rahnella aquatilis

Levan, fructo-oligosaccharides and fructosyl derivatives were formed from sucrose using recombinant levansucrase from Rahnella aquatilis. Levan formation was optimal at 30 °C resulting 57 % of the theoretical yield. The more suitable substrate concentration for levan formation was 200 g sucrose/L. Oligosaccharides was accumulated selectively at high substrate concentration. The increase of levan and oligosaccharides formation was not achieved by adding water-miscible organic solvents. Alkyl fructosides were synthesized from various alcohols as fructosyl acceptors by R. aquatilis levansucrase. © Rapid Science Ltd. 1998     

Molecular and functional characterization of a levansucrase from the sourdough isolate Lactobacillus sanfranciscensis TMW 1.392

Exopolysaccharides (EPS) produced in situ by sourdough lactobacilli affect rheological properties of dough as well as bread quality and may serve as prebiotics. The aim of this study was to characterize EPS-formation by Lactobacillus sanfranciscensis TMW 1.392 at the molecular level. A levansucrase gene from L. sanfranciscensis TMW 1.392 encompassing 2,300 bp was sequenced. This levansucrase is predicted to be a cell-wall associated protein of 879 amino acids with a relative molecular weight (M_R) of 90,000. The levansucrase gene was heterologously expressed in Escherichia coli and purified to homogeneity. The recombinant enzyme exhibited transferase and hydrolase activities and produced glucose, fructose, 1-kestose and levan from sucrose; truncation of the N-terminal domain did not affect catalytic activity. Kestose formation was enhanced relative to fructose and levan formation by low temperature or high sucrose levels. During growth in wheat doughs, strain TMW 1.392 utilized sucrose to form fructose, 1-kestose, and fructan, whereas a levansucrase deletion mutant, L. sanfranciscensis TMW 1392lev, lost the ability to hydrolyze sucrose, and did not produce fructan or 1-kestose. These results indicate that, in L. sanfranciscensis TMW 1.392, sucrose metabolism and formation of fructan and 1-kestose is dependent on the activity of a single enzyme, levansucrase.     

Gene cloning, characterization, and heterologous expression of levansucrase from Bacillus amyloliquefaciens

Although levan produced by Bacillus amyloliquefaciens is known to have efficient immunostimulant property which gives 100% survival of common carp when infected with Aeromonas hydrophila, no detailed reports are available describing kinetic studies of d-glucose production and levan formation. In this study, we cloned and characterized the enzymatic kinetics using levansucrase expressed in Escherichia coli. Optimum pH for d-glucose production and levan formation was 6.0 and 8.0, respectively, whereas optimum temperature was 30°C and 4°C, respectively. The K _m and V _max values for levansucrase were calculated to be 47.81 mM sucrose and 57.47 μmole/min mg protein, respectively. Prominent expression of levansucrase was obtained through xylose induction in Bacillus megaterium, where most of the His₆-tagged protein was secreted into the culture broth, giving levansucrase activity of 12,906 U/l. Response-surface methodology (RSM) was further employed to optimize the fermentation conditions and improve the level of levansucrase production. Maximum levansucrase activity of 20,251 U/l was obtained in 12 h of fermentation carried out at 28°C, starting induction with 0.735% xylose when A ₆₀₀ was 1.2, which was 1.6- and 62-fold higher than those obtained in the nonoptimized conditions for the recombinant strain and the native strain, respectively.     

Production of levan using recombinant levansucrase immobilized on hydroxyapatite

Levansucrase of Zymomonas mobilis was immobilized onto the surface of hydroxyapatite by ionic binding. Optimum conditions for the immobilization were: pH 6.0, 4 h of immobilization reaction time, and 20 U of enzyme/g of matrix. The enzymatic and biochemical properties of the immobilized enzyme were similar to those of the native enzyme, especially towards the effect of salts and detergents. The immobilized enzyme showed sucrose hydrolysis activity higher as that of the native enzyme, but levan formation activity was 70% of the native enzyme. HPLC analysis of levan produced by immobilized enzyme showed the presence of two different types of levan: high-molecular-weight levan and low-molecular-weight levan. The proportion of low-molecular-weight levan to total levan produced by the immobilized enzyme was much higher than that with the native enzyme, indicating that immobilized levansucrase could be applied to produce low-molecular-weight levan. Immobilized levansucrase retained 65% of the original activity after 6 times of repeated uses and 67% of the initial activity after 40 d when stored at 4 °C.     

Intrinsic Levanase Activity of Bacillus subtilis 168 Levansucrase (SacB)

Levansucrase catalyzes the synthesis of fructose polymers through the transfer of fructosyl units from sucrose to a growing fructan chain. Levanase activity of Bacillus subtilis levansucrase has been described since the very first publications dealing with the mechanism of levan synthesis. However, there is a lack of qualitative and quantitative evidence regarding the importance of the intrinsic levan hydrolysis of B. subtilis levansucrase and its role in the levan synthesis process. Particularly, little attention has been paid to the long-term hydrolysis products, including its participation in the final levan molecules distribution. Here, we explored the hydrolytic and transferase activity of the B. subtilis levansucrase (SacB) when levans produced by the same enzyme are used as substrate. We found that levan is hydrolyzed through a first order exo-type mechanism, which is limited to a conversion extent of around 30% when all polymer molecules reach a structure no longer suitable to SacB hydrolysis. To characterize the reaction, Isothermal Titration Calorimetry (ITC) was employed and the evolution of the hydrolysis products profile followed by HPLC, GPC and HPAEC-PAD. The ITC measurements revealed a second step, taking place at the end of the reaction, most probably resulting from disproportionation of accumulated fructo-oligosaccharides. As levanase, levansucrase may use levan as substrate and, through a fructosyl-enzyme complex, behave as a hydrolytic enzyme or as a transferase, as demonstrated when glucose and fructose are added as acceptors. These reactions result in a wide variety of oligosaccharides that are also suitable acceptors for fructo-oligosaccharide synthesis. Moreover, we demonstrate that SacB in the presence of levan and glucose, through blastose and sucrose synthesis, results in the same fructooligosaccharides profile as that observed in sucrose reactions. We conclude that SacB has an intrinsic levanase activity that contributes to the final levan profile in reactions with sucrose as substrate.     

Microbial production of levansucrase for synthesis of fructooligosaccharides and levan

A newly isolated thermophilic bacterial strain from Tunisian thermal source was identified as Bacillus sp. and was selected for its ability to produce extracellular levansucrase. Following the optimization of carbon source, nitrogen source, temperature and initial pH of the growth medium in submerged liquid cultures. In fact, sucrose was found to be a good inducer of levansucrase enzymes. The optimal temperature and pH of the levansucrase were 50°C and 6.5, respectively and its activity increased four folds in the presence of 50mM Fe(2+). This enzyme exhibited a remarkable stability and retained 100% of its original activity at 50°C for more than 1h at pH 6.5. The half-life of the enzyme was 1h at 90°C. Crude enzyme of Bacillus sp. rich in levansucrase was established for the synthesis of fructooligosaccharides and levan. Bacillus sp. could therefore be considered as a satisfactory and promising producer of thermostable levansucrases. Contrary to other levansucrases, the one presented in the current study was able to produce high levels of levan with high molecular weight at 50°C and having an important effect as a hypoglycemic agent which was demonstrated in our previous publications (Dahech et al., 2011 [25]) and as a hypo-cholesterolemic agent which will be investigated in further research.     

Some properties of levansucrase of Bacillus natto stabilized with periodate oxidized yeast glucomannan

It was observed that levansucrase from Bacillus natto became unstable and was easily inactivated when the salts were removed from the enzyme solution, while the enzyme was stable for long time in a buffered saline. After modification with periodate oxidized yeast glucomannan, the enzyme increased thermal stability up to 45°C, in which it conserved more than 90% of its activity after 15 min treatement. The optimum temperature was also shifted from 40°C in the case of original enzyme to 50°C for the modified enzyme after 10 min reaction time. The half-life time increased significantly from 9 min to 55 min at 50°C, however it increased from 30 min and 22 min respectively at 40°C and 45°C to more than 1 h at the same temperature. The content of carbohydrates of modified enzyme was 25% that increases the molecular weight from 57 KDa to 80 KDa. The products from sucrose by the modified enzyme were the same as the case using original enzyme. Namely, the products confirmed were levan and 3 kestoses (6-, 1-, and neo-kestose).     

Synthesis and characterization of fructooligosaccharides using levansucrase with a high concentration of sucrose

A method for synthesizing branched fructo-oligosaccharides (BFOS) with a high concentration of sucrose (1–3 M) was developed using levansucrase prepared fromLeuconostoc mesenteroides B-1355C. The degree of polymerization of oligosaccharides synthesized according to the present method ranged from 2 to over 15. The synthesized BFOS were stable at a pH ranges of 2 to 4 under 120°C. The percentage of BFOS in the reaction digest was 95.7% (excluding monosaccharides; 4.3% was levan). BFOS reduced the insoluble glucan formation byStreptococcus sobrinus on the surfaces of glass vials or stainless steel wires in the presence of sucrose. They also reduced the growth and acid productions ofS. sobrinus. Oligosaccharides can be used as sweeteners for foods such as beverages requiring thermo-and acid-stable properties and as potential inhibitors of dental caries. The first two authors contributed equally to this work.     

Purification and biochemical characterization of a native invertase from the hydrogen-producing Thermotoga neapolitana (DSM 4359)

This is the first report describing the purification and enzymatic properties of a native invertase (β-D-fructosidase) in Thermotogales. The invertase of the hydrogen-producing thermophilic bacterium Thermotoga neapolitana DSM 4359 (hereby named Tni) was a monomer of about 47 kDa having an amino acid sequence quite different from other invertases studied up to now. Its properties and substrates specificity let us classify this protein as a solute-binding protein with invertase activity. Tni was specific for the fructose moiety and the enzyme released fructose from sucrose and raffinose and the fructose polymer inulin was hydrolyzed in an endo-type fashion. Tni had an optimum temperature of 85°C at pH 6.0. At temperatures of 80–85°C, the enzyme retained at least 50% of its initial activity during a 6 h preincubation period. Tni had a K _m and k _cat /K _m values (at 85°C and pH 6.0) of about 14 mM and 5.2 × 10⁸ M⁻¹ s⁻¹, respectively. Dedicated to the memory of Prof. R. A. Nicolaus, founder of the Institute (1968).     

Levan-type FOS production using a Bacillus licheniformis endolevanase

In the present work we describe an enzymatic production method to obtain β2-6 fructose oligosaccharides (levan-type FOS) through a sequential reaction in which a bacterial endolevanase is applied to levan produced from sucrose by bacterial levansucrases. A putative gene encoding an endolevanase, designated as LevBl, was identified through a bioinformatics search, isolated from a strain of Bacillus licheniformis IBt1 from our own collection and expressed in Escherichia coli. LevB1 showed a specific activity of 1.8 U/mg protein at 35 °C in 50 mM phosphate buffer pH 6.0. A first order kinetic behavior was found when up to 150 g/L of low molecular weight levan (8.3 kDa) was used as the substrate. The product profile was determined by HPAEC-PAD and consisted of levan-type FOS with a polymerization degree between 2 and 8, with levanbiose as the major product after long reaction times. Yields of 97% of levan-type FOS were obtained when 1.0 U/mL of LevB1 reacted with 100 g/L of levan produced by the levansucrase from Bacillus subtilis. Finally, it was observed that levan-type FOS are efficiently fermented by probiotic lactic acid bacteria.     

NMR structural study of fructans produced by Bacillus sp. 3B6, bacterium isolated in cloud water

Bacillus sp. 3B6, bacterium isolated from cloud water, was incubated on sucrose for exopolysaccharide production. Dialysis of the obtained mixture (MWCO 500) afforded dialyzate (DIM) and retentate (RIM). Both were separated by size exclusion chromatography. RIM afforded eight fractions: levan exopolysaccharide (EPS), fructooligosaccharides (FOSs) of levan and inulin types with different degrees of polymerization (dp 2–7) and monosaccharides fructose:glucose = 9:1. Levan was composed of two components with molecular mass ∼3500 and ∼100 kDa in the ratio 2.3:1. Disaccharide fraction contained difructose anhydride DFA IV. 1-Kestose, 6-kestose, and neokestose were identified as trisaccharides in the ratio 2:1:3. Fractions with dp 4–7 were mixtures of FOSs of levan (2,6-βFruf) and inulin (1,2-βFruf) type. DIM separation afforded two dominant fractions: monosaccharides with fructose: glucose ratio 1:3; disaccharide fraction contained sucrose only. DIM trisaccharide fraction contained 1-kestose, 6-kestose, and neokestose in the ratio1.5:1:2, penta and hexasaccharide fractions contained FOSs of levan type (2,6-βFruf) containing α-glucose. In the pentasaccharide fraction also the presence of a homopentasaccharide composed of 2,6-linked βFruf units only was identified. Nystose, inulin (1,2-βFruf) type, was identified as DIM tetrasaccharide. Identification of levan 2,6-βFruf and inulin 1,2-βFruf type oligosaccharides in the incubation medium suggests both levansucrase and inulosucrase enzymes activity in Bacillus sp. 3B6.     

Characterization of a thermostable lipase showing loss of secondary structure at ambient temperature

A gene encoding extracellular lipase was cloned and characterized from metagenomic DNA extracted from hot spring soil. The recombinant gene was expressed in E. coli and expressed protein was purified to homogeneity using hydrophobic interactions chromatography. The mature polypeptide consists of 388 amino acids with apparent molecular weight of 43 kDa. The enzyme displayed maximum activity at 50°C and pH 9.0. It showed thermal stability up to 40°C without any loss of enzyme activity. Nearly 80% enzyme activity was retained at 50°C even after incubation for 75 min. However above 50°C the enzyme displayed thermal instability. The half life of the enzyme was determined to be 5 min at 60°C. Interestingly the CD spectroscopic study carried out in the temperature range of 25–95°C revealed distortion in solution structure above 35°C. However the intrinsic tryptophan fluorescence spectroscopic study revealed that even with the loss of secondary structure at 35°C and above the tertiary structure was retained. With p-nitrophenyl laurate as a substrate, the enzyme exhibited a K _m , V _max and K _cat of 0.73 ± 0.18 μM, 239 ± 16 μmol/ml/min and 569 s⁻¹ respectively. Enzyme activity was strongly inhibited by CuCl₂, HgCl₂ and DEPC but not by PMSF, eserine and SDS. The protein retained significant activity (~70%) with Triton X-100. The enzyme displayed 100% activity in presence of 30% n-Hexane and acetone.     

Synthesis of levan in water-miscible organic solvents

The synthesis of levan using a levansucrase from a strain of Bacillus subtilis was studied in the presence of the water-miscible solvents: acetone, acetonitrile and 2-methyl-2-propanol (2M2P). It was found that while the enzyme activity is only slightly affected by acetone and acetonitrile, 2M2P has an activating effect increasing the total activity 35% in 40-50% (v/v) 2M2P solutions at 30 degrees C. The enzyme is highly stable in water at 30 degrees C; however, incubation in the presence of 15 and 50% (v/v) 2M2P reduced the half-life time to 23.6 and 1.8 days, respectively. This effect is reversed in 83% 2M2P, where a half-life time of 11.8 days is observed. The presence of 2M2P in the system increases the transfer/hydrolysis ratio of levansucrase. As the reaction proceeds with 10% (w/v) sucrose in 50/50 water/2M2P sucrose is converted to levan and an aqueous two-phase system (2M2P/Levan) is formed and more sucrose can be added in a fed batch mode. It is shown that high molecular weight levan is obtained as an hydrogel and may be easily recovered from the reaction medium. However, when high initial sucrose concentrations (40% (w/v) in 50/50 water/2M2P) are used, an aqueous two-phase system (2M2P/sucrose) is induce, where the synthesized levan has a similar molecular weight distribution as in water and remains in solution.