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.     

Purification and Some Properties of a New Levanase from Bacillus sp. No. 71

A levanase from Bacillus sp. was purified to a homogeneous state. The enzyme had a molecular weight of 135,000 and an isoelectric point of pH 4.7. The enzyme was most active at pH 6.0 and 40°C, stable from pH 6.0 to 10.0 for 20 hr of incubation at 4°C and up to 30°C for 30 min of incubation at pH 6.0. The enzyme activity was inhibited by Ag ⁺, Hg^{2 +}, Cu^{2 +}, Fe^{3 +}, Pb²⁺, and p-chloromercuribenzoic acid. The enzyme hydrolyzed levan and phlein endowise to produce levanheptaose as a main product. The limit of hydrolysis of levan and phlein were 71% and 96%, respectively.     

The molecular structure of low and high molecular weight levans synthesized by levansucrase

The levan synthesized by Bacillus subtilis levansucrase in the presence of alcohols was of only high molecular weight, while in solutions of high ionic strength only low molecular weight (MW) levan was produced. The addition of low MW levan to the enzyme reaction mixture at low ionic strength stimulated synthesis of a high MW levan, but the levan added was not incorporated into this high MW levan. Methylation analysis revealed that low MW levans contained glucose, which was isolated as 2,3,46-tetra-O-methyl alditol acetate showing that the glucose units existed as terminal residues. The molecular weight of levan estimated on the basis of glucose content coincided with that determined by the gel filtration method. Methylation analysis also revealed that the number of fructose residues of the linear fraction linked by leads to 6(F)2 leads to type bonds was 22 for levan with a molecular weight of (8.4(-22)) x 10(3), while it was 11 for that of 2,000 x 10(3). The number of (formula: see text) type branched residues increased with increase in the molecular weight of the levan synthesized.     

Enzymic synthesis of levan and fructo-oligosaccharides by <Emphasis Type="Italic">Bacillus circulans</Emphasis> and improvement of levansucrase stability by carbohydrate coupling

Bacillus circulans was able to produce extracellular levansucrase using sucrose as carbon source optimally at 35°C. The enzymic synthesis of levan and fructo-oligosaccharides was studied using a 50% ethanol fraction of crude extract. The molecular weight of the synthesized levan was markedly affected by sucrose concentration, the molecular weight of levan decreased with increased sucrose concentration up to 32% whereby fructo-oligosaccharides were isolated. Temperature and the reaction time clearly affected the conversion of fructose to levan with molecular weight values ranging from 10 to 38 kDa. Identification of levan indicated that fructose was the building unit of the levan obtained. Thermal and pH stabilities of B. circulans levansucrase could be improved by enzyme glycosylation using sodium metaperiodate treatment. Chemical modification provides additional points of attachment of the enzyme to the support which offered the modified enzyme greater stabilization than did the free enzyme. The modified enzyme exhibited thermal tolerance up to 50°C, where it retained 88.25% of its activity, while the free enzyme only retained 64.55% of its original activity. The half-life significantly increased from 130 min for the free enzyme to 347 min for the modified enzyme at 50°C, however, it increased from 103 min for the free enzyme to 210 min for the modified enzyme at 60°C. Other properties i.e., the response to some metal ions as well as the ability to convert higher substrate levels and tolerance to an extension of the reaction periods were also improved upon modification. Obviously, the results obtained outlined the conditions leading to the formation of important high or low molecular weight or levan and fructo-oligosaccharides suitable for different industrial applications.     

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.     

Levans. II. Light-scattering and sedimentation data of Streptococcus salivarius Levan in water.

The molecular weights and radii of gyration of Streptococcus salivarius levan fractions were obtained from light-scattering measurements in water. Sedimentation coefficients and partial specific volumes of the fractions were also obtained. Double logarithmic plots of [η] versus M?_w and S⁰ versus M?_w yielded slopes having values of 0.17 and 0.62, respectively. The data and various calculated parameters show that levan from Streptococcus salivarius is highly branched and behaves hydrodynamically as a compact particle of spherical symmetry.     

Probing the critical residues for intramolecular fructosyl transfer reaction of a levan fructotransferase

Levan fructotransferase (LFTase) preferentially catalyzes the transfructosylation reaction in addition to levan hydrolysis, whereas other levan-degrading enzymes hydrolyze levan into a levan-oligosaccharide and fructose. Based on sequence comparisons and enzymatic properties, the fructosyl transfer activity of LFTase is proposed to have evolved from levanase. In order to probe the residues that are critical to the intramolecular fructosyl transfer reaction of the Microbacterium sp. AL-210 LFTase, an error-prone PCR mutagenesis process was carried out, and the mutants that led to a shift in activity from transfructosylation towards hydrolysis of levan were screened by the DNS method. After two rounds of mutagenesis, TLC and HPLC analyses of the reaction products by the selected mutants revealed two major products; one is a di-D-fructose- 2,6':6,2'-dianhydride (DFAIV) and the other is a levanbiose. The newly detected levanbiose corresponds to the reaction product from LFTase lacking transferring activity. Two mutants (2-F8 and 2-G9) showed a high yield of levanbiose (38-40%) compared with the wild-type enzyme, and thus behaved as levanases. Sequence analysis of the individual mutants responsible for the enhanced hydrolytic activity indicated that Asn-85 was highly involved in the transfructosylation activity of LFTase.     

Formation of levan and sorbitol from sucrose by Zymomonas mobilis

Summary Ethanol yields produced by Zymomonas strains from sucrose are significantly lower than from glucose or fructose. The low yield is a consequence of the formation of both levan and sorbitol as by-products. Most of the levan is in a non-precipitable form, indicating low molecular weight. Formation of sorbitol was observed with both the Zymomonas strains studied. The measured amounts of levan and sorbitol were 8% and 11% of the original sucrose content, respectively.     

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.     

Molecular cloning and expression in Escherichia coli of an exo-levanase gene from the endophytic bacterium Gluconacetobacter diazotrophicus SRT4

Gluconacetobacter diazotrophicus produces levan from sucrose by a secreted levansucrase (LsdA). A levanase-encoding gene (lsdB), starting 51 bp downstream of the lsdA gene, was cloned from strain SRT4. The lsdB gene (1605 bp) encodes a protein (calculated molecular mass 58.4 kDa) containing a putative 36-amino-acid signal peptide at the N-terminus. The deduced amino acid sequence shares 34%, 33%, 32%, and 29% identities with levanases from Actinomyces naeslundii, Bacillus subtilis, Paenibacillus polymyxa, and Bacteroides fragilis, respectively. The lsdB expression in Escherichia coli under the control of the T7 RNA polymerase promoter resulted in an active enzyme which hydrolyzed levan, inulin, 1-kestose, raffinose, and sucrose, but not melezitose. Levanase activity was maximal at pH 6.0 and 30°C, and it was not inhibited by the metal ion chelator EDTA or the denaturing agents dithiothreitol and β-mercaptoethanol. The recombinant LsdB showed a fourfold higher rate of hydrolysis on levan compared to inulin, and the reaction on both substrates resulted in the successive liberation of the terminal fructosyl residues without formation of intermediate oligofructans, indicating a non-specific exo-levanase activity. Received: 27 August 2001 / Accepted: 15 October 2001     

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.     

Molecular and enzymatic characterization of a levan fructotransferase from Microbacterium sp. AL-210

Microbacterium sp. AL-210 producing a novel levan fructotransferase (LFTase) was screened from soil samples. The LFTase was purified to homogeneity by (NH4)2SO4 fractionation, column chromatography on Resource Q, and Superdex 200HR. The molecular weight of the purified enzyme was estimated to be approximately 46 kDa by both SDS-PAGE and gel filtration, and the enzyme's isoelectric point was pH 4.8. The major product produced from the levan hydrolysis by the enzyme reaction was identified by atmospheric pressure ionization mass spectrometry and NMR analysis as di-D-fructose-2,6':6,2'-dianhydride (DFA IV). The optimum pH and temperature for DFA IV production were 7.0 and 40 degrees C, respectively. The enzyme was stable at a pH range 7.0-8.0 and up to 40 degrees C. The enzyme activity was inhibited by FeCl2 and AgNO3. The enzyme converted the levan to DFA IV, with a conversion yield of approximately 44%. A gene encoding the LFTase (lftM) from Microbacterium sp. AL-210 was cloned and sequenced. The nucleotide sequence included an ORF of 1593 nucleotides, which is translated into a protein of 530 amino acid residues. The predicted amino acid sequence of the enzyme shared 79% of the identity and 86% of the homology with that of Arthrobacter nicotinovorans GS-9.     

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.     

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.     

Cultivation of Zymomonas mobilis 113 S at Different Mixing Regimes and their Influence on the Levan Formation

The Zymomonas mobilis 113 S strain was cultivated in a bioreactor with a working volume of 1.4 l at different stirring regimes in a 15% initial sucrose medium. The levan obtained in the fermentation process was analyzed by gel filtration. Because the sucrose/biomass ratio in the fermentation broth decreased to below 300 g/g, the insufficient concentration of sucrose might have decreased the concentration of levan. Besides the growth characteristics of the population, the mixing intensity and flow structure were also found to influence the molecular mass of levan. At 600 rpm, the microorganisms produced levan with a molecular mass lower than at 300 rpm. The stirring of a fermentation broth with levan without cells at 300 rpm and 900 rpm showed changes in the molecular mass approximately at 20 kD and 5 kD. The size of eddies in the fermenter was supposed to determine the size of a levan molecule. Because the size of the eddy may be compared with that of a levan molecule, it explains the decline in the molecular mass of levan.     

Studies on the Extracellular Polysaccharides (EPS) Produced in vitro by Pseudomonas phaseolicola I. Indications for a Polysaccharide Resembling Alginic Acid in Seven P. syringae Pathovars

Extracellular polysaccharides (EPS) produced by Pseudomonas syringae pv. phaseolicola are obviously composed of two main components: the long known levan consisting of fructose, and a mannuronan consisting mainly of mannuronic acid (manA), thus resembling alginic acid (alginate). The identification of manA was established by TLC utilizing different developing systems, and by cellulose acetate electrophoresis in different buffers. References were authentic uronic acids and hydrolyzed authentic alginate. A rough quantification of the “alginate” present in crude EPS was achieved with a selective colour reaction which largely excluded compounds other than uronic acids. Levan was only synthesized with sucrose as primary carbon source. When grown on several other sugars and related compounds “alginate” was the predominant component of the EPS. Additionally, rhamnose, fucose, glucose and amino sugars were found in some instances in hydrolysates of crude EPS, suggesting the release of lipopolysaccharides (LPS) from the bacterial cell walls during culture. Growth on carbon sources not related to sugars resulted in these “LPS” as the main constituent of EPS. After cultivation with sucrose, the “alginate” was restricted to the “slime” fraction of the EPS. In the “capsular” fraction, levan was predominating. A screening program revealed the capacity to synthesize the “alginate” in six additional P. syringae pathovars: pisi, lachrymans, aptata, tomato, syringae, and glycinea. All of the strains tested so far produced levan from sucrose, however, the “alginate” was formed not by all of them. There was a tendency that fresh isolates produced more “alginate” than strains subcultured for an extended time in vitro. This was also true for the total amount of EPS.     

Hydrolysis of poly(alkylene amidophosphate)s containing amino acid or peptide residues in the side groups. Kinetics and selectivity of hydrolysis

Kinetics of hydrolysis of poly(alkylene amidophosphate)s with amino acids or dipeptides as the side groups was studied by 31P NMR at pH 1.5, 6.5, and 8.5. The direction of hydrolysis and the relative rate coefficients of breaking P-O bonds in the main chain and P-N bonds in the side groups depend strongly on the pH of the medium of hydrolysis. The P-N (amide) bond hydrolyzes much faster than the P-O (ester) bond in acidic and close to neutral conditions (negligible P-O hydrolysis), whereas above pH > or = 8.5 these differences are much smaller. For instance, for 4-Ala the rate coefficients of hydrolysis are equal (in H2O at 37 degrees C and pH 8.5) to 1.9 x 10(-8) s(-1) and 1.0 x 10(-9) s(-1) for the P-N and P-O bonds, respectively, quite different from the values found for the low molecular model 2 (0 and 1.4 x 10(-7) s(-1), respectively).     

Development of Bifidobacterium lactis Bb 12 on β‐(2,6)‐Linked Fructan‐Containing Substrate

β‐(2,1)‐linked fructan of plant origin (inulin) and the related oligosaccharides (FOS) as non‐digestible carbohydrates, i.e., potent prebiotics, can stimulate the growth of various probiotic lactic acid bacteria, including a number of bifidobacteria strains. The related β‐(2,6)‐linked fructans of microbial origin (levan and FOS), however, have scarcely been investigated in this respect. Therefore, the bifidogenic properties of various fructans, i.e., inulin, levan, fructooligosaccharides (FOS) and fructan syrup (FS), were tested as glucose substitutes in MRS media and were compared concerning their effect on the commercial strain Bifidobacterium lactis Bb 12. Although glucose was the preferred substrate for growth and biomass formation, FS exhibited a comparable cell growth (8.4 × 10⁷ counts/mL and 1.0 × 10⁷ counts/mL, respectively) and acidification power (84 °T and 74 °T, respectively) during 48 h of fermentation, as well as an increase in lactic acid and decrease in acetic acid formation. Bifidobacterium lactis Bb 12 did not utilize inulin as a sole carbon source as judged from the 60 % decrease in cell count and the insignificant (0.1 pH unit) acidification of the growth medium, whereas levan provided a noticeable increase in cell count and acidification (0.4 pH units) during 48 h of fermentation. FOS preparation appeared to be a satisfactory carbon source for this strain, but lower acidification power (56 °T) and cell counts were observed as compared to glucose‐ or FOS‐containing media (2.6 % and 22 %, respectively). The products obtained under conditions of mild lactic acid hydrolysis of levan (37 °C, pH 3.3, 24 h) enhanced the cell count (7–10 %) and acidification power (by a factor of 2.7) of Bifidobacterium lactis Bb 12.     

By-products in the fermentation of sucrose by different Zymomonas-strains

Summary Eight Zymomonas strains were compared with respect to their sucrose hydrolysing activity and subsequent ethanol, levan and sorbitol formation. The ethanol yields obtained were within narrow limits, 0.40–0.43 g·g^-1 of sucrose. The distribution of by-products differed significantly between the strains tested. A low sucrose hydrolysis rate seemed to be associated with the formation of levan and a high sucrose hydrolysis rate with the formation of sorbitol through accumulation of monomeric sugars. Fructo-oligomers consisting of two fructose and one glucose unit represented the greatest loss of sucrose in the fermentation conditions used.     

Influence of weak and covalent bonds on formation and hydrolysis of gelatin networks

The relative influence of physical and chemical bonds to overall gel properties are explored in gelatin gels. Physical, chemical, chemical-physical, and physical-chemical gels are obtained by cooling the protein solution and/or by transglutaminase reaction. Each type of network is characterized by rheology and polarimetry. It is shown that the overall properties as well as the dynamics inside the gels are dependent upon the order of formation and on the relative amount of triple helices and covalent bonds. Enzyme hydrolysis of covalent gels is slower than that of physical gels, as confirmed by the kinetics of helix release and degradation. A scheme is proposed to explain the results at both the physicochemical and the molecular levels.