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.     

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.     

The K296-D320 region of recombinant levansucrase BA-SacB can affect the sensitivity of Escherichia coli host to sucrose

Purpose

A mutant BA-SacB-Del encoding BA-SacB minus K296-D320 region was constructed to analyze its effect on catalytic characteristics of the enzyme as well as help deepen understanding of the catalytic mechanism of BA-SacB and even proteins of GH68 family.

Methods

Based on the comparison of levansucrases from Bacillus amyloliquefaciens (BA-SacB) and Sphingopyxis macrogoltabida (SM-Lev), a mutant BA-SacB-Del encoding BA-SacB minus K296-D320 region was constructed and its effect on catalytic characteristics of the enzyme was analyzed.

Results

Deletion of this region would undoubtedly affect the conserved structure (i.e., central negatively charged cavity surrounded by five antiparallel β-strands) shared by the GH68 family. Therefore, Escherichia coli-expressing mutant BA-SacB-Del could more efficiently catalyze the production of levan in media containing high concentration of sucrose, which is unrealizable for BA-SacB.

Conclusions

This result should be valuable for understanding this conditional lethal mechanism. Therefore, this study should be very valuable for understanding the catalytic mechanism of BA-SacB and even proteins of the GH68 family. More importantly, levan can be conveniently produced by direct fermentation of sucrose-containing media with E. coli-expressing BA-SacB-Del which is not sensitive to sucrose.

     

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.     

Effects of oil reservoir conditions on the production of water‐insoluble Levan by Bacillus licheniformis

Bacillus licheniformis produced a water‐insoluble levan which has potential application as a selective plugging agent in microbial enhanced oil recovery (MEOR). The microorganism grew on sucrose, glucose, and fructose but produced levan only on sucrose. Plugging may thus be selectively controlled in the reservoir by substrate manipulation. B. licheniformis and a crude preparation of its extracellular enzymes were evaluated for their ability to produce levan under reservoir conditions. Oil reservoirs which have a temperature of less than 55°C, a pH between 6 and 9, a pressure less than 500 atm, and a salt concentration of 4% or less are potentially suitable. Examples of such reservoir conditions are found in Lloydminster on the Alberta‐Saskatchewan border, one of the largest Canadian oil reserves.     

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.     

Unraveling the role of flexible coil near calcium binding site of levansucrase on thermostability and product profile via proline substitution and molecular dynamics simulations

Due to its bioactivity and versatile applications, levan has appeared as a promising biomaterial. Levansucrase is responsible for the conversion of sucrose into levan. With the goal of enhancing levan production, the strategy for enhancing the stability of levansucrase is being intensively studied. To make proteins more stable under high temperatures, proline, the most rigid residue, can be introduced into previously flexible regions. Herein, G249, D250, N251, and H252 on the flexible coil close to the calcium binding site of Bacillus licheniformis levansucrase were replaced with proline. Mutations at G249P greatly enhance both the enzyme's thermodynamic and kinetic stability, while those at H252P improve solely the enzyme's kinetic stability. GPC analysis revealed that G249P synthesize more levan, but H252P generate primarily oligosaccharides. Molecular dynamics simulations (MD) and MM/GBSA analysis revealed that G249P mutation increased not only the stability of levansucrase, but also affinity toward fructan.     

Characterization of levan produced by Serratia sp.

Levan was produced by a newly isolated bacterium from soil, taxonomically identified as a Serratia sp. This is the first report of levan production by Serratia sp. The levan was digested by levanase, which cannot hydrolyze β-2,1 linkages and the remaining substrate was analyzed by NMR. It was found that this levan had less β-2,1 linkage than other microbial levans, and that the structure was quite different from the levan produced by other bacteria such as genus Bacillus.     

Biosynthesis of Levan,a Bacterial Extracellular Polysaccharide,in the Yeast Saccharomyces cerevisiae

Levans are fructose polymers synthesized by a broad range of micro-organisms and a limited number of plant species as non-structural storage carbohydrates. In microbes, these polymers contribute to the formation of the extracellular polysaccharide (EPS) matrix and play a role in microbial biofilm formation. Levans belong to a larger group of commercially important polymers, referred to as fructans, which are used as a source of prebiotic fibre. For levan, specifically, this market remains untapped, since no viable production strategy has been established. Synthesis of levan is catalysed by a group of enzymes, referred to as levansucrases, using sucrose as substrate. Heterologous expression of levansucrases has been notoriously difficult to achieve in Saccharomyces cerevisiae. As a strategy, this study used an invertase (Δsuc2) null mutant and two separate, engineered, sucrose accumulating yeast strains as hosts for the expression of the levansucrase M1FT, previously cloned from Leuconostoc mesenteroides. Intracellular sucrose accumulation was achieved either by expression of a sucrose synthase (Susy) from potato or the spinach sucrose transporter (SUT). The data indicate that in both Δsuc2 and the sucrose accumulating strains, the M1FT was able to catalyse fructose polymerisation. In the absence of the predicted M1FT secretion signal, intracellular levan accumulation was significantly enhanced for both sucrose accumulation strains, when grown on minimal media. Interestingly, co-expression of M1FT and SUT resulted in hyper-production and extracellular build-up of levan when grown in rich medium containing sucrose. This study presents the first report of levan production in S. cerevisiae and opens potential avenues for the production of levan using this well established industrial microbe. Furthermore, the work provides interesting perspectives when considering the heterologous expression of sugar polymerizing enzymes in yeast.     

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.     

Sucrose medium osmolality as a regulator of anabolic and catabolic parameters in Zymomonas culture

This study focuses on the growth of Zymomonas mobilis strain 113 S and its ethanol and levan production under the conditions of increasing sucrose medium osmolality caused by NaCl, KCl, sorbitol or maltose. The increase in medium osmolality (700–1,500 mosml/kg) was accompanied by the inhibition of growth (growth rate, biomass yield) and ethanol production (specific productivity and yield) In contrast, levan synthesis was less affected or even stimulated and, as a consequence, levan specific productivity was increased significantly. A decrease in the anabolic growth parameters correlated with a parallel inhibition of glucose-6-P dehydrogenase and alcohol dehydrogenase (isoenzyme ADH II) activities. A significant inverse linear relationship (r = ? 0.932, 1 ? P = 0.01) was observed between the values of the specific productivities of ethanol and levan. This relationship was confirmed independently by a controlled reduction of growth and ethanol productivity (3.75–4.75 mM sodiumbisulphite as an acceptor of acetaldehyde formed in the pyruvate decarboxylase reaction). As further support of this relationship, a significant inverse correlation was observed between levan specific productivity and ATP concentration in Zymomonas mobilis cells, most probably demonstrating that a reduced level of energetic metabolism is favourable for levan production.     

Levan Enhances Associated Growth of Bacteroides,Escherichia, Streptococcus and Faecalibacterium in Fecal Microbiota

The role of dietary fiber in supporting healthy gut microbiota and overall well-being of the host has been revealed in several studies. Here, we show the effect of a bacterial polyfructan levan on the growth dynamics and metabolism of fecal microbiota in vitro by using isothermal microcalorimetry. Eleven fecal samples from healthy donors were incubated in phosphate-buffered defined medium with or without levan supplementation and varying presence of amino acids. The generation of heat, changes in pH and microbiota composition, concentrations of produced and consumed metabolites during the growth were determined. The composition of fecal microbiota and profile of metabolites changed in response to substrate (levan and amino acids) availability. The main products of levan metabolism were acetic, lactic, butyric, propionic and succinic acids and carbon dioxide. Associated growth of levan-degrading (e.g. Bacteroides) and butyric acid-producing (e.g. Faecalibacterium) taxa was observed in levan-supplemented media. The study shows that the capacity of levan and possibly also other dietary fibers/prebiotics to modulate the composition and function of colon microbiota can be predicted by using isothermal microcalorimetry of fecal samples linked to metabolite and consortia analyses.     

Production of a high molecular weight levan by Bacillus paralicheniformis,an industrially and agriculturally important isolate from the buffalo grass rhizosphere

A new exopolysaccharide (EPS) producing Gram-positive bacterium was isolated from the rhizosphere of Bouteloua dactyloides (buffalo grass) and its EPS product was structurally characterized. The isolate, designated as LB1-1A, was identified as Bacillus paralicheniformis based on 16S rRNA gene sequence and phylogenetic tree analysis. The EPS produced by LB1-1A was identified as a levan, having β(2?→?6) linked backbone with β(2?→?1) linkages at the branch points (4.66%). The isolate LB1-1A yielded large amount (~?42 g/l) of levan having high weight average molecular weight (Mw) of 5.517?×?10⁷ Da. The relatively low degree of branching and high molecular weight of this levan makes B. paralicheniformis LB1-1A a promising candidate for industrial applications.

     

Exopolymer Diversity and the Role of Levan in Bacillus subtilis Biofilms

Exopolymeric substances (EPS) are important for biofilm formation and their chemical composition may influence biofilm properties. To explore these relationships the chemical composition of EPS from Bacillus subtilis NCIB 3610 biofilms grown in sucrose-rich (SYM) and sucrose-poor (MSgg and Czapek) media was studied. We observed marked differences in composition of EPS polymers isolated from all three biofilms or from spent media below the biofilms. The polysaccharide levan dominated the EPS of SYM grown biofilms, while EPS from biofilms grown in sucrose-poor media contained significant amounts of proteins and DNA in addition to polysaccharides. The EPS polymers differed also in size with very large polymers (Mw>2000 kDa) found only in biofilms, while small polymers (Mw<200 kD) dominated in the EPS isolated from spent media. Biofilms of the eps knockout were significantly thinner than those of the tasA knockout in all media. The biofilm defective phenotypes of tasA and eps mutants were, however, partially compensated in the sucrose-rich SYM medium. Sucrose supplementation of Czapek and MSgg media increased the thickness and stability of biofilms compared to non-supplemented controls. Since sucrose is essential for synthesis of levan and the presence of levan was confirmed in all biofilms grown in media containing sucrose, this study for the first time shows that levan, although not essential for biofilm formation, can be a structural and possibly stabilizing component of B. subtilis floating biofilms. In addition, we propose that this polysaccharide, when incorporated into the biofilm EPS, may also serve as a nutritional reserve.     

Levan production byBacillus polymyxa

Summary A levan-producing bacterium was isolated from soils and its characteristics for polysaccharide synthesis were studied. A series of enrichment and plating techniques enabled the isolation of a levan-producing bacterium from closely related contaminants. Cultural and physiological characteristics of the isolate identified the organism an a strain ofBacillus polymyxa. The organism produced about 40 g extracellular polysaccharide per liter of sucrose medium, which was about three times more yield than levan obtained from known levan producers. The highest amount of polysaccharide was on a 8% sucrose medium. Hydrolysis of the product showed that the polysaccharide consisted entirely ofd-fructose, and¹³C.n.m.r. spectra confirmed that the product was levan, a fructose polymer linked by B-(26) fructofuranosyl linkage.     

Production of microbial levan from sucrose,sugarcane juice and beet molasses

Summary Bacillus polymyxa (NRRL-18475) produced a levan-type fructan (B, 26 fructofuranoside) when grown on sucrose, sugarcane juice, and sugarbeet molasses. The organism converted about 46% of the fructose moiety of sucrose to levan when grown on sucrose medium, however, the yields of levan from sugarcane juice and beet molasses were much less than sucrose solution. Such sugarcane juice and beet molasses can be made a good substrate for levan production by various modifications. Adding peptone to sugarcane juice or passing beet molasses through a column of gel filtration media improved levan yield to a level almost comparable to that obtained from sucrose.     

Molasses as fermentation substrate for levan production by <Emphasis Type="Italic">Halomonas</Emphasis> sp.

Levan is a homopolymer of fructose with many outstanding properties like high solubility in oil and water, strong adhesiveness, good biocompatibility, and film-forming ability. However, its industrial use has long been hampered by costly production processes which rely on mesophilic bacteria and plants. Recently, Halomonas sp. AAD6 halophilic bacteria were found to be the only extremophilic species producing levan at high titers in semi-chemical medium containing sucrose, and in this study, pretreated sugar beet molasses and starch molasses were both found to be feasible substitutes for sucrose. Five different pretreatment methods and their combinations were applied to both molasses types. Biomass and levan concentrations reached by the Halomonas sp. AAD6 cells cultivated on 30 g/L of pretreated beet molasses were 6.09 g dry cells/L and 12.4 g/L, respectively. When compared with literature, Halomonas sp. was found to stand out with its exceptionally high levan production yields on available fructose. Molecular characterization and monosaccharide composition studies confirmed levan-type fructan structure of the biopolymers. Rheological properties under different conditions pointed to the typical characteristics of low viscosity and pseudoplastic behaviors of the levan polymers. Moreover, levan polymer produced from molasses showed high biocompatibility and affinity with both cancerous and non-cancerous cell lines.     

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.     

Serine substitution for cysteine residues in levansucrase selectively abolishes levan forming activity

Levansucrase is responsible for levan formation during sucrose fermentation of Zymomonas mobilis, and this decreases the efficiency of ethanol production. As thiol modifying agents decrease levan formation, a role for cysteine residues in levansucrase activity has been examined using derivatives of Z. mobilis levansucrase that carry serine substitutions of cysteine at positions 121, 151 or 244. These substitutions abolished the levan forming activity of levansucrase whilst only halving its activity in sucrose hydrolysis. Thus, polymerase and hydrolase activities of Z. mobilis levansucrase are separate and have different requirements for the enzyme's cysteine residues.