Levan-type Fructooligosaccharides Research Articles

Discovery and Enzymatic Screening of Genome-Mined Microbial Levanases to Produce Second-Generation β-(2,6)-Fructooligosaccharides: Catalytic Properties.

Evidence suggests that β-(2,6)-levan-type fructooligosaccharides (FOSs) possess higher prebiotic potential and selectivity than their β-(2,1)-inulin-type counterparts. The focus of the present work was to develop an enzymatic approach for the synthesis of levan-type FOSs, employing levanases (EC 3.2.1.65), specifically those performing endo-hydrolysis on levans. To identify new levanases, a selection of candidates was obtained via in silico exploration of the levanase family biodiversity through a sequence-driven approach. A collection of 113 candidates was screened according to their specific activities on low- and high-molecular-weight (MW) levan as well as thermal stability. The most active levanases were able to hydrolyze both types of levan with similar efficiency. This ultimately revealed 10 active, highly evolutionary distant and diverse candidate levanases, which demonstrated preferential hydrolysis of levan over inulin. The end-product profile differed significantly depending on levanase with levanbiose, levantriose, and levantetraose being the major FOSs. Among them, the catalytic properties of 5 selected potential new levanases (LEV9 from Belliella Baltica, LEV36 from Dyadobacter fermentans, LEV37 from Capnocytophaga ochracea, LEV79 from Vibrio natriegens, LEV91 from Paenarthrobacter aurescens) were characterized, especially in terms of pH and temperature profiles, thermal stability, and kinetic parameters. The identification of these novel levanases is expected to contribute to the production of levan-type FOSs with properties surpassing those of commercial preparations.

Simultaneous enzyme production, Levan-type FOS synthesis and sugar by-products elimination using a recombinant Pichia pastoris strain expressing a levansucrase-endolevanase fusion enzyme

BackgroundAlthough Levan-type fructooligosaccharides (L-FOS) have been shown to exhibit prebiotic properties, no efficient methods for their large-scale production have been proposed. One alternative relies on the simultaneous levan synthesis from sucrose, followed by endolevanase hydrolysis. For this purpose, several options have been described, particularly through the synthesis of the corresponding enzymes in recombinant Escherichia coli. Major drawbacks still consist in the requirement of GRAS microorganisms for enzyme production, but mainly, the elimination of glucose and fructose, the reaction by-products.ResultsThe expression of a fusion enzyme between Bacillus licheniformis endolevanase (LevB1) and B. subtilis levansucrase (SacB) in Pichia pastoris cultures, coupled with the simultaneous synthesis of L-FOS from sucrose and the elimination of the residual monosaccharides, in a single one-pot process was developed. The proof of concept at 250 mL flask-level, resulted in 8.62 g of monosaccharide-free L-FOS and 12.83 gDCW of biomass, after 3 successive sucrose additions (30 g in total), that is a 28.7% yield (w L-FOS/w sucrose) over a period of 288 h.At a 1.5 L bioreactor-level, growth considerably increased and, after 59 h and two sucrose additions, 72.9 g of monosaccharide-free L-FOS and 22.77 gDCW of biomass were obtained from a total of 160 g of sucrose fed, corresponding to a 45.5% yield (w L-FOS/w sucrose), 1.6 higher than the flask system. The L-FOS obtained at flask-level had a DP lower than 20 fructose units, while at bioreactor-level smaller oligosaccharides were obtained, with a DP lower than 10, as a consequence of the lower endolevanase activity in the flask-level.ConclusionWe demonstrate here in a novel system, that P. pastoris cultures can simultaneously be used as comprehensive system to produce the enzyme and the enzymatic L-FOS synthesis with growth sustained by sucrose by-products. This system may be now the center of an optimization strategy for an efficient production of glucose and fructose free L-FOS, to make them available for their application as prebiotics. Besides, P. pastoris biomass also constitutes an interesting source of unicellular protein.

Cross-linked enzyme aggregates (combi-CLEAs) derived from levansucrase and variant inulosucrase are highly efficient catalysts for the synthesis of levan-type fructooligosaccharides

Efficient and convenient access to short to medium chain length levan-type fructooligosaccharides (LFOS) is needed in order realise the nutritional potential of this class of bioactive oligosaccharides. While LFOS are synthesised by fructansucrase enzymes, these reactions are routinely associated with high molecular weight fructan polymer formation. Recent studies have shown that FOS production can be enhanced by the combination of levansucrase and inulosucrase in a one-pot reaction. In the present study, the novel mixed enzyme cross-linked enzyme aggregates (combi-CLEAs) based on levansucrase (Lev) and N543A variant inulosucrase (Inu) were prepared by ammonium sulfate precipitation, followed by glutaraldehyde cross-linking. The effect of Lev and Inu ratio on the activity of combi-CLEAs was explored. The results showed that >70% of total sucrase activity was recovered after immobilization and that the combi-CLEAs produced high amounts of LFOS (degree of polymerisation 3 to 21), while high molecular weight polysaccharide production was much reduced. Biochemical characterisation indicated that the optimum pH and temperature of combi-CLEAs (pH 5.5 and 50ᵒC, respectively) were comparable to those of free enzyme; however, the stability of the enzyme was improved. In addition, these combi-CLEAs have operational stability for several reaction cycles, which makes them very attractive for biotechnology applications.

Discrimination of three varieties of Codonopsis Radix based on fingerprint profiles of oligosaccharides by high performance liquid chromatography- evaporative light scattering detector combined with multivariate analysis

Codonopsis Radix (CR) is a plant that is important in the practice of traditional Chinese medicine (TCM). The Chinese Pharmacopoeia 2020 records dried roots prepared from three varieties of Campanulaceae plants under the designation CR (“Dang-shen” in Chinese), including Codonopsis pilosula (Franch.) Nannf (C. pilosula), Codonopsis pilosula Nannf.var.modesta (Nannf.) L81. T. Shen (C. pilosula var. modesta) and Codonopsis tangshen Oliv (C. tangshen). As major constituents of CR, oligosaccharides might contribute to its clinical efficacy except for other known active compounds, yet the differences in the oligosaccharide profiles of these three varieties of CR remain incompletely understood. In the present study, 135 samples from these different CR varieties were harvested, and oligosaccharide fingerprints for these samples were characterized via HPLC-ELSD, with 19 common peaks being matched. Oligosaccharides were further identified through the combination of electrospray ionization MS/MS (ESI-MS/MS) with nuclear magnetic resonance (NMR). Principal component analysis and linear discriminant analysis (LDA) approaches were then used to compare the oligosaccharide profiles of these three CR varieties. These analyses ultimately revealed that CR was compared with principal component analysis (PCA) and linear discriminant (LDA) methods. The analyses ultimately revealed that these CR samples contained high levels of inulin- and levan-type fructooligosaccharides (FOS), with variations in the relative levels of these FOS compounds among the three analyzed CR varieties. Through the combined analysis of oligosaccharide fingerprints and LDA results, it was possible to differentiate among these CR varieties, with an accurate classification rate of 96.3% and a cross-validation rate of 95.6%. Together, these results highlight a valuable approach to the classification and identification of different CR varieties.

Functional Characterization of Recombinant Endo-Levanase (LevBk) from Bacillus koreensis HL12 on Short-Chain Levan-Type Fructooligosaccharides Production.

Levan-type fructooligosaccharides (L-FOSs) are a prominent class of non-digestible oligosaccharides with potential as nutritional prebiotics. Endo-levanase, which randomly hydrolyzes β-(2,6)-linkages in fructans, is a promising enzyme for short-chain FOS production. In this work, a recombinant levanase (LevBk) from Bacillus koreensis strain HL12 was characterized. Soluble LevBk protein was produced in Escherichia coli BL21(DE3) system at 40mg/L of culture medium. Based on sequence and structural analysis, LevBk was classified as a member of endo-levanase in GH32 family containing N-terminal substrate binding pocket and C-terminal β-sandwich domains. LevBk optimally worked at 45°C, pH 6.0 with the specific activity of 2.43 U/mg. Based on enzymatic hydrolysis, short-chain L-FOSs with degree of polymerization (DP) of 2-4 were produced from hydrolysis of timothy grass levan under optimal conditions for 9-24h. With its ability to produce L-FOSs with specific chain lengths, LevBk could be attractively applied for converting of levan containing material to high value-added sweetener in the biorefinery industry.

Engineering the β-Fructofuranosidase Fru6 with Promoted Transfructosylating Capacity for Fructooligosaccharide Production.

Levan-type fructooligosaccharides (FOS) exhibit enhanced health-promoting prebiotic effects on gut microbiota. The wild type (WT) of β-fructofuranosidase Fru6 could mainly yield 6-ketose. Semirational design and mutagenesis of Fru6 were exploited to promote the transfructosylating capacity for FOS. The promising variants not only improved the formation of 6-kestose but also newly produced tetrasaccharides of 6,6-nystose and 1,6-nystose (a new type of FOS), and combinatorial mutation boosted the production of 6-kestose and tetrasaccharides (39.9 g/L 6,6-nystose and 4.6 g/L 1,6-nystose). Molecular docking and molecular dynamics (MD) simulation confirmed that the mutated positions reshaped the pocket of Fru6 to accommodate bulky 6-kestose in a reactive conformation with better accessibility for tetrasaccharides formation. Using favored conditions, the variant S165A/H357A could yield 6-kestose up to 335 g/L, and tetrasaccharides (6,6-nystose and 1,6-nystose) reached a high level of 121.1 g/L (134.5 times of the mutant S423A). The β-(2,6)-linked FOS may show the potential application for the prebiotic ingredients.

Levan-type fructooligosaccharides synthesis by novel levansucrase-inulosucrase fusion enzyme

A novel strategy to enhance the yield of levan-type fructooligosaccharide (LFOS) was recently introduced, whereby levansucrase and inulosucrase reactions were coupled together in one pot. In order to simplify the process, in the present study we report the first example of a recombinant levansucrase-inulosucrase fusion protein and investigate its impact on LFOS production. Sequences for levansucrase from Bacillus amyloliquefaciens KK9 and inulosucrase from Lactobacillus reuteri 121 were fused genetically with a flexible eighteen residue glycine-serine peptide linker. SDS-PAGE analysis showed that the molecular weight of obtained fusion protein is approximately of 120 kDa, corresponding to the expected fusion protein molecular weight. Kinetic analysis revealed that after protein combination, the kinetic parameters of both enzymes are slightly changed. Biochemical characterization revealed that fusion did not affect the optimum pH and temperature for catalysis, but significantly change the stability of the enzyme. HPAEC-PAD analysis and enzymatic hydrolysis assays demonstrated that the fusion enzyme produced the desired higher yield of LFOS compared to those of individual levansucrases.

Crystal Structure of Levansucrase from the Gram-Negative Bacterium Brenneria Provides Insights into Its Product Size Specificity.

Microbial levansucrases (LSs, EC 2.4.1.10) have been widely studied for the synthesis of β-(2,6)-fructans (levan) from sucrose. LSs synthesize levan-type fructo-oligosaccharides, high-molecular-mass levan polymer or combinations of both. Here, we report crystal structures of LS from the G--bacterium Brenneria sp. EniD 312 (Brs-LS) in its apo form, as well as of two mutants (A154S, H327A) targeting positions known to affect LS reaction specificity. In addition, we report a structure of Brs-LS complexed with sucrose, the first crystal structure of a G--LS with a bound substrate. The overall structure of Brs-LS is similar to that of G-- and G+-LSs, with the nucleophile (D68), transition stabilizer (D225), and a general acid/base (E309) in its active site. The H327A mutant lacks an essential interaction with glucosyl moieties of bound substrates in subsite +1, explaining the observed smaller products synthesized by this mutant. The A154S mutation affects the hydrogen-bond network around the transition stabilizing residue (D225) and the nucleophile (D68), and may affect the affinity of the enzyme for sucrose such that it becomes less effective in transfructosylation. Taken together, this study provides novel insights into the roles of structural elements and residues in the product specificity of LSs.

A genomic perspective on the potential of termite-associated Cellulosimicrobium cellulans MP1 as producer of plant biomass-acting enzymes and exopolysaccharides.

BackgroundLignocellulose is a renewable and enormous biomass resource, which can be degraded efficiently by a range of cocktails of carbohydrate-active enzymes secreted by termite gut symbiotic bacteria. There is an urgent need to find enzymes with novel characteristics for improving the conversion processes in the production of lignocellulosic-based products. Although various studies dedicated to the genus Cellulosimicrobium as gut symbiont, genetic potential related to plant biomass-acting enzymes and exopolysaccharides production has been fully untapped to date.MethodsThe cellulolytic bacterial strain MP1 was isolated from termite guts and identified to the species level by phenotypic, phylogenetic, and genomic analysis. To further explore genes related to cellulose and hemicellulose degradation, the draft genome of strain MP1 was obtained by using whole-genome sequencing, assembly, and annotation through the Illumina platform. Lignocellulose degrading enzymes and levan production in the liquid medium were also examined to shed light on bacterial activities.ResultsAmong 65 isolates obtained, the strain MP1 was the most efficient cellulase producer with cellulase activity of 0.65 ± 0.02 IU/ml. The whole genome analysis depicted that strain MP1 consists of a circular chromosome that contained 4,580,223 bp with an average GC content of 73.9%. The genome comprises 23 contigs including 67 rRNA genes, three tRNA genes, a single tmRNA gene, and 4,046 protein-coding sequences. In support of the phenotypic identification, the 16S rRNA gene sequence, average nucleotide identity, and whole-genome-based taxonomic analysis demonstrated that the strain MP1 belongs to the species Cellulosimicrobium cellulans. A total of 30 genes related to the degradation of cellulases and hemicellulases were identified in the C. cellulans MP1 genome. Of note, the presence of sacC1-levB-sacC2-ls operon responsible for levan and levan-type fructooligosaccharides biosynthesis was detected in strain MP1 genome, but not with closely related C. cellulans strains, proving this strain to be a potential candidate for further studies. Endoglucanases, exoglucanases, and xylanase were achieved by using cheaply available agro-residues such as rice bran and sugar cane bagasse. The maximum levan production by C. cellulans MP1 was 14.8 ± 1.2 g/l after 20 h of cultivation in media containing 200 g/l sucrose. To the best of our knowledge, the present study is the first genome-based analysis of a Cellulosimicrobium species which focuses on lignocellulosic enzymes and levan biosynthesis, illustrating that the C. cellulans MP1 has a great potential to be an efficient platform for basic research and industrial exploitation.

Computational Design of Oligosaccharide-Producing Levansucrase from Bacillus licheniformis RN-01 to Increase Its Stability at High Temperature.

Levan-type fructooligosaccharides (LFOs) and levan can potentially be used as ingredients in prebiotics, skincare products, and antitumor agents. The Y246S mutant of Bacillus licheniformis RN-01 levansucrase (oligosaccharide-producing levansucrase, OPL) was reported to productively synthesize LFOs; however, OPL's thermostability is low at high temperatures. To enhance OPL structural stability, this study employed molecular dynamics (AMBER) to identify a highly flexible region, as measured by its average root-mean-square fluctuation (RMSF) value, on the OPL surface and computational protein design (Rosetta) to rigidify and increase favorable interactions to increase its structural stability. AMBER identified region nine (residues 277-317) as a highly flexible region that was selected for design because it has the highest number of residues and the second-highest average RMSF, and it is farthest from the active site. Rosetta designed 14 mutants with the best ΔΔG value in each position, where three mutants have better ΔG than OPL. To determine whether their flexibilities and stabilities are lower than those of OPL, all 14 designed mutants were simulated at high temperature (500 K), and we found that K296E, G309S, and A310W mutants were predicted to be more stable and could retain their native structures better than OPL. Our results suggest that enhanced structural stabilities of these mutants are caused by increased hydrogen bond strengths of the designed residues and their neighboring residues. This study designed K296E, G309S, and A310W mutants of OPL with high potential for stability improvement, and they could potentially be used for the effective production of LFOs.

Efficient substrate accessibility of cross-linked levanase aggregates using dialdehyde starch as a macromolecular cross-linker

Cross-linked enzyme aggregates (CLEAs) are influenced by mass diffusion limitations such as the degree of molecular cross-linking attained, which affects substrate accessibility. Thus, this study seeks to improve substrate accessibility using macromolecular cross-linkers in cross-linked levanase aggregates (CLLAs) formation for levan-type fructooligosaccharides (L-FOS) production. Dialdehyde starch-tapioca (DAST) was successfully developed and used to cross-link levanase to form CLLAs-D and with bovine serum albumin (BSA) to form CLLAs-DB which showed activity recoveries of 65.6% and 81.6%, respectively. After cross-linking, the pH (6–10) and thermal stability (30–40 °C) increased, and organic solvent tolerance resulted in the activation of CLLAs. Likewise, CLLAs-DB had higher substrate affinity and accessibility and a higher effectiveness factors than CLLAs-D. The total L-FOS yield of CLLAs-DB (78.9% (w/v)) was higher than that of CLLAs-D (62.4% (w/v)). Therefore, as a cross-linker, DAST may have application prospects as a promising and green biocatalyst for product formation.

Computational design of oligosaccharide producing levansucrase from Bacillus licheniformis RN-01 to improve its thermostability for production of levan-type fructooligosaccharides from sucrose

Levansucrase catalyzes production of levan and levan-type fructooligosaccharides (LFOs) with potential applications in food and pharmaceutical industries such as prebiotics and anti-tumor agents. Previous study found that Y246S mutant of Bacillus licheniformis RN-01 levansucrase (oligosaccharide producing levansucrase, OPL) could effectively produce LFOs but its thermostability is limited at high temperature. In this study, molecular dynamics (MD) and computational protein design were used to create mutants with higher thermostability than OPL by rigidifying highly flexible residues on enzyme surface. MD results show that highly flexible residues suitable for design are K82, N83, D179, and Q308. Two approaches were employed to improve their interactions by allowing them to be amino acids that could potentially form favorable interactions with their neighboring residues or natural amino acids except G, P and C. Flexibilities of designed residues of K82H, N83R, Q308S and K82H/N83R mutants are lower than those of OPL. Experimental results show that characteristics and product patterns of designed mutants are relatively similar to those of OPL. K82H/N83R mutant has higher thermostability than OPL with 1.7-fold increase in t1/2. Circular dichroism result suggests that designed mutations do not drastically affect secondary structures. This study shows how computational technique can engineer enzyme for thermostability improvement.

Computational design of Bacillus licheniformis RN-01 levansucrase for control of the chain length of levan-type fructooligosaccharides

Levansucrase (LS) from Gram-positive bacteria generally produces a large quantity of levan polymer, a polyfructose with glucose at the end (GFn) but a small quantity of levan-type fructooligosaccharides (LFOs). The properties of levan and LFOs depend on their chain lengths, thereby determining their potential applications in food and pharmaceutical industries such as prebiotics and anti-tumor agents. Therefore, an ability to redesign and engineer the active site of levansucrase for synthesis of products with desired degree of polymerization (DP) is very beneficial. We employed computational protein design, docking and molecular dynamics to redesign and engineer the active site of Bacillus licheniformis RN-01 levansucrase for production of LFOs with DP up to five (GF4), using two approaches: 1) blocking oligosaccharide binding track of GF3-LS complex with large aromatic residues and 2) eliminating hydrogen bond interactions between terminal glucose of GF4 and side chains of binding residues of GF4-LS complex. The designed enzymes and their product patterns from these two approaches were experimentally characterized. The experimental results show that the first approach was successful in creating N251W and N251W/K372Y mutants that synthesized LFOs with DP up to five. This work illustrates how computer-aided approaches can offer novel opportunities to engineer enzymes for desired products.

An overview of levan-degrading enzyme from microbes.

Functional carbohydrates are ideal substitutes for table sugar and make up a large share of the worldwide functional food market because of their numerous physiological benefits. Growing attention has been focused on levan, a β-(2,6) fructan that possesses more favorable physicochemical properties, such as lower intrinsic viscosity and greater colloidal stability, than β-(2,1) inulin. Levan can be used not only as a functional carbohydrate but also as feedstock for the production of levan-type fructooligosaccharides (L-FOSs). Three types of levan-degrading enzymes (LDEs), including levanase (EC 3.2.1.65), β-(2,6)-fructan 6-levanbiohydrolase (LF2ase, EC 3.2.1.64), and levan fructotransferase (LFTase, EC 4.2.2.16), play significant roles in the biological production of L-FOSs. These three enzymes convert levan into different L-FOSs, levanbiose, and difructose anhydride IV (DFA IV), respectively. The prebiotic properties of both L-FOSs and DFA IV have been confirmed in recent years. Although levanase, LF2ase, and LFTase belong to the same O-glycoside hydrolase 32 family (GH32), their catalytic properties and product spectra differ significantly. In this paper, recent studies on these LDEs are reviewed, including those investigating microbial source and catalytic properties. Additionally, comparisons of LDEs, including those of their differing cleavage behavior and applications for different L-FOSs, are presented in detail.

Frucooligosaccharides purification: Complexing simple sugars with phenylboronic acid

Prebiotic fructooligosaccharides (FOS) are currently obtained by enzymatic reaction with fructosyltransferases (FTFs) using sucrose as both donor and acceptor. In these reactions glucose results as the most abundant by-product, arising from each fructosyl transfer event and, together with fructose, because of the inherent hydrolytic activity of the FTFs. As FOS are mainly used as prebiotic in nutraceutical foods, the reduction or total elimination of monosaccharides is required. In this work the selective elimination of monosaccharides from a synthetic FOS mixture was achieved through the selective complexation of glucose and fructose with phenyl boronic acid (PBAc) followed by ethyl-acetate extraction. The process was applied to a complex mixture of FOS obtained in an enzymatic synthesis reaction containing 40% glucose, 15.8% fructose and 35% of FOS, elimination of the sugars was achieved through 3:1 molar reactions, resulting in a levan-type FOS product with 97% purity.

Enhancement of the Brenneria sp. levansucrase thermostability by site-directed mutagenesis at Glu404 located at the “-TEAP-” residue motif

Levansucrase (EC 2.1.4.10, LS) has been used in the production of levan and levan-type fructooligosaccharides from sucrose; however, development of further application is restricted due to its poor thermostability. The LS from Brenneria sp. EniD312 was engineered using a structure-guided approach. Residue Glu404 was located in the “-TEAP-” motif and varied among LSs with different thermostabilities. Site-directed mutagenesis was performed in Glu404 and thermostability was evaluated by measuring the half-life and structural melting temperature (Tm) of the wild-type LS and its Glu404-mutant variants. The optimal temperature for the Glu404 mutants was similar to that of the wild-type enzyme, however, the Tm of E404 L mutant was enhanced by 2.8 °C and the half-life was increased by 12.5- and 1.3- fold at 35 and 45 °C, respectively. The other mutants E404 W, E404 V, E404I, and E404 F also showed a pronounced increase in Tm and thermostability. Finally, the improvement of thermostability of LS through mutation in Glu404 belonging to the “-TEAP”- motif could be ascribed to the change of microenvironment in the LS structure. The change of the micro-environment mainly included the enhanced structural stability between two β-hairpins and the elevated hydrophobic interactions in the overall protein structure. This work proposes new insights into the thermostabilization mechanism of other LSs.

Levan-type fructooligosaccharides synthesis by a levansucrase-endolevanase fusion enzyme (LevB1SacB)

We describe here the enzymatic production of levan type-fructooligosaccharides (L-FOS) with a DP from 2 to 10, through simultaneous synthesis and hydrolysis reactions. This was accomplished by LevB1SacB, a new enzyme resulting from the fusion of SacB, a levansucrase from Bacillus subtilis and LevB1, an endolevanase from B. licheniformis. In the fusion enzyme, SacB retains its catalytic behavior with a decrease in kcat from 164 to 108s−1. LevB1 in LevB1SacB kinetic behavior improves considerably reaching saturation with levan and following Michaelis-Menten kinetics, quite differently from the previously reported first order kinetic behavior. We also report that LevB1SacB or both enzymes (LevB1 & SacB) at equimolar concentrations in simultaneous reactions result in an optimal, wide and diverse L-FOS profile, including 6-kestose, levanbiose and blastose among other L-FOS and 1-kestose, which accumulates as by-product of SacB levan synthesis. Yields of around 40% (w/w) were obtained from 600g/l sucrose with either LevB1SacB or LevB1 & SacB. The reaction was successfully scaled up to a stirred 2l bioreactor.

Replica exchange molecular dynamics simulations reveal the structural and molecular properties of levan-type fructo-oligosaccharides of various chain lengths

BackgroundLevan and levan-type fructo-oligosaccharides (LFOs) have various potential applications in pharmaceutical and food industries due to their beneficial properties such as their low intrinsic viscosity and high water solubility. Previous studies showed that they exhibited prebiotic effects, anti-inflammatory and anti-tumor activities against Sarcoma-180 tumor cells of human. Despite their various potential applications, the structural and molecular properties of LFOs of various chain lengths are not well understood.ResultsWe employed the replica-exchange molecular dynamics simulations method (REMD) in AMBER14 to elucidate structural and molecular properties of LFOs with chain lengths of 5 (LFO5), 10 (LFO10) and 15 (LFO15) residues in two models of generalized Born implicit solvent (GBHCT and GBOBC1). For LFO10 and LFO15, four distinct conformations (helix-like, partial helix, zig-zag and random structures) were characterized by their upper-middle and lower-middle torsions. For LFO5, two distinct conformations (partial helix and random structures) were characterized by their middle torsion and molecular angle of residues 1, 3 and 5. To determine hydrogen bonds important for the formation of helix-like structures of LFO10 and LFO15, occurrence frequencies of hydrogen bonds were analyzed, and the O6(i)--H3O(i+1) hydrogen bond was found with the highest frequency, suggesting its importance in helix formation. Among three dihedral angles between two fructosyl units [ϕ (O5’-C2’-O6-C6), ψ (C2’-O6-C6-C5) and ω (O6-C6-C5-C4)], dihedral angle distributions showed that ω was the most flexible dihedral angle and probably responsible for conformational differences of LFOs.ConclusionsOur study provides important insights into the structural and molecular properties of LFOs, which tend to form helical structures as the chain length increases from 5 to 15 residues. This information could be beneficial for the selection of LFOs with appropriate lengths and properties for pharmaceutical and biological applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-016-1182-7) contains supplementary material, which is available to authorized users.

Immobilization and stabilization of levansucrase biocatalyst of high interest for the production of fructooligosaccharides and levan

AbstractBACKGROUNDLevansucrase (LS)‐catalyzed‐transfructosylation reaction is a potential approach for the synthesis of fructooligosaccharides (FOSs) and levan as health promoting compounds. This biocatalytic approach is hindered by low thermal stability of LS and its high rate of hydrolysis. In the present study, LS from Bacillus amyloliquefaciens was immobilized onto modified and unmodified epoxy‐activated supports (Eupergit® C; Sepabeads®) and on modified cross‐linked‐agarose beads, to increase its thermal stability and modulate its reaction selectivity (hydrolysis/transfructosylation).RESULTSLS bound to Sepabeads® HA (98.8%) and glyoxyl agarose‐IDA/Cu (67%) retained high initial activity along with good immobilization yields. The thermal stability results indicated that glyoxyl agarose‐IDA/Cu and glyoxyl agarose, provided the greatest thermal stability with factors of 14 and 106 times, respectively. Immobilization through Sepabeads® HA increased the ratio of transfructosylation/hydrolysis by 2.3 times, although it did not promote the stabilization of LS. Immobilization on glyoxyl agarose‐IDA/Cu provided a good compromise of all three properties: retention of activity (67.0%), transfructosylation/hydrolysis ratio (120%) and thermal stability (stability factor of 13.6).CONCLUSIONThe stabilization of LS through immobilization contributes to its potential use commercially. With an increasingly stable enzyme, further work will be directed towards altering LS reaction specificity towards levan and levan‐type FOSs. © 2015 Society of Chemical Industry

Recent novel applications of levansucrases.

Levansucrase catalyzes three distinct reactions depending on the fructosyl acceptor molecule, including polymerization, transfructosylation, and hydrolysis. As a key biocatalyst in the synthesis of levan and levan-type fructooligosaccharides, levansucrase has been widely and intensively studied. Due to the promising physiological effects of levan and levan-type fructooligosaccharides, they exhibit great potential in food and pharmaceutical industries. Another important point that attracts much attention is the wide substrate specificity of levansucrase toward monosaccharides, disaccharides, and aromatic and alkyl alcohols, producing diverse sucrose analogues, hetero-oligosaccharides (especially lactosucrose), and interesting fructosides. The present article summarizes and discusses the recent applications of microbial levansucrase in detail.