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Levan is a type of fructan with β-(2, 6) fructosyl linkage. In recent years, levan has been used in the medicinal, food and chemical industries for its excellent physicochemical properties of good thermal stability, low viscosity, pseudoplasticity, and non-swelling in water. The levansucrase is key enzyme responsible for biosynthesis of levan from substrate sucrose. In the present study cell lysate of Escherichia coli was used as support for convenient immobilization of Ba-SacB (i.e., Ba-SacB-Cell-Lysate) under the action of dopamine. Compared with the free Ba-SacB, the Ba-SacB-Cell-Lysate showed surprisingly enhanced pH stability in the pH range 6–9, and improved relative activities in the pH ranges 4–5 and 7–10. With the presence of 10% (v/v) butyl alcohol, 5mmol/L Fe3+, and after vortex for 90 min, the Ba-SacB-Cell-Lysate showed up to 14.32 times, 4.63 times, and 9.2 times higher relative activities than the free Ba-SacB. The Ba-SacB-Cell-Lysate kept nearly 81% of the residual activity after 9 cycles of reuse. The fructo-oligosaccharide syrup (FOS) was efficiently produced through the catalysis of levanase with the substrate of levan produced by Ba-SacB-Cell-Lysate, and the preparation conditions were optimized by Box-Behnken response surface methodology. [ABSTRACT FROM AUTHOR]

The biocatalytic approach based on Levansucrase (LS)-catalyzed transfructosylation reaction is of particular interest for the synthesis of well-defined FOSs and β-(2−6)-levan. The present study aims to characterize the catalytic properties, kinetics, and end-product profiles of new LSs derived from Novosphingobium aromaticivoran s, Vibrio natriegens, Gluconobacter oxydans, and Burkholderia graminis. The optimal conditions for achieving the highest ratio of transfructosylation to hydrolysis was achieved were identified as follows: N. aromaticivorans LS1 (T 45 °C, pH 6, E a 33.88 kJ/mol), V. natriegens LS2 (T= 45 °C, pH 5, E a = 12.74 kJ/mol), B. graminis LS3 (T= 35 °C, pH 7, E a = 20.72 kJ/mol), and G. oxydans LS4 (T= 30 °C, pH 5, E a = 51.85 kJ/mol). The kinetic study revealed that LS from G. oxydans LS4 had the highest catalytic efficiency for the transfructosylation of sucrose (112.96 s−1M−1) and raffinose (1142.7 s−1 M−1) than their hydrolysis (39.15 and 16.5 s−1M−1 respectively). Our results highlighted the end-product profile of each LS using sucrose and raffinose as donor substrates. V. natriegens LS2 and B. graminis LS3-catalyzed sucrose bioconversion reaction resulted in the synthesis of more diverse FOSs. When compared to FOSs, all LSs catalyzing sucrose and raffinose bioconversion produced high yields of oligolevans/levans. [Display omitted] • Catalytic properties and optimum conditions for achieving the highest transfructosylation to hydrolysis ratio were identified for 4 novel levansucrases (LSs). • LS from G. oxydans exhibited the highest catalytic efficiency for the transfructosylation of sucrose and raffinose. • All LSs exhibited higher catalytic efficiency towards transfructosylation of raffinose than its hydrolysis. • V. natriegens and B. graminis LSs catalyzing sucrose transfructosylation led to diverse fructooligosaccharides (FOSs). • All LSs catalyzing sucrose and raffinose bioconversion led to high yields of oligolevans/levans than FOSs. [ABSTRACT FROM AUTHOR]

Oligofructans are a combination of fructo-oligosaccharides and fructose polymers that are linked by β-glycosidic bonds. Because of their distinctive properties and potential health benefits, they are widely used in many industries. This study optimized microbial oligofructans fermentation using Bacillus subtilis TISTR 001. Fermentative parameters (pH, temperature, shaking rate, sucrose concentration, and fermentation time) were investigated in a one-factor-at-a-time (OFAT) experiment using surface response methodology to determine the predicted model and optimization. Different carbon sources (sucrose, raw sugar, sugarcane juice, and molasses) were studied under the optimized conditions. The results from the Box–Behnken experimental design with four factors and three levels and the predicted model showed the optimum fermentation conditions were 35°Brix of substrate concentration at pH 6.5, 32.5 °C, and 250 rpm shaking speed for 48 h to obtain 88.64 g/L of oligofructans. The predicted oligofructans content (88.64 g/L) was higher than the actual production using sucrose (75.86 g/L), which was the best substrate for oligofructans production. Raw sugar and sugarcane juice could be alternative substrates for oligofructans production based on these optimized conditions, while molasses had the lowest production due to its impurities that negatively affected oligofructans production. [ABSTRACT FROM AUTHOR]

During raw sugarcane processing, a significant portion of lost sucrose is attributable to microbial degradation. Sucrose consumption by many bacteria is also linked to the production of exopolysaccharides (EPS) such as dextrans and fructans. These resulting EPS cause operational challenges during raw sugar manufacturing. Here, we report the characterization of EPS from a fructan-forming Gluconobacter japonicus bacterium that we previously isolated from a Louisiana sugarcane factory. The genome sequencing revealed the presence of two encoded levansucrase genes, lsrA and lsrB. One levansucrase, LsrB, was detected in the secreted protein fraction of G. japonicus LASM12 by QTOF LC-MS. The spotting assays indicated that G. japonicus produces EPS using sucrose and raffinose as substrates. The G. japonicus EPS correlated with levan fructan commercial standards by 1H-NMR, and with the characteristic carbohydrate fingerprint region for FTIR spectra, confirming that the G. japonicus EPS is levan fructan. The glycosyl composition and glycosyl linkage analysis revealed a linear β-2,6-fructofuranosyl polysaccharide with occasional (5.7%) β-2,1-fructofuranosyl branches. The gel permeation chromatography of the levan fructan EPS showed two main peaks at 4.5 kDa and 8 kDa and a very minor peak at 500 kDa. G. japonicus was identified as a producer of levan fructan. These findings will be useful for future studies aimed at evaluating the impact of levan fructans on sugar crop processing, which have been historically underestimated in industry. [ABSTRACT FROM AUTHOR]

Prebiotics play an important role in biological processes, which has stimulated great interest in their synthesis. This paper reports a levansucrase from Bacillus subtilis , demonstrating high catalytic selectivity for the synthesis of prebiotic lactosucrose with sucrose as a donor and lactose as an acceptor. The product was purified and the structure was characterized. The effects of lactosucrose on the proliferation and acid production of bifidobacterium and lactobacillus were better than that of lactulose and galactooligosaccharides(GOS). The short chain fatty acid (SCFA) content in the fermentation broth was also measured. It contains large amounts of SCFAs that act roles in intestinal homeostasis. The lactosucrose affected inflammatory responses in lipopolysaccharide-induced RAW 264.7 macrophages since they suppressed inflammatory cytokines TNF-α, IL-1β, and IL-6 well as compared to the control. Also, lactosucrose had no cytotoxicity in the concentration range of 10 mg/mL. These results indicate that lactosucrose was effective in promoting the proliferation of intestinal probiotics and have anti-inflammatory activity. Therefore, lactosucrose has positive prebiotic activity. [Display omitted] • Enzymatic production is a promising strategy for preparing functional lactosucrose. • Lactosucrose exhibits high prebiotic activity. • Lactosucrose has an obvious anti-inflammatory effect and low cytotoxicity. [ABSTRACT FROM AUTHOR]

Copyright of China Condiment is the property of China Condiment and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Oligofructans are fructose-containing molecules joined by beta-glycosidic bonds. They are notable in functional and health-improving products and widely used in many industries. Their beneficial health properties have led to more research and development. Oligofructans can be produced using levansucrase-producing microbial fermentation with a sucrose-based substrate. Levansucrase is an important enzyme for production through sucrose hydrolysis and transfructosylation. In the present study, the comparative efficiency of carbon sources and the optimum conditions for oligofructans fermentation by Bacillus subtilis TISTR 001 were studied. Three carbon sources (sucrose, sugarcane juice, and molasses) and four substrate concentrations (15, 20, 25, and 30 °Brix) were studied under fermentation at pH 6.8, 30 °C, and 150 rpm for 168 h. The results showed that all substrates could be a suitable carbon source and had the same trends for both the production of levansucrase and oligofructans that were positively correlated with the substrate concentration. Oligofructans formation was confirmed by the changes in the sugars (sucrose, glucose, and fructose) and the produced oligofructans (kestose, nystose, 1-fructofuranosyl-D-nystose, and levan). Cane juice at 30°Brix was found as the best carbon source with the highest levansucrase activity (2.81 × 107 Unit/mL) at 48 h and the maximum content of oligofructans (87.6 g/L) at 60 h with the kinetic parameters: specific growth rate of 0.12 h−1, substrate consumption of 2.93 g/L/h, production yield of 0.36 g/g reducing sugar, and productivity of 1.10 g/L/h. These results identified the optimum conditions for efficient oligofructans production that should assist further research on their health benefits and future applications. [ABSTRACT FROM AUTHOR]

Abstract Background Although 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. Results The 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. Conclusion We 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.

Copyright of Journal of Kirkuk University for Agricultural Sciences is the property of Republic of Iraq Ministry of Higher Education & Scientific Research (MOHESR) and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The biological production of levan by levansucrase (LS, EC 2.4.1.10) has aroused great interest in the past few years. Previously, we identified a thermostable levansucrase from Celerinatantimonas diazotrophica (Cedi-LS). A novel thermostable LS from Pseudomonas orientalis (Psor-LS) was successfully screened using the Cedi-LS template. The Psor-LS showed maximum activity at 65 °C, much higher than the other LSs. However, these two thermostable LSs showed significantly different product specificity. When the temperature was decreased from 65 to 35 °C, Cedi-LS tended to produce high-molecular-weight (HMW) levan. By contrast, Psor-LS prefers to generate fructooligosaccharides (FOSs, DP ≤ 16) rather than HMW levan under the same conditions. Notably, at 65 °C, Psor-LS would produce HMW levan with an average Mw of 1.4 × 106 Da, indicating that a high temperature might favor the accumulation of HMW levan. In summary, this study allows a thermostable LS suitable for HMW levan and levan-type FOSs production simultaneously. [ABSTRACT FROM AUTHOR]

During raw sugarcane processing, a significant portion of lost sucrose is attributable to microbial degradation. Sucrose consumption by many bacteria is also linked to the production of exopolysaccharides (EPS) such as dextrans and fructans. These resulting EPS cause operational challenges during raw sugar manufacturing. Here, we report the characterization of EPS from a fructan-forming Gluconobacter japonicus bacterium that we previously isolated from a Louisiana sugarcane factory. The genome sequencing revealed the presence of two encoded levansucrase genes, lsrA and lsrB. One levansucrase, LsrB, was detected in the secreted protein fraction of G. japonicus LASM12 by QTOF LC-MS. The spotting assays indicated that G. japonicus produces EPS using sucrose and raffinose as substrates. The G. japonicus EPS correlated with levan fructan commercial standards by 1H-NMR, and with the characteristic carbohydrate fingerprint region for FTIR spectra, confirming that the G. japonicus EPS is levan fructan. The glycosyl composition and glycosyl linkage analysis revealed a linear β-2,6-fructofuranosyl polysaccharide with occasional (5.7%) β-2,1-fructofuranosyl branches. The gel permeation chromatography of the levan fructan EPS showed two main peaks at 4.5 kDa and 8 kDa and a very minor peak at 500 kDa. G. japonicus was identified as a producer of levan fructan. These findings will be useful for future studies aimed at evaluating the impact of levan fructans on sugar crop processing, which have been historically underestimated in industry.

Background: Although 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. Results: The 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. Conclusion: We 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. [ABSTRACT FROM AUTHOR]

Levan has attracted interest due to the potential health benefits associated with its prebiotic, biological, and functional properties. However, the production of levan is expensive due to its high resource requirements. With the growing demand for levan, it is vital to determine suitable cultivation condition for its production and reduce costs accordingly. The present study characterized the enzyme levansucrase produced by a novel strain of Bacillus siamensis and optimized the conditions for the biosynthesis of levansucrase and levan. The crude levansucrase enzyme production by B. siamensis was induced at a specific temperature in a medium containing different concentrations of sucrose, fructose, and glucose to evaluate transfructosylation and hydrolysis activities. Crude levansucrase significantly increased transfructosylation relative to hydrolysis activity at 37 °C in a medium containing 20% (w/v) sucrose. Both transfructosylation and hydrolysis activities were inhibited in glucose and fructose containing medium. Purification and characterization of the levansucrase were performed by precipitating the enzyme with ammonium sulfate solution, purified anion-exchange chromatography, and analyzed by Sodium Dodecyl Sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The results showed the molecular weight of the enzyme to be approximately 30 kDa with specific activity at 15.95 U/mg, corresponding to a protein purification efficiency of 11.47 and a yield of 78.75%. The optimal culture condition for the purified-levansucrase activity for levan biosynthesis was obtained at 37 °C after 48 h, at pH 6.0 in 50 mM phosphate buffer and 20% (w/v) sucrose. The study demonstrated the optimized condition for levan biosynthesis utilizing the B. siamensis that can serve as a model for various commercial and industrial applications for efficient levan production.

The synthesis of novel prebiotics has gained attention due to a greater interest in intestinal health. In this study, lactosucrose was produced by levansucrase (LS, EC 2.4.1.10) catalyzing the transfructosylation of sucrose and lactose with whey (WP) and milk permeate (MP) as lactose sources. Selected functionalized supports were investigated for the immobilization of LSs from Gluconobacter oxydans (LS1) and Vibrio natriegens (LS2) by heterofunctional attachment. The highest immobilization protein yields of 94 % and 87 % and retained activities of 55 % and 98 %, respectively, were achieved upon the immobilization of LS1 and LS2 on RelizymeTM EP403/S functionalized with iminodiacetic acid (IDA)-cupric ions (Cu2+). The immobilization of LSs enhanced the reaction selectivity towards transfructosylation. Moreover, post-immobilization treatments (alkaline pH incubation, polyethylenimine (PEI) cross-linking) impacted the enzyme activity of the immobilized enzymes resulting in lower retained activities (11–74 %) but allowed them to achieve a higher thermal stabilization (up to 53 times increase), particularly with the high pH treated immobilized V. natriegens LS2. The identified optimal conditions for lactosucrose synthesis with high selectivity by free and immobilized RelizymeTM EP403/IDA-Cu immobilized LS2 resulted in 117 and 101 g/L, respectively, using WP as a lactose source. Lastly, the RelizymeTM EP403/IDA-Cu immobilized LS2 was successfully reused 3 consecutive times. [Display omitted] • Levansucrases (LSs) showed higher efficiency for transfructosylation bioconversion upon immobilisation. • Relizyme™ EP403 support functionalized with IDA-Cu provided the highest immobilization efficiency of LSs. • Post-immobilization treatments (high pH & PEI crosslinking) provided further thermal stabilization of LSs. • The highest bioconversion of dairy by-products into lactosucrose was achieved with immobilized V. natriegens LS2. • Reaction time and substrate ratio were significant in modulating bioconversion of whey permeate into lactosucrose. [ABSTRACT FROM AUTHOR]

Human beings consume probiotics and prebiotics because of the health benefits they provide. Lactobacillus gasseri is a probiotic microorganism native to humans which produces glycolytic enzymes directed at the hydrolysis of soluble fibers as prebiotic fructans. In this work, levansucrase (LevG) and inulosucrase (InuGB) from L. gasseri were used to predict the ability of L. gasseri to interact with linear and branched fructans prebiotics (inulin and agavins). AlphaFold and SWISS-MODEL were the servers used for tertiary structure prediction of LevG and InuGB, and fructans with different degrees of polymerization (DP2, DP4 DP6, DP8, DP12 and DP20) were used to generate ligandenzyme molecular dockings by AutodockVina, GlycoTorchVina, and Autodock FR software. The best affinity energies obtained by molecular docking were obtained with agavin and inulin with a DP of 6 and 8 units. Aspartic acid, glutamic acid, asparagine and arginin are the main amino acids involved in the interaction with these substrates. At the same time, the binding pocket shows hydrophilic characteristics; GlycoTorchVina was the best software for protein-ligand docking. These results demonstrate the ability of L. [ABSTRACT FROM AUTHOR]

Endophytic bacteria refer to bacteria which promote plant growth via direct and indirect mechanisms. Three endophytic bacteria isolated from Jerusalem artichoke exhibited plant growth induction and inulin production. These bacteria had functions of fructan degradation and synthesis from inulinase and levansucrase, respectively. Rossellomorea aquimaris 3.13 and Priestia megaterium 3.5 obtained inulinase/levanase enzyme with inulin and levan as substrates; enzyme production showed the optimum conditions in 1% inulin medium of 35 °C, pH 7.0. Bacillus velezensis 5.18 and Priestia megaterium 3.5 had inulosucrase/levansucrase enzyme with sucrose as a major carbon source; the enzyme had optimum temperature and pH conditions of 30 °C and pH 7.0, respectively. A combination of carbon sources had effect on decreasing enzyme activity; in addition, co-inoculation of bacteria showed a slight difference in enzyme production compared with single inoculation. The inulosucrase/levansucrase was produced earlier in co-culture containing bacteria with inulinase activity. Plant fructan synthesis was involved in 1-SST and 1-FFT, while 1-FEH encoded inulin degradation; these genes were evaluated in Jerusalem artichoke inoculated with the endophytic bacteria to quantify gene expression level using qPCR. All genes expressed in low levels at early stage of growth, responding to all endophytic bacteria. Significantly, Bacillus velezensis 5.18 induced all genes of the plant at 65 days of inoculation; Rossellomorea aquimaris 3.13 induced 1-FFT while Priestia megaterium 3.5 induced 1-SST. [ABSTRACT FROM AUTHOR]

Sucrases can modify numerous carbohydrates, and short-chain oligosaccharides produced by the unique transfructosylation activity of levansucrases are promising candidates for the growing sugar substitute market. These compounds could counteract the increasing number of diseases associated with the consumption of high-calorie sugars. Thus, there is great interest in the characterization of novel levansucrases. The commonly used method for sucrase activity determination is to quantify d -glucose released in the sucrose-splitting reaction. This is usually done in a discontinuous mode, i.e., several samples taken from the sucrase reaction are applied to a separately performed d -glucose determination (e.g., GOPOD assay). Employing the newly isolated levansucrase LevS KK21 from Pseudomonas sp. KK21, the feasibility of a one-pot sucrase characterization was investigated by combining sucrase reaction and GOPOD-based d -glucose determination into a single, continuous assay (Real-time GOPOD). The enzyme was characterized with respect to kinetic parameters, ion dependency, pH value, and reaction temperature in a comparative approach employing Real-time GOPOD and HPLC. High data consistency for all investigated enzyme parameters demonstrated that current processes for sucrase characterization can be considerably accelerated by the continuous assay while maintaining data validity. However, the assay was not applicable at acidic pH, as decolorization of the quinoneimine dye formed during the GOPOD reaction was observed. Overall, the study presents valuable data on the potentials of real-time sucrase activity assessment for an accelerated discovery and characterization of interesting enzymes such as the hereby introduced levansucrase LevS KK21. Progress in sucrase discovery will finally foster the development of health-promoting sucrose substitutes. [Display omitted] • A novel levansucrase (LevS KK21) from a sugar beet isolate was investigated. • To simplify sucrase characterization a new activity detection assay was developed. • Real-time GOPOD enables continuous assessment of sucrase activity in microplate format. • Real-time GOPOD is highly reliable, rapid, inexpensive, and high throughput-compatible. • Sucrase characterization and bioprocess monitoring are simplified by Real-time GOPOD. [ABSTRACT FROM AUTHOR]

Continue to hypothesize that honey is a storehouse of beneficial bacteria, and the majority of these isolates are levansucrase producers. Accordingly, ten bacterial strains were isolated from different honey sources. Four honey isolates that had the highest levansucrase production and levan yield were identified by the partial sequencing of the 16S rRNA gene as Achromobacter sp. (10A), Bacillus paralicheniformis (2M), Bacillus subtilis (9A), and Bacillus paranthracis (13M). The cytotoxicity of the selected isolates showed negative blood hemolysis. Also, they are sensitive to the tested antibiotics (Amoxicillin + Flucloxacillin, Ampicillin, Gentamicin, Benzathine benzylpenicillin, Epicephin, Vancomycin, Amikacin, and Zinol). The isolates had strong alkaline stability (pHs 9, 11) and were resistant to severe acidic conditions (29–100 percent). The tested isolates recorded complete tolerance to both H2O2 and the bile salt (0.3% Oxgall powder) after 24 h incubation. The cell-free supernatant of the examined strains had antifungal activities against C. Albicans with varying degrees. Also, isolates 2M and 13M showed strong activities against S. aureus. The isolates showed strong adhesion and auto-aggregation capacity. Isolate 10A showed the highest antioxidant activity (91.45%) followed by 2M (47.37%). The isolates recorded different catalase and protease activity. All isolates produced cholesterol oxidase and lipase with different levels. Besides, the four isolates reduced LDL (low-density lipoprotein) to different significant values. The cholesterol-reducing ability varied not only for strains but also for the time of incubation. The previous results recommended these isolates be used safely in solving the LDL problem. [ABSTRACT FROM AUTHOR]

Levansucrase LevS from Leuconostoc mesenteroides B-512F is a multidomain fructansucrase (MD-FN) that contains additional domains (ADs) to the catalytic domain. However, the understanding of the effect that these ADs have on enzyme activity remains vague. To this aim, structure-function relationship studies of these LevS ADs were performed by evaluating both biochemical properties and the enzymatic capacity of truncated versions of LevS. Joint participation of the N- and C-terminal domains is essential for stability, activity, specificity, and polymerization processes. Specifically, the N-terminal region is involved in stability, while the transition region plays an essential role in the transfructosylation reaction and polymer elongation. Based on our results, we suggest that ADs interact with each other, adopting a U-shaped topology. The importance of these ADs observed in the MD-FN of the Leuconostocaceae family is not shared by the Lactobacillaceae family. Phylogenetic analysis of LevS AD suggests that MD-FN from Lactobacillaceae and Leuconostocaceae have different evolutionary origins. This is the first study on the structure-function relationship of multidomain levansucrases from the Leuconostocaceae family. Our results point towards the functional role of AD in MD-FN and its involvement in fructan synthesis. [ABSTRACT FROM AUTHOR]

Copyright of China Brewing is the property of China Brewing Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Abstract Background Levan is a well-known homopolymer of fructose composed predominantly of β-(2, 6) fructofuranosyl linkages in the backbone with occasional β-(2, 1) linkages in the branch chains with varied applications. However, high production cost due to low yield of microbial levan has become a bottleneck for its practical applications. Furthermore, factors affecting the molecular mass of the synthesized levan by Leuconostoc spp. during prolonged cultivation is not fully elucidated. Methods The cultivation condition for Leuconostoc citreum BD1707 to synthesize levan was optimized by single-factor experiments and subsequently with response surface methodology (RSM). The average molecular weight (Mw) of levan synthesized by the strain L.citreum BD1707 under the optimized cultivation conditions was monitored by high-performance size exclusion chromatography (HPSEC). Finally, the enzyme with levan-degrading activity was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Results The levan yield of BD1707 reached 34.86 g/L with a corresponding productivity of 7.47 g/L/d under the optimal cultivation conditions deduced by RSM, i.e., cultivation at 26 °C and 200 rpm for 112 h in tomato juice supplemented with 172 g/L sucrose with an initial pH value of 6.12. The Mw of levan reached a peak value of 2.320 × 107 Da at 6 h of cultivation under the optimized cultivation conditions and then gradually decreased to 8.809 × 106 Da after 120 h of cultivation. Conclusion The levan yield of the strain L.citreum BD1707 could be sufficiently enhanced via cultivation condition optimization. The decrease in molecular mass of the synthesized levan was attributed predominantly to the hydrolytic activity of levansucrase secreted by L.citreum BD1707 during cultivation, with an estimated Mw of 130 KD by SDS-PAGE, while the effect of acid hydrolysis could be nearly neglected.

Actinobacteria occupy an important position among alternative enzyme sources with their ability to growth in extreme conditions, their potential to produce large amounts of enzymes, biochemical diversity and compatibility with genetic manipulations. In this study, the selection of suitable producer microorganisms for transglutaminase, ß-galactosidase and levansucrase enzymes was aimed, and the enzyme production capabilities of 46 actinobacterial isolates were determined. The ability of these actinobacterial isolates to produce related enzymes was determined by scanning the genome sequences deposited in "GenBank using Rapid Annotation using Subsystem Technology Version 2.0". Subsequently, the enzyme production capabilities of these isolates with relevant gene were determined by Hydroximate Method for transglutaminase (with the Paper Disc Method), the ONPG method for ß-galactosidase and the mucoid structure formation phenotype for levansucrase. By bioinformatics scanning, it was determined that all isolates contained a "transglutaminase-like enzyme" gene region and by qualitative screening, 9 isolates with different species and that can grow rapidly in medium were identified as potential isolates. A levansucrase-production gene was determined in only 2 isolates (Micromonospora sp. KC 721 and Micromonospora sp. KC213) but none of these showed activity in agar and broth medium. The presence of ß-galactosidase enzyme production gene was detected in 38 isolates. As a result of the qualitative test, 17 isolates with more intense color, different species and that can grow rapidly in medium were selected as potential ß-galactosidase-producers and determined as actinobacterial isolates with a potential to be used in industrial scale enzyme production for different biotechnological applications. [ABSTRACT FROM AUTHOR]

This study examined the potential of levan from the halophilic bacterium Bacillus licheniformis BK1 as a nanoparticle system for protein immobilization. Levan produced by B. licheniformis BK1 was obtained by incubating the bacterium in the optimized Belghith medium, containing 15% (w/v) sucrose, 7.5% (w/v) NaCl and pH 8, in a rotary shaker at 150 rpm for 16 h, at 40 °C. The structure of the levan produced was verified by FTIR and NMR. It appeared that the levan had the same structure as that from Erwinia herbicola. The obtained levan was then used as a nanoparticle system to immobilize BSA and lysozyme proteins. The BSA-nanoparticle had a non-spherical shape with a surface charge of about -4.72 mV and a size distribution in the range of 83–298 nm. In contrast, the lysozyme-nanoparticle exhibited more spherical shapes with a surface charge of -2.57 mV and 206–285 nm size distribution. The efficiency of immobilization was about 74.26% and 81.77% for BSA and lysozyme, respectively. The study thus shows that levan produced by B. licheniformis BK1 can be used as a nanoparticle system for protein immobilization.

Levan produced by Gluconobacter spp. has great potential in biotechnological applications. However, Gluconobacter spp. can synthesize organic acids during fermentation, resulting in environmental acidification. Few studies have focused on the effects of environmental acidification on levan synthesis. This study revealed that the organic acids, mainly gluconic acid (GA) and 2-keto-gluconic acid (2KGA) secreted by Gluconobacter sp. MP2116 created a highly acidic environment (pH < 3) that inhibited levan biosynthesis. The levansucrase derived from strain MP2116 had high enzyme activity at pH 4.0 ∼ pH 6.5. When the ambient pH was less than 3, the enzyme activity decreased by 67 %. Knocking out the mgdh gene of membrane-bound glucose dehydrogenase (mGDH) in the GA and 2KGA synthesis pathway in strain MP2116 eliminated the inhibitory effect of high acid levels on levansucrase function. As a result, the levan yield increased from 7.4 g/l (wild-type) to 18.8 g/l (Δ mgdh ) during fermentation without pH control. This study provides a new strategy for improving levan production by preventing the inhibition of polysaccharide synthesis by environmental acidification., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

• Immobilized Z. mobilis converted sucrose to ethanol that was consumed by C. cohnii. • Levansucrase (sacB) and ndh -negative double mutant Zm6- ndh-sacB was constructed. • Immobilized Zm6- ndh-sacB converted sucrose to ethanol aerobically with a high yield. • Fed-batch with Zm6- ndh-sacB fermentation medium and cocultivations were carried out. • Syntrophy with immobilized Zm6- ndh-sacB yielded C. cohnii biomass with up to 3% DHA. Marine heterotrophic dinoflagellate Crypthecodinium cohnii is an aerobic oleaginous microorganism that accumulates intracellular lipid with high content of 4,7,10,13,16,19-docosahexaenoic acid (DHA), a polyunsaturated ω−3 (22:6) fatty acid with multiple health benefits. C. cohnii can grow on glucose and ethanol, but not on sucrose or fructose. For conversion of sucrose-containing renewables to C. cohnii DHA, we investigated a syntrophic process, involving immobilized cells of ethanologenic bacterium Zymomonas mobilis for fermenting sucrose to ethanol. The non-respiring, NADH dehydrogenase-deficient Z. mobilis strain Zm6- ndh , with high ethanol yield both under anaerobic and aerobic conditions, was taken as the genetic background for inactivation of levansucrase (sacB). SacB mutation eliminated the levan-forming activity on sucrose. The double mutant Zm6- ndh - sacB cells were immobilized in Ca alginate, and applied for syntrophic conversion of sucrose to DHA of C. cohnii , either taking the ethanol-containing fermentation medium from the immobilized Z. mobilis for feeding to the C. cohnii fed-batch culture, or directly coculturing the immobilized Zm6- ndh - sacB with C. cohnii on sucrose. Both modes of cultivation produced C. cohnii CCMP 316 biomass with DHA content around 2–3 % of cell dry weight, corresponding to previously reported results for this strain on glucose. [ABSTRACT FROM AUTHOR]

Pancreatic cancer is the fourth most lethal cancer type worldwide. Due to multiple levan applications including anticancer activities, studies related to levansucrase production are of interest. To our knowledge, levan effect on pancreatic cancer cells has not been tested previously. In this work, among eighteen bacterial honey isolates, Bacillus subtilis MT453867 showed the highest levan yield (33 g/L) and levansucrase production (8.31 U/mL). One-factor-at-a-time technique increased levansucrase activity by 60% when MgSO 4 was eliminated. The addition of 60 g/L banana peels enhanced the enzyme activity (192 U/mL). Placket Burman design determined the media composition for maximum levan yield (54.8 g/L) and levansucrase production (505 U/mL). The identification of levan was confirmed by thin-layer chromatography, Fourier-Transform Infrared spectrometric analysis, 13C-nuclear-magnetic resonance, and 1H-nuclear-magnetic resonance. Both crude and dialyzed levan completely inhibited the pancreatic cancer cell line at 100 ppm with no cytotoxicity on the normal retinal cell line. The LD 50 of crude levan was 4833 mg/kg body weight. Levan had strong antioxidant activity and significantly reduced the expression of CXCR4 and MCM7 genes in pancreatic cancer cells with significant DNA fragmentation. In conclusion, Bacillus subtilis MT453867 levan is a promising adjunct to pancreatic-anticancer agents with both anti-cancer and chemoprotective effects. [Display omitted] [ABSTRACT FROM AUTHOR]

• The general distribution and property of fructosyl-transferases was presented. • Structure, reaction mechanism and product specificity of enzymes were discussed. • A variety of modification to alter the final product profiles was shown. • Application of fructosyl-transferases towards tailor-made product was promising. Carbohydrate-active enzymes are accountable for the synthesis and degradation of glycosidic bonds among diverse carbohydrates. Fructosyl-transferases represent a subclass of these enzymes, employing sucrose as a substrate to generate fructooligosaccharides (FOS) and fructan polymers. This category primarily includes levansucrase (LS, EC 2.4.1.10), inulosucrase (IS, EC 2.4.1.9), and β-fructofuranosidase (Ffase, EC 3.2.1.26). These three enzymes possess a similar five-bladed β-propeller fold and employ an anomer-retaining reaction mechanism mediated by nucleophiles, transition state stabilizers, and general acids/bases. However, they exhibit distinct product profiles, characterized by variations in linkage specificity and molecular mass distribution. Consequently, this article comprehensively explores recent advancements in the catalytic characteristics, structural features, reaction mechanisms, and product specificity of levansucrase, inulosucrase, and β-fructofuranosidase (abbreviated as LS, IS, and Ffase, respectively). Furthermore, it discusses the potential for modifying catalytic properties and product specificity through structure-based design, which enables the rational production of custom fructan and FOS. [ABSTRACT FROM AUTHOR]

[Display omitted] • A large amount of glucose is produced as a byproduct during levan synthesis. • The surface-engineered Y. lipolytica displaying levansucrase was constructed. • Simultaneous production of levan and lipids by the engineered yeast strain. • The byproduct glucose was converted to single cell oil. Currently, levan is attracting attention due to its promising applications in the food and biomedical fields. Levansucrase synthesizes levan by polymerizing the fructosyl unit in sucrose. However, a large amount of the byproduct glucose is produced during this process. In this paper, an engineered oleaginous yeast (Yarrowia lipolytica) strain was constructed using a surface display plasmid containing the LevS gene of Gluconobacter sp. MP2116. The levansucrase activity of the engineered yeast strain reached 327.8 U/g of cell dry weight. The maximal levan concentration (58.9 g/l) was achieved within 156 h in the 5-liter fermentation. Over 81.2 % of the sucrose was enzymolyzed by the levansucrase, and the byproduct glucose was converted to 21.8 g/l biomass with an intracellular oil content of 25.5 % (w/w). The obtained oil was comprised of 91.3 % long-chain fatty acids (C 16 -C 18). This study provides new insight for levan production and comprehensive utilization of the byproduct in levan biosynthesis. [ABSTRACT FROM AUTHOR]

Bacterial canker disease caused by Pseudomonas syringae pv. actinidiae (Psa) biovar 3 involved all global interest since 2008. We have found that in Psa3 genome, similarly to other P. syringae , there are three putative genes, lscα , lscβ and lscγ , coding for levansucrases. These enzymes, breaking the sucrose moiety and releasing glucose can synthetize the fructose polymer levan, a hexopolysaccharide that is well known to be part of the survival strategies of many different bacteria. Considering lscα non-coding because of a premature stop codon, in the present work we cloned and expressed the two putatively functional levansucrases of Psa3, lscβ and lscγ, in E. coli and characterized their biochemical properties such as optimum of pH, temperature and ionic strength. Interestingly, we found completely different behaviour for both sucrose splitting activity and levan synthesis between the two proteins; lscγ polymerizes levan quickly at pH 5.0 while lscβ has great sucrose hydrolysis activity at pH 7.0. Moreover, we demonstrated that at least in vitro conditions, they are differentially expressed suggesting two distinct roles in the physiology of the bacterium. [Display omitted] • Pseudomonas syringae pv. actinidiae biovar 3 possesses three levansucrase genes. • Lscα gene is not functional. • Lscβ and lscγ shows different sucrose splitting and polymerization properties. • Lscβ and lscγ genes show differential expression. [ABSTRACT FROM AUTHOR]

Levansucrase LevS from Leuconostoc mesenteroides B-512F is a multidomain fructansucrase (MD-FN) that contains additional domains (ADs) to the catalytic domain. However, the understanding of the effect that these ADs have on enzyme activity remains vague. To this aim, structure-function relationship studies of these LevS ADs were performed by evaluating both biochemical properties and the enzymatic capacity of truncated versions of LevS. Joint participation of the N- and C-terminal domains is essential for stability, activity, specificity, and polymerization processes. Specifically, the N-terminal region is involved in stability, while the transition region plays an essential role in the transfructosylation reaction and polymer elongation. Based on our results, we suggest that ADs interact with each other, adopting a U-shaped topology. The importance of these ADs observed in the MD-FN of the Leuconostocaceae family is not shared by the Lactobacillaceae family. Phylogenetic analysis of LevS AD suggests that MD-FN from Lactobacillaceae and Leuconostocaceae have different evolutionary origins. This is the first study on the structure-function relationship of multidomain levansucrases from the Leuconostocaceae family. Our results point towards the functional role of AD in MD-FN and its involvement in fructan synthesis.

Background: Levan is a well-known homopolymer of fructose composed predominantly of β-(2, 6) fructofuranosyl linkages in the backbone with occasional β-(2, 1) linkages in the branch chains with varied applications. However, high production cost due to low yield of microbial levan has become a bottleneck for its practical applications. Furthermore, factors affecting the molecular mass of the synthesized levan by Leuconostoc spp. during prolonged cultivation is not fully elucidated. Methods: The cultivation condition for Leuconostoc citreum BD1707 to synthesize levan was optimized by single-factor experiments and subsequently with response surface methodology (RSM). The average molecular weight (Mw) of levan synthesized by the strain L.citreum BD1707 under the optimized cultivation conditions was monitored by high-performance size exclusion chromatography (HPSEC). Finally, the enzyme with levan-degrading activity was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Results: The levan yield of BD1707 reached 34.86 g/L with a corresponding productivity of 7.47 g/L/d under the optimal cultivation conditions deduced by RSM, i.e., cultivation at 26 °C and 200 rpm for 112 h in tomato juice supplemented with 172 g/L sucrose with an initial pH value of 6.12. The Mw of levan reached a peak value of 2.320 × 107 Da at 6 h of cultivation under the optimized cultivation conditions and then gradually decreased to 8.809 × 106 Da after 120 h of cultivation. Conclusion: The levan yield of the strain L.citreum BD1707 could be sufficiently enhanced via cultivation condition optimization. The decrease in molecular mass of the synthesized levan was attributed predominantly to the hydrolytic activity of levansucrase secreted by L.citreum BD1707 during cultivation, with an estimated Mw of 130 KD by SDS-PAGE, while the effect of acid hydrolysis could be nearly neglected. [ABSTRACT FROM AUTHOR]

This study characterizes the acceptor specificity of levansucrases (LSs) from Gluconobacter oxydans (LS1), Vibrio natriegens (LS2), Novosphingobium aromaticivorans (LS3), and Paraburkholderia graminis (LS4) using sucrose as fructosyl donor and selected phenolic compounds and carbohydrates as acceptors. Overall, V. natriegens LS2 proved to be the best biocatalyst for the transfructosylation of phenolic compounds. More than one fructosyl unit could be attached to fructosylated phenolic compounds. The transfructosylation of epicatechin by P. graminis LS4 resulted in the most diversified products, with up to five fructosyl units transferred. In addition to the LS source, the acceptor specificity of LS towards phenolic compounds and their transfructosylation products were found to greatly depend on their chemical structure: the number of phenolic rings, the reactivity of hydroxyl groups and the presence of aliphatic chains or methoxy groups. Similarly, for carbohydrates, the transfructosylation yield was dependent on both the LS source and the acceptor type. The highest yield of fructosylated-trisaccharides was Erlose from the transfructosylation of maltose catalyzed by LS2, with production reaching 200 g/L. LS2 was more selective towards the transfructosylation of phenolic compounds and carbohydrates, while reactions catalyzed by LS1, LS3 and LS4 also produced fructooligosaccharides. This study shows the high potential for the application of LSs in the glycosylation of phenolic compounds and carbohydrates., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Levansucrases synthesize levans and fructooligosaccharides, which are of interest in the food and pharmaceutical industry. Leuconostoc mesenteroides Lm 17 produces levansucrase of about 120 kDa. The encoding gene from this strain was cloned and expressed in Escherichia coli BL21(DE3). The cloned gene encodes a 1022 amino acids long levansucrase with 96% identity to levansucrase LevS from L. mesenteroides NRRL B-512F strain. The induction and expression of the levansucrase gene were performed at temperatures between 15 °C and 37 °C, and concentration of the inducer isopropyl-β-D-thiogalactopyranoside from 0.1 to 2.0 (mmol/L)-1. We report for the first time recombinant expression of a levansucrase gene at a low temperature of induction, after cell biomass accumulation at 37 °C. The highest enzyme activity of 1.90 (U/mg)-1 was measured in TB medium at 18 °C temperature of induction, and concentration of the inducer from 0.1 to 1.0 (mmol/L)-1. The in situ analysis of the purified enzyme showed an active band of about 120 kDa, similar to the one produced by the native strain. The purified enzyme has a temperature optimum at 35 °C, pH optimum at 5.5, and Km = 64 (mmol/L)-1 of sucrose.

Mutation S164A largely affects the transfructosylation properties of Bacillus subtilis levansucrase (SacB). The variant uses acceptors such as glucose and short levans with an average molecular weight of 7.6 kDa more efficiently than SacB, leading to the enhanced synthesis of medium and high molecular weight polymer and a blasto-oligosaccharide series with a polymerization degree of 2–10. A 3-fold increase in blasto-oligosaccharides yield is provoked by the modified interplay between the variant and glucose. Despite its modified product specificity, protein-carbohydrate and protein-protein interactions are still a major factor affecting size and distribution of levan molecular weight. This study highlights the importance of critical factors such as protein concentration in the analysis of wild-type and mutagenized levansucrases. Docking experiments with the crystal structures of SacB and variant S164A – the latter obtained at a 2.6 Å resolution – identified unreported potential binding subsites for fructosyl moieties on the surface of both enzymes. • SacB variant S164A favors the synthesis of blasto-oligosaccharides. • The presence of short levan improves SacB S164A mutant for transfructosylation. • Protein-protein and levan-protein interactions may be critical for levan elongation. • Mutation S164A specifically affects polymerization rate. • SacB Wt and variant S164A share polymer elongation mechanism. [ABSTRACT FROM AUTHOR]

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 t 1/2. 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. [ABSTRACT FROM AUTHOR]

Many bacteria and archaea produce the polydisperse fructose polymer levan from sucrose upon biofilm formation via extracellular levansucrases (EC 2.4.1.10). We have investigated levansucrase-release and -activities as well as molecular size of the levan formed by the acetic acid bacterium Gluconobacter albidus TMW 2.1191 at varying environmental pH conditions to obtain insight in the ecological role of its constitutively expressed levansucrase and the produced levan. A buffer system was established enabling the recovery of levansucrase-containing supernatants from preincubated cell suspensions at pH 4.3–pH 5.7. The enzyme solutions were used to produce levans at different pH values and sucrose concentrations. Finally, the amounts and size distributions of the produced levans as well as the corresponding levansucrase activities were determined and correlated with each other. The data revealed that the levansucrase was released into the environment independently of its substrate sucrose, and that more levansucrase was released at pH ≥ 5.0. The glucose release and formation of high molecular weight levans (> 3.5 kDa) from 0.1 M initial sucrose was comparable between pH ~ 4.3–5.7 using equal amounts of released levansucrase. Hence, this type of levansucrase appears to be structurally adapted to changes in the extracellular pH and to exhibit a similar total activity over a wide acidic pH range, while it produced higher amounts of larger levan molecules at higher production pH and sucrose concentrations. These findings indicate the physiological adaptation of G. albidus TMW 2.1191 to efficient colonisation of sucrose-rich habitats via released levansucrases despite changing extracellular pH conditions in course of acid formation. [ABSTRACT FROM AUTHOR]

The rising global population continues to threaten the world's food security. The discovery of new technologies to produce food of nutritional and functional properties is urgently needed. One beneficial food to humans of known nutritional value is the prebiotic levan. To address the problem, the present work is aimed at isolating levansucrase enzyme-producing microorganisms from traditional fermented food in Thailand. Bacterial colony morphology was observed for mucoidal consistency on culture plates. Isolated colonies were characterized morphologically by gram staining methods. Dinitrosalicylic acid (DNS) and thin-layer chromatography (TLC) reported the highest microbial enzyme activity of 8.51 IU/ml at 12 hours via hydrolysis and frutotransferase activities. Structural characterization of levan via Fourier-transform infrared spectroscopy (FTIR) and 1H and 13C nuclear magnetic resonance (NMR) spectroscopy showed β-(2,6)-fructofuranose linkages. The highest enzyme activity was exhibited by bacterium B-6 identified as Bacillus siamensis NR 11274.1 based on the 16s rDNA gene sequence analyses. Thus, the isolated bacterium from the traditional food was confirmed to produce levansucrase enzyme of high industrial importance for the synthesis of levan as a functional food. [ABSTRACT FROM AUTHOR]

Levan and inulin are two types of fructan. Levan is composed of β-(2, 6) fructosyl linkage and inulin is composed of β-(2, 1) linkage. Both levan and inulin have been accepted and applied in the food, medicinal and chemical industries for their outstanding physicochemical properties in recent years. Microbial levansucrase and inulosucrase are key enzymes responsible for the synthesis of fructan from sucrose. In this review, levansucrase and inulosucrase are discussed together for the first time regarding the evolutionary relationships, bacteria origin, crystal structure, product-forming mechanism and commercial applications. Particularly, some insights into the product specificity about levansucrase and inulosucrase as well as the mechanism for product elongation for fructan are also discussed in the article. [ABSTRACT FROM AUTHOR]

Levan is an enzymatically synthesized fructose polymer with widely reported structural heterogeneity depending on the producing levansucrase, the reaction conditions employed for its synthesis and the characterization techniques. We studied here the specific properties of levan produced by recombinant levansucrase from B. subtilis 168 (Bs -SacB), often characterized as a bimodal distribution, that is, a mixture of low and high molecular weight levan. We found significant differences between both levans in terms of the already reported molecular weight, size and morphology using different analytical methods. The low molecular weight levan consists of a non-uniform polymer ranging from 50 to 230 kDa, synthesized through a non-processive mechanism that can spontaneously form spherical nanoparticles in the reaction medium. In contrast, high molecular weight levan is a uniform polymer, most probably synthesized through a processive mechanism, with an average molecular weight of 30,750 kDa and a poorly defined nano-structure. This is the first report exploring differences in morphology between low and high molecular weight levans. Our findings demonstrate that only the low molecular weight levan forms spherical nanoparticles in the reaction medium and that high molecular weight levan is mainly composed of a 33,000 kDa fraction with a microgel behavior. [Display omitted] [ABSTRACT FROM AUTHOR]

Levan is a biopolymer with many different uses. Temperature is an important parameter in biopolymer synthesis. Herein, levan production was carried out from Bacillus haynesii , a thermophilic microorganism, in the temperature range of 4 °C-95 °C. The highest levan production was measured as 10.9 g/L at 37 °C. The synthesized samples were characterized by FTIR and NMR analysis. The particle size of the levan samples varied between 153 and 824.4 nm at different temperatures. In levan samples produced at high temperatures, the water absorption capacity is higher in accordance with the particle size. Irregularities were observed in the surface pores at temperatures of 60 °C and above. The highest emulsion capacity of 83.4 % was measured in the sample synthesized at 4 °C. The antioxidant activity of all levan samples synthesized at different temperatures was measured as 84 % on average. All synthesized levan samples showed antibacterial effect on pathogenic bacteria. In addition, levan synthesized at 45 °C showed the highest antimicrobial effect on E. coli ATCC 35218 with an inhibition zone of 21.3 ± 1.82 mm. Antimicrobial activity against yeast sample C. albicans , was measured only in levan samples synthesized at 80 °C, 90 °C, 95 °C temperatures. Levan synthesized from Bacillus haynesii at low and high temperatures showed differences in characterization and bioactivity. [Display omitted] • Levan was synthesized by Bacillus haynesii at temperatures between 4 °C and 95 °C. • Levan synthesized at different temperatures has different characteristic properties. • Levan synthesized at high temperature has a higher particle size. • All the synthesized levan samples were antibacterial and antioxidant. [ABSTRACT FROM AUTHOR]

Levansucrase (LS) from Gram-positive bacteria generally produces a large quantity of levan polymer, a polyfructose with glucose at the end (GF n) 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 (GF 4), using two approaches: 1) blocking oligosaccharide binding track of GF 3 -LS complex with large aromatic residues and 2) eliminating hydrogen bond interactions between terminal glucose of GF 4 and side chains of binding residues of GF 4 -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. [ABSTRACT FROM AUTHOR]

BACKGROUND: To increase the low residual activity of levansucrase during long‐time processing, an enhancement of its weak thermostability is needed. Here, the effect of metal ions and polyethylene glycol (PEG) on the thermostability of levansucrase from Brenneria sp. EniD312 were studied and evaluated. The residual activity was determined and the protein structure was evaluated by circular dichroism spectrum, fluorescence intensity (FI), and surface hydrophobicity (S0). RESULTS: As a result of incubation with 10% (w/v) PEG 4000, the enzyme activity was increased by 1.24‐fold. After incubation with 5% PEG 4000 for 6 h, the residual activity at 35 and 45 °C was decreased to 55% and 60% of the initial activity, with an increase of 1.2‐ and 3.3‐fold than the wild‐type enzyme. Furthermore, the random coil content of enzyme was decreased from 53% of the wild‐type enzyme to 33.9% of the PEG pre‐incubated enzyme. Additionally, the FI was maximally increased and the S0 was decreased from 117 to 69. CONCLUSION: All of these results suggested that after incubation with PEG 4000, the secondary and tertiary structure of wild‐type enzyme could be greatly maintained and then its thermostability could be increased. This study was the first report on the enhancement of levansucrase thermostability by PEG incubation and might be a good guideline to other researches on levansucrase. © 2019 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Levan is a fructose polymer with diverse applications in the food and medical industries. In this study, levansucrase from Rahnella aquatilis (RaLsrA) was hyper-secreted using a Saccharomyces cerevisiae protein secretion system. An optimal secretion signal, a translation fusion partner (TFP) containing an N-terminal 98 amino acid domain from a mitochondrial inner membrane protein, UTH1, was employed to secrete approximately 50 U/mL of bioactive RaLsrA into culture media with 63% secretion efficiency by fed-batch fermentation. Although the purified RaLsrA was useful for enzymatic conversion of high-molecular-weight levan of approximately 3.75 × 106 Da, recombinant yeast secreting RaLsrA could produce levan more efficiently by microbial fermentation. In a 50-L scale fermenter, 76-g/L levan was directly converted from 191-g/L sucrose by recombinant yeast cells, attaining an 80% conversion yield and 3.17-g/L/h productivity. Thus, we developed a cost-effective and industrially applicable production system for food-grade levan. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Screening of 18 bacterial honey isolates revealed that all the isolates were levansucrase producers. The most potent isolate that achieved the highest activity (45.66 U/ml) was identified as Bacillus subtilis NRC based on morphological examination and 16S rRNA. The results recorded the necessity of starch (5 g/L), baker's yeast (12.5 g/L), and AlCl3 (5 mM) in improvement of the enzyme productivity. The Bacillus subtilis levansucrase was eluted as a single protein in one purification step. The enzyme molecular weight was (14 kDa). It showed its optimum activity at 45°C and could retain 60% of its activity after incubation at 50°C for 2 h. Its optimum activity was obtained at pH 8.2 and the enzyme showed great pH stability in both acidic and alkaline ranges. Unlike, most levansucrases all tested metals had an adverse effect in enzyme activity. The enzyme had antioxidant activities and were characterized as spherical micro‐ and nanoparticles by transmission electron microscopy. The effect of growth conditions and medium composition in levan structure and its fibrinolytic activity was evaluated. [ABSTRACT FROM AUTHOR]

• Fructans are versatile homopolymers with an enormous potential in a wide variety of industrial sectors. • Levansucrases produce fructans of diverse molecular weight, polydispersity and branching ratio. • Traditional and novel strategies to control levan molecular weight are reviewed. • Protein interactions, chemical modifications and protein engineering effects on levan biosynthesis are discussed. • An in-depth analysis of the distinctive structural hallmarks of bacterial levansucrases is provided. The physicochemical properties and biological activity of levan, a generic term given to oligo- and polysaccharides consisting of fructose units linked predominantly by β(2-6) bonds, are attributable to both its size and structural complexity. Branching in β(2-1) contributes to diversify levan structures and properties. There is a broad spectrum of applications for levan and accordingly it has been the subject of several comprehensive reviews. A thorough analysis focused on the product specificity of enzymes from the Glycoside-Hydrolase family 68 that synthesize levan is however missing. We analyze here traditional and novel strategies to manipulate bacterial levansucrases in favor of the generation of low- or high-molecular weight levan, including site directed mutagenesis and chemical engineering. A comparison of highly variable structural elements of levansucrases is presented in the context of their capacity to synthesize saccharides of different sizes, employing the levansucrases from Bacillus subtilis and Bacillus megaterium as references. [ABSTRACT FROM AUTHOR]

Abstract Erwinia tasmaniensis is an epiphytic bacterium related to the plant pathogen Erwinia amylovora , the etiological agent of fire blight. In this study the levansucrase from E. tasmaniensis (EtLsc) has been compared with the homologous enzyme from E. amylovora (EaLsc). We characterized the enzymatic activity and compared the products profile of both enzymes by High Performance Anion Exchange Chromatography coupled with Pulsed Amperometric Detector (HPAEC-PAD). Moreover we determined the crystal structure of EtLsc to understand the structural peculiarity causing the different product profiles of the two homologues. EtLsc exhibits increased efficiency in the production of FOS, resulting in a better catalyst for biotechnological synthesis than EaLsc. Based on our results, we propose that the role of this enzyme in the life cycle of the two bacteria is most likely related to survival, rather than linked to pathogenicity in E. amylovora. [ABSTRACT FROM AUTHOR]