Improving the activity of an inulosucrase by rational engineering for the efficient biosynthesis of low-molecular-weight inulin.

Inulin, a widely recognized prebiotic, has diverse applications across various industrial sectors. Although inulin is primarily produced through plant extraction, there is growing interest in enzymatic synthesis as an alternative. The enzymatic production of inulin from sucrose, which yields polymers with degrees of polymerization similar to those of plant-derived inulin, shows potential as a viable replacement for traditional extraction methods. In this study, an inulosucrase from Neobacillus bataviensis was identified, demonstrating a non-processive mechanism specifically tailored for synthesizing inulin with polymerization degrees ranging from 3 to approximately 40. The enzyme exhibited optimal activity at pH 6.5 and 55 °C, efficiently producing inulin with a yield of 50.6%. Ca2+ can improve the activity and thermostability of this enzyme. To enhance catalytic total activity, site-directed and truncated mutagenesis techniques were applied, resulting in the identification of a mutant, T149S, displaying a significant 57% increase in catalytic total activity. Molecular dynamics simulations unveiled that the heightened flexibility observed in three surface regions positively influenced enzymatic activity. This study not only contributes to the theoretical foundation for inulosucrase engineering but also presents a potential avenue for the production of inulin.

Hydrolysis-transglycosylation of sucrose and production of ß-(2→1)-fructan by inulosucrase from Neobacillus drentensis 57N.

Inulin, ß-(2→1)-fructan, is a beneficial polysaccharide used as a functional food ingredient. Microbial inulosucrases (ISs), catalyzing ß-(2→1)-transfructosylation, produce ß-(2→1)-fructan from sucrose. In this study, we identified a new IS (NdIS) from the soil isolate, Neobacillus drentensis 57N. Sequence analysis revealed that, like other Bacillaceae ISs, NdIS consists of a glycoside hydrolase family 68 domain and shares most of the 1-kestose-binding residues of the archaeal IS, InuHj. Native and recombinant NdIS were characterized. NdIS is a homotetramer. It does not require calcium for activity. High performance liquid chromatography and 13C-nuclear magnetic resonance indicated that NdIS catalyzed the hydrolysis and ß-(2→1)-transfructosylation of sucrose to synthesize ß-(2→1)-fructan with chain lengths of 42 or more residues. The rate dependence on sucrose concentration followed hydrolysis-transglycosylation kinetics, and a 50% transglycosylation ratio was obtained at 344 m m sucrose. These results suggest that transfructosylation from sucrose to ß-(2→1)-fructan occurs predominantly to elongate the fructan chain because sucrose is an unfavorable acceptor.

Molecular and biochemical characteristics of inulosucrase InuBK from Alkalihalobacillus krulwichiae JCM 11691.

Information about the inulosucrase of nonlactic acid bacteria is scarce. We found a gene encoding inulosucrase (inuBK) in the genome of the Gram-positive bacterium Alkalihalobacillus krulwichiae JCM 11691. The inuBK open reading frame encoded a protein comprising 456 amino acids. We expressed His-tagged InuBK in culture medium using a Brevibacillus system. The optimal pH and temperature of purified InuBK were 7.0-9.0 and 50-55 °C, respectively. The findings of high-performance anion-exchange chromatography, nuclear magnetic resonance spectroscopy, and high-performance size-exclusion chromatography with multiangle laser light scattering showed that the polysaccharide produced by InuBK was an inulin with a molecular weight of 3806, a polydispersity index (PI) of 1.047, and fructosyl chain lengths with 3-27 degrees of polymerization. The size of InuBK was smaller than commercial inulins, and the PI of the inulin that it produced was lower.

Recent advances in Levansucrase and Inulosucrase: evolution, characteristics, and application.

Levan and inulin are two types of fructan. Levan is composed of ß-(2, 6) fructosyl linkage and inulin is composed of ß-(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.

Synthesis and structural characterization of raffinosyl-oligofructosides upon transfructosylation by Lactobacillus gasseri DSM 20604 inulosucrase.

A new process based on enzymatic synthesis of a series of raffinose-derived oligosaccharides or raffinosyl-oligofructosides (RFOS) with degree of polymerization (DP) from 4 to 8 was developed in the presence of raffinose. This process involves a transfructosylation reaction catalyzed by an inulosucrase from Lactobacillus gasseri DSM 20604 (IS). The main synthesized RFOS were structurally characterized by nuclear magnetic resonance (NMR). According to the elucidated structures, RFOS consist of ß-2,1-linked fructose unit(s) to raffinose: α-D-galactopyranosyl-(1 → 6)-α-D-glucopyranosyl-(1↔2)-ß-D-fructofuranosyl-((1 ← 2)-ß-D-fructofuranoside)n (where n refers to the number of transferred fructose moieties). The maximum yield of RFOS was 33.4 % (in weight respect to the initial amount of raffinose) and was obtained at the time interval of 8-24 h of transfructosylation reaction initiated with 50 % (w/v) of raffinose. Results revealed the high acceptor and donor affinity of IS towards raffinose, being fairly comparable with that of sucrose for the production of fructooligosaccharides (FOS), including when both carbohydrates coexisted (sucrose/raffinose mixture, 250 g L(-1) each). The production of RFOS was also attempted in the presence of sucrose/melibiose mixtures; in this case, the predominant acceptor-product formed was raffinose followed by a minor production of a series of oligosaccharides with varying DP. The easiness of RFOS synthesis and the structural similarities with both raffinose and fructan series of oligosaccharides warrant the further study of the potential bioactive properties of these unexplored oligosaccharides.

A review of fructosyl-transferases from catalytic characteristics and structural features to reaction mechanisms and product specificity.

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 ß-fructofuranosidase (Ffase, EC 3.2.1.26). These three enzymes possess a similar five-bladed ß-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 ß-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.

Multistrategy Engineering of an Inulosucrase to Enhance the Activity and Thermostability for Efficient Production of Microbial Inulin.

Inulin has found commercial applications in the pharmaceutical, nutraceutical, and food industries due to its beneficial health effects. The enzymatic biosynthesis of microbial inulin has garnered increasing attention. In this study, molecular modification was applied to Lactobacillus mulieris UMB7800 inulosucrase, an enzyme that specifically produces high-molecular weight inulin, to enhance its catalytic activity and thermostability. Among the 18 variable regions, R5 was identified as a crucial region significantly impacting enzymatic activity by replacing it with more conserved sequences. Site-directed mutagenesis combined with saturated mutagenesis revealed that the mutant A250 V increased activity by 68%. Additionally, after screening candidate mutants by rational design, four single-point mutants, S344D, H434P, E526D, and G531P, were shown to enhance thermostability. The final combinational mutant, M5, exhibited a 66% increase in activity and a 5-fold enhancement in half-life at 55 °C. These findings are significant for understanding the catalytic activity and thermostability of inulosucrase and are promising for the development of microbial inulin biosynthesis platforms.

Characterization of a processive inulosucrase from Lactobacillus mulieris for efficient biosynthesis of high-molecular-weight inulin.

Inulin has been determined to have many exceptional properties and functions and has been used in the food and pharmaceutical fields. Recently, microbial high-molecular-weight inulin synthesized from sucrose by inulosucrase attracted much attention. In this study, a novel inulosucrase from Lactobacillus mulieris was constructed, overexpressed, purified, and identified. The recombinant enzyme displayed the maximum activity at pH 6.0 and 55 °C, and it exhibited high thermostability below 45 °C. After optimizing the production conditions, the conversion rate from 100 g/L sucrose to inulin reached 31 %, meanwhile, the maximum molecular weight of produced inulin reached 3.21 × 106 g/mol. The truncated IS showed a "processive" transfructosylation process, only synthesizing a small number of short-chain oligosaccharides with polymerization degrees below 6, which was in favor of the accumulation of high-molecular-weight inulin. Given this, L. mulieris inulosucrase might be a good potential candidate for the industrial production of high-molecular-weight inulin.

Production of fructan synthesis/hydrolysis of endophytic bacteria involved in inulin production in Jerusalem artichoke.

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.

High-yield production and purification of prebiotic inulin-type fructooligosaccharides.

Due to the health-promoting effects and functional properties of inulin-type fructooligosaccharides (I-FOS), the global market for I-FOS is constantly growing. Hence, there is a continuing demand for new, efficient biotechnological approaches for I-FOS production. In this work, crude inulosucrase InuGB-V3 from Lactobacillus gasseri DSM 20604 was used to synthesize I-FOS from sucrose. Supplementation with 1 mM CaCl2, a pH of 3.5-5.5, and an incubation temperature of 40 °C were found to be optimal production parameters at which crude inulosucrase showed high conversion rates, low sucrose hydrolysis, and excellent stability over 4 days. The optimal process conditions were employed in cell-free bioconversion reactions. By elevating the substrate concentration from 570 to 800 g L-1, the I-FOS concentration and the synthesis of products with a low degree of polymerization (DP) could be increased, while sucrose hydrolysis was decreased. Bioconversion of 800 g L-1 sucrose for 20 h resulted in an I-FOS-rich syrup with an I-FOS concentration of 401 ± 7 g L-1 and an I-FOS purity of 53 ± 1% [w/w]. I-FOS with a DP of 3-11 were synthesized, with 1,1-kestotetraose (DP4) being the predominant transfructosylation product. The high-calorie sugars glucose, sucrose, and fructose were removed from the generated I-FOS-rich syrup using activated charcoal. Thus, 81 ± 5% of the initially applied I-FOS were recovered with a purity of 89 ± 1%.

Synergistic enzyme cocktail between levansucrase and inulosucrase for superb levan-type fructooligosaccharide synthesis.

Inulosucrase (ISC) and levansucrase (LSC) utilise sucrose and produce inulin- and levan-type fructans, respectively. This study aims to propose a new strategy to improve levan-type fructooligosaccharide (L-FOS) production. The effect of ISC/ LSC -mixed reaction was elucidated on L-FOS production. The presence of ISC in the LSC reaction significantly leads to the higher production of L-FOSs as the main products. Furthermore, the different ratios between ISC and LSC affected the distribution of L-FOSs. A greater amount of ISC compared to LSC promoted the synthesis of short-chain L-FOSs. Conversely, when LSC was increased, the synthesis of longer-chain L-FOSs was enhanced. The addition of trisaccharide mixtures obtained from either a single ISC or LSC reaction could enhance L-FOSs synthesis in the LSC reaction. Analysis of these trisaccharides revealed that most species of the oligosaccharides were similar, with 1-kestose being the major one. The supplement of only 1-kestose in the LSC reaction showed similar results to those of the reaction in the presence of trisaccharide mixtures. Moreover, the results were supported by molecular dynamics simulations. This work not only provides an improvement in L-FOS production but also revealed and supported some insights into the mechanism of fructansucrases.

Directionally modulating the product chain length of an inulosucrase by semi-rational engineering for efficient production of 1-kestose.

Microbial inulosucrase as a transfructosylation tool has been used to produce inulin and inulin-type fructooligosaccharides with various polymerization degrees. Tailor-made oligosaccharides could be generated by inulosucrase via chain length modulation. In this study, a semi-rational design based on the modeled structure of Lactobacillus reuteri 121 inulosucrase was carried out to screen and construct variants. The residues Arg541 and Arg544 were determined to be significant to the product chain elongation of L. reuteri 121 inulosucrase. The variant R544W altered the product specificity of inulosucrase and produced short-chain fructooligosaccharides with 1-kestose as the main component. Molecular dynamic simulations verified an increased binding free energy of variant R544W with 1-kestose than the wild-type enzyme with 1-kestose. After optimization, 1-kestose and total short-chain fructooligosaccharides production reached approximately 206 g/L and 307 g/L, respectively. This study suggests the great potential of variant R544W in the biotransformation from sucrose to functional sugar.

Functional and Molecular Characterization of the Halomicrobium sp. IBSBa Inulosucrase.

Fructans are fructose-based (poly)saccharides with inulin and levan being the best-known ones. Thanks to their health-related benefits, inulin-type fructans have been under the focus of scientific and industrial communities, though mostly represented by plant-based inulins, and rarely by microbial ones. Recently, it was discovered that some extremely halophilic Archaea are also able to synthesize fructans. Here, we describe the first in-depth functional and molecular characterization of an Archaeal inulosucrase from Halomicrobium sp. IBSBa (HmcIsc). The HmcIsc enzyme was recombinantly expressed and purified in Escherichia coli and shown to synthesize inulin as proven by nuclear magnetic resonance (NMR) analysis. In accordance with the halophilic lifestyle of its native host, the enzyme showed maximum activity at very high NaCl concentrations (3.5 M), with specific adaptations for that purpose. Phylogenetic analyses suggested that Archaeal inulosucrases have been acquired from halophilic bacilli through horizontal gene transfer, with a HX(H/F)T motif evolving further into a HXHT motif, together with a unique D residue creating the onset of a specific alternative acceptor binding groove. This work uncovers a novel area in fructan research, highlighting unexplored aspects of life in hypersaline habitats, and raising questions about the general physiological relevance of inulosucrases and their products in nature.

High-resolution method for isocratic HPLC analysis of inulin-type fructooligosaccharides.

In recent decades, strategies to improve human health by modulating the gut microbiota have developed rapidly. One of the most prominent is the use of prebiotics, which can lead to a higher abundance of health-promoting microorganisms in the gut. Currently, oligosaccharides dominate the prebiotic sector due to their ability to promote the growth and activity of probiotic bacteria selectively. Extensive efforts are made to develop effective production strategies for the synthesis of prebiotic oligosaccharides, including the use of microbial enzymes. Within the genus Lactobacillus, several inulosucrases have been identified, which are suitable for the synthesis of prebiotic inulin-type fructooligosaccharides (inulin-FOS). In this study, a truncated version of the inulosucrase from Lactobacillus gasseri DSM 20604 was used for the efficient synthesis of inulin-FOS. Product titers of 146.2 ±â€¯7.4 g inulin-FOSL-1 were achieved by the catalytic activity of the purified recombinant protein InuGB-V3. A time and resource-saving HPLC method for rapid analysis of inulin-FOS in isocratic mode was developed and optimized, allowing baseline separated analysis of inulin-FOS up to a degree of polymerization (DP) of five in less than six minutes. Long-chain inulin-FOS with a DP of 17 can be analyzed in under 45 min. The developed method offers the advantages of isocratic HPLC analysis, such as low flow rates, high sensitivity, and the use of a simple, inexpensive chromatographic setup. Furthermore, it provides high-resolution separation of long-chain inulin-FOS, which can usually only be achieved with gradient systems.

Improving the catalytic behaviors of Lactobacillus-derived fructansucrases by truncation strategies.

Fructansucrases (FSs), including inulosucrase (IS) and levansucrase (LS), are the members of the Glycoside Hydrolase family 68 (GH68) enzymes. IS and LS catalyze the polymerization of the fructosyl moiety from sucrose to inulin- and levan-type fructans, respectively. Lactobacillus-derived FSs have relatively extended N- and C-terminal sequences. However, the functional roles of these sequences in their enzymatic properties and fructan biosynthesis remain largely unknown. Limosilactobacillus reuteri (basionym: Lactobacillus reuteri) 121 could produce both IS and LS, abbreviated as Lare121-IS and Lare121-LS, respectively. In this study, it was found that the terminal truncation displayed an obvious effect on their activities and the N-terminal truncated variants, Lare121-ISΔ177-701 and Lare121-LSΔ154-686, displayed the highest activities. Melting temperature (Tm) and the thermostability at 50 °C were measured to evaluate the stability of various truncated versions, revealing the different effects of N-terminal on the stability. The average molecular weight and polymerization degree of the fructans produced by different truncated variants did not change considerably, indicating that N-terminal truncation had low influence on fructan biosynthesis. In addition, it was found that N-terminal truncation could also improve the activity of other reported FSs from Lactobacillus species.

[Characterization of inulosucrase and the enzymatic synthesis of inulin].

As a type of prebiotics and dietary fiber, inulin performs plenty of significant physiological functions and is applied in food and pharmaceutical fields. Inulosucrase from microorganisms can use sucrose as the substrate to synthesize inulin possessing higher molecular weight than that from plants. In this work, a hypothetical gene coding inulosucrase was selected from the GenBank database. The catalytic domain was remained by N- and C- truncation strategies, constructing the recombinant plasmid. The recombinant plasmid was expressed in E. coli expression system, and after purifying the crude enzyme by Ni²âº affinity chromatography, a recombinant enzyme with a molecular weight of approximately 65 kDa was obtained. The optimal pH and temperature of the recombinant enzyme were 5.5 and 45 °C, respectively, when sucrose was used as the sole substrate. The activity of this enzyme was inhibited by various metal ions at different degrees. After purifying the produced polysaccharide, nuclear magnetic resonance analysis was used to determine that the polysaccharide was inulin connected by ß-(2,1) linkages. Finally, the conditions for the production of inulin were optimized. The results showed that the inulin production reached the maximum, approximately 287 g/L after 7 h, when sucrose concentration and enzyme dosage were 700 g/L and 4 U/mL, respectively. The conversion rate from sucrose to inulin was approximately 41%.

Crystal structure of an inulosucrase from Halalkalicoccus jeotgali B3T, a halophilic archaeal strain.

Several archaea harbor genes that code for fructosyltransferase (FTF) enzymes. These enzymes have not been characterized yet at structure-function level, but are of extreme interest in view of their potential role in the synthesis of novel compounds for food, nutrition, and pharmaceutical applications. In this study, 3D structure of an inulin-type fructan producing enzyme, inulosucrase (InuHj), from the archaeon Halalkalicoccus jeotgali was resolved in its apo form and with bound substrate (sucrose) molecule and first transglycosylation product (1-kestose). This is the first crystal structure of an FTF from halophilic archaea. Its overall five-bladed ß-propeller fold is conserved with previously reported FTFs, but also shows some unique features. The InuHj structure is closer to those of Gram-negative bacteria, with exceptions such as residue E266, which is conserved in FTFs of Gram-positive bacteria and has possible role in fructan polymer synthesis in these bacteria as compared to fructooligosaccharide (FOS) production by FTFs of Gram-negative bacteria. Highly negative electrostatic surface potential of InuHj, due to a large amount of acidic residues, likely contributes to its halophilicity. The complex of InuHj with 1-kestose indicates that the residues D287 in the 4B-4C loop, Y330 in 4D-5A, and D361 in the unique α2 helix may interact with longer FOSs and facilitate the binding of longer FOS chains during synthesis. The outcome of this work will provide targets for future structure-function studies of FTF enzymes, particularly those from archaea.

Improving the Thermostability and Catalytic Activity of an Inulosucrase by Rational Engineering for the Biosynthesis of Microbial Inulin.

Thermostability and enzymatic activity are two vital indexes determining the application of an enzyme on an industrial scale. A truncated inulosucrase, Laga-ISΔ138-702, from Lactobacillus gasseri showed high catalysis activity. To further enhance its thermostability and activity, multiple sequence alignment (MSA) and rational design based on the modeled structure were performed. Variants A446E, S482A, I614M, and A627S were identified with an improved denaturation temperature (Tm) of more than 1 °C. A combinational mutation method was further carried out to explore the synergistic promotion effects of single-point mutants. Additionally, 33 residues at the N-terminus were truncated to construct mutant M4N-33. The half-life of M4N-33 at 55 °C increased by 120 times compared to that of Laga-ISΔ138-702, and the relative activity of M4N-33 increased up to 152% at the optimal pH and temperature (pH 5.5 and 60 °C). Molecular dynamics (MD) simulations illustrated the decreased b-factor of the surface loop of M4N-33.

Efficient production of inulin and oligosaccharides using thermostable inulosucrase from Lactobacillus jensenii.

Fructan, inulin and levan, have been applied in many industrial fields because of their versatile properties and functions. Recently, microbial fructan has attracted much attention. In this study, a novel inulosucrase from Lactobacillus jensenii JV-V16 was constructed by truncating partial sequence. The truncated inulosucrase was overexpressed, purified and identified. The optimized pH and temperature for transfructosylation were pH 6.0 and 45 °C, respectively. The inulosucrase showed high thermostability and the half-life at 60 °C was 14.5 h. It also exhibited high transfructosylation capability, resulting in a high ratio of transfructosylation activity to hydrolysis activity. After optimizing the conditions of inulin production, 278.4 g/L inulin was obtained from 600 g/L sucrose with an approximately 46% conversion rate from sucrose to inulin. Additionally, acceptor reaction was attempted to explore the transfructosylation capability of the enzyme and some novel saccharides were detected, indicating the potential application in the synthesis of prebiotics.

Temperature-dependent inulin nanoparticles synthesized by Lactobacillus reuteri 121 inulosucrase and complex formation with flavonoids.

Inulin nanoparticles (INNPs) are a biocompatible material which has a potential application for enhancing solubility and preventing degradation of compounds. In this work, we demonstrated that INNPs could be synthesized from sucrose using inulosucrase from Lactobacillus reuteri 121. Noticeably, dynamic light scattering (DLS) analysis showed that the derived INNPs exhibited uniformity in size, which was easily controlled by the reaction temperature. The effect of enzyme and sucrose concentration, as well as reaction time, was explored. Moreover, the solubility of INNPs in various organic solvents was also investigated, and we found that the INNPs were freely regenerated in water even though they had precipitated by organic solvents. Essentially, we demonstrated that the derived INNPs could be applied for flavonoid encapsulation. The solubility and stability of quercetin and fisetin in the INNPs complexes was higher than those of free compounds. These results make the INNPs very promising for many applications.