Production of prebiotic enriched maple syrup through enzymatic conversion of sucrose into fructo-oligosaccharides.

Maple syrup, a popular natural sweetener has a high content of sucrose, whose consumption is linked to different health issues such as obesity and diabetes. Hence, within this paper, the conversion of sucrose to prebiotics (fructo-oligosaccharides, FOS) was proposed as a promising approach to obtaining a healthier, value-added product. Enzymatic conversion was optimized with respect to key experimental factors, and thereafter derived immobilized preparation of fructosyltransferase (FTase) from Pectinex® Ultra SP-L (FTase-epoxy Purolite, 255 IU/g support) was successfully utilized to produce novel functional product in ten consecutive reaction cycles. The product, obtained under optimal conditions (60 °C, 7.65 IU/mL, 12 h), resulted in 56.0% FOS, 16.7% sucrose, and 27.3% monosaccharides of total carbohydrates, leading to a 1.6-fold reduction in caloric content. The obtained products` prebiotic potential toward the probiotic strain Lactobacillus plantarum 299v was demonstrated. The changes in physico-chemical and sensorial characteristics were esteemed as negligible.

Innovative application of a novel and thermostable inulin fructotransferase from Arthrobacter sp. ISL-85 to fructan inulin in burdock root to improve nutrition.

Inulin fructotransferase converts prebiotic polysaccharide inulin to difructose anhydride III, known for its numerous beneficial physiological effects. While previous studies focused on using inulin extracts under optimal conditions, this study delves into the enzyme's behavior when dealing with more complex food materials, inulin-rich burdock root, which possesses greater nutritional value but may influence the enzymatic reaction. An inulin fructotransferase from Arthrobacter sp. ISL-85 was identified and characterized, which has the highest activity of 783 U mg-1 at pH 6.5 and 65 °C and remains stable even up to 80 °C. When applied to inulin-rich burdock root (pH 4.7) at 80 °C for 2 h, the enzyme yielded 4.1 g of difructose anhydride III, concurrently increasing fructo-oligosaccharides. This study demonstrates the potential of this enzyme as a valuable tool for efficiently processing inulin within whole food materials under high temperatures. Such an approach could pave the way for enhancing nutrition and promoting health benefits.

Similarities and differences of nano-sized levan synthesized by Bacillus haynesii at low and high temperatures: Characterization and bioactivity.

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.

Rational design of a self-assembly promoting fusion domain enhances high molecular weight levan synthesis by levansucrase SacB.

The catalytic product of levansucrase from Bacillus subtilis (SacB) is mainly composed of 10 % high molecular weight levan (HMW, ~2000 kDa) and 90 % low molecular weight levan (LMW, ~7000 Da). In order to achieve efficient production of food hydrocolloid, high molecular weight levan (HMW), with the help of molecular dynamics simulation software, a protein self-assembly element, Dex-GBD, was found and fused with the C-terminus of SacB to construct a novel fusion enzyme, SacB-GBD. The product distribution of SacB-GBD was reversed compared with SacB, and the proportion of HMW in the total polysaccharide was significantly increased to >95 %. We then confirmed that the self-assembly was responsible for the reversal of the SacB-GBD product distribution by the simultaneous modulation of SacB-GBD particle size and product distribution by SDS. The hydrophobic effect may be the main driver of self-assembly as analyzed by molecular simulations and hydrophobicity determination. Our study provides an enzyme source for the industrial production of HMW and provides a new theoretical basis for guiding the molecular modification of levansucrase towards the size of the catalytic product.

Chemo-Enzymatic Synthesis of Non-ribosomal Macrolactams by a Penicillin-Binding Protein-Type Thioesterase.

Penicillin-binding protein-type thioesterases (PBP-type TEs) are an emerging family of non-ribosomal peptide cyclases. PBP-type TEs exhibit distinct substrate scopes from the well-exploited ribosomal peptide cyclases and traditional non-ribosomal peptide cyclases. Their unique properties, as well as their stand-alone nature, highlight PBP-type TEs as valuable candidates for development as biocatalysts for peptide macrocyclization. Here in this chapter, we describe the scheme for the chemoenzymatic synthesis of non-ribosomal macrolactam by SurE, a representative member of PBP-type TEs.

Successful Manipulation of the Product Spectrum of the Erwinia amylovora Levansucrase by Modifying the Residues around loop1, Loop 3, and Loop 4.

Levansucrase (LS, EC 2.4.1.10) catalyzes the synthesis of levan by successively transferring the fructosyl moiety from sucrose to an elongated fructan chain. Although the product distribution of LS from Erwinia amylovora (Ea-LS) was studied under different sucrose concentrations, the effect of residues on the product formation is yet unknown. The first levanhexaose-complexed structure of LS from Bacillus subtilis (Bs-SacB) provided information on the oligosaccharide binding sites (OB sites), from +1 to +4 subsites. Since Ea-LS would efficiently produce fructooligosaccharides, a substitution mutation of OB sites in Bs-SacB and the corresponding residues of Ea-LS were conducted to investigate how these mutants would influence the product distribution. As a result, a series of mutants with different product spectrum were obtained. Notably, the mutants of G98E, V151F, and N200T around loop 1, loop 3, and loop 4 all showed a significant increase in both the molecular mass and the yield of high-molecular-mass levan, suggesting that the product profile of Ea-LS was significantly modified.

Production and bioactivities of nanoparticulated and ultrasonic-degraded levan generated by Erwinia tasmaniensis levansucrase in human osteosarcoma cells.

Levan is a bioactive polysaccharide that can be synthesized by various microorganisms. In this study, the physicochemical properties and bioactivity of levan synthesized by recombinant levansucrase from Erwinia tasmaniensis were investigated. The synthesis conditions, including the enzyme concentration, substrate concentration, and temperature, were optimized. The obtained levan generally appeared as a cloudy suspension. However, it could transform into a hydrogel at concentrations exceeding 10 % (w/v). Then, ultrasonication was utilized to reduce the molecular weight and increase the bioavailability of levan. Dynamic light scattering (DLS) and gel permeation chromatography (GPC) indicated that the size of levan was significantly decreased by ultrasonication, whereas Fourier transform infrared spectroscopy, 1H-nuclear magnetic resonance, and X-ray powder diffraction revealed that the chemical structure of levan was not changed. Finally, the bioactivities of both levan forms were examined using human osteosarcoma (Saos-2) cells. The result clearly illustrated that sonicated levan had higher antiproliferative activity in Saos-2 cells than original levan. Sonicated levan also activated Toll-like receptor expression at the mRNA level. These findings suggested the important beneficial applications of sonicated levan for the development of cancer therapies.

Production of Human ABC Transporters and Oligosaccharyltransferase Complexes for Structural Studies.

Structural studies of membrane proteins require high-quality samples. The target proteins should not only be pure and homogeneous but should also be active and allow the capture of a functionally relevant state. Here we present optimized methods for the expression and purification of human ABC transporters and oligosaccharyltransferase (OST) complexes that can be used for high-resolution structure determination using single-particle cryo-electron microscopy (cryo-EM). The protocols are based on the generation of stable cell lines that enable tetracycline-inducible expression of the target proteins. For the multidrug exporter ABCB1, we describe a protocol for reconstitution into nanodiscs and evaluation of the ATPase activity in the presence of drugs. For human OST, we describe a strategy for the purification of OST-A and OST-B complexes, including techniques to evaluate their integrity and activity using in vitro glycosylation assays. These protocols can be adapted for the production of other human ABC transporters and multimeric membrane protein complexes.

Structural characterization of levan synthesized by a recombinant levansucrase and its application as yogurt stabilizers.

Levan, a class of fructan with complex structure, has been found to have potential as a prebiotic. In this study, the polysaccharide was produced by in vitro catalysis (Cata-lev), structural analysis proved it was levan that possessed a branching degree of about 15% and molecular weight of 2.54 × 106 Da. Furthermore, SEM observation showed that Cata-lev was porous with loose flaky structure. Subsequently, the effects of five Cata-lev concentrations on the fermentation process, texture, nutraceutical, and sensory characteristics of set yogurts were investigated and compared to Fructooligosaccharides (FOS). The addition of Cata-lev could significantly (P < 0.05) increase the water holding capacity (WHC) of fermented yogurt (over 77%). The Cata-lev treatment groups exhibited superior system stability (Zeta and WHC) than FOS. Overall, this study expanded knowledge about levan structure and its potential utilization as a yogurt stabilizer.

Complete secretion of recombinant Bacillus subtilis levansucrase in Pichia pastoris for production of high molecular weight levan.

Three signal peptides from α-mating factor (α-MF), inulinase (INU) and native levansucrase (LS) were compared for secretion efficiency of Bacillus subtilis levansucrase SacB-T305A in Pichia pastoris GS115. The first complete secretion of bacterial levansucrase in yeasts under methanol induction was achieved while using α-MF signal. The secreted recombinant Lev(α-MF) proved to be glycosylated by combination of NanoLC-MS/MS and Endo H digestion. Interestingly, glycosylation not only improved significantly the polymerase thermostability, but also reversed the products profiles to favor synthesis of high molecular weight (HMW) levan which accounted for approximately 73 % to total levan-type polysaccharides. It indicated for the first time that the glycosylation of recombinant B. subtilis levansucrase affected significantly the products molecular weight distribution. It also provided a promising enzymatic way to effectively product HMW levan from sucrose resources.

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

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

Discovery of Glycosyltransferases Involved in the Biosynthesis of Ligupurpuroside B.

In this study, we report the characterization of three glycosyltransferases involved in the biosynthesis of ligupurpuroside B, a complex acylated phenolic glycoside in Ligustrum robustum. UGT85AF8 catalyzed the formation of salidroside from tyrosol. UGT79G7, an osmanthuside A 1,3-rhamnosyltransferase, and UGT79A19, an osmanthuside B 1,4-rhamnosyltransferase, sequentially converted osmanthuside A into ligupurpuroside B. Orthologs of UGT79G7 were also discovered from other plants producing verbascoside. These rhamnosyltransferases expand the toolbox for the biosynthesis of natural products with various sugar chains.

Exploiting pglB Oligosaccharyltransferase-Positive and -Negative Campylobacter jejuni and a Multiprotease Digestion Strategy to Identify Novel Sites Modified by N-Linked Protein Glycosylation.

Campylobacter jejuni is a bacterial pathogen encoding a unique N-linked glycosylation (pgl) system that mediates attachment of a heptasaccharide to N-sequon-containing membrane proteins by the PglB oligosaccharyltransferase (OST). Many targets of PglB are known, yet only a fraction of sequons are experimentally confirmed, and site occupancy remains elusive. We exploited pglB-positive (wild-type; WT) and -negative (ΔpglB) proteomes to identify potential glycosites. The nonglycosylated forms of known glycopeptides were typically increased in protein normalized abundance in ΔpglB relative to WT and restored by pglB reintroduction (ΔpglB::pglB). Sequon-containing peptide abundances were thus consistent with significant site occupancy in the presence of the OST. Peptides with novel sequons were either unaltered (likely not glycosylated) or showed abundance consistent with known glycopeptides. Topology analysis revealed that unaltered sequons often displayed cytoplasmic localization, despite originating from membrane proteins. Novel glycosites were confirmed using parallel multiprotease digestion, LC-MS/MS, and FAIMS-MS to define the glycoproteomes of WT and ΔpglB::pglBC. jejuni. We identified 142 glycosites, of which 32 were novel, and 83% of sites predicted by proteomics were validated. There are now 166 experimentally verified C. jejuni glycosites and evidence for occupancy or nonoccupancy of 31 additional sites. This study serves as a model for the use of OST-negative cells and proteomics for highlighting novel glycosites and determining occupancy in a range of organisms.

Active site variants in STT3A cause a dominant type I congenital disorder of glycosylation with neuromusculoskeletal findings.

Congenital disorders of glycosylation (CDGs) form a group of rare diseases characterized by hypoglycosylation. We here report the identification of 16 individuals from nine families who have either inherited or de novo heterozygous missense variants in STT3A, leading to an autosomal-dominant CDG. STT3A encodes the catalytic subunit of the STT3A-containing oligosaccharyltransferase (OST) complex, essential for protein N-glycosylation. Affected individuals presented with variable skeletal anomalies, short stature, macrocephaly, and dysmorphic features; half had intellectual disability. Additional features included increased muscle tone and muscle cramps. Modeling of the variants in the 3D structure of the OST complex indicated that all variants are located in the catalytic site of STT3A, suggesting a direct mechanistic link to the transfer of oligosaccharides onto nascent glycoproteins. Indeed, expression of STT3A at mRNA and steady-state protein level in fibroblasts was normal, while glycosylation was abnormal. In S. cerevisiae, expression of STT3 containing variants homologous to those in affected individuals induced defective glycosylation of carboxypeptidase Y in a wild-type yeast strain and expression of the same mutants in the STT3 hypomorphic stt3-7 yeast strain worsened the already observed glycosylation defect. These data support a dominant pathomechanism underlying the glycosylation defect. Recessive mutations in STT3A have previously been described to lead to a CDG. We present here a dominant form of STT3A-CDG that, because of the presence of abnormal transferrin glycoforms, is unusual among dominant type I CDGs.

The structure of an archaeal oligosaccharyltransferase provides insight into the strict exclusion of proline from the N-glycosylation sequon.

Oligosaccharyltransferase (OST) catalyzes oligosaccharide transfer to the Asn residue in the N-glycosylation sequon, Asn-X-Ser/Thr, where Pro is strictly excluded at position X. Considering the unique structural properties of proline, this exclusion may not be surprising, but the structural basis for the rejection of Pro residues should be explained explicitly. Here we determined the crystal structure of an archaeal OST in a complex with a sequon-containing peptide and dolichol-phosphate to a 2.7 Å resolution. The sequon part in the peptide forms two inter-chain hydrogen bonds with a conserved amino acid motif, TIXE. We confirmed the essential role of the TIXE motif and the adjacent regions by extensive alanine-scanning of the external loop 5. A Ramachandran plot revealed that the ring structure of the Pro side chain is incompatible with the ϕ backbone dihedral angle around -150° in the rigid sequon-TIXE structure. The present structure clearly provides the structural basis for the exclusion of Pro residues from the N-glycosylation sequon.

Substrate specificities and reaction kinetics of the yeast oligosaccharyltransferase isoforms.

Oligosaccharyltransferase (OST) catalyzes the central step in N-linked protein glycosylation, the transfer of a preassembled oligosaccharide from its lipid carrier onto asparagine residues of secretory proteins. The prototypic hetero-octameric OST complex from the yeast Saccharomyces cerevisiae exists as two isoforms that contain either Ost3p or Ost6p, both noncatalytic subunits. These two OST complexes have different protein substrate specificities in vivo. However, their detailed biochemical mechanisms and the basis for their different specificities are not clear. The two OST complexes were purified from genetically engineered strains expressing only one isoform. The kinetic properties and substrate specificities were characterized using a quantitative in vitro glycosylation assay with short peptides and different synthetic lipid-linked oligosaccharide (LLO) substrates. We found that the peptide sequence close to the glycosylation sequon affected peptide affinity and turnover rate. The length of the lipid moiety affected LLO affinity, while the lipid double-bond stereochemistry had a greater influence on LLO turnover rates. The two OST complexes had similar affinities for both the peptide and LLO substrates but showed significantly different turnover rates. These data provide the basis for a functional analysis of the Ost3p and Ost6p subunits.

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.

Fructosyltransferase production by Aspergillus oryzae BM-DIA using solid-state fermentation and the properties of its nucleotide and protein sequences.

Fructosyltransferase (FTase) catalyzes the transfer of a fructosyl group to a sucrose molecule or a fructooligosaccharide (FOS) when a FOS with a longer chain is formed. Production of FTase by two Aspergillus species and its mixture was exploited using solid-state fermentation (SSF) and employing agave sap as substrate. The maximum FTase activity (1.59 U/mL) by Aspergillus oryzae was obtained after 24 h, using a temperature of 30 °C, with an inoculum of 2 × 107 spores/mL. The nucleotide sequence coding for the fructosyltransferase showed 1494 bp and encodes for a protein of 498 amino acids. The hypothetical molecular tertiary structure of Aspergillus oryzae BM-DIA FTase showed the presence of structural domains, such as a five-bladed beta-propeller domain characteristic of GH (glycoside hydrolase) and C terminal, which forms a beta-sandwich module. This study contributes to the knowledge of stability, compatibility, and genetic expression of Aspergillus oryzae BM-DIA under SSF bioprocess conditions for industrial production of fructosyltransferase.

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.

Lscß and lscγ, two novel levansucrases of Pseudomonas syringae pv. actinidiae biovar 3, the causal agent of bacterial canker of kiwifruit, show different enzymatic properties.

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, lscß 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, lscß 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 lscß 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.