Structural elements determining the transglycosylating activity of glycoside hydrolase family 57 glycogen branching enzymes.

Glycoside hydrolase family 57 glycogen branching enzymes (GH57GBE) catalyze the formation of an α-1,6 glycosidic bond between α-1,4 linked glucooliogosaccharides. As an atypical family, a limited number of GH57GBEs have been biochemically characterized so far. This study aimed at acquiring a better understanding of the GH57GBE family by a systematic sequence-based bioinformatics analysis of almost 2500 gene sequences and determining the branching activity of several native and mutant GH57GBEs. A correlation was found in a very low or even no branching activity with the absence of a flexible loop, a tyrosine at the loop tip, and two ß-strands.

Identification of Thermotoga maritima MSB8 GH57 α-amylase AmyC as a glycogen-branching enzyme with high hydrolytic activity.

AmyC, a glycoside hydrolase family 57 (GH57) enzyme of Thermotoga maritima MSB8, has previously been identified as an intracellular α-amylase playing a role in either maltodextrin utilization or storage polysaccharide metabolism. However, the α-amylase specificity of AmyC is questionable as extensive phylogenetic analysis of GH57 and tertiary structural comparison suggest that AmyC could actually be a glycogen-branching enzyme (GBE), a key enzyme in the biosynthesis of glycogen. This communication presents phylogenetic and biochemical evidence that AmyC is a GBE with a relatively high hydrolytic (α-amylase) activity (up to 30% of the total activity), creating a branched α-glucan with 8.5% α-1,6-glycosidic bonds. The high hydrolytic activity is explained by the fact that AmyC has a considerably shorter catalytic loop (residues 213-220) not reaching the acceptor side. Secondly, in AmyC, the tryptophan residue (W 246) near the active site has its side chain buried in the protein interior, while the side chain is at the surface in Tk1436 and Tt1467 GBEs. The putative GBEs from three other Thermotogaceae, with very high sequence similarities to AmyC, were found to have the same structural elements as AmyC, suggesting that GH57 GBEs with relatively high hydrolytic activity may be widespread in nature.

Biochemical Characterization of the Lactobacillus reuteri Glycoside Hydrolase Family 70 GTFB Type of 4,6-α-Glucanotransferase Enzymes That Synthesize Soluble Dietary Starch Fibers.

4,6-α-Glucanotransferase (4,6-α-GTase) enzymes, such as GTFB and GTFW of Lactobacillus reuteri strains, constitute a new reaction specificity in glycoside hydrolase family 70 (GH70) and are novel enzymes that convert starch or starch hydrolysates into isomalto/maltopolysaccharides (IMMPs). These IMMPs still have linear chains with some α1→4 linkages but mostly (relatively long) linear chains with α1→6 linkages and are soluble dietary starch fibers. 4,6-α-GTase enzymes and their products have significant potential for industrial applications. Here we report that an N-terminal truncation (amino acids 1 to 733) strongly enhances the soluble expression level of fully active GTFB-ΔN (approximately 75-fold compared to full-length wild type GTFB) in Escherichia coli. In addition, quantitative assays based on amylose V as the substrate are described; these assays allow accurate determination of both hydrolysis (minor) activity (glucose release, reducing power) and total activity (iodine staining) and calculation of the transferase (major) activity of these 4,6-α-GTase enzymes. The data show that GTFB-ΔN is clearly less hydrolytic than GTFW, which is also supported by nuclear magnetic resonance (NMR) analysis of their final products. From these assays, the biochemical properties of GTFB-ΔN were characterized in detail, including determination of kinetic parameters and acceptor substrate specificity. The GTFB enzyme displayed high conversion yields at relatively high substrate concentrations, a promising feature for industrial application.

A GH57 4-α-glucanotransferase of hyperthermophilic origin with potential for alkyl glycoside production.

4-α-Glucanotransferase (GTase) enzymes (EC 2.4.1.25) modulate the size of α-glucans by cleaving and reforming α-1,4 glycosidic bonds in α-glucans, an essential process in starch and glycogen metabolism in plants and microorganisms. The glycoside hydrolase family 57 enzyme (GTase57) studied in the current work catalyzes both disproportionation and cyclization reactions. Amylose was converted into cyclic amylose (with a minimum size of 17 glucose monomers) as well as to a spectrum of maltodextrins, but in contrast to glycoside hydrolase family 13 cyclodextrin glucanotransferases (CGTases), no production of cyclodextrins (C6-C8) was observed. GTase57 also effectively produced alkyl-glycosides with long α-glucan chains from dodecyl-ß-D-maltoside and starch, demonstrating the potential of the enzyme to produce novel variants of surfactants. Importantly, the GTase57 has excellent thermostability with a maximal activity at 95 °C and an activity half-life of 150 min at 90 °C which is highly advantageous in this manufacturing process suggesting that enzymes from this relatively uncharacterized family, GH57, can be powerful biocatalysts for the production of large head group glucosides from soluble starch.

Mechanical heterogeneity in a soft biomaterial niche controls BMP2 signaling.

The extracellular matrix is known to impact cell function during regeneration by modulating growth factor signaling. However, how the mechanical properties and structure of biomaterials can be used to optimize the cellular response to growth factors is widely neglected. Here, we engineered a macroporous biomaterial to study cellular signaling in environments that mimic the mechanical stiffness but also the mechanical heterogeneity of native extracellular matrix. We found that the mechanical interaction of cells with the heterogeneous and non-linear deformation properties of soft matrices (E < 5 kPa) enhances BMP-2 growth factor signaling with high relevance for tissue regeneration. In contrast, this effect is absent in homogeneous hydrogels that are often used to study cell responses to mechanical cues. Live cell imaging and in silico finite element modeling further revealed that a subpopulation of highly active, fast migrating cells is responsible for most of the material deformation, while a second, less active population experiences this deformation as an extrinsic mechanical stimulation. At an overall low cell density, the active cell population dominates the process, suggesting that it plays a particularly important role in early tissue healing scenarios where cells invade tissue defects or implanted biomaterials. Taken together, our findings demonstrate that the mechanical heterogeneity of the natural extracellular matrix environment plays an important role in triggering regeneration by endogenously acting growth factors. This suggests the inclusion of such mechanical complexity as a design parameter in future biomaterials, in addition to established parameters such as mechanical stiffness and stress relaxation.

Gluco-oligomers initially formed by the reuteransucrase enzyme of Lactobacillus reuteri 121 incubated with sucrose and malto-oligosaccharides.

The probiotic bacterium Lactobacillus reuteri 121 produces a complex, branched (1 → 4, 1 → 6)-α-D-glucan as extracellular polysaccharide (reuteran) from sucrose (Suc), using a single glucansucrase/glucosyltransferase (GTFA) enzyme (reuteransucrase). To gain insight into the reaction/product specificity of the GTFA enzyme and the mechanism of reuteran formation, incubations with Suc and/or a series of malto-oligosaccharides (MOSs) (degree of polymerization (DP2-DP6)) were followed in time. The structures of the initially formed products, isolated via high-performance anion-exchange chromatography, were analyzed by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry and 1D/2D (1)H/(13)C NMR spectroscopy. Incubations with Suc only, acting as both donor and acceptor, resulted in elongation of Suc with glucose (Glc) units via alternating (α1 → 4) and (α1 → 6) linkages, yielding linear gluco-oligosaccharides up to at least DP ~ 12. Simultaneously with the ensemble of oligosaccharides, polymeric material was formed early on, suggesting that alternan fragments longer than DP ~ 12 have higher affinity with the GTFA enzyme and are quickly extended, yielding high-molecular-mass branched reuteran (4 × 10(7) Da). MOSs (DP2-DP6) in the absence of Suc turned out to be poor substrates. Incubations of GTFA with Suc plus MOSs as substrates resulted in preferential elongation of MOSs (acceptors) with Glc units from Suc (donor). This apparently reflects the higher affinity of GTFA for MOSs compared with Suc. In accordance with the GTFA specificity, most prominent products were oligosaccharides with an (α1 → 4)/(α1 → 6) alternating structure.

4,6-α-Glucanotransferase activity occurs more widespread in Lactobacillus strains and constitutes a separate GH70 subfamily.

Family 70 glycoside hydrolase glucansucrase enzymes exclusively occur in lactic acid bacteria and synthesize a wide range of α-D-glucan (abbreviated as α-glucan) oligo- and polysaccharides. Of the 47 characterized GH70 enzymes, 46 use sucrose as glucose donor. A single GH70 enzyme was recently found to be inactive with sucrose and to utilize maltooligosaccharides [(1→4)-α-D-glucooligosaccharides] as glucose donor substrates for α-glucan synthesis, acting as a 4,6-α-glucanotransferase (4,6-αGT) enzyme. Here, we report the characterization of two further GH70 4,6-αGT enzymes, i.e., from Lactobacillus reuteri strains DSM 20016 and ML1, which use maltooligosaccharides as glucose donor. Both enzymes cleave α1→4 glycosidic linkages and add the released glucose moieties one by one to the non-reducing end of growing linear α-glucan chains via α1→6 glycosidic linkages (α1→4 to α1→6 transfer activity). In this way, they convert pure maltooligosaccharide substrates into linear α-glucan product mixtures with about 50% α1→6 glycosidic bonds (isomalto/maltooligosaccharides). These new α-glucan products may provide an exciting type of carbohydrate for the food industry. The results show that 4,6-αGTs occur more widespread in family GH70 and can be considered as a GH70 subfamily. Sequence analysis allowed identification of amino acid residues in acceptor substrate binding subsites +1 and +2, differing between GH70 GTF and 4,6-αGT enzymes.

Thermus thermophilus glycoside hydrolase family 57 branching enzyme: crystal structure, mechanism of action, and products formed.

Branching enzyme (EC 2.4.1.18; glycogen branching enzyme; GBE) catalyzes the formation of α1,6-branching points in glycogen. Until recently it was believed that all GBEs belong to glycoside hydrolase family 13 (GH13). Here we describe the cloning and expression of the Thermus thermophilus family GH57-type GBE and report its biochemical properties and crystal structure at 1.35-Å resolution. The enzyme has a central (ß/α)(7)-fold catalytic domain A with an inserted domain B between ß2 and α5 and an α-helix-rich C-terminal domain, which is shown to be essential for substrate binding and catalysis. A maltotriose was modeled in the active site of the enzyme which suggests that there is insufficient space for simultaneously binding of donor and acceptor substrates, and that the donor substrate must be cleaved before acceptor substrate can bind. The biochemical assessment showed that the GH57 GBE possesses about 4% hydrolytic activity with amylose and in vitro forms a glucan product with a novel fine structure, demonstrating that the GH57 GBE is clearly different from the GH13 GBEs characterized to date.

Structural characterization of linear isomalto-/malto-oligomer products synthesized by the novel GTFB 4,6-α-glucanotransferase enzyme from Lactobacillus reuteri 121.

Recently, a novel glucansucrase (GS)-like gene (gtfB) was isolated from the probiotic bacterium Lactobacillus reuteri 121 and expressed in Escherichia coli. The purified recombinant GTFB enzyme was characterized and turned out to be inactive with sucrose, the natural GS substrate. Instead, GTFB acted on malto-oligosaccharides (MOSs), thereby yielding elongated gluco-oligomers/polymers containing besides (α1 â†’ 4) also (α1 â†’ 6) glycosidic linkages, and it was classified as a 4,6-α-glucanotransferase. To gain more insight into its reaction specificity, incubations of the GTFB enzyme with a series of MOSs and their corresponding alditols [degree of polymerization, DP2(-ol)-DP7(-ol)] were carried out, and (purified) products were structurally analyzed with matrix-assisted laser desorption ionization time-of-flight mass spectrometry and one-/two-dimensional (1)H and (13)C nuclear magnetic resonance spectroscopy. With each of the tested malto-oligomers, the GTFB enzyme yielded series of novel linear isomalto-/malto-oligomers, in the case of DP7 up to DP >35.

4,6-α-glucanotransferase, a novel enzyme that structurally and functionally provides an evolutionary link between glycoside hydrolase enzyme families 13 and 70.

Lactobacillus reuteri 121 uses the glucosyltransferase A (GTFA) enzyme to convert sucrose into large amounts of the α-D-glucan reuteran, an exopolysaccharide. Upstream of gtfA lies another putative glucansucrase gene, designated gtfB. Previously, we have shown that the purified recombinant GTFB protein/enzyme is inactive with sucrose. Various homologs of gtfB are present in other Lactobacillus strains, including the L. reuteri type strain, DSM 20016, the genome sequence of which is available. Here we report that GTFB is a novel α-glucanotransferase enzyme with disproportionating (cleaving α1→4 and synthesizing α1→6 and α1→4 glycosidic linkages) and α1→6 polymerizing types of activity on maltotetraose and larger maltooligosaccharide substrates (in short, it is a 4,6-α-glucanotransferase). Characterization of the types of compounds synthesized from maltoheptaose by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), methylation analysis, and 1-dimensional ¹H nuclear magnetic resonance (NMR) spectroscopy revealed that only linear products were made and that with increasing degrees of polymerization (DP), more α1→6 glycosidic linkages were introduced into the final products, ranging from 18% in the incubation mixture to 33% in an enriched fraction. In view of its primary structure, GTFB clearly is a member of the glycoside hydrolase 70 (GH70) family, comprising enzymes with a permuted (ß/α)8 barrel that use sucrose to synthesize α-D-glucan polymers. The GTFB enzyme reaction and product specificities, however, are novel for the GH70 family, resembling those of the GH13 α-amylase type of enzymes in using maltooligosaccharides as substrates but differing in introducing a series of α1→6 glycosidic linkages into linear oligosaccharide products. We conclude that GTFB represents a novel evolutionary intermediate between the GH13 and GH70 enzyme families, and we speculate about its origin.

GtfC Enzyme of Geobacillus sp. 12AMOR1 Represents a Novel Thermostable Type of GH70 4,6-α-Glucanotransferase That Synthesizes a Linear Alternating (α1 → 6)/(α1 → 4) α-Glucan and Delays Bread Staling.

Starch-acting α-glucanotransferase enzymes are of great interest for applications in the food industry. In previous work, we have characterized various 4,6- and 4,3-α-glucanotransferases of the glycosyl hydrolase (GH) family 70 (subfamily GtfB), synthesizing linear or branched α-glucans. Thus far, GtfB enzymes have only been identified in mesophilic Lactobacilli. Database searches showed that related GtfC enzymes occur in Gram-positive bacteria of the genera Exiguobacterium, Bacillus, and Geobacillus, adapted to growth at more extreme temperatures. Here, we report characteristics of the Geobacillus sp. 12AMOR1 GtfC enzyme, with an optimal reaction temperature of 60 °C and a melting temperature of 68 °C, allowing starch conversions at relatively high temperatures. This thermostable 4,6-α-glucanotransferase has a novel product specificity, cleaving off predominantly maltose units from amylose, attaching them with an (α1 → 6)-linkage to acceptor substrates. In fact, this GtfC represents a novel maltogenic α-amylase. Detailed structural characterization of its starch-derived α-glucan products revealed that it yielded a unique polymer with alternating (α1 → 6)/(α1 → 4)-linked glucose units but without branches. Notably, this Geobacillus sp. 12AMOR1 GtfC enzyme showed clear antistaling effects in bread bakery products.

Inulin and levan synthesis by probiotic Lactobacillus gasseri strains: characterization of three novel fructansucrase enzymes and their fructan products.

Fructansucrase enzymes polymerize the fructose moiety of sucrose into levan or inulin fructans, with beta(2-6) and beta(2-1) linkages, respectively. Here, we report an evaluation of fructan synthesis in three Lactobacillus gasseri strains, identification of the fructansucrase-encoding genes and characterization of the recombinant proteins and fructan (oligosaccharide) products. High-performance anion-exchange chromatography and nuclear magnetic resonance analysis of the fructo-oligosaccharides (FOS) and polymers produced by the L. gasseri strains and the recombinant enzymes revealed that, in situ, L. gasseri strains DSM 20604 and 20077 synthesize inulin (and oligosaccharides) and levan products, respectively. L. gasseri DSM 20604 is only the second Lactobacillus strain shown to produce inulin polymer and FOS in situ, and is unique in its distribution of FOS synthesized, ranging from DP2 to DP13. The probiotic bacterium L. gasseri DSM 20243 did not produce any fructan, although we identified a fructansucrase-encoding gene in its genome sequence. Further studies showed that this L. gasseri DSM 20243 gene was prematurely terminated by a stop codon. Exchanging the stop codon for a glutamine codon resulted in a recombinant enzyme producing inulin and FOS. The three recombinant fructansucrase enzymes characterized from three different L. gasseri strains have very similar primary protein structures, yet synthesize different fructan products. An interesting feature of the L. gasseri strains is that they were unable to ferment raffinose, whereas their respective recombinant enzymes converted raffinose into fructan and FOS.

Engineering of cyclodextrin glucanotransferases and the impact for biotechnological applications.

Cyclodextrin glucanotransferases (CGTases) are industrially important enzymes that produce cyclic alpha-(1,4)-linked oligosaccharides (cyclodextrins) from starch. Cyclodextrin glucanotransferases are also applied as catalysts in the synthesis of glycosylated molecules and can act as antistaling agents in the baking industry. To improve the performance of CGTases in these various applications, protein engineers are screening for CGTase variants with higher product yields, improved CD size specificity, etc. In this review, we focus on the strategies employed in obtaining CGTases with new or enhanced enzymatic capabilities by searching for new enzymes and improving existing enzymatic activities via protein engineering.

Digestion kinetics of low, intermediate and highly branched maltodextrins produced from gelatinized starches with various microbial glycogen branching enzymes.

Twenty-four branched maltodextrins were synthesized from eight starches using three thermostable microbial glycogen branching enzymes. The maltodextrins have a degree of branching (DB) ranging from 5 % to 13 %. This range of products allows us to explore the effect of DB on the digestibility, which was quantified under conditions that mimic the digestion process in the small intestine. The rate and extent of digestibility were analyzed using the logarithm of the slope method, revealing that the branched maltodextrins consist of a rapidly and slowly digestible fraction. The amount of slowly digestible maltodextrin increases with an increasing DB. Surprisingly, above 10 % branching the fraction of slowly digestible maltodextrin remains constant. Nevertheless, the rate of digestion of the slowly digestible fraction was found to decline with increasing DB and shorter average internal chain length. These observations increase the understanding of the structural factors important for the digestion rate of branched maltodextrins.

Directed evolution of enzymes: Library screening strategies.

Directed evolution has become the preferred engineering approach to generate tailor-made enzymes. The method follows the design guidelines of nature: Darwinian selection of genetic variants. This review discusses the different stages of directed evolution experiments with the focus on developments in screening and selection procedures.

The evolution of cyclodextrin glucanotransferase product specificity.

Cyclodextrin glucanotransferases (CGTases) have attracted major interest from industry due to their unique capacity of forming large quantities of cyclic alpha-(1,4)-linked oligosaccharides (cyclodextrins) from starch. CGTases produce a mixture of cyclodextrins from starch consisting of 6 (alpha), 7 (beta) and 8 (gamma) glucose units. In an effort to identify the structural factors contributing to the evolutionary diversification of product specificity amongst this group of enzymes, we selected nine CGTases from both mesophilic, thermophilic and hyperthermophilic organisms for comparative product analysis. These enzymes displayed considerable variation regarding thermostability, initial rates, percentage of substrate conversion and ratio of alpha-, beta- and gamma-cyclodextrins formed from starch. Sequence comparison of these CGTases revealed that specific incorporation and/or substitution of amino acids at the substrate binding sites, during the evolutionary progression of these enzymes, resulted in diversification of cyclodextrin product specificity.

Elimination of competing hydrolysis and coupling side reactions of a cyclodextrin glucanotransferase by directed evolution.

Thermoanaerobacterium thermosulfurigenes cyclodextrin glucanotransferase primarily catalyses the formation of cyclic alpha-(1,4)-linked oligosaccharides (cyclodextrins) from starch. This enzyme also possesses unusually high hydrolytic activity as a side reaction, thought to be due to partial retention of ancestral enzyme function. This side reaction is undesirable, since it produces short saccharides that are responsible for the breakdown of the cyclodextrins formed, thus limiting the yield of cyclodextrins produced. To reduce the competing hydrolysis reaction, while maintaining the cyclization activity, we applied directed evolution, introducing random mutations throughout the cgt gene by error-prone PCR. Mutations in two residues, Ser-77 and Trp-239, on the outer region of the active site, lowered the hydrolytic activity up to 15-fold with retention of cyclization activity. In contrast, mutations within the active site could not lower hydrolytic rates, indicating an evolutionary optimized role for cyclodextrin formation by residues within this region. The crystal structure of the most effective mutant, S77P, showed no alterations to the peptide backbone. However, subtle conformational changes to the side chains of active-site residues had occurred, which may explain the increased cyclization/hydrolysis ratio. This indicates that secondary effects of mutations located on the outer regions of the catalytic site are required to lower the rates of competing side reactions, while maintaining the primary catalytic function. Subsequent functional analysis of various glucanotransferases from the superfamily of glycoside hydrolases also suggests a gradual evolutionary progression of these enzymes from a common 'intermediate-like' ancestor towards specific transglycosylation activity.

Characterization of the GH13 and GH57 glycogen branching enzymes from Petrotoga mobilis SJ95 and potential role in glycogen biosynthesis.

Glycogen is a highly branched α-glucan polymer widely used as energy and carbon reserve by many microorganisms. The branches are introduced by glycogen branching enzymes (EC 2.4.1.18), that are classified into glycoside hydrolase families 13 (GH13) and 57 (GH57). Most microorganisms have typically only a single glycogen branching enzyme (gbe) gene. Only a few microorganisms carry both GH13 and GH57 gbe genes, such as Petrotoga mobilis and Mycobacterium tuberculosis. Here we report the basic characteristics of the GH13 and GH57 GBE of P. mobilis, both heterologously expressed in E. coli. The GH13 GBE has a considerably higher branching activity towards the linear α-glucan amylose, and produces a highly branched α-glucan with a high molecular weight which is very similar to glycogen. The GH57 GBE, on the contrary, makes a much smaller branched α-glucan. While the GH13 GBE acts as a classical glycogen branching enzyme involved in glycogen synthesis, the role of GH57 GBE remains unclear.

Synthesis of highly branched α-glucans with different structures using GH13 and GH57 glycogen branching enzymes.

Glycogen branching enzymes (GBEs) convert starch into branched α-glucan polymers. To explore if the amylose content of substrates effects the structure of the branched α-glucans, mixtures of amylose and amylopectin were converted by four thermophilic GBEs. The degree of branching and molecular weight of the products increased with an increasing percentage of amylose with the GH57 GBEs of Thermus thermophilus and Thermococcus kodakarensis, and the GH13 GBEs of Rhodothermus marinus and Petrotoga mobilis. The only exception is that the degree of branching of the Petrotoga mobilis GBE products is not influenced by the amylose content. A second difference is the relatively high hydrolytic activity of two GH57 GBEs, while the two GH13 GBEs have almost no hydrolytic activity. Moreover, the two GH13 GBEs synthesize branched α-glucans with a narrow molecular weight distribution, while the two GH57 GBEs products consist of two or three molecular weight fractions.

Higher Chain Length Distribution in Debranched Type-3 Resistant Starches (RS3) Increases TLR Signaling and Supports Dendritic Cell Cytokine Production.

SCOPE: Resistant starches (RSs) are classically considered to elicit health benefits through fermentation. However, it is recently shown that RSs can also support health by direct immune interactions. Therefore, it has been hypothesized that the structural traits of RSs might impact the health benefits associated with their consumption. METHODS AND RESULTS: Effects of crystallinity, molecular weight, and chain length distribution of RSs are determined on immune Toll-like receptors (TLRs), dendritic cells (DCs), and T-cell cytokines production. To this end, four type-3 RSs (RS3) are compared, namely Paselli WFR, JD150, debranched Etenia, and Amylose fraction V, which are extracted from potatoes and enzymatically modified. Dextrose equivalent seems to be the most important feature influencing immune signaling via activation of TLRs. TLR2 and TLR4 are most strongly stimulated. Especially Paselli WFR is a potent activator of multiple receptors. Moreover, the presence of amylose, even to residual levels, enhances DC and T-cell cytokine responses. Paselli WFR and Amylose fraction V influence T-cell polarization. CONCLUSIONS: It has been shown here that chain length and particularly dextrose equivalent are critical features for immune activation. This knowledge might lead to tailoring and design of immune-active RS formulations.