text:
|Home | About | Journals | Submit | Contact Us | Français|
Lactic acid bacteria (LAB) employ sucrase-type enzymes to convert sucrose into homopolysaccharides consisting of either glucosyl units (glucans) or fructosyl units (fructans). The enzymes involved are labeled glucansucrases (GS) and fructansucrases (FS), respectively. The available molecular, biochemical, and structural information on sucrase genes and enzymes from various LAB and their fructan and α-glucan products is reviewed. The GSand FS enzymes are both glycoside hydrolase enzymes that act on the same substrate (sucrose) and catalyze (retaining) transglycosylation reactions that result in polysaccharide formation, but they possess completely different protein structures. GS enzymes (family GH70) are large multidomain proteins that occur exclusively in LAB. Their catalytic domain displays clear secondary-structure similarity with α-amylase enzymes (family GH13), with a predicted permuted (β/α)8 barrel structure for which detailed structural and mechanistic information is available. Emphasis now is on identification of residues and regions important for GS enzyme activity and product specificity (synthesis of α-glucans differing in glycosidic linkage type, degree and type of branching, glucan molecular mass, and solubility). FS enzymes (family GH68) occur in both gram-negative and gram-positive bacteria and synthesize β-fructan polymers with either β-(2→6) (inulin) or β-(2→1) (levan) glycosidic bonds. Recently, the first high-resolution three-dimensional structures have become available for FS (levansucrase) proteins, revealing a rare five-bladed β-propeller structure with a deep, negatively charged central pocket. Although these structures have provided detailed mechanistic insights, the structural features in FS enzymes dictating the synthesis of either β-(2→6) or β-(2→1) linkages, degree and type of branching, and fructan molecular mass remain to be identified.
Extracellular polysaccharides (exopolysaccharides) (EPS) are commonly found in bacteria and microalgae and less frequently in yeasts and fungi (39, 142, 160, 168, 217). Several lactic acid bacteria (LAB), including species of Lactobacillus, are known to produce EPS. Depending on their composition and mechanism of biosynthesis, EPS are divided in two classes: heteropolysaccharides and homopolysaccharides. Heteropolysaccharides consist of multiple sugar types and are synthesized by the combined action of multiple different types of glycosyltransferase enzymes (39). In contrast, homopolysaccharides are synthesized from the sole substrate sucrose by the action of one sucrase enzyme. Sucrase-type enzymes synthesize polysaccharides consisting of either glucose sugar residues (glucans) or fructose residues (fructans).
Homopolysaccharide synthesis in LAB has mainly been studied in oral streptococci and Leuconostoc spp. (124, 126, 149, 153). Because of their clearly established role in formation of dental caries (7) Streptococcus mutans and Streptococcus sanguis strains have been subject to a number of studies (18, 100, 109, 157, 159). Interestingly, there is increasing evidence that a number of Lactobacillus species are also associated with advanced stages of dental caries (26). Both glucans and fructans (see below) formed by oral streptococci (and lactobacilli) apparently have major influences on the formation of dental plaque. They are involved in adherence of bacteria to each other and to the tooth surface, modulating diffusion of substances through plaque, and occasionally serving as extracellular energy reserves (29, 41, 141, 162). Alternatively, these polymers may protect microbial cells against desiccation, phagocytosis and phage attack, antibiotics or toxic compounds, predation by protozoans, and osmotic stress (20).
In general, glucans and/or fructans can be used as viscosifying, stabilizing, emulsifying, sweetening, gelling, or water-binding agents, in the food as well as in the nonfood industries (40, 51, 66, 190, 217, 218). Certain oligosaccharides (e.g., fructo-oligosaccharides, isomaltooligosaccharides, and lactulose) and polysaccharides (e.g., fructans) are used as prebiotic food additives (14, 15, 50, 84, 151, 164). Additionally, oligosaccharides containing α-(1→2) glucosidic bonds are in some cases used as feed additives (127).
Over the years a large number of glucansucrase and fructansucrase genes and enzymes have been identified by cloning, reverse genetics, and various enzyme activity assays. Enzymes synthesizing α-glucan polymers, glucansucrases (GS), are limited to LAB while enzymes synthesizing fructans, fructansucrases (FS), are present in gram-positive and gram-negative bacteria (33; http://afmb.cnrs-mrs.fr/CAZY/). Fructan biosynthesis also is known to occur in plants and fungi and involves a set of enzymes which are evolutionarily related to sucrose-hydrolyzing enzymes (invertases). They are clearly different from their bacterial counterparts (75, 106, 205, 216). Although the GS and FS enzymes perform very similar reactions on the same substrates (see below), they do not share a high amino acid sequence similarity, and differ strongly in protein structures.
The properties of GS of Streptococcus and Leuconostoc spp. (124, 126, 149, 154) and FS of LAB (126) have been reviewed previously. In view of the many recent developments in the understanding of the structure-function relationships of these sucrase enzymes, including GS and FS enzymes from lactobacilli, an overview of current knowledge of the sucrase field of research is presented here, with a focus on sucrase enzymes from LAB.
According to the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology, the following GS enzymes are classified based on the reaction catalyzed and the product specificity: dextransucrase (sucrose:1,6-α-d-glucan-6-α-d-glucosyltransferase, EC 184.108.40.206) and alternansucrase [sucrose:1,6(1,3)-α-d-glucan-6(3)-α-d-glucosyltransferase, EC 220.127.116.11]. At present, the mutan-(sucrose:1,3-α-d-glucan-3-α-d-glucosyltransferase) and reuteransucrase [sucrose:1,4(6)-α-d-glucan-4(6)-α-d-glucosyltransferase] enzymes mentioned are classified together with dextransucrase enzymes in EC 18.104.22.168. Also, two FS enzymes are distinguished now, based on the different products synthesized: inulosucrase (sucrose:2,1-β-d-fructan-1-β-d-fructosyltransferase, EC 22.214.171.124) and levansucrase (sucrose:2,6-β-d-fructan-6-β-d-fructosyltransferase, EC 126.96.36.199).
As described above, glucan- and fructan-synthesizing enzymes have been referred to as glucansucrase/glucosyltransferase and fructansucrase/fructosyltransferase, respectively (33; http://afmb.cnrs-mrs.fr/CAZY/). Since glucosyltransferase and fructosyltransferase refer to enzyme activities that are widely found in nature for enzymes that catalyze the transfer of, for instance, glucosyl and fructosyl sugar units, they are not descriptive for the substrate and product specificity of sucrase-type enzymes. Therefore, in this review we only use glucansucrases and fructansucrases for enzymes synthesizing homopolysaccharides from sucrose.
In another classification system, glycoside hydrolase (GH) enzymes have been divided into 100 different families based on their amino acid sequences (33; http://afmb.cnrs-mrs.fr/CAZY/). In view of their (different) sequence similarities, GS and FS have been included in the families GH70 and GH68, respectively. Evolutionarily, structurally, and mechanistically related families are further grouped into clans. Enzymes from the families GH32 (consisting mainly of plant and fungal fructosyltransferases) and GH68 comprise clan GH-J. The members of clan GH-J possess a five-bladed β-propeller structure (33; http://afmb.cnrs-mrs.fr/CAZY/), with three identical catalytic (Asp, Glu, and Asp) residues, and use a retaining reaction mechanism. Enzymes from families GH13 (mainly starch modifying enzymes) and GH70 and GH77 (4-α-glucanotransferases) constitute clan GH-H (also known as α-amylase superfamily) containing an α-amylase-type catalytic (β/α)8-barrel, a catalytic machinery with the catalytic Asp, Glu, and Asp residues at strands β4, β5, and β7, respectively, and a retaining reaction mechanism (see below).
The nomenclature for enzymes that synthesize polymers containing predominantly one linkage type is relatively straightforward, for instance, dextransucrase and levansucrase. The nomenclature for an enzyme synthesizing comparable numbers of different glycosidic linkages is a dilemma, since it could be assigned to multiple classes. An example is the GtfL enzyme from Streptococcus salivarius ATCC 25975 (Table (Table1).1). Similar problems might arise for FS, with several enzymes already synthesizing both β-(2→6) and β-(2→1) glycosidic bonds (Table2). This nomenclature problem is the more pressing since recent GS enzyme engineering work has revealed that a limited number of amino acid substitutions may cause large changes in glucosidic linkages in products synthesized by (mutant) sucrase enzymes (96) (see below). In view of their similar structures and reaction mechanisms, we propose that sucrase enzymes synthesizing multiple glycosidic linkages in their products in future be labeled glucansucrases (GS) and fructansucrases (FS), respectively.
Microbial glucansucrase enzymes (GH70) exclusively synthesize α-glucan polymers. Synthesis of these polymers has been observed in four different genera of LAB: Streptococcus, Leuconostoc, Weissella, and Lactobacillus (95, 98, 124, 176, 197, 198, 209).
GS are large, extracellular proteins with average molecular masses of 160,000 Da. A large number of GS genes have been identified; for an overview consult the CAZY website (http://afmb.cnrs-mrs.fr/CAZY/). With the release of newly sequenced LAB genomes the list of known or putative LAB GS genes requires updates. An example is a putative GS gene similar to gtfD from S. mutans GS-5 present in the genome sequence of Oenococcus oeni (http://genome.jgi-psf.org/mic_home.html). It should be emphasized that GS genes and enzymes have not been reported outside LAB. The reason for this limited distribution of GS genes and enzymes in LAB is unknown.
GS enzymes synthesize various glucans differing in the type of glucosidic linkages, degree and type of branching, length of the glucan chains, molecular mass, and conformation of the polymers. All these properties strongly contribute to specific polysaccharide properties such as solubility, rheology, and other physical characteristics (124). Many factors, including growth medium, temperature, incubation time, sucrose concentration used, and the presence of polysaccharide-degrading enzymes influence the molecular mass, structure, and physical characteristics of the polymers synthesized by a specific organism (85, 124).
Nevertheless, the information for glucosidic bond specificity (and other characteristics) must be encoded somewhere in the enzyme amino acid sequence and protein structure. Cracking the code for these product specificities is one of the clear challenges in GS enzyme research (see below).
GS enzymes cleave the glycosidic bond of their substrate sucrose and couple a glucose unit to a growing glucan (polyglucose) chain (transglucosylation), water (hydrolysis), or other acceptor substrate (acceptor reaction). The energy released by cleavage of the energy-rich glycosidic bond in sucrose is used for synthesis of new glucosidic bonds. Dextran is synthesized according to the following reaction:
There are many examples of GS enzymes that synthesize linear glucans with α-(1→6) glucosidic linkages (dextran). GS enzymes introducing other glucosidic linkages are discussed below. There are also many examples of GS enzymes that catalyze the formation of two different glucosidic linkages. This results either in a (highly) branched glucan product, based on a branching type of reaction, or in a linear glucan with two different (alternating) glucosidic bonds. Unfortunately, detailed structural information for both the GS proteins and their glucan products is still lacking.
Depending on the main glucosidic linkages present in their glucan, four different types of α-glucans synthesized by LAB are recognized: dextran, mutan, alternan, and reuteran.
Pasteur (143) discovered the microbial origin of the jellification of cane sugar syrups. The product causing the jellification was named dextran due to its positive rotatory power. The corresponding extracellular enzyme was named dextransucrase (73). A common feature of all dextrans is the abundance of α-(1→6) linkages with some branching points at position 2, 3, or 4. Dextrans are produced by, for instance, Leuconostoc mesenteroides strains (20).
Guggenheim (63) showed that water-insoluble glucan from S. mutans OMZ176 contained a high proportion (up to 90%) of α-(1→3) glucosidic linkages. He proposed the name mutan for this polymer. The corresponding GS enzyme became named accordingly as mutansucrase. Mutan polymers are mainly produced by various streptococci (65).
Côté and Robyt isolated an α-glucan polymer from L. mesenteroides NRRL B-1355 composed of alternating α-(1→6) and α-(1→3) glucosidic linkages. To distinguish between the dextran [95% α-(1→6)] also synthesized by this strain, they named this polymer alternan and the corresponding GS enzyme alternansucrase (32).
Recently, a new type of glucan was identified from Lactobacillus reuteri 121 containing mainly α-(1→4) glucosidic linkages (208). The glucan product was named reuteran and the corresponding GS enzyme reuteransucrase (99). Reuteran, synthesized by L. reuteri 121, is a glucan with α-(1→4), α-(1→6) glucosidic bonds and α-(1→4,6) branching points (97).
Finally, an α-glucan containing, besides large numbers of α-(1→6) linkages, a substantial number of α-(1→2) linkages is produced by two different L. mesenteroides strains: NRRL B-1299 and a mutant strain (R510) of NRRL B-1355 (17, 184).
A few LAB strains carry multiple GS genes (gtf) in their genome sequence (Table (Table1).1). In order to elucidate which enzyme activities are encoded, these genes were cloned and heterologously expressed. The distribution of glucosidic linkages has been elucidated for the glucans synthesized from sucrose by heterologously produced (mostly using Escherichia coli as the host) GS enzymes from (i) seven Streptococcus strains (13 GS enzymes, synthesizing either dextran or mutan polymers) (67, 90, 124), (ii) four Leuconostoc strains (seven GS enzymes, synthesizing mainly dextran polymers, but also one alternan, and a GS synthesizing large numbers of α-(1→2,6) branch points) (4, 17, 55, 124, 135), and (iii) seven Lactobacillus strains (seven GS enzymes, synthesizing mainly dextrans, but also reuteran, and a highly branched mutan) (94, 95, 97) (Table (Table11).
Koepsell et al. (89) observed that in the presence of sucrose and saccharide acceptor substrates such as maltose, isomaltose, and O-α-methylglucoside, GS enzymes shift from glucan synthesis towards the synthesis of oligosaccharides (the acceptor reaction). Most acceptor reaction studies have been performed using saccharides (5, 42, 43, 53, 94, 99) or saccharide derivatives as substrates (31, 36, 150). However, aromatic compounds (e.g., catechine) and salicyl alcohol have also been shown to act as acceptor substrates (114, 221). GS enzymes cannot use sucrose itself as an acceptor substrate.
Based on the deduced amino acid sequences, GS enzymes are composed of four distinct structural domains from the N to the C terminus (Fig. (Fig.1):1): (i) a signal peptide, (ii) an N-terminal stretch of highly variable amino acids, (iii) a highly conserved catalytic and/or sucrose binding domain of about 1,000 amino acids, and (iv) a C-terminal domain that is composed of a series of tandem repeats which is thought to be involved in glucan binding (61, 161). Characterization of several heterologously produced and purified GS enzymes has clearly shown that they are able to introduce multiple glucosidic bonds in their products, (largely) based on the single catalytic domain present (Table (Table1).1). Where studied, this was corroborated by the the composition of the polymer synthesized by the LAB host (Table (Table1).1). An exception to this rule is the GS from L. mesenteroides NRRL B-1299 (DSRE), which carries an additional C-terminally located catalytic domain (17) (Table (Table1).1). Evidence has been provided that this additional C-terminal domain is responsible for introduction of the branch points in the product synthesized (47).
All GS of LAB are extracellular enzymes and the N termini of these enzymes contain a signal peptide for protein secretion (36 to 40 amino acids) (Fig. (Fig.1).1). The stretch of amino acids between the signal peptide and the core region of GS is highly variable both in composition and in length (200 to 700 amino acids).
Different repeating units have been identified in the variable domains of GS enzymes from various LAB organisms (17, 56, 79, 95, 97) (Table (Table1).1). The functions of the N-terminal variable domain (and repeats) have remained unclear. Deletion studies of the complete N-terminal variable domain in GTFI of Streptococcus downei MFe28 demonstrated that it does not play a significant role in glucan structure determination (116). Additional N-terminal deletions resulted in drastic loss of enzyme activity, also indicating that the N-terminal part of the catalytic core had been deleted (122). The relatively large N-terminal variable domain of GTFA of L. reuteri 121 is important for activity with sucrose, but its deletion has only minor effects on glucan product characteristics (99).
Detailed structural data for GS proteins of family GH70 is lacking at present. However, secondary-structure prediction studies of the catalytic domain, corroborated by circular dichroism experiments, have shown that GS proteins possess a (β/α)8 barrel structure as found in glycosidases from family GH13, e.g., with α-amylase, cyclodextrin glucanotransferase (CGTase) and amylosucrase (38, 111, 117). The (β/α)8-barrel structure motif of GS is presumably circularly permuted and is characterized by the presence of eight β-sheets (E1 to E8) located in the core of the protein, alternating with eight α-helices (H1 to H8) located at the surface of the protein (Fig. (Fig.2)2) (111).
The four conserved regions (I to IV) of amino acids identified in the members of family GH13 (191) are also present in GS proteins. However, as a consequence of the circular permutation, region I in GS enzymes is located C-terminally of regions II to IV (Fig. (Fig.11 and and2).2). The seven amino acid residues that are fully conserved in family GH13 are also present and fully conserved in the GH70 family, except His122 (Taka-amylase A numbering), which is replaced by Gln in all GS enzymes (110) (Gln1514, GTFA L. reuteri 121 numbering; Fig. Fig.11 and and2)2) (111, 121).
Since GS enzymes and the enzymes of family GH13 share a related structural fold and a similar catalytic triad of amino acid residues (see below), the wealth of structure-function information uncovered for family GH13 enzymes may serve as a template to deepen our understanding of the mode of action of GS enzymes. Most members of family GH13 act on starch, e.g., α-amylase and CGTase. Whereas α-amylases generally hydrolyze α-(1→4) glucosidic bonds, CGTases mainly catalyze transglycosylation reactions, synthesizing unique circular α-(1→4)-linked oligosaccharides from starch (cyclodextrins) (146). Amylosucrase is the only enzyme from family GH13 that uses sucrose as a substrate to synthesize α-glucan polymers containing α-(1→4) glucosidic linkages (3, 182). For family GH13 enzymes, high-resolution three-dimensional structures are available, often in complex with substrates, inhibitors, or products (207). The three-dimensional structures of amylosucrase (also in complex with sucrose, oligosaccharides, and with a covalently bound glucopyranosyl moiety) resemble those of other proteins of family GH13 (80, 115, 181, 183).
Sequence alignments, mutagenesis studies, and three-dimensional structure analyses within family GH13 have resulted in identification of the amino acid residues involved in catalysis (204). Enzymes of family GH13 contain three amino acids with Bacillus carboxyl groups crucial for catalysis, Asp229/Asp286, Glu257/Glu328, and Asp328/Asp393 (CGTase of B. circulans 251 numbering and amylosucrase of Neisseria polysaccharea numbering) present at or near C-terminal β-strands 4, 5, and 7 (using the structure element numbering of family GH13 proteins) (Fig. (Fig.11 and and2)2) (204). Three corresponding residues, present in similar locations in the secondary structure, are also essential for activity in family GH70 (GS) enzymes (38, 99).
Within both families GH13 and GH70 Asp1024 (catalytic nucleophile, GTFA L. reuteri 121 numbering) (Fig. (Fig.1D)1D) is involved in the formation of the covalent glucosyl-enzyme complex (80, 111, 128, 129, 204). Its mutagenesis in family GH13 and family GH70 enzymes resulted in drastic loss of enzyme activity (38, 83, 88, 99, 119, 165). Site-directed mutagenesis studies in family GH13 and family GH70 enzymes have also provided evidence for the crucial roles of the other two invariable residues in catalysis (acid/base catalyst, Glu1061) (Fig. (Fig.1E)1E) and, transition state stabilizer, Asp1133 (Fig. (Fig.1F)1F) (GTFA Lactobacillus reuteri 121 numbering) (Fig. (Fig.11 and and2)2) (38, 88, 99, 111, 165).
Besides these three catalytic residues, the active site of members of the α-amylase superfamily contains two His amino acid residues important for activity and proposed to be involved in stabilization of the transition state (111, 131, 165). One of these His residues (His327 in B. circulans 251 CGTase and His392 in N. polysaccharea amylosucrase) is also conserved in GS enzymes (His1132 in L. reuteri 121 GTFA; Fig. Fig.1F).1F). Its mutation (His661Arg in L. mesenteroides NRRL B-512F DSRS and His561Gly in S. mutans GS-5 GTFB) resulted in very low residual enzyme activities (119, 202). This residue thus may play a similar role in GS enzymes.
The other His residue (His140 in B. circulans 251 CGTase and His187 in N. polysaccharea amylosucrase), involved in transition state stabilization (131, 165), is replaced by Gln in all GS enzymes known (Gln1514 in L. reuteri 121 GTFA; Fig. Fig.1G)1G) (111, 121). Its mutation, Gln937His, in GTFI of S. downei MFe28 resulted in drastic but not complete loss of activity (121). The authors concluded that Gln937 plays no direct role in cleavage of sucrose and in formation of the covalent glucosyl-enzyme intermediate, but may be important for transition state stabilization (121). Mutagenesis of Gln937Asn in GTFI also resulted in reduced activity and modified distribution of oligodextran and nigero-oligosaccharide products (121). Analogously, in CGTase, mutation of the corresponding His residue (His140) resulted in lower activity and altered product formation (131).
No GS enzyme mutagenesis data are available for the other two of the seven fully conserved residues, Arg1022 (Fig. (Fig.1D)1D) and Asp1509 (Fig. (Fig.1G).1G). However, mutagenesis of the corresponding residues in CGTase (Arg227 and Asp135, CGTase B. circulans 251 numbering) resulted in drastically decreased activities (103). Analysis of mutants in these conserved GS amino acid residues is required to elucidate their true function.
The structural similarities between GS and the enzymes of family GH13 (see above) allow rational identification of regions important for enzyme activity and product specificity. Based on a comparison of sugar-binding acceptor subsites in family GH13 enzymes (111), the locations of three regions putatively involved in acceptor substrate binding in GS enzymes were identified. These were C-terminal of the catalytic residues Asp1024 (GTFA numbering, region II, Fig. Fig.1D)1D) and Glu1061 (region III, Fig. Fig.1E).1E). A third acceptor substrate binding region was identified on the basis of mutagenesis studies with different GS enzymes, involving amino acid residues 1134, 1135, 1136, 1138, and 1142 (GTFA L. reuteri 121 numbering). This region is located C-terminal of the catalytic residue Asp1133 (region IV, Fig. Fig.1F),1F), determining the solubility of the glucan products and the ratio of α-(1→3) versus α-(1→6) and α-(1→4) versus α-(1→6) glucosidic linkages in the polymer (96, 123, 149, 173).
For CGTase, amylosucrase, neopullulanase, acarviosyltransferase, and the branching enzyme of family GH13 (3, 13, 96, 101, 102, 104, 105, 206) and GS enzymes of family GH70, these regions have been proven to be important for product formation, giving further insights into the structural relatedness of GS and family GH13 enzymes (see below).
The effects of different amino acid substitutions in the (putative) acceptor substrate binding regions and other conserved regions in the catalytic core of LAB GS enzymes have been investigated in the past years. In many cases the mutant enzyme products and activities have been thoroughly characterized. Below, we present a structured summary of the effects of these mutations.
(a) Affecting glucosidic linkage type. Site-directed mutagenesis allowed identification of several GS regions influencing glucosidic linkage specificity in glucan and oligosaccharide products, apart from Gln937 (see above). Below, the results for a number of mutations done in amino acids in regions II and IV, by analogy to family GH13 most likely representing acceptor substrate binding subsites, are summarized (Fig. (Fig.1).1). Derivatives of the L. reuteri 121 GTFA protein containing mutation Pro1026Val (Fig. (Fig.1D)1D) showed a clear change in the oligosaccharide and glucan products synthesized (96). Mutations in the tripeptide immediately following Asp1133, the putative transition state-stabilizing residue in GTFA of L. reuteri 121 (Asn1134Ser:Asn1135Glu:Ser1136Val; Fig. Fig.1F),1F), resulted in a drastic increase in α-(1→6) glucosidic linkages (~40%) and a drastic decrease in α-(1→4) linkages (~40%) in the polymer synthesized compared to the wild type (96). Also, a quintuple mutant was constructed, by combination of this triple amino acid mutant (Asn1134Ser:Asn1135Glu:Ser1136Val) with a double mutant (Pro1026Val:Ile1029Val) located in and near region II (Fig. (Fig.1D),1D), resulting in an even further enhanced ratio of α-(1→6) to α-(1→4) glucosidic linkages in its glucan (and also oligosaccharide) products (96). Mutation Thr667Arg (Fig. (Fig.1F)1F) in DSRS of L. mesenteroides NRRL B-512F resulted in 8% more α-(1→3) linkages in the dextran product (149).
(b) Affecting glucan solubility. Besides the glucosidic linkage type, polymers may be classified based on their solubility properties. This characteristic is undoubtly related to the structure of the polymers (linkage type, branching, and size) and thus reflects intrinsic enzyme properties.
In different GS enzymes, mutations in the residue located five amino acids behind the catalytic Asp1133 (Asp1138 GTFA of L. reuteri 121 numbering and equivalent to Thr667Arg in DSRS of L. mesenteroides NRRL B-512F) resulted in a shift in glucan solubility (Fig. (Fig.1F).1F). Mutation Thr589Glu in GTFD of S. mutans GS-5 lowered the amount of soluble glucan synthesized from 86 to 2%, and the number of α-(1→3) linkages in the insoluble mutan product synthesized also decreased from 76 to 38% (173). The reverse shift, from a completely insoluble glucan to more soluble glucan synthesis, was observed when the similar amino acid residue was mutated in GTFB (mutant Asp567Thr) of S. mutans GS-5 (increase in soluble glucan from 0 to 24%) and in GTFI (mutant Asp569Thr) of S. downei MFe28, analysis of the soluble fraction showed production of an α-(1→6)-linked glucan (123, 173).
The effects of substitutions in a number of other amino acids on glucan solubility have also been reported: Asp457Asn (C-terminal of conserved region II), Asp571Lys (Fig. (Fig.1F),1F), Lys779Gln and Lys1014Thr (not shown), all in GTFB of S. mutans GS-5, and Asn471Asp in GTFD of S. mutans GS-5 (173). These mutations resulted in an increase of soluble glucan synthesis from 0 to 37%, from 0 to 18%, from 0 to 3%, and from 0 to 14%, and insoluble glucan synthesis from 14 to 38%, respectively (173). The most marked effect was achieved (from 0 to 73% soluble glucan) when the six single GTFB mutants were combined (173). Mutagenesis of GTFB Val412Ile and GTFC Val438Ile resulted in enhanced insoluble glucan synthesis of about 10 to 20%, whereas soluble glucan synthesis by these enzymes was significantly lower than for the wild type (28) (Fig. (Fig.1C1C).
It is known that water-insoluble glucans contain mainly α-(1→3) linkages and soluble glucans mainly contain α-(1→6) glucosidic linkages (173). Unfortunately, the linkage types, degrees of branching, and sizes of the glucans synthesized by these GTFB and GTFD site-directed mutants have not been reported; conceivably, changes in either may have an effect on glucan solubility (173). Detailed analysis of these polymers may provide further insights on the structure-function relationships of GS enzymes.
(c) Affecting enzyme activity. Within the four conserved regions (I to IV) present in the catalytic core of GS enzymes, apart from the catalytic residues, other amino acid residues important for enzyme activity have been identified. Mutation of the highly conserved Trp491Gly in GTFB of S. mutans GS-5 (Fig. (Fig.1E)1E) resulted in complete loss of enzyme activity (202) (Fig. (Fig.1).1). A double mutation in GTFA of L. reuteri 121, His1065Ser:Ala1066Ser, showed a very strong change in enzyme activity, but no influence on glucosidic linkage specificity (96). It was concluded that these residues are located further away from the (putative) −1 and +1 sugar binding subsites (96).
The GS-5 catalytic core commonly starts with two to four conserved Tyr residues (Fig. (Fig.1A).1A). Conversion of one to four of these Tyr residues, at positions 169 to 172 in GTFB of S. mutans GS-5, into Ala residues had little effect on overall activity. Only the adhesiveness of the glucan products synthesized was altered by these mutations, suggesting that changes had been introduced in the glucan structure (202).
Using antibodies and chemical modification two regions, separate from the four conserved (I to IV) regions and upstream of conserved region II, that were important for activity were identified. The first of these regions extends from amino acids 368 to 382 in GTFC of S. mutans GS-5 (Fig. (Fig.1B),1B), and the second region corresponds to GTFC residues 435 to 453 (27, 37, 57) (Fig. (Fig.1C).1C). Subsequent mutational analysis of the first region in GTFI allowed identification of several residues (mutants Trp344Leu, Glu349Leu, and His355Val) that are important for activity (122) (Fig. (Fig.1B).1B). Important amino acid residues in the second region have also been identified. Substitution of Asp511 and Asp513 of DSRS from L. mesenteroides NRRL B-512F in Asn residues resulted in complete loss and in a strong decrease in glucan- and oligosaccharide-synthesizing activities, respectively (119). Similar residues in GTFB from S. mutans GS-5 (Asp411 and Asp413) and GTFC from S. mutans GS-5 (Asp437 and Asp439) have been mutated into Asn residues, also affecting glucan-synthesizing activity (28). Site-directed mutagenesis of GTFB (Glu422Gln) and GTFC (Glu448Gln) resulted in 40% reduced glucan-synthesizing activity (28, 119) (Fig. (Fig.1C1C).
Several studies have shown that the C-terminal domain of GS is involved in glucan binding. Therefore, it has been designated the glucan binding domain (GBD) (1, 82, 87, 99, 108, 120, 172). For a few GS enzymes, evidence has been presented that the C-terminal domain is also involved in determining the structure of the synthesized glucan (99, 214). In addition, the C-terminal GBD appears to be necessary for GS activity (99, 132). However, deletion of a large part of the C-terminal GBD had little effect on enzyme activity in, e.g., GTFI of S. downei MFe28 (116). Some GS enzymes with deletions at the C-terminal end retained hydrolytic activity, but glucan binding and synthesizing properties had become lost (56).
The precise role of the GBD domain in enzyme catalysis remains largely unknown. The GBD may be of importance for polymer chain growth. It has been suggested that the C-terminal domain plays a facilitating role in the transfer of products from the catalytic site (120).
The C-terminal domain of all reported GS is composed of a series of repeating units which have been divided into four classes, A, B, C, and D (Table (Table3).3). Site-directed mutagenesis studies of conserved amino acid residues in different A repeats of GFTI of S. downei MFe28 suggested that they do not contribute equally to the overall binding of dextran or that corresponding residues in different repeats may differ in their contributions (171).
Within these different repeats, a common conserved stretch of amino acids, designated the YG repeat, can be distinguished (58) (Table (Table3).3). The number, class, and distribution of these repeats are specific for each enzyme (124). L. mesenteroides NRRL B-512F dextransucrase contains, besides A and C repeats, N repeats, which have not been identified in streptococcal GS. These N repeats are not highly conserved but possess the main characteristics of YG repeats (120). Alternansucrase from L. mesenteroides NRRL B-1355 contains a single A repeat and distinct short repeats, DG(X)4APY (4).
Compared to the GBDs of other GS enzymes (average length, 400 to 500 amino acids), GTFA and GTFB from L. reuteri 121, GTFML1 from L. reuteri ML1, GTFO from L. reuteri ATCC 55730, and GTF180 from L. reuteri 180 possess relatively short GBDs of 134 to 263 amino acids (Fig. (Fig.1)1) (95, 97). The GBDs of L. reuteri GS enzymes lack A, B, C, and D repeats and consist of several (less well) conserved YG repeats (94, 95, 97). Also the putative GBDs of GTFKg15 from Lactobacillus sakei Kg15, GTFKg3 from Lactobacillus fermentum Kg3, and GTF33 from Lactobacillus parabuchneri 33 lack A, B, C, and D repeats and consist of various numbers of conserved and less-well-conserved YG repeating units. GTF33 contains, besides the 17 YG repeats, two unique repeating units designated KYQ that show no significant similarity to any protein motif present in the databases (Table (Table3).3). GTFKg15 possesses an additional stretch of amino acids at the end of its putative GBD showing similarity to part of a putative extracellular matrix binding protein from Streptococcus pyogenes M1 (95).
The catalytic mechanism of GS enzymes is complicated and not yet fully understood. There are several aspects complicating the elucidation of the reaction mechanism: various glucans as well as oligosaccharides are synthesized and a sucrose hydrolysis reaction may also occur. Furthermore, many GS enzymes also catalyze branching reactions, resulting in synthesis of different types of glucosidic linkages.
Two alternative mechanisms have been proposed for the glucan chain growth. Nonreducing end elongation involves the presence of one amino acid residue (an Asp or Glu) acting as a nucleophilic group and another residue acting as a proton donor. The glucan chain grows by successive insertions of glucose units between the catalytic site of the enzyme and the reducing end of the glucan polymer. Reducing end elongation occurs in two steps involving two sucrose binding sites (nucleophiles): (i) the nucleophilic sites attack two sucrose molecules to give two covalent glucosyl-enzyme intermediates and (ii) the C-6 hydroxyl of one of the glucosyl intermediates makes a nucleophilic attack onto C-1 of the other glucosyl intermediate to form an α-(1→6) glucosidic linkage and an isomaltosyl intermediate. The newly released nucleophilic site attacks another sucrose molecule to give a new glucosyl-enzyme intermediate. This symmetrical and alternative role of the two sucrose binding sites results in growth of the glucan chain by its reducing end (124, 153, 155). This mechanism, however, appears more and more unlikely for GS enzymes in view of the single covalent intermediate identified (128) and mutational identification of a single catalytic triad (38, 99). Moreover, only one active site has been identified in amylosucrase (GH13) (3,183).
The amylosucrase enzyme (from family GH13) from N. polysaccharea, synthesizing a (short) glucan polymer from sucrose, uses a double displacement mechanism, similar to that of other family GH13 enzymes. This mechanism involves the formation of a covalent glucosyl-enzyme intermediate (confirmed by three-dimensional structural data) (3, 80, 204). In a subsequent step this glucose moiety is transferred onto a water molecule (overall resulting in sucrose hydrolysis) or onto a hydroxyl group of a sugar acceptor (transglucosylation reaction). As for amylosucrase, again only one site capable of making a covalent bond with the glucose moiety, originating from the breakdown of sucrose, has clearly been identified in GS enzymes (high performance liquid chromatography analysis of tryptic digests of a trapped covalent glucosyl-GS enzyme intermediate) (80, 128).
At first a processive mechanism (polymer chain remains bound to the enzyme) was suggested for polymer formation by amylosucrase (145). A more detailed analysis showed that amylosucrase polymer formation is nonprocessive (release of the polymer chain after each glucose residue transfer and presence of intermediate oligosaccharides) (3). Early evidence indicates that GS glucan polymer synthesis proceeds in a processive manner, since oligosaccharide reaction intermediates cannot be detected and high-molecular-mass polysaccharide products are obtained at early reaction times (16, 44, 201). However, it cannot be excluded that the use of more sensitive analytical techniques of GS reaction products could reveal the presence of oligosaccharide reaction intermediates (indicative of a nonprocessive mechanism) (3).
Analysis of the three-dimensional structural information for amylosucrase proteins (GH13) with bound sucrose and oligosaccharide substrates and products provided convincing evidence for the use of a non-reducing-end elongation mechanism (3). Various studies of product formation by GS enzymes incubated with sucrose and acceptor substrates showed (e.g., conversion of maltose into panose by GTFA of L. reuteri ) that GS also use sucrose to elongate their oligosaccharide acceptor substrates at the nonreducing end (5, 42, 95, 99, 121, 130). Mutant data for GTFA of L. reuteri 121 showed similar changes in glucosidic bond specificity with both oligosaccharide and polysaccharide products (96). Both amylosucrase and GS enzymes thus appear to employ a non-reducing-end elongation mechanism for oligosaccharide and polymer synthesis.
Further studies need to focus on elucidation of the three-dimensional structures of GS enzymes, ideally with substrate-enzyme and/or product-enzyme complexes. This may allow elucidation of the exact reaction mechanism of family GH70 enzymes and identification of structural features determining differences in product specificity such as (i) number and position of sugar binding donor and acceptor subsites, (ii) residues involved in substrate/product binding, (iii) glucosidic bond specificity, and (iv) degree of branching.
Bacterial FS synthesize either levan (levansucrase) or inulin (inulosucrase). Inulosucrase enzymes are exclusively present in LAB, while levansucrase enzymes are widely distributed in both gram-positive and gram-negative bacteria. At the amino acid level, the levansucrases of gram-positive and those of gram-negative bacteria show low similarity (about 20%). In general, FS enzymes of LAB origin are larger than their non-LAB counterparts. The levansucrase of S. salivarius ATCC 13419 is a particularly large enzyme with a molecular mass of 140 kDa (136). Most of the research has been performed on levansucrases, in particular on enzymes from Bacillus spp. (12, 107, 189, 195) and Zymomonas spp. (71, 187, 219, 220), and, to a lesser extent, Lactobacillus spp. (196, 211, 213), Streptococcus spp. (59, 174), and Gluconacetobacter spp. (6) species. In order to provide a state-of-the-art view of the FS field of research, work performed on non-LAB FS is also discussed.
Bacterial FS are extracellular enzymes that cleave the glycosidic bond of their substrate sucrose (and in some cases also raffinose) and use the energy released to couple a fructose unit to (i) a growing fructan (either levan or inulin) chain (transfructosylation), (ii) to sucrose, (iii) to water (hydrolysis), or (iv) to another acceptor (such as raffinose). Because sucrose is used as the acceptor in the initial priming reaction, bacterial fructans contain a nonreducing glucose unit at the end of the chain (52). In the initial reaction of FS, the fructose of a sucrose molecule is coupled by the enzyme to another nonreducing fructose with a free primary alcohol at position C-2, acting as an acceptor substrate, e.g., sucrose, raffinose, or a fructan molecule (35, 152). This is also referred to as the priming reaction. In subsequent steps, the enzyme elongates the primer. A clear difference between FS and GS enzymes is the fact that GS enzymes cannot use sucrose as an acceptor but rather the cleaved glucose residue (see above).
In general, LAB produce two types of fructans using FS (see also Table Table2):2): levans, consisting mainly of β-(2→6)-linked fructose residues, occasionally containing β-(2→1)-linked branches, and inulin-type fructans, with β-(2→1)-linked fructose residues, with β-(2→6)-linked branches. Levan production has been reported for streptococci (19, 70, 178), L. mesenteroides (156), L. reuteri 121 (208, 210), and Lactobacillus sanfranciscensis (92, 93). Lactobacillus frumenti, Lactobacillus pontis, Lactobacillus panis and Weissella confusa were also found to produce fructans, but their fructan binding types have not been determined (92, 93, 198). Inulin production by LAB has been observed in some cariogenic S. mutans and S. salivarius strains (45, 158, 174), Leuconostoc citreum CW28 (138), and L. reuteri 121 (212, 213).
The molecular masses of the fructans produced (if determined) show a large variation, from 2 × 104 to 50 × 106 Da (Table (Table2).2). There are some reports that the molecular mass of the fructan produced is dependent on growth and incubation conditions, e.g., the temperature, salinity, and sucrose concentration used (10, 192, 193).
All known bacterial FS catalyze fructose transfer from sucrose (or raffinose) to a number of acceptors other than the fructan polymer. Examples of possible acceptors are water (hydrolysis of sucrose) and sucrose and raffinose (yielding a tri- or tetrasaccharide, respectively), short-chain acylalcohols, various mono- to tetrasaccharides (22, 86, 194), and sorbitol (144). The ability of L. sanfranciscensis levansucrase to use raffinose, maltotriose, maltose, xylose, or raffinose as fructosyl acceptors, leading to the formation of a range of heterooligosaccharides, has been reported recently by Tieking et al. (199). Additionally, high performance liquid chromatography analysis of the reaction products of this levansucrase with 0.4 M raffinose as the fructose donor and acceptor revealed the presence of tetra-, penta-, and hexasaccharides (GalGF2 to GalGF4; Gal is galactose) in addition to melibiose (GalG), raffinose, kestose, and nystose. This proves the ability of the enzyme to use raffinose not only as an acceptor but also as a donor of fructose moieties.
Although the reactions performed by FS and GS are similar with respect to the use of sucrose as the substrate, the proteins involved do not share sequence similarity. Based on deduced amino acid sequences, the overall FS structure is divided into four regions (Fig. (Fig.3):3): (i) a signal peptide, (ii) an N-terminal stretch that varies in length, (iii) a conserved catalytic core of about 500 amino acids that is shared between all family GH68 members (carbohydrate-active enzyme website: http://afmb.cnrs-mrs.fr/CAZY) (33), and (iv) a C-terminal stretch of various lengths, in some cases with a cell wall binding domain (LPXTG; see below).
Since bacterial FS are extracellular enzymes, they contain an N-terminal signal sequence that targets these enzymes for secretion (Fig. (Fig.3).3). The signal peptide-containing precursor is cleaved upon secretion of FS by gram-positive bacteria (11, 107, 147, 189, 195, 211). The N-terminal domain (Fig. (Fig.3)3) varies in size between the FS and no function has yet been assigned to this domain.
Most work on structure-function relationships of the core region has been performed on non-LAB FS enzymes. The results obtained for non-LAB FS enzymes have (in part) been corroborated by work performed on LAB FS (see below). Therefore, the results for non-LAB FS are also discussed below.
Recently, high-resolution crystal structures of the non-LAB Bacillus subtilis SacB levansucrase (at 1.5 Å) and a sucrose-bound inactive mutant of the same enzyme (at 2.1 Å) have been described. Both structures show a rare five-fold β-propeller topology with a deep, negatively charged central pocket (113). This topology differs strongly from the (β/α)8 barrel identified in family GH13 enzymes and putatively assigned to GS enzymes of family GH70 (see above). The central pocket is composed mostly of residues belonging to highly conserved sequence motifs, including invariant acidic residues Asp86 (Fig. (Fig.3A),3A), Asp247 (Fig. (Fig.3E),3E), and Glu342 (Fig. (Fig.3G).3G). As with GS and the enzymes of family GH13, these residues form the catalytic triad (nucleophile, transition state stabilizer, and acid-base catalyst, respectively). This has been proven by the mutational analysis of these residues and their equivalents in L. reuteri 121, the Inu and Lev enzymes (140).
Recently, a three-dimensional structure has been reported for the levansucrase enzyme of the non-LAB Gluconacetobacter diazotrophicus (112). The three-dimensional structure of this enzyme displays the same five-bladed β-propeller architecture as the B. subtilis levansucrase enzyme. The three-dimensional positions of the three catalytic residues of both levansucrases are superimposable, indicating strong structural relatedness of these enzymes.
Based on the B. subtilis SacB (113) and G. diazotrophicus LsdA (112) three-dimensional structures, residues directly involved in binding of sucrose in the active site and constituting the −1 and +1 sugar binding subsites have been identified (nomenclature according to Davies et al. ). In short, cleavage of the substrate (e.g., sucrose) takes place between subsites −1 and +1, and the enzyme forms a covalent intermediate with the cleaved substrate (i.e., fructose) at subsite −1. Subsequently, the cleaved substrate is coupled to the acceptor molecule (e.g., fructan).
Figure Figure44 shows the main amino acids involved in FS catalysis. Based on the available three-dimensional structures and sequence alignment of family GH68 proteins, residues creating subsite −1 have been identified in B. subtilis SacB, G. diazotrophicus LsdA, and L. reuteri 121 inulosucrase (Inu) and levansucrase (Lev) (Fig. (Fig.4).4). Strikingly, subsite +1 differs among the enzymes from family GH68. These differences at the +1 subsite distinguish FS enzymes from gram-positive (SacB and Inu) and gram-negative (LsdA) bacteria. They do not, however, distinguish between polymerizing (SacB) and oligomerizing (LsdA and Inu) or inulin (Inu)/levan (SacB, LsdA) synthesizing enzymes (72, 193, 213).
The three-dimensional structures of the B. subtilis and G. diazotrophicus levansucrases have provided important insights into the functional roles of several conserved amino acid residues in this core region (112, 113). Two regions that are highly conserved among FS and sucrose-hydrolyzing enzymes, invertases, have been designated sucrose binding boxes (SBB) (Fig. 3B and D). Asp312, located between SBB1 and SBB2 (Fig. (Fig.3C)3C) of S. salivarius ATCC 25975 levansucrase, is most likely involved in determining acceptor recognition or stabilizing of a β-turn in the protein (185). Analysis of the three-dimensional structure of B. subtilis SacB revealed that Asp312 indeed forms a 180° reverse β-turn between SBB1 and SBB2 and is located on the surface of the protein, far from the active site (113).
Several independent studies revealed that transglycosylation and hydrolysis reactions could be modulated separately by mutagenesis or even by changing reaction conditions. Selective inhibition of transglycosylation activity of Zymomonas mobilis levansucrase has been reported by Senthilcumar et al. (169). Point mutations in the “sucrose binding box” in levansucrase of Z. mobilis caused significant changes in the transglycosylation efficiency of the enzyme (220). The transglycosylation versus hydrolysis ratio could be also be altered by immobilization of the enzyme on hydroxyapatite, thereby mimicking in vivo conditions, where levansucrases are attached to the cell wall of bacteria or to the tooth surface. The activity of immobilized B. subtilis levansucrase was directed mainly towards its polymerizing activity (23).
Mutation of Arg331 in the B. subtilis levansucrase (Fig. (Fig.3H)3H) yielded enzymes that were differently affected in transfructosylation activity depending on the substitution chosen; three variants (Arg331Lys, Arg331Ser, and Arg331Leu) lost the ability to synthesize levan and were only able to produce the trisaccharide kestose. It has been suggested that the side chain of Arg331 could act as a proton donor in the bifunctional catalysis (22). Analysis of the three-dimensional structure of the B. subtilis levansucrase showed, however, that this Arg331 residue is structurally involved in the acceptor binding site (113). Similar conclusions were drawn from a His296-mutagenized Z. mobilis levansucrase enzyme (220).
A pressing question is what structural features in FS enzymes determine their specificity for synthesis of either β-(2→6) or β-(2→1) linkages and the sizes of the fructans produced. At present, no three-dimensional structural information has been published for inulosucrase enzymes. The G. diazotrophicus and B. subtilis levansucrase enzymes synthesize fructo-oligosaccharides and levan polymers, respectively. Further investigation of both three-dimensional structures might give clues to the difference in product profiles of these enzymes. The lack of structural data for complexes between FS and their fructosyl acceptors makes it difficult to understand what determines the polymer versus short oligosaccharide synthesis ratio.
Analysis of the three-dimensional structure of B. subtilis levansucrase has provided evidence for a presence of a bound metal ion, most likely Ca2+ (113). Asp339 of B. subtilis levansucrase was identified as one of the residues coordinating Ca2+ ions in the enzyme structure. The functional role of this residue has been studied by mutating equivalent amino acid residues in the inulosucrase and levansucrase from L. reuteri, Asp520Asn and Asp500Asn, respectively (139). Both mutants showed a decreased optimal temperature and the apparent affinity for Ca2+ binding was reduced significantly, 1,600-fold and 35-fold, respectively. Also, the S. salivarius ATCC 27975 levansucrase enzyme is dependent on calcium ions for enzyme activity (78).
Sequence alignment of family GH68 members revealed that residues involved in binding of the calcium are conserved in most enzymes from gram-positive bacteria, but are absent in proteins of gram-negative origin (139). Most likely a disulfide bridge present in the three-dimensional structure of G. diazotrophicus (gram-negative) plays a similar role to the calcium binding site identified in FS enzymes from gram-positive bacteria (112).
As with GS, the C-terminal domain of FS enzymes might affect product size and/or enzyme specificity. The only evidence for such a claim is the observation that a C-terminal enlargement of the FS enzyme from the non-LAB B. subtilis was shown to produce a larger fructan polymer which was mainly due to an increase in branches in the levan (24).
A different function for the C terminus of FS is the attachment of these enzymes to the cell wall of its producing organism. A common C-terminal LPXTG cell wall-anchoring motif (49, 133, 134) (Fig. (Fig.3)3) is found in both inulosucrase (213) and levansucrase (211) from L. reuteri 121. A motif resembling the LPXTG cell wall-anchoring motif is present in the cell wall-associated S. salivarius ATCC 25975 levansucrase (148). Proteins displayed on the bacterial surface may have various functions for the bacterial cell. For Staphylococcus aureus, surface proteins are thought to play a major role in the infection process in humans (200). Surface proteins from urogenital Lactobacillus spp. mediate adhesion to tissue cells (77, 177) and play a role in the maintenance of a healthy urogenital microbiota. Cell-associated homopolysaccharides, produced by sucrase enzymes anchored to the cell surface, may also be involved in adherence of the organism to a surface, such as teeth and the intestinal mucosa (159).
A detailed biochemical characterization of FS enzyme reactions is complicated by the fact that FS generate new fructan molecules, which in turn can be used as acceptor substrates. Accordingly, a multiple chain elongation mechanism in which the fructose residues are added randomly to all fructan acceptor molecules has been proposed (25). The nature of the fructosyl acceptor, except water, thus changes as the reaction proceeds. Kinetic and chemical studies of the levansucrase of B. subtilis further suggest that each fructose unit is added one at a time onto an acceptor molecule (25).
Most FS enzymes follow Michaelis-Menten kinetics for the hydrolysis and transferase reactions. Exceptions to this rule are the L. reuteri 121 inulosucrase and levansucrase enzymes and L. sanfranciscensis levansucrase, which cannot be saturated by their substrate, sucrose (in the case of the L. reuteri 121 levansucrase, only at 50°C) (196, 211, 212). This phenomenon has also been observed for some plant enzymes that synthesize inulin polymers (91). Low-molecular-mass fructans accelerate the rate of polysaccharide formation and increase the fructan-to-free fructose ratio (46, 212). This may partly explain the unusual reaction kinetics observed for the above-described enzymes.
A two-step mechanism has been proposed for catalysis by FS enzymes. They contain an acidic group and a nucleophilic group essential for transfructosylation (180). The first identification of a nucleophilic group responsible for binding to the fructose moiety, determined by a covalent intermediate of substrate and enzyme, was in 1976 by Chambert and Gonzy-Treboul for the B. subtilis levansucrase (21). Information about the position and identity of this Asp residue has been determined from the three-dimensional crystal structure of the B. subtilis levansucrase (113). An Asp amino acid residue in the conserved RDP motif across families GH68 and GH32 (Fig. (Fig.3E)3E) has been shown to be involved in the stabilization of the transition state by analysis of the B. subtilis levansucrase three-dimensional structure (113). Mutation of this Asp residue resulted in dramatic decreases of catalytic activities in both L. reuteri 121 FS (140) and the S. salivarius levansucrase enzymes (185).
Based on the observation that the polymerizing activity of the enzyme could be modulated separately from the oligomerization activity, a reaction mechanism involving two active sites has been proposed for the S. mutans FS (188). However, both levansucrase three-dimensional structures provide evidence for only a single active site (112, 113).
Several putative FS-encoding genes have been identified in the genome sequences of Streptococcus, Leuconostoc, and Lactobacillus strains (Table (Table4).4). Although their sizes are quite divergent, their core regions are all around 450 amino acids long. Since the characteristic residues of the sucrose binding boxes and the catalytic triad are present, these genes may very well encode active FS enzymes. The putative Lactobacillus johnsonii and Lactobacillus gasserii FS genes (Table (Table4)4) show strong amino acid sequence similarity (62% identitity and 75% similarity over 709 amino acids and 61% identitity and 74% similarity over 663 amino acids, respectively) to the L. reuteri 121 inulosucrase, which might indicate their inulosucrase identity.
Mining newly released genome sequences might reveal new types of sucrase genes. An example of such a new type of sucrase gene is islA of Leuconostoc citreum CW28 (Table (Table2),2), a hybrid between a GS and an FS gene (137). Its N-terminal region is similar to the variable region of alternansucrase from L. mesenteroides NRRL B-1355, its catalytic domain is similar to the core region of FS from various bacteria, and its C-terminal domain displays similarity to the GBD from alternansucrase (see above). Other exciting possibilities are enzymes that synthesize new types of polymers, such as a fructan with alternating β-(2→1)- and β-(2→6)-linked fructose residues or even a polymer containing combinations of glucose and fructose residues.
Lactic acid bacteria are a promising source of polysaccharides and oligosaccharides for health, food, and nutritional applications. LAB synthesize a large diversity of glucans and fructans, homopolysaccharides that vary, for instance, in molecular mass, glycosidic linkages, solubility, and degree ofbranching. These parameters determine to a large extent the functional properties of both glucan and fructan polymers. As reviewed in this paper, information about structure-function relationships in the sucrase-type enzymes that synthesize these polymers from sucrose is still limited but increasing rapidly.
Most progress has been made with respect to FS enzymes. Recently, two high-resolution three-dimensional structures of the B. subtilis and G. diazotrophicus levansucrases (family GH68) have been reported (112, 113), providing strong promise for rapid further progress. The levansucrase and inulosucrase enzymes characterized are highly similar in their primary amino acid sequences and the chemical reactions they catalyze but synthesize rather different fructan polysaccharides (levan and inulin). Elucidation of an inulosucrase three-dimensional structure and further detailed biochemical comparisons of (mutant) inulosucrase and levansucrase enzymes may provide clear insights into the structure-function relationships of these FS enzymes. Questions to be answered are (i) which factors determine the fructan binding type specificity, (ii) what determines the fructan molecular mass, and (iii) what is the underlying reaction mechanism. Subsequently, mutant enzymes may be constructed, synthesizing (hybrid) fructans with specific sizes and/or containing a specific distribution of glycosidic binding types.
No three-dimensional crystal structures are available yet for glucansucrase enzymes (family GH70). Their overall sequence and secondary-structure similarity with α-amylase-type enzymes (family GH13), however, has already allowed identification of putative acceptor substrate binding subsites and rational construction of mutant GS enzymes synthesizing glucans with clearly different glucosidic bond profiles (96) and solubilities (123, 173).
Further breakthroughs in this field are expected in the years ahead, with enzyme engineering approaches increasingly allowing construction of mutant GS and FS enzymes and the discovery of new types of sucrase enzymes from genome sequences providing exciting possibilities for the synthesis of tailor-made glucan, fructan polysaccharides, and oligosaccharides for a range of applications.
We thank Thijs Kaper for assistance in creating Fig. Fig.44 and the referees for critically reading the manuscript.
We were financially supported by the University of Groningen, TNO Quality of Life, Centre for Carbohydrate Bioprocessing, and by the EET program of the Dutch government (project number KT 97029).


text:
A CONMAN who faked his own death and fled to Thailand to avoid being caught has been jailed.
Stephen Kellaway and his wife, Nelli, scammed Hammersmith and Fulham Council out of almost £15,000 while living in Uxbridge Road, Shepherd’s Bush.
While under investigation in 2008, the couple travelled to Russia – where Nelli had her breasts enlarged and Stephen, 53, is thought to have faked his own death by bribing a mortuary worker to plant his passport on the body of a tramp.
Nelli was arrested on the day that she flew back to the UK carrying what she claimed were her husband’s cremated remains in an urn.
She was later convicted of fraud and money laundering but escaped with a suspended sentence.
Stephen Kellaway made his way to Thailand using a passport he allegedly secured using the birth certificate of a dead child.
After two years on the run, he was tracked to an airport in Bangkok in 2011 where he had been sleeping rough.
Kellaway handed himself into the Thai authorities and was detained and flown back to London in December, when he was charged with benefit and identity fraud.
He admitted four charges at Harrow Crown Court on February 16 and was sentenced to 32 months in prison at Croydon Crown Court today.
The council’s residents’ services leader, Greg Smith, said: “This rogue went to extraordinary lengths to avoid detection.
“This case shows that wherever you flee to you will eventually be tracked down and made to pay for your crimes. We will be doing everything we can to seize this man’s assets.”
The pair’s scam originally came to light after Stephen Kellaway submitted a claim for housing and council tax benefit in March 2008.
Among several offences, he did not mention rental income from a property in Camden, or nearly £200,000 he and his wife had in savings. He also said he was a single parent when he was actually living with his wife.
He also failed to state that Nelli had more than £80,000 cash in undeclared bank accounts.
Hammersmith and Fulham Council also prosecuted on behalf of Richmond Council, from whom Kellaway swindled £28,000 while living in that borough.
Since her conviction, Nelli Kellaway has been made to pay back £55,000 of defrauded money.

domain: news


text:
|by chrissy n
Today I watched the denizens of Harmony try to free Charity from the frozen pond. I found the sequence thrilling,
exciting, particularly, seeing the performers serve more ham than the deli clerk at Safeway.
The story was riveting, and with Miguel in the water, tomorrow's installment holds the promise of more excitement.
But viewers shouldn't hold their breath, because tomorrow will be a rerun of sorts. Same scenario; different face.
And repeats rarely pack the same wallop as the original.
For my money, melodrama has no consequences, nothing that will make viewers hang around after the heroine has been
removed from the train tracks, and taken to safety.
Stories about the human condition always keep viewers enthralled. For example, the sex starved Ivy has spent the
last few days trying to seduce good guy, Sam.
Iv pulled out all the stops, drugging Grace, presenting herself to Sam au naturel, scratching the man's back, pulling
down his drawstring pants. And for all her efforts, she got spurned.
For now, I am cheering, "Yeaaa." Sam proves that there are some good guys in the world. But this is sudsy
television, so the weather vane may shift directions, and Sam could become involved in a stormy affair with Ivy.
Viewers will have to stay tune.
But the Sam/Ivy story has consequences, foresight, questions.
Somewhere down the road, Sam and Grace, and Grace and Ivy will verbally slug it out about the fateful skiing junket.
Sam threatened to expose Ivy's perfidy, but he won't.
Grace will tearfully lambaste Sam for keeping it a secret. She will demand to know why Sam never told her about
Ivy's misdeeds. "Sam, that crazy woman drugged me. What if I had had an allergic reaction to whatever it was
she gave me? Why didn't you tell me?"
For spite, Ivy could lie and viciously say, "Sam and I made passionate love while you slept in the next room.
Sam was ravenous. I think having you so nearby heightened his excitement."
Grace could haul off and punch her. The dramatic possibilities are endless.
A writer can get considerable mileage out of non melodramatic tales.
Melodrama is like a gin and tonic. It can make the viewer high, get their adrenaline going. Flush them, but the
next day, it's gone.
As a writer in training, I have always been ambivalent about kidnappings and hostage situations--though I have
written them. Perils of Pauline type stories are devoid of any long term consequences.
A writer needs stories that are like the Energizer Bunny.

domain: artistic


Role of Water-Energy Nexus in Water Service Provision in Lebanon
The interrelation between energy and water, known as the water-energy nexus, though well established and understood by most practitioners, has been ignored by policy makers in developing energy and water policies. This has led to the rise of complex challenges, especially in Lebanon, due to the fact that policies and strategies in both sectors have often been developed in isolation of each other. This resulted in policy fragmentation that has negatively affected the sustainable development of both sectors.
The study conducted by IFI’s Climate Change Program aims to address the issues raised above through an examination of the role of energy, particularly electricity, in Lebanon’s water and wastewater service provision. This includes an investigation and analysis of the associated socio-economic impacts of W-E Nexus mismanagement on communities in Lebanon, and a look at how organizations working in the water and energy sector communicate amongst each other, through a social network analysis.
The first phase of the study comprised of a two-step energy analysis involving a walk-through survey followed by an in-depth energy audit and assessment were conducted for 62 water and wastewater facilities mostly operated by the four water establishments in Lebanon. The intended aim was to develop a clear picture of the extent that energy impacts the provision of water and wastewater services as well as the potential for energy efficiency opportunities within the audit and similar facilities. The analysis was performed throughout the country for 39 water pumping stations and 23 wastewater treatment plants. The conducted Energy Audit revealed the challenges faced by the water establishments in covering energy cost and day-to-day maintenance operation of water pumping stations and WWTPs. The audit showed that it is important to include energy efficiency at the core of the design of new water and wastewater facilities and the rehabilitation of existing facilities. Another key finding was that medium-to-large-scale WWTPs are more energy efficient than small-scale plants and this should be well integrated in selection of treatment facilities. Further, it is important to evaluate projects in terms of energy consumption; operational expenditure and potential energy efficiency measures included in them to reduce energy costs borne by the water establishments.
With the hypothesis that the interlinkages between water and energy and the absence of an integrated policy has ramifications that trickle down to the citizens, a social impact assessment (SIA) in a selected case study was conducted. The SIA highlighted the need for better cooperation and communication between the municipalities and the WEs and for the need to rebuild the trust between the citizens and the water establishments in order to break the vicious cycle of low bills collection rate and no cost recovery by the establishments. The energy audit and social impact assessment highlighted the fact that the interrupted power supply and the resulting water shortages have negative social and economic consequences for the ordinary citizens who are burdened with extra costs in their attempt to secure water from alternative source. It was shown that residents of a town facing such onerous costs are willing to pay higher tariffs as long as water supply is consistent in quality and quantity.
Taking into consideration the results of the social impact assessment and the gap in communication observed between water users and their water providers (Water Establishments); a social network analysis (SNA) was conducted to assess the forms of communication between stakeholders involved in the water and energy sector in Lebanon. A social network analysis (SNA) is a process that identifies and studies the interlinkages and connections between various stakeholders in a specific network. The SNA showed numerous communication problems between governmental institutions, especially between municipalities, water establishments, and the MoEW. It also showed that there are stakeholders which dominate communication within the network creating unnecessary bottlenecks. Additionally, it was shown that some stakeholders are not operating according to their mandates with some shirking policy making responsibilities while others intrude into this practice. Therefore, to improve the flow of communication – knowledge, expertise, funding, etc. – it is imperative that the strong ties that are monopolizing this flow be weakened to develop a network of weak ties between all stakeholders that allows for information and data sharing, breaching new technological ideas and opportunities and facilitate drafting policies and their enforcement.


Prices, promotions, styles and availability may vary by store and online.
Exclusive RealTree AP HD Camo
This .22 LR semi-automatic rifle is shrouded in exclusive RealTree AP HD Camo to keep you AND your gun concealed in the field. 18.5" barrel. 10-shot capacity.


text:
They supply uncomplicated and interactive gameplay, plus generous payout potential, so it’s no surprise they are a gamers’ favourite. Players were not prosecuted for playing in these overseas online gambling establishments. 21. co.uk now provides an amazing range of on-line sporting activities betting markets to bet on, any place you are, at any time! We give you with the very best markets included on the greatest events around the world, from football betting odds to today’s Horse Racing betting and also much more. We wished to give our clients the best gaming experience right to their mobile or tablet computer. Our web site additionally supplies a similar mobile experience.
Giving up one’s hand is also an option in some online blackjack games. Do bear in mind, nevertheless, increasing down and splitting entails making added, side bets. In regards to gameplay, video casino poker resembles slots, only this sort of online casino game offers gamers a more amazing experience as it permits decision-making which can affect the outcome. Video texas hold’em is a favorite amongst millions of casino fans due to the fact that it can really give them a positive assumption considered that they utilize correct method when playing their hands. Ports consist of a significant percent of the video gaming collections of all on-line casino sites. This is anything however a coincidence, however, given that the demand for this kind of game is absolutely unprecedented.
We do our utmost to offer you with an enjoyable experience, and tools to ensure you can play our video games in a secure way. Log into your account, and afterwards click on the ‘cashier’ section. You’ll after that see the alternative to click withdraw, and after that to choose your favored repayment technique. You’ll normally get your payment from our UK online gambling establishment in a couple of days.
This credentials nonetheless was never ever in position in connection with on-line gambling as it was only interested in gaming in casinos suggesting that anybody might subscribe as well as begin gambling online. My 20 complimentary spins were unlocked right after I deposited. The terms state they can only be used on Publication of Dead, which I found a little restricting, however still great, provided body is not a negative game. I do nonetheless desire the complimentary spins max payouts weren’t limited to simply ₤ 20. The Online Gambling enterprise’s sign-up offer is Wager ₤ 10 Obtain a ₤ 30 Free Bet, which means that if you down payment as well as wager ₤ 10 you will certainly receive a ₤ 30 single risk totally free bet, which can be used up in arms of 1.5+ within 7 days. There’s a welcome perk of approximately ₤ 100 plus 100 totally free rotates on guide of Dead video game, together with other promos such as everyday rewards and complimentary rotates, and also a cashback alternative.
Casinostugan fined EUR2.49 m by Swedish Video gaming Inspectorate as well as provided a warning for repetitively supplying perks in breach of regional pc gaming regulations. Online casinos can additionally include player monitoring systems that evaluate just how much cash as well as time. a gamer takes into each game. This allows them to act emphatically to control problematic gaming if certain behavioral patterns are found. As an example, blockchain is an advanced modern technology that has actually heavily influenced the gambling sector, and also the application of cryptocurrency in betting has actually made the video game faster as well as more secure. Casino.org is the globe’s leading independent online gaming authority, providing relied on online casino information, guides, reviews and information since 1995. We’ve opened a new line of enquiry to look further into challenges that people deal with when they try to withdraw their money after video gaming or betting online (whether as component of a promotion or otherwise).
Choose Titles With A Low “House Edge”
The wagering demands are iced up at 1x for all bonus cash money which offers you the upper hand when you win. You can also locate day-to-day discounts like Satisfied Hour Slots with 2x payouts on chosen video games as well as increases on particular table games. The stand-alone Fanduel Gambling enterprise was introduced to legal states in 2021 after being a prevalent sports wagering operation considering that 2018. FanDuel on-line gambling enterprise attributes slot video games from top companies like IGT, NetEnt, Microgaming, and much more.
Best Option In-play Wagering Websites Might 2023: Live-betting Bookmakers In The Uk
Play timeless card and table games, gambling enterprise poker variations or a series of Slots consisting of those you will not locate anywhere else. Whether it’s a deposit match, free spins, cash-back, or no-deposit offer, a great reward can make all the distinction to gameplay. With no-deposit perks being the gold requirement of welcome promotions, BetMGM online casino takes the cake with a $25 perk simply for subscribing. It may sound little, however you can bet it when as well as maintain whatever you win.
You require to be a minimum of 18 years old to discover our gaming horizon as component of our dedication to encourage responsible gambling. As we discussed earlier in the web page, we cover a selection of various betting topics varying from banking on sports via to playing gambling enterprise video games. Each topic has it’s very own section on the site with recommendations of betting websites. Those, that are willing to provide their abilities and also gambling establishment understanding a shot in a game versus a real, flesh-and-blood croupier, can conveniently do so from the convenience of their environments. The majority of well-known pc gaming drivers have committed Real-time Dealer sections on their web sites. These are house to a variety of standard essentials like baccarat, casino poker, roulette, and blackjack which are all organized by friendly and eye-catching croupiers in actual time.
Only perk funds count in the direction of betting contribution. 꽁머니 need to be used within 30 days, rotates within 10 days. Slots have actually long been just one of one of the most prominent online casino video games to play online, and also with modern rewards, bonus rounds and fancy motifs it’s not tough to see why. Our gambling establishment port selection below at 21. co.uk is considerable, to claim the least, and also it’s risk-free to say we have something for every person. You’ll discover some of the biggest names in casino pc gaming, like Jammin Jars, as well as Dessert Treasure Trove UK.
By doing this, you will verify that your info is precise and you are a real individual qualified to wager at a UK casino site. You even get to essentially scrape the surface of a card, so the experience is as practical as it can get. You no more have to find a newsstand or a shop to obtain your cards. Play anytime, anywhere, right on your PC or mobile. Sports betting is most likely the most preferred kind of gambling in Germany after the lottery game, particularly banking on football video games, which is ubiquitous. This is not surprising seeing as German teams are among the most effective in the world, as well as have the most dedicated fanbase.

domain: hobby


text:
Good nutrition is an important aspect of living a happy and healthy life. It is the foundation for your body to have the various nutrients necessary for energy, rest, and a sharp mind. There are many simple ways to incorporate good nutrition into your lifestyle to help you maintain a high level of activity.
Calcium is one of the most important things our bodies need to perform optimally. Whether you're nine years old or forty-nine years old, everyone needs calcium. At younger ages, calcium aids in the building of healthy bones. At older ages, our bones begin to deteriorate. Calcium can slow and even stop that process.
Proper nutrition includes eating plenty of vegetables and fruits each day. You should have about nine to thirteen servings of vegetables and fruits each day per the USDA. While it might sound like quite a bit, it isn't at all difficult to work them into your diet. Drink a glass of orange juice for breakfast of add tomato sauce to your spaghetti.
Make sure that you are getting enough vitamin E in your diet. Vitamin E is a great antioxidant that helps maintain the health of cell membranes. It has potential benefits in the protection of the heart against disease. Vitamin E is also important to the health of your eyes and plays a role in the prevention of cataracts.
Try not to buy into the fact that fruit juices and vegetable juices are a healthy beverage option. Many store bought varieties are riddled with sugar, sodium, and other additives that make them no better than something like a soda. It is best to stick to whole fruits. However, if you do desire fruit juice, the best way is to make your own with a juicer and fresh fruit and vegetables.
One aspect of healthy eating is sugar reduction. Many people believe that replacing soda with fruit juice is a good nutritional choice. This is not always true, since many commercial fruit juices contain high amounts of sugar. As you can see, being aware of what you are eating and drinking can make a big difference in your overall health.
23 Surprising Weight-Loss Tips That Are Actually Doable
The truth is, there's no one simple trick that will work for every person, in every culture, in every life situation. So run away, fast, from someone who tells you otherwise. 23 Surprising Weight-Loss Tips That Are Actually Doable
You can be very creative with food without your children even realizing it. Chop up some extra vegetables, and throw them in your chili. You can ever puree fruit, and add it to a smoothie, and your child will think they are having a nice cold dessert.
For best nutrition, don't make the mistake of thinking that brown eggs are somehow magically better than white eggs. Egg companies want you to think so; the mystique of brown eggs allow them to charge you up to 25% more than for white ones. But in reality, the differences are only shell-deep. White eggs are every bit as nutritious as their brown-shelled cousins, and are almost always cheaper.
More and more people are discovering that they are gluten intolerant or completely allergic to it with celiacs disease. Gluten is the protein found in wheat, barley, rye and other grains. If you have a sensitive stomach, or digestive issues, check with your doctor to see if you might need to be tested.
When baking, use whole wheat flour rather than white to up the nutrition value. This kind of flour has more nutrients and fiber, along with much less processing.
Tocopherols, which are commonly known as Vitamin E, are very important to one's diet. It is used as a signaling molecule by the body, as well as performing an important function as an antioxidant. http://felonypark36salina.blogminds.com/set-fitness-goals-and-reach-then-by-following-these-tips-3259893
is commonly found in wheat germ, safflower oil, and sunflower oils. Tocotrienols, which are in the same family, are even better antioxidants, and are found in wheat germ and some nuts and grains.
Even the people that eat the most healthy foods will enjoy a good dessert. collagen booster side effects
can be just as gratifying when done properly. For example, take a calcium enriched fat-free yogurt and top it with cinnamon, some frozen berries, or nutty granola. A graham cracker or shortbread cookie crumbled on top of the yogurt would also be delicious.
Want an idea on how to have good nutrition in your life? Make sure you have breakfast every day! Breakfast is necessary to maintain balance in your body and for it to produce the energy it needs to get through the day. It also makes you less likely to overeat later in the day and provides support for your brain and body to function.
By buying produce at a local farmers market or from a farm stand that one knows of in the area, an individual can get a variety of locally grown fruits and vegetables. Not only will one be getting the nutritional benefits from the fruits and vegetables but they will be supporting their local farmers.
Eating peanut butter can be an affordable way for a person to add more protein for a better nutritional balance. The peanut butter can be bought in chunky or creamy varieties and made into many different foods including sandwiches, shakes, crackers and anything one can dream up. Peanut butter has many benefits to offer nutritionally.
In this article we have discussed some tips that can make it easier for athletes to maintain a nutritionally sound diet. Proper nutrition is an essential part of any fitness program and without it, you will never see the results you're hoping for. Keep http://felonysecond98angeline.shotblogs.com/accomplish-physical-fitness-and-enjoy-your-life-3025078
in mind, and follow them daily for optimal results from your fitness routine.

domain: food


text:
sliding bathroom door wall mount sliding door bathroom sliding bathroom door uk.
glass entry door double glass front entry doors front door decor.
curved firepit benches curved fire pit benches with backs seating und chairs best ideas on curved fire pit benches.
teenage girl bedroom teen girls bedroom blush navy teenage girl comforter bed sets.
modern exterior light fixtures modern outdoor light fixture outdoor wall mount led light fixtures modern exterior lights fixtures modern outdoor mid century modern outdoor light fixtures.
tension pole lamp home decor in downers grove interior decorating classifieds.
mid century modern media console contemporary media consoles mid century modern media stand mid century modern media console elegant contemporary stand mid century modern media console 80.
28 x 80 door in x in white 2 panel shaker solid core pine interior 28 x 80 door rough opening.
hollywood regency furniture regency furniture on a budget love the faux bamboo style hollywood regency furniture west palm beach.
exposed rafter ceiling full size of decorating cakes with flowers for meme pumpkins toddlers next came the roof exposed rafter porch ceiling.

domain: design


text:
If the USC Trojans are to consider the 2011 season a success, they'll need to win at least three of their remaining four games, beginning with tonight's visit to chilly Folsom Field and the University of Colorado.
Speaking of Colorado, one of the major carrots for the Buffaloes joining the Pac-12 was the opportunity to recruit more intensely throughout California, specifically Southern California. By aggressively lobbying to be placed in the Pac-12 South Division, the Buffs got an annual trip into SoCal to showcase their program.
The Buffaloes have 29 Californians listed on their roster this season. Conversely, the Trojans have just one player from the state of Colorado, walk-on corner Allen Noble from Denver Eaglecrest High School by way of Mt. San Antonio Community College.

domain: sports


text:
OFF ROAD CARAVANS
Solo, a Couple or Family Off Road Caravans
At NEWGEN Caravans we specialise in being off road caravan manufacturers, so naturally, our range is built using the latest in cutting-edge off-road caravan design and engineering. We start with an aluminium frame and finish the walls, roof, and floor with fibreglass sandwich panels.
The result is a full off-road caravan that is both lightweight and extremely durable. If you’re an off-road explorer who wants to travel through rough terrain around Australia without sacrificing comfort or style, you’re in the right place. Welcome to your ultimate chill-out zone!
Request A Brochure
What makes an Off Road Caravan?
The design and manufacturing of an off-road caravan must take into consideration a few fundamentals to ensure it can handle Australian landscapes.
- The coupling needs to be able to articulate in any direction so your caravan follows your tow vehicle through rough conditions.
- Independent suspension allows your caravans wheels to raise and lower independently. This is a must as the terrain you’ll be driving over won’t always be even.
- Large all-terrain wheels offer greater ground clearance, durability and wear resistance than standard or mud-terrain tyres.
- A fully welded aluminium frame is ideal as it strikes the perfect balance of being lightweight and durable, and won’t rust or rot over time.
- The caravans’ cabinetry and furniture should be reinforced into the frame so they are protected when your caravan is twisting and bouncing over Australia’s harsh conditions.
Off Road Caravans Exterior Features
Our range of off-road caravans are adventure ready with features like:
- Higher ground clearance than your average on-road or semi off-road caravan
- 2 x 95ltr water tanks to keep you in the great outdoors for longer
- Pedders TrakRyder 4×4 Heavy Duty Shock Absorbers and Springs to handle the rough terrain around Australia
- Black checker plate protection around the caravan
Off Road Caravans Interior Features
Our range of off-road caravans come standard with creature comforts like:
- A separate toilet and shower
- Reverse-cycle air conditioning for your comfort
- 24″ Smart TV for when the weather turns
- 171ltr 2-door fridge/freezer for storing plenty of food and drinks
Ready to Journey into the Great Outdoors?
Every one of our off road caravans is designed to meet the specific needs of its owner. Whether you’re looking to get off the beaten track and do some off-grid camping on long weekends, or a full-time living space for travelling all over this beautiful country we call Australia, there’s an off-road caravan in our range that’s perfect for you. Download a brochure below or build your own caravan and receive a quote today!


text:
Program available at/via: [Online (full)]
Department Overview: Turf Management offers training for a wide variety of industries from golf courses to sports fields to parks departments with a focus on environmental awareness and appreciation. The study of turf management includes: turfgrass application, installation and management; equipment operation and maintenance; and irrigation system installation. Students in the program have the opportunity to participate in a work experience as part of the program to gain relevant hands-on and field experience to prepare for the industry. The program can be completed fully or partially online depending on student learning preference. The Turf Management curriculum is reviewed by an advisory board composed of local and regional industry members.
Program Level Outcomes:
- To provide a variety of courses in order to prepare students for careers in turf management.
- Provide relevant training through hands-on and field experience to prepare the students for industry.
- To encourage students to explore and develop critical thinking and creative thinking.
- To help students develop and perfect communication skills.
- To assist students in understanding and using the concepts of each course.
- To develop increased environmental awareness and appreciation.
- To help students develop and perfect the most efficient use of natural resources.
Degrees: The Associate of Applied Science (AAS) degree in Turf Management is a two-year degree program that prepares students for work in the industry. A one-year certificate in Turf Management is also available. The program can be completed fully or partially online depending on student learning preference.
Industry Description: The turf management industry has shown a steady growth rate in job opportunities for trained turf professionals. The turf management sector employs individuals as groundskeepers, golf course maintenance supervisors, parks grounds supervisors, sports field supervisors, turf landscape technicians, turf grass specialists, and sod farm managers.
Entrance Requirements: Students may enter the program fall, winter, or spring quarter, however, due to course sequencing, it is recommended to begin in the fall. WWCC has an open admissions process. It’s free to apply and everyone who applies is accepted. Applications are accepted year-round! Apply online.
Other Information: For additional information including regional employment data, completion rates, student characteristics, and employment see http://www.careerbridge.wa.gov.

domain: agronomy


text:
Fears of a blaze developing at The Bay Academy in Sandown have been dowsed, with smoke from a nearby bonfire being blamed for the alert tonight (Friday).
3 retained appliances from Sandown, Shanklin and Ryde, the aerial ladder platform from Ryde and the Incident Command Unit from Newport were all mobilised to the school on The Fairway at 20:27.
It was reported that smoke could be seen coming from the secondary school building, however, the first crews on scene have been able to quickly ascertain that there is no fire present, despite the smell of burning.
Firefighters have identified that the smoke lingering in the air is likely coming from a nearby bonfire in a residential area.
Crews are standing down and returning to station having double checked the area.


text:
Gents, move aside - The women are taking to the field in record numbers. Jess Rice of WildHERness joins Gun Talk's Kevin "KJ" Jarnagin to discuss the importance of getting new female hunters into the wilds, and empowering women by teaching them outdoor skills so they don't have to rely on anyone else, barriers to getting involved, and more. This Gun Talk Hunt is proudly supported by ATN and Pyramyd Air.
For more videos like this one, subscribe to Gun Talk at www.guntalktv.com, on Gun Talk's Roku, Amazon Fire TV, Apple TV, Android TV, iOS app, Android app, Chromecast, or find Gun Talk on YouTube, Facebook, or Instagram.
Copyright ©2021 Freefire Media, LLC


text:
Not my type font was created with a real typewriter. To be specific it was on Oliver courier typewriter that I bought at an antiques market.
I originally bought it for one of my image packs but it seemed to make sense to create a typeface from it.
I created 3 versions of every letter and number that get progressively bolder. If you enable contextual alternatives in your design application the letters will change automatically as you type.
The aim was to make it look more random, like a real typewriter.
Lot of detail
I went to great trouble to keep the detail from my original scans. I've yet to see another typewriter font that has the same amount of detail as this one.
The font includes:
- One style
- Uppercase & Lowercase letters with three Alternatives
- Numbers with three alternatives
- Basic punctuation & Symbols
- Font format is .OTF
If you want your design to have that nostalgic type effect this is perfect. It's old fashioned and retro with just a hint of grunge.
Optimum SizeAny Size
Commercial LicenseFurther Information


text:
Fourty-four thousand hospital and long-term care workers in BC have a tentative new labour deal with the province.
More than 90 percent of the workers are members of the Hospital Employees Union who still have to ratify the pact.
No specific word yet on wages, but the union says it includes initiatives to tackle the high rate of injuries and violence workers face.
“We’re pleased that health employers and government have agreed to work with us to tackle workplace injuries and violence at a system-wide level,” says Jennifer Whiteside, HEU secretary-business manager . “It’s the only way we’ll achieve sustainable and significant health and safety improvements for our members.”
It also restores contracting out protections for first time in more than 16 years.
Full details of the agreement which include compensation increases in line with the government’s negotiating mandate for public sector employers, will be provided to union members in advance of ratification votes which are yet to be scheduled.
The tentative agreement was reached after nine-weeks of talks which began in early September.
Workers covered by the agreement work in hundreds of job classifications in hospitals and health clinics, long-term care homes, corporate offices and supply warehouses.
They work in direct patient and resident care as well as in support services, technical, clerical and trades and maintenance areas.
In addition to HEU, the Facilities Bargaining Association includes workers in nine other unions, including the B.C. Government and Service Employees’ Union and the International Union of Operating Engineers.


text:
Pure Foods is an innovator in the specialty food movement focused on providing better for you substitutes in consumers’ favorite categories.Read More
Unibe branding agency added new tastes of curd glazed cheese “3K2K” brand.
Mielizia is a brand of Conapi, the national consortium of beekeepers in Italy and the most important cooperative of beekeepers in Europe.Read More
It is only natural that Greek yogurt reigns supreme in its birthplace, Greece. Countless options from big and small producers from all over the country vie for a very demanding and increasingly educated audience. Avramidis Family is a small-scale dairy with big plans.Read More
Levital exists on Kazakhstan market more than 5 years and locally is main competitor of Activia (Danone).Read More
Brand identity design for a milk bar in New Zealand, serving a variety of locally produced organic dairy products.Read More
This is a yogurt range for kids from 3 years old. Kids study and discover the world through simple forms, they better comprehend abstract, simple, geometric and pure shapes and colors.Read More
The family farm of Cheburashkini Brothers is a challenging project for the Russian market.Read More
"Dairy Culture" is positioned as a brand of modern high-quality dairy products to improve the quality of life. Related aspects of the brand’s culture are high technological solutions and present-day and advanced approach to dairy products, constant improvement of its consumer characteristics.Read More
Gammeldags is the oldest Danish ice cream brand, created in 1933. Gammeldags is more than just an ice cream brand.Read More
“Verhovye” is a dairy brand developed by Mildberry for a business alliance of X5 Retail Group (Russia) and Nesvizh Baby Food Plant (Belarus). "Verhovye” are created together by farmers, producers, and retailers that united to control the full production cycle from the farm to the shelf and provide consumers with the best quality dairy produce.Read More
Flaugnarde is a family-run company born from traditional family recipes. It brings exciting new premium dessert products to the Australian market.Read More
Delightly asked us for a digital and outdoor campaign to introduce their new brand… We created a ‘light’ look and feel, clearly highlighted the benefits, and developed a distinct cheeky tone of voice… delightly does it!Read More
Wilcox’s Ice Cream is “Vermont’s Original Ice Cream,” started in 1928 by the Wilcox brothers of Manchester, Vermont.Read More
Unibe branding agency re-launched milk brand “3K2K” bio-yogurts.Read More
Müller Light launches its first Italian-style super creamy yoghurt, Amore, offering health-conscious customers a special moment of ‘me time’ indulgence that is fat free.Read More
Organic puree – a new product line from the baby food Brand MAMUKO.Read More
SILK MILK is a concept design packaging and naming made in vintage style for farm organic natural milk.
The uniqueness is that it is an experiment in applying the vintage design style for milk, which in itself is unusual, but pleasant.
The project is a packaging design for a fictive organic milk producer company, the base of the design is the cow-shaped pattern, I have used real cow fur to the texture.Read More
Pinker is a brand for dairy products. In the beginning of project, we wanted to create a distinctive concept for this product.Read More