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

Leemhuis, Hans; Dijkman, Willem P.; Dobruchowska, Justyna M.; Pijning, Tjaard; Grijpstra, Pieter; Kralj, Slavko; Kamerling, Johannis P.; Dijkhuizen, Lubbert

doi:10.1007/s00253-012-3943-1

Cited by 57 publications

(75 citation statements)

References 52 publications

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“…1). In a previous study, we reported that GtfB-like proteins form a small cluster clearly separate from the glucansucrase-type enzymes, with just six members (12). Because of the increased number of genome sequences available as of June 2015, the GtfB-like 4,6-␣-glucanotransferase GH70 subfamily contains 22 proteins.…”

Section: Resultsmentioning

confidence: 99%

“…Small signals corresponding to 6-substituted reducingend glucose residues (R␣ H-1, ␦ 5.241; R␤ H-1, ␦ 4.670) were also detected. These trace amounts of 6-substituted reducing-end glucose units are also found in the 1 H NMR spectra of the product mixtures produced by the GtfB-type enzyme, indicating that these 4,6-␣-glucanotransferases use glucose as an acceptor substrate and elongate it with one ␣1¡6-linked glucose residue at the nonreducing site (12). In summary, the 1 H-NMR spectra of the products generated by GtfC presented signals characteristic of the linear isomalto-/malto-oligomer products also synthesized by GtfB (11); thus, GtfC acts as a 4,6-␣-glucanotransferase.…”

Section: Figmentioning

confidence: 95%

“…Instead, GtfB enzymes act on starch/ maltodextrin substrates catalyzing a 4,6-␣-glucanotransferase reaction, cleaving ␣1¡4 linkages and introducing ␣1¡6 linkages in linear product chains. This results in the synthesis of isomalto/ malto-polysaccharides (IMMPs) that are digested not by human enzymes but by the gut microbiota, thus acting as a soluble starch dietary fiber (10)(11)(12)(13)(14). GtfB enzymes have a domain organization similar to that of glucansucrases but likely differ in the architecture of their active site (12).…”

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The Exiguobacteriumsibiricum 255-15 GtfC Enzyme Represents a Novel Glycoside Hydrolase 70 Subfamily of 4,6-α-Glucanotransferase Enzymes

Gangoiti

Pijning

Dijkhuizen

2016

Appl Environ Microbiol

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105

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The glycoside hydrolase 70 (GH70) family originally was established for glucansucrase enzymes found solely in lactic acid bacteria synthesizing ␣-glucan polysaccharides from sucrose (e.g., GtfA). In recent years, we have characterized GtfB and related Lactobacillus enzymes as 4,6-␣-glucanotransferase enzymes. These GtfB-type enzymes constitute the first GH70 subfamily of enzymes that are unable to act on sucrose as a substrate but are active with maltodextrins and starch, cleave ␣1¡4 linkages, and synthesize linear ␣1¡6-glucan chains. The GtfB disproportionating type of activity results in the conversion of malto-oligosaccharides into isomalto/malto-polysaccharides with a relatively high percentage of ␣1¡6 linkages. This paper reports the identification of the members of a second GH70 subfamily (designated GtfC enzymes) and the characterization of the Exiguobacterium sibiricum 255-15 GtfC enzyme, which is also inactive with sucrose and displays 4,6-␣-glucanotransferase activity with maltooligosaccharides. GtfC differs from GtfB in synthesizing isomalto/malto-oligosaccharides. Biochemically, the GtfB-and GtfCtype enzymes are related, but phylogenetically, they clearly constitute different GH70 subfamilies, displaying only 30% sequence identity. Whereas the GtfB-type enzyme largely has the same domain order as glucansucrases (with ␣-amylase domains A, B, and C plus domains IV and V), this GtfC-type enzyme differs in the order of these domains and completely lacks domain V. In GtfC, the sequence of conserved regions I to IV of clan GH-H is identical to that in GH13 (I-II-III-IV) but different from that in GH70 (II-III-IV-I because of a circular permutation of the (␤/␣) 8 barrel. The GtfC 4,6-␣-glucanotransferase enzymes thus represent structurally and functionally very interesting evolutionary intermediates between ␣-amylase and glucansucrase enzymes.T he starch-and sucrose-acting enzymes of the glycoside hydrolase 13 (GH13) and GH70 families are evolutionarily related, displaying similar protein folds and activity mechanisms (retaining, covalent intermediate, double displacement mechanism, three catalytic residues), constituting clan GH-H (http://www .cazy.org) (B. Svensson and Š. Janeček, glycoside hydrolase family 13 in CAZypedia, available at http://www.cazypedia.org/, accessed 2 June 2015; M. Remaud-Simeon, glycoside hydrolase family 70 in CAZypedia, available at http://www.cazypedia.org/, accessed 2 June 2015). They differ in their overall activities, degrading or modifying ␣-glucan substrates (starch, maltodextrins, GH13) (1, 2) or synthesizing ␣-glucan products (from sucrose, GH70) (3), e.g., by the Lactobacillus reuteri 121 GtfA enzyme (4). GH13 proteins have three domains (A, B, and C) with a common catalytic (␤/␣) 8 fold (triosephosphate isomerase [TIM] barrel); their active site is located in an open cavity between the A and B domains (5, 6). GH70 proteins share this domain organization, but their (␤/ ␣) 8 barrel is circularly permuted. Moreover, they possess unique domains IV and V (7). Recently, we ha...

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Section: Resultsmentioning

confidence: 99%

Section: Figmentioning

confidence: 95%

mentioning

confidence: 99%

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The Exiguobacteriumsibiricum 255-15 GtfC Enzyme Represents a Novel Glycoside Hydrolase 70 Subfamily of 4,6-α-Glucanotransferase Enzymes

Gangoiti

Pijning

Dijkhuizen

2016

Appl Environ Microbiol

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105

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“…For years the GH70 family has been mainly known to house glucansucrases. Recently, a new subgroup was disclosed that contains 4,6-␣ glucanotransferases, a type of ␣-transglucosylases that uses ␣-(134)-linked glucans as a donor instead of sucrose (42,43). According to our biochemical and phylogenetic analyses, branching sucrases could also constitute a new GH70 subgroup.…”

Section: Characterization Of Two New Putative Branching Sucrases and mentioning

confidence: 99%

Characterization of the First α-(1→3) Branching Sucrases of the GH70 Family

Vuillemin

Claverie

Brison

et al. 2016

Journal of Biological Chemistry

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Leuconostoc citreum NRRL B-742 has been known for years to produce a highly ␣-(133)-branched dextran for which the synthesis had never been elucidated. In this work a gene coding for a putative ␣-transglucosylase of the GH70 family was identified in the reported genome of this bacteria and functionally characterized. From sucrose alone, the corresponding recombinant protein, named BRS-B, mainly catalyzed sucrose hydrolysis and leucrose synthesis. However, in the presence of sucrose and a dextran acceptor, the enzyme efficiently transferred the glucosyl residue from sucrose to linear ␣-(136) dextrans through the specific formation of ␣-(133) linkages. To date, BRS-B is the first reported ␣-(133) branching sucrase. Using a suitable sucrose/dextran ratio, a comb-like dextran with 50% of ␣-(133) branching was synthesized, suggesting that BRS-B is likely involved in the comb-like dextran produced by L. citreum NRRL B-742. In addition, data mining based on the search for specific sequence motifs allowed the identification of two genes putatively coding for branching sucrases in the genome of Leuconostoc fallax KCTC3537 and Lactobacillus kunkeei EFB6. Biochemical characterization of the corresponding recombinant enzymes confirmed their branching specificity, revealing that branching sucrases are not only found in L. citreum species. According to phylogenetic analyses, these enzymes are proposed to constitute a new subgroup of the GH70 family.Numerous lactic acid bacteria from the Leuconostoc genus isolated from different habitats, such as sugar juice, fermenting vegetables, or dairy products, have long been known to produce slimes in sucrose solution (1, 2). These slimy compounds were rapidly characterized as dextrans, homopolymers of ␣-D-glucopyranosyl units mainly linked by ␣-(136) linkages. Dextrans with a high content of ␣-(136) linkages and a low degree of branching found their first industrial applications in the 1940s as a source of synthetic blood volume expanders (3). These findings motivated Jeanes et al. (22) to investigate the diversity of polymers produced during sucrose fermentation by different strains of lactic acid bacteria. A total of 96 strains were screened, and their dextrans were structurally characterized. In this study one strain, Leuconostoc mesenteroides NRRL B-742, also known as L. mesenteroides ATCC 13146 and renamed Leuconostoc citreum NRRL B-742 (4), received particular attention. Indeed, this strain, first isolated from a can of spoiled tomatoes in 1927 (1), was shown to produce two types of dextrans that differed by their structures and degree of solubility from sucrose. The less soluble polymer contained 75% of ␣-(136) linkages and 15% of ␣-(134) linkages, whereas the more soluble polymer displayed an uncommon comb-like structure consisting of a linear backbone of ␣-(136)-D-glucopyranosyl residues grafted with one ␣-(133)-linked glucosyl unit on every glucosyl moiety (5-9). Concomitantly with those findings, dextrans were shown to be synthesized by ␣-transglucosylases, commonly named g...

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“…It has been proposed that conversion occurs mainly by the stepwise addition of single glucose moieties onto the nonreducing end of an ␣-glucan, introducing linear chains with ␣1¡6 linkages (15-18). The 4,6-␣-GTase enzymes have strong potential for slowly digestible starch or soluble dietary fiber production in the starch industry (19).The expression yields of all three studied 4,6-␣-GTase enzymes in Escherichia coli are rather low, and most of the protein accumulates in inclusion bodies (18,20); to obtain more active protein, strategies to use denatured refolded GTFB protein, or nonclassical inclusion body preparations, have been tested. The biochemical and catalytic properties of these enzymes, e.g., their hydrolysis and transferase activities, have not been characterized yet because of a lack of suitable quantitative assays.…”

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confidence: 99%

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

Bai

Kaaij

Leemhuis

et al. 2015

Appl Environ Microbiol

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c 4,6-␣-Glucanotransferase (4,6-␣-GTase) enzymes, such as GTFB and GTFW of Lactobacillus reuteri strains, constitute a new reaction specificity in glycoside hydrolase family 70 (GH70) and are novel enzymes that convert starch or starch hydrolysates into isomalto/maltopolysaccharides (IMMPs). These IMMPs still have linear chains with some ␣1¡4 linkages but mostly (relatively long) linear chains with ␣1¡6 linkages and are soluble dietary starch fibers. 4,6-␣-GTase enzymes and their products have significant potential for industrial applications. Here we report that an N-terminal truncation (amino acids 1 to 733) strongly enhances the soluble expression level of fully active GTFB-⌬N (approximately 75-fold compared to full-length wild type GTFB) in Escherichia coli. In addition, quantitative assays based on amylose V as the substrate are described; these assays allow accurate determination of both hydrolysis (minor) activity (glucose release, reducing power) and total activity (iodine staining) and calculation of the transferase (major) activity of these 4,6-␣-GTase enzymes. The data show that GTFB-⌬N is clearly less hydrolytic than GTFW, which is also supported by nuclear magnetic resonance (NMR) analysis of their final products. From these assays, the biochemical properties of GTFB-⌬N were characterized in detail, including determination of kinetic parameters and acceptor substrate specificity. The GTFB enzyme displayed high conversion yields at relatively high substrate concentrations, a promising feature for industrial application. Starch is the second-most-abundant carbohydrate on earth and a major dietary carbohydrate for humans; as a storage carbohydrate it is present in seeds, roots, and tubers of plants (1). It consists of ␣-glucan polymers with ␣1¡4 linkages and a low percentage of ␣1¡6 linkages, in the form of amylose and branched amylopectin (2). Starches are applied in various industrial products such as food, paper, and textiles, often after processing by physical, chemical, or enzymatic treatment (3-6).Dietary fibers and low-glycemic-index (low-GI) food are considered healthy food contributing to our long-term well-being (7,8). Of all the nutritional types of starch, slowly digestible starch with low GI has drawn the strongest interest. Annealing/heatmoisture treatment, recrystallization, and enzymatic treatment are recognized approaches to obtain slowly digestible starch (9-11). Slowly digestible starch materials prepared by physical processing suffer losses upon boiling; therefore, structural modifications through enzymatic treatment of starch are more desirable. In the human digestive system, the ␣1¡6 linkages in starch are hydrolyzed at a lower rate than ␣1¡4 linkages (12, 13). Branching enzymes, alone or in combination with ␤-amylase, are used to increase the percentage of ␣1¡4,6 branches in starches (12)(13)(14).The 4,6-␣-glucanotransferase (4,6-␣-GTase) enzymes, such as GTFB, GTFW, and GTFML4, of Lactobacillus reuteri strains constitute a subfamily of glycoside hydrolase family 70 (GH70); GH70 ...

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

Cited by 57 publications

References 52 publications

The Exiguobacteriumsibiricum 255-15 GtfC Enzyme Represents a Novel Glycoside Hydrolase 70 Subfamily of 4,6-α-Glucanotransferase Enzymes

The Exiguobacteriumsibiricum 255-15 GtfC Enzyme Represents a Novel Glycoside Hydrolase 70 Subfamily of 4,6-α-Glucanotransferase Enzymes

Characterization of the First α-(1→3) Branching Sucrases of the GH70 Family

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

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