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

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105

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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confidence: 99%

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

Gangoiti

Pijning

Dijkhuizen

2016

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105

“…Unlike GSes, 4,6-␣-GTases are not sucrose-acting enzymes but rather starch-converting enzymes, capable of converting (1¡4)-␣-D-gluco-oligosaccharides into dietary fiber isomalto/maltopolysaccharides (IMMPs). 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)(16)(17)(18). The 4,6-␣-GTase enzymes have strong potential for slowly digestible starch or soluble dietary fiber production in the starch industry (19).…”

mentioning

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

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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 ...

“…1A). L. reuteri was chosen as our model LAB strain, as it is an endogenous mammalian gut strain more adapted to human colonization than other LAB species involved in food preparation, and much is known on the structure of EPS produced by this strain (25,30,31). Additionally, it is used as a probiotic supplement, such as treatment of Helicobacter pylori infections and infant ailments such as colic (32,33).…”

Section: Methodsmentioning

confidence: 99%

“…1A) (31, 34). Thus, three forms of L. reuteri EPS were used for this study: LrEPS from L. reuteri 121 grown on sucrose, which consists of both levan and reuteran, reuteran only, which was produced by the mutant L. reuteri 121 strain 35-5 lacking the levansucrase enzyme grown on sucrose (25,35), and IMMP from wild-type L. reuteri 121 grown on maltodextrins (31). B. thetaiotaomicron grown in the presence of 5 mg/ml LrEPS demonstrated only moderate growth on LrEPS (Fig.…”

Section: Methodsmentioning

confidence: 99%

Differential Metabolism of Exopolysaccharides from Probiotic Lactobacilli by the Human Gut Symbiont Bacteroides thetaiotaomicron

Bueren

Saraf

Martens

et al. 2015

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bProbiotic microorganisms are ingested as food or supplements and impart positive health benefits to consumers. Previous studies have indicated that probiotics transiently reside in the gastrointestinal tract and, in addition to modulating commensal species diversity, increase the expression of genes for carbohydrate metabolism in resident commensal bacterial species. In this study, it is demonstrated that the human gut commensal species Bacteroides thetaiotaomicron efficiently metabolizes fructan exopolysaccharide (EPS) synthesized by probiotic Lactobacillus reuteri strain 121 while only partially degrading reuteran and isomalto/malto-polysaccharide (IMMP) ␣-glucan EPS polymers. B. thetaiotaomicron metabolized these EPS molecules via the activation of enzymes and transport systems encoded by dedicated polysaccharide utilization loci specific for ␤-fructans and ␣-glucans. Reduced metabolism of reuteran and IMMP ␣-glucan EPS molecules may be due to reduced substrate binding by components of the starch utilization system (sus). This study reveals that microbial EPS substrates activate genes for carbohydrate metabolism in B. thetaiotaomicron and suggests that microbially derived carbohydrates provide a carbohydrate-rich reservoir for B. thetaiotaomicron nutrient acquisition in the gastrointestinal tract.T rillions of bacteria inhabit the gut, imparting symbiotic effects that benefit the overall health and well-being of individuals (1). While the human genome has approximately 30,000 genes, the human microbiome contributes an additional 3 to 9 million gene products (termed the gut metagenome) that contribute to functionalities in human lifestyle (2). One prominent example is the human utilization of carbohydrates. Carbohydrates provide a substantial proportion of the daily energy intake of an individual. However, humans have the ability to metabolize only very few dietary carbohydrate compounds, such as lactose, starch, and sucrose, by enzymes either present in saliva (amylases) or anchored to the epithelial wall of the small intestine with glycosylphosphatidylinositol (GPI) anchors (such as invertases and lactases). All other carbohydrates that traverse the gastrointestinal (GI) tract have the potential to be metabolized by commensal gut bacteria, where it is estimated that gut microbiota provide an additional 30,000 enzymes which facilitate the breakdown of carbohydrates in the gut (3).Approximately 30% of resident human gut microbiota are from the Bacteriodetes phylum. These Gram-negative, obligate anaerobes are well armed with a repertoire of carbohydrate-degrading enzymes for harvesting carbohydrate nutritional resources. One species, Bacteroides thetaiotaomicron, devotes approximately 18% of its genome to carbohydrate foraging (4). Discrete polysaccharide utilization loci (PULs) within its genome are upregulated in response to carbohydrates dependent on the source. Most carbohydrate sources for B. thetaiotaomicron nutrient acquisition originate from dietary sources (such as nondigestible plant polysaccharides)...