Mining novel starch-converting Glycoside Hydrolase 70 enzymes from the Nestlé Culture Collection genome database: The Lactobacillus reuteri NCC 2613 GtfB

Abstract

The Glycoside hydrolase (GH) family 70 originally was established for glucansucrases of lactic acid bacteria (LAB) converting sucrose into α-glucan polymers. In recent years we have identified 3 subfamilies of GH70 enzymes (designated GtfB, GtfC and GtfD) as 4,6-α-glucanotransferases, cleaving (α1 → 4)-linkages in maltodextrins/starch and synthesizing new (α1 → 6)-linkages. In this work, 106 putative GtfBs were identified in the Nestlé Culture Collection genome database with ~2700 genomes, and the L. reuteri NCC 2613 one was selected for further characterization based on variations in its conserved motifs. Using amylose the L. reuteri NCC 2613 GtfB synthesizes a low-molecular-mass reuteran-like polymer consisting of linear (α1 → 4) sequences interspersed with (α1 → 6) linkages, and (α1 → 4,6) branching points. This product specificity is novel within the GtfB subfamily, mostly comprising 4,6-α-glucanotransferases synthesizing consecutive (α1 → 6)-linkages. Instead, its activity resembles that of the GtfD 4,6-α-glucanotransferases identified in non-LAB strains. This study demonstrates the potential of large-scale genome sequence data for the discovery of enzymes of interest for the food industry. The L. reuteri NCC 2613 GtfB is a valuable addition to the starch-converting GH70 enzyme toolbox. It represents a new evolutionary intermediate between families GH13 and GH70, and provides further insights into the structure-function relationships of the GtfB subfamily enzymes.

Biotechnological potential of novel glycoside hydrolase family 70 enzymes synthesizing α-glucans from starch and sucrose

“…An inventory of the GH70 enzymes present in the Nestlé Culture Collection genome database resulted in identification of 106 putative GtfB enzymes. Only seven of these GtfB-like protein sequences displayed further differences in motifs II and IV (Gangoiti et al, 2017c), namely at the same positions (1029, 1137 and 1140, Gtf180 numbering) as observed for L. fermentum GtfB 4,3-α-GTase (see Fig. 2C, D for more details).…”

“…α-GTase(Kralj et al, 2011;Leemhuis et al, 2013a;Bai et al, 2015), the L. fermentum 4,3-α-GTase(Gangoiti et al, 2017b), and the L. reuteri NCC 2613 GtfB 4,6-α-GTase(Gangoiti et al, 2017c).…”

Synthesis of novel α-glucans with potential health benefits through controlled glucose release in the human gastrointestinal tract

“…Lactobacillus reuteri NCC 2613 GtfB (Gangoiti, van Leeuwen, Meng, et al 2017 was found to encode two GH70 enzymes (named DSRE and BRS-A) specialized in (a1!2) transglycosylation from sucrose to a dextran acceptor substrate (Brison et al 2012;Passerini et al 2015). The gluco-oligosaccharides containing (a1!2) linkages synthesized by L. mesenteroides NRRL B-1299 DSRE were found to be highly resistant to the action of digestive enzymes in both humans and animals (Valette et al 1993).…”

“…In recent years, novel GH70 enzymes inactive on sucrose, but displaying clear disproportionating activity on starch/ maltodextrin substrates, have been identified (Table 3) (Gangoiti et al 2015;Gangoiti, Pijning, and Dijkhuizen 2016;Gangoiti, Lamothe, et al 2017;Gangoiti, van Leeuwen, Gerwig, et al 2017;Gangoiti, van Leeuwen, Meng, et al 2017;Kralj et al 2011;Leemhuis, Dijkman, et al 2013). Regarding their substrate specificity, these starch-converting GH70 enzymes resemble GH13 family enzymes also acting on starch-like substrates, however, they share higher amino acid sequence similarity with GS from the GH70 family.…”

“…It has been proposed that evolution from an ancestor a-amylase to present-day GS occurred via these GtfB-, GtfC-, and GtfD-like intermediates (Gangoiti et al 2015;Gangoiti, Pijning, and Dijkhuizen 2016;Gangoiti, van Leeuwen, Gerwig, et al 2017;Kralj et al 2011). In addition to their scientific relevance, GtfB, GtfC and GtfD type of enzymes represent very interesting and powerful enzymatic tools for the conversion of the starch present in food matrices into soluble novel dietary fibers and/or slowly-digestible carbohydrates, and thus, they have gained substantial interest for the development of healthier starchy food products (Gangoiti, van Leeuwen, Meng, et al 2017). However, only few starch-converting GH70 enzymes have been characterized so far.…”