An Extracellular Cell-Attached Pullulanase Confers Branched α-Glucan Utilization in Human Gut Lactobacillus acidophilus

Abstract

Of the few predicted extracellular glycan-active enzymes, glycoside hydrolase family 13 subfamily 14 (GH13_14) pullulanases are the most common in human gut lactobacilli. These enzymes share a unique modular organization, not observed in other bacteria, featuring a catalytic module, two starch binding modules, a domain of unknown function, and a C-terminal surface layer association protein (SLAP) domain. Here, we explore the specificity of a representative of this group of pullulanases, Lactobacillus acidophilus Pul13_14 (LaPul13_14), and its role in branched ␣-glucan metabolism in the well-characterized Lactobacillus acidophilus NCFM, which is widely used as a probiotic. Growth experiments with L. acidophilus NCFM on starch-derived branched substrates revealed a preference for ␣-glucans with short branches of about two to three glucosyl moieties over amylopectin with longer branches. Cell-attached debranching activity was measurable in the presence of ␣-glucans but was repressed by glucose. The debranching activity is conferred exclusively by LaPul13_14 and is abolished in a mutant strain lacking a functional LaPul13_14 gene. Hydrolysis kinetics of recombinant LaPul13_14 confirmed the preference for short-branched ␣-glucan oligomers consistent with the growth data. Curiously, this enzyme displayed the highest catalytic efficiency and the lowest K m reported for a pullulanase. Inhibition kinetics revealed mixed inhibition by ␤-cyclodextrin, suggesting the presence of additional glucan binding sites besides the active site of the enzyme, which may contribute to the unprecedented substrate affinity. The enzyme also displays high thermostability and higher activity in the acidic pH range, reflecting adaptation to the physiologically challenging conditions in the human gut.IMPORTANCE Starch is one of the most abundant glycans in the human diet. Branched ␣-1,6-glucans in dietary starch and glycogen are nondegradable by human enzymes and constitute a metabolic resource for the gut microbiota. The role of healthbeneficial lactobacilli prevalent in the human small intestine in starch metabolism remains unexplored in contrast to colonic bacterial residents. This study highlights the pivotal role of debranching enzymes in the breakdown of starchy branched ␣-glucan oligomers (␣-limit dextrins) by human gut lactobacilli exemplified by Lactobacillus acidophilus NCFM, which is one of the best-characterized strains used as probiotics. Our data bring novel insight into the metabolic preference of L. acidophilus for ␣-glucans with short ␣-1,6-branches. The unprecedented affinity of the debranching enzyme that confers growth on these substrates reflects its adaptation to the nutrient-competitive gut ecological niche and constitutes a potential advantage in cross-feeding from human and bacterial dietary starch metabolism.

An 1,4-α-glucosyltransferase defines a new maltodextrin catabolism scheme inLactobacillus acidophilus

“…Lactobacilli are industrially important and diverse bacteria that colonize a multitude of ecological niches including gastrointestinal tracts of humans and animals. L. acidophilus and closely related species from the acidophilus group are adapted to the small intestine of humans, where α-glucans from starch break-down are an abundant metabolic resource (6, 19).…”

“…MOS produced from starch and glycogen degradation by human digestive enzymes, other bacteria or by the extracellular pullulanase ( La Pul13_14; (19)) are internalised by specific transporters. An ATP-binding cassette transporter is conserved in the locus in Lactobacillus , but likely defect in the L. acidophilus NCFM due to the presence of a transposase (19). Odd numbered MOS are degraded into M3, whereas even numbered MOS are degraded to M2 by La GH13_20.…”

“…Recently, we have shown that the utilization of short branched α-glucans from starch degradation by L. acidophilus NCFM is conferred by a cell-attached 1,6-α-debranching enzyme ( La Pul13_14) (19). The MOS products from this enzyme or from human starch breakdown are likely internalized by an ATP-binding cassette transporter, which is conserved in MOS utilization gene clusters in lactobacilli together with amylolytic and catabolic enzymes (gene locus tags LBA1864−LBA1874 in L. acidophilus NCFM) (Fig.…”

Comparative genomics of human Lactobacillus crispatus isolates reveals genes for glycosylation and glycogen degradation: Implications for in vivo dominance of the vaginal microbiota

“…The identified gene putatively encodes a pullulanase type I enzyme belonging to the glycoside hydrolase family 13 [38]. Its closest ortholog is an extracellular cell-attached pullulanase found in L. acidophilus [32]. The L. crispatus pullulanase gene described here carries three conserved domains, comprising an N-terminal carbohydrate-binding module family 41, a catalytic module belonging to the pullulanase super family and a C-terminal bacterial surface layer protein (SLAP) [39] (Figure 6).…”

“…All of these deletions are upstream of the carbohydrate-binding module in a sequence encoding a putative signal peptide. Furthermore, the presence of a SLAP-domain suggests that this enzyme is assigned to the outermost S-layer of the cell wall and is hence expected to be capable of degrading extracellular glycogen [32]. Further functional experiments are needed to fully characterize this pullulanase enzyme and to assess whether it degrades intra- or extracellular glycogen.…”