Substrate recognition mode of a glycoside hydrolase family 42 ß-galactosidase from Bifidobacterium longum subspecies infantis (BiBga42A) revealed by crystallographic and mutational analyses.

Aim: Bifidobacterium longum subsp. infantis uses a glycoside hydrolase (GH) family 42 ß-galactosidase (BiBga42A) for hydrolyzing lacto-N-tetraose (LNT), which is the most abundant core structure of human milk oligosaccharides (HMOs). As such, BiBga42A represents one of the pivotal enzymes underpinning the symbiosis between bifidobacteria and breastfed infants. Despite its importance, the structural basis underlying LNT hydrolysis by BiBga42A is not understood. Moreover, no substrate-complexed structures are available to date for GH42 family members. Methods: X-ray crystallography was used to determine the structures of BiBga42A in the apo- and liganded forms. The roles of the amino acid residues that were presumed to be involved in catalysis and substrate recognition were examined by a mutational study, in which kinetic parameters of each mutant were determined using 4-nitrophenyl-ß-D-galactoside, lacto-N-biose I, LNT, and lacto-N-neotetraose (LNnT) as substrates. Conservation of those amino acid residues was examined among structure-determined GH42 ß-galactosidases. Results: Crystal structures of the wild-type enzyme complexed with glycerol, the E160A/E318A double mutant complexed with galactose (Gal), and the E318S mutant complexed with LNT were determined at 1.7, 1.9, and 2.2 Å resolutions, respectively. The LNT molecule (excluding the Gal moiety at subsite +2) bound to the E318S mutant is recognized by an extensive hydrogen bond network and several hydrophobic interactions. The non-reducing end Gal moiety of LNT adopts a slightly distorted conformation and does not overlap well with the Gal molecule bound to the E160A/E318A mutant. Twelve of the sixteen amino acid residues responsible for LNT recognition and catalysis in BiBga42A are conserved among all homologs including ß-1,6-1,3-galactosidase (BlGal42A) from Bifidobacterium animalis subsp. lactis. Conclusion: BlGal42A is active on 3-ß-galactobiose similarly to BiBga42A but is inactive on LNT. Interestingly, we found that the entrance of the catalytic pocket of BlGal42A is narrower than that of BiBga42A and seems not easily accessible from the solvent side due to the presence of two bulky amino acid side chains. The specificity difference may reflect the structural difference between the two enzymes.

Production of Lacto-N-biose I Using Crude Extracts of Bifidobacterial Cells.

Lacto-N-biose I (LNB) is supposed to represent the bifidus factor in human milk oligosaccharides, and can be practically produced from sucrose and GlcNAc using four bifidobacterial enzymes, 1,3-ß-galactosyl-N-acetylhexosamine phosphorylase, sucrose phosphorylase, UDP-glucose-hexose 1-phosphate uridylyltransferase, and UDP-glucose 4-epimerase, recombinantly produced by Escherichia coli. Here the production of LNB by the same enzymatic method without using genetically modified enzymes to consider the use of LNB for a food ingredient was reported. All four enzymes were produced as the intracellular enzymes of Bifidobacterium strains. The mixture of the crude extracts contained all four enzymes, with other enzymes interfering with the LNB production, namely, phosphoglucomutase, fructose 6-phosphate phosphoketolase, and glycogen phosphorylase. The first two interfering enzymes were selectively inactivated by heat treatment at 47 °C for 1 h in the presence of pancreatin, and glycogen phosphorylase was disabled by hydrolyzing its possible acceptor molecules using glucoamylase. Finally, 91 % of GlcNAc was converted into LNB in the 100-mL reaction mixture containing 300 mM GlcNAc.

Next-generation prebiotic promotes selective growth of bifidobacteria, suppressing Clostridioides difficile.

Certain existing prebiotics meant to facilitate the growth of beneficial bacteria in the intestine also promote the growth of other prominent bacteria. Therefore, the growth-promoting effects of ß-galactosides on intestinal bacteria were analyzed. Galactosyl-ß1,4-l-rhamnose (Gal-ß1,4-Rha) selectively promoted the growth of Bifidobacterium. Bifidobacterium longum subsp. longum 105-A (JCM 31944) has multiple solute-binding proteins belonging to ATP-binding cassette transporters for sugars. Each strain in the library of 11 B. longum subsp. longum mutants, in which each gene of the solute-binding protein was disrupted, was cultured in a medium containing Gal-ß1,4-Rha as the sole carbon source, and only the BL105A_0502 gene-disruption mutant showed delayed and reduced growth compared to the wild-type strain. BL105A_0502 homolog is highly conserved in bifidobacteria. In a Gal-ß1,4-Rha-containing medium, Bifidobacterium longum subsp. infantis JCM 1222T, which possesses BLIJ_2090, a homologous protein to BL105A_0502, suppressed the growth of enteric pathogen Clostridioides difficile, whereas the BLIJ_2090 gene-disrupted mutant did not. In vivo, administration of B. infantis and Gal-ß1,4-Rha alleviated C. difficile infection-related weight loss in mice. We have successfully screened Gal-ß1,4-Rha as a next-generation prebiotic candidate that specifically promotes the growth of beneficial bacteria without promoting the growth of prominent bacteria and pathogens.

Large scale production of lacto-N-biose I, a building block of type I human milk oligosaccharides, using sugar phosphorylases.

Human milk oligosaccharides (HMOs) have drawn attention for their contribution to the explosive bifidobacterial growth in the intestines of neonates. We found that bifidobacteria can efficiently metabolize lacto-N-biose I (LNB), the major building blocks of HMOs, and we have developed a method to synthesize LNB by applying this system. We produced LNB on a kilogram scale by the method. This proved that, among the enterobacteria, only bifidobacteria can assimilate LNB, and provided the data that supported the explosive growth of bifidobacteria in neonates. Furthermore, we were also able to reveal the structure of LNB crystal and the low stability for heating at neutral pH, which has not been clarified so far. In this paper, using bifidobacteria and LNB as examples, I describe the research on oligosaccharide synthesis that was conducted by utilizing a sugar metabolism.Abbreviations: LNB: lacto-N-biose I; GNB: galacto-N-biose; HMOs: human milk oligosaccharides; GLNBP: GNB/LNB phosphorylase; NahK: N-acetylhexosamine 1-kinase; GalT: UDP-glucose-hexose-1-phosphate uridylyltransferase; GalE: UDP-glucose 4-epimerase; SP: sucrose phosphorylase.

Structural basis for broad substrate specificity of UDP-glucose 4-epimerase in the human milk oligosaccharide catabolic pathway of Bifidobacterium longum.

Infant gut-associated bifidobacteria has a metabolic pathway that specifically utilizes lacto-N-biose I (Gal-ß1,3-GlcNAc) and galacto-N-biose (Gal-ß1,3-GalNAc) from human milk and mucin glycans. UDP-glucose 4-epimerase (GalE) from Bifidobacterium longum (bGalE) catalyzes epimerization reactions of UDP-Gal into UDP-Glc and UDP-GalNAc into UDP-GlcNAc with the same level of activity that is required to send galacto-hexoses into glycolysis. Here, we determined the crystal structures of bGalE in three ternary complex forms: NAD+/UDP, NAD+/UDP-GlcNAc, and NAD+/UDP-Glc. The broad specificity of bGalE was explained by structural features of the binding pocket for the N-acetyl or C2 hydroxy group of the substrate. Asn200 is located in a pocket of the C2 group, and its side chain adopts different conformations in the complex structures with UDP-Glc and UDP-GlcNAc. On the other side, Cys299 forms a large pocket for the C5 sugar ring atom. The flexible C2 pocket and the large C5 pocket of bGalE are suitable for accommodating both the hydroxy and N-acetyl groups of the substrate during sugar ring rotation in the catalytic cycle. The substrate specificity and active site structure of bGalE were distinct from those of Esherichia coli GalE but similar to those of human GalE.

Structural insights into the difference in substrate recognition of two mannoside phosphorylases from two GH130 subfamilies.

In Ruminococcus albus, 4-O-ß-D-mannosyl-D-glucose phosphorylase (RaMP1) and ß-(1,4)-mannooligosaccharide phosphorylase (RaMP2) belong to two subfamilies of glycoside hydrolase family 130. The two enzymes phosphorolyze ß-mannosidic linkages at the nonreducing ends of their substrates, and have substantially diverse substrate specificity. The differences in their mechanism of substrate binding have not yet been fully clarified. In the present study, we report the crystal structures of RaMP1 with/without 4-O-ß-D-mannosyl-d-glucose and RaMP2 with/without ß-(1→4)-mannobiose. The structures of the two enzymes differ at the +1 subsite of the substrate-binding pocket. Three loops are proposed to determine the different substrate specificities. One of these loops is contributed from the adjacent molecule of the oligomer structure. In RaMP1, His245 of loop 3 forms a hydrogen-bond network with the substrate through a water molecule, and is indispensible for substrate binding.

Characterization and crystal structure determination of ß-1,2-mannobiose phosphorylase from Listeria innocua.

Glycoside hydrolase family 130 consists of phosphorylases and hydrolases for ß-mannosides. Here, we characterized ß-1,2-mannobiose phosphorylase from Listeria innocua (Lin0857) and determined its crystal structures complexed with ß-1,2-linked mannooligosaccharides. ß-1,2-Mannotriose was bound in a U-shape, interacting with a phosphate analog at both ends. Lin0857 has a unique dimer structure connected by a loop, and a significant open-close loop displacement was observed for substrate entry. A long loop, which is exclusively present in Lin0857, covers the active site to limit the pocket size. A structural basis for substrate recognition and phosphorolysis was provided.

An inverting ß-1,2-mannosidase belonging to glycoside hydrolase family 130 from Dyadobacter fermentans.

The glycoside hydrolase family (GH) 130 is composed of inverting phosphorylases that catalyze reversible phosphorolysis of ß-D-mannosides. Here we report a glycoside hydrolase as a new member of GH130. Dfer_3176 from Dyadobacter fermentans showed no synthetic activity using α-D-mannose 1-phosphate but it released α-D-mannose from ß-1,2-mannooligosaccharides with an inversion of the anomeric configuration, indicating that Dfer_3176 is a ß-1,2-mannosidase. Mutational analysis indicated that two glutamic acid residues are critical for the hydrolysis of ß-1,2-mannotriose. The two residues are not conserved among GH130 phosphorylases and are predicted to assist the nucleophilic attack of a water molecule in the hydrolysis of the ß-D-mannosidic bond.

Open-close structural change upon ligand binding and two magnesium ions required for the catalysis of N-acetylhexosamine 1-kinase.

Infant gut-associated bifidobacteria possess a metabolic pathway to utilize lacto-N-biose (Gal-ß1,3-GlcNAc) and galacto-N-biose (Gal-ß1,3-GalNAc) from human milk and glycoconjugates specifically. In this pathway, N-acetylhexosamine 1-kinase (NahK) catalyzes the phosphorylation of GlcNAc or GalNAc at the anomeric C1 position with ATP. Crystal structures of NahK have only been determined in the closed state. In this study, we determined open state structures of NahK in three different forms (apo, ADP complex, and ATP complex). A comparison of the open and closed state structures revealed an induced fit structural change defined by two rigid domains. ATP binds to the small N-terminal domain, and binding of the N-acetylhexosamine substrate to the large C-terminal domain induces a closing conformational change with a rotation angle of 16°. In the nucleotide binding site, two magnesium ions bridging the α-γ and ß-γ phosphates were identified. A mutational analysis indicated that a residue coordinating both of the two magnesium ions (Asp228) is essential for catalysis. The involvement of two magnesium ions in the catalytic machinery is structurally similar to the catalytic structures of protein kinases and aminoglycoside phosphotransferases, but distinct from the structures of other anomeric kinases or sugar 6-kinases. These findings help to elucidate the possible evolutionary adaptation of substrate specificities and induced fit mechanism.

Functional reassignment of Cellvibrio vulgaris EpiA to cellobiose 2-epimerase and an evaluation of the biochemical functions of the 4-O-ß-D-mannosyl-D-glucose phosphorylase-like protein, UnkA.

The aerobic soil bacterium Cellvibrio vulgaris has a ß-mannan-degradation gene cluster, including unkA, epiA, man5A, and aga27A. Among these genes, epiA has been assigned to encode an epimerase for converting D-mannose to D-glucose, even though the amino acid sequence of EpiA is similar to that of cellobiose 2-epimerases (CEs). UnkA, whose function currently remains unknown, shows a high sequence identity to 4-O-ß-D-mannosyl-D-glucose phosphorylase. In this study, we have investigated CE activity of EpiA and the general characteristics of UnkA using recombinant proteins from Escherichia coli. Recombinant EpiA catalyzed the epimerization of the 2-OH group of sugar residue at the reducing end of cellobiose, lactose, and ß-(1→4)-mannobiose in a similar manner to other CEs. Furthermore, the reaction efficiency of EpiA for ß-(1→4)-mannobiose was 5.5 × 10(4)-fold higher than it was for D-mannose. Recombinant UnkA phosphorolyzed ß-D-mannosyl-(1→4)-D-glucose and specifically utilized D-glucose as an acceptor in the reverse reaction, which indicated that UnkA is a typical 4-O-ß-D-mannosyl-D-glucose phosphorylase.

Facile enzymatic synthesis of sugar 1-phosphates as substrates for phosphorylases using anomeric kinases.

Three sugar 1-phosphates that are donor substrates for phosphorylases were produced at the gram scale from phosphoenolpyruvic acid and the corresponding sugars by the combined action of pyruvate kinase and the corresponding anomeric kinases in good yields. These sugar 1-phosphates were purified through two electrodialysis steps. α-D-Galactose 1-phosphate was finally isolated as crystals of dipotassium salts. α-D-Mannose 1-phosphate and 2-acetamido-2-deoxy-α-D-glucose 1-phosphate were isolated as crystals of bis(cyclohexylammonium) salts.

Discovery of two ß-1,2-mannoside phosphorylases showing different chain-length specificities from Thermoanaerobacter sp. X-514.

We characterized Teth514_1788 and Teth514_1789, belonging to glycoside hydrolase family 130, from Thermoanaerobacter sp. X-514. These two enzymes catalyzed the synthesis of 1,2-ß-oligomannan using ß-1,2-mannobiose and d-mannose as the optimal acceptors, respectively, in the presence of the donor α-d-mannose 1-phosphate. Kinetic analysis of the phosphorolytic reaction toward 1,2-ß-oligomannan revealed that these enzymes followed a typical sequential Bi Bi mechanism. The kinetic parameters of the phosphorolysis of 1,2-ß-oligomannan indicate that Teth514_1788 and Teth514_1789 prefer 1,2-ß-oligomannans containing a DP ≥3 and ß-1,2-Man2, respectively. These results indicate that the two enzymes are novel inverting phosphorylases that exhibit distinct chain-length specificities toward 1,2-ß-oligomannan. Here, we propose 1,2-ß-oligomannan:phosphate α-d-mannosyltransferase as the systematic name and 1,2-ß-oligomannan phosphorylase as the short name for Teth514_1788 and ß-1,2-mannobiose:phosphate α-d-mannosyltransferase as the systematic name and ß-1,2-mannobiose phosphorylase as the short name for Teth514_1789.

Characterization of a thermophilic 4-O-ß-D-mannosyl-D-glucose phosphorylase from Rhodothermus marinus.

4-O-ß-D-Mannosyl-D-glucose phosphorylase (MGP), found in anaerobes, converts 4-O-ß-D-mannosyl-D-glucose (Man-Glc) to α-D-mannosyl phosphate and D-glucose. It participates in mannan metabolism with cellobiose 2-epimerase (CE), which converts ß-1,4-mannobiose to Man-Glc. A putative MGP gene is present in the genome of the thermophilic aerobe Rhodothermus marinus (Rm) upstream of the gene encoding CE. Konjac glucomannan enhanced production by R. marinus of MGP, CE, and extracellular mannan endo-1,4-ß-mannosidase. Recombinant RmMGP catalyzed the phosphorolysis of Man-Glc through a sequential bi-bi mechanism involving ternary complex formation. Its molecular masses were 45 and 222 kDa under denaturing and nondenaturing conditions, respectively. Its pH and temperature optima were 6.5 and 75 °C, and it was stable between pH 5.5-8.3 and below 80 °C. In the reverse reaction, RmMGP had higher acceptor preferences for 6-deoxy-D-glucose and D-xylose than R. albus NE1 MGP. In contrast to R. albus NE1 MGP, RmMGP utilized methyl ß-D-glucoside and 1,5-anhydro-D-glucitol as acceptor substrates.

Distinct substrate specificities of three glycoside hydrolase family 42 ß-galactosidases from Bifidobacterium longum subsp. infantis ATCC 15697.

Glycoside hydrolase family 42 (GH42) includes ß-galactosidases catalyzing the release of galactose (Gal) from the non-reducing end of different ß-d-galactosides. Health-promoting probiotic bifidobacteria, which are important members of the human gastrointestinal tract microbiota, produce GH42 enzymes enabling utilization of ß-galactosides exerting prebiotic effects. However, insight into the specificity of individual GH42 enzymes with respect to substrate monosaccharide composition, glycosidic linkage and degree of polymerization is lagging. Kinetic analysis of natural and synthetic substrates resembling various milk and plant galactooligosaccharides distinguishes the three GH42 members, Bga42A, Bga42B and Bga42C, encoded by the probiotic B. longum subsp. infantis ATCC 15697 and revealed the glycosyl residue at subsite +1 and its linkage to the terminal Gal at subsite -1 to be key specificity determinants. Bga42A thus prefers the ß1-3-galactosidic linkage from human milk and other ß1-3- and ß1-6-galactosides with glucose or Gal situated at subsite +1. In contrast, Bga42B very efficiently hydrolyses 4-galactosyllactose (Galß1-4Galß1-4Glc) as well as 4-galactobiose (Galß1-4Gal) and 4-galactotriose (Galß1-4Galß1-4Gal). The specificity of Bga42C resembles that of Bga42B, but the activity was one order of magnitude lower. Based on enzyme kinetics, gene organization and phylogenetic analyses, Bga42C is proposed to act in the metabolism of arabinogalactan-derived oligosaccharides. The distinct kinetic signatures of the three GH42 enzymes correlate to unique sequence motifs denoting specific clades in a GH42 phylogenetic tree providing novel insight into GH42 subspecificities. Overall, the data illustrate the metabolic adaptation of bifidobacteria to the ß-galactoside-rich gut niche and emphasize the importance and diversity of ß-galactoside metabolism in probiotic bifidobacteria.

Directed evolution to enhance thermostability of galacto-N-biose/lacto-N-biose I phosphorylase.

Galacto-N-biose/lacto-N-biose I phosphorylase (GLNBP) is the key enzyme in the enzymatic production of lacto-N-biose I. For the purpose of industrial use, we improved the thermostability of GLNBP by evolutionary engineering in which five substitutions in the amino acid sequence were selected from a random mutagenesis GLNBP library constructed using error-prone polymerase chain reaction. Among them, C236Y and D576V mutants showed considerably improved thermostability. Structural analysis of C236Y revealed that the hydroxyl group of Tyr236 forms a hydrogen bond with the carboxyl group of E319. The C236Y and D576V mutations together contributed to the thermostability. The C236Y/D576V mutant exhibited 20°C higher thermostability than the wild type.

Discovery of cellobionic acid phosphorylase in cellulolytic bacteria and fungi.

A novel phosphorylase was characterized as new member of glycoside hydrolase family 94 from the cellulolytic bacterium Xanthomonas campestris and the fungus Neurospora crassa. The enzyme catalyzed reversible phosphorolysis of cellobionic acid. We propose 4-O-ß-D-glucopyranosyl-D-gluconic acid: phosphate α-D-glucosyltransferase as the systematic name and cellobionic acid phosphorylase as the short names for the novel enzyme. Several cellulolytic fungi of the phylum Ascomycota also possess homologous proteins. We, therefore, suggest that the enzyme plays a crucial role in cellulose degradation where cellobionic acid as oxidized cellulolytic product is converted into α-D-glucose 1-phosphate and D-gluconic acid to enter glycolysis and the pentose phosphate pathway, respectively.

Discovery of ß-1,4-D-mannosyl-N-acetyl-D-glucosamine phosphorylase involved in the metabolism of N-glycans.

A gene cluster involved in N-glycan metabolism was identified in the genome of Bacteroides thetaiotaomicron VPI-5482. This gene cluster encodes a major facilitator superfamily transporter, a starch utilization system-like transporter consisting of a TonB-dependent oligosaccharide transporter and an outer membrane lipoprotein, four glycoside hydrolases (α-mannosidase, ß-N-acetylhexosaminidase, exo-α-sialidase, and endo-ß-N-acetylglucosaminidase), and a phosphorylase (BT1033) with unknown function. It was demonstrated that BT1033 catalyzed the reversible phosphorolysis of ß-1,4-D-mannosyl-N-acetyl-D-glucosamine in a typical sequential Bi Bi mechanism. These results indicate that BT1033 plays a crucial role as a key enzyme in the N-glycan catabolism where ß-1,4-D-mannosyl-N-acetyl-D-glucosamine is liberated from N-glycans by sequential glycoside hydrolase-catalyzed reactions, transported into the cell, and intracellularly converted into α-D-mannose 1-phosphate and N-acetyl-D-glucosamine. In addition, intestinal anaerobic bacteria such as Bacteroides fragilis, Bacteroides helcogenes, Bacteroides salanitronis, Bacteroides vulgatus, Prevotella denticola, Prevotella dentalis, Prevotella melaninogenica, Parabacteroides distasonis, and Alistipes finegoldii were also suggested to possess the similar metabolic pathway for N-glycans. A notable feature of the new metabolic pathway for N-glycans is the more efficient use of ATP-stored energy, in comparison with the conventional pathway where ß-mannosidase and ATP-dependent hexokinase participate, because it is possible to directly phosphorylate the D-mannose residue of ß-1,4-D-mannosyl-N-acetyl-D-glucosamine to enter glycolysis. This is the first report of a metabolic pathway for N-glycans that includes a phosphorylase. We propose 4-O-ß-D-mannopyranosyl-N-acetyl-D-glucosamine:phosphate α-D-mannosyltransferase as the systematic name and ß-1,4-D-mannosyl-N-acetyl-D-glucosamine phosphorylase as the short name for BT1033.

In vitro comparative evaluation of the impact of lacto-N-biose I, a major building block of human milk oligosaccharides, on the fecal microbiota of infants.

Lacto-N-biose I (LNB) is a potential factor for the selective growth of bifidobacteria. We previously reported that the species of bifidobacteria predominant in infant intestines might use LNB. We aimed to assess the prebiotic properties of LNB in comparison to other oligosaccharides using an in vitro fermentation system. Stool samples from formula-fed infants were inoculated with media containing a sole carbon source of 1% LNB, lactulose, raffinose, galactooligosaccharide, or mannanoligosaccharides. LNB significantly increased the total bifidobacterial population similarly to other oligosaccharides, but induced a significantly higher level of Bifidobacterium bifidum in comparison to other oligosaccharides. Furthermore, significantly lower concentrations of lactic acid and significantly higher concentrations of acetic acid were produced in cultures containing LNB in comparison to cultures that contained other oligosaccharides. In conclusion, LNB might have a beneficial effect on the fecal microbiota of infants and is a potential prebiotic for application in infant foods or supplements.

Characterization of a laminaribiose phosphorylase from Acholeplasma laidlawii PG-8A and production of 1,3-ß-D-glucosyl disaccharides.

We identified a glycoside hydrolase family 94 homolog (ACL0729) from Acholeplasma laidlawii PG-8A as a laminaribiose (1,3-ß-D-glucobiose) phosphorylase (EC 2.4.1.31). The recombinant ACL0729 produced in Escherichia coli catalyzed phosphorolysis of laminaribiose with inversion of the anomeric configuration in a typical sequential bi bi mechanism releasing α-D-glucose 1-phosphate and D-glucose. Laminaritriose (1,3-ß-D-glucotriose) was not an efficient substrate for ACL0729. The phosphorolysis is reversible, enabling synthesis of 1,3-ß-D-glucosyl disaccharides by reverse phosphorolysis with strict regioselectivity from α-D-glucose 1-phosphate as the donor and suitable monosaccharide acceptors (D-glucose, 2-deoxy-D-arabino-hexopyranose, D-xylose, D-glucuronic acid, 1,5-anhydro-D-glucitol, and D-mannose) with C-3 and C-4 equatorial hydroxyl groups. The D-glucose and 2-deoxy-D-arabino-hexopyranose caused significantly strong competitive substrate inhibition compared with other glucobiose phosphorylases reported, in which the acceptor competitively inhibited the binding of the donor substrate. By contrast, none of the examined disaccharides served as acceptor in the synthetic reaction.

Characterization of a bacterial laminaribiose phosphorylase.

Bacterial laminaribiose phosphorylase (LBP(bac)) was first identified and purified from cell-free extract of Paenibacillus sp. YM-1. It phosphorolyzed laminaribiose into α-glucose 1-phosphate and glucose, but did not phosphorolyze other glucobioses. It slightly phosphorolyzed laminaritriose and higher laminarioligosaccharides. The specificity of the degree of polymerization of the substrate was clearly different from that of the enzyme of Euglena gracilis (LBP(Eug)): LBP(bac) was more specific to laminaribiose than LBP(Eug). It showed acceptor specificity in reverse phosphorolysis similar to LBP(Eug). Cloning of the gene encoding LBP(bac) (lbpA) has revealed that LBP(bac) is a member of the glucoside hydrolase family 94, which includes cellobiose phosphorylase, cellodextrin phosphorylase, and N,N'-diacetylchitobiose phosphorylase. The genes that encode the components of an ATP-binding cassette sugar transporter specific to laminarioligosaccharides were identified upstream of lbpA, suggesting that the role of LBP(bac) is to utilize laminaribiose generated outside the cell. This role is different from that of LBP(Eug), which participates in the utilization of paramylon, the intracellular storage 1,3-ß-glucan.