Cellobiose 2-epimerase (CE) reversibly converts glucose residue to mannose residue at the reducing end of β-1,4-linked oligosaccharides. It efficiently produces epilactose carrying prebiotic properties from lactose, but the utilization of known CEs is limited due to thermolability. We focused on thermoholophilic Rhodothermus marinus JCM9785 as a CE producer, since a CE-like gene was found in the genome of R. marinus DSM4252. CE activity was detected in the cell extract of R. marinus JCM9785. The deduced amino acid sequence of the CE gene from R. marinus JCM9785 (RmCE) was 94.2% identical to that from R. marinus DSM4252. The N-terminal amino acid sequence and tryptic peptide masses of the native enzyme matched those of RmCE. The recombinant RmCE was most active at 80 °C at pH 6.3, and stable in a range of pH 3.2–10.8 and below 80 °C. In contrast to other CEs, RmCE demonstrated higher preference for lactose over cellobiose

Cellobiose 2-epimerase (CE), found mainly in anaerobes, reversibly converts D-glucose residues at the reducing end of β-1,4-linked oligosaccharides to D-mannose residues. In this study, we characterized CE-like proteins from various aerobes (Flavobacterium johnsoniae NBRC 14942, Pedobacter heparinus NBRC 12017, Dyadobacter fermentans ATCC 700827, Herpetosiphon aurantiacus ATCC 23779, Saccharophagus degradans ATCC 43961, Spirosoma linguale ATCC 33905, and Teredinibacter turnerae ATCC 39867), because aerobes, more easily cultured on a large scale than anaerobes, are applicable in industrial processes. The recombinant CE-like proteins produced in Escherichia coli catalyzed epimerization at the C2 position of cellobiose, lactose, epilactose, and β-1,4-mannobiose, whereas N-acetyl-D-glucosamine, N-acetyl-D-mannosamine, D-glucose, and D-mannose were inert as substrates. All the CEs, except for P. heparinus CE, the optimum pH of which was 6.3, showed highest activity at weakly alkaline pH. CEs from D. fermentans, H. aurantiacus, and S. linguale showed higher optimum temperatures and thermostability than the other enzymes analyzed. The enzymes from D. fermentans, S. linguale, and T. turnerae showed significantly high k(cat) and K(m) values towards cellobiose and lactose. Especially, T. turnerae CE showed a very high k(cat) value towards lactose, an attractive property for the industrial production of epilactose, which is carried out at high substrate concentrations.

An ␣-glucosidase (HaG) with the following unique properties was isolated from Halomonas sp. strain H11: (i) high transglucosylation activity, (ii) activation by monovalent cations, and (iii) very narrow substrate specificity. The molecular mass of the purified HaG was estimated to be 58 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). HaG showed high hydrolytic activities toward maltose, sucrose, and p-nitrophenyl ␣-D-glucoside (pNPG) but to almost no other disaccharides or malto-oligosaccharides higher than trisaccharides. HaG showed optimum activity to maltose at 30°C and pH 6.5. Monovalent cations such as K ؉ , Rb ؉ , Cs ؉ , and NH 4 ؉ increased the enzymatic activity to 2-to 9-fold of the original activity. These ions shifted the activity-pH profile to the alkaline side. The optimum temperature rose to 40°C in the presence of 10 mM NH 4 ؉ , although temperature stability was not affected. The apparent K m and k cat values for maltose and pNPG were significantly improved by monovalent cations. Surprisingly, k cat /K m for pNPG increased 372-to 969-fold in their presence. HaG used some alcohols as acceptor substrates in transglucosylation and was useful for efficient synthesis of ␣-D-glucosylglycerol. The efficiency of the production level was superior to that of the previously reported enzyme Aspergillus niger ␣-glucosidase in terms of small amounts of by-products. Sequence analysis of HaG revealed that it was classified in glycoside hydrolase family 13. Its amino acid sequence showed high identities, 60%, 58%, 57%, and 56%, to Xanthomonas campestris WU-9701 ␣-glucosidase, Xanthomonas campestris pv. raphani 756C oligo-1,6-glucosidase, Pseudomonas stutzeri DSM 4166 oligo-1,6-glucosidase, and Agrobacterium tumefaciens F2 ␣-glucosidase, respectively. ␣ -Glucosidase (EC 3.2.1.20, ␣-D-glucoside glucohydrolase) is a ubiquitous enzyme widely distributed in microorganisms, plants, and animals. It catalyzes liberation of ␣-glucose from the nonreducing ends of ␣-glucosides such as malto-oligosaccharides and sucrose. Its substrate specificity is significantly diverse, depending on the enzyme origins, and the enzymes are classified into three groups on the basis of their substrate specificities (10). Group I enzymes are more active toward heterogeneous substrates, represented by sucrose and synthetic glycosides such as p-nitrophenyl ␣-D-glucoside (pNPG) and phenyl ␣-D-glucoside, than toward homogeneous substrates such as maltose. Group II enzymes, known as maltases, prefer homogeneous substrates to heterogeneous substrates. Like group II enzymes, group III enzymes mainly hydrolyze homogeneous substrates but have high activity toward long-chain substrates like glycogen and soluble starch.According to the sequence-based classification of glycoside hydrolase (GH), ␣-glucosidases are generally classified into two families, GH families 13 (subfamily 30) and 31 (9). Enzymes from bacteria, some yeast such as Saccharomyces cerevisiae, and insects are mainly classified into the former family, and those...

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