Takeshi Senoura

New microbial mannan catabolic pathway that involves a novel mannosylglucose phosphorylase.

The consecutive genes BF0771-BF0774 in the genome of Bacteroides fragilis NCTC 9343 were found to constitute an operon. The functional analysis of BF0772 showed that the gene encoded a novel enzyme, mannosylglucose phosphorylase that catalyzes the reaction, 4-O-β-d-mannopyranosyl-d-glucose+Pi→mannose-1-phosphate+glucose. Here we propose a new mannan catabolic pathway in the anaerobe, which involves 1,4-β-mannanase (BF0771), a mannobiose and/or sugar transporter (BF0773), mannobiose 2-epimerase (BF0774), and mannosylglucose phosphorylase (BF0772), finally progressing to glycolysis. This pathway is distributed in microbes such as Bacteroides, Parabacteroides, Flavobacterium, and Cellvibrio.

Prebiotic Properties of Epilactose

We recently reported that cellobiose 2-epimerase from Ruminococcus albus effectively converted lactose to epilactose. In this study, we examined the biological effects of epilactose on intestinal microbiota, bile acid metabolism, and postadministrative plasma glucose by animal tests. Dietary supplementation with epilactose or fructooligosaccharide (4.5% each) increased cecal wall weight and cecal contents and decreased the pH of the cecal contents in Wistar-ST rats. The number of total anaerobes tended to be greater in rats fed epilactose and fructooligosaccharide than in those fed the control diet. Lactobacilli and bifidobacteria were more numerous in rats fed epilactose and fructooligosaccharide diets than in those fed the control diet. Analysis of clone libraries of 16S rRNA suggests that supplementation with epilactose did not induce the proliferation of harmful bacteria belonging to classes Clostridia or Bacteroidetes. Epilactose, as well as fructooligosaccharide, inhibited the conversion of primary bile acids to secondary bile acids, which are suggested to be promoters of colon cancer. In addition, oral administration of epilactose did not elevate the plasma glucose concentration in ddY mice. These results clearly indicate that epilactose is a promising prebiotic. We also showed that cellobiose 2-epimerase converted lactose in cow milk and a spray-dried ultrafiltrate of cheese whey to epilactose. Cellobiose 2-epimerase may increase the value of dairy products by changing lactose to epilactose possessing prebiotic properties.

Cloning and sequencing of the gene for cellobiose 2-epimerase from a ruminal strain of Eubacterium cellulosolvens

Cellobiose 2-epimerase (CE; EC 5.1.3.11) is known to catalyze the reversible epimerization of cellobiose to 4-O-beta-D-glucopyranosyl-D-mannose in Ruminococcus albus cells. Here, we report a CE in a ruminal strain of Eubacterium cellulosolvens for the first time. The nucleotide sequence of the CE had an ORF of 1218 bp (405 amino acids; 46 963.3 Da). The CE from E. cellulosolvens showed 44-54% identity to N-acyl-D-glucosamine 2-epimerase-like hypothetical proteins in the genomes of Coprococcus eutactus, Faecalibacterium prausnitzii, Clostridium phytofermentans, Caldicellulosiruptor saccharolyticus, and Eubacterium siraeum. Surprisingly, it exhibited only 46% identity to a CE from R. albus. The recombinant enzyme expressed in Escherichia coli was purified by two-step chromatography. The purified enzyme had a molecular mass of 46.7 kDa and exhibited optimal activity at around 35 degrees C and pH 7.0-8.5. In addition to cello-oligosaccharides, it converted lactose to epilactose (4-O-beta-D-galactopyranosyl-D-mannose).

Identification of the cellobiose 2-epimerase gene in the genome of Bacteroides fragilis NCTC 9343.

Cellobiose 2-epimerase (CE, EC 5.1.3.11) catalyzes the reversible epimerization of cellobiose to 4-O-β-D-glucopyranosyl-D-mannose. In this study, we found a CE gene in the genome sequence of non-cellulolytic Bacteroides fragilis NCTC 9343. The recombinant enzyme, expressed in Escherichia coli cells, catalyzed a hydroxyl stereoisomerism at the C-2 positions of the reducing terminal glucose and at the mannose moiety of cello-oligosaccharides, lactose, β-mannobiose (4-O-β-D-mannopyranosyl-D-mannose), and globotriose [O-α-D-galactopyranosyl-(1→4)-O-β-D-galactopyranosyl-(1→4)-D-glucose]. The CE from B. fragilis showed less than 40% identity to reported functional CEs. It exhibited 44–63% identities to N-acyl-D-glucosamine 2-epimerase-like hypothetical proteins of unknown function in bacterial genome sequences of the phyla Firmicutes, Bacteroidetes, Proteobacteria, Chloroflexi, and Verrucomicrobia. On the other hand, it showed less than 26% identity to functional N-acyl-D-glucosamine 2-epimerases. Based on the amino acid homology and phylogenetic positions of the functional epimerases, we emphasize that many genes for putative N-acyl-D-glucosamine 2-epimerases and related hypothetical proteins of unknown function reported to date in the bacterial genomes should be annotated as CE-like proteins or putative CEs.

Effects of Epilactose on Calcium Absorption and Serum Lipid Metabolism in Rats

Epilactose (4-O-beta-galactopyranosyl-D-mannnose) is a rare disaccharide in cow milk that can be synthesized from lactose by the cellobiose 2-epimerase of Ruminococcus albus. In this study, we examined the biological activities of epilactose using male Wistar-ST rats. The apparent rates of calcium and magnesium absorption of rats fed epilactose and fructooligosaccharide diets were greater than those fed control and lactose diets, accompanied by greater weight gain of the cecal wall and higher levels of short-chain fatty acids and other organic acids. Epilactose also increased the calcium absorption in everted small intestinal sacs. In addition, the levels of plasma total cholesterol and nonhigh-density lipoprotein cholesterol were lower in epilactose-fed rats. These results indicate that epilactose promotes calcium absorption in the small intestine and possibly lowers the risk of arteriosclerosis. Cecal microbes may efficiently utilize epilactose and contribute to these biological activities.

Differential Chain-Length Specificities of Two Isoamylase-Type Starch-Debranching Enzymes from Developing Seeds of Kidney Bean

Plant isoamylase-type starch-debranching enzymes (ISAs) hydrolyze α-1,6-linkages in α-1,4/α-1,6-linked polyglucans. Two ISAs, designated PvISA1/2 and PvISA3, were purified from developing seeds of kidney bean by ammonium sulfate fractionation and several column chromatographic procedures. The enzymes displayed different substrate specificities for polyglucans: PvISA1/2 showed broad chain-length specificities, whereas PvISA3 liberated specific chains with a DP of 2 to 4.

Characterization of Starch Synthase I and II Expressed in Early Developing Seeds of Kidney Bean (Phaseolus vulgaris L.)

Plant starch synthase (SS) contributes to the elongation of glucan chains during starch biosynthesis and hence plays an essential role in determining the fine structure of amylopectin. To elucidate the role of SS activity in the formation of amylopectin in kidney bean (Phaseolus vulgaris L.), a study was undertaken to isolate cDNA clones for SS and to characterize the enzymatic properties of the coded recombinant enzymes. Two SS cDNAs, designated pvss1 and pvss21, which were isolated from early developing seeds, encoded SSI and SSII (designated PvSSI and PvSSII-1) that displayed significant identity (more than 65%) with other SSI and SSII members, respectively. RNA gel blot analysis indicated that both transcripts accumulate in leaves and developing seeds at the early stage. Immunoblot analysis with antisera raised against both recombinant proteins (rPvSSI and rPvSSII-1) showed that the accumulation of both proteins parallels the gene expression profiles, although both were detectable only in starch-granule fractions. Recombinant enzymes expressed by Escherichia coli cells showed distinct chain-length specificities for the extension of glucan chains. Our results suggest that these SS isozymes for synthesis of transitory starch are also responsible for synthesis of storage starch in early developing seeds of kidney bean.

Enzymatic characterization of starch synthase III from kidney bean (Phaseolus vulgaris L.)

In plants and green algae, several starch synthase isozymes are responsible for the elongation of glucan chains in the biosynthesis of amylose and amylopectin. Multiple starch synthase isozymes, which are classified into five major classes (granule‐bound starch synthases, SSI, SSII, SSIII, and SSIV) according to their primary sequences, have distinct enzymatic properties. All the starch synthase isozymes consist of a transit peptide, an N‐terminal noncatalytic region (N‐domain), and a C‐terminal catalytic region (C‐domain). To elucidate the enzymatic properties of kidney bean (Phaseolus vulgaris L.) SSIII and the function of the N‐domain of kidney bean SSIII, three recombinant proteins were constructed: putative mature recombinant SSIII, recombinant kidney bean SSIII N‐domain, and recombinant kidney bean SSIII C‐domain. Purified recombinant kidney bean SSIII displayed high specific activities for primers as compared to the other starch synthase isozymes from kidney bean. Kinetic analysis showed that the high specific activities of recombinant kidney bean SSIII are attributable to the high kcat values, and that the Km values of recombinant kidney bean SSIII C‐domain for primers were much higher than those of recombinant kidney bean recombinant SSIII. Recombinant kidney bean SSIII and recombinant kidney bean SSIII C‐domain had similar chain‐length specificities for the extension of glucan chains, indicating that the N‐domain of kidney bean SSIII does not affect the chain‐length specificity. Affinity gel electrophoresis indicated that recombinant kidney bean SSIII and recombinant kidney bean SSIII N‐domain have high affinities for amylose and amylopectin. The data presented in this study provide direct evidence for the function of the N‐domain of kidney bean SSIII as a carbohydrate‐binding module.

Identification and distribution of cellobiose 2-epimerase genes by a PCR-based metagenomic approach

Cellobiose 2-epimerase (CE) catalyzes the reversible epimerization of cellobiose to 4-O-β-d-glucopyranosyl-d-mannose. By using a PCR-based metagenomic approach, 71 ce-like gene fragments were obtained from wide-ranging environmental samples such as sheep rumen, soils, sugar beet extracts, and anaerobic sewage sludge. The frequency of isolation of the fragments similar to known sequences varied depending on the nature of the samples used. The ce-like genes appeared to be widely distributed in environmental bacteria belonging to the phyla Bacteroidetes, Chloroflexi, Dictyoglomi, Firmicutes, Proteobacteria, Spirochaetes, and Verrucomicrobia. The phylogenetic analysis suggested that the cluster of CE and CE-like proteins was functionally and evolutionarily separated from that of N-acetyl-d-glucosamine 2-epimerase (AGE) and AGE-like proteins. Two ce-like genes containing full-length ORFs, designated md1 and md2, were obtained by PCR and expressed in Escherichia coli. The recombinant mD1 and mD2 exhibited low Km values and high catalytic efficiencies (kcat/Km) for mannobiose compared with cellobiose, suggesting that they should be named mannobiose 2-epimerase, which is involved in a new mannan catabolic pathway we proposed.