Hiromi Murakami

Purification and Characterization of a Carbohydrate : Acceptor Oxidoreductase from Paraconiothyrium sp. That Produces Lactobionic Acid Efficiently

A carbohydrate:acceptor oxidoreductase from Paraconiothyrium sp. was purified and characterized. The enzyme efficiently oxidized β-(1→4) linked sugars, such as lactose, xylobiose, and cellooligosaccharides. The enzyme also oxidized maltooligosaccharides, D-glucose, D-xylose, D-galactose, L-arabinose, and 6-deoxy-D-glucose. It specifically oxidized the β-anomer of lactose. Molecular oxygen and 2,6-dichlorophenol indophenol were reduced by the enzyme as electron acceptors. The Paraconiothyrium enzyme was identified as a carbohydrate:acceptor oxidoreductase according to its specificity for electron donors and acceptors, and its molecular properties, as well as the N-terminal amino acid sequence. Further comparison of the amino acid sequences of lactose oxidizing enzymes indicated that carbohydrate:acceptor oxidoreductases belong to the same group as glucooligosaccharide oxidase, while they differ from cellobiose dehydrogenases and cellobiose:quinone oxidoreductases.

Optimization of Lactobionic Acid Production by Acetobacter orientalis Isolated from Caucasian Fermented Milk, “Caspian Sea Yogurt”

We have reported that lactobionic acid is produced from lactose by Acetobacter orientalis in traditional Caucasian fermented milk. To maximize the application of lactobionic acid, we investigated favorable conditions for the preparation of resting A. orientalis cells and lactose oxidation. The resting cells, prepared under the most favorable conditions, effectively oxidized 2–10% lactose at 97.2 to 99.7 mol % yield.

Purification and Some Properties of a New Levanase from Bacillus sp. No. 71.

A levanase from Bacillus sp. was purified to a homogeneous state. The enzyme had a molecular weight of 135,000 and an isoelectric point of pH 4.7. The enzyme was most active at pH 6.0 and 40°C, stable from pH 6.0 to 10.0 for 20 hr of incubation at 4°C and up to 30°C for 30 min of incubation at pH 6.0. The enzyme activity was inhibited by Ag (+), Hg(2 +), Cu(2 +), Fe(3 +), Pb(2+), and p-chloromercuribenzoic acid. The enzyme hydrolyzed levan and phlein endowise to produce levanheptaose as a main product. The limit of hydrolysis of levan and phlein were 71% and 96%, respectively.

Purification and Characterization of a Novel α-Glucuronidase from Aspergillus niger Specific for O-α-D-Glucosyluronic Acid α-D-Glucosiduronic Acid

A new α-glucuronidase that specifically hydrolyzed O-α-D-glucosyluronic acid α-D-glucosiduronic acid (trehalose dicarboxylate, TreDC) was purified from a commercial enzyme preparation from Aspergillus niger, and its properties were examined. The enzyme did not degrade O-α-D-glucosyluronic acid α-D-glucoside, O-α-D-glucosyluronic acid β-D-glucosiduronic acid, O-α-D-glucosyluronic acid-(1→2)-β-D-fructosiduronic acid, p-nitrophenyl-O-α-D-glucosiduronic acid, methyl-O-α-D-glucosiduronic acid, or 6-O-α-(4-O-α-D-glucosyluronic acid)-D-glucosyl-β-cyclodextrine. Furthermore, it showed no activity on α-glucuronyl linkages of 4-O-methyl-D-glucosyluronic acid-α-(1→2)-xylooligosaccharides, derived from xylan, a supposed substrate of α-glucuronidases. The molecular mass of the enzyme was estimated to be 120 kDa by gel filtration and 58 kDa by SDS–PAGE suggesting, the enzyme is composed of two identical subunits. It was most active at pH 3.0–3.5 and at 40 °C. It was stable in pH 2.0–4.5 and below 30 °C. It hydrolyzed O-α-D-glucosyluronic acid α-D-glucosiduronic acid to produce α- and β-anomers of D-glucuronic acid in an equimolar ratio. This result suggests that inversion of the anomeric configuration of the substrate is involved in the hydrolysis mechanism.

Synthesis of Novel Heterobranched β-Cyclodextrins Having β-D-N-Acetylglucosaminyl-Maltotriose on the Side Chain

From a mixture of N-acetylglucosaminyl-β-cyclodextrin (GlcNAc-βCD) and lactose, β-D-galactosyl-GlcNAc-βCD (Gal-GlcNAc-βCD) was synthesized by the transfer action of β-galactosidase. GlcNAc-maltotriose (Glc3) and Gal-GlcNAc-Glc3 were produced with hydrolysis of GlcNAc-βCD by cyclodextrin glycosyltransferase, and Gal-GlcNAc-βCD by bacterial saccharifying α-amylase respectively. Finally, GlcNAc-Glc3-βCD and Gal-GlcNAc-Glc3-βCD were synthesized in 5.2% and 3.5% yield when Klebsiella pneumoniae pullulanase was incubated with the mixture of GlcNAc-Glc3 and βCD, or Gal-GlcNAc-Glc3 and βCD respectively. The structures of GlcNAc-Glc3-βCD and Gal-GlcNAc-Glc3-βCD were analyzed by FAB-MS and NMR spectroscopy and identified as 6-O-α-(63-O-β-D-N-acetylglucosaminyl-maltotriosyl)-βCD, and 6-O-α-(4-O-β-D-galactopyranosyl-63-O-β-D-N-acetylglucosaminyl-maltotriosyl)-βCD respectively.

Characterization of Dimers of Hydroquinone Glucosides Produced by Peroxidase-Catalyzed Polymerization

4′-Hydroxyphenyl α-glucoside and 4′-hydroxyphenyl β-glucoside were polymerized with horseradish peroxidase. The isolated dimers were found to have linkages at C3′ of the hydroxyphenyl moieties and proved to be fluorescent. Low accumulation of oligomers was attributed to increasing electrochemical reactivity with polymerization degrees, which were expected from the levels of highest occupied molecular orbital.

Lactobionic and cellobionic acid production profiles of the resting cells of acetic acid bacteria

Lactobionic acid was produced by acetic acid bacteria to oxidize lactose. Gluconobacter spp. and Gluconacetobacter spp. showed higher lactose-oxidizing activities than Acetobacter spp. Gluconobacter frateurii NBRC3285 produced the highest amount of lactobionic acid per cell, among the strains tested. This bacterium assimilated neither lactose nor lactobionic acid. At high lactose concentration (30%), resting cells of the bacterium showed sufficient oxidizing activity for efficient production of lactobionic acid. These properties may contribute to industrial production of lactobionic acid by the bacterium. The bacterium showed higher oxidizing activity on cellobiose than that on lactose and produced cellobionic acid. Graphical abstract Oxidation of lactose and cellobiose by acetic acid bacteria produced lactobionic and cellobionic acid, respectively. Lactobionic acid was effectively produced under high-lactose conditions.

Preparation of branched cyclomaltoheptaose with 3-O-α-l-fucopyranosyl-α-d-mannopyranose and changes in fucosylation of HCT116 cells treated with the fucose-modified cyclomaltoheptaose

From a mixture of 4-nitrophenyl α-L-fucopyranoside and D-mannopyranose, 3-O-α-L-fucopyranosyl-D-mannopyranose was synthesised through the transferring action of α-fucosidase (Sumizyme PHY). 6(I),6(IV)-Di-O-(3-O-α-L-fucopyranosyl-α-D-mannopyranosyl)-cyclomaltoheptaose {8, 6(I),6(IV)-di-O-[α-L-Fuc-(1→3)-α-D-Man]-βCD} was chemically synthesised using the trichloroacetimidate method. The structures were confirmed by MS and NMR spectroscopy. A cell-based assay using the fucosyl βCD derivatives, including the newly synthesised 8, showed that derivatives with two branches of the α-L-Fuc or α-L-Fuc-(1→3)-α-D-Man residues possessed slight growth-promoting effects and lower toxicity in HCT116 cells compared to those with one branch. These compounds may be useful as drug carriers in targeted drug delivery systems.

Studies on Levan-Degrading Enzymes from Microorganisms

Levan is a microbial polyfructan; constructed from β-2, 6-fructofuranosyl main chain and β-2, 1-fructof uranosyl linked side, chains. The enzymatic synthesis of levan has been well studied for several decades. No detailed report has been made, however, to describe the degradation of levan and levan degrading enzymes; most previous reports were on unpurified enzymes. This paper deals with screenings, enzymatic properties, and classification of newly obtained levanases.