Efficient Synthesis of β-Glucose 1-Phosphate through Enzymatic Phosphorolysis and Baker's Yeast Fermentation.

β-Glucose 1-phosphate (βGlc1P) is a donor substrate in the synthesis of various α-glucosides by glycoside phosphorylases belonging to the glycoside hydrolase family 65. This study presents an efficient synthesis of βGlc1P combining enzymatic phosphorolysis of inexpensive maltose and baker's yeast fermentation to bias the equilibrium toward maltose phosphorolysis by removing released glucose. Mass production of βGlc1P was obtained in a 2 L reaction mixture initially containing 500 mM maltose and inorganic phosphate, with a yield of 76 %.

Molecular Basis of Absorption at 340 nm of 3-Ketoglucosides under Alkaline Conditions.

Transient absorption at 340 nm under alkaline conditions has long been used to detect the presence of 3-keto--glycosides without understanding the molecular basis of the absorbance. The time course of for the alkaline treatment of 3-ketolevoglucosan, an intramolecular 3-keto--glycoside, was investigated to identify the three products generated through alkaline treatment. By comparing the spectra of these compounds under neutral and alkaline conditions, we identified 1,5-anhydro-D--hex-1-en-3-ulose (2-hydroxy-3-keto-D-glucal) as being the compound responsible for the absorption.

Substrate recognition mode of a glycoside hydrolase family 42 β-galactosidase from subspecies (Bga42A) revealed by crystallographic and mutational analyses.

subsp. uses a glycoside hydrolase (GH) family 42 β-galactosidase (Bga42A) for hydrolyzing lacto--tetraose (LNT), which is the most abundant core structure of human milk oligosaccharides (HMOs). As such, Bga42A represents one of the pivotal enzymes underpinning the symbiosis between bifidobacteria and breastfed infants.

Extracellular oil production by Rhodotorula paludigena BS15 for biorefinery without complex downstream processes.

To realize biomass refinery without complex downstream processes, we extensively screened for microbial strains that efficiently produce extracellular oil from sugars. Rhodotorula paludigena (formerly Rhodosporidium paludigenum) BS15 was found to efficiently produce polyol esters of fatty acids (PEFAs), which mainly comprised of 3-acetoxypalmitic acid and partially acetylated mannitol/arabinitol. To evaluate the performance of this strain, fed-batch fermentation was demonstrated on a flask scale, and 110 g/L PEFA and 103 g/L dry cells were produced in 12 days.

Automatic Calculation of the Kinetic Parameters of Enzymatic Reactions with Their Standard Errors Using Microsoft Excel.

We created a Microsoft Excel file, Enzyme_Kinetics_Calculator, which includes macro programs that automatically calculates kinetic parameters for typical kinetic equations of enzymatic reactions, accompanied by their standard errors, by minimizing the residual sum of squares thereof. The []- plot is automatically drawn with the theoretical lines and, similarly, the 1/[]-1/ plot in the case of linear theoretical lines. Enzyme_Kinetics_Calculator is available as a supplementary file for this paper (see J.

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

Lacto--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-β-galactosyl--acetylhexosamine phosphorylase, sucrose phosphorylase, UDP-glucose-hexose 1-phosphate uridylyltransferase, and UDP-glucose 4-epimerase, recombinantly produced by . 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 strains.

Priority effects shape the structure of infant-type Bifidobacterium communities on human milk oligosaccharides.

Bifidobacteria are among the first colonizers of the infant gut, and human milk oligosaccharides (HMOs) in breastmilk are instrumental for the formation of a bifidobacteria-rich microbiota. However, little is known about the assembly of bifidobacterial communities. Here, by applying assembly theory to a community of four representative infant-gut associated Bifidobacterium species that employ varied strategies for HMO consumption, we show that arrival order and sugar consumption phenotypes significantly affected community formation.

Discovery of solabiose phosphorylase and its application for enzymatic synthesis of solabiose from sucrose and lactose.

Glycoside phosphorylases (GPs), which catalyze the reversible phosphorolysis of glycosides, are promising enzymes for the efficient production of glycosides. Various GPs with new catalytic activities are discovered from uncharacterized proteins phylogenetically distant from known enzymes in the past decade. In this study, we characterized Paenibacillus borealis PBOR_28850 protein, belonging to glycoside hydrolase family 94.

Diversification of a Fucosyllactose Transporter within the Genus .

Human milk oligosaccharides (HMOs), which are natural bifidogenic prebiotics, were recently commercialized to fortify formula milk. However, HMO assimilation phenotypes of bifidobacteria vary by species and strain, which has not been fully linked to strain genotype. We have recently shown that specialized uptake systems, particularly for the internalization of major HMOs (fucosyllactose [FL]), are associated with the formation of a -rich gut microbial community.

Generation of 3-deoxypentulose by the isomerization and β-elimination of 4-O-substituted glucose and fructose.

Aldose-ketose isomerization is commonly used to prepare rare oligosaccharides such as maltulose (4-O-α-d-glucopyranosyl-d-fructose) and lactulose (4-O-β-d-galactopyranosyl-d-fructose). However, both sugars are degraded under alkaline conditions via β-elimination, while their subsequent benzylic acid rearrangement leads to the formation of isosaccharinic acids. Here, we investigated the behavior of maltose and maltulose upon heating in phosphate buffer solution at pH 7.

Conversion of levoglucosan into glucose by the coordination of four enzymes through oxidation, elimination, hydration, and reduction.

Levoglucosan (LG) is an anhydrosugar produced through glucan pyrolysis and is widely found in nature. We previously isolated an LG-utilizing thermophile, Bacillus smithii S-2701M, and suggested that this bacterium may have a metabolic pathway from LG to glucose, initiated by LG dehydrogenase (LGDH). Here, we completely elucidated the metabolic pathway of LG involving three novel enzymes in addition to LGDH.

Alkoxycarbonyl elimination of 3-O-substituted glucose and fructose by heat treatment under neutral pH.

3-O-Substituted reducing aldoses are commonly unstable under heat treatment at neutral and alkaline pH. In this study, to evaluate the decomposition products, nigerose (3-O-α-d-glucopyranosyl-d-glucose) and 3-O-methyl glucose were heated at 90 °C in 100 mM sodium phosphate buffer (pH 7.5).

Effect of C-6 Methylol Groups on Substrate Recognition of Glucose/Xylose Mixed Oligosaccharides by Cellobiose Dehydrogenase from the Basidiomycete .

Cellobiose dehydrogenase (CDH) is a flavocytochrome catalyzing oxidation of the reducing end of cellobiose and cellooligosaccharides, and has a key role in the degradation of cellulosic biomass by filamentous fungi. Here, we use a lineup of glucose/xylose-mixed β-1,4-linked disaccharides and trisaccharides, enzymatically synthesized by means of the reverse reaction of cellobiose phosphorylase and cellodextrin phosphorylase, to investigate the substrate recognition of CDH. We found that CDH utilizes β-D-xylopyranosyl-(1→4)-D-glucopyranose (Xyl-Glc) as an electron donor with similar and values to cellobiose.

Varied Pathways of Infant Gut-Associated to Assimilate Human Milk Oligosaccharides: Prevalence of the Gene Set and Its Correlation with Bifidobacteria-Rich Microbiota Formation.

The infant's gut microbiome is generally rich in the genus. The mother's milk contains natural prebiotics, called human milk oligosaccharides (HMOs), as the third most abundant solid component after lactose and lipids, and of the different gut microbes, infant gut-associated bifidobacteria are the most efficient in assimilating HMOs. Indeed, the fecal concentration of HMOs was found to be negatively correlated with the fecal abundance of in infants.

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-β1,3-GlcNAc) and galacto-N-biose (Gal-β1,3-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.

Epimerization and Decomposition of Kojibiose and Sophorose by Heat Treatment under Neutral pH Conditions.

We evaluated the stabilities of kojibiose and sophorose when heated under neutral pH conditions. Kojibiose and sophorose epimerized at the C-2 position of glucose on the reducing end, resulting in the production of 2--α-D-glucopyranosyl-D-mannose and 2--β-D-glucopyranosyl-D-mannose, respectively. Under weak alkaline conditions, kojibiose was decomposed due to heating into its mono-dehydrated derivatives, including 3-deoxy-2,3-unsaturated compounds and bicyclic 3,6-anhydro compounds.

Sharing of human milk oligosaccharides degradants within bifidobacterial communities in faecal cultures supplemented with Bifidobacterium bifidum.

Gut microbiota of breast-fed infants are generally rich in bifidobacteria. Recent studies show that infant gut-associated bifidobacteria can assimilate human milk oligosaccharides (HMOs) specifically among the gut microbes. Nonetheless, little is known about how bifidobacterial-rich communities are shaped in the gut.

Identification, functional characterization, and crystal structure determination of bacterial levoglucosan dehydrogenase.

Levoglucosan is the 1,6-anhydrosugar of d-glucose formed by pyrolysis of glucans and is found in the environment and industrial waste. Two types of microbial levoglucosan metabolic pathways are known. Although the eukaryotic pathway involving levoglucosan kinase has been well-studied, the bacterial pathway involving levoglucosan dehydrogenase (LGDH) has not been well-investigated.

Expression and Characterization of Recombinant Sucrose Phosphorylase.

SPase is widely used in the food, cosmetics, and pharmaceutical industries. Previously, a SPase gene was cloned from Bifidobacterium longum JCM1217 and constructed into Escherichia coli BL21. In this paper, its expression conditions were optimized.

Synthesis of 3-Keto-levoglucosan Using Pyranose Oxidase and Its Spontaneous Decomposition via β-Elimination.

3-Keto-levoglucosan (3ketoLG) has been postulated to be the product of a reaction catalyzed by levoglucosan dehydrogenase (LGDH), a bacterial enzyme involved in the metabolism of levoglucosan (LG). To investigate the LG metabolic pathway catalyzed by LGDH, 3ketoLG is needed. However, 3ketoLG has not been successfully isolated from the LGDH reaction.