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

AbstractGlycoside 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. Screening of acceptor substrates for reverse phosphorolysis, in which α-d-glucose 1-phosphate was used as the donor substrate, revealed that the recombinant PBOR_28850 produced in Escherichia coli specifically utilized d-galactose as an acceptor and produced solabiose (β-d-Glcp-(1 → 3)-d-Gal). This indicates that PBOR_28850 is a new GP, solabiose phosphorylase. PBOR_28850 catalyzed the phosphorolysis and synthesis of solabiose through a sequential bi-bi mechanism involving the formation of a ternary complex. The production of solabiose from lactose and sucrose has been established. Lactose was hydrolyzed to d-galactose and d-glucose by β-galactosidase. Phosphorolysis of sucrose and synthesis of solabiose were then coupled by adding sucrose, sucrose phosphorylase, and PBOR_28850 to the reaction mixture. Using 210 mmol lactose and 280 mmol sucrose, 207 mmol of solabiose was produced. Yeast treatment degraded the remaining monosaccharides and sucrose without reducing solabiose. Solabiose with a purity of 93.7% was obtained without any chromatographic procedures.

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Sucrose Phosphorylase and Related Enzymes in Glycoside Hydrolase Family 13: Discovery, Application and Engineering

International Journal of Molecular Sciences ◽

10.3390/ijms21072526 ◽

2020 ◽

Vol 21 (7) ◽

pp. 2526 ◽

Cited By ~ 4

Author(s):

Jorick Franceus ◽

Tom Desmet

Keyword(s):

Glycoside Hydrolase ◽

Enzyme Engineering ◽

Glycoside Hydrolase Family ◽

Carbohydrate Active Enzymes ◽

Sucrose Phosphorylase ◽

Engineering Studies ◽

The Past ◽

Hydrolase Family ◽

Glycoside Hydrolase Family Gh13 ◽

Made In

Sucrose phosphorylases are carbohydrate-active enzymes with outstanding potential for the biocatalytic conversion of common table sugar into products with attractive properties. They belong to the glycoside hydrolase family GH13, where they are found in subfamily 18. In bacteria, these enzymes catalyse the phosphorolysis of sucrose to yield α-glucose 1-phosphate and fructose. However, sucrose phosphorylases can also be applied as versatile transglucosylases for the synthesis of valuable glycosides and sugars because their broad promiscuity allows them to transfer the glucosyl group of sucrose to a diverse collection of compounds other than phosphate. Numerous process and enzyme engineering studies have expanded the range of possible applications of sucrose phosphorylases ever further. Moreover, it has recently been discovered that family GH13 also contains a few novel phosphorylases that are specialised in the phosphorolysis of sucrose 6F-phosphate, glucosylglycerol or glucosylglycerate. In this review, we provide an overview of the progress that has been made in our understanding and exploitation of sucrose phosphorylases and related enzymes over the past ten years.

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

Scientific Reports ◽

10.1038/s41598-020-77133-8 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Yuya Kuritani ◽

Kohei Sato ◽

Hideo Dohra ◽

Seiichiro Umemura ◽

Motomitsu Kitaoka ◽

...

Keyword(s):

Escherichia Coli ◽

Recombinant Proteins ◽

Metabolic Pathway ◽

Glycoside Hydrolase ◽

Gene Locus ◽

Glycoside Hydrolase Family ◽

Sequential Reaction ◽

Layer Chromatography ◽

Hydrolysis Of ◽

Hydrolase Family

AbstractLevoglucosan (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. In the S-2701M genome, three genes expected to be involved in the LG metabolism were found in the vicinity of the LGDH gene locus. These four genes including LGDH gene (lgdA, lgdB1, lgdB2, and lgdC) were expressed in Escherichia coli and purified to obtain functional recombinant proteins. Thin layer chromatography analyses of the reactions with the combination of the four enzymes elucidated the following metabolic pathway: LgdA (LGDH) catalyzes 3-dehydrogenation of LG to produce 3-keto-LG, which undergoes β-elimination of 3-keto-LG by LgdB1, followed by hydration to produce 3-keto-d-glucose by LgdB2; next, LgdC reduces 3-keto-d-glucose to glucose. This sequential reaction mechanism resembles that proposed for an enzyme belonging to glycoside hydrolase family 4, and results in the observational hydrolysis of LG into glucose with coordination of the four enzymes.

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A New β-Galactosidase from the Antarctic Bacterium Alteromonas sp. ANT48 and Its Potential in Formation of Prebiotic Galacto-Oligosaccharides

Marine Drugs ◽

10.3390/md17110599 ◽

2019 ◽

Vol 17 (11) ◽

pp. 599 ◽

Cited By ~ 4

Author(s):

Li ◽

Zhu ◽

Xing

Keyword(s):

Escherichia Coli ◽

Molecular Docking ◽

Glycoside Hydrolase ◽

Intestinal Flora ◽

Glycoside Hydrolase Family ◽

Initial Activity ◽

Antarctic Bacterium ◽

Encoding Gene ◽

The Antarctic ◽

Hydrolase Family

As an important medical enzyme, β-galactosidases catalyze transgalactosylation to form prebiotic Galacto-Oligosaccharides (GOS) that assist in improving the effect of intestinal flora on human health. In this study, a new glycoside hydrolase family 2 (GH2) β-galactosidase-encoding gene, galA, was cloned from the Antarctic bacterium Alteromonas sp. ANT48 and expressed in Escherichia coli. The recombinant β-galactosidase GalA was optimal at pH 7.0 and stable at pH 6.6–7.0, which are conditions suitable for the dairy environment. Meanwhile, GalA showed most activity at 50 °C and retained more than 80% of its initial activity below 40 °C, which makes this enzyme stable in normal conditions. Molecular docking with lactose suggested that GalA could efficiently recognize and catalyze lactose substrates. Furthermore, GalA efficiently catalyzed lactose degradation and transgalactosylation of GOS in milk. A total of 90.6% of the lactose in milk could be hydrolyzed within 15 min at 40 °C, and the GOS yield reached 30.9%. These properties make GalA a good candidate for further applications.

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