Primeverose

beta-Diglycosidases from microorganisms as industrial biocatalysts: biochemical characteristics and potential applications.[Pubmed:30116842]

Appl Microbiol Biotechnol. 2018 Oct;102(20):8717-8723.

Flavonoid glycoside degradation can proceed through two alternative enzymatic pathways: one that is mediated by monoglycosidases, and the other catalyzed by a diglycosidase. beta-Diglycosidase performs the flavonoid deglycosylation in a single reaction. The characterized beta-diglycosidase activities recognize the following disaccharidic sugar moieties: beta-Primeverose, acuminose, vicianose, and beta-rutinose. The present paper reviews the biochemical characteristics and potential industrial applications of microbial beta-diglycosidases that break down plant diglycoconjugated flavonoids.

Crystal structures of beta-primeverosidase in complex with disaccharide amidine inhibitors.[Pubmed:24753293]

J Biol Chem. 2014 Jun 13;289(24):16826-34.

beta-Primeverosidase (PD) is a disaccharide-specific beta-glycosidase in tea leaves. This enzyme is involved in aroma formation during the manufacturing process of oolong tea and black tea. PD hydrolyzes beta-primeveroside (6-O-beta-d-xylopyranosyl-beta-d-glucopyranoside) at the beta-glycosidic bond of Primeverose to aglycone, and releases aromatic alcoholic volatiles of aglycones. PD only accepts Primeverose as the glycone substrate, but broadly accepts various aglycones, including 2-phenylethanol, benzyl alcohol, linalool, and geraniol. We determined the crystal structure of PD complexes using highly specific disaccharide amidine inhibitors, N-beta-primeverosylamidines, and revealed the architecture of the active site responsible for substrate specificity. We identified three subsites in the active site: subsite -2 specific for 6-O-beta-d-xylopyranosyl, subsite -1 well conserved among beta-glucosidases and specific for beta-d-glucopyranosyl, and wide subsite +1 for hydrophobic aglycone. Glu-470, Ser-473, and Gln-477 act as the specific hydrogen bond donors for 6-O-beta-d-xylopyranosyl in subsite -2. On the other hand, subsite +1 was a large hydrophobic cavity that accommodates various aromatic aglycones. Compared with aglycone-specific beta-glucosidases of the glycoside hydrolase family 1, PD lacks the Trp crucial for aglycone recognition, and the resultant large cavity accepts aglycone and 6-O-beta-d-xylopyranosyl together. PD recognizes the beta-primeverosides in subsites -1 and -2 by hydrogen bonds, whereas the large subsite +1 loosely accommodates various aglycones. The glycone-specific activity of PD for broad aglycone substrates results in selective and multiple release of temporally stored alcoholic volatile aglycones of beta-primeveroside.

Expression and biochemical characterization of beta-primeverosidase and application of beta-primeverosylamidine to affinity purification.[Pubmed:18256510]

Biosci Biotechnol Biochem. 2008 Feb;72(2):376-83.

Beta-primeverosidase (PD) is a family 1 glycosidase catalyzing the hydrolysis of beta-primeverosides (6-O-beta-D-xylopyranosyl-beta-D-glucopyranosides) to release a disaccharide Primeverose. To investigate how PD recognizes the disaccharide moiety of beta-primeverosides, the recombinant PD was expressed by a baculovirus-insect cell system. The recombinant PD was secreted from High Five cells and was properly modified with N-glycosylation and correct cleavage at the N-terminal signal peptide. The recombinant PD exhibited high substrate specificity to beta-primeverosides in terms of the glycone moiety, consistently with the substrate specificity of native PD from Camellia sinensis. Next, beta-glycosylamidines were synthesized as substrate analog inhibitors. Beta-primeverosylamidine strongly inhibited PD activity, but beta-glucosylamidine did not. Hence beta-primeverosylamidine is an ideal chemical tool for probing disaccharide recognition in the active site of PD. An affinity adsorbent for PD was prepared using beta-primeverosylamidine as a ligand. Affinity chromatography gave large amounts of PD with high purity, permitting crystallographic study.

Characteristic fragmentation patterns of the trimethylsilyl and trimethylsilyl-oxime derivatives of various saccharides as obtained by gas chromatography coupled to ion-trap mass spectrometry.[Pubmed:18061601]

J Chromatogr A. 2008 Jan 4;1177(1):183-9.

The fragmentation patterns of selected glycosidic linkage containing non-reducing (methylmannoside, methylgalactoside, lactitol, sucrose, trehalose, raffinose, erlose, melezitose) and reducing saccharides (maltose, cellobiose, lactose, melibiose, palatinose, Primeverose, rutinose) have been compared as their trimethylsilyl and as their trimethylsilyl-oxime derivatives. Fragmentation characteristics of the glycosidic linkage containing trimethylsilyl-oxime derivatives have been investigated at the first time: these spectra are not available in the official libraries (NIST, Wiley). Applying gas chromatography-ion trap mass spectrometry, informative fragments of high masses with high intensities have been obtained. Results confirmed characteristic differences between the simple trimethylsilyl derivative providing non-reducing glycosides and the trimethylsilyl, syn and antioxime species. Fragmentation starts at the glycosidic linkage resulting in the case of the non-reducing di- and trisaccharides in two, identical fragments of ring structure, with the abundant selective fragment ion at m/z=361. In the case of reducing disaccharides fragmentation provides two different moieties: one moiety of ring structure at m/z=361, and one of the open chain trimethylsilyl-oxime moiety with two special fragment ions at m/z=361 and at m/z=538. These fragmentation patterns proved to be independent on the position of the glycosidic linkage. Distribution of the selective fragment ions, obtained from their total ion current elutions, was evaluated on a quantitative basis, expressed in percentages of the total of ions formed. Reproducibility in the formation of these selective fragment ions, depending on their amount to be fragmented, proved to be proper for identification and quantitation purposes, equally. On this basis, in addition to the authentic ones, also two reducing disaccharides (Primeverose and rutinose), as authentic compounds not available on the market, were identified and quantified in natural matrices.

Isolation and characterization of a beta-primeverosidase-like enzyme from Penicillium multicolor.[Pubmed:16556987]

Biosci Biotechnol Biochem. 2006 Mar;70(3):691-8.

p-Nitrophenyl and eugenyl beta-primeveroside (6-O-beta-D-xylopyranosyl-beta-D-glucopyranoside) hydrolytic activity was found in culture filtrate from Penicillium multicolor IAM7153, and the enzyme was isolated. The enzyme was purified as a beta-primeverosidase-like enzyme by precipitation with ammonium sulfate followed by successive chromatographies on Phenyl Sepharose, Mono Q, and beta-galactosylamidine affinity columns. The molecular mass was estimated to be 50 kDa by SDS-PAGE and gel filtration. The purified enzyme was highly specific toward the substrate p-nitrophenyl beta-primeveroside, which was cleaved in an endo-manner into Primeverose and p-nitrophenol, but a series of beta-primeveroside as aroma precursors were hydrolyzed only slightly as substrates for the enzyme. In analyses of its hydrolytic action and kinetics, the enzyme showed narrow substrate specificity with respect to the aglycon and glycon moieties of the diglycoside. We conclude that the present enzyme is a kind of beta-diglycosidase rather than beta-primeverosidase.

Cloning of beta-primeverosidase from tea leaves, a key enzyme in tea aroma formation.[Pubmed:12481100]

Plant Physiol. 2002 Dec;130(4):2164-76.

A beta-primeverosidase from tea (Camellia sinensis) plants is a unique disaccharide-specific glycosidase, which hydrolyzes aroma precursors of beta-primeverosides (6-O-beta-D-xylopyranosyl-beta-D-glucopyranosides) to liberate various aroma compounds, and the enzyme is deeply concerned with the floral aroma formation in oolong tea and black tea during the manufacturing process. The beta-primeverosidase was purified from fresh leaves of a cultivar for green tea (C. sinensis var sinensis cv Yabukita), and its partial amino acid sequences were determined. The beta-primeverosidase cDNA has been isolated from a cDNA library of cv Yabukita using degenerate oligonucleotide primers. The cDNA insert encodes a polypeptide consisting of an N-terminal signal peptide of 28 amino acid residues and a 479-amino acid mature protein. The beta-primeverosidase protein sequence was 50% to 60% identical to beta-glucosidases from various plants and was classified in a family 1 glycosyl hydrolase. The mature form of the beta-primeverosidase expressed in Escherichia coli was able to hydrolyze beta-primeverosides to liberate a Primeverose unit and aglycons, but did not act on 2-phenylethyl beta-D-glucopyranoside. These results indicate that the beta-primeverosidase selectively recognizes the beta-primeverosides as substrates and specifically hydrolyzes the beta-glycosidic bond between the disaccharide and the aglycons. The stereochemistry for enzymatic hydrolysis of 2-phenylethyl beta-primeveroside by the beta-primeverosidase was followed by (1)H-nuclear magnetic resonance spectroscopy, revealing that the enzyme hydrolyzes the beta-primeveroside by a retaining mechanism. The roles of the beta-primeverosidase in the defense mechanism in tea plants and the floral aroma formation during tea manufacturing process are also discussed.

Isolation and characterization of a beta-primeverosidase-like endo-manner beta-glycosidase from Aspergillus fumigatus AP-20.[Pubmed:12036053]

Biosci Biotechnol Biochem. 2002 Apr;66(4):801-7.

A novel beta-glycosidase-producing microorganism was isolated from soil and identified as Aspergillus fumigatus AP-20 based on its taxonomical characteristics. The enzyme was found to be an extracellular protein in the culture of the isolated fungus and was purified 88-fold by fractionation with ammonium sulfate followed by successive column chromatographies on phenyl-Sepharose HP and Mono P HR. The molecular mass was estimated to be 47 kDa by SDS-PAGE and the isoelectric point to be pH 6.0 by isoelectric focusing. The purified enzyme was highly specific for a substrate, p-nitrophenyl beta-primeveroside (6-O-beta-D-xylopyranosyl-beta-D-glucopyranoside), which was cleaved in an endo-manner into Primeverose and p-nitrophenol.

Enzymatic synthesis and characterization of 6-O-Beta-D-xylopyranosyl-2-acetamido-2-deoxy-D-glucopyranose, a structural analog of primeverose.[Pubmed:9821268]

Carbohydr Res. 1998 Sep;311(1-2):79-83.

The synthesis of the disaccharide 6-O-beta-D-xylopyranosyl-2-acetamido-2-deoxy-D-glucopyranose (N-acetylprimeverosamine), structurally related to the natural disaccharide 6-O-beta-D-xylopyranosyl-D-glycopyranose (Primeverose), was obtained via a transglycosylation reaction catalyzed by a crude preparation of beta-D-xylosidase from Aspergillus niger, using p-nitrophenyl beta-D-xylopyranoside as the donor and 2-acetamido-2-deoxy-D-glucopyranose as the acceptor. The yield of the reaction was 36% on a molar basis with respect to the donor. The chemical identity of the product was assessed by HPLC, ionspray mass spectrometry and NMR spectroscopy.