Acremonium sp. diglycosidase-aid chemical diversification: valorization of industry by-products.

The fungal diglycosidase α-rhamnosyl-ß-glucosidase I (αRßG I) from Acremonium sp. DSM 24697 catalyzes the glycosylation of various OH-acceptors using the citrus flavanone hesperidin. We successfully applied a one-pot biocatalysis process to synthesize 4-methylumbellipheryl rutinoside (4-MUR) and glyceryl rutinoside using a citrus peel residue as sugar donor. This residue, which contained 3.5 % [w/w] hesperidin, is the remaining of citrus processing after producing orange juice, essential oil, and peel-juice. The low-cost compound glycerol was utilized in the synthesis of glyceryl rutinoside. We implemented a simple method for the obtention of glyceryl rutinoside with 99 % yield, and its purification involving activated charcoal, which also facilitated the recovery of the by-product hesperetin through liquid-liquid extraction. This process presents a promising alternative for biorefinery operations, highlighting the valuable role of αRßG I in valorizing glycerol and agricultural by-products. KEYPOINTS: • αRßG I catalyzed the synthesis of rutinosides using a suspension of OPW as sugar donor. • The glycosylation of aliphatic polyalcohols by the αRßG I resulted in products bearing a single rutinose moiety. • αRßG I catalyzed the synthesis of glyceryl rutinoside with high glycosylation/hydrolysis selectivity (99 % yield).

Biosynthesis of Menaquinone in E.†coli: Identification of an Elusive Isomer of SEPHCHC.

In the biosynthesis of menaquinone in bacteria, the thiamine diphosphate-dependent enzyme MenD catalyzes the decarboxylative carboligation of α-ketoglutarate and isochorismate to (1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxycyclohex-3-ene-1-carboxylate (SEPHCHC). The regioisomer of SEPHCHC, namely (1R,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxycyclohex-2-ene-1-carboxylate (iso-SEPHCHC), has been considered as a possible product, however, its existence has been doubtful due to a spontaneous elimination of pyruvate from SEPHCHC to 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC). In this work, the regioisomer iso-SEPHCHC was distinguished from SEPHCHC by liquid chromatography-tandem mass spectrometry. Iso-SEPHCHC was purified and identified by NMR spectroscopy. Just as SEPHCHC remained hidden as a MenD product for more than two decades, its regioisomer iso-SEPHCHC has remained until now.

Peculiarities and systematics of microbial diglycosidases, and their applications in food technology.

Diglycosidases are endo-ß-glucosidases that hydrolyze the heterosidic linkage of diglycoconjugates, thereby releasing in a single reaction the disaccharide and the aglycone. Plant diglycosidases belong to the glycoside hydrolase family 1 and are associated with defense mechanisms. Microbial diglycosidases exhibit higher diversity-they belong to the families 3, 5, and 55-and play a catabolic role. As diglycoconjugates are widespread in the environments, so are the microbial diglycosidases, which allow their utilization as nutritional source and carbon recycling. In the last 10 years, six microbial diglycosidases have been sequenced, and for two of them, the three-dimensional structure has been elucidated. This knowledge allowed the identification of their diverse phylogenetic origin, and gave insights into the understanding of the substrate specificity. Here, the last advances and the applications of microbial diglycosidases are reviewed. KEY POINTS: • Substrate specificity and phylogenetic relationships of diglycosidases are reviewed. • On-going and potential applications of diglycosidases are discussed.

The flavonoid degrading fungus Acremonium sp. DSM 24697 produces two diglycosidases with different specificities.

AbstractDiglycosidases hydrolyze the heterosidic linkage of diglycoconjugates, releasing the disaccharide and the aglycone. Usually, these enzymes do not hydrolyze or present only low activities towards monoglycosylated compounds. The flavonoid degrading fungus Acremonium sp. DSM 24697 produced two diglycosidases, which were termed 6-O-α-rhamnosyl-ß-glucosidase I and II (αRßG I and II) because of their function of releasing the disaccharide rutinose (6-O-α-L-rhamnosyl-ß-D-glucose) from the diglycoconjugates hesperidin or rutin. In this work, the genome of Acremonium sp. DSM 24697 was sequenced and assembled with a size of ~ 27 Mb. The genes encoding αRßG I and II were expressed in Pichia pastoris KM71 and the protein products were purified with apparent molecular masses of 42 and 82 kDa, respectively. A phylogenetic analysis showed that αRßG I grouped in glycoside hydrolase family 5, subfamily 23 (GH5), together with other fungal diglycosidases whose substrate specificities had been reported to be different from αRßG I. On the other hand, αRßG II grouped in glycoside hydrolase family 3 (GH3) and thus is the first GH3 member that hydrolyzes the heterosidic linkage of rutinosylated compounds. The substrate scopes of the enzymes were different: αRßG I showed exclusive specificity toward 7-O-ß-rutinosyl flavonoids, whereas αRßG II hydrolyzed both 7-O-ß-rutinosyl- and 3-O-ß-rutinosyl- flavonoids. None of the enzymes displayed activity toward 7-O-ß-neohesperidosyl- flavonoids. The recombinant enzymes also exhibited transglycosylation activities, transferring rutinose from hesperidin or rutin onto various alcoholic acceptors. The different substrate scopes of αRßG I and II may be part of an optimized strategy of the original microorganism to utilize different carbon sources.

Alteration of the Route to Menaquinone towards Isochorismate-Derived Metabolites.

Chorismate and isochorismate constitute branch-point intermediates in the biosynthesis of many aromatic metabolites in microorganisms and plants. To obtain unnatural compounds, we modified the route to menaquinone in Escherichia coli. We propose a model for the binding of isochorismate to the active site of MenD ((1R,2S, 5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxycyclohex-3-ene-1-carboxylate (SEPHCHC) synthase) that explains the outcome of the native reaction with α-ketoglutarate. We have rationally designed variants of MenD for the conversion of several isochorismate analogues. The double-variant Asn117Arg-Leu478Thr preferentially converts (5S,6S)-5,6-dihydroxycyclohexa-1,3-diene-1-carboxylate (2,3-trans-CHD), the hydrolysis product of isochorismate, with a >70-fold higher ratio than that for the wild type. The single-variant Arg107Ile uses (5S,6S)-6-amino-5-hydroxycyclohexa-1,3-diene-1-carboxylate (2,3-trans-CHA) as substrate with >6-fold conversion compared to wild-type MenD. The novel compounds have been made accessible in vivo (up to 5.3 g L-1 ). Unexpectedly, as the identified residues such as Arg107 are highly conserved (>94 %), some of the designed variations can be found in wild-type SEPHCHC synthases from other bacteria (Arg107Lys, 0.3 %). This raises the question for the possible natural occurrence of as yet unexplored branches of the shikimate pathway.

Enzyme-mediated transglycosylation of rutinose (6-O-α-l-rhamnosyl-d-glucose) to phenolic compounds by a diglycosidase from Acremonium sp. DSM 24697.

The structure of the carbohydrate moiety of a natural phenolic glycoside can have a significant effect on the molecular interactions and physicochemical and pharmacokinetic properties of the entire compound, which may include anti-inflammatory and anticancer activities. The enzyme 6-O-α-rhamnosyl-ß-glucosidase (EC 3.2.1.168) has the capacity to transfer the rutinosyl moiety (6-O-α-l-rhamnopyranosyl-ß-d-glucopyranose) from 7-O-rutinosylated flavonoids to hydroxylated organic compounds. This transglycosylation reaction was optimized using hydroquinone (HQ) and hesperidin as rutinose acceptor and donor, respectively. Since HQ undergoes oxidation in a neutral to alkaline aqueous environment, the transglycosylation process was carried out at pH values ≤6.0. The structure of 4-hydroxyphenyl-ß-rutinoside was confirmed by NMR, that is, a single glycosylated product with a free hydroxyl group was formed. The highest yield of 4-hydroxyphenyl-ß-rutinoside (38%, regarding hesperidin) was achieved in a 2-h process at pH 5.0 and 30 °C, with 36 mM OH-acceptor and 5% (v/v) cosolvent. Under the same conditions, the enzyme synthesized glycoconjugates of various phenolic compounds (phloroglucinol, resorcinol, pyrogallol, catechol), with yields between 12% and 28% and an apparent direct linear relationship between the yield and the pKa value of the aglycon. This work is a contribution to the development of convenient and sustainable processes for the glycosylation of small phenolic compounds.

Screening and quantification of the enzymatic deglycosylation of the plant flavonoid rutin by UV-visible spectrometry.

The enzymatic deglycosylation of the plant flavonoid rutin (quercetin-3-O-(6-O-α-l-rhamnopyranosyl-ß-d-glucopyranoside) is usually assessed by means of high performance liquid chromatography (HPLC). We have developed a spectrophotometric method for the quantification of the released quercetin. After the enzymatic reaction, quercetin is extracted with ethyl acetate, and subsequently oxidized under basic conditions. The absorbance of quercetin autooxidation products at 320nm was correlated with the quercetin concentration by linear regression (molar extinction coefficient 23.2 (±0.3)×103M-1cm-1). With this method, rutin-deglycosylation activity in buckwheat flour and a commercial naringinase was measured, and showed no significant differences with the results obtained by HPLC. The convenience of this method resides on the enzymatic activity quantification using the natural substrate by UV-visible spectrometry. Moreover, the simplicity and speed of analysis allows its application for a large number of samples.

Bacteria as source of diglycosidase activity: Actinoplanes missouriensis produces 6-O-α-L-rhamnosyl-ß-D-glucosidase active on flavonoids.

Bacteria represent an underexplored source of diglycosidases. Twenty-five bacterial strains from the genera Actinoplanes, Bacillus, Corynebacterium, Microbacterium, and Streptomyces were selected for their ability to grow in diglycosylated flavonoids-based media. The strains Actinoplanes missouriensis and Actinoplanes liguriae exhibited hesperidin deglycosylation activity (6-O-α-L-rhamnosyl-ß-D-glucosidase activity, EC 3.2.1.168), which was 3 to 4 orders of magnitude higher than the corresponding monoglycosidase activities. The diglycosidase production was confirmed in A. missouriensis by zymographic assays and NMR analysis of the released disaccharide, rutinose. The gene encoding the 6-O-α-L-rhamnosyl-ß-D-glucosidase was identified in the genome sequence of A. missouriensis 431(T) (GenBank accession number BAL86042.1) and functionally expressed in Escherichia coli. The recombinant protein hydrolyzed hesperidin and hesperidin methylchalcone, but not rutin, which indicates its specificity for 7-O-rutinosylated flavonoids. The protein was classified into the glycoside hydrolase family 55 (GH55) in contrast to the known eukaryotic diglycosidases, which belong to GH1 and GH5. These findings demonstrate that organisms other than plants and filamentous fungi can contribute to an expansion of the diglycosidase toolbox.

Cytochrome P450-catalyzed regio- and stereoselective phenol coupling of fungal natural products.

For almost 100 years, phenoxy radical coupling has been known to proceed in nature. Because of the linkage of their molecular halves (regiochemistry) and the configuration of the biaryl axis (stereochemistry), biaryls are notoriously difficult to synthesize. Whereas the intramolecular enzymatic coupling has been elucidated in detail for several examples, the bimolecular intermolecular coupling could not be assigned to one single enzyme in the biosynthesis of axially chiral biaryls. As these transformations often take place regio- and stereoselectively, enzyme-catalyzed control is reasonable. We now report the identification and expression of fungal cytochrome P450 enzymes that catalyze regio- and stereoselective intermolecular phenol couplings. The cytochrome P450 enzyme KtnC from the kotanin biosynthetic pathway of Aspergillus niger was expressed in Saccharomyces cerevisiae. The recombinant cells catalyzed the coupling of the monomeric coumarin 7-demethylsiderin both regio- and stereoselectively to the 8,8'-dimer P-orlandin, a precursor of kotanin. The sequence information obtained from the kotanin biosynthetic gene cluster was used to identify in silico a similar gene cluster in the genome of Emericella desertorum, a producer of desertorin A, the 6,8'-regioisomer of orlandin. The cytochrome P450 enzyme DesC was also expressed in S. cerevisiae and was found to regio- and stereoselectively catalyze the coupling of 7-demethylsiderin to M-desertorin A. Our results show that fungi use highly specific cytochrome P450 enzymes for regio- and stereoselective phenol coupling. The enzymatic activities of KtnC and DesC are relevant for an understanding of the mechanism of this important biosynthetic step. These results suggest that bimolecular phenoxy radical couplings in nature can be catalyzed by phenol-coupling P450 heme enzymes, which might also apply to the plant kingdom.

Polyethyleneimine coating of magnetic particles increased the stability of an immobilized diglycosidase.

The diglycosidase, α-rhamnosyl-ß-glucosidase, from Acremonium sp. DSM24697 was immobilized by adsorption and cross-linking onto polyaniline-iron (PI) particles. The immobilization yield and the immobilization efficiency were relatively high, 31.2% and 8.9%, respectively. However, the heterogeneous preparation showed lower stability in comparison with the soluble form of the enzyme in operational conditions at 60 °C. One parameter involved in the reduced stability of the heterogeneous preparation was the protein metal-catalyzed oxidation achieved by iron traces supplied from the support. To overcome the harmful effect, iron particles were coated with polyethyleneimine (PEI; 0.84 mg/g) previously for the immobilization of the catalyst. The increased stability of the catalyst was correlated with the amount of iron released from the support. Under operational conditions, the uncoated particles lost between 76% and 52% activity after two cycles of reuse, whereas the PEI-coated preparation reduced 45-28% activity after five cycles of reuse in the range of pH 5.0-10, respectively. Hence, polymer coating of magnetic materials used as enzyme supports might be an interesting approach to improve the performance of biotransformation processes.

Quantification of hesperidin in citrus-based foods using a fungal diglycosidase.

A simple enzymatic-spectrophotometric method for hesperidin quantification was developed by means of a specific fungal enzyme. The method utilises the diglycosidase α-rhamnosyl-ß-glucosidase (EC 3.2.1.168) to quantitatively hydrolyse hesperidin to hesperetin, and the last is measured by its intrinsic absorbance in the UV range at 323 nm. The application of this method to quantify hesperidin in orange (Citrus sinensis) juices was shown to be reliable in comparison with the standard method for flavonoid quantification (high performance liquid chromatography, HPLC). The enzymatic method was found to have a limit of quantification of 1.8 µM (1.1 mg/L) hesperidin, similar to the limit usually achieved by HPLC. Moreover, it was feasible to be applied to raw juice, without sample extraction. This feature eliminated the sample pre-treatment, which is mandatory for HPLC, with the consequent reduction of the time required for the quantification.

Transglycosylation specificity of Acremonium sp. α-rhamnosyl-ß-glucosidase and its application to the synthesis of the new fluorogenic substrate 4-methylumbelliferyl-rutinoside.

Transglycosylation potential of the fungal diglycosidase α-rhamnosyl-ß-glucosidase was explored. The biocatalyst was shown to have broad acceptor specificity toward aliphatic and aromatic alcohols. This feature allowed the synthesis of the diglycoconjugated fluorogenic substrate 4-methylumbelliferyl-rutinoside. The synthesis was performed in one step from the corresponding aglycone, 4-methylumbelliferone, and hesperidin as rutinose donor. 4-Methylumbelliferyl-rutinoside was produced in an agitated reactor using the immobilized biocatalyst with a 16% yield regarding the sugar acceptor. The compound was purified by solvent extraction and silica gel chromatography. MALDI-TOF/TOF data recorded for the [M+Na](+) ions correlated with the theoretical monoisotopic mass (calcd [M+Na](+): 507.44 m/z; obs. [M+Na](+): 507.465 m/z). 4-Methylumbelliferyl-rutinoside differs from 4-methylumbelliferyl-glucoside in the rhamnosyl substitution at the C-6 of glucose, and this property brings about the possibility to explore in nature the occurrence of endo-ß-glucosidases by zymographic analysis.

α-Rhamnosyl-ß-glucosidase-catalyzed reactions for analysis and biotransformations of plant-based foods.

Most aroma compounds exist in vegetal tissues as disaccharide conjugates, rutinose being an abundant sugar moiety in grapes. The availability of aroma precursors would facilitate analytical analysis of plant-based foods. The diglycosidase α-rhamnosyl-ß-glucosidase from Acremonium sp. DSM 24697 efficiently transglycosylated the rutinose moiety from hesperidin to 2-phenylethanol, geraniol, and nerol in an aqueous-organic biphasic system. 2-Phenethyl rutinoside was synthesized up to millimolar level with an 80% conversion regarding the donor hesperidin. The hydrolysis of the synthesized aroma precursors was not detected in an aqueous medium. However, in the presence of ethanol as a sugar acceptor, the enzyme was able to transfer the disaccharide residue forming the alkyl-rutinoside. The aroma precursors were significantly hydrolyzed (up to 3-4% in 2 h at 30 °C), which indicated the potential use of the enzyme for biotechnological applications, for example, in aroma modulation of fermented foods.

Dose-dependent significance of monosaccharides on intracellular alpha-L-rhamnosidase activity from Pseudoalteromonas sp.

Intracellular alpha-L-rhamnosidase (EC 3.2.1.40) from the psychrotolerant Pseudoalteromonas sp. 005NJ showed a dose-dependent inhibition for L-rhamnose (IC(50) = 20 mM) and D-ribose (IC(50) = 95 mM), whereas D-glucose and L-fucose presented a lower inhibition, with IC(50) values as high as >0.5 and >0.2 M, respectively. On the other hand, D-fructose enhanced enzyme activity threefold, reaching a plateau of maximum specific activity between 0.2 and 0.4 M of this monosaccharide. Both effects, low inhibition and stimulation, caused by key fruit sugars (glucose and fructose), make this biocatalyst an interesting system in terms of its potential application for debittering fruit juices.