Deglycosylation Activity Research Articles

Functional characterization, structural basis, and protein engineering of a rare flavonoid 2′-O-glycosyltransferase from Scutellaria baicalensis

Glycosylation is an important post-modification reaction in plant secondary metabolism, and contributes to structural diversity of bioactive natural products. In plants, glycosylation is usually catalyzed by UDP-glycosyltransferases. Flavonoid 2′-O-glycosides are rare glycosides. However, no UGTs have been reported, thus far, to specifically catalyze 2′-O-glycosylation of flavonoids. In this work, UGT71AP2 was identified from the medicinal plant Scutellaria baicalensis as the first flavonoid 2′-O-glycosyltransferase. It could preferentially transfer a glycosyl moiety to 2′-hydroxy of at least nine flavonoids to yield six new compounds. Some of the 2′-O-glycosides showed noticeable inhibitory activities against cyclooxygenase 2. The crystal structure of UGT71AP2 (2.15 Å) was solved, and mechanisms of its regio-selectivity was interpreted by pKa calculations, molecular docking, MD simulation, MM/GBSA binding free energy, QM/MM, and hydrogen‒deuterium exchange mass spectrometry analysis. Through structure-guided rational design, we obtained the L138T/V179D/M180T mutant with remarkably enhanced regio-selectivity (the ratio of 7-O-glycosylation byproducts decreased from 48% to 4%) and catalytic efficiency of 2′-O-glycosylation (kcat/Km, 0.23 L/(s·μmol), 12-fold higher than the native). Moreover, UGT71AP2 also possesses moderate UDP-dependent de-glycosylation activity, and is a dual function glycosyltransferase. This work provides an efficient biocatalyst and sets a good example for protein engineering to optimize enzyme catalytic features through rational design.

Spontaneous and site-specific immobilization of PNGase F via spy chemistry.

Protein N-glycosylation plays a critical role in a wide range of biological processes, and aberrant N-glycosylation is frequently associated with various pathological states. For global N-glycosylation analysis, N-glycans are typically released from glycoproteins mediated by endoglycosidases, primarily peptide N-glycosidase F (PNGase F). However, conventional N-glycan release by in-solution PNGase F is time-consuming and nonreusable. Although some immobilization methods can save time and reduce the enzyme dosage, including affinity interaction and covalent binding, the immobilized PNGase F by these traditional methods may compromises the immobilized enzyme's stability and biofunction. Therefore, a new approach is urgently needed to firmly and steadily immobilize PNGase F. To meet this demand, we have developed a spontaneous and site-specific way to immobilize PNGase F onto magnetic nanoparticles via Spy chemistry. The magnetic nanoparticles were synthesized and modified with SpyTag as a solid surface. The PNGase F fused with SpyCatcher can then be site-specifically and covalently immobilized onto this solid phase, forming a firm isopeptide bond via self-catalysis between the SpyTag peptide and SpyCatcher. Importantly, the immobilization process mediated by mild spy chemistry does not result in PNGase F inactivation; and allows immobilized PNGase F to rapidly release various types of glycans (high-mannose, sialylated, and hybrid) from glycoproteins. Moreover, the immobilized PNGase F exhibited good deglycosylation activity and facilitated good reusability in consecutive reactions. Deglycosylation of clinical samples was completed by the immobilized PNGase F as fast as several minutes.

Isomorphic Fluorescent Nucleosides Facilitate Real-Time Monitoring of RNA Depurination by Ribosome Inactivating Proteins.

Ribosome-inactivating proteins, a family of highly cytotoxic proteins, interfere with protein synthesis by depurinating a specific adenosine residue within the conserved α-sarcin/ricin loop of eukaryotic ribosomal RNA. Besides being biological warfare agents, certain RIPs have been promoted as potential therapeutic tools. Monitoring their deglycosylation activity and their inhibition in real time have remained, however, elusive. Herein, we describe the enzymatic preparation and utility of consensus RIP hairpin substrates in which specific G residues, next to the depurination site, are surgically replaced with tz G and th G, fluorescent G analogs. By strategically modifying key positions with responsive fluorescent surrogate nucleotides, RIP-mediated depurination can be monitored in real time by steady-state fluorescence spectroscopy. Subtle differences observed in preferential depurination sites provide insight into the RNA folding as well as RIPs' substrate recognition features.

Hydrogen/Deuterium Exchange Mass Spectrometry with Integrated Electrochemical Reduction and Microchip-Enabled Deglycosylation for Epitope Mapping of Heavily Glycosylated and Disulfide-Bonded Proteins.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a recognized method to study protein conformational dynamics and interactions. Proteins encompassing post-translational modifications (PTMs), such as disulfide bonds and glycosylations, present challenges to HDX-MS, as disulfide bond reduction and deglycosylation is often required to extract HDX information from regions containing these PTMs. In-solution deglycosylation with peptide-N4-(N-acetyl-β-d-glucosaminyl)-asparagine amidase A (PNGase A) or PNGase H+ combined with chemical reduction using tris-(2-carboxyethyl)phosphine (TCEP) has previously been used for HDX-MS analysis of disulfide-linked glycoproteins. However, this workflow requires extensive manual sample preparation and consumes large amounts of enzyme. Furthermore, large amounts of TCEP and glycosidases often result in suboptimal liquid chromatography-mass spectrometry (LC-MS) performance. Here, we compare the in-solution activity of PNGase A, PNGase H+, and the newly discovered PNGase Dj under quench conditions and immobilize them onto thiol-ene microfluidic chips to create HDX-MS-compatible immobilized microfluidic enzyme reactors (IMERs). The IMERS retain deglycosylation activity, also following repeated use and long-term storage. Furthermore, we combine a PNGase Dj IMER, a pepsin IMER, and an electrochemical cell to develop an HDX-MS setup capable of efficient online disulfide-bond reduction, deglycosylation, and proteolysis. We demonstrate the applicability of this setup by mapping the epitope of a monoclonal antibody (mAb) on the heavily disulfide-bonded and glycosylated sema-domain of the tyrosine-protein kinase Met (SD c-Met). We achieve near-complete sequence coverage and extract HDX data to identify regions of SD c-Met involved in mAb binding. The described methodology thus presents an integrated and online workflow for improved HDX-MS analysis of challenging PTM-rich proteins.

General and Robust Chemoenzymatic Method for Glycan-Mediated Site-Specific Labeling and Conjugation of Antibodies: Facile Synthesis of Homogeneous Antibody-Drug Conjugates.

Site-specific labeling and conjugation of antibodies are highly desirable for fundamental research and for developing more efficient diagnostic and therapeutic methods. We report here a general and robust chemoenzymatic method that permits a one-pot site-specific functionalization of antibodies. A series of selectively modified disaccharide oxazoline derivatives were designed, synthesized, and evaluated as donor substrates of different endoglycosidases for antibody Fc glycan remodeling. We found that among several endoglycosidases tested, wild-type endoglycosidase from Streptococcus pyogenes of serotype M49 (Endo-S2) exhibited remarkable activity in transferring the functionalized disaccharides carrying site-selectively modified azide, biotin, or fluorescent tags to antibodies without hydrolyzing the resulting transglycosylation products. This discovery, together with the excellent Fc deglycosylation activity of Endo-S2 on recombinant antibodies, allowed direct labeling and functionalization of antibodies in a one-pot manner without the need of intermediate and enzyme separation. The site-specific introduction of varied numbers of azide groups enabled a highly efficient synthesis of homogeneous antibody-drug conjugates (ADCs) with a precise control of the drug-to-antibody ratio (DAR) ranging from 2 to 12 via a copper-free strain-promoted click reaction. Cell viability assays showed that ADCs with higher DARs were more potent in killing antigen-overexpressed cells than the ADCs with lower DARs. This new method is expected to find applications not only for antibody-drug conjugation but also for cell labeling, imaging, and diagnosis.

Combined isoflavones biotransformation increases the bioactive and antioxidant capacity of soymilk.

Isoflavones are phenolic secondary metabolites mainly occurring in soy and soybean products. Compared to glycoside forms, isoflavone aglycones present higher biological activities. This study evaluated the potential of microbial and enzymatic treatments in biotransformed isoflavones in their biologically active forms in soymilk. Seven different cultures of lactic acid bacteria and bifidobacteria associated with the action of immobilized tannase enzyme were screened for isoflavone glycoside biotransformation ability. The biotransformed soymilk samples were characterized regarding isoflavone profile, total phenolic content, and in vitro antioxidant activities. All bacterial strains showed a good growth capacity in soymilk matrix and produced β-glucosidase enzyme, which hydrolyzed isoflavone glycosides into aglycones in soymilk after 24h of fermentation. The microbial fermentation followed by tannase reaction (FT processes) resulted in the highest increase of bioactive aglycones (10.3- to 13.1-fold for daidzein, 10.4- to 12.3-fold for genistein, and 3.8- to 4.7-fold for glycitein), compared to control soymilk. Further, FT processes enhanced the total phenolic content (53-70%) and antioxidant activity by ORAC (69-102%) and FRAP (49-71%) assays of the soymilk matrix. Therefore, the combination of microbial fermentation and tannase treatment is a promising strategy to obtain a fermented soy product rich in bioactive isoflavones with greater health-promoting potential. KEY POINTS: • Bacterial cultures and tannase enzyme displayed isoflavone deglycosylation activity. • The addition of tannase following the fermentation maximized the isoflavone conversion. • Increased isoflavone aglycones contributed to the improved antioxidant activity of soymilk.

Deglycosylation by the Acidic Glycosidase PNGase H+ Enables Analysis of N-Linked Glycoproteins by Hydrogen/Deuterium Exchange Mass Spectrometry.

Hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) has become an important method to study the structural dynamics of proteins. However, glycoproteins represent a challenge to the traditional HDX-MS workflow for determining the deuterium uptake of the protein segments that contain the glycan. We have recently demonstrated the utility of the glycosidase PNGase A to enable HDX-MS analysis of N-glycosylated protein regions. Here, we have investigated the use of the acidic glycosidase PNGase H+, which has a pH optimum at 2.6, to efficiently deglycosylate N-linked glycosylated peptides during HDX-MS analysis of glycoproteins. Our results show that PNGase H+ retains high deglycosylation activity at HDX quench conditions. When used in an HDX-MS workflow, PNGase H+ allowed the extraction of HDX data from all five glycosylated regions of the serpin α1-antichymotrypsin. We demonstrate that PNGase A and PNGase H+ are capable of similar deglycosylation performance during HDX-MS analysis of α1-antichymotrypsin and the IgG1 antibody trastuzumab (TZ). However, PNGase H+ provides broader specificity and greater tolerance to the disulfide-bond reducing agent TCEP, while PNGase A offers advantages in terms of commercial availability and purity. Overall, our findings demonstrate the unique features of PNGase H+ for improving conformational analysis of glycoproteins by HDX-MS, in particular, challenging glycoproteins containing both glycosylations and disulfide bonds.

Cloning, expression and characterization of a new endo-β-N-acetylglucosaminidase from Streptomyces alfalfae

Endo-β-N-acetylglucosaminidase is used widely in the glycobiology studies and industries. In this study, a new endo-β-N-acetylglucosaminidase, designated as Endo SA, was cloned from Streptomyces alfalfae ACCC 40021 and expressed in Escherichia coli BL21 (DE3). The purified recombinant Endo SA exhibited the maximum activity at 35 ºC and pH 6.0, good thermo/pH stability and high specific activity (1.0×10⁶ U/mg). It displayed deglycosylation activity towards different protein substrates. These good properties make EndoSA a potential tool enzyme and industrial biocatalyst.

Loss of N-Glycanase 1 Alters Transcriptional and Translational Regulation in K562 Cell Lines.

N-Glycanase 1 (NGLY1) deficiency is an ultra-rare, complex and devastating neuromuscular disease. Patients display multi-organ symptoms including developmental delays, movement disorders, seizures, constipation and lack of tear production. NGLY1 is a deglycosylating protein involved in the degradation of misfolded proteins retrotranslocated from the endoplasmic reticulum (ER). NGLY1-deficient cells have been reported to exhibit decreased deglycosylation activity and an increased sensitivity to proteasome inhibitors. We show that the loss of NGLY1 causes substantial changes in the RNA and protein landscape of K562 cells and results in downregulation of proteasomal subunits, consistent with its processing of the transcription factor NFE2L1. We employed the CMap database to predict compounds that can modulate NGLY1 activity. Utilizing our robust K562 screening system, we demonstrate that the compound NVP-BEZ235 (Dactosilib) promotes degradation of NGLY1-dependent substrates, concurrent with increased autophagic flux, suggesting that stimulating autophagy may assist in clearing aberrant substrates during NGLY1 deficiency.

A shrimp glycosylase protein, PmENGase, interacts with WSSV envelope protein VP41B and is involved in WSSV pathogenesis

Viral glycoproteins are expressed by many viruses, and during infection they usually play very important roles, such as receptor attachment or membrane fusion. The mature virion of the white spot syndrome virus (WSSV) is unusual in that it contains no glycosylated proteins, and there are currently no reports of any glycosylation mechanisms in the pathogenesis of this virus. In this study, we cloned a glycosylase, mannosyl-glycoprotein endo-β-N-acetylglucosaminidase (ENGase, EC 3.2.1.96), from Penaeus monodon and found that it was significantly up-regulated in WSSV-infected shrimp. A yeast two-hybrid assay showed that PmENGase interacted with both structural and non-structural proteins, and GST-pull down and co-immunoprecipitation (Co-IP) assays confirmed its interaction with the envelope protein VP41B. In the WSSV challenge tests, the cumulative mortality and viral copy number were significantly decreased in the PmEngase-silenced shrimp, from which we conclude that shrimp glycosylase interacts with WSSV in a way that benefits the virus. Lastly, we speculate that the deglycosylation activity of PmENGase might account for the absence of glycosylated proteins in the WSSV virion.

Simultaneous Optimal Production of Flavonol Aglycones and Degalloylated Catechins from Green Tea Using a Multi-Function Food-Grade Enzyme

(1) Background: Green tea (GT) contains well-known phytochemical compounds; namely, it is rich in flavan-3-ols (catechins) and flavonols comprising all glycoside forms. These compounds in GT might show better biological activities after a feasible enzymatic process, and the process on an industrial scale should consider enzyme specificity and cost-effectiveness. (2) Methods: In this study, we evaluated the most effective method for the enzymatic conversion of flavonoids from GT extract. One enzyme derived from Aspergillus niger (molecular weight 80–90 kDa) was ultimately selected, showing two distinct but simultaneous activities: intense glycoside hydrolase activity via deglycosylation and weak tannin acyl hydrolase activity via degalloylation. (3) Results: The optimum conditions for producing flavonol aglycones were pH 4.0 and 50 °C. Myricetin glycosides were cleaved 3.7–7.0 times faster than kaempferol glycosides. Flavonol aglycones were produced effectively by both enzymatic and hydrochloride treatment in a time-course reaction. Enzymatic treatment retained 80% (w/w) catechins, whereas 70% (w/w) of catechins disappeared by hydrochloride treatment. (4) Conclusions: This enzymatic process offers an effective method of conditionally producing flavonol aglycones and de-galloylated catechins from conversion of food-grade enzyme.

Efficient Enzyme-Assisted Extraction and Conversion of Polydatin to Resveratrol From Polygonum cuspidatum Using Thermostable Cellulase and Immobilized β-Glucosidase.

Resveratrol, a bioactive compound in high quantities in Polygonum cuspidatum, has well-known health benefits. However, it mainly exists in its glycosidic form, polydatin, in plants. To increase the production of resveratrol for various uses in medicine, foods, and cosmetics, an efficient deglycosylation technique is needed for converting polydatin into resveratrol. We screened a new cellulolytic strain of Bacillus from herb compost, and we optimized parameters within the fermentation process using response surface methodology with a Box–Behnken design. The yield of cellulase reached 2701.08 U/L, corresponding to values that were 5.4 times as high as those under unoptimized conditions. The Bacillus cellulase possessed good thermostablity and was stable under both acidic and neutral conditions. The cellulase was then used in the pretreatment of P. cuspidatum root. After incubation at 50°C for 4 h with shaking at 150 rpm, the contents of piceid and resveratrol were determined to be 7.60 ± 0.15 and 9.72 ± 0.29 mg/g, respectively. To obtain complete deglycosylation, immobilized β-glucosidase (bgl2238) was added to the cellulase-treated extracts of P. cuspidatum root to convert residual polydatin into resveratrol. After the first cycle, the contents of piceid and resveratrol were determined to be 0 and 13.69 ± 0.30 mg/g, respectively. Moreover, enzyme activity showed little loss during up to 4 consecutive cycles. These results demonstrated that the immobilized β-glucosidase possessed high deglycosylation activity and outstanding operational stability. The mixture of Bacillus cellulase and immobilized bgl2238 appears promising as a means to increase the supply of resveratrol in the medicine market worldwide.

Expression, purification, and characterization of highly active endo-α-N-acetylgalactosaminidases expressed by silkworm-baculovirus expression system

Abstract The O-glycosidase, endo-α-N-acetylgalactosaminidase from Enterococcus faecalis (endoEF) catalyzes the cleavage of core 1 and core 3 type O-linked disaccharides between GalNAc and serine or threonine residues from glycoproteins. The endoEF has broad substrate specificity and thus is extensively utilized for the structural and functional analysis of the O-linked glycans. In this study, we expressed and purified the recombinant endoEF (rEndoEF) by using the silkworm-baculovirus expression vector system (Silkworm-BEVS) and confirmed the deglycosylation activity of rEndoEF targeting reporter glycoproteins, which was equivalent to the commercial O-glycosidase. Thus, our study provides important clues to produce highly active rEndoEF O-glycosidases employing silkworm-BEVS as an alternative.

Fermentation of commercial soy beverages with lactobacilli and bifidobacteria strains featuring high β-glucosidase activity

Abstract An increase of isoflavone aglycone content in soy foods can be attained through fermentation with food-grade bacteria. In this study, two commercial soy beverages with distinctive chemical composition (AS and VS) were fermented by strains of lactobacilli (eight strains) and bifidobacteria (two strains) with a high β-glucosidase activity. Along fermentation, growth of the strains and isoflavone deglycosylation in the soy beverages were monitored. Large differences in growth, aglycone content and chemical parameters in AS and VS beverages fermented by different species and strains were observed. Isoflavone glycosides were completely transformed into their corresponding aglycones by most strains during fermentation of AS beverage, whereas large amounts of undeglycosilated isoflavones were still present in fermented VS. Four strains showing strong deglycosylation activity and appropriate technological properties (Lactobacillus casei LP71, Lactobacillus plantarum E112, Lactobacillus rhamnosus E41 and Bifidobacterium pseudocatenulatum C35) were proposed as industrial starters to improve functionality in soy-based fermented foods.

Expression and characterization of recombinant l-asparaginase from Pseudomonas fluorescens

l-asparaginase (E.C. 3.5.1.1), an anti-cancer drug has been used in the treatment of acute lymphoblastic leukemia. A novel source of l-asparaginase from Pseudomonas fluorescens was addressed in the present studies. The ANS gene in Pseudomonas fluorescens MTCC 8127 which produces l-asparaginase was cloned and expressed in E. coli BL21 (DE3). The expressed recombinant protein (PfAns) which was purified, showed l-asparaginase activity. The enzyme was further characterized. The pH and temperature optima were found to be 7.5 and 37 °C. The recombinant enzyme was stable to temperature and pH. The enzyme was homotetramer with the molecular weight of the monomer being 35 kDa and a whole protein molecular weight of 140 kDa. The purified l-asparaginase had a specific activity of 26 U/mg with a Km and Vmax of 0.050 M and 4.032 mol−1min. The enzyme exhibited a high affinity towards l-asparagine and showed a very minimal activity with glutamine as a substrate. The enzyme activity was inhibited by PMSF, suggesting the presence of serine at the active site. The presence of Mg2+ enhanced PfAns activity by 49%, and SDS strongly inhibited the enzyme activity. The in vitro half-life of the recombinant enzyme was ∼40 h. The enzyme demonstrated deglycosylation activity which could exhibit an additional barrier for proliferating cancer cells.

Mannose and fructose metabolism in red blood cells during cold storage in SAGM.

Alternate sugar metabolism during red blood cell (RBC) storage is not well understood. Here we report fructose and mannose metabolism in RBCs during cold storage in SAGM and the impact that these monosaccharides have on metabolic biomarkers of RBC storage lesion. RBCs were stored in SAGM containing uniformly labeled 13 C-fructose or 13 C-mannose at 9 or 18 mmol/L concentration for 25 days. RBCs and media were sampled at 14 time points during storage and analyzed using ultraperformance liquid chromatography-mass spectrometry. Blood banking quality assurance measurements were performed. Red blood cells incorporated fructose and mannose during cold storage in the presence of glucose. Mannose was metabolized in preference to glucose via glycolysis. Fructose lowered adenosine triphosphate (ATP) levels and contributed little to ATP maintenance when added to SAGM. Both monosaccharides form the advanced glycation end product glycerate. Mannose activates enzymes in the RBC that take part in glycan synthesis. Fructose or mannose addition to RBC SAGM concentrates may not offset the shift in metabolism of RBCs that occurs after 10 days of storage. Fructose and mannose metabolism at 4°C in SAGM reflects their metabolism at physiologic temperature. Glycerate excretion is a measure of protein deglycosylation activity in stored RBCs. No cytoprotective effect was observed upon the addition of either fructose or mannose to SAGM.

Characterization of an extracellular β-glucosidase from Dekkera bruxellensis for resveratrol production

Polygonum cuspidatum is a widely grown crop with a rich source of polydatin (also called piceid) for resveratrol production. Resveratrol is produced from piceid via enzymatic cleavage of the sugar moiety of piceid. In this study, Dekkera bruxellensis mutants were selected based on their high p-nitrophenyl-β-d-glucopyranoside and piceid conversion activities. The enzyme responsible for piceid conversion was a heterodimeric protein complex that was predominantly secreted to the extracellular medium and consisted of two subunits at an equal ratio with molecular masses of 30.5 kDa and 48.3 kDa. The two subunits were identified as SCW4p and glucan-β-glucosidase precursor in D. bruxellensis. Both proteins were individually expressed in Saccharomyces cerevisiae exg1Δ mutants, which lack extracellular β-glucosidase activity, to confirm each protein's enzymatic activities. Only the glucan-β-glucosidase precursor was shown to be a secretory protein with piceid deglycosylation activity. Our pilot experiments of piceid bioconversion demonstrate the possible industrial applications for this glucan-β-glucosidase precursor in the future.

Biotransformation of Rutin Using Crude Enzyme from Rhodopseudomonas palustris.

Rhodopseudomonas palustris was selected for the ability to grow in diglycosylated flavonoids-based media, exhibited deglycosylation activity. The present study aimed to investigate the ability of a crude enzyme from Rhodopseudomonas palustris to transform rutin. Our results showed the crude enzyme was found to transform rutin to quercetin via isoquercitrin. The maximum enzyme activities were observed at pH 7.0, 25 °C, and rutin concentration of 1.0mgmL- 1. Under optimal conditions, 13.11μM rutin was biotransformed into 6.86μM isoquercitrin and 11.64μM quercetin after 11 and 21h, respectively. The study demonstrates an eco-friendly and potential economically viable 'green' conversion route to convert rutin to isoquercitrin and quercetin, which is of great interest, considering the therapeutic applications of isoquercitrin and quercetin. The specific biotransformation of rutin to isoquercitrin and quercetin, using the crude enzyme from Rhodopseudomonas palustris, may potentially serve as a new method for industrial production of isoquercitrin and quercetin.

Enzymatic deglycosylation of flavonoids in deep eutectic solvents-aqueous mixtures: paving the way for sustainable flavonoid chemistry

The low solubility of glycosylated flavonoids represents a hurdle to conduct efficient enzymatic deglycosylations in aqueous media. To overcome this drawback, environmentally-unfriendly dimethylsulfoxide (DMSO) is typically used as co-solvent. Using a specific diglycosidase from Acremonium sp. DSM24697 for the deglycosylation of the rutinosylated flavonoid (hesperidin) as model reaction, this communication explores the use of (non-hazardous and biodegradable) deep eutectic solvents (DESs) as co-solvents in flavonoid biocatalysis. The enzymatic deglycosylation was observed when DES composed of choline chloride and glycerol or ethylene-glycol was used at proportions of up to 40% (DES-Buffer, v/v), displaying a promising framework to combine enhanced flavonoid solubilities and high enzymatic activities. The deglycosylation activity significantly increased when the single DES components – glycerol and ethylene-glycol – were added (e.g. 140% of enzyme activity at glycerol at 40% v/v), whereas deleterious effects were observed when choline chloride was solely added, presumably due to its chaotropic effect. Future research opportunities may be envisaged in the genetic design to evolve more robust biocatalysts, and in tailoring DES to deliver more enzyme-compatible solvents.

Bacteria as source of diglycosidase activity: Actinoplanes missouriensis produces 6-O-α-L-rhamnosyl-β-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-β-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-β-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.