Structure-Guided Immobilization of an Evolved Unspecific Peroxygenase.

Unspecific peroxygenases (UPOs) are highly promiscuous biocatalyst with self-sufficient mono(per)oxygenase activity. A laboratory-evolved UPO secreted by yeast was covalently immobilized in activated carriers through one-point attachment. In order to maintain the desired orientation without compromising the enzyme's activity, the S221C mutation was introduced at the surface of the enzyme, enabling a single disulfide bridge to be established between the support and the protein. Fluorescence confocal microscopy demonstrated the homogeneous distribution of the enzyme, regardless of the chemical nature of the carrier. This immobilized biocatalyst was characterized biochemically opening an exciting avenue for research into applied synthetic chemistry.

Optimization of Regioselective α-Glucosylation of Hesperetin Catalyzed by Cyclodextrin Glucanotransferase.

The regioselective α-glucosylation of hesperetin was achieved by a transglycosylation reaction catalyzed by cyclodextrin glucanotransferase (CGTase) from Thermoanaerobacter sp. using soluble starch as glucosyl donor. By combining mass spectrometry (ESI-TOF) and 2D-NMR analysis, the main monoglucosylated derivative was fully characterized (hesperetin 7-O-α-d-glucopyranoside). In order to increase the yield of monoglucoside, several reaction parameters were optimized: Nature and percentage of cosolvent, composition of the aqueous phase, glucosyl donor, temperature, and the concentrations of hesperetin and soluble starch. Under the optimal conditions, which included the presence of 30% of bis(2-methoxyethyl) ether as cosolvent, the maximum concentration of monoglucoside was approximately 2 mM, obtained after 24 h of reaction. To our knowledge, this is the first report of direct glucosylation of hesperetin employing free enzymes instead of whole cells.

Enzymatic Synthesis of a Novel Pterostilbene α-Glucoside by the Combination of Cyclodextrin Glucanotransferase and Amyloglucosidase.

The synthesis of a novel α-glucosylated derivative of pterostilbene was performed by a transglycosylation reaction using starch as glucosyl donor, catalyzed by cyclodextrin glucanotransferase (CGTase) from Thermoanaerobacter sp. The reaction was carried out in a buffer containing 20% (v/v) DMSO to enhance the solubility of pterostilbene. Due to the formation of several polyglucosylated products with CGTase, the yield of monoglucoside was increased by the treatment with a recombinant amyloglucosidase (STA1) from Saccharomyces cerevisiae (var. diastaticus). This enzyme was not able to hydrolyze the linkage between the glucose and pterostilbene. The monoglucoside was isolated and characterized by combining ESI-MS and 2D-NMR methods. Pterostilbene α-d-glucopyranoside is a novel compound. The α-glucosylation of pterostilbene enhanced its solubility in water to approximately 0.1 g/L. The α-glucosylation caused a slight loss of antioxidant activity towards ABTS˙⁺ radicals. Pterostilbene α-d-glucopyranoside was less toxic than pterostilbene for human SH-S5Y5 neurons, MRC5 fibroblasts and HT-29 colon cancer cells, and similar for RAW 264.7 macrophages.

Micro-scale procedure for enzyme immobilization screening and operational stability assays.

OBJECTIVE: A simple and inexpensive methodology, based on the use of micro-centrifuge filter tubes, is proposed for establishing the best enzyme immobilization conditions. RESULTS: The immobilized biocatalyst is located inside the filter holder during the whole protocol, thus facilitating the incubations, filtrations and washings. This procedure minimizes the amount of enzyme and solid carrier needed, and allows exploring different immobilization parameters (pH, buffer concentration, enzyme/carrier ratio, incubation time, etc.) in a fast manner. The handling of immobilized enzymes using micro-centrifuge filter tubes can also be applied to assess the apparent activity of the biocatalysts, as well as their reuse in successive batch reaction cycles. The usefulness of the proposed methodology is shown by the determination of the optimum pH for the immobilization of an inulinase (Fructozyme L) on two anion-exchange polymethacrylate resins (Sepabeads EC-EA and Sepabeads EC-HA). CONCLUSION: The micro-scale procedure described here will help to overcome the lack of guidelines that usually govern the selection of an immobilization method, thus favouring the development of stable and robust immobilized enzymes that can withstand harsh operating conditions in industry.

Strategy for the Enzymatic Acylation of the Apple Flavonoid Phloretin Based on Prior α-Glucosylation.

The acylation of flavonoids serves as a means to alter their physicochemical properties, enhance their stability, and improve their bioactivity. Compared with natural flavonoid glycosides, the acylation of nonglycosylated flavonoids presents greater challenges since they contain fewer reactive sites. In this work, we propose an efficient strategy to solve this problem based on a first α-glucosylation step catalyzed by a sucrose phosphorylase, followed by acylation using a lipase. The method was applied to phloretin, a bioactive dihydrochalcone mainly present in apples. Phloretin underwent initial glucosylation at the 4'-OH position, followed by subsequent (and quantitative) acylation with C8, C12, and C16 acyl chains employing an immobilized lipase from Thermomyces lanuginosus. Electrospray ionization-mass spectrometry (ESI-MS) and two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) confirmed that the acylation took place at 6-OH of glucose. The water solubility of C8 acyl glucoside closely resembled that of aglycone, but for C12 and C16 derivatives, it was approximately 3 times lower. Compared with phloretin, the radical scavenging capacity of the new derivatives slightly decreased with 2,2-diphenyl-1-picrylhydrazyl (DPPH) and was similar to 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•+). Interestingly, C12 acyl-α-glucoside displayed an enhanced (3-fold) transdermal absorption (using pig skin biopsies) compared to phloretin and its α-glucoside.

Widening the pH activity profile of a fungal laccase by directed evolution.

Unnatural selection: A fungal laccase was tailored by directed evolution to be active at neutral/alkaline pH. After five generations, the final mutant showed a broader pH profile while retaining 50 to 80 % of its activity at neutral pH.

Analysis of fermentation selectivity of purified galacto-oligosaccharides by in vitro human faecal fermentation.

The in vitro fermentation of several purified galacto-oligosaccharides (GOS), specifically the trisaccharides 4'-galactosyl-lactose and 6'-galactosyl-lactose and a mixture of the disaccharides 6-galactobiose and allolactose, was carried out. The bifidogenic effect of GOS at 1% (w/v) was studied in a pH-controlled batch culture fermentation system inoculated with healthy adult human faeces. Results were compared with those obtained with a commercial GOS mixture (Bimuno-GOS). Changes in bacterial populations measured through fluorescence in situ hybridization and short-chain fatty acid (SCFA) production were determined. Bifidobacteria increased after 10-h fermentation for all the GOS substrates, but the changes were only statistically significant (P<0.05) for the mixture of disaccharides and Bimuno-GOS. Acetic acid, whose formation is consistent with bifidobacteria metabolism, was the major SCFA synthesized. The acetate concentration at 10 h was similar with all the substrates (45-50 mM) and significantly higher than the observed for formic, propionic and butyric acids. All the purified GOS could be considered bifidogenic under the assayed conditions, displaying a selectivity index in the range 2.1-3.0, which was slightly lower than the determined for the commercial mixture Bimuno-GOS.

Endothelial progenitor cells as biomarkers of diabetes-related cardiovascular complications.

Diabetes mellitus (DM) constitutes a chronic metabolic disease characterized by elevated levels of blood glucose which can also lead to the so-called diabetic vascular complications (DVCs), responsible for most of the morbidity, hospitalizations and death registered in these patients. Currently, different approaches to prevent or reduce DM and its DVCs have focused on reducing blood sugar levels, cholesterol management or even changes in lifestyle habits. However, even the strictest glycaemic control strategies are not always sufficient to prevent the development of DVCs, which reflects the need to identify reliable biomarkers capable of predicting further vascular complications in diabetic patients. Endothelial progenitor cells (EPCs), widely known for their potential applications in cell therapy due to their regenerative properties, may be used as differential markers in DVCs, considering that the number and functionality of these cells are affected under the pathological environments related to DM. Besides, drugs commonly used with DM patients may influence the level or behaviour of EPCs as a pleiotropic effect that could finally be decisive in the prognosis of the disease. In the current review, we have analysed the relationship between diabetes and DVCs, focusing on the potential use of EPCs as biomarkers of diabetes progression towards the development of major vascular complications. Moreover, the effects of different drugs on the number and function of EPCs have been also addressed.

Evolving thermostability in mutant libraries of ligninolytic oxidoreductases expressed in yeast.

BACKGROUND: In the picture of a laboratory evolution experiment, to improve the thermostability whilst maintaining the activity requires of suitable procedures to generate diversity in combination with robust high-throughput protocols. The current work describes how to achieve this goal by engineering ligninolytic oxidoreductases (a high-redox potential laccase -HRPL- and a versatile peroxidase, -VP-) functionally expressed in Saccharomyces cerevisiae. RESULTS: Taking advantage of the eukaryotic machinery, complex mutant libraries were constructed by different in vivo recombination approaches and explored for improved stabilities and activities. A reliable high-throughput assay based on the analysis of T50 was employed for discovering thermostable oxidases from mutant libraries in yeast. Both VP and HRPL libraries contained variants with shifts in the T50 values. Stabilizing mutations were found at the surface of the protein establishing new interactions with the surrounding residues. CONCLUSIONS: The existing tradeoff between activity and stability determined from many point mutations discovered by directed evolution and other protein engineering means can be circumvented combining different tools of in vitro evolution.

Immobilization of the glucose isomerase from Caldicoprobacter algeriensis on Sepabeads EC-HA and its efficient application in continuous High Fructose Syrup production using packed bed reactor.

The glucose isomerase GICA from Caldicoprobacter algeriensis was immobilized by ionic adsorption on polymethacrylate carriers (Sepabeads EC-EA and EC-HA) or covalent attachment to glyoxal agarose. The Sepabeads EC-HA yielded the highest recovery of activity (89%). The optimum temperature and pH of immobilized GICA were 90 °C and 7.0, respectively, similar to the corresponding values of free enzyme. Nevertheless, the adsorbed enzyme displayed higher relative activity at acidic pH, greater thermostability, and better storage stability, compared to the free form. Moreover, the immobilized enzyme showed an excellent operational stability, in 15 successive 3 h reaction cycles at 85 °C under a batch reactor, preserving 83% of its initial activity. Interestingly, a continuous process for High Fructose Syrup (HFS) production was established with the adsorbed GICA using a packed bed reactor during eleven days at 70 °C. HPAEC-PAD analysis showed a maximum bioconversion rate of 49% after 48 h of operation.

Selective Synthesis of Galactooligosaccharides Containing ß(1→3) Linkages with ß-Galactosidase from Bifidobacterium bifidum (Saphera).

The transglycosylation activity of a novel commercial ß-galactosidase from Bifidobacterium bifidum (Saphera) was evaluated. The optimal conditions for the operation of this enzyme, measured with o-nitrophenyl-ß-d-galactopyranoside, were 40 °C and pH around 6.0. Although at low lactose concentrations the property of this enzyme was basically hydrolytic, an increase of lactose concentration to 400 g/L resulted in a significant formation (107.2 g/L, 27% yield) of prebiotic galactooligosaccharides (GOS). The maximum amount of GOS was obtained at a lactose conversion of approximately 90%, which contrasts with other ß-galactosidases, for which the highest GOS yield is achieved at 40-50% lactose conversion. Using high-performance anion-exchange chromatography with pulsed amperometric detection, semipreparative high-performance liquid chromatography-hydrophilic interaction liquid chromatography, mass spectrometry, and 1D and 2D NMR, we determined the structure of most of the GOS synthesized by this enzyme. The main identified products were Gal-ß(1→3)-Gal-ß(1→4)-Glc (3'-O-ß-galactosyl-lactose), Gal-ß(1→6)-Glc (allolactose), Gal-ß(1→3)-Glc (3-galactosyl-glucose), Gal-ß(1→3)-Gal (3-galactobiose), and the tetrasaccharide Gal-ß(1→3)-Gal-ß(1→3)-Gal-ß(1→4)-Glc. In general, B. bifidum ß-galactosidase showed a tendency to form ß(1→3) linkages followed by ß(1→6) and more scarcely ß(1→4).

Deciphering the molecular specificity of phenolic compounds as inhibitors or glycosyl acceptors of ß-fructofuranosidase from Xanthophyllomyces dendrorhous.

Enzymatic glycosylation of polyphenols is a tool to improve their physicochemical properties and bioavailability. On the other hand, glycosidic enzymes can be inhibited by phenolic compounds. In this work, we studied the specificity of various phenolics (hydroquinone, hydroxytyrosol, epigallocatechin gallate, catechol and p-nitrophenol) as fructosyl acceptors or inhibitors of the ß-fructofuranosidase from Xanthophyllomyces dendrorhous (pXd-INV). Only hydroquinone and hydroxytyrosol gave rise to the formation of glycosylated products. For the rest, an inhibitory effect on both the hydrolytic (H) and transglycosylation (T) activity of pXd-INV, as well as an increase in the H/T ratio, was observed. To disclose the binding mode of each compound and elucidate the molecular features determining its acceptor or inhibitor behaviour, ternary complexes of the inactive mutant pXd-INV-D80A with fructose and the different polyphenols were analyzed by X-ray crystallography. All the compounds bind by stacking against Trp105 and locate one of their phenolic hydroxyls making a polar linkage to the fructose O2 at 3.6-3.8 Å from the C2, which could enable the ulterior nucleophilic attack leading to transfructosylation. Binding of hydroquinone was further investigated by soaking in absence of fructose, showing a flexible site that likely allows productive motion of the intermediates. Therefore, the acceptor capacity of the different polyphenols seems mediated by their ability to make flexible polar links with the protein, this flexibility being essential for the transfructosylation reaction to proceed. Finally, the binding affinity of the phenolic compounds was explained based on the two sites previously reported for pXd-INV.

Effect of α-Glucosylation on the Stability, Antioxidant Properties, Toxicity, and Neuroprotective Activity of (-)-Epigallocatechin Gallate.

(-)-Epigallocatechin gallate (EGCG), the predominant catechin (≥50%) in green tea (Camellia sinensis), displays several bioactive properties but its stability and bioavailability are low. In this work, the properties of two α-glucosyl derivatives of EGCG (3'- and 7-O-α-D-glucopyranoside), obtained by enzymatic synthesis, were assessed. The α-glucosylation enhanced the pH and thermal stability of EGCG. The analysis of scavenging activity toward ABTS·+ radicals showed that the α-glucosylation at C-7 of A-ring caused a higher loss of antioxidant activity compared with the sugar conjugation at C-3' of B-ring. The 3'-glucoside also showed higher potential to alleviate intracellular reactive oxygen species (ROS) levels and to boost REDOX activity. The toxicity of EGCG and its monoglucosides was tested in human SH-S5Y5 neurons, RAW 264.7 macrophages, MRC5 fibroblasts, and HT-29 colon cancer cells. Interestingly, the 3'-O-α-D-glucoside increased the viability of neural cells in vitro (2.75-fold at 100 µM) in the presence of H2O2, whilst EGCG gave rise only to a 1.7-fold enhancement. In conclusion, the α-glucoside of EGCG at C-3' has a great potential for nutraceutical, cosmetic and biomedical applications.

Altering the laccase functionality by in vivo assembly of mutant libraries with different mutational spectra.

The generation of diversity for directed protein evolution experiments shows an important bottleneck in the in vitro random mutagenesis protocols. Most of them are biased towards specific changes that eventually confer a predicted and conservative mutational spectrum, limiting the exploration of the vast protein space. The current work describes a simple methodology to in vivo recombine mutant libraries with different nucleotide bias created by in vitro methods. This in vivo assembly was based on the accurate physiology of Saccharomyces cerevisiae, which as host, provided its high homologous recombination frequency to shuffle the libraries in a nonmutagenic way. The fungal thermophilic laccase from Myceliophthora thermophila expressed in S. cerevisiae was submitted to this protocol under the selective pressure of high concentrations of organic solvents. Mutant 2E9 with approximately 3-fold better kinetics than parent type showed two consecutive amino acid changes (G614D -GGC/GAC- and E615K -GAG/AAG-) because of the in vivo shuffling of the mutant libraries. Both mutations are located in the C-terminal tail that is specifically processed at the Golgi during the maturation of the protein by the Kex2 protease. Notoriously, the oxygen consumption at the T2/T3 trinuclear copper cluster was altered and the catalytic copper at the T1 site was perturbed showing differences in its redox potential and geometry. The change in the isoelectric point of C-terminal extension upon mutations seems to affect the folding of the protein at the posttranslational processing steps providing new insights in the significance of the C-terminal tail for the functionality of the ascomycete laccases.

Combinatorial saturation mutagenesis of the Myceliophthora thermophila laccase T2 mutant: the connection between the C-terminal plug and the conserved (509)VSG(511) tripeptide.

A mutant laccase from the Ascomycete Myceliophthora thermophila has been submitted to iterative cycles of combinatorial saturation mutagenesis through in vivo overlap extension in Saccharomyces cerevisiae. Over 180,000 clones were explored, among which the S510G mutant revealed a direct interaction between the conserved (509)VSG(511) tripeptide, located in the neighborhood of the T1 site, and the C-terminal plug. The K(m)(O)(2) value of the mutant increased 1.5-fold, and the electron transfer pathway between the reducing substrate and the T1 copper ion was altered, improving the catalytic efficiency towards non-phenolic and phenolic substrates by about 3- and 8-fold. Although the geometry at the T1 site was perturbed by the mutation, paradoxically the laccase redox potential was not significantly altered. Together, the results obtained in this study suggest that the (509)VSG(511) tripeptide may play a hitherto unrecognized role in regulating the traffic of oxygen through the C-terminal plug, the latter blocking access to the T2/T3 copper cluster in the native enzyme.

In vitro evolution of a fungal laccase in high concentrations of organic cosolvents.

Fungal laccases are remarkable green catalysts that have a broad substrate specificity and many potential applications in bioremediation, lignocellulose processing, organic synthesis, and more. However, most of these transformations must be carried out at high concentrations of organic cosolvents in which laccases undergo unfolding, thereby losing their activity. We have tailored a thermostable laccase that tolerates high concentrations of cosolvents, the genetic product of five rounds of directed evolution expressed in Saccharomyces cerevisiae. This evolved laccase--R2 variant--was capable of resisting a wide array of cosolvents at concentrations as high as 50% (v/v). Intrinsic laccase features such as the redox potential and the geometry of catalytic copper varied slightly during the course of the molecular evolution. Some mutations at the protein surface stabilized the laccase by allowing additional electrostatic and hydrogen bonding to occur.

Engineering and Applications of fungal laccases for organic synthesis.

Laccases are multi-copper containing oxidases (EC 1.10.3.2), widely distributed in fungi, higher plants and bacteria. Laccase catalyses the oxidation of phenols, polyphenols and anilines by one-electron abstraction, with the concomitant reduction of oxygen to water in a four-electron transfer process. In the presence of small redox mediators, laccase offers a broader repertory of oxidations including non-phenolic substrates. Hence, fungal laccases are considered as ideal green catalysts of great biotechnological impact due to their few requirements (they only require air, and they produce water as the only by-product) and their broad substrate specificity, including direct bioelectrocatalysis.Thus, laccases and/or laccase-mediator systems find potential applications in bioremediation, paper pulp bleaching, finishing of textiles, bio-fuel cells and more. Significantly, laccases can be used in organic synthesis, as they can perform exquisite transformations ranging from the oxidation of functional groups to the heteromolecular coupling for production of new antibiotics derivatives, or the catalysis of key steps in the synthesis of complex natural products. In this review, the application of fungal laccases and their engineering by rational design and directed evolution for organic synthesis purposes are discussed.

Efficient α-Glucosylation of Epigallocatechin Gallate Catalyzed by Cyclodextrin Glucanotransferase from Thermoanaerobacter Species.

The glycosylation of plant polyphenols may modulate their solubility and bioavailability and protect these molecules from oxygen, light degradation, and during gastrointestinal transit. In this work, the synthesis of various α-glucosyl derivatives of (-)-epigallocatechin gallate, the predominant catechin in green tea, was performed in water at 50 °C by a transglycosylation reaction catalyzed by cyclodextrin glycosyltransferase from Thermoanaerobacter sp. The molecular weight of reaction products was determined by high-performance liquid chromatography coupled to mass spectrometry. Using hydrolyzed potato starch as a glucosyl donor, two main monoglucosides were obtained with conversion yields of 58 and 13%, respectively. The products were isolated and chemically characterized by combining two-dimensional nuclear magnetic resonance methods. The major derivative was epigallocatechin gallate 3'- O-α-d-glucopyranoside (1), and the minor derivative was epigallocatechin gallate 7- O-α-d-glucopyranoside (2).

Lipase-Catalyzed Synthesis of Fatty Acid Esters of Trisaccharides.

Carbohydrate fatty acid esters have a broad spectrum of applications in the food, cosmetic, and pharmaceutical industries. The enzyme-catalyzed acylation is significantly more selective than the chemical process and is carried out at milder conditions. Compared with mono- and disaccharides, the acylation of trisaccharides has been less studied. However, trisaccharide esters display notable bioactive properties, probably due to the higher hydrophilicity of the sugar head group. In this chapter, we describe the acylation of two trisaccharides, maltotriose and 1-kestose, catalyzed by different immobilized lipases, using vinyl esters as acyl donors. To illustrate the potential of such compounds, the antitumor activity of 6″-O-palmitoyl-maltotriose is shown.

Directed -in vitro- evolution of Precambrian and extant Rubiscos.

Rubisco is an ancient, catalytically conserved yet slow enzyme, which plays a central role in the biosphere's carbon cycle. The design of Rubiscos to increase agricultural productivity has hitherto relied on the use of in vivo selection systems, precluding the exploration of biochemical traits that are not wired to cell survival. We present a directed -in vitro- evolution platform that extracts the enzyme from its biological context to provide a new avenue for Rubisco engineering. Precambrian and extant form II Rubiscos were subjected to an ensemble of directed evolution strategies aimed at improving thermostability. The most recent ancestor of proteobacteria -dating back 2.4 billion years- was uniquely tolerant to mutagenic loading. Adaptive evolution, focused evolution and genetic drift revealed a panel of thermostable mutants, some deviating from the characteristic trade-offs in CO2-fixing speed and specificity. Our findings provide a novel approach for identifying Rubisco variants with improved catalytic evolution potential.