Simple Enzyme Research Articles

Biomimetic nanozymes with tunable peroxidase activity based on peptides co-assembly for colorimetric sensing of uric acid

The construction of simple and efficient artificial enzymes is of great importance in the fields of sensing and catalysis. In this work, peptide nanotubes (CA-PNTs) co-assembled from two short peptides were non-covalently combined with hemin to obtain a series of CA-PNTs/hemin composites as biomimetic peroxidase materials. Due to the introduction of histidine and arginine in the short peptides simulating the microenvironment near the hydrophobic cavity in the activity center of the natural enzyme, CA-PNTs/hemin shows enhanced peroxidase activity compared with free hemin and the single short peptide self-assembled peptide nanotubes-supported hemin complex, and the enzyme activity of the material could be regulated by simply adjusting the proportion of co-assembled short peptides. Further investigation of the catalytic mechanism demonstrates that CA-PNTs/hemin can effectively bind to H2O2 to form active intermediates and catalyze the decomposition of H2O2 to generate superoxide radicals (O2•−), thus promoting the whole enzymatic reaction. Combined with an enzyme cascade reaction and enzyme catalytic chromogenic system, a colorimetric method based on CA-PNTs/hemin for uric acid (UA) sensing was constructed. The strategy shows a linear range of 0.5–80 μM with a low limit of detection (0.2 μM) and good selectivity, and has been successfully applied to the determination of UA in human urine. A portable platform for UA detection was further developed by conjugating the strategy with smartphones.

Impact of microelectrode geometry and surface finish on enzymatic biosensor performance

Food production, environmental protection, biotechnology, and medicine, all depend on biosensors for measuring specific biological molecules. Among biosensors, enzymatic electrochemical biosensors have received particular attention due to their simple operation and enzymes’ inherent specificity. Researchers have extensively explored novel materials and biological designs to improve performance (i.e., sensitivity, linearity, limit of detection, and response time). However, in comparison, physical and geometrical design has not received the same attention. To this end, we compared platinum (Pt) microelectrodes with circular or fractal geometry with the same surface area (2D geometry). We also studied the effect of 3D geometry by nanostructuring both circular and fractal microelectrodes via electroplating Pt black. Fractal Pt black microelectrodes displayed the highest current density, charge storage capacity, and sensitivity towards H2O2. Next, we immobilized glucose oxidase onto various microelectrode geometries by microcontact stamping. Fractal Pt and Pt black glucose biosensors were 91.7 % and 83.3 % more sensitive than circular counterparts. Circular and fractal Pt black glucose biosensors were 63.0 % and 55.9 % more sensitive than Pt counterparts. Fractal geometry also provided better linearity and limit of detection. We modified enzyme layer thickness through multiple layer stamping and found a trade-off whereby increasing thickness increased sensitivity but also increased response time. Lastly, we developed a COMSOL Multiphysics numerical model to interpret the amperometric data and the impact of physical design on critical parameters. The work here will serve as a guideline for improving enzymatic electrochemical and other biosensors via physical design, which is simpler to modify than material or biological design.

Multi-compartmentalized electrochemical sensing platforms for monitoring cascade enzymatic reactions

Designing an electrochemical biosensor with the required features is a complex process involving multiple factors. For instance, nanocomposite materials used ensure the adaptation of the sensor to specific parameters on demand. But beyond making more versatile sensors, these materials likely offer excellent sensing platforms to mimic biological processes (e.g. cell communication) on the electrode surface. For this, developed methods are needed to integrate non-conductive nanoreactors for molecular communication into a biocompatible matrix on electrode surfaces with the request of spatially separated and controlled enzyme localization. Here, we present a novel reduced graphene oxide hybrid material to immobilize polymeric nanoreactors supported by an alginate network as a matrix on the electrode surface. The possibility of introducing cascade reactions in an electrochemical biosensor has the advantage of broadening the target substrates as well as their selectivity using enzymes in different nanocompartments (=compartmentalization). The polymeric vesicles allow the loading of enzymes or artificial enzymes, offering active center retention and long-term enzyme stability. Firstly, the inclusion of spatially separated polymeric active nanocompartments into the matrix on the electrode surface is used to monitor a simple enzyme reaction. However, the study’s accomplishment lies in its successful ability to monitor an enzyme cascade reaction, one producing and the other consuming hydrogen peroxide, hosting natural and artificial enzymes. This proof-of-concept generates an important contribution to the design of analytical platforms capable of detecting complex environments or even monitoring communication between cell-like structures, extremely useful for fields such as synthetic biology, biomimetics and advanced biosensors.

The progress of clinical research on the detection of 1,5-anhydroglucitol in diabetes and its complications.

1,5-Anhydroglucitol (1,5-AG) is sensitive to short-term glucose fluctuations and postprandial hyperglycemia, which has great potential in the clinical application of diabetes as a nontraditional blood glucose monitoring indicator. A large number of studies have found that 1,5-AG can be used to screen for diabetes, manage diabetes, and predict the perils of diabetes complications (diabetic nephropathy, diabetic cardiovascular disease, diabetic retinopathy, diabetic pregnancy complications, diabetic peripheral neuropathy, etc.). Additionally, 1,5-AG and β cells are also associated with each other. As a noninvasive blood glucose monitoring indicator, salivary 1,5-AG has much more benefit for clinical application; however, it cannot be ignored that its detection methods are not perfect. Thus, a considerable stack of research is still needed to establish an accurate and simple enzyme assay for the detection of salivary 1,5-AG. More clinical studies will also be required in the future to confirm the normal reference range of 1,5-AG and its role in diabetes complications to further enhance the blood glucose monitoring system for diabetes.

The genetic landscape of a metabolic interaction

While much prior work has explored the constraints on protein sequence and evolution induced by physical protein-protein interactions, the sequence-level constraints emerging from non-binding functional interactions in metabolism remain unclear. To quantify how variation in the activity of one enzyme constrains the biochemical parameters and sequence of another, we focus on dihydrofolate reductase (DHFR) and thymidylate synthase (TYMS), a pair of enzymes catalyzing consecutive reactions in folate metabolism. We use deep mutational scanning to quantify the growth rate effect of 2696 DHFR single mutations in 3 TYMS backgrounds under conditions selected to emphasize biochemical epistasis. Our data are well-described by a relatively simple enzyme velocity to growth rate model that quantifies how metabolic context tunes enzyme mutational tolerance. Together our results reveal the structural distribution of epistasis in a metabolic enzyme and establish a foundation for the design of multi-enzyme systems.

Frustration can Limit the Adaptation of Promiscuous Enzymes Through Gene Duplication and Specialisation

Virtually all enzymes catalyse more than one reaction, a phenomenon known as enzyme promiscuity. It is unclear whether promiscuous enzymes are more often generalists that catalyse multiple reactions at similar rates or specialists that catalyse one reaction much more efficiently than other reactions. In addition, the factors that shape whether an enzyme evolves to be a generalist or a specialist are poorly understood. To address these questions, we follow a three-pronged approach. First, we examine the distribution of promiscuity in empirical enzymes reported in the BRENDA database. We find that the promiscuity distribution of empirical enzymes is bimodal. In other words, a large fraction of promiscuous enzymes are either generalists or specialists, with few intermediates. Second, we demonstrate that enzyme biophysics is not sufficient to explain this bimodal distribution. Third, we devise a constraint-based model of promiscuous enzymes undergoing duplication and facing selection pressures favouring subfunctionalization. The model posits the existence of constraints between the catalytic efficiencies of an enzyme for different reactions and is inspired by empirical case studies. The promiscuity distribution predicted by our constraint-based model is consistent with the empirical bimodal distribution. Our results suggest that subfunctionalization is possible and beneficial only in certain enzymes. Furthermore, the model predicts that conflicting constraints and selection pressures can cause promiscuous enzymes to enter a ‘frustrated’ state, in which competing interactions limit the specialisation of enzymes. We find that frustration can be both a driver and an inhibitor of enzyme evolution by duplication and subfunctionalization. In addition, our model predicts that frustration becomes more likely as enzymes catalyse more reactions, implying that natural selection may prefer catalytically simple enzymes. In sum, our results suggest that frustration may play an important role in enzyme evolution.

Murburn concept in cellular function and bioenergetics, Part 1: Understanding murzymes at the molecular level

Bioenergetics is the study of how life-activities are powered within the cell. This also deals with the interactive exchange of matter/radiation between cellular components and their environment, and the accompanying changes thereof. The acclaimed bioenergetics paradigm has relied on “electron transport chains” and selective/stoichiometric electrogenic “ion-pumping” mediated by vectorial protein-embedded membranes. Therein, an electrochemical gradient was deemed to be the driving force for chemical reactions leading to ATP production, physical thermogenesis by uncoupling proteins, and complex electromechanical processes like information relay along the axon. On one hand, this vitally deterministic perception requires the membrane proteins to “intelligently” manipulate ion-fluxes and generate/harness an electrochemical gradient by a gambit-type logic. At the other hand, it also seeks that the same gradient should cyclically control the membrane-proteins’ activity. Our recent pursuits have questioned such traditional perspectives and advocated the alternate explanation of murburn concept, leading to a revamping of the macroscopic treatments of overall thermodynamic, kinetic, mechanistic, and evolutionary (probability) considerations. The current review aims to consolidate the murburn paradigm of bioenergetics, wherein murzymes initiate redox processes by effective charge separation and diffusible reactive species formation, enabling cells to work as simple chemical engines. Herein, we discuss the reaction chemistry of some simple enzyme systems and also delve into protein complex arrays mediated powering routines like mitochondrial respiration-thermogenesis and chloroplast-centered photosynthesis. Furthermore, we remark that the “water–ion–molecules” phase continuum is actually discretized into dynamically fluctuating coacervates and express concern over the marginalization of sound chemico-physical ideas by the bioenergetics community.

Exploring the Dynamics of Simple Inhibition Systems in Continuous Stirred-Tank Reactor: Mathematical Modelling and Bifurcation Analysis

The simple enzyme inhibition systems consist of competitive inhibition, noncompetitive inhibition and uncompetitive inhibition. In this work, we incorporated these simple inhibition systems in the continuous stirred-tank reactor (CSTR) and analysed the models using some techniques from dynamical systems and bifurcation analysis. Our aim is to investigate the behaviours of such systems and compare their overall dynamics. The phase portrait is constructed to simulate possible behaviours such as stable steady states, stable limit cycle, bistability between the steady state and the stable limit cycle and bistability between two steady states. The systems undergo bifurcational changes in dynamics as enzyme concentration, dilution rate and proportional control constant are varied. Moreover, we conducted a codimension two bifurcation analysis to examine the joint effects of dilution rate and proportional control constant on the systems behaviours. Our results revealed distinct dynamics for each inhibition system. Increasing the dilution rate led to a transition from low to high substrate concentrations, with competitive inhibition showing the highest tipping (or bifurcation) point where dynamical regimes change due to intense substrate-inhibitor competition. Elevating enzyme concentration reduced substrate concentration, particularly in non-competitive inhibition systems due to higher conversion rates. Furthermore, the proportional control constant had varying effects depending on the specific inhibition system. These findings emphasize the on the combined influences of distinct chemical procoesses in controlling reactor heat and optimizing bioprocess efficiency, considering the unique characteristics of each inhibition system. Overall, the dynamical study on these simple inhibition systems enables us to improve our understanding on the chemical processes involving enzymes with multiple types of inhibitors and may give some insights in its controlling process.

Quantitative Analysis of The High-Yield Hydrolysis of Kelp by Laminarinase and Alginate Lyase.

Kelp is an abundant, farmable biomass-containing laminarin and alginate as major polysaccharides, providing an excellent model substrate to study their deconstruction by simple enzyme mixtures. Our previous study showed strong reactivity of the glycoside hydrolase family 55 during hydrolysis of purified laminarin, raising the question of its reactivity with intact kelp. In this study, we determined that a combination of a single glycoside hydrolase family 55 β-1,3-exoglucanase with a broad-specificity alginate lyase from the polysaccharide lyase family 18 gives efficient hydrolysis of untreated kelp to a mixture of simple sugars, that is, glucose, gentiobiose, mannitol-end glucose, and mannuronic and guluronic acids and their soluble oligomers. Quantitative assignments from nanostructure initiator mass spectrometry (NIMS) and 2D HSQC NMR spectroscopy and analysis of the reaction time-course are provided. The data suggest that binary combinations of enzymes targeted to the unique polysaccharide composition of marine biomass are sufficient to deconstruct kelp into soluble sugars for microbial fermentation.

Reaction-Induced Molecular Dancing and Boosted Diffusion of Enzymes

A novel mechanism of reaction-induced active molecular motion, not involving any kind of self-propulsion, is proposed and analyzed. Because of the momentum exchange with the surrounding solvent, conformational transitions in mechano-chemical enzymes are accompanied by motions of their centers of mass. As we show, in combination with rotational diffusion, such repeated reciprocal motions generate an additional random walk - or molecular dancing - and hence boost translational diffusion of an enzyme. A systematic theory of this phenomenon is developed, using as an example a simple enzyme model of a rigid two-state dumbbell. To support the analysis, numerical simulations are performed. Our conclusion is that the phenomenon of molecular dancing could underlie the observations of reaction-induced diffusion enhancement in enzymes. Major experimental findings, such as the occurrence of leaps, the anti-chemotaxis, the linear dependence on the reaction turnover rate and on the rate of energy supply, become thus explained. Moreover, the dancing behavior is possible in other systems, natural and synthetic, too. In the future, interesting biotechnology applications may be developed using such effects.

Damage-free remote SF6 plasma-treated CNTs for facile fabrication of electrochemical enzyme biosensors

Remote SF6 plasma treatment of CNTs is performed to graft hydrophobic fluorine on the surface of CNTs. Fluorinated CNTs (F-CNTs) allow for the facile adsorption of enzyme molecules via hydrophobic interaction between F-CNTs and the hydrophobic patches of enzyme molecules. The loading of glucose oxidase (GOx) on F-CNTs correlates well with plasma treatment time. Interestingly, the Rct value of GOx/F-CNTs decreases when CNTs are treated with plasma for a short time such as 1 and 5 min, which is due to weakly-bound chemisorbed fluorine molecules on F-CNTs (semi-ionic C-F). As an example, the Rct value of GOx/F-CNT electrode with 1 min plasma treatment is 0.57 times lower than that of GOx/CNTs, while increasing its sensitivity by 2.5 times. Upon extending the treatment time such as 30, 45 and 60 min, the XPS results reveal the dominance of covalently-attached fluorine, leading to marginally-increased resistance and sensitivity. It is worthy of noting this work is the first report of simple enzyme adsorption on remote-plasma treated CNT surfaces together with its detailed mechanism studies, especially for the binding of enzyme molecules semi-ionic fluorine.

Enzyme-Catalyzed Hydrogen-Deuterium Exchange between Environmental Pollutants and Enzyme-Regulated Endogenous Metabolites.

Environmental pollutants can disrupt the homeostasis of endogenous metabolites in organisms, leading to metabolic disorders and syndromes. However, it remains highly challenging to efficiently screen for critical biological molecules affected by environmental pollutants. Herein, we found that enzyme could catalyze hydrogen-deuterium (H-D) exchange between a deuterium-labeled environmental pollutant [D38-bis(2-ethylhexyl) phthalate (D38-DEHP)] and several groups of enzyme-regulated metabolites [cardiolipins (CLs), monolysocardiolipins (MLCLs), phospholipids (PLs), and lysophospholipids (LPLs)]. A high-throughput scanning identified the D-labeled endogenous metabolites in a simple enzyme [phospholipase A2 (PLA2)], enzyme mixtures (liver microsomes), and living organisms (zebrafish embryos) exposed to D38-DEHP. Mass fragmentation and structural analyses showed that similar positions were D-labeled in the CLs, MLCLs, PLs, and LPLs, and this labeling was not attributable to natural metabolic transformations of D38-DEHP or incorporation of its D-labeled side chains. Molecular docking and competitive binding analyses revealed that DEHP competed with D-labeled lipids for binding to the active site of PLA2, and this process mediated H-D exchange. Moreover, competitive binding of DEHP against biotransformation enzymes could interfere with catabolic or anabolic lipid metabolism and thereby affect the concentrations of endogenous metabolites. Our findings provide a tool for discovering more molecular targets that complement the known toxic endpoints of metabolic disruptors.

Performance of a Flow-Through Enzyme Reactor Prepared from a Silica Monolith and an α-Poly(D-Lysine)-Enzyme Conjugate.

Horseradish peroxidase (HRP) is covalently bound in aqueous solution to polycationic α-poly(D-lysine) chains of ≈1000 repeating units length, PDL, via a bis-aryl hydrazone bond (BAH). Under the experimental conditions used, about 15 HRP molecules are bound along the PDL chain. The purified PDL-BAH-HRP conjugate is very stable when stored at micromolar HRP concentration in a pH 7.2 phosphate buffer solution at 4°C. When a defined volume of such a conjugate solution of desired HRP concentration (i.e., HRP activity) is added to a macro- and mesoporous silica monolith with pore sizes of 20-30µm as well as below 30nm, quantitative and stable noncovalent conjugate immobilization is achieved. The HRP-containing monolith can be used as flow-through enzyme reactor for bioanalytical applications at neutral or slightly alkaline pH, as demonstrated for the determination of hydrogen peroxide in diluted honey. The conjugate can be detached from the monolith by simple enzyme reactor washing with an aqueous solution of pH 5.0, enabling reloading with fresh conjugate solution at pH 7.2. Compared to previously investigated polycationic dendronized polymer-enzyme conjugates with approximately the same average polymer chain length, the PDL-BAH-HRP conjugate appears to be equally suitable for HRP immobilization on silica surfaces.

PHAPE: a plasmid for production of DNA size marker ladders for gel electrophoresis.

DNA size markers (also known as 'molecular weight markers' or 'DNA ladders') are an essential tool when using gel electrophoresis to identify and purify nucleic acids. However, the cost of these DNA ladders is not insignificant and, over time, impinges on the funds available for research and training in molecular biology. Here, we describe a method for the generation of 'pHAPE', a plasmid from which a variety of DNA ladders can be generated via simple restriction enzyme digestions. The pHAPE plasmid can be generated by mutagenesis of the commonly used pBluescript II SK+ phagemid followed by insertion of a 7141 bp sequence (comprised of three smaller, synthetic fragments). Our use of pHAPE allows us some small relief from the ever-rising costs of performing molecular biology experiments ('Don't worry, pHAPE').

A streamlined process for discovery and characterization of inhibitors against phenylalanyl-tRNA synthetase of Mycobacterium tuberculosis.

Aminoacyl-tRNA synthetases (aaRSs) catalyze aminoacylation of tRNAs to produce aminoacyl-tRNAs for protein synthesis. Bacterial aaRSs have distinctive features, play an essential role in channeling amino acids into biomolecular assembly, and are vulnerable to inhibition by small molecules. The aaRSs continue to be targets for potential antibacterial drug development. The first step of aaRS reaction is the activation of amino acid by hydrolyzing ATP to form an acyladenylate intermediate with the concomitant release of pyrophosphate. None-radioactive assays usually measure the rate of ATP consumption or phosphate generation, offering advantages in high-throughput drug screening. These simple aaRS enzyme assays can be adapted to study the mode of inhibition of natural or synthetic aaRS inhibitors. Taking phenylalanyl-tRNA synthetase (PheRS) of Mycobacterium tuberculosis (Mtb) as an example, we describe a process for identification and characterization of Mtb PheRS inhibitor.

Direct electrochemistry of hemoglobin/peptide-carbon nanotube modified electrode for hydrogen peroxide biosensing

Mediatorless enzymatic biosensors based on direct electron transfer show promise due to their high stability and remarkable electrocatalytical performance. In this study, the direct electrochemistry of a glassy carbon electrode modified with hemoglobin (Hb) embedded in a hybrid layer of self-assembling EFK8 peptide and single-walled carbon nanotubes (SWNTs) was investigated. Electrochemical studies in 0.1 M phosphate buffer (PB) revealed that the immobilized Hb did not denature and retained its bio-catalytic activity. The resulting biosensor was used to detect and measure the hydrogen peroxide (H2O2) concentration in 0.1 M PB (pH 7.0), yielding a linear detection range from 20 to 960 µM and a detection limit of 7.54 µM (S/N = 3). The EFK8-SWNT hybrid layer shows promise as a biocompatible layer for simple non-covalent enzyme immobilization as the basis for future mediatorless enzymatic biosensors.

Structural Insights into (Tere)phthalate-Ester Hydrolysis by a Carboxylesterase and Its Role in Promoting PET Depolymerization.

TfCa, a promiscuous carboxylesterase from Thermobifida fusca, was found to hydrolyze polyethylene terephthalate (PET) degradation intermediates such as bis(2-hydroxyethyl) terephthalate (BHET) and mono-(2-hydroxyethyl)-terephthalate (MHET). In this study, we elucidated the structures of TfCa in its apo form, as well as in complex with a PET monomer analogue and with BHET. The structure-function relationship of TfCa was investigated by comparing its hydrolytic activity on various ortho- and para-phthalate esters of different lengths. Structure-guided rational engineering of amino acid residues in the substrate-binding pocket resulted in the TfCa variant I69W/V376A (WA), which showed 2.6-fold and 3.3-fold higher hydrolytic activity on MHET and BHET, respectively, than the wild-type enzyme. TfCa or its WA variant was mixed with a mesophilic PET depolymerizing enzyme variant [Ideonella sakaiensis PETase (IsPETase) PM] to degrade PET substrates of various crystallinity. The dual enzyme system with the wild-type TfCa or its WA variant produced up to 11-fold and 14-fold more terephthalate (TPA) than the single IsPETase PM, respectively. In comparison to the recently published chimeric fusion protein of IsPETase and MHETase, our system requires 10% IsPETase and one-fourth of the reaction time to yield the same amount of TPA under similar PET degradation conditions. Our simple dual enzyme system reveals further advantages in terms of cost-effectiveness and catalytic efficiency since it does not require time-consuming and expensive cross-linking and immobilization approaches.

Reaction‐controlled microgel‐lipase co‐stabilized compartmentalized emulsion for recyclable biodiesel production

AbstractPickering emulsions have been widely used for biphasic catalysis in the past decade. However, it remains a great challenge to achieve simple product collection and enzyme recovery. Poly(N‐isopropylacrylamide) (PNIPAM)‐based microgels can endow Pickering emulsions with stimuli‐responsiveness, while most microgel‐stabilized emulsions are oil‐in‐water (O/W) type and not ideal for interfacial catalysis. Besides, altering temperature or pH value for demulsification is time‐ and energy‐consuming and may cause irreversible deactivation of enzymes. In this work, inverse water‐in‐oil (W/O) Pickering emulsions were formed using hexanoic acid‐swollen microgels as the sole emulsifiers. When lipase was added in the water phase, stable oil‐in‐water‐in‐oil (O/W/O) Pickering double emulsions could be formed through one‐step emulsification, owing to the synergistic effect of the hydrophobic microgels and hydrophilic lipase at the interface. Compared with other biphasic systems, such double emulsion systems represent a desirable platform for highly efficient biodiesel production because of the ultra‐high interfacial areas and fast mass transport between two phases. More importantly, the switchable transition between hydrophobicity/hydrophilicity of microgels is controlled by the catalytic reaction. Therefore, double emulsions demulsify spontaneously when substrates are used up without the need for energy input or loss of enzymatic activity, enabling the facile collection of products and demonstrating the excellent recyclability of the biphasic catalysis system.

Studies on the Rapid and Simple DNA Extraction Method, Antibacterial Activity and Enzyme Activity Involved in Plant Biomass Conversion by Cookeina sulcipes and C. tricholoma (Cup Fungi)

Cookeina sulcipes and C. tricholoma are a cup fungi (Ascomycota) collected in Saraburi, Thailand. The fungi have been isolated, cultured and confirmed as respective species. For morphology, both Cookeina sp. are white mycelium and the growth rate on potato dextrose agar (PDA) result present C. sulcipes is faster than C. tricholoma. The molecular characterization from a rapid and simple DNA extraction method that is modified based on thermolysis method, The DNA extraction is finish in thirty minutes and efficiency to continuous with polymerase chain reaction (PCR) amplification to fungi species level identification. The DNA sequence from internal transcribed spacer (ITS) gene regions by universal primer pairs ITS5/ITS4 is effective to confirm Cookeina species level that C. sulcipes has 616 bp and C. tricholoma has 570 bp. Including, DNA sequence of large subunit (LSU) gene regions by universal primer pairs LROR/LR5 is generate that C. sulcipes has 912 bp and C. tricholoma has 906 bp. The cultures are screened for antibacterial activity by agar plug diffusion method and found that both isolates have been no activity against test strains (Bacillus subtilis, Escherichia coli, Kocuria rhizophila (Micrococcus luteus), Pseudomonas aeruginosa, Staphylococcus aureus and S. epidermidis). In a preliminary screening test of enzymes involved in plant biomass breakdown by agar plate method, both Cookeina sp. show cellulolytic and hemicellulolytic enzymatic activity, and manganese peroxidase (MnP) productivity. In contrast, only C. sulcipes had additional laccase activity. Neither isolate generate pectinolytic and lignin peroxidase (LiP) activities. Thus, Cookeina spp. proved the potentiality to break down lignocelluloses.

A Redox-Controlled Substrate Engineering Strategy for Site-Specific Enzymatic Fucosylation.

Fucosylation is one of the most common modifications of oligo-N-acetyllactosamine (oligo-LacNAc) glycans. However, none of known fucosyltransferases (FucTs) could install the α1,3-linked fucose to the oligo-LacNAc substrates in a site-specific manner. Here, we report a facile and general redox-controlled substrate engineering strategy for the site-specific α1,3-fucosylation of complex glycans containing multiple LacNAc units. This strategy takes advantage of an operationally simple oxidation enzyme module by using galactose oxidase (GOase) to convert the LacNAc unit into oxidized C6'-aldehyde LacNAc sequence, which is not a good substrate for recombinant α1,3-FucT from Helicobacter pylori strain 26695 (Hpα1,3FucT), enabling the site-specific α1,3-fucosylation at intact LacNAc sites. The general applicability and robustness of this strategy were demonstrated by the synthesis of a variety of structurally well-defined fucosides of linear and branched O- and N-linked glycans.