EnzymeML: seamless data flow and modeling of enzymatic data.

The design of biocatalytic reaction systems is highly complex owing to the dependency of the estimated kinetic parameters on the enzyme, the reaction conditions, and the modeling method. Consequently, reproducibility of enzymatic experiments and reusability of enzymatic data are challenging. We developed the XML-based markup language EnzymeML to enable storage and exchange of enzymatic data such as reaction conditions, the time course of the substrate and the product, kinetic parameters and the kinetic model, thus making enzymatic data findable, accessible, interoperable and reusable (FAIR). The feasibility and usefulness of the EnzymeML toolbox is demonstrated in six scenarios, for which data and metadata of different enzymatic reactions are collected and analyzed. EnzymeML serves as a seamless communication channel between experimental platforms, electronic lab notebooks, tools for modeling of enzyme kinetics, publication platforms and enzymatic reaction databases. EnzymeML is open and transparent, and invites the community to contribute. All documents and codes are freely available at https://enzymeml.org .

Laccase catalysis for the synthesis of bioactive compounds.

The demand for compounds of therapeutic value is increasing mainly because of new applications of bioactive compounds in medicine, pharmaceutical, agricultural, and food industries. This has necessitated the search for cost-effective methods for producing bioactive compounds and therefore the intensification of the search for enzymatic approaches in organic synthesis. Laccase is one of the enzymes that have shown encouraging potential as biocatalysts in the synthesis of bioactive compounds. Laccases are multicopper oxidases with a diverse range of catalytic activities revolving around synthesis and degradative reactions. They have attracted much attention as potential industrial catalysts in organic synthesis mainly because they are essentially green catalysts with a diverse substrate range. Their reaction only requires molecular oxygen and releases water as the only by-product. Laccase catalysis involves the abstraction of a single electron from their substrates to produce reactive radicals. The free radicals subsequently undergo homo- and hetero-coupling to form dimeric, oligomeric, polymeric, or cross-coupling products which have practical implications in organic synthesis. Consequently, there is a growing body of research focused on the synthetic applications of laccases such as organic synthesis, hair and textile dyeing, polymer synthesis, and grafting processes. This paper reviews the major advances in laccase-mediated synthesis of bioactive compounds, the mechanisms of enzymatic coupling, structure-activity relationships of synthesized compounds, and the challenges that might guide future research directions.

Laccase applications in biofuels production: current status and future prospects.

The desire to reduce dependence on the ever diminishing fossil fuel reserves coupled with the impetus towards green energy has seen increased research in biofuels as alternative sources of energy. Lignocellulose materials are one of the most promising feedstocks for advanced biofuels production. However, their utilisation is dependent on the efficient hydrolysis of polysaccharides, which in part is dependent on cost-effective and benign pretreatment of biomass to remove or modify lignin and release or expose sugars to hydrolytic enzymes. Laccase is one of the enzymes that are being investigated not only for potential use as pretreatment agents in biofuel production, mainly as a delignifying enzyme, but also as a biotechnological tool for removal of inhibitors (mainly phenolic) of subsequent enzymatic processes. The current review discusses the major advances in the application of laccase as a potential pretreatment strategy, the underlying principles as well as directions for future research in the search for better enzyme-based technologies for biofuel production. Future perspectives could include synergy between enzymes that may be required for optimal results and the adoption of the biorefinery concept in line with the move towards the global implementation of the bioeconomy strategy.

Future prospects in mRNA vaccine development.

The recent advancements in messenger ribonucleic acid (mRNA) vaccine development have vastly enhanced their use as alternatives to conventional vaccines in the prevention of various infectious diseases and treatment of several types of cancers. This is mainly due to their remarkable ability to stimulate specific immune responses with minimal clinical side effects. This review gives a detailed overview of mRNA vaccines currently in use or at various stages of development, the recent advancements in mRNA vaccine development, and the challenges encountered in their development. Future perspectives on this technology are also discussed.

Gelation profile of laccase-crosslinked Bambara groundnut (Vigna subterranea) protein isolate.

Enzymatic crosslinking has gained attention in improving plant protein heat-induced gels, which are composed of weak network structures. The aim of the study was to investigate the effect of laccase crosslinking on the rheological and microstructural properties of heat-induced Bambara groundnut protein gels. The rheological properties of laccase-modified Bambara groundnut protein isolate (BPI1) gel formed in situ were investigated. Changes in viscoelastic properties were monitored during heating and cooling ramps and gel structure fingerprints were analyzed by frequency sweep. Laccase addition induced an initial protein structure breakdown (G″>G') at an enzyme dose-dependent (1-3 U/g) before gel formation and stabilization. Gel point temperatures were significantly decreased from 85°C to 29°C (∼3 folds) with increasing laccase activity (0 to 3 U/g protein, respectively). For laccase crosslinked gels, G' was substantially greater than G" (>1 log) with no dependency on angular frequency, which suggests the formation of relatively well-structured gels. The highest gel strength (tan δ of 0.09, G* of 555.51 kPa & An of 468.04 kPa) was recorded at a laccase activity of 2 U/g protein and the gels formed at this activity appeared homogeneous with compact lath sheet-like structure. The crosslinking effects of laccase were corroborated by the decrease in thiol and phenolic contents as well as the crosslinking of amino acids in model reactions. Overall, the use of laccase improved gel properties and significantly altered the gelation profile of BPI. Laccase-modified Bambara groundnut protein gels have potential to be used in food texture improvement and development of new food products. For instance, they can be used in plant-based milk products such as yoghurt and cheese.

Current Trends and Prospects for Application of Green Synthesized Metal Nanoparticles in Cancer and COVID-19 Therapies.

Cancer and COVID-19 have been deemed as world health concerns due to the millions of lives that they have claimed over the years. Extensive efforts have been made to develop sophisticated, site-specific, and safe strategies that can effectively diagnose, prevent, manage, and treat these diseases. These strategies involve the implementation of metal nanoparticles and metal oxides such as gold, silver, iron oxide, titanium oxide, zinc oxide, and copper oxide, formulated through nanotechnology as alternative anticancer or antiviral therapeutics or drug delivery systems. This review provides a perspective on metal nanoparticles and their potential application in cancer and COVID-19 treatments. The data of published studies were critically analysed to expose the potential therapeutic relevance of green synthesized metal nanoparticles in cancer and COVID-19. Although various research reports highlight the great potential of metal and metal oxide nanoparticles as alternative nanotherapeutics, issues of nanotoxicity, complex methods of preparation, biodegradability, and clearance are lingering challenges for the successful clinical application of the NPs. Thus, future innovations include fabricating metal nanoparticles with eco-friendly materials, tailor making them with optimal therapeutics for specific disease targeting, and in vitro and in vivo evaluation of safety, therapeutic efficiency, pharmacokinetics, and biodistribution.

Enhancing the expression of recombinant small laccase in Pichia pastoris by a double promoter system and application in antibiotics degradation.

Low-expression levels remain a challenge in the quest to use the small laccase (rSLAC) as a viable catalyst. In this study, a recombinant Pichia pastoris strain (rSLAC-GAP-AOX) producing rSLAC under both AOX and GAP promoters (located in two different plasmids) was generated and cultivated in the presence of methanol and mixed feed (methanol:glycerol). Induction with methanol resulted in a maximum laccase activity of 1200 U/L for rSLAC-GAP-AOX which was approximately 2.4-fold higher than rSLAC-AOX and 5.1-fold higher than rSLAC-GAP. The addition of methanol:glycerol in a stoichiometric ratio of 9:1 consistently improved biomass and led to a 1.5-fold increase in rSLAC production as compared to induction with methanol alone. The rSLAC removed 95% of 5 mg/L ciprofloxacin (CIP) and 99% of 100 mg/L tetracycline (TC) in the presence of a mediator. Removal of TC resulted in complete elimination of antibacterial activity while up to 48% reduction in antibacterial activity was observed when CIP was removed. Overall, the present study highlights the effectiveness of a double promoter system in enhancing SLAC production.

Enzymatic treatment of phenolic pollutants by a small laccase immobilized on APTES-functionalised magnetic nanoparticles.

In this study, we have successfully synthesized magnetic nanoparticles (MNPs), functionalised them by silanization and used them for the covalent immobilization of a recombinant small laccase (rSLAC) from Streptomyces coelicolor. The immobilized recombinant laccase (MNP-rSLAC) was subsequently used for the treatment of phenol, 4-chlorophenol (4-CP) and 4-fluorophenol (4-FP). The enzyme completely degraded 80 µg/mL of the selected phenolic compounds within 2 h in the presence of a natural mediator, acetosyringone. The MNP-rSLAC retained > 73% of initial activity (2,6-dimethoxyphenol as substrate) after 10 catalytic cycles and could be easily recovered from the reaction mixture by the application of magnetic field. Furthermore, immobilised rSLAC exhibited better storage stability than its free counterpart. The Michaelis constant (Km) value for the immobilised rSLAC was higher than free rSLAC, however the maximum velocity (Vmax) of the immobilised SLAC was similar to that of the free rSLAC. Growth inhibition studies using Escherichia coli showed that rSLAC-mediated treatment of phenolic compounds reduced the toxicity of phenol, 4-CP and 4-FP by 90, 60 and 55%, respectively. Interestingly, the presence of selected metal ions (Co2+, Cu2+, Mn2+) greatly enhanced the catalytic activity of rSLAC and MNP-rSLAC. This study indicates that immobilized small laccase (MNP-rSLAC) has potential for treating wastewater contaminated with phenolic compounds. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02854-0.

Phytochemical Content, Antioxidant, Alpha-Glucosidase Inhibitory and Antibacterial Activities of Spineless Cactus Pear Cultivars.

Variation in cultivars can influence plant biological activities. This study aimed to identify superior cultivars while determining the variability in the phytochemical content, antioxidant, alpha-glucosidase inhibitory and antibacterial activities of cladode extracts from selected spineless Burbank cactus pear (Opuntia ficus-indica and Opuntia robusta) cultivars. Total phenolic and flavonoid contents were determined using the Folin-Ciocalteu and aluminum chloride spectrophotometric methods, respectively. Antioxidant activity was investigated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging and ß-carotene linoleic acid assays. Alpha-glucosidase inhibition was determined using a spectrophotometric method and antibacterial activity using a non-polar (petroleum ether) and polar (50% methanol) extracts against two Gram-positive and two Gram-negative bacteria. Significant variation in phytochemical content, antioxidant, antidiabetic and antibacterial activities was observed amongst the cultivars. Alpha-glucosidase inhibitory activity varied widely with IC50 values ranging from 0.06 to 1.85 mg/mL. Radical scavenging activity of Polypoly cultivar was about seven fold higher than that recorded in other cultivars with low activity. Turpin and Berg x Mexican cultivars had the highest total phenolic and flavonoid contents, whilst the non-polar extract of Turpin also exhibited higher antibacterial activity against Bacillus subtilis and Escherichia coli. Sicilian Indian Fig was amongst the cultivars with a higher antioxidant activity, whilst also showing a strong inhibition against B. subtilis and E. coli. Polypoly cultivar demonstrated strong antioxidant and antidiabetic activities while its polar extract showed the highest total antibacterial activity against B. subtilis. The cultivar Malta was superior in terms of its antibacterial potency and efficacy against B. subtilis, Staphylococcus aureus and E. coli. The potential of using spineless cactus pear cladodes as a functional food with antioxidant, antidiabetic and antibacterial properties against pathogenic food spoilage bacteria in place of synthetic compounds was established. The significance of cultivar selection to increase this potential was highlighted.

Mechanistic insights into laccase-mediated functionalisation of lignocellulose material.

Recent emerging studies on the grafting mechanisms of functional molecules onto complex lignocellulose moieties have shown useful insights and possibilities in opening new frontiers in the enzymatic development of multifunctional polymers. Thanks to these studies which have demonstrated in principle the ability of laccases to mediate the coupling of antimicrobial compounds, hydrophobic molecules, including application processes for the development of fibreboards, particle boards, laminates etc. Further, laccase mediated grafting strategies developed using small reactive molecules e.g. phenolic amines which impart reactive properties to an inert polymer demonstrates the remarkable opportunities of enzyme meditated functionalization of polymers. Therefore recent studies focusing on understanding the mechanistic basis of the coupling mechanisms in order to make meaningful contribution to the development of new processes and products are a welcome development.

Transglutaminase-mediated crosslinking of Bambara groundnut protein hydrogels: Implications on rheological, textural and microstructural properties.

Interest in plant protein-based hydrogels with desirable strength has been increasing in recent years. In this study, Bambara groundnut protein isolate (BPI) was crosslinked with transglutaminase (TGase) (0 - 25 U/g protein) during gelation and rheological, textural and microstructural properties of the resulting hydrogels were investigated. Treatment with TGase up to 15 U/g protein resulted in the formation of hydrogels with small pores and an organised homogeneous network. G' of TGase-treated BPI hydrogels was more than ten-fold higher than G" throughout the frequency range of 0-100 rad/s, suggesting dominance of the elastic like behaviour. BPI hydrogel with the highest G' (6967 Pa) and hardness (5.60 N) was formed at 15 U/g protein of TGase activity. The hydrogel had a high distribution ß-sheets (53.52%) and α-helixes (26.17%) as compared to the ß-turns and random coils. However, a further increase in TGase activity did not improve the hydrogel properties. Transglutaminase mediated crosslinking of BPI hydrogel was demonstrated by the reduction in amine and thiol groups and the formation of a new protein band (56 kDa) in crosslinked hydrogels. Overall, TGase promoted the formation of a strong gel with an organised network.

Production and characterisation of a novel actinobacterial DyP-type peroxidase and its application in coupling of phenolic monomers.

The extracellular peroxidase from Streptomyces albidoflavus BSII#1 was purified to near homogeneity using sequential steps of acid and acetone precipitation, followed by ultrafiltration. The purified peroxidase was characterised and tested for the ability to catalyse coupling reactions between selected phenolic monomer pairs. A 46-fold purification of the peroxidase was achieved, and it was shown to be a 46 kDa haem peroxidase. Unlike other actinobacteria-derived peroxidases, it was only inhibited (27 % inhibition) by relatively high concentrations of sodium azide (5 mM) and was capable of oxidising eleven (2,4-dichlorophenol, 2,6-dimethoxyphenol, 4-tert-butylcatechol, ABTS, caffeic acid, catechol, guaiacol, l-DOPA, o-aminophenol, phenol, pyrogallol) of the seventeen substrates tested. The peroxidase remained stable at temperatures of up to 80 °C for 60 min and retained >50 % activity after 24 h between pH 5.0-9.0, but was most sensitive to incubation with hydrogen peroxide (H2O2; 0.01 mM), l-cysteine (0.02 mM) and ascorbate (0.05 mM) for one hour. It was significantly inhibited by all organic solvents tested (p ≤ 0.05). The Km and Vmax values of the partially purified peroxidase with the substrate 2,4-DCP were 0.95 mM and 0.12 mmol min-1, respectively. The dyes reactive blue 4, reactive black 5, and Azure B, were all decolourised to a certain extent: approximately 30 % decolourisation was observed after 24 h (1 µM dye). The peroxidase successfully catalysed coupling reactions between several phenolic monomer pairs including catechin-caffeic acid, catechin-catechol, catechin-guaiacol and guaiacol-syringaldazine under the non-optimised conditions used in this study. Genome sequencing confirmed the identity of strain BSII#1 as a S. albidoflavus strain. In addition, the genome sequence revealed the presence of one peroxidase gene that includes the twin arginine translocation signal sequence of extracellular proteins. Functional studies confirmed that the peroxidase produced by S. albidoflavus BSII#1 is part of the dye-decolourising peroxidase (DyP-type) family.

Antioxidant activity assay based on laccase-generated radicals.

A novel antioxidant activity assay was developed using laccase-oxidized phenolics. In a three-step approach, phenolic compounds were first oxidized by laccase. Laccase was then inhibited using 80% (v/v) methanol which also stabilized the oxidized phenolics which were then used to measure antioxidant activities of ascorbic acid and Trolox. From a number of laccase-oxidized phenolics screened for potential use in the measurement of antioxidant activities, syringaldazine emerged the best, giving results comparable to the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, which is currently used in conventional methods. Like DPPH radicals, two moles of stoichiometric oxidized syringaldazine were reduced by one mole of either ascorbic acid or Trolox. For the first time we show that antioxidant activity can be correlated to oxygen consumption by laccase. Reduction of one molecule of oxygen corresponded to oxidation of four molecules of syringaldazine which in turn is reduced by two molecules of Trolox or ascorbic acid. This study therefore demonstrates the great potential of using laccase-oxidized syringaldazine for the measurement of antioxidant activity.

Opuntia (Cactaceae) plant compounds, biological activities and prospects - A comprehensive review.

Opuntia species are utilized as local medicinal interventions for chronic diseases and as food sources mainly because they possess nutritional properties and biological activities. The Opuntia plant is distributed worldwide and has great economic potential. Differences in Opuntia species phytochemical composition exist between wild and domesticated species, and within species. Opuntia aerial and underground parts exhibit beneficial properties due to their phenolic content, other antioxidants (for example ascorbate), pigments (carotenoids, betalains), and other unidentified components. This work comprehensively reviews the phytochemical composition of the different aerial and underground plant parts of Opuntia species. The applications of Opuntia compounds and their biological activities are also discussed. Other topical aspects covered include Opuntia spp. taurine composition, Opuntia side effects, Opuntia by-products valorisation and the role of Opuntia spp. in tackling antimicrobial resistance. Although biological activities have been extensively researched, much less information is available on reaction mechanisms, herbal mixtures toxicology and commercialisation prospects - aspects which should be considered for future research in this area.

Laccase-mediated crosslinking of gluten-free amadumbe flour improves rheological properties.

The absence of gluten in gluten-free flours presents a challenge to their application in baking. Enzymatic modification of the protein and polysaccharides may result in a network that mimics gluten. In the current study, the effects of laccase on the rheological properties of amadumbe dough were investigated. Thiol and total phenolic contents of dough decreased by up to 28% and 93%, respectively, as laccase activity was increased (0-3 U/g flour). Both G' and G″ of laccase-treated dough increased significantly due to laccase-catalysed cross-linking of proteins and polysaccharides esterified with phenolics, as demonstrated by relevant model reactions. Tan δ decreased with increase in laccase activity indicating an increase in the elastic character of the dough. The improvement in dough viscoelasticity may enable the retention of adequate carbon dioxide during proofing and production of more acceptable gluten-free bread.

Secretory expression of recombinant small laccase from Streptomyces coelicolor A3(2) in Pichia pastoris.

This work reports for the first time the secretory expression of the small laccase (SLAC) from Streptomyces coelicolor A3(2) in Pichia pastoris. Using an AOX1 promoter and α factor as a secretion signal, the recombinant P. pastoris harbouring the laccase gene (rSLAC) produced high titres of extracellular laccase (500 ±â€¯10 U/l), which were further increased seven fold by pre-incubation at 80 °C for 30 min. The enzyme (∼38 kDa) had an optimum activity at 80 °C, but optimum pH varied with substrate used. Km values for ABTS, SGZ and 2,6-DMP were 142.85 µM, 10 µM and 54.55 µM and the corresponding kcat values were 60.6 s-1, 25.36 s-1 and 27.84 s-1, respectively. The t1/2 values of the rSLAC at 60 °C, 70 °C, 80 °C were 60 h, 32 h and 10 h, respectively. The enzyme deactivation energy (Ed) was 117.275 kJ/mol while ΔG, ΔH and ΔS for thermal inactivation of the rSLAC were all positive. The rSLAC decolourised more than 90% of Brilliant Blue G and Trypan Blue dye in 6 h without the addition of a mediator. High titres of SLAC expressed in P. pastoris enhance its potential for various industrial applications.

Composition, thermal and rheological properties of polysaccharides from amadumbe (Colocasia esculenta) and cactus (Opuntia spp.).

The extensive application of hydrocolloids in the food industry, coupled with their short supply and shortcomings, has led to the ongoing search for alternative sources. In this study, the compositional, rheological and thermal properties of amadumbe and cactus mucilages were investigated. The mucilages had a similar qualitative composition of monosaccharides and amino acids, except for the absence of rhamnose in amadumbe mucilage. Fractionation of amadumbe and cactus mucilages on an anion-exchange column yielded four and three fractions, respectively. The fractions eluting with protein showed no ß-elimination, suggesting stronger glycosylation bonds such as those in arabinogalactan proteins (AGPs). There was no evidence of thermal depolymerisation of the mucilages up to 195 °C. Cactus mucilage showed a pseudoplastic flow behaviour whilst amadumbe mucilage showed a Newtonian flow behaviour at up to 5% (w/v) concentrations. Amadumbe mucilage may be a potential emulsifier, whilst cactus mucilage can potentially be used as a thickening or emulsifying agent.

Versatility of oxidoreductases in the remediation of environmental pollutants.

Enzymatic transformation of recalcitrant and other pollutants is a promising eco-friendly alternative to physico-chemical methods in environmental remediation. This review summarizes some of the significant advances in applications of oxidative enzymes for treatment of xenobiotics. The review also discusses some of the underlying principles and enzyme reaction mechanisms in the detoxification or removal of xenobiotic compounds such as dyes, phenolic compounds, nitroaromatic compounds and polyaromatic hydrocarbons, as a way of remediating contaminated soils or wastewaters.

Novel, biocatalytically produced hydroxytyrosol dimer protects against ultraviolet-induced cell death in human immortalized keratinocytes.

Compounds derived from botanicals, such as olive trees, have been shown to possess various qualities that make them function as ideal antioxidants and, in doing so, protect them against the damaging effect of ultraviolet (UV)-derived oxidative stress. The aim of this study was to biocatalytically synthesize a dimeric product (compound II) from a known botanical, 3-hydroxytyrosol, and test it for its antioxidant ability using a human immortalized keratinocyte cell line (HaCaT). 2,2-Diphenyl-picryhydrazyl (DPPH) antioxidant assays showed 33 and 86.7% radical scavenging activity for 3-hydroxytyrosol and its dimer, respectively. The ferric-reducing antioxidant power (FRAP) assay corroborated this by showing a 3-fold higher antioxidant activity for the dimer than 3-hydroxytyrosol. Western blot analyses, showing cells exposed to 500 µM of the dimeric product when ultraviolet A (UVA)-irradiated, increased the anti-apoptotic protein Bcl-2 expression by 16% and reduced the pro-apoptotic protein Bax by 87.5%. Collectively, the data show that the dimeric product of 3-hydroxytyrosol is a more effective antioxidant and could be considered for use in skin-care products, health, and nutraceuticals.

Enzymatic synthesis of lignin-siloxane hybrid functional polymers.

This study combines the properties of siloxanes and lignin polymers to produce hybrid functional polymers that can be used as adhesives, coating materials, and/or multifunctionalized thin-coating films. Lignin-silica hybrid copolymers were synthesized by using a sol-gel process. Laccases from Trametes hirsuta were used to oxidize lignosulphonates to enhance their reactivity towards siloxanes and then were incorporated into siloxane precursors undergoing a sol-gel process. In vitro copolymerization studies using pure lignin monomers with aminosilanes or ethoxytrimethylsilane and analysis by ²9Si NMR spectroscopy revealed hybrid products. Except for kraft lignin, an increase in lignin concentration positively affected the tensile strength in all samples. Similarly, the viscosity generally increased in all samples with increasing lignin concentration and also affected the curing time.