Harnessing a Biocatalyst to Bioremediate the Purification of Alkylglycosides.

As the world moves towards net-zero carbon emissions, the development of sustainable chemical manufacturing processes is essential. Within manufacturing, purification by distillation is often used, however this process is energy intensive and methods that could obviate or reduce its use are desirable. Developed herein is an alternative, oxidative biocatalytic approach that enables purification of alkyl monoglucosides (essential bio-based surfactant components). Implementing an immobilised engineered alcohol oxidase, a long-chain alcohol by-product derived from alkyl monoglucoside synthesis (normally removed by distillation) is selectively oxidised to an aldehyde, conjugated to an amine resin and then removed by simple filtration. This affords recovery of the purified alkyl monoglucoside. The approach lays a blueprint for further development of sustainable alkylglycoside purification using biocatalysis and, importantly, for refining other important chemical feedstocks that currently rely on distillation.

A Novel Biosensor for the Detection of Glucose Concentration Using the Dual-Peak Long Period Grating in the Near- to Mid-Infrared.

In this article, we demonstrate an improved efficient fibre sensor with a high sensitivity to measure glucose concentrations in the physiological range of human beings, operating in a broad spectral bandwidth from the near- to mid-infrared. The sensor consists of a dual-peak long period grating (DPLPG) with a period of 150 µm inscribed in an optical fibre with a diameter of 80 µm. The investigation of sensing for refractive index results in a sensitivity of ~-885.7 nm/refractive index unit (RIU) and ~2008.6 nm/RIU in the range of 1.30-1.44. The glucose measurement is achieved by the immobilisation of a layer of enzyme of glucose oxidase (GOD) onto the fibre surface for the selective enhancement of sensitivity for glucose. The sensor can measure glucose concentrations with a maximum sensitivity of -36.25 nm/(mg/mL) in the range of 0.1-3.0 mg/mL. To the best of our knowledge, this is the highest sensitivity ever achieved for a measurement of glucose with a long period grating-based sensor, indicating its potential for many applications including pharmaceutical, biomedical and food industries.

Computing Arithmetic Functions Using Immobilised Enzymatic Reaction Networks.

Living systems use enzymatic reaction networks to process biochemical information and make decisions in response to external or internal stimuli. Herein, we present a modular and reusable platform for molecular information processing using enzymes immobilised in hydrogel beads and compartmentalised in a continuous stirred tank reactor. We demonstrate how this setup allows us to perform simple arithmetic operations, such as addition, subtraction and multiplication, using various concentrations of substrates or inhibitors as inputs and the production of a fluorescent molecule as the readout.

High-level extracellular production and immobilisation of methyl parathion hydrolase from Plesiomonas sp. M6 expressed in Pichia pastoris.

Methyl parathion hydrolase (MPH) hydrolyses methyl parathion efficiently and specifically. Herein, we produced MPH from Plesiomonas sp. M6 using a Pichia pastoris multi-copy expression system. The original signal peptide sequence of the target gene was removed, and a modified coding sequence was synthesised. Multi-copy expression plasmids containing MPH were constructed using pHBM905BDM, and used to generate recombinant strains containing 1, 2, 3 or 4 copies of the MPH gene. The results showed that a higher target gene copy number increased the production of recombinant MPH (MPH-R), as anticipated. The expression level of the recombinant strain containing four copies of the MPH gene was increased to 1.9 U/ml using 500 ml shake flasks, and the specific activity was 15.8 U/mg. High-density fermentation further increased the target protein yield to 18.4 U/ml. Several metal ions were tested as additives, and Ni2+, Co2+ and Mg2+ at a concentration of 1 mM enhanced MPH-R activity by 196%, 201% and 154%, respectively. Enzyme immobilisation was then applied to overcome the difficulties in recovery, recycling and long-term stability associated with the free enzyme. Immobilised MPH-R exhibited significantly enhanced thermal and long-term stability, as well as broad pH adaptability. In the presence of inhibitors and chelating agents such as sodium dodecyl sulphate (SDS), immobilised MPH-R displayed 2-fold higher activity than free MPH-R, demonstrating its potential for industrial application.

A novel framework for the cell-free enzymatic production of glucaric acid.

Glucaric acid (GlucA) is a valuable glucose-derived chemical with promising applications as a biodegradable and biocompatible chemical in the manufacturing of plastics, detergents and drugs. Recently, there has been a significant focus on producing GlucA microbially (in vivo) from renewable materials such as glucose, sucrose and myo-inositol. However, these in vivo GlucA production processes generally lack efficiency due to toxicity problems, metabolite competition and suboptimal enzyme ratios. Synthetic biology and accompanying cell-free biocatalysis have been proposed as a viable approach to overcome many of these limitations. However, cell-free biocatalysis faces its own limitations for industrial applications due to high enzyme costs and cofactor consumption. We have constructed a cell-free GlucA pathway and demonstrated a novel framework to overcome limitations of cell-free biocatalysis by i) the combination of both thermostable and mesophilic enzymes, ii) incorporation of a cofactor regeneration system and iii) immobilisation and recycling of the pathway enzymes. The cell-free production of GlucA was achieved from glucose-1-phosphate with a titre of 14.1 ±â€¯0.9 mM (3.0 ±â€¯0.2 g l-1) and a molar yield of 35.2 ±â€¯2.3% using non-immobilised enzymes, and a titre of 8.1 ±â€¯0.2 mM (1.70 ±â€¯0.04 g l-1) and a molar yield of 20.2 ±â€¯0.5% using immobilised enzymes with a total reaction time of 10 h. The resulting productivities (0.30 ±â€¯0.02 g/h/l for free enzymes and 0.170 ±â€¯0.004 g/h/l for immobilised enzymes) are the highest productivities so far reported for glucaric acid production using a synthetic enzyme pathway.

Electrospun poly(methyl methacrylate)/polyaniline fibres as a support for laccase immobilisation and use in dye decolourisation.

Novel electrospun poly(methyl methacrylate)/polyaniline electrospun fibres were produced, characterised, modified, and used as a support for laccase immobilisation by two methods: adsorption and covalent binding. Effective deposition of laccase by both methods was confirmed by FTIR and CLSM results. Nevertheless, the main objective of the study was to select the most favourable immobilisation conditions and prepare heterogeneous biocatalysts with the best possible catalytic properties. The highest relative activity of enzymes immobilised by adsorption and covalent binding were obtained after 1 h of immobilisation using laccase solution at a concentration of 1 mg/mL, at pH 5 and 25 °C. It was found that the immobilised enzymes, which were present in amounts of 110 mg/g and 185 mg/g for systems with adsorbed and covalently bonded laccase respectively, exhibited slightly lower substrate affinity, and in consequence also a lower maximum reaction rate, than the free enzyme. The stability of laccase improved significantly upon immobilisation: both heterogeneous biocatalysts retained over 80% relative activity even after 10 repeated catalytic cycles and 30 days of storage. The obtained systems were used for decolourisation of Remazol Brilliant Blue R dye from a model aqueous solution, resulting in removal efficiencies of 87% and 58% using adsorbed and covalently bonded laccase, respectively. The described approach to the removal of textile dye from model solution is significant for the sustainable and environmentally friendly decolourisation of various compounds from wastewater.

An AGT-based protein-tag system for the labelling and surface immobilization of enzymes on E. coli outer membrane.

The use of natural systems, such as outer membrane protein A (OmpA), phosphoporin E (PhoE), ice nucleation protein (INP), etc., has been proved very useful for the surface exposure of proteins on the outer membrane of Gram-negative bacteria. These strategies have the clear advantage of unifying in a one-step the production, the purification and the in vivo immobilisation of proteins/biocatalysts onto a specific biological support. Here, we introduce the novel Anchoring-and-Self-Labelling-protein-tag (ASLtag), which allows the in vivo immobilisation of enzymes on E. coli surface and the labelling of the neosynthesised proteins with the engineered alkylguanine-DNA-alkyl-transferase (H5) from Sulfolobus solfataricus. Our results demonstrated that this tag enhanced the overexpression of thermostable enzymes, such as the carbonic anhydrase (SspCA) from Sulfurihydrogenibium yellowstonense and the ß-glycoside hydrolase (SsßGly) from S. solfataricus, without affecting their folding and catalytic activity, proposing a new tool for the improvement in the utilisation of biocatalysts of biotechnological interest.

Accelerated CO2 Hydration with Thermostable Sulfurihydrogenibium azorense Carbonic Anhydrase-Chitin Binding Domain Fusion Protein Immobilised on Chitin Support.

Carbonic anhydrases (CAs) represent a group of enzymes that catalyse important reactions of carbon dioxide hydration and dehydration, a reaction crucial to many biological processes and environmental biotechnology. In this study we successfully constructed a thermostable fusion enzyme composed of the Sulfurihydrogenibium azorense carbonic anhydrase (Saz_CA), the fastest CA discovered to date, and the chitin binding domain (ChBD) of chitinase from Bacillus circulans. Introduction of ChBD to the Saz_CA had no major impact on the effect of ions or inhibitors on the enzymatic activity. The fusion protein exhibited no negative effects up to 60 °C, whilst the fusion partner appears to protect the enzyme from negative effects of magnesium. The prepared biocatalyst appears to be thermally activated at 60 °C and could be partially purified with heat treatment. Immobilisation attempts on different kinds of chitin-based support results have shown that the fusion enzyme preferentially binds to a cheap, untreated chitin with a large crystallinity index over more processed forms of chitin. It suggests significant potential economic benefits for large-scale deployment of immobilised CA technologies such as CO2 utilisation or mineralisation.

Application of ß-glucuronidase-immobilised silica gel formulation to microfluidic platform for biotransformation of ß-glucuronides.

OBJECTIVE: To improve the efficiency of reactions of ß-glucuronidase (GUS)-assisted glucuronic acid (GluA) removal within a microfluidic system. RESULTS: ß-glucuronidase from Helix pomatia was immobilised and characterised in silica-based sol-gel monoliths. Efficiency of the GUS-doped silica monoliths was tested for hydrolysis of p-Nitrophenyl-ß-D-glucuronide (pNP-GluA) in both ml-scaled medium via batch reactions and microfluidic environment via continuous-flow reactions. In the microfluidic platform, within a duration of 150 min of continuous operation (flow rate: 1 µL/min), the obtained highest pNP yield was almost 50% higher than that of the corresponding batchwise reaction. However, increased flow rates (3, 5, and 10 µL/min) resulted in lower conversion yields compared to 1 µL/min. The microfluidic platform demonstrated continuous hydrolytic activity for 7 days with considerable reaction yields while using a small amount of the enzyme. CONCLUSION: These results revealed that usage of the microreactors has considerable potential to efficiently obtain bioactive GluA-free aglycons from various plant-derived ß-glucuronides for pharmaceutical applications.

Effect of alginate microencapsulation on the catalytic efficiency and in vitro enzyme-prodrug therapeutic efficacy of cytosine deaminase and of recombinant E. coli expressing cytosine deaminase.

Cytosine deaminase (CD) catalyses the enzymatic conversion of the non-toxic prodrug 5-fluorocytosine (5-FC) to the potent chemotherapeutic form, 5-fluorouracil (5-FU). Intratumoral delivery of CD localises chemotherapy dose while reducing systemic toxicity. Encapsulation in biocompatible microcapsules immunoisolates CD and protects it from degradation. We report on the effect of alginate encapsulation on the catalytic and functional activity of isolated CD and recombinant E. coli engineered to express CD (E. coli(CD)). Alginate microcapsules containing either CD or Escherichia coli(CD) were prepared using ionotropic gelation. Conversion of 5-FC to 5-FU was quantitated in unencapsulated and encapsulated CD/E. coli(CD) using spectrophotometry, with a slower rate of conversion observed following encapsulation. Both encapsulated CD/5-FC and E. coli(CD)/5-FC resulted in cell kill and reduced proliferation of 9 L rat glioma cells, which was comparable to direct 5-FU treatment. Our results show that encapsulation preserves the therapeutic potential of CD and E. coli(CD) is equally effective for enzyme-prodrug therapy.

Enzyme encapsulation in magnetic chitosan-Fe3O4 microparticles.

Two simple procedures for the preparation of magnetic chitosan enzyme microparticles have been investigated and used for the immobilisation of endophytic fungus Cercospora kikuchii lipase as model enzyme. In the first case, lipase was entrapped in Fe3O4-chitosan microparticles by cross-linking method, while in the second case magnetic immobilised derivatives were produced using spray drying. Immobilised enzymes showed high enzyme activity retention and stability during storage without significant loss of activity. Glutaraldehyde Fe3O4-chitosan powders presented a higher lipase activity retention and storage stability than the others preparations. However, the immobilised derivatives produced by cross-linking showed higher enzyme activity after reuse cycles. The results proved that the magnetic Fe3O4-chitosan microparticles are an effective support for the enzyme immobilisation since the immobilised lipase showed best properties than the free form.

Study of different reaction schemes for the enzymatic synthesis of polyglycerol polyricinoleate.

BACKGROUND: Different strategies for the solvent-free enzymatic production of polyglycerol polyricinoleate (PGPR) were explored in an attempt to simplify and improve the process. Besides the conventional procedure (obtaining polyricinoleic acid, followed by its esterification with polyglycerol), two alternative methods are proposed: (1) reversing the synthesis order, i.e. esterification of polyglycerol with ricinoleic acid and then the condensation of ricinoleic acid with the previously obtained polyglycerol ester; and (2) the enzymatic synthesis of PGPR in a single-step process. RESULTS: The reaction sequences were carried out in an open-air reactor with free and immobilised lipases (triacylglycerol acylhydrolases, E.C. 3.1.1.3): Candida rugosa lipase to obtain polyricinoleic acid and Rhizopus oryzae lipase for the esterification of polyglycerol with the carboxyl group of ricinoleic or polyricinoleic acid. A co-immobilised derivative containing both lipases was used to catalyse the single-stage scheme. The three processes were carried out in a vacuum reactor, obtaining in every case PGPR that complied with the legal specifications of the European Community and recommendations provided in the Food Chemical Codex. CONCLUSION: The results demonstrate that all three protocols are viable for the enzymatic synthesis of PGPR and require similar reaction times. The single-stage scheme is easier to carry out.

Peroxygenase-Catalysed Sulfoxidations in Non-Aqueous Media.

Chiral sulfoxides are valuable building blocks in asymmetric synthesis. However, the biocatalytic synthesis of chiral sulfoxides is still challenged by low product titres. Herein, we report the use of peroxygenase as a catalyst for asymmetric sulfoxidation under non-aqueous conditions. Upon covalent immobilisation, the peroxygenase showed stability and activity under neat reaction conditions. A large variety of sulfides was converted into chiral sulfoxides in very high product concentration with moderate to satisfactory optical purity (e. g. 626 mM of (R)-methyl phenyl sulfoxide in approx. 89 % ee in 48 h). Further polishing of the ee value via cascading methionine reductase A (MsrA) gave>99 % ee of the sulfoxide. The robustness of the enzymes and high product titer is superior to the state-of-the-art methodologies. Gram-scale synthesis has been demonstrated. Overall, we demonstrated a practical and facile catalytic method to synthesize chiral sulfoxides.

Recent Advances and Perspectives on Food-Grade Immobilisation Systems for Enzymes.

The use of enzyme immobilisation is becoming increasingly popular in beverage processing, as this method offers significant advantages, such as enhanced enzyme performance and expanded applications, while allowing for easy process termination via simple filtration. This literature review analysed approximately 120 articles, published on the Web of Science between 2000 and 2023, focused on enzyme immobilisation systems for beverage processing applications. The impact of immobilisation on enzymatic activity, including the effects on the chemical and kinetic properties, recyclability, and feasibility in continuous processes, was evaluated. Applications of these systems to beverage production, such as wine, beer, fruit juices, milk, and plant-based beverages, were examined. The immobilisation process effectively enhanced the pH and thermal stability but caused negative impacts on the kinetic properties by reducing the maximum velocity and Michaelis-Menten constant. However, it allowed for multiple reuses and facilitated continuous flow processes. The encapsulation also allowed for easy process control by simplifying the removal of the enzymes from the beverages via simple filtration, negating the need for expensive heat treatments, which could result in product quality losses.

Effects of potential inducers to enhance laccase production and evaluating concomitant enzyme immobilisation.

This work investigated non-polar solvent hexane and polar solvents methanol and ethanol as inducers besides a well-known inducer, copper, for laccase production with and without mesoporous silica-covered plastic packing under sterilised and unsterilised conditions. The potential of waste-hexane water, which is generated during the mesoporous silica production process, was also investigated as a laccase inducer. During the study, the free and immobilised laccase activity on the packing was measured. The results showed that the highest total laccase activity, approximately 10,000 Units, was obtained under sterilised conditions with 0.5 mM copper concentration. However, no immobilised laccase activity was detected except in the copper and ethanol sets under unsterilised conditions. The maximum immobilised laccase activity of the sets that used waste hexane as an inducer was 1.25 U/mg packing. According to its significant performance, waste hexane can be an alternative inducer under sterilised conditions. Concomitant immobilised packing showed satisfactory laccase activities and could be a promising method to reduce operation costs and improve the cost-efficiency of enzymatic processes in wastewater treatment plants.

The Influence of the Structure of Selected Polymers on Their Properties and Food-Related Applications.

Every application of a substance results from the macroscopic property of the substance that is related to the substance's microscopic structure. For example, the forged park gate in your city was produced thanks to the malleability and ductility of metals, which are related to the ability of shifting of layers of metal cations, while fire extinguishing powders use the high boiling point of compounds related to their regular ionic and covalent structures. This also applies to polymers. The purpose of this review is to summarise and present information on selected food-related biopolymers, with special attention on their respective structures, related properties, and resultant applications. Moreover, this paper also highlights how the treatment method used affects the structure, properties, and, hence, applications of some polysaccharides. Despite a strong focus on food-related biopolymers, this review is addressed to a broad community of both material engineers and food researchers.

Protein surface engineering and interaction studies of maltogenic amylase towards improved enzyme immobilisation.

A combined strategy of computational, protein engineering and cross-linked enzyme aggregates (CLEAs) approaches was performed on Bacillus lehensis G1 maltogenic amylase (Mag1) to investigate the preferred amino acids and orientation of the cross-linker in constructing stable and efficient biocatalyst. From the computational analysis, Mag1 exhibited the highest binding affinity towards chitosan (-7.5 kcal/mol) and favours having interactions with aspartic acid whereas glutaraldehyde was the least favoured (-3.4 kcal/mol) and has preferences for lysine. A total of eight Mag1 variants were constructed with either Asp or Lys substitutions on different secondary structures surface. Mutant Mag1-mDh exhibited the highest recovery activity (82.3%) in comparison to other Mag1 variants. Mutants-CLEAs exhibited higher thermal stability (20-30% activity) at 80 °C whilst Mag1-CLEAs could only retain 9% of activity at the same temperature. Reusability analysis revealed that mutants-CLEAs can be recovered up to 8 cycles whereas Mag1-CLEAs activity could only be retained for up to 6 cycles. Thus, it is evident that amino acids on the enzyme's surface play a crucial role in the construction of highly stable, efficient and recyclable CLEAs. This demonstrates the necessity to determine the preferential amino acid by the cross-linkers in advance to facilitate CLEAs immobilisation for designing efficient biocatalysts.

Characterisation and optimisation of a novel laccase from Sulfitobacter indolifex for the decolourisation of organic dyes.

Laccases are multi­copper oxidases that possess the potential for industrial wastewater treatments. In this study, a putative laccase from Sulfitobacter indolifex was recombinantly produced and characterised. The enzyme was found to be stable and active at low to ambient temperature and across a range of pH conditions. The ability of the putative bacterial laccase to catalyse the decolourisation of seven common industrial dyes was also examined. Our results showed that the putative laccase could efficiently decolourise Indigo Carmine, Coomassie Brilliant Blue R-250, Congo Red, Malachite Green and Alizarin in the presence of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as a redox mediator. Furthermore, the use of enzyme immobilisation technology to improve the operational stability and reusability of the putative laccase was also investigated. We found that immobilising the enzyme through the cross-linked enzyme aggregate method significantly improved its tolerance towards extreme pH as well as the presence of organic solvents. This work expands the arsenal of bacterial laccases available for the bioremediation of dye-containing wastewater.

Functionalisable Epoxy-rich Electrospun Fibres Based on Renewable Terpene for Multi-Purpose Applications.

New bio-based polymers capable of either outperforming fossil-based alternatives or possessing new properties and functionalities are of relevant interest in the framework of the circular economy. In this work, a novel bio-based polycarvone acrylate di-epoxide (PCADE) was used as an additive in a one-step straightforward electrospinning process to endow the fibres with functionalisable epoxy groups at their surface. To demonstrate the feasibility of the approach, poly(vinylidene fluoride) (PVDF) fibres loaded with different amounts of PCADE were prepared. A thorough characterisation by TGA, DSC, DMTA and XPS showed that the two polymers are immiscible and that PCADE preferentially segregates at the fibre surface, thus developing a very simple one-step approach to the preparation of ready-to-use surface functionalisable fibres. We demonstrated this by exploiting the epoxy groups at the PVDF fibre surface in two very different applications, namely in epoxy-based carbon fibre reinforced composites and membranes for ω-transaminase enzyme immobilisation for heterogeneous catalysis.

Data-driven enzyme immobilisation: a case study using DNA to immobilise galactose oxidase.

Biocatalysis has the potential to enable green chemistry. New methods of enzyme immobilisation will be required to improve enzyme stability, product purification, and compatibility of different enzymes in the same reaction conditions. Deoxyribonucleic acid (DNA) stands out among supramolecular scaffolds, as simple Watson-Crick base-pairing rules can be used to rationally design a unique nanoscale environment around each individual enzyme in a cascade. Enhancements of enzyme activity and stability on DNA nanostructures have previously been reported, but never in the context of industrially relevant chemical syntheses or reaction conditions. Here, the authors show DNA can enhance the activity and stability of a galactose oxidase mutant, which could be used in a cascade to produce bioplastics from lignin. The enzyme was enhanced in the cell-free extract, which to their knowledge has not been shown before for any enzymes on DNA. This is significant because crude biocatalytic reactions are vastly more cost-effective. This opens the door to further work on multienzyme cascades by tuning the properties of individual enzymes.