Efficient Biocatalytic Degradation of Pollutants by Enzyme‐Releasing Self‐Propelled Motors

The first example of a self‐propelled tubular motor that releases an enzyme for the efficient biocatalytic degradation of chemical pollutants is demonstrated. How the motors are self‐propelled by the Marangoni effect, involving simultaneous release of SDS surfactant and the enzyme remediation agent (laccase) in the polluted sample, is illustrated. The movement induces fluid convection and leads to the rapid dispersion of laccase into the contaminated solution and to a dramatically accelerated biocatalytic decontamination process. The greatly improved degradation efficiency, compared to quiescent solutions containing excess levels of the free enzyme, is illustrated for the efficient biocatalytic degradation of phenolic and azo‐type pollutants. The high efficiency of the motor‐based decontamination approach makes it extremely attractive for a wide‐range of remediation processes in the environmental, defense and public health fields.     

Transforming Inert Cycloalkanes into α,ω-Diamines by Designed Enzymatic Cascade Catalysis

Aliphatic α,ω-diamines (DAs) are important monomer precursors that are industrially produced by energy-intensive, multistage chemical reactions that are harmful to the environment. Therefore, the development of sustainable green DA synthetic routes is highly desired. Herein, we report an efficient one-pot in vivo biocatalytic cascade for the transformation of cycloalkanes into DAs with the aid of advanced techniques, including the RetroBioCat tool for biocatalytic route design, enzyme mining for finding appropriate enzymes and microbial consortia construction for efficient pathway assembly. As a result, DAs were successfully produced by the designed microbial consortia-based biocatalytic system. In particular, the highest biosynthesis productivity record of 1,6-hexanediamine was achieved when using either cyclohexanol or cyclohexane as a substrate. Thus, the developed biocatalytic process provides a promising alternative to the dominant industrial process for manufacturing DAs.     

Research activities in the field of enzyme engineering in the framework of the russian state scientific program “novel methods in bioengineering”

The article describes the research activities in the field of enzyme engineering in Russia. The discussion is focused on fundamental studies of biocatalytic processes that expand utilization of enzymes, biocatalytic synthesis of organic products from renewable raw mate rials, enzymes for hydrolysis of cellulose and lignocellulose materials, immobilized cells, new enzyme-based drugs, enzymes in fine organic synthesis, bioanalytic devices, biosensors, and biofuels.     

Reaching New Biocatalytic Reactivity Using Continuous Flow Reactors

The use of flow reactors in biocatalysis has increased significantly in recent years. Chemists have begun to design flow systems that even allow new biocatalytic reactions to take place. This concept article will focus on the design of flow systems that have allowed enzymes to go beyond their limits in batch. The case is made for moving towards fully continuous systems. With flow chemistry increasingly seen as an enabling technology for automated synthesis, and with advancements in AI-assisted enzyme design, there is a real possibility to fully automate the development and implementation of a continuous biocatalytic processes. This will lead to significantly improved enzyme processes for synthesis.     

In Vivo Biocatalytic Cascades Featuring an Artificial-Enzyme-Catalysed New-to-Nature Reaction**

Artificial enzymes utilizing the genetically encoded non-proteinogenic amino acid p-aminophenylalanine (pAF) as a catalytic residue are able to react with carbonyl compounds through an iminium ion mechanism to promote reactions that have no equivalent in nature. Herein, we report an in vivo biocatalytic cascade that is augmented with such an artificial enzyme-catalysed new-to-nature reaction. The artificial enzyme in this study is a pAF-containing evolved variant of the lactococcal multidrug-resistance regulator, designated LmrR_V15pAF_RMH, which efficiently converts benzaldehyde derivatives produced in vivo into the corresponding hydrazone products inside E. coli cells. These in vivo biocatalytic cascades comprising an artificial-enzyme-catalysed reaction are an important step towards achieving a hybrid metabolism.     

Visualisation of the local bio-electrocatalytic activity in biofuel cell cathodes by means of redox competition scanning electrochemical microscopy (RC-SECM)

Optimisation of biocatalytic systems for the electroreduction of molecular O₂ in biofuel cell cathodes implies screening of the catalytic activity of enzyme/redoxpolymer assemblies. Os-complex modified electrodeposition polymers are suggested for linking bilirubin oxidase catalysed O₂ reduction via an electron hopping sequence along the redox polymer to the electrode. They can be non-manually precipitated on electrode surfaces by electrochemically induced pH modulation. Cyclic voltammetry provides a good estimation of the electrocatalytic activity of a redox polymer/enzyme modified electrode surface. In addition, scanning electrochemical microscopy operating in redox competition mode (RC-SECM) supplies images of the spatial distribution of the biocatalytic activity.     

Biocatalytic deacylation studies on tetra-O-acyl-β-D-xylofuranosyl nucleosides: synthesis of xylo-LNA monomers

A Novozyme-435 catalytic methodology has been developed for selective deacylation of one of the acyloxy functions involving a primary -OH group over the other acyloxy functions involving primary and secondary -OH groups in 4'-C-acyloxymethyl-2',3',5'-tri-O-acyl-β-D-xylofuranosyl nucleosides. Optimization of the biocatalytic reaction revealed that tetra-O-butanoyl-β-D-xylofuranosyl nucleosides are the best substrates for the enzyme. The possibility of acyl migration during enzymatic deacylation reactions has been ruled out by carrying out biocatalytic deacylation reactions on mixed esters of 4'-C-hydroxymethyl-2',3',5'-tri-O-acetyl-β-D-xylofuranosyl nucleosides. The developed methodology has been used for the efficient synthesis of xylo-LNA monomers T, U, A, and C in good yields.     

Nanoengineered ligninolytic enzymes for sustainable lignocellulose biorefinery

Lignin is a key structural component of lignocellulosic biomass with immense potential to replace non-renewable and environmentally unfriendly fossil resources. Structural recalcitrance, heterogeneity, and multifaceted composition of lignin are the major impediments to its gainful biotransformation to a spectrum of bio-based products, biomaterials, and specialty chemicals. In contrast to physicochemical methods, harnessing the biocatalytic potential of the robust ligninolytic armory is considered a greener and more sustainable way for lignin biorefinery. Immobilization of ligninolytic enzymes on different nanoengineered support matrices resulted in designing nanobiocatalytic system with intensified catalytic performance and long-term stability for efficient lignocellulosic biomass valorization. Enzyme incorporation on magnetic nanostructures additionally facilitates facile separation, recovery, and reusability of magnetic nanobiocatalysts. Therefore, developing and implementing immobilized ligninolytic enzyme-based nanoengineered biocatalytic systems constitutes a prodigious and eco-sustainable option to catalyze the deconstruction of lignocellulosic biomass. The multi-enzyme nano-biocatalytic system offers the advantage of direct substrate conversion into the product in a single step owing to its concurrent biocatalytic attributes. This opinion article spotlights current achievements and state-of-the-art developments in engineering ligninolytic enzymes to create a novel biocatalytic system to create greener and sustainable lignocellulose biorefineries ranging from the production of biomaterials to bioenergy.     

Adsorption and catalytic properties of enzymes on the surface of silicalite-1

Microporous hydrophobic silicalite-1 was used as a carrier for immobilization of different enzymes, such as horseradish peroxidase, calf intestinal alkaline phosphatase and two β-galactosidases of different origin, to create heterogeneous biocatalytic systems. The peculiarities of enzyme adsorption on the surface of silicalite-1, as well as catalytic properties of the obtained systems compared to enzyme activity in solution and on the surface of other carriers, are discussed.     

聚邻苯二胺膜电极中辣根过氧化物酶的电子传递

利用酸的电化学固定法制备含辣根过氧化物酶的聚邻苯二胺膜电极，研究其伏安行为及对Ｈ２Ｏ２还原的生物电催化作用，结果表明，在所述生物电催化反应中酶与聚合物基质 直接电子传递，但对新制的酶电极而言，电聚合时生成并包埋在酶膜中的寡聚体可作为电子传递体加速氧化态酶的再生，根据酶电极电流响应实验曲线的拟合，发现经态酶的再生速度随是极电位的变化表观上符合Ｔａｆｅｌ关系式，提出了酶反应学参数的测定方法。     

Biocatalysis made to order

Recombinant DNA technology is now being explored to engineer enzyme molecules. It has many far-reaching applications in biocatalytic processes of enzyme engineering. The facts have pursued certain important industrial, biomedical, and environmental problems. These current excitements are mainly focused on the basis of gene cloning and in vitro mutagenesis for overproduction and redesigning of enzymes, as well as their probable implications in industry, antibiotic research, and waste degradation.     

A General Strategy for Site‐Directed Enzyme Immobilization by Using NiO Nanoparticle Decorated Mesoporous Silica

Mesoporous materials have recently gained much attention owing to their large surface area, narrow pore size distribution, and superior pore structure. These materials have been demonstrated as excellent solid supports for immobilization of a variety of proteins and enzymes for their potential applications as biocatalysts in the chemical and pharmaceutical industries. However, the lack of efficient and reproducible methods for immobilization has limited the activity and recyclability of these biocatalysts. Furthermore, the biocatalysts are usually not robust owing to their rapid denaturation in bulk solvents. To solve these problems, we designed a novel hybrid material system, mesoporous silica immobilized with NiO nanoparticles (SBA‐NiO), wherein enzyme immobilization is directed to specific sites on the pore surface of the material. This yielded the biocatalytic species with higher activity than free enzyme in solution. These biocatalytic species are recyclable with minimal loss of activity after several cycles, demonstrating an advantage over free enzymes.     

Expanding the organic toolbox: a guide to integrating biocatalysis in synthesis

This critical review presents an introduction to biocatalysis for synthetic chemists. Advances in biocatalysis of the past 5 years illustrate the breadth of applications for these powerful and selective catalysts in conducting key reaction steps. Asymmetric synthesis of value-added targets and other reaction types are covered, with an emphasis on pharmaceutical intermediates and bulk chemicals. Resources of interest for the non-initiated are provided, including specialized websites and service providers to facilitate identification of suitable biocatalysts, as well as references to recent volumes and reviews for more detailed biocatalytic procedures. Challenges related to the application of biocatalysts are discussed, including how 'green' a biocatalytic reaction may be, and trends in biocatalyst improvement through enzyme engineering are presented (152 references).     

Enzyme-catalyzed bio-pumping of electrons into au-nanoparticles: a surface plasmon resonance and electrochemical study

The enzyme glucose oxidase (GOx) is reconstituted on a flavin adenin dinucleotide (FAD, 1) cofactor-functionalized Au-nanoparticle (Au-NP), 1.4 nm, and the GOx/Au-NP hybrid is linked to a bulk Au-electrode by a short dithiol, 1,4-benzenedithiol (2), or a long dithiol, 1,9-nonanedithiol (3), monolayer. The reconstituted GOx/Au-NP hybrid system exhibits electrical communication between the enzyme redox cofactor and the Au-NP core. Because the thiol monolayers provide a barrier for electron tunneling, the electron transfer occurring upon the biocatalytic oxidation of glucose results in the Au-NPs charging. The charging of the Au-NPs alters the plasma frequency and the dielectric constant of the Au-NPs, thus leading to the changes of the dielectric constant of the interface. These are reflected in pronounced shifts of the plasmon angle, theta(P), in the surface plasmon resonance (SPR) spectra. As the biocatalytic charging phenomenon is controlled by the concentration of glucose, the changes in the theta(P) values correlate with the concentration of glucose. The biocatalytic charging process is characterized by following the differential capacitance of the GOx/Au-NP interface and by monitoring the potential generated on the bulk Au-electrode. The charging of the GOx/Au-NPs is also accomplished in the absence of glucose by the application of an external potential on the electrode, that resulted in similar plasmon angle shifts. The results allowed us to estimate the number of electrons stored per Au-NP at variable concentrations of glucose in the presence of the two different thiol linkers.     

Evaluation of performance and stability of biocatalytic redox films constructed with different copper oxygenases and osmium-based redox polymers

We are interested in investigating the applications of biocatalytic mediated reduction of oxygen by oxygenases in films on electrode surfaces, as such reactions can form the basis for biosensors or biocatalytic fuel cell development. Here we present approaches aimed at improving the stability and signal output of such films. These include selection of oxygen reducing biocatalysts which are active under physiological conditions and development of redox mediators which offer the opportunity to tailor the mediator to each enzyme. It was found that for each enzyme Melanocarpus albomyces laccase (MaL), Trametes hirsutus laccase (ThL) or bilirubin oxidase (MvBOD) it was the biocatalytic films mediated by Os(2,2′-bipyridine)₂Cl·PVI that not only generated the highest current densities compared to Os(4,4′-dimethyl-2,2′-bipyridine)₂Cl·PVI and Os(4,4′-dichloro-2,2′-bipyridine)₂Cl·PVI, but also proved to be the most stable over 48 h. Under physiological conditions electrodes constructed from MvBOD generated the highest initial current densities for each of the osmium redox polymers, however these films proved to be the least stable over 48 h. Stability could be improved using surface pre-treatment.     

Enzyme catalysis in organic solvents: a promising field for optical biosensing

The ability of enzymes to work in non-aqueous media offers new and almost unexploited possibilities for the development of new optical biosensors. The advantages of performing biocatalytic reactions in non-aqueous media are discussed in relation to their possible application in optical biosensor design. Attention is focused on the factors that influence enzymatic catalysis in organic solvents, including the role of enzyme-associated water, criteria for solvent selection and the alteration of enzyme specificity. Recent examples of relevant applications and future prospects of organic-phase optical biosensing are discussed.     

Not-XOR (NXOR) Logic Gate Realized with Enzyme-Catalyzed Reactions: Optical and Electrochemical Signal Transduction

The studied enzyme-based biocatalytic system mimics NXOR Boolean logic gate, which is a logical operator that corresponds to equality in Boolean algebra. It gives the functional value true ( 1 ) if both functional arguments (input signals) have the same logical value ( 0 , 0 or 1 , 1 ), and false ( 0 ) if they are different ( 0 , 1 or 1 , 0 ). The output signal producing reaction is catalyzed by pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH), which is inhibited at acidic and basic pH values. Two other reactions catalyzed by esterase and urease produce acetic acid and ammonium hydroxide, respectively, shifting solution pH from the optimum pH for PQQ-GDH to acidic and basic values ( 1 , 0 and 0 , 1 input combinations, respectively), thus switching the enzyme activity off (output 0 ). When the input signals are not applied ( 0 , 0 combination) or both applied compensating each other ( 1 , 1 combination) the optimum pH is preserved, thus keeping PQQ-GDH running at the high rate (output 1 ). The biocatalytic cascade mimicking the NXOR gate was characterized optically and electrochemically. In the electrochemical experiments the PQQ-GDH enzyme communicated electronically with a conducting electrode support, thus resulting in the electrocatalytic current when signal combinations 0 , 0 and 1 , 1 were applied. The logic gate operation, when it was realized electrochemically, was also extended to the biomolecular release controlled by the gate. The release system included two electrodes, one performing the NXOR gate and another one activated for the release upon electrochemically stimulated alginate hydrogel dissolution. The studied system represents a general approach to the biocatalytic realization of the NXOR logic gate, which can be included in different catalytic cascades mimicking operation of concatenated gates in sophisticated logic circuitries.     

Biocatalytic Strategy for Highly Diastereo‐ and Enantioselective Synthesis of 2,3‐Dihydrobenzofuran‐Based Tricyclic Scaffolds

2,3‐Dihydrobenzofurans are key pharmacophores in many natural and synthetic bioactive molecules. A biocatalytic strategy is reported here for the highly diastereo‐ and enantioselective construction of stereochemically rich 2,3‐dihydrobenzofurans in high enantiopurity (>99.9% de and ee), high yields, and on a preparative scale via benzofuran cyclopropanation with engineered myoglobins. Computational and structure‐reactivity studies provide insights into the mechanism of this reaction, enabling the elaboration of a stereochemical model that can rationalize the high stereoselectivity of the biocatalyst. This information was leveraged to implement a highly stereoselective route to a drug molecule and a tricyclic scaffold featuring five stereogenic centers via a single‐enzyme transformation. This work expands the biocatalytic toolbox for asymmetric C–C bond transformations and should prove useful for further development of metalloprotein catalysts for abiotic carbene transfer reactions.     

Enzyme Shielding in an Enzyme‐thin and Soft Organosilica Layer

The fragile nature of most enzymes is a major hindrance to their use in industrial processes. Herein, we describe a synthetic chemical strategy to produce hybrid organic/inorganic nanobiocatalysts; it exploits the self‐assembly of silane building blocks at the surface of enzymes to grow an organosilica layer, of controlled thickness, that fully shields the enzyme. Remarkably, the enzyme triggers a rearrangement of this organosilica layer into a significantly soft structure. We demonstrate that this change in stiffness correlates with the biocatalytic turnover rate, and that the organosilica layer shields the enzyme in a soft environment with a markedly enhanced resistance to denaturing stresses.     

Biocatalytic production of tetrahydroisoquinolines

The promiscuity of the enzyme norcoclaurine synthase is described. This biocatalyst yielded a diverse array of substituted tetrahydroisoquinolines by cyclizing dopamine with various acetaldehydes in a Pictet-Spengler reaction. This enzymatic reaction may provide a biocatalytic route to a range of tetrahydroisoquinoline alkaloids.