Biocatalysis Research Articles

The Effects of Commercial Pesticide Formulations on the Function of In Vitro and In Vivo Assay Systems: A Comparative Analysis

Pesticides are commonly used in agriculture and are an important factor of food security for humankind. However, the overuse of pesticides can harm non-target organisms, and, thus, it is vital to comprehensively study their effects on the different metabolic pathways of living organisms. In the present study, enzyme-inhibition-based assays have been used to investigate the effects of commercial pesticide formulations on the key enzymes of the organisms, which catalyze a wide variety of metabolic reactions (protein catabolism, lactic acid fermentation, alcohol metabolism, the conduction of nerve impulses, etc.). Assay conditions have been optimized, and the limitations of the methods used in the study, which are related to the choice of the solvent for commercial pesticide formulations and optical effects occurring when commercial pesticide formulations are mixed with solutions of enzymes and substrates of assay systems, have been revealed. The effects of commercial pesticide formulations on simple chemoenzymatic assay systems (single-enzyme reactions) have been compared to their effects on complex multicomponent molecular systems (multi-enzyme reactions) and organisms (luminescent bacterium). The in vitro assay systems have shown higher sensitivity to pesticide exposure than the in vivo assay system. The sensitivity of the in vitro assay systems increases with the elongation of the chain of conjugated chemoenzymatic reactions. The effects exerted by commercial pesticide formulations with the same active ingredient but produced by different manufacturers on assay system functions have been found to differ from each other.

Chemoenzymatic conversion of glycerol to lactic acid and glycolic acid

Catalytic valorization of raw glycerol derived from biodiesel into high-value chemicals has attracted great attention. Here, we report chemoenzymatic cascade reactions that convert glycerol to lactic acid and glycolic acid. In the enzymatic step, a coenzyme recycling system was developed to convert glycerol into 1,3-dihydroxyacetone (DHA) with a yield of 92.3% in potassium phosphate buffer (300 mM, pH 7.1) containing 100 mM glycerol, 2 mM NAD+, 242 U/mL glycerol dehydrogenase-GldA and NADH oxidase-SpNoxK184R at 30 °C. Subsequently, NaOH or NaClO2 catalyzes the formation of lactic acid and glycolic acid from DHA. The high yield of lactic acid (72.3%) and glycolic acid (78.2%) verify the benefit of the chemoenzymatic approaches.Graphical

NMR as a readout to monitor and restore the integrity of complex chemoenzymatic reactions

The non-invasive nature of NMR offers a means to monitor biochemical reactions in situ at the atomic level. We harness this advantage to monitor a complex chemoenzymatic reaction that sequentially modifies reagents and loads the product on a nonribosomal peptide synthetase carrier protein. We present a protocol including a pulse sequence that permits to assess both the integrity of reagents and the completion of each step in the reaction, thus alleviating otherwise time-consuming and costly approaches to debug and repeat inefficient reactions. This study highlights the importance of NMR as a tool to establish reliable and reproducible experimental conditions in biochemical studies.

Chemo-enzymatic total syntheses of bis-tetrahydroisoquinoline alkaloids and systematic exploration of the substrate scope of SfmC

Chemo-enzymatic hybrid process merging enzymatic cascade reactions of designed substrates with chemo- and site-selective synthetic manipulations allowed the chemo-enzymatic total syntheses of jorunnamycin A and saframycin A, in just 4 and 5 pots processes, respectively. Artificial variants with installation of various amino acids in place of l-Ala were systematically generated, demonstrating the substantially broader substrate scope of SfmC. This chemo-enzymatic hybrid process can considerably minimize the number of steps, labor, and time, and thus offer a rapid and versatile means to generate therapeutically important natural products and their derivatives.

Pickering emulsion droplet-based biomimetic microreactors for continuous flow cascade reactions

A continuous flow cascade of multi-step catalytic reactions is a cutting-edge concept to revolutionize stepwise catalytic synthesis yet is still challenging in practical applications. Herein, a method for practical one-pot cascade catalysis is developed by combining Pickering emulsions with continuous flow. Our method involves co-localization of different catalytically active sub-compartments within droplets of a Pickering emulsion yielding cell-like microreactors, which can be packed in a column reactor for continuous flow cascade catalysis. As exemplified by two chemo-enzymatic cascade reactions for the synthesis of chiral cyanohydrins and chiral ester, 5 − 420 fold enhancement in the catalysis efficiency and as high as 99% enantioselectivity were obtained even over a period of 80 − 240 h. The compartmentalization effect and enriching-reactant properties arising from the biomimetic microreactor are theoretically and experimentally identified as the key factors for boosting the catalysis efficiency and for regulating the kinetics of cascade catalysis.

New 19F NMR methodology reveals structures of molecules in complex mixtures of fluorinated compounds.

Although the number of natural fluorinated compounds is very small, fluorinated pharmaceuticals and agrochemicals are numerous. 19F NMR spectroscopy has a great potential for the structure elucidation of fluorinated organic molecules, starting with their production by chemical or chemoenzymatic reactions, through monitoring their structural integrity, to their biotic and abiotic transformation and ultimate degradation in the environment. Additionally, choosing to incorporate 19F into any organic molecule opens a convenient route to study reaction mechanisms and kinetics. Addressing limitations of the existing 19F NMR techniques, we have developed methodology that uses 19F as a powerful spectroscopic spy to study mixtures of fluorinated molecules. The proposed 19F-centred NMR analysis utilises the substantial resolution and sensitivity of 19F to obtain a large number of NMR parameters, which enable structure determination of fluorinated compounds without the need for their separation or the use of standards. Here we illustrate the 19F-centred structure determination process and demonstrate its power by successfully elucidating the structures of chloramination disinfectant by-products of a single mono-fluorinated phenolic compound, which would have been impossible otherwise. This novel NMR approach for the structure elucidation of molecules in complex mixtures represents a major contribution towards the analysis of chemical and biological processes involving fluorinated compounds.

Synthesis of silver and gold nanoparticles-enzyme-polymer conjugate hybrids as dual-activity catalysts for chemoenzymatic cascade reactions.

Novel hybrids containing silver or gold nanoparticles have been synthesized in aqueous media and at room temperature using enzymes or tailor-made enzyme-polymer conjugates, which directly induced the formation of inorganic silver or gold species. The choice of pH, protein, or bioconjugate strongly affected the final metallic nanoparticles hybrid formation. Using Candida antarctica lipase (CALB) in a solution, nanobiohybrids containing Ag2O nanoparticles of 9 nm average diameter were obtained. The use of tailor-made bioconjugates, for example, the CALB modified with dextran-aspartic acid polymer (Dext6kDa), resulted in a nanobiohybrid containing smaller Ag(0)/Ag2O nanoparticles. In the case of nanobiohybrids based on gold, Au(0) species were found in all cases. The Au-CALB hybrid contained spherical nanoparticles with 18 nm average diameter size, with a minor range of larger ones (>100 nm) while the AuNPs-CALB-Dext6kDa hybrid was formed by much smaller nanoparticles (9 nm, minor range of 22 nm), and also nanorods of 20-30/40-50 nm length. Using Thermomyces lanuginosus lipase (TLL), apart from the nanoparticle formation, nanoflowers with a diameter range of 100-200 nm were obtained. All nanobiohybrids maintained (dual) enzymatic and metallic activities. For instance, these nanobiohybrids exhibited exquisite dual-activity for hydrolysis/cycloisomerization cascades starting from allenic acetates. By merging the transition metal reactivity with the inherent lipase catalysis, allenic acetates directly converted to the corresponding O-heterocycles in enantiopure form catalysed by AgNPs-CALB-Dext6kDa, taking advantage of a kinetic resolution/cyclization pathway. These results showed the high applicability of these novel hybrids, offering new opportunities for the design of novel reaction cascades.

Biodegradation Of Iron Industry And Gold Industrial Wastewater By White Rot Fungi – Calocybe Indica & Agaricus Bisporus Comparative Study

Increasing discharge and improper management of liquid and solid industrial wastes have created a great concern among industrialists and the scientific community over their economic treatment and safe disposal. Hence, there is a growing need for the development of novel, efficient, eco-friendly, and cost-effective approach for the remediation for these industries released into the environment and to safeguard the ecosystem. In this regard, recent advances in wastewater of heavy metal have propelled bioremediation as a prospective alternative to conventional techniques. Heavy metals are toxic and dangerous to the ecosystem. White rot fungi (WRF) are versatile and robust organisms having enormous potential for oxidative bioremediation of a variety of toxic chemical pollutants due to high tolerance to toxic substances in the environment. The decolorization and detoxification potential of WRF can be harnessed thanks to emerging knowledge of the physiology of these organisms as well as of the bio catalysis and stability characteristics of their enzymes. This knowledge will need to be transformed into reliable and robust waste treatment processes.

Multicatalytic Hybrid Materials for Biocatalytic and Chemoenzymatic Cascades—Strategies for Multicatalyst (Enzyme) Co-Immobilization

During recent decades, the use of enzymes or chemoenzymatic cascades for organic chemistry has gained much importance in fundamental and industrial research. Moreover, several enzymatic and chemoenzymatic reactions have also served in green and sustainable manufacturing processes especially in fine chemicals, pharmaceutical, and flavor/fragrance industries. Unfortunately, only a few processes have been applied at industrial scale because of the low stabilities of enzymes along with the problematic processes of their recovery and reuse. Immobilization and co-immobilization offer an ideal solution to these problems. This review gives an overview of all the pathways for enzyme immobilization and their use in integrated enzymatic and chemoenzymatic processes in cascade or in a one-pot concomitant execution. We place emphasis on the factors that must be considered to understand the process of immobilization. A better understanding of this fundamental process is an essential tool not only in the choice of the best route of immobilization but also in the understanding of their catalytic activity.

Biocatalysis

Biocatalysis

Segmentierte Flow‐Prozesse zur Überwindung von Limitierungen der Ganzzell‐Biokatalyse in Gegenwart von organischen Lösungsmitteln

AbstractIn der modernen Prozessentwicklung spielt Biokatalyse eine immer gewichtigere Rolle, wobei die Benutzung von Ganzzellkatalysatoren häufig eine bevorzugte Form dieser Biokatalysatoren darstellt. Die Anwendung von Ganzzellkatalysatoren in organischen zweiphasigen Batch‐Synthesen ist jedoch oft durch Einschränkungen im Hinblick auf Stofftransfer, Emulsionsbildung, aufwändiger Aufarbeitung und damit verbundenen geringen Ausbeuten beeinträchtigt. In diesem Beitrag zeigen wir, dass unter Zuhilfenahme von segmentierten Flow‐Prozessen eine effiziente Ganzzellkatalyse in zweiphasigen Medien ermöglicht wird. Die Leistungsfähigkeit solcher segmentierter Flow‐Prozesse ist dabei exemplarisch an drei unterschiedlichen Biotransformationen dargestellt. Zum Beispiel wurde in einem solchen Flow‐Prozess eine 3‐fache Steigerung des Umsatzes von 34 % auf >99 % verbunden mit einer erheblich vereinfachten Aufarbeitung erzielt, die zu einer 1.5‐fachen Steigerung der Ausbeute von 44 % auf 65 % im Vergleich zum analogen Batch‐Prozess führte.

Modular biocatalysis for polyamines

Modular biocatalysis for polyamines

Asymmetric reduction of 2-chloro-3-oxo-ester into enantiomerically high pure diltiazem precursor by a Candida ketoreductase

• A highly stereoselective reductase towards the 2-chloro-3-oxo-ester identified. • Highest space-time yield (46 g L −1 d −1 ) reported in bio-reduction of 1a . • Nearly enantiopure (2 R ,3 S )-MPGM obtained through chemo-enzymatic synthesis. Methyl (2 R ,3 S )-3-(4-methoxyphenyl)glycidate [(2 R ,3 S )-MPGM] is an advanced chiral synthon for the synthesis of the cardiovascular drug diltiazem. It can be easily accessed by cyclizing the reduction products of methyl 2-chloro-3-(4-methoxyphenyl)-3-oxo-propanoate ( 1a ). Herein, we report an identified carbonyl reductase ( Cp KR) from Candida parapsilosis that displayed an excellent stereoselectivity toward the keto substituent at the C 3 -position of the 2-chloro-3-oxo-ester 1a . The engineered Escherichia coli cells harboring Cp KR gene were directly applied for the asymmetric reduction of keto ester 1a with a space-time yield of 46 g L −1 d −1 , which represents the highest productivity in bio-reduction of 1a reported so far. The isolated chiral alcohol products were then applied to the chemical synthesis of (2 R ,3 S )-MPGM in 99% ee and a total yield of 76% in the two-step chemo-enzymatic reactions, which far exceeded the maximum theoretical yield (50%) of the existing industrial process based on a lipase-catalyzed resolution of racemic MPGM. This work provides a promising eco-friendly and cost-effective route toward industrial synthesis of pharmaceutically relevant diltiazem.

Special Issue on “Applied Biocatalysis in Europe: A Sustainable Tool for Improving Life Quality”

Applied biocatalysis, i [...]

Hybrid chemoenzymatic heterogeneous catalyst prepared in one step from zeolite nanocrystals and enzyme-polyelectrolyte complexes.

The combination of inorganic heterogeneous catalysts and enzymes, in so-called hybrid chemoenzymatic heterogeneous catalysts (HCEHCs), is an attractive strategy to effectively run chemoenzymatic reactions. Yet, the preparation of such bifunctional materials remains challenging because both the inorganic and the biological moieties must be integrated in the same solid, while preserving their intrinsic activity. Combining an enzyme and a zeolite, for example, is complicated because the pores of the zeolite are too small to accommodate the enzyme and a covalent anchorage on the surface is often ineffective. Herein, we developed a new pathway to prepare a nanostructured hybrid catalyst built from glucose oxidase and TS-1 zeolite. Such hybrid material can catalyse the in situ biocatalytic formation of H2O2, which is subsequently used by the zeolite to trigger the epoxidation of allylic alcohol. Starting from an enzymatic solution and a suspension of zeolite nanocrystals, the hybrid catalyst is obtained in one step, using a continuous spray drying method. While enzymes are expectedly unable to resist the conditions used in spray drying (temperature, shear stress, etc.), we leverage on the preparation of “enzyme–polyelectrolyte complexes” (EPCs) to increase the enzyme stability. Interestingly, the use of EPCs also prevents enzyme leaching and appears to stabilize the enzyme against pH changes. We show that the one-pot preparation by spray drying gives access to hybrid chemoenzymatic heterogeneous catalysts with unprecedented performance in the targeted chemoenzymatic reaction. The bifunctional catalyst performs much better than the two catalysts operating as separate entities. We anticipate that this strategy could be used as an adaptable method to prepare other types of multifunctional materials starting from a library of functional nanobuilding blocks and biomolecules.

REAÇÕES EM CASCATA ENZIMÁTICA, QUIMIOENZIMÁTICA E FOTOENZIMÁTICA: PERSPECTIVAS PARA UMA SÍNTESE ORGÂNICA MAIS SUSTENTÁVEL

ENZYMATIC, CHEMOENZYMATIC AND PHOTOENZYMATIC CASCADE REACTIONS: PERSPECTIVES FOR A MORE SUSTAINABLE ORGANIC SYNTHESIS. Chemicals are rarely the result of a single transformation but rather the consequence of several individual reaction steps. Classically, these individual steps are carried out in sequence, including isolation and purification of the intermediate products. However, the reasons for choosing to carry out cascade reactions can be multiple, such as (i) the chance to prepare unstable and/or toxic intermediates, which are consumed directly in a subsequent step, (ii) the option to shift the equilibrium of a reaction by removing the co (product), or (iii) simply bypass the tedious work of isolating and purifying intermediates, saving reagents, solvents, time and money, and still generating less waste. Therefore, it is not surprising that so-called cascade reactions are gaining considerable interest in academic and industrial research. The purpose of this review is to show recent, promising research and new trends, the potential and current limitations of cascade reactions in organic synthesis that involves at least one enzymatic step in the cascade. This review will address cascades involving multi enzymatic, chemoenzymatic, and photoenzymatic cascades, where all steps are performed in one reaction vessel simultaneously or sequentially and the cascades in the regime of continuous flow.

Esterification of a waste cooking oil using metal-hybrid catalysts for chemoenzymatic reactions

Waste cooking oil (WCO) serve as a cheap feedstocks for biodiesel production. However, the high acidity of this oil disqualifies it from using alkaline transesterification as the synthesis reaction for biodiesel production. Thus, the integration of enzymatic and chemical catalysts to form a hybrid catalyst for reducing oil acidity is a new option in the biodiesel industry. In the present study, five types of metal oxides and Candida rugosa lipase (CRL) were sequentially co-immobilized on the Alumina (Al2O3) for esterification of free fatty acid (FFA) to fatty acid methyl esters (FAMEs).The lowest FFA content achieved was 1.10% using CRL-CaO-Al2O3 for 24 h esterification reaction, correspond to a conversion rates of 66.94%. The FAMEs conversion of the treated WCO indicates that the hybrid catalyst also can perform transesterification of triglycerides in the oil. Among all the hybrid catalysts, CRL-MgO-Al2O3 shows the highest FAME conversion of 69.10%. The best immobilization time was recorded at 7 h which successfully reduced the FFA content until 1.39%. The application of heterogeneous enzyme-metal hybrid catalysts promise a better insight in the biodiesel industry in terms of its simplicity and catalyst recovery.

Ancestral L-amino acid oxidases for deracemization and stereoinversion of amino acids

L-amino acid oxidases (LAAOs) can be applied to convert racemic amino acids to D-isomers, which are potential precursors of pharmaceuticals. However, this application is hampered by the lack of available stable and structure-determined LAAOs. In this study, we attempt to address this limitation by utilizing two ancestral LAAOs: AncLAAO-N4 and AncLAAO-N5. AncLAAO-N4 has the highest thermal and temporal stabilities among the designed LAAOs that can be used for deracemization and stereoinversion. AncLAAO-N5 can provide X-ray crystal structures, which are helpful to reveal substrate recognition and reaction mechanisms of LAAOs at the molecular level. Next, we attempted to improve activity of AncLAAO-N4 toward L-Val through a semi-rational protein engineering method. Three variants with enhanced activity toward L-Val were obtained. Taken together, we believe that the activity and substrate selectivity of AncLAAOs give them the potential to be key enzymes in various chemoenzymatic reactions.

Improved soluble expression and use of recombinant human renalase.

Electrochemical bioreactor systems have enjoyed significant attention in the past few decades, particularly because of their applications to biobatteries, artificial photosynthetic systems, and microbial electrosynthesis. A key opportunity with electrochemical bioreactors is the ability to employ cofactor regeneration strategies critical in oxidative and reductive enzymatic and cell-based biotransformations. Electrochemical cofactor regeneration presents several advantages over other current cofactor regeneration systems, such as chemoenzymatic multi-enzyme reactions, because there is no need for a sacrificial substrate and a recycling enzyme. Additionally, process monitoring is simpler and downstream processing is less costly. However, the direct electrochemical reduction of NAD(P)+ on a cathode may produce adventitious side products, including isomers of NAD(P)H that can act as potent competitive inhibitors to NAD(P)H-requiring enzymes such as dehydrogenases. To overcome this limitation, we examined how nature addresses the adventitious formation of isomers of NAD(P)H. Specifically, renalases are enzymes that catalyze the oxidation of 1,2- and 1,6-NAD(P)H to NAD(P)+, yielding an effective recycling of unproductive NAD(P)H isomers. We designed several mutants of recombinant human renalase isoform 1 (rhRen1), expressed them in E. coli BL21(DE3) to enhance protein solubility, and evaluated the activity profiles of the renalase variants against NAD(P)H isomers. The potential for rhRen1 to be employed in engineering applications was then assessed in view of the enzyme’s stability upon immobilization. Finally, comparative modeling was performed to assess the underlying reasons for the enhanced solubility and activity of the mutant enzymes.

Construction of chemoenzymatic cascade reactions for bridging chemocatalysis and Biocatalysis: Principles, strategies and prospective

• Superiorities and challenges of bridging bio- and chemo-catalysis were summarized. • Principles and strategies to overcome incompatibility were highlighted. • Chemo-bio nanoreactors, temporal and spatial compartmentalization were introduced. • The application of chemoenzymatic cascade reactions was elucidated. • The prospects on the future development of chemoenzymatic catalysis were analyzed. Multi-step one-pot chemoenzymatic cascade reactions ( mo -CECRs) bridging chemo- and bio-catalysis can not only accomplish chemical transformations with minimized purification steps, low cost and improved stereochemical control, but also enhance reactivity and outcomes through the cooperative effect of multiple catalysts. Despite the attractiveness of the concept which meets the principles of green and sustainable chemistry, the establishment of such catalytic networks faces important challenges due to the incompatibilities between different catalytic disciplines. The purpose of this review is to chart progress on mo -CECRs, including temporal compartmentalization, spatial compartmentalization and chemo-bio nanoreactors, with an emphasis on platform methods and technologies involved. The perspectives on future challenges and strategies in this burgeoning research are declared. This review may provide guidance for the design of new and robust mo -CECRs.