Biocatalytic Oxidation Research Articles

Back Cover: Development of Continuous Flow Systems to Access Secondary Amines Through Previously Incompatible Biocatalytic Cascades (Angew. Chem. Int. Ed. 34/2021)

Compartmentalised flow reactors allow biocatalytic cascades that would be impossible using standard methods, as described by Sebastian C. Cosgrove, Sabine L. Flitsch, and co-workers in their Research Article on page 18660. Carbonyl intermediates were continuously generated via biocatalytic oxidation, then converted into different amine products using immobilised aminating enzymes. The use of flow equipment allowed exquisite control of the paths and enabled a range of new biocatalytic cascades to be constructed, paving the way for fully automated continuous biocatalytic cascades.

Chemoenzymatic Total Synthesis of Natural Products.

The total synthesis of structurally complex natural products has challenged and inspired generations of chemists and remains an exciting area of active research. Despite their history as privileged bioactivity-rich scaffolds, the use of natural products in drug discovery has waned. This shift is driven by their relatively low abundance hindering isolation from natural sources and the challenges presented by their synthesis. Recent developments in biocatalysis have resulted in the application of enzymes for the construction of complex molecules. From the inception of the Narayan lab in 2015, we have focused on harnessing the exquisite selectivity of enzymes alongside contemporary small molecule-based approaches to enable concise chemoenzymatic routes to natural products.We have focused on enzymes from various families that perform selective oxidation reactions. For example, we have targeted xyloketal natural products through a strategy that relies on a chemo- and site-selective biocatalytic hydroxylation. Members of the xyloketal family are characterized by polycyclic ketal cores and demonstrate potent neurological activity. We envisioned assembling a representative xyloketal natural product (xyloketal D) involving a biocatalytically generated ortho-quinone methide intermediate. The non-heme iron (NHI) dependent monooxygenase ClaD was used to perform the benzylic hydroxylation of a resorcinol precursor, the product of which can undergo spontaneous loss of water to form an ortho-quinone methide under mild conditions. This intermediate was trapped using a chiral dienophile to complete the total synthesis of xyloketal D.A second class of biocatalytic oxidation that we have employed in synthesis is the hydroxylative dearomatization of resorcinol compounds using flavin-dependent monooxygenases (FDMOs). We anticipated that the catalyst-controlled site- and stereoselectivity of FDMOs would enable the total synthesis of azaphilone natural products. Azaphilones are bioactive compounds characterized by a pyranoquinone bicyclic core and a fully substituted chiral carbon atom. We leveraged the stereodivergent reactivity of FDMOs AzaH and AfoD to achieve the enantioselective synthesis of trichoflectin enantiomers, deflectin 1a, and lunatoic acid. We also leveraged FDMOs to construct tropolone and sorbicillinoid natural products. Tropolones are a structurally diverse class of bioactive molecules characterized by an aromatic cycloheptatriene core bearing an α-hydroxyketone moiety. We developed a two-step biocatalytic cascade to the tropolone natural product stipitatic aldehyde using the FDMO TropB and a NHI monooxygenase TropC. The FDMO SorbC obtained from the sorbicillin biosynthetic pathway was used in the concise total synthesis of a urea sorbicillinoid natural product.Our long-standing interest in using enzymes to carry out C-H hydroxylation reactions has also been channeled for the late-stage diversification of complex scaffolds. For example, we have used Rieske oxygenases to hydroxylate the tricyclic core common to paralytic shellfish toxins. The systemic toxicity of these compounds can be reduced by adding hydroxyl and sulfate groups, which improves their properties and potential as therapeutic agents. The enzymes SxtT, GxtA, SxtN, and SxtSUL were used to carry out selective C-H hydroxylation and O-sulfation in saxitoxin and related structures. We conclude this Account with a discussion of existing challenges in biocatalysis and ways we can currently address them.

Enzyme Cascade Reaction Involving Lytic Polysaccharide Monooxygenase and Dye-Decolorizing Peroxidase for Chitosan Functionalization.

In situ H2O2 generation systems are efficient for H2O2-dependent biocatalytic oxidation reactions. Here, we report that lytic polysaccharide monooxygenases (LPMOs), copper-dependent polysaccharide monooxygenases, can efficiently supply H2O2 in situ to dye-decolorizing peroxidases (DyPs) using substrate gallic acid (GA) for chitosan functionalization. The maximum grafting ratio induced by the cascade reaction was significantly higher than that achieved by a reaction with initial exogenous H2O2. The maximum grafting ratio was obtained with 12 g/L GA, 5.6 mg/L DyP, 20-30 mg/L LPMO, and pH 4.5-5.0. UV-vis, Fourier transform infrared (FT-IR), and nuclear magnetic resonance (1H NMR) spectroscopy confirmed GA grafting onto chitosan. X-ray diffraction (XRD) analysis and thermogravimetric analysis (TGA) indicated that GA-chitosan conjugates had lower thermal stability and crystallinity than chitosan. The GA-chitosan conjugates had significantly higher antioxidant activity than chitosan. This study supplies a green and high-efficiency approach to achieve an enzymatic cascade reaction for chitosan functionalization and has potential applications in H2O2-dependent biocatalytic oxidation reactions.

Whole-cell screening of oxidative enzymes using genetically encoded sensors.

Biocatalysis is increasingly used for synthetic purposes in the chemical and especially the pharmaceutical industry. Enzyme discovery and optimization which is frequently needed to improve biocatalytic performance rely on high-throughput methods for activity determination. These methods should ideally be generic and applicable to entire enzyme families. Hydrogen peroxide (H2O2) is a product of several biocatalytic oxidations and its formation can serve as a proxy for oxidative activity. We designed a genetically encoded sensor for activity measurement of oxidative biocatalysts via the amount of intracellularly-formed H2O2. A key component of the sensor is an H2O2-sensitive transcriptional regulator, OxyR, which is used to control the expression levels of fluorescent proteins. We employed the OxyR sensor to monitor the oxidation of glycerol to glyceraldehyde and of toluene to o-cresol catalysed by recombinant E. coli expressing an alcohol oxidase and a P450 monooxygenase, respectively. In case of the P450 BM3-catalysed reaction, we additionally monitored o-cresol formation via a second genetically encoded sensor based on the phenol-sensitive transcriptional activator, DmpR, and an orthogonal fluorescent reporter protein. Single round screens of mutant libraries by flow cytometry or by visual inspection of colonies on agar plates yielded significantly improved oxidase and oxygenase variants thus exemplifying the suitability of the sensor system to accurately assess whole-cell oxidations in a high-throughput manner.

Applicability of Recombinant Laccases From the White-Rot Fungus Obba rivulosa for Mediator-Promoted Oxidation of Biorefinery Lignin at Low pH.

Utilization of lignin-rich side streams has been a focus of intensive studies recently. Combining biocatalytic methods with chemical treatments is a promising approach for sustainable modification of lignocellulosic waste streams. Laccases are catalysts in lignin biodegradation with proven applicability in industrial scale. Laccases directly oxidize lignin phenolic components, and their functional range can be expanded using low-molecular-weight compounds as mediators to include non-phenolic lignin structures. In this work, we studied in detail recombinant laccases from the selectively lignin-degrading white-rot fungus Obba rivulosa for their properties and evaluated their potential as industrial biocatalysts for the modification of wood lignin and lignin-like compounds. We screened and optimized various laccase mediator systems (LMSs) using lignin model compounds and applied the optimized reaction conditions to biorefinery-sourced technical lignin. In the presence of both N–OH-type and phenolic mediators, the O. rivulosa laccases were shown to selectively oxidize lignin in acidic reaction conditions, where a cosolvent is needed to enhance lignin solubility. In comparison to catalytic iron(III)–(2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) oxidation systems, the syringyl-type lignin units were preferred in mediated biocatalytic oxidation systems.

Disposable Capacitive Biosensor for Dopamine Sensing

AbstractThe present paper reports fabrication of a disposable tyrosinase (Tyr) biosensor for rapid detection of dopamine. Tyr immobilized polyaniline/carbon nanotubes/cellulose nanocrystals (Tyr@PANI/CNTs/CNC) conductive film was fabricated on polyvinyl acetate (PVA) transparency and characterized by scanning electron microscopy (SEM) and cyclic voltammetry (CV). Tyr catalyzed dopamine oxidation to o‐dopaquinone was analysed by CV and capacitance was recorded. PANI/CNTs/CNC film acted as a suitable enzyme support which also showed its synergistic effect in accelerating the biocatalytic oxidation reaction. Tyr biosensor exhibited excellent reproducibility, and specificity towards dopamine with correlation coefficient (R2) of 0.9508 and limit of detection (LOD) of 1.57 nM within linear concentration range of 7–1000 mM. The study suggested practical utilization of disposable biosensor towards dopamine detection in biological fluids.

Photoelectrochemically-assisted biofuel cell constructed by redox complex and g-C3N4 coated MWCNT bioanode

Herein, we report a membraneless glucose and air photoelectrochemical biofuel cell (PBFC) with a visible light assisted photobioanode. Flavin adenine dinucleotide dependent glucose dehydrogenase (FADGDH) was immobilized on the combined photobioanode for the visible light assisted glucose oxidation (GCE|MWCNT|g-C3N4|Ru-complex|FADGDH) with a quinone mediated electron transfer. Bilirubine oxidase (BOx) immobilized on MWCNT coated GCE (GCE|BOx) was used as the cathode with direct electron transfer (DET). An improvement of biocatalytic oxidation current was observed by 6.2% due in part to the light-driven electron-transfer. The large oxidation currents are probably owing to the good contacting of the immobilized enzymes with the electrode material and the utilization of light assisted process. Under the visible light, the photobioanode shows an anodic photocurrent of 1.95 μA cm2 at attractively low potentials viz. −0.4 vs Ag/AgCl. The lower-lying conduction band of g-C3N4 as compared to Ru-complexes decreases the rate of hole and electron recombination and enhances the charge transportation. The bioanode shows maximum current density for glucose oxidation up to 6.78 μA cm−2 at 0.2 V vs Ag/AgCl at pH:7. The performance of three promising Ru-complexes differing in chemical and redox properties were compared as electron mediators for FADGDH. Upon illumination, the PBFC delivered a maximum power density of 28.5 ± 0.10 μW cm−2 at a cell voltage of +0.4 V with an open circuit voltage of 0.64 V.

Optimization of Alcohol Dehydrogenase for Industrial Scale Oxidation of Lactols.

Alcohol dehydrogenases (ADH) are widely used to enantioselectively reduce ketones to chiral alcohols, but their application in industrial scale oxidations is rare. Reasons are the need for an NAD(P)+ cofactor regeneration system, often low performance in oxidative reactions and the limited substrate scope of ADHs. ADHA from Candida magnoliae DSMZ 70638 is identified to efficiently catalyze the regio-selective hydroxy-lactone oxidations to hydroxy-lactones. Hydroxy-lactones are common intermediates in industrial processes to cholesterol lowering (va)statin drugs. A biocatalytic aliphatic hydroxy-lactone oxidation process is developed using pure oxygen as oxidant reaching volumetric productivities of up to 12g L-1 h-1 , product concentrations of almost 50g L-1 and 95% reaction yield. For co-factor recycling a previously engineered, water-forming NAD(P)H-oxidase from Streptococcus mutans is used. The process is scaled up to industrial pilot plant scale and it could be demonstrated that ADH catalyzed oxidations can be developed to efficient and safe processes. However, the ADHA wild-type enzyme is not productive enough in chlorolactol oxidation. Therefore, enzyme engineering and multi-parameter screening is successfully applied to optimize the enzyme for the target reaction. The optimized ADHA variant shows a 17-fold higher oxidative activity, a 26°C increased stability and is applied to develop an efficient chlorolactol oxidation process.

Biocatalytic Oxidation of Alcohols

Enzymatic methods for the oxidation of alcohols are critically reviewed. Dehydrogenases and oxidases are the most prominent biocatalysts, enabling the selective oxidation of primary alcohols into aldehydes or acids. In the case of secondary alcohols, region and/or enantioselective oxidation is possible. In this contribution, we outline the current state-of-the-art and discuss current limitations and promising solutions.

Biocatalytic Oxidation of Aromatic Compounds by Spore-Based System

Enzyme-based catalyzed reactions are of increasing importance in organic synthesis as they are eco-friendly and allow a multitude of selective and efficient reactions in aqueous system. In this wor...

ADVANCED REMOVAL OF TRICHLOROETHYLENE FROM WATER BY SONOLYSIS, BIOCATALYTIC OXIDATION AND ULTRAFILTRATION

Trichloroethylene (TCE) causes important pollution problems of soil and groundwater in other countries even in this day when its direct application is less than the last century. This paper emphasized a modern treatment flow for trichloroethylene advanced removal from water based on four phases: sonolysis - biocatalytical oxidation - sonolysis - ultrafiltration. Following this scheme, it is possible to obtain residual TCE content in the effluent approx. 0.01 mg/L, from initial content of 0.5 mg TCE/L. Ultrasonic energy (50-200 kJ) biocatalyst dose (laccase), biocatalytic oxidation time and ultrafiltration separation capacity are the main operating parameters that control the TCE removal efficiency (max. 87%).

Process properties of an l-amino acid oxidase from Hebeloma cylindrosporum for the synthesis of phenylpyruvic acid from l-phenylalanine

The biocatalytic oxidation of amino acids represents an attractive approach towards the synthesis of α-keto acids, which are interest for various industrial applications. As l-amino acids are readily available from fermentation processes, these natural amino acids can serve as substrates in combination with an l-amino acid oxidase. Besides an aqueous phase as reaction medium, a further advantage of such a process is the utilization of air as oxidation agent. In this study, we studied the organic-synthetic properties of a literature-known recombinant l-amino acid oxidase from the fungus Hebeloma cylindrosporum with respect to its suitability to catalyze the formation of α-keto acids exemplified for the synthesis of phenylpyruvic acid starting from l-phenylalanine as a substrate. In our study the enzyme displayed a reasonable operational stability in the reaction system and as well as promising applicability data with respect to substrate and product inhibition. In a biotransformation, 20 mM of substrate were converted after 4 h reaction. The formation of undesired by-products was suppressed using a commercially available catalase enzyme.

Oxidation of aromatic and aliphatic aldehydes to carboxylic acids by Geotrichum candidum aldehyde dehydrogenase

Oxidation reaction is one of the most important and indispensable organic reactions, so that green and sustainable catalysts for oxidation are necessary to be developed. Herein, biocatalytic oxidation of aldehydes was investigated, resulted in the synthesis of both aromatic and aliphatic carboxylic acids using a Geotrichum candidum aldehyde dehydrogenase (GcALDH). Moreover, selective oxidation of dialdehydes to aldehydic acids by GcALDH was also successful.

The Effect of Dissolved Oxygen on Kinetics during Continuous Biocatalytic Oxidations

Conventional oxidation catalysts frequently exhibit limited selectivity, restricting their use in industrial syntheses. In particular, for the continuous synthesis of complex active pharmaceutical ...

Biocatalytic Oxidation of Silanes to Silanols

Key words directed evolution - silanols - silanes - cytochrome P450 monooxygenase - Escherichia coli

Biocatalytic allylic hydroxylation of unsaturated triterpenes and steroids by Bacillus megaterium CGMCC 1.1741.

In this study, we described the microbial catalyzed allylic oxidation by Bacillus megaterium CGMCC 1.1741 of three Δ12-pentacyclic triterpenes, erythrodiol (1), uvaol (2), hederagenin (3) and of four steroids including Δ5-steroids, diosgenin (4), pennogenin (5), 25(R,S)-ruscogenin (6) and Δ4-steroid, diosgenone (7). As a result, fourteen metabolites were generated with allyl hydroxyl moiety. Ten (1a-c, 2a, 2c, 3a, 5a-b, and 6a-b) of them were new natural products and their structures were determined on the basis of 1D/2D NMR and HR-MS data. Biocatalytic allylic oxidation by B. megaterium CGMCC 1.1741 is thus a potential non-toxic and efficient alternative method toward metal-mediated oxidation procedures in the synthesis of natural products and medicines.

Biocatalytic Oxidation of Saccharides in Continuous Flow

Biocatalytic Oxidation of Saccharides in Continuous Flow

Resolving the formidable barrier of oxygen transferring rate (OTR) in ultrahigh-titer bioconversion/biocatalysis by a sealed-oxygen supply biotechnology (SOS)

BackgroundThe critical issue in the competitiveness between bioengineering and chemical engineering is the products titer and the volume productivity. The most direct and effective approach usually employs high-density biocatalyst, while the weakened mass transfer and evoked foam problem accompany ultrahigh-density biocatalyst loading and substrate/product titer. In high-density obligate aerobic bioconversion, oxygen as electron acceptor is a speed-limiting step in bioprocesses, but sufficient oxygen supply will lead to the foaming which results in a significant reduction in oxygen utilization and the use of additional defoamers. In this study, we designed a novel sealed-oxygen supply (SOS) biotechnology to resolve the formidable barrier of oxygen transferring rate (OTR), for bio-based fuels and chemical production process.ResultsBased on systemic analysis of whole-cell catalysis in Gluconobacter oxydans, a novel sealed-oxygen supply technology was smartly designed and experimentally performed for biocatalytic oxidation of alcohols, sugars and so on. By a simple operation skill of automatic online supply of oxygen in a sealed stirring tank bioreactor of SOS, OTR barrier and foaming problem was resolved with great ease. We finally obtained ultrahigh-titer products of xylonic acid (XA), 3-hydroxypropionic acid (3-HPA), and erythrulose at 588.4 g/L, 69.4 g/L, and 364.7 g/L, respectively. Moreover, the volume productivity of three chemical products was improved by 150–250% compared with normal biotechnology. This SOS technology provides a promising approach to promote bioengineering competitiveness and advantages over chemical engineering.ConclusionSOS technology was demonstrated as an economic and universally applicable approach to bio-based fuels and chemicals production by whole-cell catalysis. The novel technology greatly promotes the competitiveness of bioengineering for chemical engineering, and provides a promising platform for the green and environmental use of biofuels.

Simultaneous cyclic deracemisation and stereoinversion of alcohols using orthogonal biocatalytic oxidation and reduction reactions

We developed a concurrent cyclic deracemisation approach for secondary alcohols that combines a non-stereospecific oxidation step and a stereoselective reduction step using two mutants of TeSADH that exhibit various extents of stereoselectivities.

Anoxic ammonia removal using granulated nanostructured Fe oxyhydroxides and the effect of pH, temperature and potential inhibitors on the process

Simultaneous nitrification and denitrification coupled to Fe redox cycling was achieved by activated sludge flocs sourced from a sequential batch reactor (SBR), treating domestic sewage and with less efficiency using the sludge from various environments. This novel process involves bio-catalytic oxidation of NH4+-N using nanostructured Fe oxyhydroxides under anoxic environment in a SBR. NH4+-N oxidation is associated with biogenic Fe(III) reduction and, simultaneous Fe(II) oxidation which is inturn associated with NO2−-N and/or NO3−-N reduction. The internal recycling of nanostructured Fe oxyhydroxides makes NH4+-N removal to undergo multiple cycles. The synthesized oxides were identified as FeOOH based on XPS data and have an average size of 30 nm. The NH4+-N and total nitrogen removals obtained were 73.1 ± 17.4 mg/L/d and 68.2 ± 16.9 mg/L/d, respectively. Optimum conditions for NH4+-N removal were at pH of 7 and temperature of 25−30 °C. The inhibition effects of NH4+-N, NO2−-N and S2- on anoxic NH4+-N removal and the possibility of recovery from inhibition were also studied and discussed. This process does not require oxygen and alkalinity and hence is inexpensive compared to other existing techniques.