Efficient Biocatalytic System Research Articles

Engineering of 4-hydroxyphenylacetate-3-hydroxylase from Escherichia coli for efficient biosynthesis of piceatannol

Piceatannol, a catechol compound, exhibits various health benefits, with great application value in food, medicine, and cosmetics. However, the low natural abundance of piceatannol limits its cost-effective isolation, and chemical synthesis is difficult. Here, we report an efficient biocatalytic system for the production of piceatannol from resveratrol. Structure-based semi-rational engineering was used to generate HpaBCG209F, a novel HpaBC mutant of 4-hydroxyphenylacetate-3-hydroxylase from Escherichia coli. Biocatalytic activity assays revealed that HpaBCG209F exhibits 3.12 times higher activity toward resveratrol than wild-type HpaBC. We also introduced formic acid dehydrogenase (FDH) as an NADH-regeneration system for HpaBCG209F to support piceatannol production. Co-expression of HpaBCG209F and FDH in E. coli BL21(DE3) enabled the generation of 8.23 mM piceatannol from 10.2 mM resveratrol within 9.5 h under optimized conditions, representing an 80.6% molar conversion ratio. This study therefore successfully developed a highly active HpaBC variant for hydroxylating resveratrol to piceatannol, as well as demonstrated a promising biopathway for piceatannol production.

A key O-demethylase in the degradation of guaiacol by Rhodococcus opacus PD630.

Rhodococcus opacus PD630 is a high oil-producing strain with the ability to convert lignin-derived aromatics to high values, but limited research has been done to elucidate its conversion pathway, especially the upper pathways. In this study, we focused on the upper pathways and demethylation mechanism of lignin-derived aromatics metabolism by R. opacus PD630. The results of the aromatic carbon resource utilization screening showed that R. opacus PD630 had a strong degradation capacity to the lignin-derived methoxy-containing aromatics, such as guaiacol, 3,4-veratric acid, anisic acid, isovanillic acid, and vanillic acid. The gene of gcoAR, which encodes cytochrome P450, showed significant up-regulation when R. opacus PD630 grew on diverse aromatics. Deletion mutants of gcoAR and its partner protein gcoBR resulted in the strain losing the ability to grow on guaiacol, but no significant difference to the other aromatics. Only co-complementation alone of gcoAR and gcoBR restored the strain's ability to utilize guaiacol, demonstrating that both genes were equally important in the utilization of guaiacol. In vitro assays further revealed that GcoAR could convert guaiacol and anisole to catechol and phenol, respectively, with the production of formaldehyde as a by-product. The study provided robust evidence to reveal the molecular mechanism of R. opacus PD630 on guaiacol metabolism and offered a promising study model for dissecting the demethylation process of lignin-derived aromatics in microbes.IMPORTANCEAryl-O-demethylation is believed to be the key rate-limiting step in the catabolism of heterogeneous lignin-derived aromatics in both native and engineered microbes. However, the mechanisms of O-demethylation in lignin-derived aromatic catabolism remain unclear. Notably, guaiacol, the primary component unit of lignin, lacks in situ demonstration and illustration of the molecular mechanism of guaiacol O-demethylation in lignin-degrading bacteria. This is the first study to illustrate the mechanism of guaiacol metabolism by R. opacus PD630 in situ as well as characterize the purified key O-demethylase in vitro. This study provided further insight into the lignin metabolic pathway of R. opacus PD630 and could guide the design of an efficient biocatalytic system for lignin valorization.

Selective formate production from H2 and CO2 using encapsulated whole-cells under mild reaction conditions

Biocatalytic CO2 reduction into formate is a crucial strategy for developing clean energy because formate is considered as one of the promising hydrogen storage materials for achieving net-zero carbon emissions. Here, we developed an efficient biocatalytic system to produce formate selectively by coupling two enzymatic activities of H2 oxidation and CO2 reduction using encapsulated bacterial cells of Citrobacter sp. S-77. The encapsulated whole-cell catalyst was made by living cells depositing into polyvinyl alcohol and gellan gum cross-linked by calcium ions to form hydrogel beads. Formate production using encapsulated cells was carried out under the resting state conditions in the gas mixture of H2/CO2 (70:30, v/v%). The whole-cell biocatalyst showed highly efficient and selective catalytic production of formate, reaching the specific rate of formate production of 110mmolL-1· gprotein-1·h-1 at 30°C, pH 7.0, and 0.1MPa. The encapsulated cells can be reused at least 8 times while keeping their high catalytic activities for formate production under mild reaction conditions.

Role of catalyst supports in biocatalysis

AbstractBiocatalysis utilizes enzymes and microbial cells as catalysts for a wide range of applications in biotechnology. Immobilization of biocatalysts on various materials has several advantages, including the capacity for reuse, quick reaction termination, easy biocatalyst recovery and operational stability. The present article focuses on the use of material supports for developing immobilized biocatalysts in applications related to energy, environment and chemical synthesis. The work provides a comprehensive overview of a broad class of materials, including organic, inorganic and composites, that have been shown to be prosperous candidates to support the immobilization of enzymes and microbial cells. It also highlights the properties of nanomaterial support such as large surface area and comfort compartment for immobilization. The availability of different types of materials as catalyst support provides an opportunity to understand and develop efficient biocatalytic systems. The choice of selecting a catalyst support will mostly depend on the interaction of the material with the enzyme or microbial cell. Finally, potential challenges, future approaches in developing immobilized biocatalytic systems for various applications and novel material supports are suggested. © 2022 Society of Chemical Industry (SCI).

Efficient Biocatalytic System for Biosensing by Combining Metal-Organic Framework (MOF)-Based Nanozymes and G-Quadruplex (G4)-DNAzymes.

A high catalytic efficiency associated with a robust chemical structure are among the ultimate goals when developing new biocatalytic systems for biosensing applications. To get ever closer to these goals, we report here on a combination of metal-organic framework (MOF)-based nanozymes and a G-quadruplex (G4)-based catalytic system known as G4-DNAzyme. This approach aims at combining the advantages of both partners (chiefly, the robustness of the former and the modularity of the latter). To this end, we used MIL-53(Fe) MOF and linked it covalently to a G4-forming sequence (F3TC), itself covalently linked to its cofactor hemin. The resulting complex (referred to as MIL-53(Fe)/G4-hemin) exhibited exquisite peroxidase-mimicking oxidation activity and an excellent robustness (being stored in water for weeks). These properties were exploited to devise a new biosensing system based on a cascade of reactions catalyzed by the nanozyme (ABTS oxidation) and an enzyme, the alkaline phosphatase (or ALP, ascorbic acid 2-phosphate dephosphorylation). The product of the latter poisoning the former, we thus designed a biosensor for ALP (a marker of bone diseases and cancers), with a very low limit of detection (LOD, 0.02 U L-1), which is operative in human plasma samples.

Implementation of Biocatalysis in Continuous Flow for the Synthesis of Small Cyclic Amines.

Significant progress has been made in establishing transaminases as robust biocatalysts for the green and scalable synthesis of a diverse range of chiral amines. However, very few examples on the amination of small cyclic ketones have been reported. Cyclic ketones are particularly challenging for transaminase enzymes because they do not display the well-defined small and large substituent areas that are characteristic for the bio- catalytic mechanism. In this work, we exploited the broad substrate scope of the (S)-selective transaminase from Halomonas elongata (HeWT) to develop an efficient biocatalytic system in continuous flow to generate a range of small cyclic amines which feature very often in pharmaceuticals and agrochemicals. [3] Tetrahydrofuran-3-one and other challenging prochiral ketones were rapidly (5-45 min) transformed to their corresponding amines with excellent molar conversion (94-99%) and moderate to excellent ee.

Enhancement of interfacial catalysis in a triphase reactor using oxygen nanocarriers

Multiphase catalysis is used in many industrial processes; however, the reaction rate can be restricted by the low accessibility of gaseous reactants to the catalysts in water, especially for oxygen-dependent biocatalytic reactions. Despite the fact that solubility and diffusion rates of oxygen in many liquids (such as perfluorocarbon) are much higher than in water, multiphase reactions with a second liquid phase are still difficult to conduct, because the interaction efficiency between immiscible phases is extremely low. Herein, we report an efficient triphase biocatalytic system using oil core–silica shell oxygen nanocarriers. Such design offers the biocatalytic system an extremely large water-solid-oil triphase interfacial area and a short path required for oxygen diffusion. Moreover, the silica shell stabilizes the oil nanodroplets in water and prevents their aggregation. Using oxygen-dependent oxidase enzymatic reaction as an example, we demonstrate this efficient biocatalytic system for the oxidation of glucose, choline, lactate, and sucrose by substituting their corresponding oxidase counterparts. A rate enhancement by a factor of 10–30 is observed when the oxygen nanocarriers are introduced into reaction system. This strategy offers the opportunity to enhance the efficiency of other gaseous reactants involved in multiphase catalytic reactions.

Green and scalable synthesis of chiral aromatic alcohols through an efficient biocatalytic system.

SummaryChiral aromatic alcohols have received much attention due to their widespread use in pharmaceutical industries. In the asymmetric synthesis processes, the excellent performance of alcohol dehydrogenase makes it a good choice for biocatalysts. In this study, a novel and robust medium‐chain alcohol dehydrogenase RhADH from Rhodococcus R6 was discovered and used to catalyse the asymmetric reduction of aromatic ketones to chiral aromatic alcohols. The reduction of 2‐hydroxyacetophenone (2‐HAP) to (R)‐(‐)‐1‐phenyl‐1,2‐ethanediol ((R)‐PED) was chosen as a template to evaluate its catalytic activity. A specific activity of 110 U mg−1 and a 99% purity of e.e. was achieved in the presence of NADH. An efficient bienzyme‐coupled catalytic system (RhADH and formate dehydrogenase, CpFDH) was established using a two‐phase strategy (dibutyl phthalate and buffer), which highly raised the tolerated substrate concentration (60 g l−1). Besides, a broad range of aromatic ketones were enantioselectively reduced to the corresponding chiral alcohols by this enzyme system with highly enantioselectivity. This system is of the potential to be applied at a commercial scale.

A multicomponent biocatalyst for vegetable feedstock processing

he improvement of technologies for the production of foodstuffs and dietary supplements from vegetable feedstock using natural and efficient biocatalytic systems is a promising direction in the development of agroindustrial complex. At present, enzyme preparations produced in Russia are virtually absent at the market; therewith, high cost of their imported analogs increases the prime cost of products. A study on efficiency of the domestic broad-spectrum enzyme preparation Glucanofoetidin, which was developed at the Russian Scientific Research Institute of Food Biotechnology, toward fruit and berry feedstock and corn showed an increase in the yield of extractive substances (amino acids, sugars, phenol compounds, and carotenoids), particularly the biologically active components (tocopherols, anthocyanins, and vitamins), by 7–18 % as well as the quality improvement and an increase in the yield of target products (juices and extracts) by 10–44 %. Practical importance of Glucanofoetidin consists in a more rational use of vegetable feedstock as compared to conventional technology without enzymatic treatment, and also in a considerable (twofold) shortening of the process time. The multienzyme composition of Glucanofoetidin makes it promising for use in juice, confectionary, brewing, oil-and-fat and baking industries.

Development of an efficient biocatalytic system based on bacterial laccase for the oxidation of selected 1,4-dihydropyridines

Biocatalytic oxidations mediated by laccases are gaining importance due to their versatility and beneficial environmental effects. In this study, the oxidation of 1,4-dihydropyridines has been performed using three different types of bacterial laccase-based catalysts: purified laccase from Bacillus licheniformis ATCC 9945a (BliLacc), Escherichia coli whole cells expressing this laccase, and bacterial nanocellulose (BNC) supported BliLacc catalysts. The catalysts based on bacterial laccase were compared to the commercially available Trametes versicolor laccase (TvLacc). The oxidation product of 2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate was obtained within 7–24 h with good yields (70–99%) with all three biocatalysts. The substrate scope was examined with five additional 1,4-dihydropyridines, one of which was oxidized in high yield. Whole-cell biocatalyst was stable when stored for up to 1-month at 4 °C. In addition, evidence has been provided that multicopper oxidase CueO from the E. coli expression host contributed to the oxidation efficiency of the whole-cell biocatalyst. The immobilized whole-cell biocatalyst showed satisfactory activity and retained 37% of its original activity after three biotransformation cycles.

Asp305Gly mutation improved the activity and stability of the styrene monooxygenase for efficient epoxide production in Pseudomonas putida KT2440

BackgroundStyrene monooxygenase (SMO) catalyzes the first step of aromatic alkene degradation yielding the corresponding epoxides. Because of its broad spectrum of substrates, the enzyme harbors a great potential for an application in medicine and chemical industries.ResultsIn this study, we achieved higher enzymatic activity and better stability towards styrene by enlarging the ligand entrance tunnel and improving the hydrophobicity through error-prone PCR and site-saturation mutagenesis. It was found that Asp305 (D305) hindered the entrance of the FAD cofactor according to the model analysis. Therefore, substitution with amino acids possessing shorter side chains, like glycine, opened the entrance tunnel and resulted in up to 2.7 times higher activity compared to the wild-type enzyme. The half-lives of thermal inactivation for the variant D305G at 60 °C was 28.9 h compared to only 3.2 h of the wild type SMO. Moreover, overexpression of SMO in Pseudomonas putida KT2440 with NADH regeneration was carried out in order to improve biotransformation efficiency for epoxide production. A hexadecane/buffer (v/v) biphasic system was applied in order to minimize the inactivation effect of high substrate concentrations on the SMO enzyme. Finally, SMO activities of 190 U/g CDW were measured and a total amount of 20.5 mM (S)-styrene oxide were obtained after 8 h.ConclusionsThis study offers an alternative strategy for improved SMO expression and provides an efficient biocatalytic system for epoxide production via engineering the entrance tunnel of the enzyme’s active site.

Thiographene synthesized from fluorographene via xanthogenate with immobilized enzymes for environmental remediation.

Graphene, graphene oxide and their related thiographene-, hydroxygraphene- or fluorographene-based materials have broad applications. We report on the thiol-functionalization of fluorographene via xanthogenate. Such thiographene contains 5.1 at% of sulphur in the form of thiol groups, which is the highest thiol content reported to date. Such tailored thiographene allows the immobilization of two types of enzymes. Here, we explore the functionalization of highly thiolated graphene with enzymes via physisorption or covalent linkage producing an important heterogeneous biocatalyst platform for wastewater treatment applications. Thiographene modified with a lipase from Mucor miehei can find utilization in lipid-rich wastewater treatment whereas the catalase-modified thiographene is intended for bioremediation applications. Upon increasing concentration of the thiol groups on graphene, protein loading of the catalase was increased by 16% and the ester bond cleavage activity of the thiographene-immobilized lipase was 129% that of the free lipase. We expect that such a highly active heterogeneous thiographene-based biocatalyst will find a use in water remediation applications.

Synthesis of Azobenzene Dyes Mediated by CotA Laccase.

An eco-friendly protocol for the synthesis of azobenzene dyes by oxidative coupling of primary aromatic amines is reported. As efficient biocatalytic systems, CotA laccase and CotA laccase/ABTS (2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)) enable the oxidation of various substituted anilines, in aqueous medium, ambient atmosphere and under mild reaction conditions of pH and temperature. A series of azobenzene dyes were prepared in good to excellent yields in an one-pot reaction. A mechanistic proposal for the formation of the azo derivatives is presented. Our strategy offers an alternative approach for the direct synthesis of azobenzene dyes, avoiding the harsh conditions generally required for most of the traditional synthetic methods.

Concanavalin A induced orientation immobilization of Nuclease P 1 : The effect of lectin agglutination

Abstract Orientation immobilization of enzymes has attracted intensive interest owing to the retainable specific activity and stability. Specially, glycoprotein immobilized onto Concanavalin A (Con A) modified carriers induces the orientation of the enzyme. However, the effects of the interface properties of carriers and enzymes are still not well understood yet. In this study, we synthesized the activated porous poly (styrene- divinylbenzene) resin carriers with 30 nm pore sizes and 72 m2 g−1 specific surface areas and decorated with Con A. The resultant loading capacity of NP1 on Con A modified carriers was as high as 4.02 mg g−1 wet support as a result of strong affinity between the enzyme and Con A decorated on carriers. It was found that the acid resistance, thermal stability, reusability and degradation efficiency of the immobilized enzyme on Con A modified porous carriers were significantly improved. The reduction of Km from 18.40 ± 0.55 mg mL−1 to 17.19 ± 0.51 mg mL−1 illustrated the improved substrate affinity of HA-GA-ConA-NP1. Moreover, Con A-affinity NP1 exhibited the best operational stability that only 7% of its initial activity was lost even after 9 batches repeated reaction. This work demonstrates that surface property manipulation of porous carriers and its derivatives has great potential in efficient biocatalytic systems.

Intensifying the O2-dependent heterogeneous biocatalysis: Superoxygenation of solid support from H2O2 by a catalase tailor-made for effective immobilization

Besides merely destroying H2O2, an important use of the catalase reaction, H2O2→1/2 O2+H2O, is to supply O2 to oxygenation reactions. Due to convenient spatiotemporal control over O2 release, oxygenation from H2O2 is useful in particular for enzymatic reactions confined to solid supports. Because commercial catalases are difficult to immobilize, we have developed a one-step procedure of purification and immobilization of Bordetella pertussis catalase, recombinantly produced in Escherichia coli. Fusion of the catalase to a positively charged binding module enabled effective immobilization of the chimeric enzyme on anionic support (Relisorb SP 400), giving a controllable activity loading of between 5000 and 100,000 units/g support. Use of the immobilized catalase in combination with H2O2 feeding provided O2 to the reaction of glucose oxidase in solution for a range of volumetric conversion rates (0.2–1.5mM/min). Using optical sensing to measure the O2 concentration in the liquid but also in the solid phase, we showed that internal superoxygenation of the support was made possible under these conditions, resulting in an inverted (that is, negative) O2 concentration gradient between the bulk and the particle and allowing the internal O2 concentration to exceed by up to 4-fold the limit of atmospheric-pressure air saturation in solution. By tailored immobilization of B. pertussis catalase, therefore, an efficient biocatalytic system for hydrogen peroxide conversion in porous solid support was developed. This could find application for bubble-free oxygenation of O2-dependent enzymes co-immobilized with the catalase whereby enhanced internal availability of O2 would contribute to biocatalytic reaction intensification.

Efficient nanobiocatalytic systems of nuclease P1 immobilized on PEG-NH2 modified graphene oxide: effects of interface property heterogeneity

The use of graphene oxide (GO) nanosheets for functional enzyme support has attracted intensive interest owing to their unique planar structure and intriguing physical and chemical properties. However, the detailed effects of the interface properties of GO and its functionalized derivatives on active biomolecules are not well understood. We immobilize nuclease P1, a common industrial nucleic acid production enzyme, on pristine and amino poly(ethylene glycol) (PEG-NH2) modified GO nanosheets with interface property heterogeneity using two approaches, physical adsorption and chemical crosslinking. It is demonstrated that nuclease P1 could be stable immobilized on the surface of pristine GO by physical adsorption and on the edge of modified GO nanosheets by chemical crosslinking. The resultant loading capacity of nuclease P1 on pristine GO is as high as 6.45mg/mg as a consequence of strong electrostatic and hydrophobic interactions between the enzyme and carrier. However, it is determined that the acid resistance, thermal stability, reusability and degradation efficiency of the immobilized enzyme on PEG-NH2-modified GO are obviously improved compared to those of the enzyme immobilized on pristine GO. The enhanced catalytic behavior demonstrates that GO and its derivatives have great potential in efficient biocatalytic systems.

Stability, Scalability, and Reusability of a Volume Efficient Biocatalytic System Constructed on Magnetic Nanoparticles.

This report investigates for the first time stability, scalability, and reusability characteristics of a protein nano-bioreactor useful for green synthesis of fine chemicals in aqueous medium extracting maximum enzyme efficiency. Enzyme catalysts conjugated with magnetic nanomaterials allow easy product isolation after a reaction involving simple application of a magnetic field. In this study, we examined a biocatalytic system made of peroxidase-like myoglobin (Mb), as a model protein, to covalently conjugate with poly(acrylic acid) functionalized magnetic nanoparticles (MNPs, 100 nm hydrodynamic diameter) to examine the catalytic stability, scalability, and reusability features of this bioconjugate. Application of the conjugate was effective for electrochemical reduction of organic and inorganic peroxides, and for both peroxide-mediated and electrocatalytic oxidation of the protein substrate 2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) with greater turnover rates and product yields than Mb prepared in solution or MNP alone. Mb-attached MNPs displayed extensive catalytic stability even after 4 months of storage compared to Mb present in solution. Five- and ten-fold scale up of MNPs in the bioconjugates resulted in two- and four-fold increases in protein-catalyzed oxidation products, respectively. Nearly 40% of the initial product was present even after four reuses, which is advantageous for synthesizing sufficient products with a minimal investment of precious enzymes. Thus, the results obtained in this study are highly significant in guiding cost-effective development and efficient multiple uses of enzyme catalysts for biocatalytic, electrocatalytic, and biosensing applications via magnetic nanomaterials conjugation.

An efficient biocatalytic system for floxuridine biosynthesis based on Lactobacillus animalis ATCC 35046 immobilized in Sr-alginate

An efficient and green immobilized biocatalyst is herein reported to obtain 5-fluorouracil-2′-deoxyriboside (5FUradRib), an antimetabolite known as Floxuridine, used in gastrointestinal cancer treatment.Alginate is a natural polysaccharide used in the pharmaceutical industry due to its physicochemical properties, biocompatibility and non-toxicity. Multivalent cations, exposure time and cross-linking solution concentration were optimized, being Sr2+, 2h and 0.2M the best immobilization conditions. Furthermore, compression strength, swelling ratio and fracture frequency were evaluated, improving the mechanical stability of the biocatalyst favoring a future scale-up.On the other hand, the reaction parameters for 5FUradRib biosynthesis were optimized in order to obtain an immobilized biocatalyst with enhanced activity. Thus, Lactobacillus animalis ATCC 35046 immobilized in Sr-alginate showed yields of 96% at short reaction times.The obtained biocatalyst was stable for more than 25 days in storage conditions (4°C) and could be reused at least 10 times without loss of its activity.Additionally, Sr-alginate biocatalyst stability was evaluated in different organic solvents to obtain hydrophobic compounds such as 5-bromouracil-2′-deoxyriboside (5BrUradRib), an effective radiosensitizing agent used in anti-cancer therapy, being hexane the best co-solvent.Finally, a smooth, cheap and environmentally friendly method to obtain anti-cancer drugs was developed in this study.

A cytochrome P450 class I electron transfer system from Novosphingobium aromaticivorans

Cytochrome P450 (CYP) enzymes of the CYP101 and CYP111 families from Novosphingobium aromaticivorans are heme monooxygenases that catalyze the hydroxylation of a range of terpenoid compounds. CYP101D1 and CYP101D2 oxidized camphor to 5-exo-hydroxycamphor. CYP101B1 and CYP101C1 oxidized beta-ionone to predominantly 3-R-hydroxy-beta-ionone and 4-hydroxy-beta-ionone, respectively. CYP111A2 oxidized linalool to 8-hydroxylinalool. Physiologically, these CYP enzymes could receive electrons from Arx, a [2Fe-2S] ferredoxin equivalent to putidaredoxin from the CYP101A1 system from Pseudomonas putida. A putative ferredoxin reductase (ArR) in the N. aromaticivorans genome, with high amino acid sequence homology to putidaredoxin reductase, has been over-produced in Escherichia coli and found to support substrate oxidation by these CYP enzymes via Arx with both high activity and coupling of product formation to NADH consumption. The ArR/Arx electron-transport chain has been co-expressed with the CYP enzymes in an E. coli host to provide in vivo whole-cell substrate oxidation systems that could produce up to 6.0 g L(-1) of 5-exo-hydroxycamphor at rates of up to 64 microM (gram of cell dry weight)(-1) min(-1). These efficient biocatalytic systems have potential uses in preparative scale whole-cell biotransformations.