Enzymatic Route Research Articles

Engineering a high-sugar tolerant strain of Saccharomyces cerevisiae for efficient trehalose production using a cell surface display approach

Trehalose production via a one-step enzymatic route using trehalose synthase (TreS) holds significant promise for industrial-scale applications due to its simplicity and utilization of low-cost substrates. However, the development of a robust whole-cell biocatalyst expressing TreS remains crucial for enabling practical and economically viable production. In this study, a high-sugar tolerant strain of S. cerevisiae was screened and employed as a host cell for the cell surface display of TreS from Acidiplasma aeolicum. The resultant strain, S. cerevisiae I3A, exhibited remarkable surface displayed TreS activity of 3358 U/g CDW and achieved approximately 64% trehalose yield (10.8 g/L/h productivity) from maltose. Interestingly, no glucose by-product was observed during trehalose production. The S. cerevisiae I3A cells exhibited reusability for up to 12 cycles leading to potential cost reduction of trehalose products. Therefore, our study demonstrated the development of a high-sugar tolerant S. cerevisiae strain expressing TreS on its surface as a whole-cell biocatalyst for efficient and economical trehalose production with potential applications in the food and pharmaceutical industries.

Lactose hydrolysis in sheep and cow milk with varying fat levels to evaluate enzymatic production of lactulose

AbstractThis study aimed to perform lactose hydrolysis using free and immobilised β-galactosidase in cow and sheep milk with different fat levels, followed by lactulose synthesis through the isomerisation of glucose to fructose using immobilised glucose isomerase. There was no significant difference (P > 0.05) in lactose hydrolysis (>95%) using the free enzyme in the different types of milk. In contrast, significant differences were observed for the hydrolysis of whole cow milk (WCM) and skimmed cow milk (SCM) with the immobilised enzyme (IE). WCM required a longer hydrolysis time (43 h). Thus, the fat content may have affected the lactose hydrolysis of bovine milk with immobilised β-galactosidase. No difference was observed for whole sheep milk and skimmed sheep milk hydrolysed by IE, possibly due to the presence of smaller fat globules, which did not affect the lactose hydrolysis. No significant differences were observed for lactulose synthesis in whole milk from the different species evaluated. The lactulose synthesis in cow and sheep milk by the enzymatic route may be a promising alternative, though further studies are required to optimise the production of this prebiotic in situ.

Enzymatic Routes to Designer Hemicelluloses for Use in Biobased Materials

Enzymatic Routes to Designer Hemicelluloses for Use in Biobased Materials

Enzymatic synthesis of azide by a promiscuous N-nitrosylase.

Azides are energy-rich compounds with diverse representation in a broad range of scientific disciplines, including material science, synthetic chemistry, pharmaceutical science and chemical biology. Despite ubiquitous usage of the azido group, the underlying biosynthetic pathways for its formation remain largely unknown. Here we report the characterization of an enzymatic route for de novo azide construction. We demonstrate that Tri17, a promiscuous ATP- and nitrite-dependent enzyme, catalyses organic azide synthesis through sequential N-nitrosation and dehydration of aryl hydrazines. Through biochemical, structural and computational analyses, we further propose a plausible molecular mechanism for azide synthesis that sets the stage for future biocatalytic applications and biosynthetic pathway engineering.

Evaluating the removal of the tetra-azo dye direct black-22 in Chlorella vulgaris closed-cultivation systems

ABSTRACT The removal of the tetra-azo dye Direct Black 22 (DB22) using the microalga Chlorella vulgaris was evaluated in the present study, aiming to understand the contribution of different processes (biodegradation, photodegradation, and adsorption) in the removal of this contaminant. The growth and morphological characteristics of C. vulgaris were not affected by the presence of the dye in the reaction medium. The efficiency of dye removal was 62.6 ± 1.46%, 47.7 ± 7.2% of which was attributed to photodegradation, while 13.2 ± 6.5% were associated with the contribution of the microalga by an enzymatic route and 1.7 ± 9.6% with an adsorption process. Additionally, tests with the organism Allium cepa as a bioindicator revealed that DB22 and its byproducts did not induce toxicity, but cytotoxicity and genotoxicity were induced. We observed that genotoxicity was reduced after the remediation process. Our results establish photodegradation as the primary mechanism and biodegradation as the secondary mechanism driving the removal of DB22 within a Chlorella culture. Researchers must carefully consider all aspects involved in the removal process, including photodegradation, biodegradation, and adsorption processes.

An investigation of the enzymatic oligomerization of nitro-substituted phenylene diamine: Thermal and fluorescence properties

2-nitro-p-phenylenediamine, an aromatic diamine, was studied for its oxidative oligomerization with H2O2 using enzyme-catalyzed oligomer synthesis. Characterization of molecular structures was performed utilizing ultraviolet-visible (UV-Vis) spectrophotometer, Fourier-transform infrared spectroscopy (FT-IR), and nuclear magnetic resonance (NMR) techniques, identifying phenazine-bridged oligomer resulting from the enzymatic oligomerization process. Based on the results of gel permeation chromatography (GPC) analysis, the synthesized compound was identified as being in an oligomeric form. Conversely, the number of repeating units, as determined by Mw, was found to be 28. The solvent effect on the optical features of the synthesized oligomer in polar solvents was analyzed. The degradation of phenazine-type structures in the oligomer occurred at higher temperatures than that of the monomer. Under visible light excitation, the oligomer exhibited green light emission with a quantum yield (QY) of 6.2% in N,N-dimethylformamide (DMF). 2-nitro-p-phenylenediamine was readily oxidized into an oligomer with ortho-coupled constitutional units, having a lower electrochemical band gap than the monomer, via the enzymatic oligomerization route. Scanning electron microscopy revealed that enzyme-catalyzed oxidation of monomers exhibited a spongy morphology with some pores

Chemical and enzymatic hydrolysis of alginate: a review

Abstract Alginate is a brown seaweed-based linear polysaccharide of D – mannuronic acid and L – guluronic acid residues. Hydrolysis products of the polysaccharide, specifically oligosaccharides, have been receiving increasing interest, due to their significant bioactivity and potential utilisation routes. The bioactivity of alginate oligosaccharides is closely linked to structural characteristics, namely: molecular weight, degree of polymerisation, and ratio of the monomers (the M/G ratio). Hence, potential applications (such as utilisation as a biostimulant fertilizer) depend on these parameters. This review focuses on recent advances in producing alginate oligosaccharides using chemical or enzymatic methods. The literature survey includes utilisation of these methods at both laboratory and industrial scale. For the chemical methods, we assessed the standard laboratory scale procedures of alginate oligosaccharide production, the potential of scaling up to an industrial level, and the subsequent challenges. For the enzymatic route, we provide an overview of alginate lyases and the application perspectives of enzymatic hydrolysis of alginate.

Novel enzymatic route to the synthesis of C-8 hydroxyflavonoids including flavonols and isoflavones

Flavin-dependent monooxygenases (FMOs) are a valuable group of biocatalysts that can regioselectively introduce a hydroxy group for the targeted modification of biologically active compounds. Here, we present the fdeE, the FMO from Herbaspirillum seropedicae SmR1 that is a part of the naringenin degradation pathway and is active towards a wide range of flavonoids—flavanones, flavones, isoflavones, and flavonols. Bioinformatics and biochemical analysis revealed a high similarity between the analyzed enzyme and other F8H FMOs what might indicate convergent evolutionary mechanism of flavonoid degradation pathway emergence by microorganism. A simple approach with the manipulation of the reaction environment allowed the stable formation of hydroxylation products, which showed very high reactivity in both in vivo and in vitro assays. This approach resulted in an 8-hydroxyquercetin—gossypetin titer of 0.16 g/L and additionally it is a first report of production of this compound.

An Enzymatic Prodrug-like Route to Thio and Selenoamides.

6-Thioguanine (6TG) is a clinically used antitumor agent that was rationally designed as a DNA-targeting antimetabolite, but it also occurs naturally. 6TG is a critical virulence factor produced by Erwinia amylovorans, a notorious plant pathogen that causes fire blight of pome fruit trees. The biosynthesis of the rare thioamide metabolite involves an adenylating enzyme (YcfA) and a sulfur-mobilizing enzyme (YcfC), but the mechanism of sulfur transfer and putative intermediates have remained elusive. Through dissection and in vitro reconstitution of the thionation process using diverse substrates, we uncover an intermediate, prodrug-like thio-conjugate and elucidate the precise enzyme functions. YcfA not only adenylates GMP but also transfers the mercapto group of l-cysteine to the activated carbonyl. A designated C-S lyase (YcfC) then cleaves the resulting S-adduct to yield the thioamide. This pathway is distinct from canonical tRNA sulfur modifications and known enzymatic peptide thionations. By exploring a wide range of substrate surrogates, we exploited the tolerance of the enzyme pair to produce even a seleno analog. This study provides valuable insight into a previously unexplored area of bacterial thioamide formation and lays the groundwork for synthetic biology approaches to produce thioamide antimetabolites.

Enzymatic Routes for Chiral Amine Synthesis: Protein Engineering and Process Optimization.

Chiral amines are essential motifs in pharmaceuticals, agrochemicals, and specialty chemicals. While traditional chemical routes to chiral amines often lack stereoselectivity and require harsh conditions, biocatalytic methods using engineered enzymes can offer high efficiency and selectivity under sustainable conditions. This review discusses recent advances in protein engineering of transaminases, oxidases, and other enzymes to improve catalytic performance. Strategies such as directed evolution, immobilization, and computational redesign have expanded substrate scope and enhanced efficiency. Furthermore, process optimization guided by techno-economic assessments has been crucial for establishing viable biomanufacturing routes. Combining state-of-the-art enzyme engineering with multifaceted process development will enable scalable, economical enzymatic synthesis of diverse chiral amine targets.

Valorizing fungal diversity for the degradation of fluoroquinolones

Continued widespread use of antibiotics, especially fluoroquinolones, raises environmental concerns, as its driving bacterial resistance and disrupts microbial ecosystems. Here we investigate the biodegradation of ten fluoroquinolone antibiotics (six for medical use and four for veterinary use) by ligninolytic fungi, including Trametes versicolor, Bjerkandera adusta, Porosterum spadiceum, Irpex lacteus, Pleuroteus ostreatus, Phanerochaete chrysosporium, Pycnoporus cinnabarinus, Ganoderma lucidum, and Gloeophyllum trabeum. The results show significant variations between strains in the efficiency of antibiotic transformation. B. adusta and P. spadiceum were the fungi that most efficiently reduced antibiotic concentrations and were able to totally degrade eight and six antibiotics, respectively, within a 15-day period. T. versicolor and P. ostreatus also showed the ability to effectively degrade antibiotics. Specifically, T. versicolor degraded six out of the ten fluoroquinolone antibiotics by more than 70 %, while P. ostreatus degraded the tested antibiotics between 43 % and 100 %. The remaining antibiotic activity did not always correlate with a reduction in antibiotic concentrations, which points to the presence of post-transformation antimicrobial metabolites. This study also explores the potential mechanisms used by these fungi to remove selected models of fluroquinolones via enzymatic routes, such as oxidation by laccases, heme-peroxidases, and cytochrome P450, or via adsorption on fungal biomass.

Bacterial cellulose nanocrystals obtained through enzymatic and acidic routes: A comparative study of their main properties and in vitro biological responses

Cellulose nanocrystals (CNCs) are crystalline domains isolated from cellulosic fibers. They have been utilized in a wide range of applications, such as reinforcing fillers, antibacterial agents and manufacturing of biosensors. Whitin this context, the aim of this work was to obtain and analyze CNCs extracted from bacterial nanocellulose (BNC) using two distinct methods combined with milling pre-treatment: an acidic hydrolysis using 64 % sulfuric acid and an enzymatic hydrolysis using a commercial cellulase enzyme mixture. The CNCs obtained from the enzymatic route (e-CNCs) were observed to be spherical nanoparticles with diameter of 56 ± 11 nm. In contrast, the CNCs from the acid hydrolysis (a-CNCs) appeared as needle-shaped nanoparticles with a high aspect ratio with lengths/widths of 158 ± 64 nm/11 ± 2 nm. The surface zeta potential (ZP) of the a-CNCs was −30,8 mV, whereas the e-CNCs has a potential of +2.70 ± 3.32 mV, indicating that a-CNCs consisted of negatively charged particles with higher stability in solution. Although the acidic route resulted in nanocrystals with a slightly higher crystallinity index compared to the enzymatic route, e-CNCs was found to be more thermally stable than BNC and a-CNCs. Here, we also confirmed the safety of a-CNCs and e-CNCs using L929 cell line. Lastly, this article describes two different CNCs synthesis approaches that leads to the formation of nanoparticles with different dimensions, morphology and unique physicochemical properties. To the best of our knowledge, this is the first study to yield spherical nanoparticles as a result of BNC enzymatic treatment.

In situ immobilization of Cal B enzyme in mesoporous materials of the MCM-48 type using different ionic solids: synthesis and application in esterification reactions

The ionic solids [C16MI]Cl and [C14MI]Cl were used as structure directors on MCM-48 mesoporous support with immobilized CALB lipase for the synthesis of esters from geraniol. Significant results were obtained in the synthesis of geranyl acetate and geranyl butyrate. Enzyme concentration, temperature and molar ratio of substrates were parameters that affect the synthesis yield. The highest temperature (60°C) and highest enzyme concentration (13%) conditions showed the best ester conversion results. For both esters, the ionic liquid [C16MI]Cl showed better conversion results and reached a value above 38% of conversion. Furthermore, the support tested in the present study for application in ester synthesis is cost-effective. In addition, the enzymatic route to obtain the esters presents mild reaction conditions and the results show potential for application in industrial applications.

Whole-cell catalytic production of pseudouridine by recombinant Escherichia coli

Pseudouridine is the most abundant modified nucleoside found in non-coding RNA and is widely used in biological and pharmaceutical fields. However, current methods for pseudouridine production suffer from drawbacks such as complex procedures, low efficiency and high costs. This study presents a novel enzymatic cascade reaction route in Escherichia coli, enabling the whole-cell catalytic synthesis of pseudouridine from uridine. Initially, a metabolic pathway was established through plasmid-mediated overexpression of endogenous pseudouridine-5-phosphase glycosidase, ribokinase, and ribonucleoside hydrolase, resulting in the accumulation of pseudouridine. Subsequently, highly active endogenous ribonucleoside hydrolase was screened to enhance uridine hydrolysis and provide more precursors for pseudouridine synthesis. Furthermore, modifications were made to the substrates and products transport pathways to increase the pseudouridine yield while avoiding the accumulation of by-product uridine. The resulting recombinant strain Ψ-7 catalyzed the conversion of 30 g/L uridine into 27.24 g/L pseudouridine in 24 h, achieving a conversion rate of 90.8% and a production efficiency of 1.135 g/(L·h). These values represent the highest reported yield and production efficiency achieved by enzymatic catalysis methods to date.

Health benefits of fraxetin: From chemistry to medicine.

Fraxetin is a bioactive molecule present in various natural plants, especially Cortex Fraxini. Evidenced outcomes in phytochemical and biological analyses for this agent are now available in the literature, but an insightful review isyet unknown. The goal of the current research is to offer a panoramic illustration of natural observation, biosynthesis, synthesis, pharmacology, and pharmacokinetics for fraxetin. Esculetin and ferulic acid acted as precursors in the enzymatic biosynthetic route, whereas fraxetin could be easily synthesized from simple phenols. A great deal of interest was obtained in using this molecule for pharmacological targets. Herein, its pharmacological value included anticancer, antioxidative, anti-inflammatory, antidiabetic, antiobesity, and antimicrobial activities, as well as the protection of the liver, neurons, heart, bone, lung, kidney, and others. Anticancer activity may involve the inhibition of proliferation, invasion, and migration, together with apoptotic induction. Health benefits from this molecule were deduced from its ability to suppress cytokines and protect the immune syndrome. Various signaling pathways, such as Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3), phosphoinositide 3 kinase (PI3K)/protein kinase B (Akt), nuclear factor kappa B (NF-κB)/NLRP3, Akt/AMPK, have been proposed for in vitro and in vivo mechanisms of action. Fraxetin is highly distributed to rat plasma and several organs. However, more pharmacokinetic studies to improve its bioavailability are needed since its solubility in water is still limited.

An enzymatic route to the synthesis of tricyclic fused hexahydrofuranofuran P2-Ligand for a series of highly potent HIV-1 protease inhibitors

We describe a stereoselective synthesis of an optically active (1R, 3aS, 5R, 6S, 7aR)-octahydro-1,6-epoxy-isobenzo-furan-5-ol derivative. This stereochemically defined heterocycle serves as a high-affinity ligand for a variety of HIV-1 protease inhibitors. The key synthetic steps involve a highly enantioselective enzymatic desymmetrization of meso-1,2(dihydroxymethyl)cyclohex-4-ene and conversion of the resulting optically active alcohol to a methoxy hexahydroisobenzofuran derivative. A substrate controlled stereoselective dihydroxylation afforded syn-1,2-diols. Oxidation of diol provided the substituted 1,2-diketone and L-Selectride reduction provided the corresponding inverted syn-1,2-diols. Acid catalyzed cyclization furnished the ligand alcohol in optically active form.

The genomic and enzymatic basis for iridoid biosynthesis in cat thyme (Teucrium marum).

Iridoids are non-canonical monoterpenoids produced by both insects and plants. An example is the cat-attracting and insect-repelling volatile iridoid nepetalactone, produced by Nepeta sp. (catmint) and aphids. Recently, both nepetalactone biosynthetic pathways were elucidated, showing a remarkable convergent evolution. The iridoid, dolichodial, produced by Teucrium marum (cat thyme) and multiple insect species, has highly similar properties to nepetalactone but its biosynthetic origin remains unknown. We set out to determine the genomic, enzymatic, and evolutionary basis of iridoid biosynthesis in T. marum. First, we generated a de novo chromosome-scale genome assembly for T. marum using Oxford Nanopore Technologies long reads and proximity-by-ligation Hi-C reads. The 610.3 Mb assembly spans 15 pseudomolecules with a 32.9 Mb N50 scaffold size. This enabled identification of iridoid biosynthetic genes, whose roles were verified via activity assays. Phylogenomic analysis revealed that the evolutionary history of T. marum iridoid synthase, the iridoid scaffold-forming enzyme, is not orthologous to typical iridoid synthases but is derived from its conserved paralog. We discovered an enzymatic route from nepetalactol to diverse iridoids through the coupled activity of an iridoid oxidase cytochrome P450 and acetyltransferases, via an inferred acylated intermediate. This work provides a genomic resource for specialized metabolite research in mints and demonstration of the role of acetylation in T. marum iridoid diversity. This work will enable future biocatalytic or biosynthetic production of potent insect repellents, as well as comparative studies into iridoid biosynthesis in insects.

Cloning and expression heterologous alanine dehydrogenase genes: Investigation of reductive amination potential of L-alanine dehydrogenases for green synthesis of alanine derivatives

Unnatural amino acids (UAAs) offer significant promise in a wide range of applications, including drug discovery, the custom design of peptides and proteins, and their utility and use as markers for monitoring molecular interactions in biological research. The synthesis of UAAs presents a formidable challenge and can be classified into two primary categories: enzymatic and chemical synthesis. Notably, the enzymatic route, specifically asymmetric synthesis, emerges as a an attractive method for procuring enantiopure UAAs with high efficiency, owing to its streamlined and concise reaction mechanism.The current study investigated the reductive amination activity mechanisms of alanine dehydrogenase (L-AlaDH), sourced from a combination of newly and previously characterized microorganisms. Our principal aim was to evaluate the catalytic efficiency of these L-AlaDH enzymes concerning a range of specific ketoacids and pyruvate to ascertain their capability for facilitating the production of both natural and unnatural amino acids. After the characterization processes, mutation points for TtAlaDH were determined and as a result of the mutations, mutants that could use ketocaproate and ketovalerate more effectively than the wild type were obtained.Among the enzymes studied, MetAlaDH exhibited the highest specific activity against pyruvate, 173 U/mg, and a KM value of 1.3 mM. VlAlaDH displayed the most favourable catalytic efficiency with a rate constant of 170 s−1mM−1. On the other hand, AfAlaDH demonstrated the highest catalytic efficiency against α-ketobutyrate (34.0 s−1mM−1) and α-ketovalerate (2.7 s−1mM−1). Of the enzymes investigated in the study, TtAlaDH exhibited the highest effectiveness among bacterial enzymes in catalyzing ketocaproate with a measured catalytic efficiency of about 0.6 s−1mM−1 and a KM value of approximately 0.3 mM. These findings provide valuable insights into the substrate specificity and catalytic performance of L-AlaDHs, enhancing our understanding of their potential applications in various biocatalytic processes.

Biocatalytic cascade to polysaccharide amination

BackgroundChitin, the main form of aminated polysaccharide in nature, is a biocompatible, polycationic, and antimicrobial biopolymer used extensively in industrial processes. Despite the abundance of chitin, applications thereof are hampered by difficulties in feedstock harvesting and limited structural versatility. To address these problems, we proposed a two-step cascade employing carbohydrate oxidoreductases and amine transaminases for plant polysaccharide aminations via one-pot reactions. Using a galactose oxidase from Fusarium graminearum for oxidation, this study compared the performance of CvATA (from Chromobacterium violaceum) and SpATA (from Silicibacter pomeroyi) on a range of oxidized carbohydrates with various structures and sizes. Using a rational enzyme engineering approach, four point mutations were introduced on the SpATA surface, and their effects on enzyme activity were evaluated.ResultsHerein, a quantitative colorimetric assay was developed to enable simple and accurate time-course measurement of the yield of transamination reactions. With higher operational stability, SpATA produced higher product yields in 36 h reactions despite its lower initial activity. Successful amination of oxidized galactomannan by SpATA was confirmed using a deuterium labeling method; higher aminated carbohydrate yields achieved with SpATA compared to CvATA were verified using HPLC and XPS. By balancing the oxidase and transaminase loadings, improved operating conditions were identified where the side product formation was largely suppressed without negatively impacting the product yield. SpATA mutants with multiple alanine substitutions besides E407A showed improved product yield. The E407A mutation reduced SpATA activity substantially, supporting its predicted role in maintaining the dimeric enzyme structure.ConclusionsUsing oxidase–amine transaminase cascades, the study demonstrated a fully enzymatic route to polysaccharide amination. Although the activity of SpATA may be further improved via enzyme engineering, the low operational stability of characterized amine transaminases, as a result of low retention of PMP cofactors, was identified as a key factor limiting the yield of the designed cascade. To increase the process feasibility, future efforts to engineer improved SpATA variants should focus on improving the cofactor affinity, and thus the operational stability of the enzyme.Graphical

Mass spectrometric methods for evaluation of voriconazole avian pharmacokinetics and the inhibition of its cytochrome P450-induced metabolism

Invasive fungal aspergillosis is a leading cause of morbidity and mortality in many species including avian species such as common ravens (Corvus corax). Methods were developed for mass spectral determination of voriconazole in raven plasma as a means of determining pharmacokinetics of this antifungal agent. Without further development, GC/MS/MS (gas chromatography-tandem quadrupole mass spectrometry) proved to be inferior to LC/MS/MS (liquid chromatography-tandem quadrupole mass spectrometry) for measurement of voriconazole levels in treated raven plasma owing to numerous heat-induced breakdown products despite protection of voriconazole functional groups with trimethylsilyl moieties. LC/MS/MS measurement revealed in multi-dosing experiments that the ravens were capable of rapid or ultrarapid metabolism of voriconazole. This accounted for the animals’ inability to raise the drug into the therapeutic range regardless of dosing regimen unless cytochrome P450 (CYP) inhibitors were included. Strategic selection of CYP inhibitors showed that of four selected compounds including cimetidine, enrofloxacin and omeprazole, only ciprofloxacin (Cipro) was able to maintain voriconazole levels in the therapeutic range until the end of the dosing period. The optimal method of administration involved maintenance doses of voriconazole at 6 mg/kg and ciprofloxacin at 20 mg/kg. Higher doses of voriconazole such as 18 mg/kg were also tenable without apparent induction of toxicity. Although most species employ CYP2C19 to metabolize voriconazole, it was necessary to speculate that voriconazole might be subject to metabolism by CYP1A2 in the ravens to explain the utility of ciprofloxacin, a previously unknown enzymatic route. Finally, despite its widespread catalog of CYP inhibitions including CYP1A2 and CYP2C19, cimetidine may be inadequate at enhancing voriconazole levels owing to its known effects on raising gastric pH, a result that may limit voriconazole solubility.