RESUMO
Enzymes play a pivotal role in the human body, but their potential is not limited to just that. Scientists have successfully modified these enzymes as nanobiocatalysts or nanozymes for industrial or commercial use, either in the food, medicine, biotech or even textile industries. These nanobiocatalysts and nanozymes offer several advantages over enzymes, like better stability, improved shelf-life, increased percentage yield, and reuse potential, which is very difficult with normal enzymes. The various techniques of NBC synthesis using immobilization techniques like adsorption, covalent binding, affinity immobilization, and entrapment methods are briefly discussed. The enzymes are either entrapped or adsorbed on the nanocarrier matrices, which can be nanofibers, nanoporous carriers, or nanocontainers as nanobiocatalysts. We also highlight the challenges the nanobiocatalyst overcomes in the industrial production of some drugs like sitagliptin, montelukast, pregabalin, and atorvastatin. Also, the inactivation of an organophosphate or opioid poisoning treating agent, SSOPOX nanohybrid, is discussed in this paper. Nanozymes are intrinsic enzyme-like compounds, and they also show wide application in themselves. Their GQD/AGNP nanohybrid shows antibacterial potential; they can also be utilized in optical sensing to detect small molecules, ions, nucleic acids, proteins, and cancer cells. In this paper, various applications of these NBCs have been discussed, and their potential applications with examples are also mentioned. Nanoenzymes can address targeted drug delivery via the controlled release of drugs to increase the efficacy of anticancer drugs that minimize damage to healthy tissue or cells.
Assuntos
Desenvolvimento de Medicamentos , Humanos , Desenvolvimento de Medicamentos/métodos , Biocatálise , Enzimas Imobilizadas/química , Técnicas Biossensoriais/métodos , Animais , Nanopartículas/química , Nanotecnologia/métodos , Portadores de Fármacos/química , Nanoestruturas/químicaRESUMO
The development and manufacture of high-quality starch are a new research focus in food science. Here, transglutaminase was used in the wet processing of glutinous rice flour to prepare customized sweet dumplings. Transglutaminase (0.2 %) lowered protein loss in wet processing and reduced the crystallinity and viscosity of glutinous rice flour. Moreover, it lowered the cracking and cooking loss of sweet dumplings after freeze-thaw cycles, and produced sweet dumplings with reduced hardness and viscosity, making them more suitable for people with swallowing difficulties. Additionally, in sweet dumplings with 0.2 % transglutaminase, the encapsulation of starch granules by the protein slowed down the digestion and reduced the final hydrolysis rate, which are beneficial for people with weight and glycemic control issues. In conclusion, this study contributes to the production of tasty, customized sweet dumplings.
Assuntos
Digestão , Farinha , Oryza , Amido , Transglutaminases , Oryza/química , Oryza/metabolismo , Transglutaminases/metabolismo , Transglutaminases/química , Farinha/análise , Amido/química , Amido/metabolismo , Manipulação de Alimentos , Humanos , Viscosidade , Culinária , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , BiocatáliseRESUMO
Biocatalytic degradation of non-hydrolyzable plastics is a rapidly growing field of research, driven by the global accumulation of waste. Enzymes capable of cleaving the carbon-carbon bonds in synthetic polymers are highly sought-after as they may provide tools for environmentally friendly plastic recycling. Despite some reports of oxidative enzymes acting on non-hydrolyzable plastics, including polyethylene or poly(vinyl chloride), the notion that these materials are susceptible to efficient enzymatic degradation remains controversial, partly driven by a general lack of studies independently reproducing previous observations. Here, we attempt to replicate two recent studies reporting that deconstruction of polyethylene and poly(vinyl chloride) can be achieved using an insect hexamerin from Galleria mellonella (so-called "Ceres") or a bacterial catalase-peroxidase from Klebsiella sp., respectively. Reproducing previously described experiments, we do not observe any activity on plastics using multiple reaction conditions and multiple substrate types. Digging deeper into the discrepancies between the previous data and our observations, we show how and why the original experimental results may have been misinterpreted.
Assuntos
Biodegradação Ambiental , Klebsiella , Polietileno , Cloreto de Polivinila , Polietileno/metabolismo , Polietileno/química , Animais , Cloreto de Polivinila/química , Cloreto de Polivinila/metabolismo , Klebsiella/enzimologia , Klebsiella/metabolismo , Catalase/metabolismo , Catalase/química , Proteínas de Insetos/metabolismo , Proteínas de Insetos/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , BiocatáliseRESUMO
Nonheme iron enzymes are versatile biocatalysts for a broad range of unique and powerful transformations, such as hydroxylation, chlorination, and epimerization as well as cyclization/ring-opening of organic molecules. Beyond their native biological functions, these enzymes are robust for engineering due to their structural diversity and high evolvability. Based on enzyme promiscuity and directed evolution as well as inspired by synthetic organic chemistry, nonheme iron enzymes can be repurposed to catalyze reactions previously only accessible with synthetic catalysts. To this end, our group has engineered a series of nonheme iron enzymes to employ non-natural radical-relay mechanisms for new-to-nature radical transformations. In particular, we have demonstrated that a nonheme iron enzyme, (4-hydroxyphenyl)pyruvate dioxygenase from streptomyces avermitilis (SavHppD), can be repurposed to enable abiological radical-relay process to access C(sp3)-H azidation products. This represents the first known instance of enzymatic radical relay azidation reactions. In this chapter, we describe the detailed experimental protocol to convert promiscuous nonheme iron enzymes into efficient and selective biocatalyst for radical relay azidation reactions. One round of directed evolution is described in detail, which includes the generation and handling of site-saturation mutagenesis, protein expression and whole-cell reactions screening in a 96-well plate. These protocol details might be useful to engineer various nonheme iron enzymes for other applications.
Assuntos
Biocatálise , Engenharia de Proteínas , Streptomyces , Engenharia de Proteínas/métodos , Streptomyces/enzimologia , Streptomyces/genética , Ferroproteínas não Heme/química , Ferroproteínas não Heme/metabolismo , Ferroproteínas não Heme/genética , 4-Hidroxifenilpiruvato Dioxigenase/genética , 4-Hidroxifenilpiruvato Dioxigenase/metabolismo , 4-Hidroxifenilpiruvato Dioxigenase/química , Azidas/química , Azidas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismoRESUMO
Rieske non-heme iron oxygenases (ROs) possess the ability to catalyze a wide range of reactions. Their ability to degrade aromatic compounds is a unique characteristic and makes ROs interesting for a variety of potential applications. However, purified ROs can be challenging to work with due to low stability and long, complex electron transport chains. Whole cell biocatalysis represents a quick and reliable method for characterizing the activity of ROs and harnessing their metabolic potential. In this protocol, we outline a step-by-step protocol for the overexpression of ROs for whole cell biocatalysis and characterization. We have utilized a caffeine-degrading, N-demethylation system, expressing the RO genes ndmA and ndmD, as an example of this method.
Assuntos
Biocatálise , Escherichia coli/genética , Escherichia coli/metabolismo , Cafeína/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/química , Complexo III da Cadeia de Transporte de Elétrons/genéticaRESUMO
The implementation of biocatalytic steroid hydroxylation processes plays a crucial role in the pharmaceutical industry due to a plethora of medicative effects of hydroxylated steroid derivatives and their crucial role in drug approval processes. Cytochrome P450 monooxygenases (CYP450s) typically constitute the key enzymes catalyzing these reactions, but commonly entail drawbacks such as poor catalytic rates and the dependency on additional redox proteins for electron transfer from NAD(P)H to the active site. Recently, these bottlenecks were overcome by equipping Escherichia coli cells with highly active variants of the self-sufficient single-component CYP450 BM3 together with hydrophobic outer membrane proteins facilitating cellular steroid uptake. The combination of the BM3 variant KSA14m and the outer membrane pore AlkL enabled exceptionally high testosterone hydroxylation rates of up to 45 U gCDW-1 for resting (i.e., living but non-growing) cells. However, a rapid loss of specific activity heavily compromised final product titers and overall space-time yields. In this study, several stabilization strategies were evaluated on enzyme-, cell-, and reaction level. However, neither changes in biocatalyst configuration nor variation of cultivation media, expression systems, or inducer concentrations led to considerable improvement. This qualified the so-far used genetic construct pETM11-ksa14m-alkL, M9 medium, and the resting-cell state as the best options enabling comparatively efficient activity along with fast growth prior to biotransformation. In summary, we report several approaches not enabling a stabilization of the high testosterone hydroxylation rates, providing vital guidance for researchers tackling similar CYP450 stability issues. A comparison with more stable natively steroid-hydroxylating CYP106A2 and CYP154C5 in equivalent setups further highlighted the high potential of the investigated CYP450 BM3-based whole-cell biocatalysts. The immense and continuously developing repertoire of enzyme engineering strategies provides promising options to stabilize the highly active biocatalysts.
Assuntos
Biocatálise , Sistema Enzimático do Citocromo P-450 , Escherichia coli , Hidroxilação , Sistema Enzimático do Citocromo P-450/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Escherichia coli/metabolismo , Escherichia coli/genética , Testosterona/metabolismo , Esteroides/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , NADPH-Ferri-Hemoproteína Redutase/metabolismo , NADPH-Ferri-Hemoproteína Redutase/genética , Estabilidade EnzimáticaRESUMO
Tyrosinases (TYR) play a key role in melanin biosynthesis by catalyzing two reactions: monophenolase and diphenolase activities. Despite low amino acid sequence homology, TYRs from various organisms (from bacteria to humans) have similar active site architectures and catalytic mechanisms. The active site of the TYRs contains two copper ions coordinated by histidine (His) residues. The catalytic mechanism of TYRs involves electron transfer between copper sites, leading to the hydroxylation of monophenolic compounds to diphenols and the subsequent oxidation of these to corresponding dopaquinones. Although extensive studies have been conducted on the structure, catalytic mechanism, and enzymatic capabilities of TYRs, some mechanistic aspects are still debated. This chapter will delve into the structure of the active site, catalytic function, and inhibition mechanism of TYRs. The goal is to improve our understanding of the molecular mechanisms underlying TYR activity. This knowledge can help in developing new strategies to modulate TYR function and potentially treat diseases linked to melanin dysregulation.
Assuntos
Domínio Catalítico , Monofenol Mono-Oxigenase , Humanos , Monofenol Mono-Oxigenase/metabolismo , Monofenol Mono-Oxigenase/química , Melaninas/metabolismo , Melaninas/biossíntese , Animais , Catálise , Biocatálise , OxirreduçãoRESUMO
Phthalic acid esters (PAE) are widely used as plasticizers and have been classified as ubiquitous environmental contaminants of primary concern. PAE have accumulated intensively in surface water, groundwater, and wastewaters; thus, PAE degradation is essential. In the present study, the ability of a saline soil bacteria (SSB)-consortium to degrade synthetic wastewater-phthalates with alkyl chains of different lengths, such as diethyl phthalate (DEP), di-n-butyl phthalate (DBP), benzyl butyl phthalate (BBP), and di (2-ethylhexyl) phthalate (DEHP) was characterized. A central composite design-response surface methodology was applied to optimize the degradation of each phthalate, where the independent variables were temperature (21-41 °C), pH (5.3-8.6) and PAE concentration (79.5-920.4 mg L-1), and Gas Chromatography-Mass Spectrometry was used to identify the metabolites generated during phthalate degradation. Optimal conditions were 31 °C, pH 7.0, and an initial PAE concentration of 500 mg L-1, where the SSB-consortium removed 84.9%, 98.47%, 99.09% and 98.25% of initial DEP, DBP, BBP, and DEHP, respectively, in 168h. A first-order kinetic model explained - the biodegradation progression, while the half-life of PAE degradation ranged from 12.8 to 29.8 h. Genera distribution of the SSB-consortium was determined by bacterial meta-taxonomic analysis. Serratia, Methylobacillus, Acrhomobacter, and Pseudomonas were the predominant genera; however, the type of phthalate directly affected their distribution. Scanning electron microscopy analysis showed that high concentrations (1000 mg L-1) of phthalates induced morphological alterations in the bacterial SSB-consortium. The metabolite profiling showed that DEP, DBP, BBP, and DEHP could be fully metabolized through the de-esterification and ß-oxidation pathways. Therefore, the SSB-consortium can be considered a potential candidate for bioremediation of complex phthalate-contaminated water resources.
Assuntos
Biodegradação Ambiental , Ésteres , Ácidos Ftálicos , Águas Residuárias , Poluentes Químicos da Água , Ácidos Ftálicos/metabolismo , Águas Residuárias/química , Ésteres/metabolismo , Poluentes Químicos da Água/metabolismo , Bactérias/metabolismo , Microbiologia do Solo , Biocatálise , Dibutilftalato/metabolismo , Plastificantes/metabolismo , Dietilexilftalato/metabolismoRESUMO
Urethanase is a promising biocatalyst for degrading carcinogen ethyl carbamate (EC) in fermented foods. However, their vulnerability to high ethanol and/or salt and acidic conditions severely limits their applications. In this study, a novel urethanase from Alicyclobacillus pomorum (ApUH) was successfully discovered using a database search. ApUH shares 49.4% sequence identity with the reported amino acid sequences. It belongs to the Amidase Signature family and has a conserved "K-S-S" catalytic triad and the characteristic "GGSS" motif. The purified enzyme overexpressed in Escherichia coli exhibits a high EC affinity (Km, 0.306 mM) and broad pH tolerance (pH 4.0-9.0), with an optimum pH 7.0. Enzyme activity remained at 58% in 12% (w/v) NaCl, and 80% in 10% (v/v) ethanol or after 1 h treatment with the same ethanol solution at 37 °C. ApUH has no hydrolytic activity toward urea. Under 30 °C, the purified enzyme (200 U/L) degraded about 15.4 and 43.1% of the EC in soy sauce samples (pH 5.0, 6.0), respectively, in 5 h. Furthermore, the enzyme also showed high activity toward the class 2A carcinogen acrylamide in foods. These attractive properties indicate their potential applications in the food industry.
Assuntos
Alicyclobacillus , Alimentos de Soja , Uretana , Alimentos de Soja/análise , Uretana/metabolismo , Uretana/química , Alicyclobacillus/enzimologia , Alicyclobacillus/genética , Alicyclobacillus/metabolismo , Concentração de Íons de Hidrogênio , Estabilidade Enzimática , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Amidoidrolases/metabolismo , Amidoidrolases/química , Amidoidrolases/genética , Cinética , Especificidade por Substrato , Carcinógenos/metabolismo , Carcinógenos/química , Cloreto de Sódio/metabolismo , Cloreto de Sódio/química , Biocatálise , Sequência de AminoácidosRESUMO
Pyridoxal phosphate (PLP), the active form of vitamin B6, is an important coenzyme in various enzyme-catalyzed reactions. PLP-dependent enzymes can catalyze a variety of chemical reactions, such as racemization, decarboxylation, ß-addition, ß-elimination, retro-aldol cleavage, transamination, and α-elimination. They are biologically synthesized a powerful tool for a variety of natural amino acids, non-natural amino acids and their related compounds. This article details the structural features and catalytic mechanisms of typical PLP-dependent enzymes such as ω-transaminase, lysine decarboxylase, threonine aldolase, and L-tyrosine phenol-lyase, and reviews the research progress in molecular modification and industrial applications of these enzymes. Finally, this article provides an outlook on the future development of PLP-dependent enzymes, including in vivo regeneration system and industrial applications of PLP cofactors, and discusses the tremendous potential of these enzymes in biocatalytic applications.
Assuntos
Fosfato de Piridoxal , Transaminases , Fosfato de Piridoxal/metabolismo , Transaminases/metabolismo , Transaminases/genética , Tirosina Fenol-Liase/metabolismo , Tirosina Fenol-Liase/genética , Glicina Hidroximetiltransferase/metabolismo , Glicina Hidroximetiltransferase/genética , BiocatáliseRESUMO
Value shaping and innovation capability improvement are two important goals of postgraduate course teaching. Biocatalysis and Enzyme Engineering is a core course for postgraduates majoring in bioengineering. Our teaching team established a "dual-drive and dual-guide" teaching model, that is, "double drive" (value-driven + innovation-driven) as the traction, this model integrated professional quality with industry needs, engineering ethics, and scientist spirit, combined theoretical teaching with research frontiers, industrial cases, and engineering practice, and incorporated thematic lectures, group peer evaluation, and review papers into course assessment. The teaching under this model achieved the teaching objectives of "double guide" (guiding ideology and ability), and improved the postgraduates' value identification, innovation awareness, engineering thinking, and ability to solve practical engineering problems.
Assuntos
Biocatálise , Engenharia de Proteínas , Ensino , Bioengenharia/educação , Educação de Pós-Graduação , Modelos Educacionais , CurrículoRESUMO
Enzyme nanoreactors are nanoscale compartments consisting of encapsulated enzymes and a selectively permeable barrier. Sequestration and colocalization of enzymes can increase catalytic activity, stability, and longevity, highly desirable features for many biotechnological and biomedical applications of enzyme catalysts. One promising strategy to construct enzyme nanoreactors is to repurpose protein nanocages found in nature. However, protein-based enzyme nanoreactors often exhibit decreased catalytic activity, partially caused by a mismatch of protein shell selectivity and the substrate requirements of encapsulated enzymes. No broadly applicable and modular protein-based nanoreactor platform is currently available. Here, we introduce a pore-engineered universal enzyme nanoreactor platform based on encapsulins-microbial self-assembling protein nanocompartments with programmable and selective enzyme packaging capabilities. We structurally characterize our protein shell designs via cryo-electron microscopy and highlight their polymorphic nature. Through fluorescence polarization assays, we show their improved molecular flux behavior and highlight their expanded substrate range via a number of proof-of-concept enzyme nanoreactor designs. This work lays the foundation for utilizing our encapsulin-based nanoreactor platform for diverse future biotechnological and biomedical applications.
Assuntos
Engenharia de Proteínas , Porosidade , Nanotecnologia , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Biocatálise , Tamanho da PartículaRESUMO
The deprotonation-reprotonation sequence introduces additional cyclization branches in terpene biosynthesis. However, the underlying mechanism remains poorly understood. In this study, we employed a combined approach of molecular dynamics (MD) simulations and site-directed mutagenesis on astellifadiene synthase EvAS from Emericella variecolor to investigate the role of a protonated S85 residue. This residue acts as a catalytic acid, previously unreported, that facilitates the reprotonation step in astellifadiene biosynthesis. Mutating S85 led to the production of a new tricyclic sesterterpene.
Assuntos
Simulação de Dinâmica Molecular , Serina , Serina/biossíntese , Serina/química , Serina/metabolismo , Biocatálise , Mutagênese Sítio-Dirigida , Sesterterpenos/química , Sesterterpenos/metabolismoRESUMO
Enzymes are making a significant impact on chemical synthesis. However, the range of chemical products achievable through biocatalysis is still limited compared to the vast array of products possible with organic synthesis. For instance, azoxy products have rarely been synthesized using enzyme catalysts. In this study, we discovered that fungal unspecific peroxygenases are promising catalysts for synthesizing azoxy products from simple aniline starting materials. The catalytic features (up to 48,450 turnovers and a turnover frequency of 6.7 s-1) and substrate transformations (up to 99% conversion with 98% chemoselectivity) highlight the synthetic potential. We propose a mechanism where peroxygenase-derived hydroxylamine and nitroso compounds spontaneously (non-enzymatically) form the desired azoxy products. This work expands the reactivity repertoire of biocatalytic transformations in the underexplored field of azoxy compound formation reactions.
Assuntos
Compostos Azo , Biocatálise , Oxigenases de Função Mista , Oxigenases de Função Mista/metabolismo , Compostos Azo/química , Compostos Azo/metabolismo , Compostos de Anilina/química , Compostos de Anilina/metabolismo , Compostos Nitrosos/química , Compostos Nitrosos/metabolismo , Hidroxilamina/química , Hidroxilamina/metabolismoRESUMO
Strain engineering plays an important role in tuning electronic structure and improving catalytic capability of biocatalyst, but it is still challenging to modify the atomic-scale strain for specific enzyme-like reactions. Here, we systematically design Pt single atom (Pt1), several Pt atoms (Ptn) and atomically-resolved Pt clusters (Ptc) on PdAu biocatalysts to investigate the correlation between atomic strain and enzyme-like catalytic activity by experimental technology and in-depth Density Functional Theory calculations. It is found that Ptc on PdAu (Ptc-PA) with reasonable atomic strain upshifts the d-band center and exposes high potential surface, indicating the sufficient active sites to achieve superior biocatalytic performances. Besides, the Pd shell and Au core serve as storage layers providing abundant energetic charge carriers. The Ptc-PA exhibits a prominent peroxidase (POD)-like activity with the catalytic efficiency (Kcat/Km) of 1.50 × 109 mM-1 min-1, about four orders of magnitude higher than natural horseradish peroxidase (HRP), while catalase (CAT)-like and superoxide dismutase (SOD)-like activities of Ptc-PA are also comparable to those of natural enzymes. Biological experiments demonstrate that the detection limit of the Ptc-PA-based catalytic detection system exceeds that of visual inspection by 132-fold in clinical cancer diagnosis. Besides, Ptc-PA can reduce multi-organ acute inflammatory damage and mitigate oxidative stress disorder.
Assuntos
Biocatálise , Catalase , Ouro , Platina , Platina/química , Ouro/química , Humanos , Catalase/química , Catalase/metabolismo , Paládio/química , Peroxidase do Rábano Silvestre/química , Peroxidase do Rábano Silvestre/metabolismo , Superóxido Dismutase/química , Superóxido Dismutase/metabolismo , Catálise , Teoria da Densidade Funcional , Nanopartículas Metálicas/químicaRESUMO
α-Lipoic acid possesses remarkable antioxidant activity; however, its poor lipid solubility greatly restricts its practical utilization. The present study was the first (i) to synthesize a novel lipophilic antioxidant of octacosanol lipoate and (ii) to assess its antioxidant potency in sunflower oil by hydrogen nuclear magnetic resonance (1H NMR) spectroscopy. In brief, octacosanol lipoate was successfully synthesized using octacosanol and lipoic acid as substrates and Candida sp. 99-125 lipase as a catalyst. The conversion of octacosanol lipoate could reach as high as 98.1% within merely 2 h, with an overall yield of 87.9%. The hydrophobicity of lipoic acid was significantly enhanced upon esterification with octacosanol. Interestingly, both traditional methods and 1H NMR analysis consistently indicated that octacosanol lipoate exhibited superior antioxidant activity compared with butyl hydroxytoluene at high temperatures. It was concluded that octacosanol lipoate has the potential to be developed into a safe and efficient natural antioxidant which can be utilized not only in daily cooking oils but also in frying oils.
Assuntos
Antioxidantes , Lipase , Óleo de Girassol , Antioxidantes/química , Antioxidantes/síntese química , Óleo de Girassol/química , Lipase/química , Lipase/metabolismo , Ácido Tióctico/química , Esterificação , Candida/enzimologia , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Biocatálise , Álcoois GraxosRESUMO
Polyols, or sugar alcohols, are widely used in the industry as sweeteners and food formulation ingredients, aiming to combat the incidence of diet-related Non-Communicable Diseases. Given the attractive use of Generally Regarded As Safe (GRAS) enzymes in both academia and industry, this study reports on an optimized process to achieve polyols transglucosylation using a dextransucrase enzyme derived from Leuconostoc mesenteroides. These enzyme modifications could lead to the creation of a new generation of glucosylated polyols with isomalto-oligosaccharides (IMOS) structures, potentially offering added functionalities such as prebiotic effects. These reactions were guided by a design of experiment framework, aimed at maximizing the yields of potential new sweeteners. Under the optimized conditions, dextransucrase first cleared the glycosidic bond of sucrose, releasing fructose with the formation of an enzyme-glucosyl covalent intermediate complex. Then, the acceptor substrate (i.e., polyols) is bound to the enzyme-glucosyl intermediate, resulting in the transfer of glucosyl unit to the tested polyols. Structural insights into the reaction products were obtained through nuclear maneic resonance (NMR) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analyses, which revealed the presence of linear α(1 â 6) glycosidic linkages attached to the polyols, yielding oligosaccharide structures containing from 4 to 10 glucose residues. These new polyols-based oligosaccharides hold promise as innovative prebiotic sweeteners, potentially offering valuable health benefits.
Assuntos
Glucosiltransferases , Leuconostoc mesenteroides , Oligossacarídeos , Glucosiltransferases/química , Glucosiltransferases/metabolismo , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Leuconostoc mesenteroides/enzimologia , Leuconostoc mesenteroides/química , Leuconostoc mesenteroides/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Polímeros/química , Polímeros/metabolismo , Biocatálise , Edulcorantes/química , Edulcorantes/metabolismo , GlicosilaçãoRESUMO
Flutriafol, a globally utilized triazole fungicide in agriculture, is typically applied as a racemic mixture, but its enantiomers differ in bioactivity and environmental impact. The synthesis of flutriafol enantiomers is critically dependent on chiral precursors: 2,2-bisaryl-substituted oxirane [(2-fluorophenyl)-2-(4-fluorophenyl)oxirane, 1a] and 1,2-diol [1-(2-fluorophenyl)-1-(4-fluorophenyl)ethane-1,2-diol, 1b]. Here, we engineered a Rhodotorula paludigensis epoxide hydrolase (RpEH), obtaining mutant Escherichia coli/RpehH336W/L360F with a 6.4-fold enhanced enantiomeric ratio (E) from 5.5 to 35.4. This enabled a gram-scale resolution of rac-1a by E. coli/RpehH336W/L360F, producing (S)-1a (98.2% ees) and (R)-1b (75.0% eep) with 44.3 and 55.7% analytical yields, respectively. As follows, chiral (S)-flutriafol (98.2% ee) and (R)-flutriafol (75.0% ee) were easily synthesized by a one-step chemocatalytic process from (S)-1a and a two-step chemocatalytic process from (R)-1b, respectively. This chemoenzymatic approach offers a superior alternative for the asymmetric synthesis of flutriafol enantiomers. Furthermore, molecular dynamics simulations revealed insight into the enantioselectivity improvement of RpEH toward bulky 2,2-bisaryl-substituted oxirane 1a.
Assuntos
Epóxido Hidrolases , Proteínas Fúngicas , Fungicidas Industriais , Rhodotorula , Triazóis , Fungicidas Industriais/química , Fungicidas Industriais/síntese química , Fungicidas Industriais/metabolismo , Triazóis/química , Triazóis/metabolismo , Triazóis/síntese química , Epóxido Hidrolases/metabolismo , Epóxido Hidrolases/genética , Epóxido Hidrolases/química , Estereoisomerismo , Rhodotorula/enzimologia , Rhodotorula/genética , Rhodotorula/química , Rhodotorula/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Biocatálise , Escherichia coli/genética , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Engenharia de ProteínasRESUMO
Less steric ketones exhibited low stereoselectivity toward M5 due to their difficulty in restricting the free rotation of the imine intermediate. An engineered enantio-complementary imine reductase from M5 was obtained with catalytic activity. We identified four key residues that play essential roles in controlling stereoselectivity. Two mutants, I149Y-W234L (up to 99%S ee) and L200M-F260M (up to 99%R ee), were achieved, showing excellent stereoselectivity toward the tested substrates, offering valuable biocatalysts for synthesizing alkylated amphetamines.
Assuntos
Anfetaminas , Iminas , Oxirredutases , Estrutura Molecular , Estereoisomerismo , Iminas/química , Oxirredutases/metabolismo , Oxirredutases/química , Anfetaminas/química , Anfetaminas/síntese química , Alquilação , Catálise , BiocatáliseRESUMO
Phenylpropanoid sucrose esters are a large and important group of natural substances with significant therapeutic potential. This work describes a pilot study of the enzymatic hydroxycinnamoylation of sucrose and its derivatives which was carried out with the aim of obtaining precursors of natural phenylpropanoid sucrose esters, e.g., vanicoside B. In addition to sucrose, some chemically prepared sucrose acetonides and substituted 3'-O-cinnamates were subjected to enzymatic transesterification with vinyl esters of coumaric, ferulic and 3,4,5-trimethoxycinnamic acid. Commercial enzyme preparations of Lipozyme TL IM lipase and Pentopan 500 BG exhibiting feruloyl esterase activity were tested as biocatalysts in these reactions. The substrate specificity of the used biocatalysts for the donor and acceptor as well as the regioselectivity of the reactions were evaluated and discussed. Surprisingly, Lipozyme TL IM catalyzed the cinnamoylation of sucrose derivatives more to the 1'-OH and 4'-OH positions than to the 6'-OH when the 3'-OH was free and the 6-OH was blocked by isopropylidene. In this case, Pentopan reacted comparably to 1'-OH and 6'-OH positions. If sucrose 3'-O-coumarate was used as an acceptor, in the case of feruloylation with Lipozyme in CH3CN, 6-O-ferulate was the main product (63%). Pentopan feruloylated sucrose 3'-O-coumarate comparably well at the 6-OH and 6'-OH positions (77%). When a proton-donor solvent was used, migration of the 3'-O-cinnamoyl group from fructose to the 2-OH position of glucose was observed. The enzyme hydroxycinnamoylations studied can shorten the targeted syntheses of various phenylpropanoid sucrose esters.