RESUMEN
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.
Asunto(s)
Digestión , Harina , Oryza , Almidón , Transglutaminasas , Oryza/química , Oryza/metabolismo , Transglutaminasas/metabolismo , Transglutaminasas/química , Harina/análisis , Almidón/química , Almidón/metabolismo , Manipulación de Alimentos , Humanos , Viscosidad , Culinaria , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , BiocatálisisRESUMEN
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.
Asunto(s)
Biocatálisis , Sistema Enzimático del Citocromo P-450 , Escherichia coli , Hidroxilación , Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Escherichia coli/metabolismo , Escherichia coli/genética , Testosterona/metabolismo , Esteroides/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , NADPH-Ferrihemoproteína Reductasa/metabolismo , NADPH-Ferrihemoproteína Reductasa/genética , Estabilidad de EnzimasRESUMEN
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.
Asunto(s)
Ingeniería de Proteínas , Porosidad , Nanotecnología , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Biocatálisis , Tamaño de la PartículaRESUMEN
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.
Asunto(s)
Anfetaminas , Iminas , Oxidorreductasas , Estructura Molecular , Estereoisomerismo , Iminas/química , Oxidorreductasas/metabolismo , Oxidorreductasas/química , Anfetaminas/química , Anfetaminas/síntesis química , Alquilación , Catálisis , BiocatálisisRESUMEN
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.
Asunto(s)
Ácidos Cumáricos , Sacarosa , Sacarosa/química , Sacarosa/metabolismo , Ácidos Cumáricos/química , Ácidos Cumáricos/metabolismo , Lipasa/metabolismo , Lipasa/química , Cinamatos/química , Cinamatos/metabolismo , Especificidad por Sustrato , Esterificación , Hidrolasas de Éster Carboxílico/metabolismo , Hidrolasas de Éster Carboxílico/química , Ésteres/química , Ésteres/metabolismo , BiocatálisisRESUMEN
In the glycerolysis process for diacylglycerol (DAG) preparation, free lipases suffer from poor stability and the inability to be reused. To address this, a cost-effective immobilized lipase preparation was developed by cross-linking macroporous resin with poly (ethylene glycol) diglycidyl ether (PEGDGE) followed by lipase adsorption. The selected immobilization conditions were identified as pH 7.0, 35 °C, cross-linking agent concentration 2.0%, cross-linking time 4 h, lipase amount 5 mg/g of support, and adsorption time 4 h. Enzymatic properties of the immobilized lipase were analyzed, revealing enhanced pH stability, thermal stability, storage stability, and operational stability post-immobilization. The conditions for immobilized enzyme-catalyzed glycerolysis to produce DAG were selected, demonstrating the broad applicability of the immobilized lipase. The immobilized lipase catalyzed glycerolysis reactions using various oils as substrates, with DAG content in the products ranging between 35 and 45%, demonstrating broad applicability. Additionally, the changes during the repeated use of the immobilized lipase were characterized, showing that mechanical damage, lipase leakage, and alterations in the secondary structure of the lipase protein contributed to the decline in catalytic activity over time. These findings provide valuable insights for the industrial application of lipase.
Asunto(s)
Diglicéridos , Estabilidad de Enzimas , Enzimas Inmovilizadas , Lipasa , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Lipasa/química , Lipasa/metabolismo , Diglicéridos/química , Concentración de Iones de Hidrógeno , Glicerol/química , Temperatura , Eurotiales/enzimología , Biocatálisis , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismoRESUMEN
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.
Asunto(s)
Biocatálisis , Ingeniería de Proteínas , Streptomyces , Ingeniería de Proteínas/métodos , Streptomyces/enzimología , Streptomyces/genética , Proteínas de Hierro no Heme/química , Proteínas de Hierro no Heme/metabolismo , Proteínas de Hierro no Heme/genética , 4-Hidroxifenilpiruvato Dioxigenasa/genética , 4-Hidroxifenilpiruvato Dioxigenasa/metabolismo , 4-Hidroxifenilpiruvato Dioxigenasa/química , Azidas/química , Azidas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismoRESUMEN
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.
Asunto(s)
Biocatálisis , Escherichia coli/genética , Escherichia coli/metabolismo , Cafeína/metabolismo , Complejo III de Transporte de Electrones/metabolismo , Complejo III de Transporte de Electrones/química , Complejo III de Transporte de Electrones/genéticaRESUMEN
The conversion of lignocellulosic feedstocks by cellulases to glucose is a critical step in biofuel production. ß-Glucosidases catalyze the final step in cellulose breakdown, producing glucose, and are often the rate-limiting step in biomass hydrolysis. The specific activity of most natural and engineered ß-glucosidase is higher on the artificial substrate p-nitrophenyl ß-d-glucopyranoside (pNPGlc) than on the natural substrate, cellobiose. We report an engineered ß-glucosidase (Q319A H0HC94) with a 1.8-fold higher specific activity (366.3 ± 36 µmol/min/mg), a 1.5-fold increase in kcat (340.8 ± 27 s-1), and a 3-fold increase in catalytic efficiency (236.65 mM-1 s-1) over H0HC94 (WT) on cellobiose. Molecular dynamic simulations and protein structure network analysis indicate that the Q319A H0HC94 active site pocket is significantly remodeled compared to the WT, leading to changes in enzyme conformation, better accessibility of cellobiose inside the active site pocket, and higher enzymatic activity. This study shows the impact of rational engineering of a nonconserved residue to increase ß-glucosidase substrate accessibility and catalytic efficiency by reducing crowding interaction between cellobiose and active site pocket residues near the gatekeeper region and increasing pocket volume and surface area. Thus, rational engineering of previously characterized enzymes could be an excellent strategy to improve cellulose hydrolysis.
Asunto(s)
Dominio Catalítico , Celobiosa , Simulación de Dinámica Molecular , Ingeniería de Proteínas , beta-Glucosidasa , Celobiosa/metabolismo , Celobiosa/química , beta-Glucosidasa/química , beta-Glucosidasa/metabolismo , beta-Glucosidasa/genética , Biocatálisis , CinéticaRESUMEN
Although cellular transport machinery is mostly ATP-driven and ATPase-dependent, there has been a recent surge in understanding colloidal transport processes relying on a nonspecific physical interaction with biologically significant small molecules. Herein, we probe the phoretic behavior of a biocolloid [composed of a Zn(II)-coordinated metallomicelle and enzymes horseradish peroxidase (HRP) and glucose oxidase (GOx)] when exposed to a concentration gradient of ATP under microfluidic conditions. Simultaneously, we demonstrate that an ATP-independent oxidative biocatalytic product formation zone can be modulated in the presence of a (glucose + ATP) gradient. We report that both directionality and extent of transport can be tuned by changing the concentration of the ATP gradient. This diffusiophoretic mobility of a submicrometer biocolloidal object for the spatial transposition of a biocatalytic zone signifies the ATP-mediated functional transportation without the involvement of ATPase. Additionally, the ability to analyze colloidal transport in microfluidic channels using an enzymatic fluorescent product-forming reaction could be a new nanobiotechnological tool for understanding transport and spatial catalytic patterning processes. We believe that this result will inspire further studies for the realization of elusive biological transport processes and target-specific delivery vehicles, considering the omnipresence of the ATP-gradient across the cell.
Asunto(s)
Adenosina Trifosfato , Biocatálisis , Glucosa Oxidasa , Peroxidasa de Rábano Silvestre , Zinc , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/química , Zinc/química , Zinc/metabolismo , Peroxidasa de Rábano Silvestre/química , Peroxidasa de Rábano Silvestre/metabolismo , Glucosa Oxidasa/química , Glucosa Oxidasa/metabolismo , Oxidación-Reducción , Coloides/químicaRESUMEN
Luteolin-7-O-glucoside(L7G), a glycosylation product of luteolin, is present in a variety of foods, vegetables, and medicinal herbs and is commonly used in dietary supplements due to its health benefits. Meanwhile, luteolin-7-O-glucoside is an indicator component for the quality control of honeysuckle in the pharmacopoeia. However, its low content in plants has hindered its use in animal pharmacological studies and clinical practice. In this study, a novel 7-O-glycosyltransferase CmGT from Cucurbita moschata was cloned, which could efficiently convert luteolin into luteolin-7-O-glucoside under optimal conditions (40 °C and pH 8.5). To further improve the catalytic efficiency of CmGT, a 3D structure of CmGT was constructed, and directed evolution was performed. The mutant CmGT-S16A-T80W was obtained by using alanine scanning and iterative saturation mutagenesis. This mutant exhibited a kcat/Km value of 772 s-1·M-1, which was 3.16-fold of the wild-type enzyme CmGT. Finally, by introducing a soluble tag and UDPG synthesis pathway, the strain BXC was able to convert 1.25 g/L of luteolin into 1.91 g/L of luteolin-7-O-glucoside under optimal conditions, achieving a molar conversion rate of 96% and a space-time yield of 27.08 mg/L/h. This study provides an efficient method for the biosynthesis of luteolin-7-O-glucoside, which holds broad application prospects in the food and pharmaceutical industry.
Asunto(s)
Biocatálisis , Cucurbita , Glucósidos , Glicosiltransferasas , Luteolina , Proteínas de Plantas , Glucósidos/metabolismo , Glucósidos/química , Glucósidos/biosíntesis , Luteolina/química , Luteolina/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/química , Glicosiltransferasas/genética , Glicosiltransferasas/metabolismo , Glicosiltransferasas/química , Cucurbita/genética , Cucurbita/enzimología , Cucurbita/química , Cucurbita/metabolismo , Clonación Molecular , Cinética , Evolución Molecular DirigidaRESUMEN
Although Nature's evolution and intelligence have gifted humankind with noteworthy enzyme candidates to simplify complex reactions with ultrafast, overselective, effortless, mild biological reactions for millions of years, their availability at minute-scale, short-range time-temperature stability, and purification costs hardly justify recycling/or reuse. Covalent immobilization, particularly via multipoint bonds, prevents denaturing, maintains activities for long-range time, pH, and temperature, and makes catalysts available for repetitive usages; which attracts researchers and industries to bring more immobilized enzyme contenders in science and commercial progressions. Inert-support activation, the most crucial step, needs appropriate activators; under mild conditions, the activator's functional group(s) still present on the activated support rapidly couples the enzyme, preventing unfolding and keeping the active site alive. This review summarizes exciting experimental advances, from the 1950s until today, in the activation strategies of various inert supports with five different surface activators, the cyanogen bromide, the isocyanate/isothiocyanate, the glutaraldehyde, the carbodiimide (with or without N-hydroxysuccinimide (NHS)), and the diazo group, for the immobilization of diverse enzymes for broader applications. These activators under mild pH (7.5 ± 0.5) and temperature (27 ± 3 °C) and ordinary stirring witnessed support activation and enzyme coupling and put off unfolding, harnessing addressable activities (CNBr: 40 ± 10%; -NâCâO/-NâCâS: 32 ± 7%; GA: 70 ± 15%; CDI: 60 ± 10%; -N+≡N: 80 ± 15%), while underprivileged stability, longevity, and reusabilities keep future investigations alive.
Asunto(s)
Biocatálisis , Enzimas Inmovilizadas , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismoRESUMEN
This study aimed to explore the capacity of immobilized lipases on the acetylation of six aglycon flavonoids, namely myricetin, quercetin, luteolin, naringenin, fisetin and morin. For this purpose, lipase B from Candida antarctica (CaLB) and lipase from Thermomyces lanuginosus (TLL) were immobilized onto the surface of ZnOFe nanoparticles derived from an aqueous olive leaf extract. Various factors affecting the conversion of substrates and the formation of monoesterified and diesterified products, such as the amount of biocatalyst and the molar ratio of the substrates and reaction solvents were investigated. Both CaLB and TLL-ZnOFe achieved 100% conversion yield of naringenin to naringenin acetate after 72 h of reaction time, while TLL-ZnOFe achieved higher conversion yields of quercetin, morin and fisetin (73, 85 and 72% respectively). Notably, CaLB-ZnOFe displayed significantly lower conversion yields for morin compared with TLL-ZnOFe. Molecular docking analysis was used to elucidate this discrepancy, and it was revealed that the position of the hydroxyl groups of the B ring on morin introduced hindrances on the active site of CaLB. Finally, selected flavonoid esters showed significantly higher antimicrobial activity compared with the original compound. This work indicated that these lipase-based nanobiocatalysts can be successfully applied to produce lipophilic derivatives of aglycon flavonoids with improved antimicrobial activity.
Asunto(s)
Enzimas Inmovilizadas , Flavonoides , Proteínas Fúngicas , Lipasa , Simulación del Acoplamiento Molecular , Flavonoides/química , Flavonoides/metabolismo , Lipasa/metabolismo , Lipasa/química , Enzimas Inmovilizadas/metabolismo , Enzimas Inmovilizadas/química , Acetilación , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Biocatálisis , Eurotiales/enzimologíaRESUMEN
This study investigated the blocking mechanism of immobilized penicillin G acylase (PGA) during the enzymatic synthesis of amoxicillin. Laboratory observations revealed that the primary cause of clogging was the crystallization of the substrate and product on the enzyme surface. Adjusting key parameters can significantly reduce clogging and improve catalytic efficiency. Methanol can decrease enzyme activity, but isopropyl alcohol cleaners can effectively remove clogs and protect enzyme activity. These findings provide an experimental foundation for optimizing the PGA immobilization process, which is crucial for achieving high efficiency and sustainability in industrial production.
Asunto(s)
Amoxicilina , Enzimas Inmovilizadas , Penicilina Amidasa , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Amoxicilina/química , Penicilina Amidasa/química , Penicilina Amidasa/metabolismo , Biocatálisis , Metanol/químicaRESUMEN
Glycoside hydrolase family 91 (GH91) inulin fructotransferase (IFTases) enables biotransformation of fructans into sugar substitutes for dietary intervention in metabolic syndrome. However, the catalytic mechanism underlying the sequential biodegradation of inulin remains unelusive during the biotranformation of fructans. Herein we present the crystal structures of IFTase from Arthrobacter aurescens SK 8.001 in apo form and in complexes with kestose, nystose, or fructosyl nystose, respectively. Two kinds of conserved noncatalytic binding regions are first identified for IFTase-inulin interactions. The conserved interactions of substrates were revealed in the catalytic center that only contained a catalytic residue E205. A switching scaffold was comprised of D194 and Q217 in the catalytic channel, which served as the catalytic transition stabilizer through side chain displacement in the cycling of substrate sliding in/out the catalytic pocket. Such features in GH91 contribute to the catalytic model for consecutive cutting of substrate chain as well as product release in IFTase, and thus might be extended to other exo-active enzymes with an enclosed bottom of catalytic pocket. The study expands the current general catalytic principle in enzyme-substrate complexes and shed light on the rational design of IFTase for fructan biotransformation.
Asunto(s)
Dominio Catalítico , Hexosiltransferasas , Inulina , Inulina/metabolismo , Inulina/química , Hexosiltransferasas/metabolismo , Hexosiltransferasas/química , Especificidad por Sustrato , Modelos Moleculares , Arthrobacter/enzimología , Catálisis , Biocatálisis , Fructanos/metabolismo , Fructanos/química , Conformación ProteicaRESUMEN
The blood glucose concentration in aquatic organisms, a crucial indicator reflecting their health status, holds significant importance for detecting glucose levels in serum in terms of processing and quality monitoring. In this study, a novel POD biomimetic enzyme (p-BEs) with horseradish peroxidase catalytic properties was designed, optimized, and its mechanism was discussed in detail. Based on this, a portable system has been developed capable of determining glucose levels in three ways: quantitatively analyzed through UV-Vis/MD, quantitatively analyzed on-site using a mobile phone RGB, and semi-quantitatively analyzed through a drip plate. Meanwhile, compared with other catalytic methods for detecting glucose, we achieved a lower limit of detection (0.03 µM) and shorter detection time (12 min), with high catalytic activity. This study provides new insights into the design of efficient and reliable cascade catalytic systems responsive to glucose, offering a low-cost, simplicity of operation method for glucose detection.
Asunto(s)
Técnicas Biosensibles , Peroxidasa de Rábano Silvestre , Técnicas Biosensibles/métodos , Peroxidasa de Rábano Silvestre/química , Peroxidasa de Rábano Silvestre/metabolismo , Glucosa/análisis , Glucemia/análisis , Catálisis , Materiales Biomiméticos/química , Límite de Detección , Biomimética/métodos , BiocatálisisRESUMEN
Cytochrome P450 (P450) enzymes dominate steroid metabolism. In general, the simple C-hydroxylation reactions are mechanistically straightforward and are generally agreed to involve a perferryl oxygen species (formally FeO3+). Several of the steroid transformations are more complex and involve C-C bond scission. We initiated mechanistic studies with several of these (i.e., 11A1, 17A1, 19A1, and 51A1) and have now established that the dominant modes of catalysis for P450s 19A1 and 51A1 involve a ferric peroxide anion (i.e., Fe3+O2¯) instead of a perferryl ion complex (FeO3+), as demonstrated with 18O incorporation studies. P450 17A1 is less clear. The indicated P450 reactions all involve sequential oxidations, and we have explored the processivity of these multi-step reactions. P450 19A1 is distributive, i.e., intermediate products dissociate and reassociate, but P450s 11A1 and 51A1 are highly processive. P450 17A1 shows intermediate processivity, as expected from the release of 17-hydroxysteroids for the biosynthesis of key molecules, and P450 19A1 is very distributive. P450 11B2 catalyzes a processive multi-step oxidation process with the complexity of a chemical closure of an intermediate to a locked lactol form.
Asunto(s)
Sistema Enzimático del Citocromo P-450 , Oxidación-Reducción , Esteroides , Sistema Enzimático del Citocromo P-450/metabolismo , Esteroides/metabolismo , Humanos , Catálisis , Animales , BiocatálisisRESUMEN
Mogrosides are natural compounds highly valued in the food sector for their exceptional sweetness. Here, we report a novel O-glycosyltransferase (UGT74DD1) from Siraitia grosvenorii that catalyzes the conversion of mogrol to mogroside IIE. Site-directed mutagenesis yielded the UGT74DD1-W351A mutant, which exhibited the new capability to transform mogroside IIE into the valuable sweetener mogroside III, but with low catalytic activity. Subsequently, using structure-guided directed evolution with combinatorial active-site saturation testing, the superior mutant M6 (W351A/Q373 K/E49H/Q335W/S278C/D17F) were obtained, which showed a 46.1-fold increase in catalytic activity compared to UGT74DD1-W351A. Molecular dynamics simulations suggested that the enhanced activity and extended substrate profiles of M6 are due to its enlarged substrate-binding pocket and strengthened enzyme-substrate hydrogen bonding interactions. Overall, we redesigned UGT74DD1, yielding mutants that catalyze the conversion of mogrol into mogroside III. This study thus broadens the toolbox of UGTs capable of catalyzing the formation of valuable polyglycoside compounds.
Asunto(s)
Glicosiltransferasas , Edulcorantes , Glicosiltransferasas/genética , Glicosiltransferasas/química , Glicosiltransferasas/metabolismo , Edulcorantes/química , Edulcorantes/metabolismo , Cucurbitaceae/química , Cucurbitaceae/enzimología , Cucurbitaceae/genética , Cucurbitaceae/metabolismo , Mutagénesis Sitio-Dirigida , Proteínas de Plantas/genética , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Biocatálisis , Dominio Catalítico , Ingeniería de Proteínas , Especificidad por Sustrato , CinéticaRESUMEN
Investigating organic carriers' utilization efficiency and bioactivity within organic-inorganic hybrid nanoflowers is critical to constructing sensitive immunosensors. Nevertheless, the sensitivity of immunosensors is interactively regulated by different classes of biomolecules such as antibodies and enzymes. In this work, we introduced a new alkaline phosphatase-antibody-CaHPO4 hybrid nanoflowers (AAHNFs) microreactor based colorimetric immunoprobe. This system integrates a biometric unit (antibody) with a signal amplification element (enzyme) through the biomineralization process. Specifically, the critical factors affecting antibody recognition activity in the formation mechanism of AAHNFs are investigated. The designed AAHNFs retain antibody recognition ability with enhanced protection for encapsulated proteins against high temperature, organic solvents, and long-term storage, facilitating the selective construction of lock structures against antigens. Additionally, a colorimetric immunosensor based on AAHNFs was developed. After ascorbic acid 2-phosphate hydrolysis by alkaline phosphatase (ALP), the generated ascorbic acid decomposes I2 to I-, inducing the localized surface plasmon resonance in the silver nanoplate, which is effectively tuned through shape conversion to develop the sensor. Further, a 3D-printed portable device is fabricated, integrated with a smartphone sensing platform, and applied to the data of collection and analysis. Notably, the immunosensor exhibits improved analytical performance with a 0.1-6.25 ng·mL-1 detection range and a 0.06 ng·mL-1 detection limit for quantitative saxitoxin (STX) analysis. The average recoveries of STX in real samples ranged from 85.9% to 105.9%. This study presents a more in-depth investigation of the recognition element performance, providing insights for improved antibody performance in practical applications.
Asunto(s)
Fosfatasa Alcalina , Colorimetría , Saxitoxina , Fosfatasa Alcalina/metabolismo , Fosfatasa Alcalina/química , Saxitoxina/análisis , Saxitoxina/química , Colorimetría/métodos , Técnicas Biosensibles/métodos , Biocatálisis , Límite de Detección , Nanoestructuras/química , Inmunoensayo/métodos , Ácido Ascórbico/química , Ácido Ascórbico/análisis , Ácido Ascórbico/análogos & derivados , Plata/químicaRESUMEN
The Hsp90 chaperone is an ATPase enzyme composed of two copies of a three-domain subunit. Hsp90 stabilizes and activates a diverse array of regulatory proteins. Substrates are bound and released by the middle domain through a clamping cycle involving conformational transitions between a dynamic open state and a compact conformationally restricted closed state. Intriguingly, the overall ATPase activity of dimeric Hsp90 can be asymmetrically enhanced through a single subunit when Hsp90 is bound to a cochaperone or when Hsp90 is composed of one active and one catalytically defunct subunit as a heterodimer. To explore the mechanism of asymmetric Hsp90 activation, we designed a subunit bearing N-terminal ATPase mutations that demonstrate increased intra- and interdomain dynamics. Using intact Hsp90 and various N-terminal and middle domain constructs, we blended 19F NMR spectroscopy, molecular dynamics (MD) simulations, and ATPase assays to show that within the context of heterodimeric Hsp90, the conformationally dynamic subunit stimulates the ATPase activity of the normal subunit. The contrasting dynamic properties of the subunits within heterodimeric Hsp90 provide a mechanistic framework to understand the molecular basis for asymmetric Hsp90 activation and its importance for the biological function of Hsp90.