Hydrolysis Of P-nitrophenyl Acetate Research Articles

Carbonic anhydrase-mimicking metal-organic frameworks built from amino acid and cadmium ions.

Built from L-histidine amino acid ligand and cadmium ions, two new 3D chiral metal-organic frameworks, [α-Cd(HIS)] (1) and [β-Cd(HIS)] (2), which contain metal-histidine bonds mimicking the structure features of carbonic anhydrase, demonstrate interesting properties of catalyzing the hydrolysis of p-nitrophenylacetate (p-NPA) to para-nitrophenol (p-NP).

Fabrication of polyphenol-etched amino acid-modified ZIF-8 and their carbonic anhydrase-like catalytic activity

Carbonic anhydrase mimics have recently emerged as promising alternative catalysts for industrial CO2 capture and utilization. Up to now, many strategies have been developed to design and prepare nanostructures with excellent carbonic anhydrase-like catalytic activity. Herein, two kinds of polyphenol molecules (tannic acid (TA) and gallic acid (GA)) were used to etch amino acid (valine (Val) and leucine (Leu)) modified ZIF-8 (AA-ZIF-8) for fabricating carbonic anhydrase mimics. The TA-treated AA-ZIF-8 (Val-ZIF-8/TA and Leu-ZIF-8/TA) possess hollow nanostructure while the GA-treated AA-ZIF-8 (Val-ZIF-8/GA and Leu-ZIF-8/GA) are decomposed into smaller nanoparticles. Zn-polyphenol coordination interaction and protons released by polyphenols are considered to be the driving force for the etching of AA-ZIF-8. Using hydrolysis of p-nitrophenyl acetate (p-NPA) as the model reaction, the carbonic anhydrase-like activity of AA-ZIF-8/polyphenol nanoparticles were evaluated and the results indicated their excellent catalytic activity. Moreover, after using AA-ZIF-8/polyphenol as catalysts, the CO2 hydration efficiency could also be enhanced. This work provides a new approach for developing carbonic anhydrase mimics, which could help for designing of materials for industrial CO2 capture.

Synthesis of ZIF-90 based nanozyme with excellent carbonic anhydrase activity

Capturing CO2 using natural carbonic anhydrase (CA) has advantages of environmental friendliness, low energy consumption, renewable and no secondary pollution. However, the high cost, poor stability and low reusability of free CA under harsh conditions limit its industrial applications. Great efforts have been devoted to developing CA mimetic nanozymes to solve these problems. In this work, ZIF-90-based nanoparticles were prepared to mimic the active center of natural CA. The excellent CA-like catalytic activity of ZIF-90 nanoparticles was confirmed by using hydrolysis of p-nitrophenyl acetate (p-NPA) as the model reaction. Moreover, modifying ZIF-90 with monoethanolamine (MEA) could further improve its affinity for CO2, which in turn enhances the CO2 uptake capacity. Moreover, the hydrolysis of CO2 was also investigated through the pH decrease of the reaction solution and the results indicated that CO2 hydration and exchange were remarkably enhanced with the aid of ZIF-90 or ZIF-90 @MEA. This approach is facile and costless, which sheds light on the development of novel catalysts for industrial CO2 capture.

Biosynthesis of diisooctyl 2,5-furandicarboxylate by Candida antarctica lipase B (CALB) immobilized on a macroporous epoxy resin.

Diisooctyl 2,5-furandicarboxylate (DEF), an ester derivative of 2,5-furandicarboxylic acid (FDCA, a bio-based platform chemical), resembles the physical and chemical properties of phthalates. Due to its excellent biodegradability, DEF is considered a safer alternative to the hazardous phthalate plasticizers. Although FDCA esters are currently mainly produced by chemical synthesis, the enzymatic synthesis of DEF is a green, promising alternative. The current study investigated the biosynthesis of DEF by Candida antarctica lipase B (CALB) immobilized on macroporous resins. Out of five macroporous resins (NKA-9, LX-1000EP, LX-1000HA, XAD-7HP, and XAD-8) evaluated, the LX-1000EP epoxy resin was identified as the best carrier for CALB, and the XAD-7HP weakly polar resin was identified as the second best. The optimal immobilization conditions were as follows: CALB (500μL) and LX-1000EP (0.1g) were incubated in phosphate butter (20mM, pH 6.0) for 10h at 35°C. The resulting immobilized CALB (EP-CALB) showed an activity of 639U/g in the hydrolysis of p-nitrophenyl acetate, with an immobilization efficiency of 87.8% and an activity recovery rate of 56.4%. Using 0.02g EP-CALB as the catalyst in 10mL toluene, and the molar ratio of 2,5-dimethyl furanediformate (1mmol/mL) and isooctyl alcohol (4mmol/mL) that was 1:4, a DEF conversion rate of 91.3% was achieved after a 24-h incubation at 50°C. EP-CALB had similar thermal stability and organic solvent tolerance compared to Novozym 435, and both were superior to CALB immobilized on the XAD-7HP resin. EP-CALB also exhibited excellent operational stability, with a conversion rate of 52.6% after 10 repeated uses. EP-CALB could be a promising alternative to Novozym 435 in the biomanufacturing of green and safe plasticizers such as DEF.

Modeling of micellar‐catalyzed bimolecular ionic reactions by a nonlinear Poisson−Boltzmann equation and an electrostatic free energy model

AbstractThe hydrolysis reactions of crystal violet (CV) and p‐nitrophenyl acetate (PNPA) by sodium hydroxide in cetyltrimethylammonium bromide (CTAB) micellar solution were studied in this work. Combined with the Boltzmann probability of ionic species in an electrostatic potential field, a model with a numerical scheme was developed based on the nonlinear Poisson−Boltzmann equation in the micelle‐cell consisting of the reactants and the surfactant. The numerical solution gives the ionic species distribution in the micelle phase and the radius of the micelle‐phase. The dielectric constant of the micelle‐phase varies in the range of 39.8−54.4. The second‐order reaction rate constants in the micelle‐phase were determined using the electrostatic and dipolar free energy models. For the basic hydrolysis of CV and PNPA, the overall second‐order rate constants are three to five times and 1.4 times greater than the corresponding values in water, respectively. The rate enhancement occurs due to the distribution of the reactive ionic species in the cell region and the enhancement of the Coulombic and dipolar interactions among the ionic and polar reactants.

Reduction-triggered Disassembly of Organic Cationic Nanorods Produces a Cysteine Protease Mimic (Miravet et al.).

The emergence of catalytic activity associated with a disassembly process is reported, reminiscent of complex biological systems. A cystine derivative with pendant imidazole groups self-assembles into cationic nanorods in the presence of the cationic surfactants cetylpyridinium chloride (CPC) or cetyltrimethylammonium bromide (CTAB). Disulfide reduction triggers nanorod disassembly and the generation of a simple cysteine protease mimic, which shows a dramatically improved catalytic efficiency in the hydrolysis of p-nitrophenyl acetate (PNPA).

Autocatalytic strategy for tuning drug release from peptide-drug supramolecular hydrogel

Peptide-drug conjugates (PDCs) composed of peptide, spacer and drug have gained extensive attention in the field of drug delivery owing to its precise control over the drug payload and architecture. However, the achievement of controllable and rapid drug release at targeted site by PDCs is still a great challenge for pharmaceutist. Herein, we introduced the histidine residue into PDCs to generate a supramolecular hydrogel via a pH-trigger strategy, which exhibited an autocatalytic effect to precisely tune drug release from PDCs hydrogel. Using indomethacin (Idm) as model drug, various PDCs (Y(Idm)EEH, Y(Idm)EEK and Y(Idm)EER) were synthesized and their self-assembling properties were investigated in terms of critical aggregation concentration (CAC), transmission electron microscopy (TEM) and rheometer. Introduction of histidine residue into PDCs presented a robust catalytic activity on the ester hydrolysis of p-nitrophenyl acetate in aqueous solution, as well conferred the autocatalytic capacity to hydrolyze the PDCs into active parent drug (Idm). Overall, we reported an autocatalytic activity of histidine residue to precisely tune drug release from PDCs hydrogels.

Hydrolytic reactivity of novel copper(II) complexes with reduced N-salicylate threonine Schiff bases: distinguishable effects of various micelles

Three new bi- or tetranuclear copper (II) complexes (complexes 1, 2 and 3) with reduced N-salicylate threonine were synthesized and characterized by single crystal X-ray diffraction, X-ray powder diffraction, UV–vis, and thermogravimetric analysis. Single-crystal diffraction analysis reveals that these complexes have similar coordination modes and conformations which were linked into multidimensional networks through some weak interactions. These three complexes were employed to promote the hydrolytic cleavage of p-nitrophenyl picolinate (PNPP) and p-nitrophenyl acetate (PNPA). Catalytic reactivities of the as-prepared complexes show that in the hydrolysis of p-nitrophenyl acetate (PNPA) these three complexes provide 51, 45, 39-fold rate acceleration as compared to spontaneous hydrolysis of PNPA at pH 7.0, respectively. Under selected conditions, excess three orders of magnitude rate enhancement were observed for the catalytic hydrolysis of another carboxylic ester, p-nitrophenyl picolinate (PNPP). Furthermore, these complexes efficiently promote the PNPA hydrolysis in micellar solution of cetyltrimethylammonium bromide (CTAB) or bis(hexadecylhexmethyl ammonium)ethane bromide (16−6−16) giving rise to excess two orders of magnitude rate enhancement which is approximate 2.0 − 4.0 times higher than that in the buffer. However, the introduction of micelles of n-lauroylsarcosine sodium (LSS) or double-chained C22/8 surfactant did not increase the rate or even inhibited the hydrolysis reaction in contrast with buffered aqueous medium.

An artificial self-assembling peptide with carboxylesterase activity and substrate specificity restricted to short-chain acid p-nitrophenyl esters.

Natural enzymes possess remarkable catalytic activity and high substrate specificity. Many efforts have been dedicated to construct artificial enzymes with high catalytic activity. However, how to mimic the exquisite substrate specificity of a natural enzyme remains challenging because of the complexity of the enzyme structure. Here, we report artificial carboxylesterases that are specific for short chain fatty acids and were constructed via peptide self-assembly. These artificial systems have esterase-like activity rather than lipase-like activity towards p-nitrophenyl esters. The designer peptides self-assembled into nanofibers with strong β-sheet character. The extending histidine units and the hydrophobic edge of the fibrillar structure collectively form the active center of the artificial esterase. These artificial esterases show substrate specificity for short-chain acids esters. Moreover, 1-isopropoxy-4-nitrobenzene could function as a competitive inhibitor of hydrolysis of p-nitrophenyl acetate for an artificial esterase.

Statistical optimisation of esterase from Salinicoccus roseus strain RF1H and its potential application in synthetic dye decolorisation

Halophilic bacteria isolated from Indian Sundarban Wetland, identified as Salinicoccus roseus strain RF1H, were screened for extracellular esterase production. Media components for esterase production under submerged fermentation were optimised using Plackett–Burman Design followed by Box-Behnken Design of Response Surface Methodology (RSM) at pH 4.0, 3.5 g/L of MgSO4, and tributyrin concentration of 1(%v/v). The regression model was significant with a determination coefficient (R 2) value of 0.826. Using RSM, the experimental value of maximum enzyme activity (2.442 UmL−1) was 1.35 fold increased. The kinetic parameters K m and V max for hydrolysis of p-nitrophenyl acetate were 9.98 mM and 2.26 µmol/min/mg respectively, establishing a high affinity for the substrate. Further, the enzyme conferred promising results in decolorisation of synthetic dyes -Malachite Green, Congo Red, Brilliant Yellow, and Orange G. Molecular docking was used to investigate the binding pockets of the esterase, enzyme-ligand interactions, and amino acids which played a key role in dye decolorisation. Docking score results and decolorisation percentage were found in good agreement. This is the first report on the biotransformation of synthetic dyes to non-toxic products using esterase from Salinicoccus roseus.

Enzymatic Production of Ecodiesel by Using a Commercial Lipase CALB, Immobilized by Physical Adsorption on Mesoporous Organosilica Materials

The synthesis of two biocatalysts based on a commercial Candida antarctica lipase B, CALB enzyme (E), physically immobilized on two silica supports, was carried out. The first support was a periodic mesoporous organosilica (PMO) and the second one was a commercial silica modified with octyl groups (octyl-MS3030). The maximum enzyme load was 122 mg enzyme/g support on PMO and 288 mg enzyme/g support on octyl-MS3030. In addition, the biocatalytic efficiency was corroborated by two reaction tests based on the hydrolysis of p-nitrophenylacetate (p-NPA) and tributyrin (TB). The transesterification of sunflower oil with ethanol was carried out over the biocatalysts synthesized at the following reaction conditions: 6 mL sunflower oil, 1.75 mL EtOH, 30 °C, 25 μL NaOH 10 N and 300 rpm, attaining conversion values over 80% after 3 h of reaction time. According to the results obtained, we can confirm that these biocatalytic systems are viable candidates to develop, optimize and improve a new methodology to achieve the integration of glycerol in different monoacylglycerol molecules together with fatty acid ethyl esters (FAEE) molecules to obtain Ecodiesel.

Covalent immobilization of carbonic anhydrase on amine functionalized alumino-Siloxane aerogel beads for biomimetic sequestration of CO2

Biological CO2 sequestration using carbonic anhydrase is the most promising and eco-friendly technique in CO2 capture technology. The present study entails biomimetic CO2 capture using Carbonic Anhydrase from bovine erythrocytes (BCA) immobilized on versatile porous alumino-siloxane hybrid aerogel beads through covalent attachment. Alumino-siloxane hybrid aerogel beads (Al/Si) were functionalized with (3-Aminopropyl)- trimethoxysilane to achieve effective loading of the enzyme. Amine functionalized hybrid beads (Al/Si-NH2) were characterized by X-Ray Diffraction (XRD), Fourier Transform Infrared (FTIR), Field Emission-Scanning Electron Microscope (FE-SEM), and Brunauer–Emmett–Teller (BET). The biocatalytic activity of BCA immobilized on amine-functionalized alumino-siloxane aerogel beads (BCA-Al/Si-NH2) was estimated by hydrolysis of p-nitrophenyl acetate (p-NPA). Kinetic parameter, km and kcat/ km values of BCA-Al/Si-NH2 hybrid beads were found to be comparable with the free enzyme. BCA immobilized Al/Si-NH2 hybrid beads possess thermal stability, pH stability, and storage stability and retained almost 90% of activity over ten consecutive reaction cycles. The carbonation capacity of BCA-Al/Si-NH2 hybrid beads is found to be 12 mg of CaCO3/17 μg of BCA. Thus, BCA immobilized on amine-functionalized alumino-siloxane aerogel beads has been found to directly promote the absorption of CO2 and store the captured CO2 as inert solid carbonates.

Rotating bed reactor packed with heterofunctional structured silica-supported lipase. Developing an effective system for the organic solvent and aqueous phase reactions

Abstract Production of specialty chemicals increasingly makes use of the enzyme catalysts in organic solvents, and Novozym 435 (N435) is among most often applied. However, its polymeric skeleton is unstable in many solvents. In this context we report the results of a more systematic study of the biocatalysts, fabricated using highly porous siliceous pellets/enzyme carriers (MH, 4 cm3 g−1 total pore volume, 310 m2 g−1 surface area), grafted with octyl (-O), amino (-A) and octyl and amino (-OA) groups, to attach the enzyme - CALB lipase. The catalysts were deployed in a rotating bed reactor (RBR) and tested in hydrolysis of p-nitrophenyl acetate, and esterification of levulinic acid. Performance of the system was compared with the same RBR filled with N435. N435 appeared the most active in both reactions, when activity was related to bulk mass of the catalysts, mainly owing to very large enzyme load. But its pore structure degraded in many typical solvents already after 24 h of soaking, whereas no significant change in MH-O and MH-OA activity was detected. Irrespective of the solvent and reaction, the highest specific activity of the enzyme featured MH-O. However, a significant enzyme leaching from MH-O observed in a hydrolytic reaction, in stark contrast to MH-OA which came second in specific activity, point to MH-OA as the catalyst of choice for hydrolytic reactions. In esterification reaction the MH-O lipase was not only most active but it was also quite stable. Therefore, MH-O appears the most suitable production system for the reactions carried out in organic solvents.

Influence of cationic surfactants and inorganic salts on the enzyme kinetic activity of Mucor javanicus lipase

AbstractIn this article, the enzyme kinetic activity of Mucor javanicus lipase (MJL) has investigated via hydrolysis of p‐nitrophenyl acetate (PNPA) in different pH environment, cationic surfactants, and inorganic salts. The influence of hydrophilicity has been studied in the presence of cationic cetyltrimethylammonium bromide (CTAB), cetyltriphenylphosphonium bromide (CTPB), and cetyldimethylethanolammonium bromide (CDMEAB) surfactants. The head group dimensions of surfactants show noteworthy contributions to enhance the catalytic activity of MJL, that is the CDMEAB surfactant, which is more efficient when compared with CTPB and CTAB. The increase in hydrophobicity of alkyltrimethylammonium bromide and alkyltriphenylphosphonium bromide in series dodecyl (C12) < tetradecyl (C14) < cetyl (C16) leads to a marked decrease in the observed enzymatic superactivity, but an opposite trend appears in case of alkyldimethylammonium bromide. The activity of MJL for the hydrolysis of PNPA was higher in the presence of sodium chloride and potassium chloride anion due to specific interactions between the lipase surface and anions of the Hofmeister effect.

A De Novo Designed Esterase with p-Nitrophenyl Acetate Hydrolysis Activity

Esterases are a large family of enzymes with wide applications in the industry. However, all esterases originated from natural sources, limiting their use in harsh environments or newly- emerged reactions. In this study, we designed a new esterase to develop a new protocol to satisfy the needs for better biocatalysts. The ideal spatial conformation of the serine catalytic triad and the oxygen anion hole at the substrate-binding site was constructed by quantum mechanical calculation. The catalytic triad and oxygen anion holes were then embedded in the protein scaffold using the new enzyme protocol in Rosetta 3. The design results were subsequently evaluated, and optimized designs were used for expression and purification. The designed esterase had significant lytic activities towards p-nitrophenyl acetate, which was confirmed by point mutations. Thus, this study developed a new protocol to obtain novel enzymes that may be useful in unforgiving environments or novel reactions.

Biocatalysis of D,L-Peptide Nanofibrillar Hydrogel.

Self-assembling peptides are attracting wide interest as biodegradable building blocks to achieve functional nanomaterials that do not persist in the environment. Amongst the many applications, biocatalysis is gaining momentum, although a clear structure-to-activity relationship is still lacking. This work applied emerging design rules to the heterochiral octapeptide sequence His–Leu–DLeu–Ile–His–Leu–DLeu–Ile for self-assembly into nanofibrils that, at higher concentration, give rise to a supramolecular hydrogel for the mimicry of esterase-like activity. The peptide was synthesized by solid-phase and purified by HPLC, while its identity was confirmed by 1H-NMR and electrospray ionization (ESI)-MS. The hydrogel formed by this peptide was studied with oscillatory rheometry, and the supramolecular behavior of the peptide was investigated with transmission electron microscopy (TEM) analysis, circular dichroism (CD) spectroscopy, thioflavin T amyloid fluorescence assay, and attenuated total reflectance (ATR) Fourier-transform infrared (FT-IR) spectroscopy. The biocatalytic activity was studied by monitoring the hydrolysis of p-nitrophenyl acetate (pNPA) at neutral pH, and the reaction kinetics followed an apparent Michaelis–Menten model, for which a Lineweaver–Burk plot was produced to determine its enzymatic parameters for a comparison with the literature. Finally, LC–MS analysis was conducted on a series of experiments to evaluate the extent of, if any, undesired peptide acetylation at the N-terminus. In conclusion, we provide new insights that allow gaining a clearer picture of self-assembling peptide design rules for biocatalysis.

Zn-triazole coordination polymers: Bioinspired carbonic anhydrase mimics for hydration and sequestration of CO2

Artificial enzyme mimics have recently emerged as alternative biocatalysts for overcoming the intrinsic fragility of natural enzyme in practical applications. However, current researches regarding mimetic enzymes are still confined to very few reaction types, with artificial oxidoreductases as dominance. Herein, inspired by nature, we designed and fabricated a range of Zn-Triazole coordination polymers (ZnTazs) that presented similar coordination structures with the active site of natural carbonic anhydrase Ⅱ (CA Ⅱ). These synthesized compounds exhibited inherently mimetic function with natural CA for catalyzing the hydrolysis of p-nitrophenyl acetate (p-NPA). Especially, the initial hydrolysis rate (V0) of p-NPA catalyzed by ZnTaz-1 (Zn5(bta)6(NO3)4 · H2O) and ZnTaz-2 (Zn3(OH)2(btca)2 · DMF · 4 H2O) reached 42.1 and 73.8 nM · s−1, respectively. Meanwhile, ZnTaz-1 and ZnTaz-2 showed favorable recyclability, and excellent stability towards various pH values and organic solvents, which are of great significance for practical employment. Moreover, they could also promote the efficient hydration and sequestration of greenhouse gas CO2 in aqueous medium. Based on this work, we aim to provide more theoretical and practical basis for rational design of CA mimics from the inspiration of natural enzyme, as well as propose a potential strategy for tackling the CO2 crisis.

Smart bilayer polymer reactor with cascade/non-cascade switching catalyst characteristics

In this work, a new method is proposed to meet the challenge of preparing new catalysts with cascade/non-cascade switching catalytic property. Inspired from “soft” characteristics and divisional isolation function in natural biological systems, this objective was accomplished by developing a new class of hydrogels made of two unique functional layers with different temperature responses where each may self-govern coupled processes at a specific temperature. This hydrogel polymer reactor exhibited almost no catalytic activity at low-temperature range (<37 °C) as both channels of bilayer hydrogel polymer catalyst were closed. At modest temperatures (between 37 °C and 50 °C), the first step of the tandem reaction (the hydrolysis of p-nitrophenyl acetate (NPA)) showed significant reactivity that arises from the relaxing of the weak polymer complexes in the hydrogel layer. This enabled NPA the access to the acidic catalytic active center of the hydrogel. At range of higher temperatures (>50 °C), the hydrogel catalytic polymer reactor further exhibited significant efficiency towards the hydrolysis reaction of NPA as well as the reduction of the intermediate product p-nitrophenol (NP). This mainly resulted from the opening of both the weak polymer complexes and the stronger polymer complexes hydrogel layers, allowing entrance to both the acidic catalytic active center and the metal nanoparticles active center. As a result, the novel hydrogel polymer reactor could be used to control cascade/non-cascade catalysis reactions. This new protocol enables efficient control of switchable tandem reactions, inspiring for difficulty to control tandem catalytic reactors.

Synthesis and structure-affinity relationship of chlorinated pyrrolidinone-bearing benzenesulfonamides as human carbonic anhydrase inhibitors.

A novel set of pyrrolidinone-based chlorinated benzenesulfonamide derivatives were synthesized and investigated for their binding affinity and selectivity against recombinant human carbonic anhydrases I-XIV using fluorescent thermal shift, p-nitrophenyl acetate hydrolysis and stopped-flow enzymatic inhibition assays. The hydrazones 10-22 prepared from 1-(2-chloro-4-sulfamoylphenyl)-5-oxopyrrolidine-3-carboxylic acid exhibited low nanomolar affinity against cancer-related CA IX (Kd in the range of 5.0-37nM). Compounds with triazole or oxadiazole groups attached directly to pyrrolidinone moiety bound all CAs weaker than compounds with more flexible tail groups. Chloro group at the meta position of benzenesulfonamide derivatives increased affinity to all CAs as compared with binding data for nonchlorinated compounds. The compounds have a potential for further development of CA inhibitors with higher selectivity for a particular CA isozyme.

Immobilisation of lipase enzyme onto bacterial magnetosomes for stain removal

Lipase was immobilized onto bacterial magnetosomes using glutaraldehyde cross-linking and confirmed by Fourier transform infrared spectrometry (FT-IR) and Scanning electron microscopy (SEM). Enzyme activity of immobilised lipase as well as free lipase was estimated by the release of p-nitro phenol due to the hydrolysis of p-nitro phenyl acetate (pNPA). The immobilisation yield of lipase onto magnetosome was found to be 88 %. The optimal pH (7) and temperature (40 °C) for activity was standardised and found to be similar to free lipase. The stored immobilized lipase maintained higher activity even after 30 days at a temperature of 4 °C whereas compared to free lipase. Immobilized lipase found to have removed vegetable oil stain and showed higher cleaning efficiency when compared to free lipase. The results suggest that bacterial magnetosome displays great potential as a support material for the immobilization of industrial enzymes such as lipase.