Water-soluble Enzyme Research Articles

Immobilization of Water-Soluble HRP within Poly-N-isopropylacrylamide Microgel Particles for Use in Organic Media

In the present work, the immobilization of enzymes within poly-N-isopropylacrylamide (p-NIPAM) microgels using the method of solvent exchange is applied to the enzyme horseradish peroxidase (HRP). When the solvent is changed from water to isopropanol, HRP is embedded within the polymer structure. After the determination of the immobilized amount of enzyme, an enhanced specific activity of the biocatalyst in isopropanol can be observed. Karl Fischer titration is used to determine the amount of water within the microgel particles before and after solvent exchange, leading to the conclusion that an "aqueous cage" remains within the polymer structure. This represents the driving force for the immobilization due to the high affinity of HRP for water. Beside, confocal laser scanning microscopy (CLSM) images show that HRP is located within the microgel network after immobilization. This gives the best conditions for HRP to be protected against chemical and mechanical stress. We were able to transfer a water-soluble enzyme to an organic phase by reaching a high catalytic activity. Hence, the method of solvent exchange displays a general method for immobilizing enzymes within p-NIPAM microgels for use in organic solvents. With this strategy, enzymes that are not soluble in organic solvents such as HRP can be used in such polar organic solvents.

Designing Efficient Oxygen Scavenging Coating Formulations for Food Packaging Applications

Modified atmosphere packaging is widely used to prolong the shelf life of many oxygen‐sensitive items including food, pharmaceuticals, work of art and so on. For food packaging applications, we investigated the effectiveness of oxygen scavenging coatings and flexographic printing ink formulations made using water soluble and water insoluble ascorbate enzyme systems. The present study summarizes the effects of various parameters on the oxygen scavenging rates and other performance aspects of the formulations. It is shown that; the initial oxygen scavenging rate can be increased by increasing the surface area of Calcium Ascorbate crystals and addition of catalyst or edible oil; addition of a catalyst like TiO2 or Alumina and an edible oil in Ascorbate and laccase‐based formulations induces a synergistic effect upon the rate of scavenging oxygen from the packages; an efficient oxygen scavenging coating formulation can be made using water soluble Calcium Ascorbate and insoluble Ascorbate Palmitate containing enzyme and substrate; designing an efficient oxygen scavenging formulation using water insoluble Ascorbate Palmitate can reduce or eliminate formation of effluent which has potential to change the aperral of food items inside the package; an adverse effect of some binders which cause a reduction in the oxygen scavenging rate can be eliminated by making blends of different binders. Copyright © 2013 John Wiley & Sons, Ltd.

Insertion of the Ca2+-Independent Phospholipase A2 into a Phospholipid Bilayer via Coarse-Grained and Atomistic Molecular Dynamics Simulations

Group VI Ca2+-independent phospholipase A2 (iPLA2) is a water-soluble enzyme that is active when associated with phospholipid membranes. Despite its clear pharmaceutical relevance, no X-ray or NMR structural information is currently available for the iPLA2 or its membrane complex. In this paper, we combine homology modeling with coarse-grained (CG) and all-atom (AA) molecular dynamics (MD) simulations to build structural models of iPLA2 in association with a phospholipid bilayer. CG-MD simulations of the membrane insertion process were employed to provide a starting point for an atomistic description. Six AA-MD simulations were then conducted for 60 ns, starting from different initial CG structures, to refine the membrane complex. The resulting structures are shown to be consistent with each other and with deuterium exchange mass spectrometry (DXMS) experiments, suggesting that our approach is suitable for the modeling of iPLA2 at the membrane surface. The models show that an anchoring region (residues 710–724) forms an amphipathic helix that is stabilized by the membrane. In future studies, the proposed iPLA2 models should provide a structural basis for understanding the mechanisms of lipid extraction and drug-inhibition. In addition, the dual-resolution approach discussed here should provide the means for the future exploration of the impact of lipid diversity and sequence mutations on the activity of iPLA2 and related enzymes.

MECHANISMS OF COMBINED ACTION OF ACE INHIBITORS AND CALCIUM ANTAGONISTS IN ARTERIAL HYPERTENSION

This review presents the modern views on the effectiveness of water-soluble angiotensin-converting enzyme (ACE) antagonists and calcium antagonists (CA), as monotherapy or as fixed-dose combination therapy, in the treatment of arterial hypertension (AH). The results of the X-ray structural analysis of crystal ACE-lisinopril complexes suggest a highly selective, strong inhibitory effect of lisinopril on various ACE forms of the hormonal renin-angiotensin-aldosterone system and tissue renin-angiotensin systems. The association between specific action mechanisms and therapeutic effects in hypertension is analysed for a third-generation CA amlodipine. The benefits of a fixed-dose combination medication Ekvator (amlodipine 5 mg plus lisinopril 10 mg) in hypertension are described, including its synergetic protective effects.

Strategies to characterize fungal lipases for applications in medicine and dairy industry.

Lipases are water-soluble enzymes that act on insoluble substrates and catalyze the hydrolysis of long-chain triglycerides. Lipases play a vital role in the food, detergent, chemical, and pharmaceutical industries. In the past, fungal lipases gained significant attention in the industries due to their substrate specificity and stability under varied chemical and physical conditions. Fungal enzymes are extracellular in nature, and they can be extracted easily, which significantly reduces the cost and makes this source preferable over bacteria. Soil contaminated with spillage from the products of oil and dairy harbors fungal species, which have the potential to secrete lipases to degrade fats and oils. Herein, the strategies involved in the characterization of fungal lipases, capable of degrading fatty substances, are narrated with a focus on further applications.

A β-cyclodextrin–chitosan complex as the immobilization matrix for horseradish peroxidase and its application for the removal of azo dyes from textile effluent

The aim of this study was to develop a system based on the use of immobilized horseradish peroxidase (HRP) for the removal of color due to azo dyes from a textile effluent. The maximum binding of HRP on a β-cyclodextrin (CD)–chitosan complex was achieved at pH 8.0. Oxidative removal of azo dyes from effluent by water-soluble and β-CD–chitosan–HRP inclusion complexes was monitored by HPLC analysis. The crosslinked peroxidase preparation made with a β-CD–chitosan complex was more active, effective and stable than the un-crosslinked and the water-soluble enzyme preparations. The maximum dye oxidation from the diluted textile effluent was observed in the presence of 0.6 mM H2O2 and 0.2 U ml−1 at pH 8.0 and at 40 °C for 2 h. A batch process with HRP was also performed, whereby complete dye removal from the effluent was obtained with a crosslinked β-CD–chitosan complex within 120 min. On the other hand, a β-CD–chitosan–HRP-based continuous bed reactor was found to be efficient for complete removal of the dye from the effluent during 15 days of operation. The reuse of the crosslinked enzyme preparations proved to be more effective in the decolorization of textile effluent than the un-crosslinked enzyme preparations. Based on a comet assay the genotoxicity associated the colored effluent was completely lost after HRP oxidation of the dye, showing the dye metabolites are not toxic.

Site-Specific Immobilization of Enzymes on Magnetic Nanoparticles and Their Use in Organic Synthesis

Magnetic nanoparticles (MNPs) are attractive materials that serve as a support for enzyme immobilization and facilitate separations by applying an external magnetic field; this could facilitate the recycling of enzymes and broaden their applications in organic synthesis. Herein, we report the methods for the immobilization of water-soluble and membrane-bound enzymes, and the activity difference between free and immobilized enzymes is discussed. Sialyltransferase (PmST1, from Pasteurella multocida ) and cytidine monophosphate (CMP)-sialic acid synthetase (CSS, from Neisseria meningitides ) were chosen as water-soluble enzymes and expressed using an intein expression system. The enzymes were site-specifically and covalently immobilized on PEGylated-N-terminal cysteine MNPs through native chemical ligation (NCL). Increasing the length of the PEG linker between the enzyme and the MNP surface increased the activity of the immobilized enzymes relative to the free parent enzymes. In addition, the use of a fluorescent acceptor tag for PmST1 affected enzyme kinetics. In contrast, sialyltransferase from Neisseria gonorrheae (NgST, a membrane-bound enzyme) was modified with a biotin-labeled cysteine at the C-terminus using NCL, and the enzyme was then assembled on streptavidin-functionalized MNPs. Using a streptavidin-biotin interaction, it was possible to immobilize NgST on a solid support under mild ligation conditions, which prevented the enzyme from high-temperature decomposition and provided an approximately 2-fold increase in activity compared to other immobilization methods on MNPs. Finally, the ganglioside GM3-derivative (sialyl-lactose derivative) was synthesized in a one-pot system by combining the use of immobilized PmST1 and CSS. The enzymes retained 50% activity after being reused ten times. Furthermore, the results obtained using the one-pot two-immobilized-enzyme system demonstrated that it can be applied to large-scale reactions with acceptable yields and purity. These features make enzyme-immobilized MNPs applicable to organic synthesis.

Possible mechanisms of the influence of hexylresorcinol on the structure-dynamic and functional properties of lysozyme protein

The influence of hexylresorcinol on the structure, equilibrium fluctuations, and functional activity of water-soluble enzyme lysozyme was studied over a wide range of hexylresorcinol concentrations. Hexylresorcinol was found to be not only a stabilizer of lysozyme. At low hexylresorcinol concentrations (2 to 10 molecules per lysozyme globule), the activity of lysozyme sharply increased; activity began to decrease as the concentration grew. The influence of hexylresorcinol on the structural, dynamic, and functional lysozyme characteristics is well described by models of preferential hydration and preferential protein interaction with hexylresorcinol. The hexylresorcinol molecule consists of hydrophobic (alkyl radical) and hydrophilic (aromatic nucleus) moieties, which has additional regulatory action on the functional activity of lysozyme. As the concentration of hexylresorcinol increases, the effect of regions with preferential hydration begins to noticeably predominate over the effect of preferential interaction with hexylresorcinol. At hexylresorcinol concentrations higher than 100 molecules per lysozyme globule, the activity of lysozyme is fully inhibited. This is caused by the preferential hydration of the protein with the displacement of hexylresorcinol from direct contacts with it. The displacement of hexylresorcinol causes the formation of high-density hexylresorcinol micelles. Dense micelles interfere with the approach of substrates to the protein and fully inhibit its functional activity. The complete inhibition of lysozyme activity occurs at hexylresorcinol concentrations lower by an order of magnitude than glycerol inhibiting concentrations.

Immobilization of lipase B within micron-sized poly-N-isopropylacrylamide hydrogel particles by solvent exchange

The aim of the present work is the use of a water soluble enzyme in an organic solvent, still with a pronounced catalytic activity. Therefore, lipase B from Candida antarctica (CalB) is immobilized within micron-sized thermosensitive p-NIPAM hydrogel particles using a solvent exchange from polar to organic solvents. The absorbed amount of CalB is investigated at different immobilization temperatures. Confocal laser scanning microscopy (CLSM) shows that CalB is homogeneously distributed within the polymer network. An enhanced specific activity of CalB in n-hexane is achieved after immobilization within the p-NIPAM microgels. In order to get information on the supply of the substrate depending on the temperature, the activity is determined at different reaction temperatures. Additionally, the system is stable in the organic solvent, namely n-hexane, and shows a good reusability.

Substrate Specificity of Low-Molecular Mass Bacterial dd-Peptidases

The bacterial DD-peptidases or penicillin-binding proteins (PBPs) catalyze the formation and regulation of cross-links in peptidoglycan biosynthesis. They are classified into two groups, the high-molecular mass (HMM) and low-molecular mass (LMM) enzymes. The latter group, which is subdivided into classes A-C (LMMA, -B, and -C, respectively), is believed to catalyze DD-carboxypeptidase and endopeptidase reactions in vivo. To date, the specificity of their reactions with particular elements of peptidoglycan structure has not, in general, been defined. This paper describes the steady-state kinetics of hydrolysis of a series of specific peptidoglycan-mimetic peptides, representing various elements of stem peptide structure, catalyzed by a range of LMM PBPs (the LMMA enzymes, Escherichia coli PBP5, Neisseria gonorrhoeae PBP4, and Streptococcus pneumoniae PBP3, and the LMMC enzymes, the Actinomadura R39 dd-peptidase, Bacillus subtilis PBP4a, and N. gonorrhoeae PBP3). The R39 enzyme (LMMC), like the previously studied Streptomyces R61 DD-peptidase (LMMB), specifically and rapidly hydrolyzes stem peptide fragments with a free N-terminus. In accord with this result, the crystal structures of the R61 and R39 enzymes display a binding site specific to the stem peptide N-terminus. These are water-soluble enzymes, however, with no known specific function in vivo. On the other hand, soluble versions of the remaining enzymes of those noted above, all of which are likely to be membrane-bound and/or associated in vivo and have been assigned particular roles in cell wall biosynthesis and maintenance, show little or no specificity for peptides containing elements of peptidoglycan structure. Peptidoglycan-mimetic boronate transition-state analogues do inhibit these enzymes but display notable specificity only for the LMMC enzymes, where, unlike peptide substrates, they may be able to effectively induce a specific active site structure. The manner in which LMMA (and HMM) DD-peptidases achieve substrate specificity, both in vitro and in vivo, remains unknown.

Designer Nanorings with Functional Cavities from Self‐Assembling β‐Sheet Peptides

β-Barrel proteins that take the shape of a ring are common in many types of water-soluble enzymes and water-insoluble transmembrane pore-forming proteins. Since β-barrel proteins perform diverse functions in the cell, it would be a great step towards developing artificial proteins if we can control the polarity of artificial β-barrel proteins at will. Here, we describe a rational approach to construct β-barrel protein mimics from the self-assembly of peptide-based building blocks. With this approach, the direction of the self-assembly process toward the formation of water-soluble β-barrel nanorings or water-insoluble transmembrane β-barrel pores could be controlled by the simple but versatile molecular manipulation of supramolecular building blocks. This study not only delineates the basic driving force that underlies the folding of β-barrel proteins, but also lays the foundation for the facile fabrication of β-barrel protein mimics, which can be developed as nanoreactors, ion- and small-molecule-selective pores, and novel antibiotics.

If the KI Is Defined by the Free Energy of Binding to P-Glycoprotein, Which Kinetic Parameters Define the IC50 for the Madin-Darby Canine Kidney II Cell Line Overexpressing Human Multidrug Resistance 1 Confluent Cell Monolayer?

From previous fits of drug transport kinetics across confluent Madin-Darby canine kidney II cell line overexpressing human multidrug resistance 1 cell monolayers, we found that a drug's binding constant to P-glycoprotein (P-gp) was significantly smaller than its IC(50) when that drug was used as an inhibitor against another P-gp substrate. We tested several IC(50) candidate functions, including the standard function, the Kalvass-Pollack function, and the efflux ratio, to determine whether any of them yielded an IC(50) = K(I), as would be expected for water-soluble enzymes. For the confluent cell monolayer, the IC(50)/K(I) ratio is greater than 1 for all candidate functions tested. From the mass action kinetic model, we have derived a simple approximate equation that shows how the IC(50)/K(I) ratio depends on the elementary rate constants from our mass action model. Thus, the IC(50) will differ between cell lines and tissues, for the same probe substrate and inhibitor, if there are different membrane concentrations of P-gp, or the probe substrate's elementary rate constants, partition coefficient, binding constant to P-gp, passive permeability, and ability to access the other transporters (if any) in the two cell lines. The mass action model and the approximate equation for the IC(50)/K(I) ratio derived here can be used to estimate the elementary rate constants needed to extrapolate in vitro drug-drug interactions for compounds to the in vivo environment.

High catalytic performances of Pseudomonas fluorescens lipase adsorbed on a new type of cyclodextrin-based nanosponges

Lipases are water-soluble enzymes that catalyze the hydrolysis of triacylglycerols (in aqueous media) or trans-esterification reactions (in microaqueous media) and are involved in a number of industrial applications. As a limit to lipase application is represented by the need for interfacial activation, the search for suitable solid supports able to fulfill this requirement is always ongoing. In the present work, we report the preliminary characterization of a system obtained by adsorbing Pseudomonas fluorescens lipase on a newly synthesized cyclodextrin-based carbonate nanosponge (CD–NS–1:4). The activity and structural stability of lipase adsorbed on this new support were evaluated by checking the effect of temperature, pH changes and organic solvents (methanol) on the enzyme structure and function, which were compared with those of the free enzyme in solution. Our data show that the non-covalent interaction of Ps. fluorescens lipase with CD–NS–1:4 results in enzyme structural and functional stabilization, as it was still active after 66 days of incubation at T ∼ 18 °C. Stabilization with respect to T, pH and the presence of organic solvent was observed as well as, unlike the solubilized enzyme, the adsorbed lipase was active at T > 40 °C, at pH 5 and after 24-h incubation with 70% (v/v) methanol (13% residual activity).

Refolding and purification of the human secreted group IID phospholipase A 2 expressed as inclusion bodies in Escherichia coli

The secreted phospholipases A 2 (sPLA 2s) are water-soluble enzymes that bind to the surface of both artificial and biological lipid bilayers and hydrolyze the membrane phospholipids. The tissue expression pattern of the human group IID secretory phospholipase A 2 (hsPLA 2-IID) suggests that the enzyme is involved in the regulation of the immune and inflammatory responses. With an aim to establish an expression system for the hsPLA 2-IID in Escherichia coli, the DNA-coding sequence for hsPLA 2-IID was subcloned into the vector pET3a, and expressed as inclusion bodies in E. coli (BL21). A protocol has been developed to refold the recombinant protein in the presence of guanidinium hydrochloride, using a size-exclusion chromatography matrix followed by dilution and dialysis to remove the excess denaturant. After purification by cation-exchange chromatography, far ultraviolet circular dichroism spectra of the recombinant hsPLA 2-IID indicated protein secondary structure content similar to the homologous human group IIA secretory phospholipase A 2. The refolded recombinant hsPLA 2-IID demonstrated Ca 2+-dependent hydrolytic activity, as measuring the release free fatty acid from phospholipid liposomes. This protein expression and purification system may be useful for site-directed mutagenesis experiments of the hsPLA 2-IID which will advance our understanding of the structure–function relationship and biological effects of the protein.

Self-Assembling of Polymer-Enzyme Conjugates at Oil/Water Interfaces

Interface-binding enzymes are desirable for biphasic reactions in that they offer simultaneous access to substrates dissolved in both phases across the interface. It has been shown that conjugating water-soluble enzymes with hydrophobic polymers facilitated the assembling of enzymes at oil/water interfaces. In this work, the interfacial assembling of alpha-chymotrypsin conjugated with polystyrene, poly(methyl methacrylate), and poly(l-lactic acid) was examined using the pendant drop method. The interface-assembling process of the conjugates from the organic phase followed a similar pattern of that of native alpha-chymotrypsin from the aqueous buffer phase, i.e., the interfacial tension decreased gradually with time. However, when the conjugates were dispersed in the form of particulates in the aqueous phase, in which the conjugate was insoluble, the assembling occurred faster and the interfacial tension quickly approached zero. It was suspected that the assembling in this case involved two steps, i.e., the adsorption of the particulates and the subsequent rearrangement, dissociation, and redispersion of the conjugates at the interface. The effect of other factors, including the polarity of organic solvent and pH and ionic strength of the aqueous phase, was evaluated. It was found that the polar solvent slightly facilitated the assembling, whereas pH and ionic strength showed minimal effects.

Enzymes Without Borders: Mobilizing Substrates, Delivering Products

Many cellular reactions involve both hydrophobic and hydrophilic molecules that reside within the chemically distinct environments defined by the phospholipid-based membranes and the aqueous lumens of cytoplasm and organelles. Enzymes performing this type of reaction are required to access a lipophilic substrate located in the membranes and to catalyze its reaction with a polar, water-soluble compound. Here, we explore the different binding strategies and chemical tricks that enzymes have developed to overcome this problem. These reactions can be catalyzed by integral membrane proteins that channel a hydrophilic molecule into their active site, as well as by water-soluble enzymes that are able to capture a lipophilic substrate from the phospholipid bilayer. Many chemical and biological aspects of this type of enzymology remain to be investigated and will require the integration of protein chemistry with membrane biology.

Vitamin B Biosynthesis in Plants

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Human sulfatases: a structural perspective to catalysis.

The sulfatase family of enzymes catalyzes hydrolysis of sulfate ester bonds of a wide variety of substrates. Seventeen genes have been identified in this class of sulfatases, many of which are associated with genetic disorders leading to reduction or loss of function of the corresponding enzymes. Amino acid sequence homology suggests that the enzymes have similar overall folds, mechanisms of action, and bivalent metal ion-binding sites. A catalytic cysteine residue, strictly conserved in prokaryotic and eukaryotic sulfatases, is post-translationally modified into a formylglycine. Hydroxylation of the formylglycine residue by a water molecule forming the activated hydroxylformylglycine (a formylglycine hydrate or a gem-diol) is a necessary step for the enzyme's sulfatase activity. Crystal structures of three human sulfatases, arylsulfatases A and B(ARSA and ARSB), and estrone/dehydroepiandrosterone sulfatase or steroid sulfatase (STS), also known as arylsulfatase C, have been determined. While ARSA and ARSB are water-soluble enzymes, STS has a hydrophobic domain and is an integral membrane protein of the endoplasmic reticulum. In this article, we compare and contrast sulfatase structures and revisit the proposed catalytic mechanism in light of available structural and functional data. Examination of the STS active site reveals substrate-specific interactions previously identified as the estrogen-recognition motif. Because of the proximity of the catalytic cleft of STS to the membrane surface, the lipid bilayer has a critical role in the constitution of the active site, unlike other sulfatases.

Changes in pectic substances and cell wall hydrolase enzymes of mangosteen (Garcinia mangostana) fruit during storage

Mangosteen (Garcinia mangostana) fruits at mature green stage with 5% red spotting were held at room temperature (31–35°C, 84 ± 2% relative humidity (RH)) and at the storage temperature of 13°C (90 ± 2% RH). Firmness, pectin content, and activities of pectin methylesterase and polygalacturonase in the pulp (aril) were monitored every 2 days. Pulp firmness rapidly decreased at room temperature from day 0 to day 4 after storage and slightly decreased thereafter, whereas pulp firmness slowly decreased at 13°C. Water soluble pectin content in pulp increased more rapidly at room temperature than at 13°C. Insoluble pectin content in pulp decreased throughout the storage time at both storage conditions. Both pectin methylesterase and polygalacturonase activities in pulp increased more rapidly at room temperature than at 13°C. Changes in water soluble pectin and cell wall hydrolase enzymes in relation to pulp softening of mangosteen fruits are discussed.

Synthesis of sn-1 functionalized phospholipids as substrates for secretory phospholipase A 2

Secretory phospholipase A 2 (sPLA 2) represents a family of small water-soluble enzymes that catalyze the hydrolysis of phospholipids in the sn-2 position liberating free fatty acids and lysophospholipids. Herein we report the synthesis of two new phospholipids ( 1 and 2) with bulky allyl-substituents attached to the sn-1 position of the glycerol backbone. The synthesis of phospholipids 1 and 2 is based upon the construction of a key aldehyde intermediate 3 which locks the stereochemistry in the sn-2 position of the final phospholipids. The aldehyde functionality serves as the site for insertion of the allyl-substituents by a zinc mediated allylation. Small unilamellar liposomes composed of phospholipids 1 and 2 were subjected to sPLA 2 activity measurements. Our results show that only phospholipid 1 is hydrolyzed by the enzyme. Molecular dynamics simulations revealed that the lack of hydrolysis of phospholipid 2 is due to steric hindrance caused by the bulky side chain of the substrate allowing only limited access of water molecules to the active site.