Hydrolytic Activity Assays Research Articles

Inhibitory Effect of Thymol on Tympanostomy Tube Biofilms of Methicillin-Resistant Staphylococcus aureus and Ciprofloxacin-Resistant Pseudomonas aeruginosa.

The formation of antibiotic-resistant strain biofilms in tympanostomy tubes results in persistent and refractory otorrhea. In the present study, we investigated the in vitro antibiofilm activity of thymol against biofilms formed by methicillin-resistant Staphylococcus aureus (MRSA) and ciprofloxacin-resistant Pseudomonas aeruginosa (CRPA), using live and dead bacterial staining and adhesion, biofilm formation, biofilm eradication, and biofilm hydrolytic activity assays. The antibiofilm activity of thymol against tympanostomy tube biofilms formed by MRSA and CRPA strains was examined using a scanning electron microscope. In response to thymol treatment, we detected significant concentration-dependent reductions in the viability and adhesion of MRSA and CRPA. Exposure to thymol also inhibited the formation of both MRSA and CRPA biofilms. Furthermore, thymol was observed to enhance the eradication of preformed mature biofilms produced by MRSA and CRPA and also promoted a reduction in the rates of MRSA and CRPA hydrolysis. Exposure to thymol eradicated extracellular polysaccharide present in the biofilm matrix produced by MRSA and CRPA. Additionally, thymol was observed to significantly eradicate MRSA and CRPA biofilms that had formed on the surface on tympanostomy tubes. Collectively, our findings indicate that thymol is an effective inhibitor of MRSA and CRPA biofilms, and accordingly has potential utility as a therapeutic agent for the treatment of biofilm-associated refractory post-tympanostomy tube otorrhea resulting from MRSA and CRPA infection.

Quantitative assessments of adenosine triphosphatase hydrolytic activity by ultrafiltration-coupled ion-pair reversed-phase high-performance liquid chromatography.

Adenosine triphosphate is a universal energy currency that can directly provide energy required for a multitude of biochemical reactions and biophysical actions through adenosine triphosphatase catalyzed hydrolysis. Adenosine triphosphatase activity is thus one important feature for the characterization of protein function and cell activity. Herein, we optimized ion-pair reversed-phase high-performance liquid chromatography technique for highly efficient separation of adenosine triphosphate, adenosine diphosphate, and adenosine monophosphate, and the method demonstrated good linearity. Moreover, by coupling a protein-removable ultrafiltration, we developed a sensitive and robust approach for quantification of adenosine triphosphatase hydrolytic activity. By this assay, we demonstrated that RecA filaments-catalyzed adenosine triphosphate hydrolysis approached a second-order reaction, and its rate constant was estimated as 0.057mM-1 min-1 . In addition, we explored the effects of DNA length on this reaction and revealed that the increase of the length of single-stranded DNA can promote the adenosine triphosphatase hydrolytic activity of RecA filaments. All these results confirm the feasibility of this new method in quantification of adenosine triphosphatase hydrolytic activity assays. Compared with previous complicated enzyme-coupled or homogeneous colorimetric measurements, the developed approach with high resolution separation allows a simple reaction system for adenosine triphosphatase assay and a sensitive detection free of interference from background noise.

Report of false positives when using zymography to assess peptidoglycan hydrolytic activity of an endopeptidase with multiple LysM domains

Zymography is a widely used technique enabling visualization of in-gel peptidase/protease hydrolytic activities. This technique is used to study the activity of bacterial peptidoglycan (PG) hydrolytic enzymes named autolysins. Zymography is particularly suited for PG autolysin characterization as bulk PG is notorious to work with due to its highly insoluble nature. This recalcitrant property of PG therefore makes the set-up of PG hydrolytic activity assay very challenging. In the course of studying the catalytic activity of the CwlS protein, a D,L NlpC/P60 endopeptidase possessing multiple LysM carbohydrate-binding domains from Bacillus subtilis, we observed a potential artifact of the zymography technique. The generation of CwlS truncated mutants impaired in their PG binding capacity presented lower apparent hydrolytic activities on zymograms. Furthermore, a catalytically dead version of CwlS, or a CwlS mutant that possesses only its LysM domains and no catalytic domain, maintained similar apparent PG hydrolytic properties as wild-type CwlS on zymograms. Additionally, a mutant harboring twelve mutations in the four LysM domains, previously demonstrated to be unable to bind PG but has a similar net positive charge as the wild-type protein also presented apparent activity on zymogram. We demonstrate in this study that zymography results, which are meant to be interpreted in favor of apparent PG hydrolytic activities, are instead reflecting impairment of gel staining probably due to the very high net positive charge of the protein.

Assessment of E. coli Expression System for Overexpression of Active Recombinant Ocriplasmin.

Purpose: Ocriplasmin (Jetrea TM) is a FDA approved recombinant enzyme utilized in the treatment of vitreomacular adhesion (VMA). This is a recombinant C-terminal fragment of human plasmin produced using yeast Pichia pastoris. Since ocriplasmin does not contain any Oor N-glycosylation or some other post-translational modifications, bacterial expression systems such as Escherichia coli could be considered as an economical host for recombinant expression. In the present study, we aimed to evaluate the efficiency of E. coli expression system for highlevel expression of recombinant ocriplasmin.Methods: The gene coding for ocriplasmin was cloned and expressed in E. coli BL21. The bacterial cells were cultured on large scale and the expressed recombinant protein was purified using Ni-NTA chromatography. Refolding of denatured ocriplasmin to active enzyme was carried out by the stepwise removal of denaturant. The identity of recombinant ocriplasmin was confirmed using western blotting and ELISA assays. The presence of the active ocriplasmin was monitored by the hydrolytic activity assay against the chromogenic substrate S-2403.Results: The final yield of E. coli BL21-produced ocriplasmin was approximately 1 mg/mL which was greater than that of P. pastoris. Using western blotting and ELISA assay, the identity of recombinant ocriplasmin was confirmed. The hydrolysis of chromogenic substrate S-2403 verified the functional activity of E. coli produced ocriplasmin.Conclusion: The results of this study indicated that E. coli could be used for high level expression of ocriplasmin. Although the recombinant protein was expressed as inclusion body, the stepwise refolding leads to the biologically active proteins.

The hydrolysis of indoxyl acetate: A versatile reaction to assay carbonic anhydrase activity by high-throughput screening

The interest in CO2 capture and conversion by biological methodologies into various beneficial products is increased. In nature, there are enzymes, with hydration activity, which catalyze the reversible hydration of the CO2 molecule and, thus, are of high interest for biotechnological applications. Such enzymes are carbonic anhydrases (CAs). Structural, functional and mutational studies have shown, that besides hydratase activity, CAs have exposed hydrolytic, particularly esterase activity, and importantly, both activities follow similar catalytic mechanisms in the same catalytic pocket. CAs activity measurement methods based on electrometric assays for hydration activity and nitrophenyl based esters for hydrolytic activity assays do not fulfill the requirements amenable for enzyme activity screening methods. By this study, we were aiming to develop an indigogenic assay method based on the esterase activity of CAs. The first time use of indoxyl acetate as a substrate for CA has shown promising results to gain simplicity, repeatability, and applicability to implement high-throughput screening methods.

CTX-M-33, a CTX-M-15 derivative conferring reduced susceptibility to carbapenems.

CTX-M-type extended-spectrum ß-lactamases (ESBL) are widespread among Enterobacterales worldwide. The most common variant is CTX-M-15 hydrolyzing ceftazidime at high rate, but sparing carbapenems. We identified here CTX-M-33, a point mutant derivative of CTX-M-15 (Asp to Ser substitution at Ambler position 109), exhibiting a low carbapenemase activity. ß-Lactamase CTX-M-33 was identified in a Klebsiella pneumoniae isolate belonging to ST405, lacking the outer membrane protein OmpK36, that was resistant to broad-spectrum cephalosporins and ß-lactam/ß-lactamase inhibitor combinations, and displayed a decreased susceptibility to carbapenems. Comparative hydrolytic activity assays showed that CTX-M-33 hydrolyzed ceftazidime at a lower level than CTX-M-15, but significantly hydrolyzed meropenem. In addition, CTX-M-33 showed higher Mutant Prevention Concentration values and wider mutant selection window in presence of meropenem, in accordance with its observed hydrolytic properties. We identified here the very first CTX-M enzyme possessing a weak carbapenemase activity, that may correspond to an emerging phenomenon when considering its possibility to evolve from the widespread ESBL CTX-M-15.

Cover Feature: Fluorescent Turn‐On Probes for the Development of Binding and Hydrolytic Activity Assays for mRNA Cap‐Recognizing Proteins (Chem. Eur. J. 27/2019)

Cover Feature: Fluorescent Turn‐On Probes for the Development of Binding and Hydrolytic Activity Assays for mRNA Cap‐Recognizing Proteins (Chem. Eur. J. 27/2019)

Fluorescent Turn‐On Probes for the Development of Binding and Hydrolytic Activity Assays for mRNA Cap‐Recognizing Proteins

The m7 G cap is a unique nucleotide structure at the 5'-end of all eukaryotic mRNAs. The cap specifically interacts with numerous cellular proteins and participates in biological processes that are essential for cell growth and function. To provide small molecular probes to study important cap-recognizing proteins, we synthesized m7 G nucleotides labeled with fluorescent tags via the terminal phosph(on)ate group and studied how their emission properties changed upon protein binding or enzymatic cleavage. Only the pyrene-labeled compounds behaved as sensitive turn-on probes. A pyrene-labeled m7 GTP analogue showed up to eightfold enhanced fluorescence emission upon binding to eukaryotic translation initiation factor 4E (eIF4E) and over 30-fold enhancement upon cleavage by decapping scavenger (DcpS) enzyme. These observations served as the basis for developing binding- and hydrolytic-activity assays. The assay utility was validated with previously characterized libraries of eIF4E ligands and DcpS inhibitors. The DcpS assay was also applied to study hydrolytic activity and inhibition of endogenous enzyme in cytoplasmic extracts from HeLa and HEK cells.

A robust nanobiocatalyst based on high performance lipase immobilized to novel synthesised poly(o-toluidine) functionalized magnetic nanocomposite: Sterling stability and application

Herein, as a promising support, a magnetic enzyme nanoformulation have been designed and fabricated by a poly-o-toluidine modification approach. Owing to the magnetic nature and the existence of amine functionalized groups, the as-synthesised poly(o-toluidine) functionalized magnetic nanocomposite (Fe3O4@POT) was employed as potential support for Candida rugosa lipase (CRL) immobilization to explore its application in fruit flavour esters synthesis. The morphology and structure of the Fe3O4@POT NC were examined through various analytical tools. Hydrolytic activity assays disclose that immobilized lipase demonstrated an activity yield of 120%. It is worth mentioning that CRL#Fe3O4@POT showed superior resistance to extremes of temperature and pH and different organic solvents in contrast to free CRL. The magnetic behaviour of the as-synthesised NC was affirmed by alternating gradient magnetometer analysis. It was found to own facile immobilization process, enhanced catalytic performance for the immobilized form which may be stretched to the immobilization of various vital industrial enzymes. Moreover, it retained improved recycling performance. After 10 cycles of repetitive uses, it still possessed around 90% of its initial activity for the hydrolytic reaction, since the enzyme-magnetic nanoconjugate was effortlessly obtained using a magnet from the reaction system. The formulated nanobiocatalyst was selected for the esterification reaction to synthesize the fruit flavour esters, ethyl acetoacetate and ethyl valerate. The immobilized lipase successfully synthesised flavour compounds in aqueous and n-hexane media having significant higher ester yields compared to free enzyme. The present work successfully combines an industrially prominent biocatalyst, CRL, and a novel magnetic nanocarrier, Fe3O4@POT, into an immobilized nanoformulation with upgraded catalytic properties which has excellent potential for practical industrial implications.

A Simple Strategy to Determine the Dependence of Membrane-Bound Pyrophosphatases on K+ as a Cofactor.

Membrane-bound pyrophosphatases (mPPases) couple pyrophosphate hydrolysis to H+ and/or Na+ pumping across membranes and are found in all domains of life except for multicellular animals including humans. They are important for development and stress resistance in plants. Furthermore, mPPases play a role in virulence of human pathogens that cause severe diseases such as malaria and African sleeping sickness. Sequence analysis, functional studies, and recently solved crystal structures have contributed to the understanding of the mPPase catalytic cycle. However, several key mechanistic features remain unknown. During evolution, several subgroups of mPPases differing in their pumping specificity and cofactor dependency arose. mPPases are classified into one of five subgroups, usually by sequence analysis. However, classification based solely on sequence has been inaccurate in several instances due to our limited understanding of the molecular mechanism of mPPases. Thus, pumping specificity and cofactor dependency of mPPases require experimental confirmation. Here, we describe a simple method for the determination of K+ dependency in mPPases using a hydrolytic activity assay. By coupling these dependency studies with site-directed mutagenesis, we have begun to build a better understanding of the molecular mechanisms of mPPases. We optimized the assay for thermostable mPPases that are commonly used as model systems in our lab, but the method is equally applicable to mesophilic mPPases with minor modifications.

Defining the enzymatic pathway for polymorphic O-glycosylation of the pneumococcal serine-rich repeat protein PsrP

Protein O-glycosylation is an important post-translational modification in all organisms, but deciphering the specific functions of these glycans is difficult due to their structural complexity. Understanding the glycosylation of mucin-like proteins presents a particular challenge as they are modified numerous times with both the enzymes involved and the glycosylation patterns being poorly understood. Here we systematically explored the O-glycosylation pathway of a mucin-like serine-rich repeat protein PsrP from the human pathogen Streptococcus pneumoniae TIGR4. Previous works have assigned the function of 3 of the 10 glycosyltransferases thought to modify PsrP, GtfA/B, and Gtf3 as catalyzing the first two reactions to form a unified disaccharide core structure. We now use in vivo and in vitro glycosylation assays combined with hydrolytic activity assays to identify the glycosyltransferases capable of decorating this core structure in the third and fourth steps of glycosylation. Specifically, the full-length GlyE and GlyG proteins and the GlyD DUF1792 domain participate in both steps, whereas full-length GlyA and the GlyD GT8 domain catalyze only the fourth step. Incorporation of different sugars to the disaccharide core structure at multiple sites along the serine-rich repeats results in a highly polymorphic product. Furthermore, crystal structures of apo- and UDP-complexed GlyE combined with structural analyses reveal a novel Rossmann-fold "add-on" domain that we speculate to function as a universal module shared by GlyD, GlyE, and GlyA to forward the peptide acceptor from one enzyme to another. These findings define the complete glycosylation pathway of a bacterial glycoprotein and offer a testable hypothesis of how glycosyltransferase coordination facilitates glycan assembly.

Transesterification of plant oils using Staphylococcus haemolyticus L62 lipase displayed on Escherichia coli cell surface using the OmpA signal peptide and EstAβ8 anchoring motif

Staphylococcus haemolyticus L62 (SHL62) lipase was displayed on the outer membrane of Escherichia coli using the OmpA signal peptide and the autotransporter EstAβ8 protein. Localization of SHL62 lipase on the outer membrane of E. coli was confirmed using immunofluorescence microscopy and flow cytometry analysis. Lipase activity of the displayed SHL62 lipase was also measured using spectrophotometry and pH titration. SHL62 lipase activity of whole cells reached 2.0U/ml culture (OD600nm of 10) when it was measured by the p-nitrophenyl caprylate assay after being induced with 1mM IPTG for 24h. The optimum temperature and pH for the lipase was 45°C and 10, respectively. Furthermore, it maintained more than 90% of maximum lipase activity at up to 50°C and in a pH range of 5–9. The hydrolytic activity assay conduted with various substrates confirmed that p-nitrophenyl caprylate and corn oil were preferred substrates among various synthetic and natural substrates, respectively. The displayed SHL62 lipase produced fatty acid esters from various alcohols and plant oils through transesterification.

RAAV2-mediated restoration of LEKTI in LEKTI-deficient cells from Netherton patients

Netherton syndrome (NS, MIM 256500) is a potential live threatening autosomal-recessive skin disorder clinically characterized by the trias of congenital erythroderma, hair shaft anomalies and atopic diathesis. It is caused by mutations in the gene SPINK5 resulting in a deficiency of its processed protein named lympho-epithelial Kazal-type related inhibitor (LEKTI). LEKTI controls the activity of several serine proteases in the skin that are involved in terminal differentiation. Loss of LEKTI results in protease hyperactivity, increased degradation of intercellular junctions, reduced stratum corneum adhesion and impaired skin barrier function. Today NS can only be treated symptomatically. Does gene transfer offer a therapeutic option for NS in the future? A recombinant adeno-associated virus type 2 vector was constructed containing the full length cDNA (rAAV2/C-SPINK5) of functional human LEKTI. Infectious virus particles were used for transfection of LEKTI-deficient-keratinocytes of NS patients in vitro. Gene transfer of SPINK5 in NS-keratinocytes led to a five-fold increase in mRNA expression of SPINK5 reaching almost 75% of normal value. The functionality of the expressed LEKTI was proven in a hydrolytic activity assay demonstrating that the activity of LEKTI after gene transfer increased closely to the level seen in keratinocytes of healthy individuals. The results provide first evidence that gene transfer of SPINK5 results in increased LEKTI activity in NS-keratinocytes, thus offering a rational to further pursue such a gene therapy approach for NS.

Serine protease inhibitor lymphoepithelial Kazal type-related inhibitor tends to be decreased in atopic dermatitis

A pathogenic role of serine protease inhibitor lymphoepithelial Kazal type-related inhibitor (LEKTI) in atopic dermatitis (AD) is currently in intense debate. Analyses of an association between genetic polymorphisms of SPINK5 and atopic diseases revealed contradictory results. Herein, we assessed the role of LEKTI in AD at an expressional and functional level. The expression of LEKTI and its inhibitory capacity was measured by real-time polymerase chain reaction and hydrolytic activity assay, respectively, in keratinocyte cell cultures of three AD patients in comparison to cultures of healthy individuals (5x) and Netherton (NS) patients (3x). Expression of LEKTI was significantly decreased in AD vs. healthy volunteers. Due to reduced protease inhibition, trypsin-like hydrolytic activity in AD was slightly increased, although not significantly. Even though the number of investigated subjects was small and hydrolytic activity was only slightly increased, the results denote that LEKTI might be diminished in AD. The study also disclosed the necessity of functional analyses in addition to genetic investigations to gain further and more detailed insights into the role of LEKTI in AD.

Microwave‐assisted immobilization of lipase from Candida rugosa on Eupergit® supports

AbstractBACKGROUND: Immobilization of lipase (triacylglycerol acylhydrolase EC 3.1.1.3) from Candida rugosa on Eupergit® C and Eupergit® C 250L was performed under microwave irradiation in order to reduce immobilization time. Lipase loading, hydrolytic activity, esterification activity and operational stability in organic solvent of immobilized lipase preparation were determined.RESULTS: The microwave‐assisted procedure resulted in a 29% lower lipase loadings, compared with immobilized lipase obtained without microwaves. In hydrolytic activity assay, lipase immobilized under microwaves exhibited a 23% higher specific activity. Slight activation of lipase by microwave‐assisted immobilization was observed, since specific activity was around 5% higher than for free lipase. Lipase of highest activity was obtained after 2 min immobilization on Eupergit® C. The same preparation exhibited high esterification activity in organic medium and a half life of 212 h was determined in multiple use assay.CONCLUSION: The application of microwave irradiation leads to reduction of immobilization time from 2 days to only 2 min. The immobilized lipase obtained has prospects for further application due to its high retained activity and stability. Copyright © 2009 Society of Chemical Industry

Structural and Biochemical Analysis of Human Pathogenic Astrovirus Serine Protease at 2.0 Å Resolution

Astroviruses are single-stranded RNA viruses with a replication strategy based on the proteolytic processing of a polyprotein precursor and subsequent release of the viral enzymes of replication. So far, the catalytic properties of the astrovirus protease as well as its structure have remained uncharacterized. In this study, the three-dimensional crystal structure of the predicted protease of human pathogenic astrovirus has been solved to 2.0 Å resolution. The protein displays the typical properties of trypsin-like enzymes but also several characteristic features: (i) a catalytic Asp-His-Ser triad in which the aspartate side chain is oriented away from the histidine, being replaced by a water molecule; (ii) a non-common conformation and composition of the S1 pocket; and (iii) the lack of the typical surface β-ribbons together with a “featureless” shape of the substrate-binding site. Hydrolytic activity assays indicate that the S1 pocket recognises Glu and Asp side chains specifically, which, therefore, are predicted to occupy the P1 position on the substrate cleavage site. The positive electrostatic potential featured by the S1 region underlies this specificity. The comparative structural analysis highlights the peculiarity of the astrovirus protease, and differentiates it from the human and viral serine proteases.

Na+-K+-ATPase in rat skeletal muscle: content, isoform, and activity characteristics.

The purpose of this study was to investigate the hypothesis that muscle Na+-K+-ATPase activity is directly related to Na+-K+-ATPase content and the content of the alpha2-catalytic isoform in muscles of different fiber-type composition. To investigate this hypothesis, tissue was sampled from soleus (Sol), red gastrocnemius (RG), white gastrocnemius (WG), and extensor digitorum longus (EDL) muscles at rest from 38 male Wistar rats weighing 413 +/- 6.0 g (mean +/- SE). Na+-K+-ATPase activity was determined in homogenates (Hom) and isolated crude membranes (CM) by the regenerating ouabain-inhibitable hydrolytic activity assay (ATPase) and the 3-O-methylfluorescein K+-stimulated phosphatase (3-O-MFPase) assay in vitro. In addition, Na+-K+-ATPase content (Bmax) and the distribution of alpha1-, alpha2-, beta1-, and beta2-isoforms were determined by [3H]ouabain binding and Western blot, respectively. For the ATPase assay, differences (P < 0.05) in enzyme activity between muscles were observed in Hom (EDL > WG) and in CM (Sol > EDL = WG). For the 3-O-MFPase assay, differences (P < 0.05) were also found for Hom (Sol > RG = EDL > WG) and CM (Sol = WG > RG). For Bmax, differences in the order of RG = EDL > Sol = WG (P < 0.05) were observed. Isoform distribution was similar between Hom and CM and indicated in CM, a greater density (P < 0.05) of alpha1 in Sol than WG and EDL (P < 0.05), but more equal distribution of alpha2 between muscles. The beta1 was greater (P < 0.05) in Sol and RG, and the beta2 was greater in EDL and WG (P < 0.05). Over all muscles, the correlation (r) between Hom 3-O-MFPase and Bmax was 0.45 (P < 0.05) and between Hom alpha2 and Bmax, 0.59 (P < 0.05). The alpha1 distribution correlated to Hom 3-O-MFPase (r = 0.79, P < 0.05) CM ATPase (r = 0.69, P < 0.005) and CM 3-O-MFPase activity (r = 0.32, P < 0.05). The alpha2 distribution was not correlated with any of the Na+-K+-ATPase activity measurements. The results indicate generally poor relationships between activity and total pump content and alpha2 isoform content of the Na+-K+-ATPase. Several factors, including the type of preparation and the type of assay, appear important in this regard.