Exopeptidase Activity Research Articles

Network pharmacology and in vitro experiments reveal the potential therapeutic effects of Scrophularia ningpoensis Hemsl in the treatment of ameloblastoma

PurposeThis study aimed to explore active ingredients in Scrophularia ningpoensis Hemsl (SNH) with potential effects on ameloblastoma (AM) using network pharmacological approach, bioinformatic gene analysis and in vitro cell experiments. MethodsThe active ingredients and their corresponding targets of SNH were identified from the Traditional Chinese Medicine Systems Pharmacology (TCMSP), as well as SwissTargetPrediction. Disease targets of AM were selected from GeneCards and DisGeNET databases. Differentially expressed genes (DEGs) of AM were identified, and Gene Ontology enrichment analysis were performed using the Gene Expression Omnibus (GEO) dataset GSE38494 through bioinformatic analysis. The STRING database platform was utilized to generate a protein–protein interaction network diagram, followed by hub gene analysis using Cytoscape software. AutoDock Vina software was used to perform molecular docking verification of the effects of the active ingredients on potential core targets. Additionally, in vitro experiments including quantitative reverse transcription polymerase chain reaction (RT-qPCR), EdU assay and CCK-8 cell proliferation assay were conducted using AM cell line AM-1 after SNH extract treatment. ResultThe study revealed that SNH contains eight active ingredients and a total of 388 drug targets, including 10 potential core targets in AM. Hub genes identified in the analysis were CCNA2, HRAS, PTGS2, PIK3CB, FGFR1, CASP3, MMP1, SLC2A1, MMP14, and MME. Molecular docking analysis demonstrated strong binding activity between key active ingredients (β-sitosterol, scropolioside A_qt, scropolioside D, scropolioside D_qt, and sugiol) and target genes (CASP3, FGFR1, HRAS, PTGS2, and SLC2A1). Gene Ontology enrichment analysis indicated that SNH exerts its effects on AM through pathways related to cellular response to abiotic stimulus, cellular response to hypoxia, and exopeptidase activity. Immunohistochemical analysis using tissue microarray showed higher expression of MMP14 and PTGS2 in AM compared to dentigerous cyst. Using AM-1 cell line, RT-qPCR results confirmed that SNH suppressed the expression of MMP14 and PTGS2 at mRNA level. Additionally, the EdUassay and CCK-8 assay indicated the inhibitory effect of SNH on the proliferation of AM-1 cells. ConclusionThese findings showed that SNH could suppress expression of MMP14 and PTGS2 and restrain the proliferation of AM. Our study highlights the potential of SNH as a promising therapeutic candidate for AM, which may provide more options for clinical treatment.

Plant cathepsin B, a versatile protease.

Plant proteases are essential enzymes that play key roles during crucial phases of plant life. Some proteases are mainly involved in general protein turnover and recycle amino acids for protein synthesis. Other proteases are involved in cell signalling, cleave specific substrates and are key players during important genetically controlled molecular processes. Cathepsin B is a cysteine protease that can do both because of its exopeptidase and endopeptidase activities. Animal cathepsin B has been investigated for many years, and much is known about its mode of action and substrate preferences, but much remains to be discovered about this potent protease in plants. Cathepsin B is involved in plant development, germination, senescence, microspore embryogenesis, pathogen defence and responses to abiotic stress, including programmed cell death. This review discusses the structural features, the activity of the enzyme and the differences between the plant and animal forms. We discuss its maturation and subcellular localisation and provide a detailed overview of the involvement of cathepsin B in important plant life processes. A greater understanding of the cell signalling processes involving cathepsin B is needed for applied discoveries in plant biotechnology.

An Unusual Two-Domain Thyropin from Tick Saliva: NMR Solution Structure and Highly Selective Inhibition of Cysteine Cathepsins Modulated by Glycosaminoglycans.

The structure and biochemical properties of protease inhibitors from the thyropin family are poorly understood in parasites and pathogens. Here, we introduce a novel family member, Ir-thyropin (IrThy), which is secreted in the saliva of Ixodes ricinus ticks, vectors of Lyme borreliosis and tick-borne encephalitis. The IrThy molecule consists of two consecutive thyroglobulin type-1 (Tg1) domains with an unusual disulfide pattern. Recombinant IrThy was found to inhibit human host-derived cathepsin proteases with a high specificity for cathepsins V, K, and L among a wide range of screened cathepsins exhibiting diverse endo- and exopeptidase activities. Both Tg1 domains displayed inhibitory activities, but with distinct specificity profiles. We determined the spatial structure of one of the Tg1 domains by solution NMR spectroscopy and described its reactive center to elucidate the unique inhibitory specificity. Furthermore, we found that the inhibitory potency of IrThy was modulated in a complex manner by various glycosaminoglycans from host tissues. IrThy was additionally regulated by pH and proteolytic degradation. This study provides a comprehensive structure-function characterization of IrThy-the first investigated thyropin of parasite origin-and suggests its potential role in host-parasite interactions at the tick bite site.

Potential elevation of exopeptidase activity of Glu-specific endopeptidase I/GluV8 mediated by hydrophobic P1′-position amino acid residue

We recently reported that the activities of dipeptidyl-peptidase (DPP)7 and DPP11, S46-family exopeptidases were significantly elevated by the presence of prime-side amino acid residues of substrates caused by an increase in kcat [Ohara-Nemoto Y. et al., J Biol Chem 298(3):101585. doi: 10.1016/j.jbc.2022]. In the present study, the effects of prime-side residues on Glu-specific endopeptidase I/GluV8 from Staphylococcus aureus were investigated using a two-step cleavage method with tetrapeptidyl-methycoumaryl-7-amide (MCA) carrying P2- to P2′-position residues coupled with DPP11 as the second enzyme. GluV8 showed maximal activity toward benzyloxycarbonyl (Z)-LLE-MCA, while the effects of hydrolysis of substrates one residue shorter, such as acetyl (Ac)-Val-Glu- and Leu-Glu-MCA, were negligible. Nevertheless, activity towards Ac-VE-|-ID-MCA, a substrate carrying P1′ and P2′ residues, emerged and reached a level 44 % of that for Z-LLE-MCA. Among 11 Ac-HAXD-MCA (X is a varied amino acid), the highest level of activity enhancement was achieved with P1′-Leu and Ile, followed by Phe, Val, Ser, Tyr, and Ala, while Gly and Lys showed scant effects. This activation order was in parallel with the hydrophobicity indexes of these amino acids. The prime-side residues increased kcat/KM primarily through a maximum 500-fold elevation of kcat as well as S46-family exopeptidases. The MEROPS substrate database also indicates a close relationship between activity and hydrophobicity of the P1′ residues in 93 N-terminal-truncated substrates, though no correlation was observed among all 4328 GluV8 entities examined. Taken together, these results are the first to demonstrate N-terminal exopeptidase activity of GluV8, considered to be prompted by hydrophobic P1′ amino acid residues.

Effect of a commercial peptidase on volatile composition and sensory properties of wheat bread crumb and crust

Flavourzyme is a commercial peptidase, primarily exhibiting exopeptidase activity. The effects of Flavourzyme addition (0–1.6 g/kg, flour basis) on chemical composition, volatile profile and sensory properties of bread crumb and crust were systematically studied. Free amino acids (from 999 to 2156 mg/kg with 1.6 g/kg Flavourzyme addition) and trichloroacetic acid-soluble small peptides in dough remarkably increased with Flavourzyme addition. Gas chromatography-mass spectrometry analysis revealed that 3-methyl-1-butanol, 3-(methylthio)-1-propanol, 2-phenylethanol, acetoin and indole enhanced in crumb after Flavourzyme addition. Meanwhile, pyrazines, benzaldehyde, 5-methyl-2-furanmethanol, maltol and 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one accumulated in crust. Descriptive sensory analysis revealed that the cheesy and alcoholic notes of crumb and the caramel, toasted and nutty notes of crust were strengthened with Flavourzyme addition. The Flavourzyme hydrolysis products enhanced yeast activity in fermentation and facilitated Maillard reactions in baking, leading to the accumulation of related volatile compounds. These compounds subsequently modified the aroma properties of crumb and crust.

The Role of Cysteine Protease Cathepsins B, H, C, and X/Z in Neurodegenerative Diseases and Cancer.

Papain-like cysteine proteases are composed of 11 human cysteine cathepsins, originally located in the lysosomes. They exhibit broad specificity and act as endopeptidases and/or exopeptidases. Among them, only cathepsins B, H, C, and X/Z exhibit exopeptidase activity. Recently, cysteine cathepsins have been found to be present outside the lysosomes and often participate in various pathological processes. Hence, they have been considered key signalling molecules. Their potentially hazardous proteolytic activities are tightly regulated. This review aims to discuss recent advances in understanding the structural aspects of these four cathepsins, mechanisms of their zymogen activation, regulation of their activities, and functional aspects of these enzymes in neurodegeneration and cancer. Neurodegenerative effects have been evaluated, particularly in Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, and neuropsychiatric disorders. Cysteine cathepsins also participate in tumour progression and metastasis through the overexpression and secretion of proteases, which trigger extracellular matrix degradation. To our knowledge, this is the first review to provide an in-depth analysis regarding the roles of cysteine cathepsins B, H, C, and X in neurodegenerative diseases and cancer. Further advances in understanding the functions of cysteine cathepsins in these conditions will result in the development of novel, targeted therapeutic strategies.

An optimized NIR fluorescent probe for visualizing DPP IV and applications in the diagnosis and therapy of inflammatory bowel diseases

Dipeptidyl peptidase IV (DPP IV), is a membrane glycoprotein with exopeptidase activity, that mediates the degradation of glucagon-like-1 (GLP-1) and plays a vital role in diabetes and inflammatory bowel diseases (IBD). In the present work, a highly selective near-infrared (NIR) fluorescent probe DSACD was developed for DPP IV, which exhibited excellent fluorescence properties with high selectivity and affinity toward DPP IV. Using DSACD realized the real-time imaging of DPPIV in various living cells and organs. Importantly, the significant upregulation of DPPIV in intestine was found during the development of IBD via visual imaging and further validated by western blot. At last, DSACD was used for the high-throughput screening the inhibitory effects of 150 kinds common herbs toward DPP IV. Demethoxycurcumin as a novel natural inhibitor for DPP IV was discovered from Curcuma Longa. Furthermore, Demethoxycurcumin exhibited an excellent bioactivity in vivo for the treatment of DSS-induced IBD. In summary, DSACD could serve as a promising tool not only for real-time visual monitoring and exploring the physiological function of DPP IV, but also provided a practical approach for rapid discovery of new potential inhibitors toward DPP IV for treatment of IBD in clinic.

Heterologous production of basidiomycetous peptidases to decrease sodium chloride in food

In the past, the topic of excessive salt consumption associated with various chronic diseases became a focus of nutritional science. Enzymes such as hydrolases and specifically peptidases are widely used in industry since they do not require cofactors, are often stable at high pH values, and have a broad cleavage specificity. Thus, a new approach to use peptidases would be the generation of L‐arginyl dipeptides, which do not have a taste on their own, but are able to enhance the salty taste. This thesis should focus on the isolation and characterisation of arginyl‐specific peptidase genes from basidiomycetes to generate these dipeptides.First, various peptidase genes from Trametes versicolor, Phanerochaete chrysosporium and Schizophyllum commune were isolated and expressed in Escherichia coli or Komagataella phaffii. The serine (ABB73029) peptidase from P. chrysosporium and the aspartate peptidase from T. versicolor (EIW62808) were successfully purified. Activity assays showed that the aspartate peptidase from T. versicolor is not an arginyl‐specific peptidase, while the analysis of cleavage sites indicates that the serine peptidase from P. chrysosporium is an exopeptidase. Next, 29 basidiomycetes were cultivated over 24 days in minimal medium with 1 % gluten and analysed regarding their endo‐ and exopeptidase activity. Out of 29 fungi, five interesting species with arginyl‐specific peptidase activity were chosen, namely Agrocybe aegerita, Fomitop‐ sis pinicola, Flammulina velutipes, Hypholoma sublateritium and Pleurotus eryngii. After purification of the culture supernatant using size exclusion chromatography, active fractions were used to hydrolyse the substrate casein, and cleavage products were analysed. Cleavage sites were only detected for A. aegerita, F. velutipes and P. eryngii. Beside arginyl‐specific cleavage sites also a high amount of undesired exopeptidase cleavage sites was determined assuming that these peptidases are probably exopeptidases rather than arginyl‐specific peptidases.Generation of salt taste enhancing dipeptides should finally be achieved with a dipeptidyl‐ peptidase V (DPPV) from Pleurotusfloridanus. PflDPPV was heterologously produced in E. coli, had optima at pH 8.5 and 60 °C and was not completely inhibited by the tested peptidase inhibitors.

Peptidomic insights on structural protein hydrolysis by exopeptidase activities and its effects on bitterness defects of dry‐cured ham

SummaryExcessive bitterness is a common problem and is related to the hydrolysis of structural proteins degradation in the dry‐cured ham industry. To better understand the effect of structural proteins (myosin, titin, troponin) degradation on sensory development of Jinhua ham, sensory attributes, exopeptidase activities, peptide fragments and cleavage sites of structural proteins were investigated in both premium and defective hams. The results of sensory evaluation and electronic tongue revealed that more than 1.5‐fold values in bitterness intensities and less than 0.67‐fold values in richness intensities were observed in defective ham, compared with premium ham. The activities of exopeptidase were determined, and the results indicated that the residual activities of TPP I in defective ham were higher, while the residual activities of LAP and AAP were lower, compared with premium ham. The large numbers of unique peptide fragments derived from myosin, titin and troponin were identified in defective ham. Several unique cleavage sites including L‐399, L‐1047 and K‐1053 for MYH 1 and MYH 4, K‐257, R‐694, K‐1374 and K‐1651 for MYH 7, and K‐92 and K‐103 for TNN C1 and TNN C2 were only presented in defective ham, which were intensely response to the excessive accumulation of peptides and the development of excessive bitterness in defective ham. This study could provide a theoretical basis for the regulation of bitterness defects in dry‐cured ham in the future.

DPP-IV Inhibitory Peptides GPF, IGL, and GGGW Obtained from Chicken Blood Hydrolysates.

Blood is a meat by-product rich in proteins with properties that can be improved after hydrolysis, making it a sustainable alternative for use in the generation of bioactive peptides. The objective of this study was to identify dipeptidyl peptidase IV (DPP-IV) inhibitory peptides obtained from different chicken blood hydrolysates prepared using combinations of four different enzymes. Best results were observed for AP (2% Alcalase + 5% Protana Prime) and APP (2% Alcalase + 5% Protana Prime + 3% Protana UBoost) hydrolysates obtaining inhibition values of 60.55 and 53.61%, respectively, assayed at a concentration of 10 mg/mL. Free amino acids were determined to establish the impact of exopeptidase activity in the samples. A total of 79 and 12 sequences of peptides were identified by liquid chromatography and mass spectrometry in tandem (LC-MS/MS) in AP and APP samples, respectively. Nine of the identified peptides were established as potential DPP-IV inhibitory using in silico approaches and later synthesized for confirmation. Thus, peptides GPF, IGL, and GGGW showed good DPP-IV inhibitory activity with IC50 values of 0.94, 2.22, and 2.73 mM, respectively. This study confirmed the potential of peptides obtained from chicken blood hydrolysates to be used as DPP-IV inhibitors and, therefore, in the control or modulation of type 2 diabetes.

Foaming and sensory properties of bovine milk protein isolate and its associated enzymatic hydrolysates

The surface tension, foaming, bitterness and colour properties of bovine milk protein isolate (MPI) and MPI hydrolysates generated with Flavourzyme™, Neutrase™ and Protamex™ were evaluated. The surface tension of the hydrolysate samples (46.6–48.3 mN m−1), measured using pendant drop shape analysis, was reduced compared with the intact protein samples (49.1–49.6 mN m−1). The foam half-life of the hydrolysates (58.1–214 s) at 25 °C was significantly reduced compared with the intact protein (783–850 s). The L∗ value of the hydrolysates (69.9–77.4) was significantly lower than the intact protein (84.3–86.0). The Flavourzyme™ hydrolysate (60 min incubation) demonstrated a mean bitterness score (2.14) comparable with the intact protein (1.52–1.57). All other hydrolysates displayed increased bitterness; Neutrase™ and Protamex™ hydrolysates had the highest mean bitterness scores (8.80–9.39). The hydrolysate foaming and sensory properties were enzyme and DH dependent; bitterness development may be minimised by using Flavourzyme™ as the hydrolytic enzyme due to the presence of exopeptidase activities.

Differential Distribution and Activity Profile of Acylpeptide Hydrolase in the Rat Seminiferous Epithelium.

Acylpeptide hydrolase (APEH) is a serine protease involved in amino acid recycling from acylated peptides (exopeptidase activity) and degradation of oxidized proteins (endoproteinase activity). This enzyme is inhibited by dichlorvos (DDVP), an organophosphate compound used as an insecticide. The role of APEH in spermatogenesis has not been established; therefore, the aim of this study was to characterize the distribution and activity profile of APEH during this process. For this purpose, cryosections of male reproductive tissues (testis and epididymis) and isolated cells (Sertoli cells, germ cells, and spermatozoa) were obtained from adult rats in order to analyze the intracellular localization of APEH by indirect immunofluorescence. In addition, the catalytic activity profiles of APEH in the different male reproductive tissues and isolated cells were quantified. Our results show that APEH is homogeneously distributed in Sertoli cells and early germ cells (spermatocytes and round spermatids), but this pattern changes during spermiogenesis. Specifically, in elongated spermatids and spermatozoa, APEH was localized in the acrosome and the principal piece. The exopeptidase activity was higher in the germ cell pool, compared to sperm and Sertoli cells, while the endoproteinase activity in epididymal homogenates was higher compared to testis homogenates at 24 h of incubation. In isolated cells, this activity was increased in Sertoli and germ cell pools, compared to spermatozoa. Taken together, these results indicate that APEH is differentially distributed in the testicular epithelium and undergoes re-localization during spermiogenesis. A possible role of APEH as a component of a protection system against oxidative stress and during sperm capacitation is discussed.

Dipeptidylamino-tripeptidylcarboxypeptidase NEMP3 and DPP3 (DPP III) are the same protein

Earlier it was shown that a group of extracellular low-specific metallopeptidases is present in the mammalian brain Kropotova and Mosevitsky (2016) [1]. These enzymes are weakly connected to the axonal ends of neurons. They were named Neuron bound Extracellular MetalloPeptidases (NEMP). The enzyme named NEMP3 turned out to be a unique exopeptidase that exhibits two activities: it removes the dipeptide from the N-end of the peptide, and it can also remove the tripeptide from the C-end of the peptide. Therefore, NEMP3 possesses the activities of dipeptidylaminopeptidase and of tripeptidylcarboxypeptidase. Mass spectrometry has revealed a homology of NEMP3 with DPP3 (DPP III, EC3.4.14.4), known as cytosolic dipeptidylaminopeptidase. We isolated DPP3 from rat and bovine liver and brain by the procedures used for this purpose by other authors. The effect of DPP3 on test peptides is the same as that of NEMP3. In particular, all DPP3 samples delete the tripeptide (AKF) from the C-end of the test peptide blocked at the N-end. The data obtained show that NEMP3 and DPP3 are the same protein (enzyme). Thus, DPP3 has two exopeptidase activities: the previously known activity of dipeptidylaminopeptidase and the activity of tripeptidylcarboxypeptidase discovered in this study. Another discovery is the extracellular activity of DPP 3 in the mammalian brain near synapses, which controls neuropeptides. DPP3 is involved in various processes, but in many cases its role remains to be clarified. The results obtained in this study will be useful for solving these questions.

Forward and reverse degradomics defines the proteolytic landscape of human knee osteoarthritic cartilage and the role of the serine protease HtrA1

Proteolytic destruction of articular cartilage, a major pathogenic mechanism in osteoarthritis (OA), was not previously investigated by terminomics strategies. We defined the degradome of human knee OA cartilage and the contribution therein of the protease HtrA1 using Terminal Amine Isotopic Labeling of Substrates (TAILS). Proteins from OA cartilage taken at knee arthroplasty (n=6) or separately, from healthy cartilage incubated in triplicate with/without active HtrA1, were labeled at natural and proteolytically cleaved N-termini by reductive dimethylation, followed by trypsin digestion, enrichment of N-terminally labeled/blocked peptides, tandem mass spectrometry and positional peptide annotation to identify cleavage sites. Biglycan proteolysis by HtrA1 was validated biochemically and Amino-Terminal Oriented Mass Spectrometry of Substrates (ATOMS) was used to define the HtrA1 cleavage sites. We identified 10,155 unique internal peptides from 2,162 proteins, suggesting at least 10,797 cleavage sites in OA cartilage. 7,635 internal peptides originated in 371 extracellular matrix/secreted components, many undergoing extensive proteolysis. Rampant ragging of protein termini suggested pervasive exopeptidase activity. HtrA1, the most abundant protease in OA cartilage, experimentally generated 323 cleavages in 109 cartilage proteins, accounting for 171 observed cleavages in the OA degradome. ATOMS identified HtrA1 cleavage sites in a selected substrate, biglycan, whose direct cleavage by HtrA1 was thus orthogonally validated. OA cartilage demonstrates widespread proteolysis by endo- and exopeptidases. HtrA1 contributes broadly to cartilage proteolysis. Forward degradomics of OA cartilage together with reverse degradomics of proteases active in OA, e.g., HtrA1, can potentially fully annotate OA proteolytic pathways and provide new biomarkers.

Effects of a commercial peptidase on rheology, microstructure, gluten properties of wheat dough and bread quality

Flavourzyme is a commercial peptidase, mainly with exopeptidase activity. In this study, effects of Flavourzyme addition (0–0.16 g/100 g) on properties of wheat dough and bread were systematically studied. Bread with a low concentration (0.01–0.08 g/100 g) of Flavourzyme addition had a large specific volume and soft texture with a uniform cell structure. The optimal Flavourzyme content was 0.04 g/100 g and the specific volume increased from 3.9 mL/g to 4.4 mL/g, the hardness decreased from 21.7 N to 13.3 N. The gas production capacity of yeast increased with low concentrations of Flavourzyme addition, promoting dough expansion. Flavourzyme had a clear hydrolysis effect on wheat gluten in dough and caused the depolymerization and unfolding of gluten structure, thus decreasing the strength and viscoelasticity of dough. Confocal laser scanning microscopy and scanning electron microscopy results further revealed that a low concentration of Flavourzyme had a slight effect on gluten. However, the gluten structure became discontinuous and open, which made the dough easily collapsed during fermentation, resulting in poor bread quality with high Flavourzyme content. The positive effects of appropriate amount of Flavourzyme addition on dough and bread properties indicated the potential of Flavourzyme as a bread improver by substituting chemical additives.

In Vitro Assays for the Bifunctional Acylpeptide Hydrolase (APEH) Enzyme from Antarctic Fish.

The bifunctional enzyme acylpeptide hydrolase (APEH) is involved in important metabolic processes both as an exopeptidase and as an endopeptidase. Hence, the growing interest in the study of this protein and the need to set up in vitro assays for its characterization. This chapter describes two in vitro assays able to detect the activities of APEH, one for the exopeptidase activity and one for the endopeptidase activity. In particular, these assays have been set up on the two APEH isoforms from Antarctic fish, characterized by a distinct functionality and marked exo- and endopeptidase activities.

Flavor Characterization of Animal Hydrolysates and Potential of Glucosamine in Flavor Modulation.

Bovine (meat and heart) and porcine (hemoglobin and plasma) raw materials were hydrolyzed by Protease A (both endo- and exopeptidase activity), with or without glucosamine added during the enzyme inactivation step. Hydrolysates were characterized via peptide analysis (yield, UV- and fluorescence scanning spectroscopy, and peptide size distribution via size exclusion chromatography), sensory evaluation, and volatile compound analysis via gas chromatography mass-spectrometry (GC-MS) to determine if glucosamine-induced Maillard reaction improved taste and flavor. Porcine hemoglobin produced the most flavor-neutral hydrolysate, and could expectedly have the broadest application in food products. Both bovine meat and -heart hydrolysates were high in umami, and thereby good candidates for savory applications. Porcine plasma hydrolysate was high in liver flavor and would be suitable for addition to certain meat products where liver flavor is desirable. All hydrolysates had low perceived bitterness. Glucosamine-induced Maillard reaction had just a minor influence on the sensory profile via an increased perception of sweet taste (p = 0.038), umami taste (p = 0.042), and yolk flavor (p = 0.038) in the hydrolysates, irrespective of raw material. Glucosamine addition had a statistically significant effect on 13 of 69 volatiles detected in the hydrolysates, but the effect was minor and raw material-specific.

Development of protease nanobiocatalysts and their application in hydrolysis of sunflower meal protein isolate

SummaryIn this study, the suitability of fumed silica nanoparticles (FNS) and its derivatives (amino‐modified FNS (AFNS), cyanuric chloride‐activated AFNS (CCAFNS) and epoxy‐modified FNS (GFNS)), for covalent immobilisation of two commercial protease preparations Alcalase® and Flavourzyme® was investigated. The highest hydrolytic activities of immobilised preparations were 25 IU g−1 support (Alcalase‐GFNS) and 2.95 IU g−1 support (Flavourzyme‐CCAFNS). Furthermore, the immobilised preparations showed 43% and 20% of initial specific activities of commercial protease preparations, respectively. Flavourzyme‐CCAFNS also exhibited the highest exopeptidase activity of 22.83 L‐pNAU g−1 support. Finally, these two nanobiocatalysts were successfully applied for hydrolysis of sunflower meal protein isolate (SMPI), providing two times higher hydrolysis yields in comparison to free enzymes, justifying the applied immobilisation process. Namely, the highest hydrolysis yield (30%) was gained by the sequential hydrolysis with Alcalase‐GFNS and Flavourzyme‐CCAFNS, which resulted in the formation of small hydrophobic and hydrophilic peptides, ≤5 kDa, confirmed by HPLC analysis and electrophoretic separation.

Extraction of Pb(II) from wheat samples via dual-frequency ultrasound-assisted enzymatic digestion and the mechanisms of its interactions with wheat proteins

A novel dual-frequency ultrasound-assisted enzymatic digestion (DUED) technique was used to extract Pb(II) from certified reference materials (CRMs) of wheat flour. Following this, the interactions of Pb(II) with wheat proteins were investigated to provide evidence for the selection of enzyme species. The results showed that the simultaneous use of α-amylase and flavourzyme resulted in the recovery of 97.9% of Pb(II) in 6 min under a 40 kHz ultrasonic bath combined with a 20 kHz ultrasonic probe. The exopeptidase activity of the flavourzyme was found to be the main contributor to the extraction of Pb(II) from the CRMs. Additionally, the proposed method exhibited a low detection limit (8.2 ng/g) and high recoveries of real samples (93.4%-112.2%) with RSD less than 7.33%. Furthermore, the oxygen-containing groups of wheat proteins, the nitrogen-containing groups of albumins and globulins, and the sulfur-containing groups of gliadins and glutenins were found to offer coordination sites for Pb(II).

In silico analysis of Naegleria fowleri cathepsin B paralogs: important drug targets.

Naegleria fowleri is a deadly human pathogen that causes primary amoebic meningoencephalitis (PAM). In this study, in silico investigations of two important N. fowleri cathepsin B paralogs, i.e., copies of genes resulting from a gene duplication event, were carried out using comparative modeling and molecular dynamics (MD) simulations. Comparative models of both paralogs showed significant architectural similarity with their template, i.e., rat cathepsin B. However, in N. fowleri cathepsin B (UniProt ID: X5D761) and putative cathepsin B (UniProt ID: M1HE19) enzymes, eleven and fifteen residues in the occluding loop regions were deleted, respectively, suggesting that these enzymes have a short occluding loop. Thus, it is concluded that N. fowleri cathepsin B and putative cathepsin B enzymes lack exopeptidase activity but possess enhanced endopeptidase activity and an affinity for macromolecular inhibitors. MD simulations further confirmed that prosegments (macromolecular inhibitors) bond more tightly with both enzymes than with wild-type cathepsin B. Additionally, a mutation was identified at an important N-glycosylation site; this mutation is believed to affect cathepsin B targeting inside the cell and make cathepsin B available in the extracellular environment. Due to this important N-glycosylation site mutation, these enzymes are secreted in the extracellular environment via an alternative, still unknown, posttranslational processing strategy. The present study is the first to predict the three-dimensional folds of N. fowleri cathepsin B paralogous enzymes, including a detailed description of the active site architecture and information about propeptide binding mode. This information can contribute to the discovery of novel and selective treatments that are effective against N. fowleri.