Subtilisin Research Articles

Controlled and rapid microreactor-assisted hydrolysis of sesame protein by a novel nanobiocatalyst; immobilized alcalase over activated sol–gel magnetic nanosupport

In this study, sesame cake was valorized to produce sesame protein hydrolysate (SPH) with improved functional and antioxidant properties using a recoverable nanomagnetic immobilized Alcalase (ALC); the process was accelerated in the microreactor through a controlled-hydrolysis technique. For this purpose, silica-coated magnetic nanoparticles (Fe3O4@SiO2) were synthesized and functionalized with tetraethoxysilane (TEOS), N-(trimethoxysilylpropyl) ethylenediamine (TMSED), and finally activated with glutaraldehyde (GLU) to produce Fe3O4@SiO2-TMSED-GLU. Immobilization efficiency of ALC was 88.78 % in the optimum conditions of ALC concentration (1.23 mg/mL), immobilization period (119.12 min), and temperature (38.93 °C). The successful synthesis of Fe3O4@SiO2-TMSED-GLU-ALC magnetic nanobiocomposite (MNBC) was approved using FTIR, XRD, VSM, FESEM, EDX, and elemental mapping. The effective immobilisation of the enzyme was responsible for the decrease in the intensity of Fe3O4 diffraction peaks (in XRD) in MNBC and a reduction in saturation magnetization (in VSM) following ALC immobilisation. Microreactor-intensified hydrolysis in the optimized conditions of MNBC = 4.85 wt%, reaction time = 54.10 s, and temperature = 46.95 °C, led to a degree of hydrolysis = 7.19 %. After 10 hydrolysis cycles, MNBC had relative activity > 80 %, due to the appropriate covalent bonding between the enzyme and the support and the short duration of the process. The produced SPH had superior foaming capacity (113.09 %) compared to sesame protein (76.34 %). The emulsion activity and emulsion stability of SPH were 28.78 m2/g and 56.43 min, respectively, which were higher than those of the sesame protein.

Genome-Wide Identification, Phylogeny and Expression Analysis of Subtilisin (SBT) Gene Family under Wheat Biotic and Abiotic Stress.

The subtilisin-like protease (SBT) family is widely known for its role in stress resistance to a number of stressors in different plant species, but is rarely studied in wheat. Subtilisin-like serine proteases (SBTs) are serine proteolytic enzymes that hydrolyze proteins into small peptides, which bind to receptors as signal molecules or ligands and participate in signal transduction. In this study, we identified 255 putative SBT genes from the wheat reference genome and then divided these into seven clades. Subsequently, we performed syntenic relation analysis, exon-intron organization, motif composition, and cis-element analysis. Further, expression analysis based on RNA-seq and tissue-specific expression patterns revealed that TaSBT gene family expression has multiple intrinsic functions during various abiotic and biotic stresses. Analysis of RNA-seq expression assays and further validation through qRT PCR suggested that some of the TaSBT genes have significant changes in expression levels during Pst interaction. TaSBT7, TaSBT26, TaSBT102, and TaSBT193 genes showed increasing expression levels during compatible and non-compatible interactions, while the expression levels of TaSBT111 and TaSBT213 showed a decreasing trend, indicating that these members of the wheat SBT gene family may have a role in wheat's defense against pathogens. In conclusion, these results expand our understanding of the SBT gene family, and provide a valuable reference for future research on the stress resistance function and comprehensive data of wheat SBT members.

Simvastatin Resistance of Leishmania amazonensis Induces Sterol Remodeling and Cross-Resistance to Sterol Pathway and Serine Protease Inhibitors.

The sterol biosynthesis pathway of Leishmania spp. is used as a pharmacological target; however, available information about the mechanisms of the regulation and remodeling of sterol-related genes is scarce. The present study investigated compensatory mechanisms of the sterol biosynthesis pathway using an inhibitor of HMG-CoA reductase (simvastatin) and by developing drug-resistant parasites to evaluate the impact on sterol remodeling, cross-resistance, and gene expression. Simvastatin-resistant L. amazonensis parasites (LaSimR) underwent reprogramming of sterol metabolism manifested as an increase in cholestane- and stigmastane-based sterols and a decrease in ergostane-based sterols. The levels of the transcripts of sterol 24-C-methyltransferase (SMT), sterol C14-α-demethylase (C14DM), and protease subtilisin (SUB) were increased in LaSimR. LaSimR was cross-resistance to ketoconazole (a C14DM inhibitor) and remained sensitive to terbinafine (an inhibitor of squalene monooxygenase). Sensitivity of the LaSimR mutant to other antileishmanial drugs unrelated to the sterol biosynthesis pathway, such as trivalent antimony and pentamidine, was similar to that of the wild-type strain; however, LaSimR was cross-resistant to miltefosine, general serine protease inhibitor N-p-tosyl-l-phenylalanine chloromethyl ketone (TPCK), subtilisin-specific inhibitor 4-[(diethylamino)methyl]-N-[2-(2-methoxyphenyl)ethyl]-N-(3R)-3-pyrrolidinyl-benzamide dihydrochloride (PF-429242), and tunicamycin. The findings on the regulation of the sterol pathway can support the development of drugs and protease inhibitors targeting this route in parasites.

Pork liver protein hydrolysates as extenders of pork patties shelf‐life

SummaryThe growing demand for the consumption of more natural products challenges the food industry to meet consumer expectations. In this regard, bioactive peptides obtained from flavourzyme, bromelain and alcalase activity were added to pork patties with the aim of improving their shelf‐life. Five treatments were prepared: control (CON, without enzymatic extract), positive control (ASA, with 150 mg kg−1 of ascorbic acid) and three batches with 250 mg kg−1 of different porcine liver hydrolysates [flavourzyme (FLA), bromelain (BRO) and alcalase (ALC)]. The analysis of raw burgers included proximate composition (moisture, protein, fat and ash contents), colour parameters (L*, a*, b*), metmyoglobin, lipid oxidation, volatile compounds profile and sensory evaluation. Hydrolysate obtained from bromelain enzyme showed the best a* parameter (5.15), the least lipid oxidation (0.63 mg MDA kg−1) and the lowest percentage of metmyoglobin (56.02%). Furthermore, this batch also presented the lowest content of aldehydes and alcohols resulting from lipid oxidation. Our results suggested that the hydrolysates obtained from bromelain activity possessed a good antioxidative activity, and therefore, they could be used as a natural antioxidant to extend the shelf‐life of pork burgers.

Subtilisin Gene Activity in Dermatophytes: A study on the Presence of the Subtilisin Gene in Trichophyton verrucosum and Microsporum gypseum in Clinical and Nonclinical Samples in Tehran, Iran.

The keratinolytic activities of dermatophyte species are accompanied by the secretion of enzymes, such as serine proteases, which are coded by the Subtilisin (SUB) genes. This study aimed to determine the presence of the SUB genes in the clinical and nonclinical samples of Trichophyton verrucosum and Microsporum gypseum. Isolation was carried out by direct and laboratory examination. Following that, for the determination of the presence of the SUB gene, polymerase chain reaction with specific primers was conducted. The frequencies of the SUB gene were observed in almost 66% of the isolates. Statistical analysis showed a significant relationship between the presence of the SUB gene and the samples collected from human, animals, and soil (p ˂0.005). The current investigation has been the first study of the presence/absence of the SUB gene in the clinical and nonclinical isolates of T. verrucosum and M. gypseum in Iran which may be a new step to perform further studies.

Production of Protein Hydrolysates Using Marine Catfish Bagre panamensis Muscle or Casein as Substrates: Effect of Enzymatic Source and Degree of Hydrolysis on Antioxidant and Biochemical Properties.

Protein hydrolysates from fishery byproducts have resulted to be nutraceutical ingredients with potential to be applied in human nutrition; however, critical quality attributes are dependent on some process parameters such as enzyme source and degree of hydrolysis. This study analyzed the biochemical properties and in vitro antioxidant activity (using DPPH, ABTS, and FRAP assays), of protein hydrolysates at 10, 20, and 30% degree of hydrolysis (DH), measured by pH-STAT and prepared from sea catfish (Bagre panamensis) muscle and casein as protein sources by treatment with alcalase (ALC) and a semi-purified protease extract (SPE) from B. panamensis intestinal tissues as enzyme sources. With SPE, the DH was reached faster than ALC regardless of the protein substrate used. Sea catfish muscle (MUSC) hydrolysate made with SPE at 30% DH showed the highest antioxidant activity (DPPH: 118.8 μmoles TE/mg; ABTS: EC50 of 1.5 mg/mL). In FRAP assay, the MUSC hydrolysates produced with SPE or ALC at 20% DH showed the higher activity (0.38 and 0.40 μmoles TE/mg, respectively). MUSC hydrolysates made with SPE contained the highest proportion of peptides with MW < 1.35 kDa and had a high protein content (72 to 78%), and almost 50% of the amino acids were essential. These results suggest that intestinal proteases and muscle of marine catfish represent a potential source to elaborate antioxidant protein hydrolysates. Our results promote the full utilization of this fish species and offer a biotechnological strategy for the management and valorization of its byproducts.

Properties of hydrolyzed α-lactalbumin, β-lactoglobulin and bovine serum albumin by the alcalase and its immunemodulation activity in Raw 264.7 cell

Properties of hydrolyzed α-lactalbumin, β-lactoglobulin and bovine serum albumin by the alcalase and its immunemodulation activity in Raw 264.7 cell

Cricket (Acheta domesticus) protein hydrolysates’ impact on the physicochemical, structural and sensory properties of tortillas and tortilla chips

Consumer interest towards consumption of more sustainable protein sources has led way towards the adoption of insect protein for human food applications. Research shows that insect proteins utilised as food ingredients are more promising to be accepted by Westerners. In this study, cricket protein hydrolysates (CPH) were produced using Alcalase (AL) and Flavourzyme (FL) proteases. The physicochemical and structural properties, as well as the sensory acceptability of corn tortillas formulated with 20% (w/w) CPH were evaluated. CPH-tortillas contained all essential amino acids, including 40% of the daily lysine requirement. In raw corn masa (dough), AL-CPH increased the elastic modulus (G’), indicating cross-linking between polymers, while FL-CPH decreased G’. Tortillas formulated with AL-CPH resulted in matrices with low hardness and extensibility values, while FL-CPH tortillas developed a strong and flexible structure, indicating differences in intermolecular interactions between the AL- and FL-CPH and the tortilla matrix in the cooked tortillas compared to the raw masa. Sensory evaluation results showed acceptability (scores&gt;6.0) towards tortillas chips formulated with 20% CPH. Enzymatic hydrolysis of cricket protein can create peptides with functional characteristics and sensory acceptability for their use as ingredients in food formulation.

Ultrasound microwave-assisted enzymatic production and characterisation of antioxidant peptides from sweet potato protein

Herein, we investigated the effects of ultrasound microwave (UM)-assisted hydrolysis using Alcalase (ALC), Flavourzyme (FLA), and their combination (ALC + FLA), on the production of sweet potato protein hydrolysates (SPPH). UM-assisted enzymatic hydrolysis significantly increased the degree of hydrolysis of SPPH compared with untreated (UN) samples. Fractions with differences in molecular weight (MW) of >10, 3–10, and < 3 kDa in SPPH from UM-assisted ALC, FLA, and ALC + FLA hydrolysis displayed higher antioxidant activities than those from UN samples. MW < 3 kDa fractions of SPPH from UM-assisted ALC and ALC + FLA hydrolysis treatments presented much stronger Fe2+-chelating activity (98.48% and 98.59%), ·OH scavenging activity (67.11% and 60.06%), and higher ORAC values (110.32 and 106.32 µg TE/mL), from which diverse peptides with potential antioxidant activities were obtained by semi-preparative HPLC and LC-MS/MS. All identified peptide sequences exhibited at least three potential antioxidant amino acids. Additionally, changes in peptide conformational structure and antioxidant amino acid composition were revealed by structure–activity relationship analysis. Thus, ultrasound microwave treatment has great potential in antioxidant peptides production.

Inclisiran for Highest-Risk Patients: A Suggestion of Hope

Monoclonal antibodies directed against proprotein convertase subtilisin–kexin type 9 (PCSK9) can reduce LDL cholesterol and lower cardiovascular risk

Production and characterisation of antioxidant peptides from sweet potato protein by enzymatic hydrolysis with radio frequency pretreatment

SummaryEffect of radio frequency (RF at 70, 80 and 90 °C) pretreatment on production and characterisation of sweet potato protein hydrolysates (SPPH) prepared using Alcalase (ALC), Protease (PRO) and the combination of ALC + PRO was investigated. RF highly improved degree of hydrolysis (DH) and enhanced antioxidant activity of all SPPH produced by ALC, PRO and the combination as compared to traditional water bath (WB) heating pretreatment (P &lt; 0.05). RF significantly increased molecular weight (MW) &lt;3 kDa peptide fraction from SPPH produced by ALC, and MW &lt;3 kDa peptide fraction from RF80‐ and RF90‐pretreated samples has higher antioxidant activity. Diverse peptides in MW &lt;3 kDa fractions with RF80 and RF90 pretreatments were identified using LC–QTOF–MS/MS, which matched the sequences of sporamins and contained antioxidant amino acids Trp, Tyr, Met, Phe and/or His. There is a great potential application of using SPPH in functional foods as a novel ingredient.

Genes Encoding Proteolytic Enzymes Fungalysin and Subtilisin in Dermatophytes of Human and Animal Origin: A Comparative Study.

Dermatophytes are among the most successful fungal pathogens in humans, but their virulence mechanisms have not yet been fully characterized. Dermatophytic fungi secrete proteases in vivo, which are responsible for fungal colonization and degradation of the keratinized tissue during infection. In the present study, we used PCR to investigate the presence of genes encoding fungalysins (MEP) and subtilisins (SUB) in three dermatophyte species whose incidence is increasing in Europe: the anthropophilic Trichophyton rubrum (n = 58), zoophilic Microsporum canis (n = 33), and Trichophyton benhamiae (n = 6). MEP2 and SUB4 genes were significantly correlated with T. rubrum; MEP3 and SUB1 were mostly frequently harbored by M. canis; and MEP1, 2, and 4 and SUB3-7 were most frequently harbored by T. benhamiae isolates (p < 0.05). Furthermore, MEP1-5 and SUB1-3 genes were significantly more prevalent among human clinical isolates of M. canis (n = 17) than among asymptomatic cat isolates of M. canis (n = 16; p < 0.05). Unidentified MEP and/or SUB genes in some isolates in the current study may suggest that other gene repertoires may be involved in the degradation of keratin. The presented analysis of the incidence of MEP and SUB virulence genes in three dermatophyte species of diverse origins provides an insight into the host-fungus interaction and dermatophyte pathogenesis.

Polyzwitterionic hydrogels as wound dressings with enzymatic debridement functionality for highly exuding wounds

AbstractWound debridement is crucial for proper wound care as it promotes fast and efficient wound healing through removal of necrotic tissue. The latter not only impairs new healthy tissue formation but also increases the odour and the wound exudate, allowing bacteria and other harmful foreign invaders to spread and infect the wound. Hydrogel wound dressings are usually applied for promoting autolytic wound debridement but this is slow and not a very efficient process. On the other hand, enzymatic products for wound debridement are either ointments or gels and they are easily washed out when used for treating highly exuding wounds. This study is an attempt to combine enzymatic debridement functionality with the high swelling ability of polyzwitterionic networks and to produce an innovative dressing with debridement functionality for the healing of highly exuding wounds. For this purpose, two polyzwitterionic hydrogels were synthesized, poly(sulfobetaine methacrylate) and poly(carboxybetaine methacrylate) hydrogels, which were loaded with the protease subtilisin DY for imparting debridement functionality. The swelling ability and mechanical properties of zwitterionic polymer (ZP) hydrogels were shown to depend on their different propensities to physical network formation. Poly(carboxybetaine methacrylate) hydrogels demonstrated better capacity for wound exudate absorption as well as for exerting higher enzymatic debridement activity. Both ZP hydrogels were shown to be non‐cytotoxic which confirms their appropriateness for direct contact with injured tissues. Thus, the newly developed ZP hydrogels demonstrate the potential to be used as new dressing materials with enzymatic debridement functionality for highly exuding wounds. © 2019 The Authors. Polymer International published by John Wiley &amp; Sons Ltd on behalf of Society of Chemical Industry.

Impact of thermal treatment and hydrolysis by Alcalase and Cynara cardunculus enzymes on the functional and nutritional value of Okara

Abstract Enzymatic hydrolysis of dried okara (autoclaved and non-autoclaved) with Alcalase (AL) and Cynara cardunculus extract (CY) was studied, assessing the impact of heat treatment and hydrolysis on potential antioxidant and antihypertensive activities of final hydrolysates. This study showed that the thermal treatment (sterilization at 121 °C, 1 atm and 15 min) facilitated the enzymatic access to substrate and increased the degree of hydrolysis (DH), especially for AL (37.9%) when compared to CY (3.6%). The antioxidant activity of dried Okara (either autoclaved or not) when hydrolysed with AL was higher (4.2 mg Trolox/mL) than that observed for CY. Additionally, the potential ACE-inhibitory activity was high for samples hydrolysed with both enzymes, however the highest ACE inhibition was also found for AL (IC50 = 9.97 μg/mL). This study allowed the identification of new peptide sequences in dried okara hydrolysed with both enzymes, and some sequences that can explain their bioactivities. The results indicate that dried okara hydrolysates can either be used as functional ingredient or as food supplement for blood pressure lowering or antioxidant applications in the future.

Production and In Vitro Gastrointestinal Digestion of Antioxidant Peptides from Enzymatic Hydrolysates of Sweet Potato Protein Affected by Pretreatment.

Effects of ultrasonication, boiling, steaming, microwaving and autoclaving pretreatments on the production of sweet potato protein hydrolysates (SPPH) by single and combined Alcalase (ALC) and Protease (PRO) were investigated, as well as antioxidant activities of SPPH subjected to in vitro gastrointestinal digestion (GID). All pretreatments significantly increased the degree of hydrolysis (DH) and antioxidant activities of SPPH by ALC, PRO and ALC + PRO in the order of autoclaving > steaming, microwaving, boiling > ultrasonication (P < 0.05). GID significantly enhanced antioxidant activities and increased MW <3kDa peptide fraction contents of all SPPH. Diverse peptides were identified as sporamin A, A precursor and sporamin B before and after GID from LC-QTOF-MS/MS analysis. Peptides with higher antioxidant amino acids of Trp, Tyr, Met, Cys, His and Phe were found after GID. There is a great potential application of SPPH as a novel food ingredient as a natural antioxidant.

Effectiveness of proteolytic enzymes to remove gluten residues and feasibility of incorporating them into cleaning products for industrial purposes

The development of protocols for efficient gluten elimination is one of the most critical aspects of any allergen management strategy in the industry. The suitability of different proteolytic enzymes to be included in a cleaning formulation that allows the effective elimination of gluten residues was studied. Alcalase (ALC), neutrase (NEUT) and flavourzyme (FLAV) were selected from in silico analysis. The presence of 1% (v/v) of linear alkylbenzene sulphonate (LAS), a common anionic detergent, improved the gluten solubility, which may favour its elimination. Chromatographic analysis showed that the three enzymes studied were able to hydrolyse gluten in the presence of LAS. The highest percentage of short peptides (< 5 kDa) was achieved with ALC, what increases the probability of reducing the gluten antigenicity. Besides, in the presence of ALC and detergent LAS have detected the lowest levels of gluten with ELISA kits. So, effective amounts of ALC and LAS were added to a cleaning formulation, where its proteolytic activity was maintained above 90% after 37 days at 4 °C and 25 °C (under dark). Preliminary validation of the effectiveness enzymatic cleaning formulation to hydrolyse gluten was performed in a ready-to-eat/frozen food company, in which previous episodes of cross-contamination with gluten have been detected. The gluten content decreased to values below 0.125 μg/100 cm2 when the cleaning formulation was tested on different surfaces with different cleaning protocols, demonstrating the high suitability of the enzymatic cleaning formulation developed.

EFFECTS OF ULTRASOUND TREATMENT ON STRUCTURAL, CHEMICAL AND FUNCTIONAL PROPERTIES OF PROTEIN HYDROLYSATE OF RAINBOW TROUT (ONCORHYNCHUS MYKISS) BY-PRODUCTS

In this study, the effects of ultrasound treatment on biochemical, physical, structural and functional properties of fish protein hydrolysate of rainbow trout (Oncorhynchus mykiss) by-products were investigated. Enzymatic hydrolysis was conducted by Alcalase 2.4 L, pH 8, 1 h at 60°C, and enzyme/substrate ratio at 0.5%. A probe-type ultrasound was used for ultrasound assisted hydrolysis (UH) process. Higher protein recovery was obtained in UH than in the conventional enzymatic hydrolysis (CH). The highest foaming capacities of CH and UH were measured as 137.5% and 152.5%, respectively (p<0.05). Overall, our data suggest that ultrasound treatment helps to improve the functional properties such as foaming capacity and stability.

Can a PCSK9 Inhibitor Prevent Events After Acute Coronary Syndrome

Proprotein convertase subtilisin–kexin type 9 (PCSK9) inhibitors reduce the risk for ischemic cardiovascular events in patients with stable

Antidrug-Antibody Formation: A Problem in PCSK9 Therapy?

Therapy based on monoclonal antibodies (mAbs) might induce unwanted immunogenicity, which may lead to minor problems, such as injection-site reactions or flu-like symptoms, or to more serious problems, such as anaphylaxis or loss of drug efficacy. Two recent manufacturer-supported reports describe antidrug-antibody formation in patients exposed to mAb-based proprotein convertase subtilisin–kexin type 9 (PCSK9) therapies. Ridker and colleagues reported data from six studies on approximately 4000 patients randomized to the experimental PCSK9 inhibitor bococizumab (a “humanized” …

Evolocumab Improves Cardiovascular Outcomes

Prior research on evolocumab — a monoclonal antibody that inhibits proprotein convertase subtilisin–kexin type 9 (PCSK9) — has shown that this now