Construction of highly active and stable recombinant nattokinase by engineered bacteria and computational design.

Nattokinase (NK) is an enzyme that has been recognized as a new potential thrombolytic drug due to its strong thrombolytic activity. However, it is difficult to maintain the enzyme activity of NK during high temperature environment of industrial production. In this study, we constructed six NK mutants with potential for higher thermostability using a rational protein engineering strategy integrating free energy-based methods and molecular dynamics (MD) simulation. Then, wild-type NK and NK mutants were expressed in Escherichia coli (E. coli), and their thermostability and thrombolytic activity were tested. The results showed that, compared with wild-type NK, the mutants Y256P, Q206L and E156F all had improved thermostability. The optimal mutant Y256P showed a higher melting temperature (Tm) of 77.4 °C, an increase of 4 °C in maximum heat-resistant temperature and an increase of 51.8 % in activity at 37 °C compared with wild-type NK. Moreover, we also explored the mechanism of the increased thermostability of these mutants by analysing the MD trajectories under different simulation temperatures.

Heterologous expression of nattokinase in E. coli: Biochemical characterization and functional analysis of fibrin binding residues.

Heterologous expression of nattokinase, a potent fibrinolytic enzyme, has been successfully carried out in various microorganisms. However, the successful expression of this enzyme as a soluble protein was not achieved in E. coli. This study delves into the expression of nattokinase in E. coli as a soluble protein followed by its biochemical characterization and functional analysis for fibrinolytic activity. E. coli BL21C41 and pET32a vector host strain with pGro7 protein chaperone induced with IPTG at 16 °C 180 rpm for 16 h enabled the production of recombinant nattokinase in soluble fraction. Enzymatic assays demonstrated its protease activity, while characterization revealed optimal catalytic conditions at 37 °C and pH 8.0, with remarkable stability over a broad pH range (6.0-10.0) and up to 50 °C. The kinetic constants were determined as follows: Km = 25.83 ± 3.43 µM, Vmax = 62.91 ± 1.68 µM/s, kcat = 38.45 ± 1.06 s-1, and kcat/Km = 1.49 × 106 M-1 s-1. In addition, the fibrinolytic activity of NK, quantified by the fibrin plate hydrolysis assay was 1038 ± 156 U/ml, with a corresponding specific activity of 1730 ± 260 U/mg and the assessment of clot lysis time on an artificial clot (1 mg) was found to be 51.5 ± 2.5 min unveiling nattokinase's fibrinolytic potential. Through molecular docking, a substantial binding energy of -6.46 kcal/mol was observed between nattokinase and fibrin, indicative of a high binding affinity. Key fibrin binding residues, including Ser300, Leu302, and Asp303, were identified and confirmed. These mutants affected specifically the fibrin binding and not the proteolytic activity of NK. This comprehensive study provides crucial conditions for the expression of protein in soluble form in E. coli and biochemical properties paving the way for future research and potential applications in medicine and biotechnology.

Peptidic boronic acids are potent cell-permeable inhibitors of the malaria parasite egress serine protease SUB1.

Malaria is a devastating infectious disease, which causes over 400,000 deaths per annum and impacts the lives of nearly half the world's population. The causative agent, a protozoan parasite, replicates within red blood cells (RBCs), eventually destroying the cells in a lytic process called egress to release a new generation of parasites. These invade fresh RBCs to repeat the cycle. Egress is regulated by an essential parasite subtilisin-like serine protease called SUB1. Here, we describe the development and optimization of substrate-based peptidic boronic acids that inhibit Plasmodium falciparum SUB1 with low nanomolar potency. Structural optimization generated membrane-permeable, slow off-rate inhibitors that prevent Pfalciparum egress through direct inhibition of SUB1 activity and block parasite replication in vitro at submicromolar concentrations. Our results validate SUB1 as a potential target for a new class of antimalarial drugs designed to prevent parasite replication and disease progression.

Quantitative at-line monitoring of enzymatic hydrolysis using benchtop diffusion nuclear magnetic resonance spectroscopy.

Benchtop diffusion nuclear magnetic resonance (NMR) spectroscopy was used to perform quantitative monitoring of enzymatic hydrolysis. The study aimed to test the feasibility of the technology to characterize enzymatic hydrolysis processes in real time. Diffusion ordered spectroscopy (DOSY) was used to measure the signal intensity and apparent self-diffusion constant of solubilized protein in hydrolysate. The NMR technique was tested on an enzymatic hydrolysis reaction of red cod, a lean white fish, by the endopeptidase alcalase at 50°C. Hydrolysate samples were manually transferred from the reaction vessel to the NMR equipment. Measurement time was approximately 3 min per time point. The signal intensity from the DOSY experiment was used to measure protein concentration and the apparent self-diffusion constant was converted into an average molecular weight and an estimated degree of hydrolysis. These values were plotted as a function of time and both the rate of solubilization and the rate of protein breakdown could be calculated. In addition to being rapid and noninvasive, DOSY using benchtop NMR spectroscopy has an advantage compared with other enzymatic hydrolysis characterization methods as it gives a direct measure of average protein size; many functional properties of proteins are strongly influenced by protein size. Therefore, a method to give protein concentration and average size in real time will allow operators to more tightly control production from enzymatic hydrolysis. Although only one type of material was tested, it is anticipated that the method should be applicable to a broad variety of enzymatic hydrolysis feedstocks.

Impact of Alcalase Hydrolysis and Simulated Gastrointestinal Digestion on the Release of Bioactive Peptides from Erythrina edulis (Chachafruto) Proteins.

Amidst increasing awareness of diet-health relationships, plant-derived bioactive peptides are recognized for their dual nutritional and health benefits. This study investigates bioactive peptides released after Alcalase hydrolysis of protein from chachafruto (Erythrina edulis), a nutrient-rich South American leguminous plant, focusing on their behavior during simulated gastrointestinal digestion. Evaluating their ability to scavenge radicals, mitigate oxidative stress, and influence immune response biomarkers, this study underscores the importance of understanding peptide interactions in digestion. The greatest contribution to the antioxidant activity was exerted by the low molecular weight peptides with ORAC values for the <3 kDa fraction of HES, GD-HES, and GID-HES of 0.74 ± 0.03, 0.72 ± 0.004, and 0.56 ± 0.01 (µmol TE/mg protein, respectively). GD-HES and GID-HES exhibited immunomodulatory effects, promoting the release of NO up to 18.52 and 8.58 µM, respectively. The findings of this study highlighted the potential of chachafruto bioactive peptides in functional foods and nutraceuticals, supporting human health through dietary interventions.

Enzymatic Hydrolysis as an Effective Method for Obtaining Wheat Gluten Hydrolysates Combining Beneficial Functional Properties with Health-Promoting Potential.

The byproduct from wheat starch production contains approximately 70% gluten (WG) and is an inexpensive but demanding protein raw material for the food industry. This study attempted to determine the optimal hydrolysis conditions for such raw material to obtain peptides combining beneficial functional characteristics with health-promoting activity. The proteases Bromelain, Alcalase, Flavourzyme, and a protease from A. saitoi were used for hydrolysis. It was shown that the tested proteases differ both in terms of the effective hydrolysis conditions of gluten and the profile of the released hydrolysates. Bromelain was particularly effective in converting gluten into peptides, combining beneficial health and functional properties. It achieved maximum activity (189 U/g) against WG at pH 6 and 60 °C, and the best-balanced peptides in terms of desired properties were released at a dose of 2.5 U/g. These peptides were free from most allergenic epitopes, effectively inhibited ACE, and, at 0.34 g, were equivalent to the approved dose of BHT. Their emulsifying activity was higher than that of gluten, and the foaming formation and stabilization potential exceeded that of ovalbumin by 10% and 19%, respectively. It seems that Bromelain-released WG hydrolysates are a promising candidate for a safe fat stabilizer and egg white substitute.

In Vitro Multi-Bioactive Potential of Enzymatic Hydrolysis of a Non-Toxic Jatropha curcas Cake Protein Isolate.

The Jatropha curcas cake, a protein-rich by-product of biofuel production, was the subject of our study. We identified and quantified the ACE inhibitory, antioxidant, and antidiabetic activities of bioactive peptides from a Jatropha curcas L. var Sevangel protein isolate. The protein isolate (20.44% recovered dry matter, 38.75% protein content, and 34.98% protein yield) was subjected to two enzyme systems for hydrolysis: alcalase (PEJA) and flavourzyme (PEJF), recording every 2 h until 8 h had passed. The highest proteolytic capacity in PEJA was reached at 2 h (4041.38 ± 50.89), while in PEJF, it was reached at 6 h (3435.16 ± 59.31). Gel electrophoresis of the PEJA and PEJF samples showed bands corresponding to peptides smaller than 10 kDa in both systems studied. The highest values for the antioxidant capacity (DPPH) were obtained at 4 h for PEJA (56.17 ± 1.14), while they were obtained at 6 h for PEJF (26.64 ± 0.52). The highest values for the antihypertensive capacity were recorded at 6 h (86.46 ± 1.85) in PEJF. The highest antidiabetic capacity obtained for PEJA and PEJF was observed at 6 h, 68.86 ± 8.27 and 52.75 ± 2.23, respectively. This is the first report of their antidiabetic activity. Notably, alcalase hydrolysate outperformed flavourzyme hydrolysate and the cereals reported in other studies, confirming its better multi-bioactivity.

Unraveling the biological potential of chicken viscera proteins: a study based on their enzymatic hydrolysis to obtain hydrolysates with antioxidant properties.

Chicken meat production has increased over the years, leading to a proportional increase in waste generation, which often contains high levels of proteins, such as viscera. Therefore, this study aimed to investigate the enzymatic hydrolysis of chicken viscera proteins as a strategy to value solid waste from the poultry industry. The hydrolysates were characterized for their antioxidant properties and molecular weight distribution. Additionally, the enzymatic hydrolysis process was scaled up from 125 mL flasks with 50 mL of protein solution to 3 L using a 6 L bioreactor. The enzymatic hydrolysis of chicken viscera proteins using a binary mixture of proteases (85.25 U/mL of each enzyme, Alcalase and Flavourzyme, totaling 170.5 U/mL) resulted in an increase of up to 245% in 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging, 353% 2,2-diphenyl-1-picryl-hydrazyl (DPPH) in radical scavenging, 69% in Ferric Reducing Antioxidant Power Assay (FRAP) and 146% in total reducing capacity (TRC). The antioxidant properties of the protein hydrolysates are preserved during the scale-up of enzymatic hydrolysis. Protein fractions smaller than 5 kDa showed the highest ABTS and DPPH radical scavenging activities, while fractions greater than 30 kDa showed the best results for the FRAP method.

Structure, physicochemical properties, and biological activities of protein hydrolysates from Zanthoxylum seed.

BACKGROUND: Zanthoxylum seed, as a low-cost and easily accessible plant protein resource, has good potential in the food industry. But protein and its hydrolysates from Zanthoxylum seed are underutilized due to the dearth of studies on them. This study aimed to investigate the structure and physicochemical and biological activities of Zanthoxylum seed protein (ZSP) hydrolysates prepared using Protamex®, Alcalase®, Neutrase®, trypsin, or pepsin. RESULTS: Hydrolysis using each of the five enzymes diminished average particle size and molecular weight of ZSP but increased random coil content. ZSP hydrolysate prepared using pepsin had the highest degree of hydrolysis (24.07%) and the smallest molecular weight (<13 kDa) and average particle size (129.80 nm) with the highest solubility (98.9%). In contrast, ZSP hydrolysate prepared using Alcalase had the highest surface hydrophobicity and foaming capacity (88.89%), as well as the lowest foam stability (45.00%). Moreover, ZSP hydrolysate prepared using Alcalase exhibited the best hydroxyl-radical scavenging (half maximal inhibitory concentration (IC50 ) 1.94 mg mL-1 ) and ferrous-ion chelating (IC50 0.61 mg mL-1 ) activities. Additionally, ZSP hydrolysate prepared using pepsin displayed the highest angiotensin-converting enzyme inhibition activity (IC50 0.54 mg mL-1 ). CONCLUSION: These data showed that enzyme hydrolysis improved the physicochemical properties of ZSP, and enzymatic hydrolysates of ZSP exhibited significant biological activity. These results provided validation for application of ZSP enzymatic hydrolysates as antioxidants and antihypertensive agents in the food or medicinal industries. © 2023 Society of Chemical Industry.

Heterologous expression of recombinant nattokinase in Escherichia coli BL21(DE3) and media optimization for overproduction of nattokinase using RSM.

Nattokinase, a serine protease, was discovered in Bacillus subtilis during the fermentation of a soybean byproduct. Nattokinase is essential for the lysis of blood clots and the treatment of cardiac diseases including atherosclerosis, thrombosis, high blood pressure, and stroke. The demand for thrombolytic drugs rises as the prevalence of cardiovascular disease rises, and nattokinase is particularly effective for the treatment of cardiovascular diseases due to its long duration of action. In this study, we cloned the nattokinase gene from the Bacillus subtilis strain into the pET32a vector and expressed the protein in the E. coli BL21(DE3) strain. The active recombinant nattokinase was purified using Ni-NTA affinity chromatography and then evaluated for fibrinolytic and blood clot lysis activity. Physiological parameters for optimizing protein production at optimal pH, temperature, IPTG concentration, and incubation time were investigated. A statistical technique was used to optimize media components for nattokinase overproduction, and Central Composite Design-Response Surface Methodology-based optimization was used to select significant components for protein production. The optimized media produced 1805.50 mg/L of expressed nattokinase and 42.80 gm/L of bacterial mass. The fibrinolytic activity obtained from refolded native protein was 58FU/mg, which was five times higher than the available orokinase drug (11FU/mg). The efficiency with which a statistical technique for media optimization was implemented improved recombinant nattokinase production and provides new information for scale - up nattokinase toward industrial applications.

The Structural Characteristics and Bioactivity Stability of Cucumaria frondosa Intestines and Ovum Hydrolysates Obtained by Different Proteases.

The study aimed to investigate the effects of alcalase, papain, flavourzyme, and neutrase on the structural characteristics and bioactivity stability of Cucumaria frondosa intestines and ovum hydrolysates (CFHs). The findings revealed that flavourzyme exhibited the highest hydrolysis rate (51.88% ± 1.87%). At pH 2.0, the solubility of hydrolysate was the lowest across all treatments, while the solubility at other pH levels was over 60%. The primary structures of hydrolysates of different proteases were similar, whereas the surface hydrophobicity of hydrolysates was influenced by the types of proteases used. The hydrolysates produced by different proteases were also analyzed for their absorption peaks and antioxidant activity. The hydrolysates of flavourzyme had ß-fold absorption peaks (1637 cm-1), while the neutrase and papain hydrolysates had N-H bending vibrations. The tertiary structure of CFHs was unfolded by different proteases, exposing the aromatic amino acids and red-shifting of the λ-peak of the hydrolysate. The alcalase hydrolysates showed better antioxidant activity in vitro and better surface hydrophobicity than the other hydrolysates. The flavourzyme hydrolysates displayed excellent antioxidant stability and pancreatic lipase inhibitory activity during gastrointestinal digestion, indicating their potential use as antioxidants in the food and pharmaceutical industries.

Identification of Antioxidative Peptides Derived from Arthrospira maxima in the Biorefinery Process after Extraction of C-Phycocyanin and Lipids.

Arthrospira maxima has been identified as a sustainable source of rich proteins with diverse functionalities and bioactivities. After extracting C-phycocyanin (C-PC) and lipids in a biorefinery process, the spent biomass still contains a large proportion of proteins with potential for biopeptide production. In this study, the residue was digested using Papain, Alcalase, Trypsin, Protamex 1.6, and Alcalase 2.4 L at different time intervals. The resulting hydrolyzed product with the highest antioxidative activity, evaluated through their scavenging capability of hydroxyl radicals, superoxide anion, 2,2-diphenyl-1-picrylhydrazyl (DPPH), and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS), was selected for further fractionation and purification to isolate and identify biopeptides. Alcalase 2.4 L was found to produce the highest antioxidative hydrolysate product after four-hour hydrolysis. Fractionating this bioactive product using ultrafiltration obtained two fractions with different molecular weights (MW) and antioxidative activity. The low-molecular-weight fraction (LMWF) with MW <3 kDa had higher DPPH scavenging activity with the IC50 value of 2.97 ± 0.33 compared to 3.76 ± 0.15 mg/mL of the high-molecular-weight fraction (HMWF) with MW >3 kDa. Two stronger antioxidative fractions (F-A and F-B) with the respective significant lower IC50 values of 0.83 ± 0.22 and 1.52 ± 0.29 mg/mL were isolated from the LMWF using gel filtration with a Sephadex G-25 column. Based on LC-MS/MS analysis of the F-A, 230 peptides derived from 108 A. maxima proteins were determined. Notably, different antioxidative peptides possessing various bioactivities, including antioxidation, were detected with high predicted scores together with in silico analyses on their stability and toxicity. This study established knowledge and technology to further value-add to the spent A. maxima biomass by optimizing hydrolysis and fraction processes to produce antioxidative peptides with Alcalase 2.4 L after two products already produced in a biorefinery. These bioactive peptides have potential applications in food and nutraceutical products.

Identification of ACE I-Inhibitory Peptides Released by the Hydrolysis of Tub Gurnard (Chelidonichthys lucerna) Skin Proteins and the Impact of Their In Silico Gastrointestinal Digestion.

Tub gurnard is a highly abundant fishery species caught as a discard in the Mediterranean Sea. This work proposes its valorisation through the release of potential antihypertensive peptides and glycosaminoglycans (GAGs) through the controlled hydrolysis of tub gurnard skin proteins. Four proteases (Esperase, Alcalase, Trypsin and Pronase E) were used to obtain potent angiotensin converting enzyme I (ACE)-inhibitory hydrolysates. Peptides and GAGs were separated and evaluated for their antihypertensive potential by fluorometry. The peptide-rich fractions derived from the Esperase and Alcalase hydrolysates showed very low IC50 values (47 and 68 µg/mL, respectively). Only the GAGs from the Trypsin and Esperase hydrolysates were relevant ACE inhibitors (63 and 52% at 1 mg/mL, respectively). The peptide composition of the most potent ACE-inhibitory fractions derived from the Esperase and Alcalase hydrolysates (IC50 values of 33 and 29 µg/mL, respectively) was analysed by RP-LC-ESI-MS/MS. The analysis suggests that the ACE-inhibitory activity is related to the peptide hydrophobicity, as well as to the presence of specific residues at any of the last four C-terminal positions. The in silico gastrointestinal digestion of these fractions yielded small peptides with antihypertensive potential.

The foaming properties of sweet potato protein hydrolysates produced by Alcalase and Ficin.

BACKGROUND: The processing of sweet potatoes generates a waste by-product rich in sweet potato protein (SPP). OBJECTIVE: In this study, the effects of the concentrations of Alcalase and Ficin, hydrolysis time and pH value on the foaming properties of SPP hydrolysates (SPPHs) determined via gas sparging method were investigated. RESULTS: The results showed that SPPH prepared by Alcalase exhibited a significantly higher foaming expansion (the highest of 576%) than that of the SPP (462%) but displayed a weaker liquid volume stability compared with SPPH hydrolyzed by Ficin. The molecular weight of SPPH prepared by Alcalase was distributed in 10-30 kDa. A good microbiological quality of the SPPH prepared by Alcalase in pH 13 has been confirmed, and it is suitable for food application with respect to its microbiological safety profile. CONCLUSIONS: SPPH (pH 13) could be further safely applied in food, especially as a food additive at low concentrations to create a better organic plant-based foaming agent for the food industry. © 2023 Society of Chemical Industry.

Stepwise Strategy to Identify Thrombin as a Hydrolytic Substrate for Nattokinase.

Nattokinase (NK) is a serine protease with a potent thrombolytic activity that possesses multiple cardiovascular disease (CVD) preventative and treatment activities. In light of its advanced beneficial cardiovascular effects and its nature as a serine protease, characterizing its biological substrates is essential for informing and ultimately delineating the molecular mechanism of its thrombolytic and anticoagulant activities that will unlock the powerful strategic design of effective therapies for CVDs. Given the efficacy of NK to break the vicious loop between inflammation, oxidative stress, and thrombosis, and the extensive role of thrombin in the loop, a stepwise computational strategy was developed to investigate the cleavage events of NK, including both a protein-protein complex model for protein substrate recognition and a protease-peptide complex model for the cleavage site identification, whereby their contact region was sited to allow for the prediction of the corresponding cleavage site that was successfully verified by both mass spectrometry (MS)-based N-terminal sequencing and various functional assays. Collectively, thrombin was predicted and identified to be a novel biological substrate of NK, which expanded the comprehensive antithrombus mechanism of NK via breaking the vicious loop between inflammation, oxidative stress, and thrombosis. This study not only provided insight into the interaction characteristics between NK and its hydrolytic substrate for a better understanding toward its catalytic mechanism but also developed a comprehensive computational strategy to elucidate the proteolytic targets of NK for the breakthrough of feature drug development.

Preparation, Identification, Molecular Docking Study and Protective Function on HUVECs of Novel ACE Inhibitory Peptides from Protein Hydrolysate of Skipjack Tuna Muscle.

To prepare bioactive peptides with high angiotensin-I-converting enzyme (ACE)-inhibitory (ACEi) activity, Alcalase was selected from five kinds of protease for hydrolyzing Skipjack tuna (Katsuwonus pelamis) muscle, and its best hydrolysis conditions were optimized using single factor and response surface experiments. Then, the high ACEi protein hydrolysate (TMPH) of skipjack tuna muscle was prepared using Alcalase under the optimum conditions of enzyme dose 2.3%, enzymolysis temperature 56.2 °C, and pH 9.4, and its ACEi activity reached 72.71% at 1.0 mg/mL. Subsequently, six novel ACEi peptides were prepared from TMPH using ultrafiltration and chromatography methods and were identified as Ser-Pro (SP), Val-Asp-Arg-Tyr-Phe (VDRYF), Val-His-Gly-Val-Val (VHGVV), Tyr-Glu (YE), Phe-Glu-Met (FEM), and Phe-Trp-Arg-Val (FWRV), with molecular weights of 202.3, 698.9, 509.7, 310.4, 425.6, and 606.8 Da, respectively. SP and VDRYF displayed noticeable ACEi activity, with IC50 values of 0.06 ± 0.01 and 0.28 ± 0.03 mg/mL, respectively. Molecular docking analysis illustrated that the high ACEi activity of SP and VDRYF was attributed to effective interaction with the active sites/pockets of ACE by hydrogen bonding, electrostatic force, and hydrophobic interaction. Furthermore, SP and VDRYF could significantly up-regulate nitric oxide (NO) production and down-regulate endothelin-1 (ET-1) secretion in HUVECs after 24 h treatment, but also abolish the negative effect of 0.5 µM norepinephrine (NE) on the generation of NO and ET-1. Therefore, ACEi peptides derived from skipjack tuna (K. pelamis) muscle, especially SP and VDRYF, are beneficial components for functional food against hypertension and cardiovascular diseases.

The effects of different protease treatments on the techno-functional, structural, and bioactive properties of bovine casein.

In this study, bovine sodium caseinate (NaCas) was hydrolyzed with four proteases, alcalase, savinase, subtilisin A, and flavourzyme. In addition to the structural changes occurred through the enzymatic hydrolysis, the solubility, oil binding capacity, zeta potential, emulsification properties, and in vitro antioxidant capacity, anti-carcinogenic and antidiabetic properties of hydrolysates were determined. FTIR combined with hierarchical cluster analysis (HCA) made in Amide I region enable to classification of the samples based on the changes of the secondary structure depending on the enzyme type and degree of fragmentation. Technological properties of NaCas were enhanced through the enzymatic hydrolysis, and those were more prominent in serine-type enzymes, regardless of the enzyme type, all hydrolysates showed high antioxidant capacities. All hydrolysates, specifically those produced by savinase and alcalase, reduced the viability of the carcinogenic Caco-2 cells in a dose-dependent manner and showed a very low level of cytotoxicity against healthy HEK-293 cells. The hydrolysis treatment made a significant contribution to the antidiabetic activity of NaCas. Particularly alcalase and savinase hydrolysates suppressed the activity of α- amylase and α- glucosidase. Therefore, the generated milk protein hydrolysates could be used in functional food developments for specific dietary purposes.

Physicochemical and functional properties of Alcalase-extracted protein hydrolysate from oil palm leaves.

BACKGROUND: The oil palm tree produces 90% of wastes and the limited usage of these wastes causes a major disposal problem in the mills. Nevertheless, these by-products have a large amount of nutritional components. Thus, the present study aimed to determine the physicochemical and functional properties of protein hydrolysates (PH) from oil palm leaves (OPL) extracted using different concentrations of Alcalase (0-10%) at 2 h of hydrolysis time. RESULTS: Fourier transform infrared spectral analyses showed that the enzymatic hydrolysis altered functional groups of OPL where a secondary amine was present in the PH. Changes were also observed in the thermal stability where the enthalpy heat obtained for PH (933.93-1142.57 J g-1 ) was much lower than OPL (7854.11 J g-1 ). The results showed that the PH extracted by 8% Alcalase exhibited absolute zeta potential, as well as a high emulsifying activity index (70.64 m2  g-1 of protein) and emulsion stability index (60.58 min). Furthermore, this PH showed higher solubility (96.32%) and emulsifying properties compared to other PHs. It is also comparable with commercial plant proteins, indicating that 8% Alcalase is an optimum concentration for hydrolysis. CONCLUSION: In summary, the physicochemical and functional properties of PH extracted from OPL showed good functional properties, suggesting that it can be used as an alternative plant protein in food industries. © 2021 Society of Chemical Industry.

The motif EXEXXXL in the cytosolic tail of the secretory human proprotein convertase PC7 regulates its trafficking and cleavage activity.

Many secretory proteins are activated by cleavage at specific sites. The proprotein convertases (PCs) form a family of nine secretory subtilisin-like serine proteases, seven of which cleave at specific basic residues within the trans-Golgi network, granules, or at the cell surface/endosomes. The seventh member, PC7, is a type-I transmembrane (TM) protein with a 97-residue-long cytosolic tail (CT). PC7 sheds human transferrin receptor 1 (hTfR1) into soluble shTfR1 in endosomes. To better understand the physiological roles of PC7, here we focused on the relationship between the CT-regulated trafficking of PC7 and its ability to shed hTfR1. Deletion of the TMCT resulted in soluble PC7 and loss of its hTfR1 shedding activity. Extensive CT deletions and mutagenesis analyses helped us zoom in on three residues in the CT, namely Glu-719, Glu-721, and Leu-725, that are part of a novel motif, EXEXXXL725, critical for PC7 activity on hTfR1. NMR studies of two 14-mer peptides mimicking this region of the CT and its Ala variants revealed that the three exposed residues are on the same side of the molecule. This led to the identification of adaptor protein 2 (AP-2) as a protein that recognizes the EXEXXXL725 motif, thus representing a potentially new regulator of PC7 trafficking and cleavage activity. Immunocytochemistry of the subcellular localization of PC7 and its Ala variants of Leu-725 and Glu-719 and Glu-721 revealed that Leu-725 enhances PC7 localization to early endosomes and that, together with Glu-719 and Glu-721, it increases the endosomal activity of PC7 on hTfR1.

Heterologous expression of nattokinase from B. subtilis natto using Pichia pastoris GS115 and assessment of its thrombolytic activity.

BACKGROUND: Nattokinase is a fibrinolytic enzyme that has huge market value as a nutritional supplement for health promotion. In order to increase nattokinase yields, fermentation conditions, strains, cultivation media, and feeding strategies have been optimized. Nattokinase has been expressed using several heterologous expression systems. Pichia pastoris heterologous expression system was the alternative. RESULTS: This report aimed to express high levels of nattokinase from B. subtilis natto (NK-Bs) using a Pichia pastoris heterologous expression system and assess its fibrinolytic activity in vivo. Multicopy expression strains bearing 1-7 copies of the aprN gene were constructed. The expression level of the target protein reached a maximum at five copies of the target gene. However, multicopy expression strains were not stable in shake-flask or high-density fermentation, causing significant differences in the yield of the target protein among batches. Therefore, P. pastoris bearing a single copy of aprN was used in shake-flask and high-density fermentation. Target protein yield was 320 mg/L in shake-flask fermentation and approximately 9.5 g/L in high-density fermentation. The recombinant nattokinase showed high thermo- and pH-stability. The present study also demonstrated that recombinant NK-Bs had obvious thrombolytic activity. CONCLUSIONS: This study suggests that the P. pastoris expression system is an ideal platform for the large-scale, low-cost preparation of nattokinase.