Natural Protease Research Articles

Green Nanotechnology Through Papain Nanoparticles: Preclinical in vitro and in vivo Evaluation of Imaging Triple-Negative Breast Tumors.

Recent advancements in nanomedicine and nanotechnology have expanded the scope of multifunctional nanostructures, offering innovative solutions for targeted drug delivery and diagnostic agents in oncology and nuclear medicine. Nanoparticles, particularly those derived from natural sources, hold immense potential in overcoming biological barriers to enhance therapeutic efficacy and diagnostic accuracy. Papain, a natural plant protease derived from Carica papaya, emerges as a promising candidate for green nanotechnology-based applications due to its diverse medicinal properties, including anticancer properties. This study presents a novel approach in nanomedicine and oncology, exploring the potential of green nanotechnology by developing and evaluating technetium-99m radiolabeled papain nanoparticles (99mTc-P-NPs) for imaging breast tumors. The study aimed to investigate the efficacy and specificity of these nanoparticles in breast cancer models through preclinical in vitro and in vivo assessments. Papain nanoparticles (P-NPs) were synthesized using a radiation-driven method and underwent thorough characterization, including size, surface morphology, surface charge, and cytotoxicity assessment. Subsequently, P-NPs were radiolabeled with technetium-99m (99mTc), and in vitro and in vivo studies were conducted to evaluate cellular uptake at tumor sites, along with biodistribution, SPECT/CT imaging, autoradiography, and immunohistochemistry assays, using breast cancer models. The synthesized P-NPs exhibited a size mean diameter of 9.3 ± 1.9 nm and a spherical shape. The in vitro cytotoxic activity of native papain and P-NPs showed low cytotoxicity in HUVEC, MDA-MB231, and 4T1 cells. The achieved radiochemical yield was 94.2 ± 3.1% that were sufficiently stable (≥90%) for 6 h. The tumor uptake achieved in the 4T1 model was 2.49 ± 0.32% IA/g at 2h and 1.51 ± 0.20% IA/g at 6h. In the spontaneous breast cancer model, 1.19 ± 0.20% IA/g at 2h and 0.86 ± 0.31% IA/g at 6h. SPECT/CT imaging has shown substantial tumor uptake of the new nanoradiopharmaceutical and clear tumor visualization. 99mTc-P-NPs exhibited a high affinity to tumoral cells confirmed by ex vivo autoradiography and immunohistochemistry assays. The findings underscore the potential of green nanotechnology-driven papain nanoparticles as promising agents for molecular imaging of breast and other tumors through SPECT/CT imaging. The results represent a substantial step forward in the application of papain nanoparticles as carriers of diagnostic and therapeutic radionuclides to deliver diagnostic/therapeutic payloads site-specifically to tumor sites for the development of a new generation of nanoradiopharmaceuticals.

Endogenous Proteases in Sea Cucumber (Apostichopus japonicas): Deterioration and Prevention during Handling, Processing, and Preservation.

The sea cucumber is an essential nutrient source and a significant economic marine resource associated with successful aquaculture. However, sea cucumbers are highly susceptible to autolysis induced by endogenous protease after postmortem, and the phenomenon of body wall "melting" occurs, which seriously affects the food quality of products and the degree of acceptance by consumers. To satisfy the growing demand for fresh or processed sea cucumbers, we must clarify the autolysis mechanism of sea cucumbers and the methods to achieve autolysis regulation. In this paper, the factors leading to the quality deterioration and texture softening of sea cucumbers are reviewed, with emphasis on enzymatic characteristics, the autolysis mechanism, the effects of autolysis on the physicochemical properties of the body wall of the sea cucumber, and the development of potential natural protease inhibitors. We aim to provide some reference in future preservation and processing processes for sea cucumbers, promote new processing and preservation technologies, and advance the sea cucumber industry's development.

Contribution of sequential pretreatments and enzyme-assisted extraction to rice bran protein yield improvement

Alcalase is commonly used in enzyme-assisted extraction of rice bran protein. However, broad handling of rice bran can affect proteins before proteolysis. Various pretreatments, including thermal stabilization, washing, defatting, alkaline extraction, and enzymatic cellulosic degradation, and combinations were identified regarding the present and possible handling processes of rice bran protein extraction to investigate their effects on the proteolysis of the protein afterward. Then, the protein extraction yield and proteolytic abilities were compared. The thermal stabilization of the rice bran strongly affects enzymatic protein extraction, which more elaborate extraction protocols can mitigate. Rice bran oil can be extracted earlier without seriously affecting the protein extraction. Washing removes native protease inhibitors and hence speeds up the protein extraction. Alkaline extraction is highly efficient but can cause protein denaturation, generating a challenging protein separation from the gel-like structure. The protein extraction with enzymatic (Ultimase) pretreatment is an efficient method that allows high hydrolysis of intact rice bran protein during the protein extraction step.

COVID-19: A case for plasma derived natural anticoagulants?

Convalescent plasma was proposed for passive immunization against COVID-19; but so far there are conflicting results and still open questions. However, besides antibodies, other plasma proteins may be good candidates for further research and application. Thromboinflammation frequently complicates severe COVID-19, and classical anticoagulants like heparins seem to have limited effect. The natural protease inhibitors antithrombin III (ATIII), α1-antitrypsin (α1-AT) and α2-macroglobulin (α2-M), which are found decreased in severe COVD-19, play a crucial role in prothrombotic and inflammatory pathways. While ATIII and α1-AT are licensed as commercially prepared therapeutic concentrates, there is no preparation of α2-M available. The diagnostic, prognostic, and even therapeutic potential of plasma protease inhibitors should be further explored.

Nanoproteases: Alternatives to Natural Protease for Biotechnological Applications.

Some nanomaterials with intrinsic protease-like activity have the advantages of good stability, biosafety, low price, large-scale preparation and unique property of nanomaterials, which are promising alternatives for natural proteases in various applications. An especial term, "nanoprotease", has been coined to stress the intrinsic proteolytic property of these nanomaterials. As a new generation of artificial proteases, they have become a burgeoning field, attracting many researchers to design and synthesize high performance nanoproteases. In this review, we summarize recent progress on all types of nanoproteases with regard of their activity, mechanism and application and introduce a new and effective strategy for engineering high-performance nanoproteases. In addition, we discuss the challenges and opportunities of nanoprotease research in the future.

Utilizing immobilized recombinant serine alkaline protease from Bacillus safensis lab418 in wound healing: Gene cloning, heterologous expression, optimization, and characterization

Microbial proteases have proven their efficiency in various industrial applications; however, their application in accelerating the wound healing process has been inconsistent in previous studies. In this study, heterologous expression was used to obtain an over-yielding of the serine alkaline protease. The serine protease-encoding gene aprE was isolated from Bacillus safensis lab 418 and expressed in E. coli BL21 (DE3) using the pET28a (+) expression vector. The gene sequence was assigned the accession number OP610065 in the NCBI GenBank. The open reading frame of the recombinant protease (aprEsaf) was 383 amino acids, with a molecular weight of 35 kDa. The yield of aprEsaf increased to 300 U/mL compared with the native serine protease (SAFWD), with a maximum yield of 77.43 U/mL after optimization conditions. aprEsaf was immobilized on modified amine-functionalized films (MAFs). By comparing the biochemical characteristics of immobilized and free recombinant enzymes, the former exhibited distinctive biochemical characteristics: improved thermostability, alkaline stability over a wider pH range, and efficient reusability. The immobilized serine protease was effectively utilized to expedite wound healing. In conclusion, our study demonstrates the suitability of the immobilized recombinant serine protease for wound healing, suggesting that it is a viable alternative therapeutic agent for wound management.

SARS-CoV-2 and Angiotensin-Converting Enzyme-2 Receptor Interaction Blocker– an In-Silico Approach

The global COVID-19 pandemic, instigated by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has led to substantial morbidity and mortality on a worldwide scale. While COVID-19 vaccines offer hope, the emergence of mutated viral strains necessitates the development of FDA-approved drugs to address future outbreaks. Objective: To examine prospective antiviral medications through an analysis of the interaction between the spike protein of SARS-CoV-2 and Angiotensin-converting enzyme-2 (ACE-2) receptors, which play a pivotal role in facilitating viral entry into host cells. Methods: Molecular docking was employed to assess the binding affinities of various protease inhibitors with ACE-2 receptors. Natural proteases, including Furin and Transmembrane serine protease 2 (TMPRSS2), cleave viral spike proteins into S1 and S2 subunits, facilitating fusion with ACE-2 receptors. We assessed the binding energies of Indinavir, Nafamostat, Fosamprenavir, Lopinavir, and Boceprevir to inhibit this interaction with a sense of optimism for their potential therapeutic applications. Results: Our findings suggest potential treatments for COVID-19, with Indinavir, Nafamostat, Fosamprenavir, Lopinavir, and Boceprevir displaying promising binding energies of -9.6 kcal/mol, -8.4 kcal/mol, -7.7 kcal/mol, and -7.5 kcal/mol, respectively. Conclusions: While promising, further clinical trials are important to potentially evaluate the efficacy and safety of these proposed drugs in combating COVID-19 and its variants.

Discovery of Protease-Activated Receptor 4 (PAR4)-Tethered Ligand Antagonists Using Ultralarge Virtual Screening.

Here, we demonstrate a structure-based small molecule virtual screening and lead optimization pipeline using a homology model of a difficult-to-drug G-protein-coupled receptor (GPCR) target. Protease-activated receptor 4 (PAR4) is activated by thrombin cleavage, revealing a tethered ligand that activates the receptor, making PAR4 a challenging target. A virtual screen of a make-on-demand chemical library yielded a one-hit compound. From the single-hit compound, we developed a novel series of PAR4 antagonists. Subsequent lead optimization via simultaneous virtual library searches and structure-based rational design efforts led to potent antagonists of thrombin-induced activation. Interestingly, this series of antagonists was active against PAR4 activation by the native protease thrombin cleavage but not the synthetic PAR4 agonist peptide AYPGKF.

Novel Stable Protease Inhibitor from Phoenix dactylifera(L.) Flowers with Antimicrobial and Antitumoral Activities.

A novel protease inhibitor isolated from date palm Phoenix dactylifera(L.) flowers (PIDF) was purified and characterized. A heat and acidic treatment step followed by ethanol precipitation and reverse-phase high-performance chromatography was applied to purify this natural protease inhibitor to homogeneity with a single band of about 19 kDa. The stability study depicted that PIDF was fully stable at 40 °C and retained 65% of its initial activity after heating at 50 °C for 24 h. Its thermal stability at 70 °C was markedly enhanced by adding calcium, bovine serum albumin, and sorbitol as well as by metal divalent cations, especially Mg2+ and Hg2+. This protease inhibitor showed high inhibitory activity against therapeutic proteases, including pepsin, trypsin, chymotrypsin, and collagenase, and acted as a potent inhibitor of some commercial microbial proteases from Aspergillus oryzae, Bacillus. sp, and Bacillus licheniformis. Moreover, a potent antibacterial spectrum against Gram (+) and Gram (-) bacterial strains and an efficient antifungal effect were observed. Its cytotoxicity toward human colorectal cancer cell LoVo and HCT-116 lines suggested that PIDF could serve as a new therapeutic target inhibiting human colorectal cancer.

Penyembuhan Luka Bakar Gel Enzim Bromelin Secara In Vivo

Bromelain is a proteolytic enzyme or natural protease found in tissues, including the stem, fruit, and leaves of pineapple (Ananas comosus var. comosus) and other plant species of the Bromeliaceae family. Bromelain is known as an efficient debdriding agent because it is beneficial in burn healing and tissue regeneration. This research aims to determine the effectiveness of bromelain enzyme for burns wounds in rabbits (Oryctolagus cuniculus). This research began with the optimization of carbopol 940 base preparations with variations in concentration, namely Formula 1 (F1) for 0,5%, Formula 2 (F2) for 1%, and Formula 3 (F3) for 1,5%. The evaluation of the gel formulation includes an organoleptic test, homogeneity test, pH test, viscosity test, adhesive force test, and spreadability test. The evaluation result indicates the base concentration in Formula 1 (F1) at 0,5% has met the requirements for a gel preparation. Formula 1 (F1) for 0,5, then combined with varying concentration of bromelain, namely Formula 1A (0,1%), Formula 1B (0,5%), and Formula 1C (1%). The gel formulation wa re-evaluated, including an organoleptic test, homogeneity test, pH test, viscosity test, adhesion test, spreadability test, irritation test, and in vivo test. In vivo effectiveness testing and irritation testing were conducted on five treatment groups, namely a positive control group (Bioplacenton®), Formula 1A (0,1%), Formula 1B (0,5%), Formula 1C (1%), and a group without treatment, then observed for 15 days. The observation data were processed using the One Way ANOVA test. Based on the results of the irritation test, Formula 1A (0,1%). Formula 1B (0,5%) and Formula 1C (1%) did not cause skin irritation. In the in vivo test, Formula 1C (1%) indicated the fastest recovery compared to other treatment groups, as evidenced by the decrease in wound diameter and visual observation, where the wound had started to close completely on day 15 of observation.

Application of a recombinant novel trypsin from Trichinella spiralis for serodiagnosis of trichinellosis

BackgroundThe excretory/secretory (ES) antigen of Trichinella spiralis muscle larvae (ML) is currently the most widely used diagnostic antigen to detect T. spiralis infection. However, this antigen has certain drawbacks, such as a complicated ES antigen preparation process and lower sensitivity during the early phase of infection. The aim of this study was to investigate the features of a novel T. spiralis trypsin (TsTryp) and evaluate its potential diagnostic value for trichinellosis.MethodsThe TsTryp gene was cloned and recombinant TsTryp (rTsTryp) expressed. Western blotting and an enzyme-linked immunosorbent assay (ELISA) were performed to confirm the antigenicity of rTsTryp. The expression pattern and distribution signature of TsTryp at various life-cycle stages of T. spiralis were analyzed by quantitative PCR, western blotting and the immunofluorescence test. An ELISA with rTsTryp and ML ES antigens was used to detect immunoglobulins G and M (IgG, IgM) in serum samples of infected mice, swine and humans. The seropositive results were further confirmed by western blot with rTsTryp and ML ES antigens.ResultsTsTryp expression was observed in diverse T. spiralis life-cycle phases, with particularly high expression in the early developmental phase (intestinal infectious larvae and adults), with distribution observed mainly at the nematode outer cuticle and stichosome. rTsTryp was identified by T. spiralis-infected mouse sera and anti-rTsTryp sera. Natural TsTryp protease was detected in somatic soluble and ES antigens of the nematode. In mice infected with 200 T. spiralis ML, serum-specific IgG was first detected by rTsTryp-ELISA at 8 days post-infection (dpi), reaching 100% positivity at 12 dpi, and first detected by ES-ELISA at 10 dpi, reaching 100% positivity at 14 dpi. Specific IgG was detected by rTsTryp 2 days earlier than by ES antigens. When specific IgG was determined in serum samples from trichinellosis patients, the sensitivity of rTsTryp-ELISA and ES antigens-ELISA was 98.1% (51/52 samples) and 94.2% (49/52 samples), respectively (P = 0.308), but the specificity of rTsTryp was significantly higher than that of ES antigens (98.7% vs. 95.4%; P = 0.030). Additionally, rTsTryp conferred a lower cross-reaction, with only three serum samples in total testing positive from 11 clonorchiasis, 20 cysticercosis and 24 echinococcosis patients (1 sample from each patient group).ConclusionsTsTryp was shown to be an early and highly expressed antigen at intestinal T. spiralis stages, indicating that rTsTryp represents a valuable diagnostic antigen for the serodiagnosis of early Trichinella infection.Graphical

Characterisation and beneficial effects of a Lupinus angustifolius protein hydrolysate obtained by immobilisation of the enzyme alcalase®.

Bioactive peptides have been considered potential components for the future functional foods and nutraceuticals generation. The enzymatic method of hydrolysis has several advantages compared to those of chemical hydrolysis and fermentation. Despite this fact, the high cost of natural and commercial proteases limits the commercialization of hydrolysates in the food and pharmacological industries. For this reason, more efficient and economically interesting techniques, such as the immobilisation of the enzyme, are gaining attention. In the present study, a new protein hydrolysate from Lupinus angustifolius was generated by enzymatic hydrolysis through the immobilisation of the enzyme alcalase® (imLPH). After the chemical and nutritional characterization of the imLPH, an in vivo study was carried out in order to evaluate the effect of 12 weeks treatment with imLPH on the plasmatic lipid profile and antioxidant status in western-diet-fed apolipoprotein E knockout mice. The immobilisation of alcalase® generated an imLPH with a degree of hydrolysis of 29.71 ± 2.11%. The imLPH was mainly composed of protein (82.50 ± 0.88%) with a high content of glycine/glutamine, arginine, and aspartic acid/asparagine. The imLPH-treatment reduced the amount of abdominal white adipose tissue, total plasma cholesterol, LDL-C, and triglycerides, as well as the cardiovascular risk indexes (CRI) -I, CRI-II, and atherogenic index of plasma. The imLPH-treated mice also showed an increase in the plasma antioxidant capacity. For the first time, this study demonstrates the beneficial in vivo effect of a lupin protein hydrolysate obtained with the alcalase® immobilised and points out this approach as a possible cost-effective solution at the expensive generation of the hydrolysate through the traditional batch conditions with soluble enzymes.

Trichinella spiralis cathepsin B bound and degraded host's intestinal type I collagen

Trichinellosis is a zoonotic parasitic disease that poses threats to human health, the meat industry, food safety, and huge financial losses. The critical stage of Trichinella spiralis (T. spiralis) infection is the invasion of intestinal larvae into the host's intestinal epithelial cells (IECs). T. spiralis Cathepsin B (TsCB) specifically interacts with IECs to facilitate the invasion of larvae. This study aims to look at how TsCB affects mouse IECs. TsCB was successfully cloned, expressed, and characterized, demonstrating its natural cysteine protease hydrolysis activity. A total of 140 proteins that interact with rTsCB were identified by GST pull-down combined with LC-MS/MS, including type I collagen, an essential component of the host's intestinal epithelial barrier system and intimately related to intestinal epithelial damage. TsCB transcription and expression levels rise, whereas type I collagen in the host's intestinal mucosa declines when the T. spiralis larvae invaded. Besides, it was discovered that TsCB bound to and degraded type I collagen of the host's intestine. This research can serve as a foundation for clarifying how T. spiralis invades the host's intestinal barrier and might provide information on potential targets for the creation of novel treatments to treat parasite illnesses.

Trichinella spiralis cathepsin L damages the tight junctions of intestinal epithelial cells and mediates larval invasion.

Cathepsin L, a lysosomal enzyme, participates in diverse physiological processes. Recombinant Trichinella spiralis cathepsin L domains (rTsCatL2) exhibited natural cysteine protease activity and hydrolyzed host immunoglobulin and extracellular matrix proteins in vitro, but its functions in larval invasion are unknown. The aim of this study was to explore its functions in T. spiralis invasion of the host's intestinal epithelial cells. RNAi significantly suppressed the expression of TsCatL mRNA and protein with TsCatL specific siRNA-302. T. spiralis larval invasion of Caco-2 cells was reduced by 39.87% and 38.36%, respectively, when anti-TsCatL2 serum and siRNA-302 were used. Mice challenged with siRNA-302-treated muscle larvae (ML) exhibited a substantial reduction in intestinal infective larvae, adult worm, and ML burden compared to the PBS group, with reductions of 44.37%, 47.57%, and 57.06%, respectively. The development and fecundity of the females from the mice infected with siRNA-302-treated ML was significantly inhibited. After incubation of rTsCatL2 with Caco-2 cells, immunofluorescence test showed that the rTsCatL2 gradually entered into the cells, altered the localization of cellular tight junction proteins (claudin 1, occludin and zo-1), adhesion junction protein (e-cadherin) and extracellular matrix protein (laminin), and intercellular junctions were lost. Western blot showed a 58.65% reduction in claudin 1 expression in Caco-2 cells treated with rTsCatL2. Co-IP showed that rTsCatL2 interacted with laminin and collagen I but not with claudin 1, e-cadherin, occludin and fibronectin in Caco-2 cells. Moreover, rTsCatL2 disrupted the intestinal epithelial barrier by inducing cellular autophagy. rTsCatL2 disrupts the intestinal epithelial barrier and facilitates T. spiralis larval invasion.

Selective protein hydrolysis catalyzed by LaCoO3 nanoparticles

Many nanoproteases contain quadrivalent metal ions and catalyze peptide-bond hydrolysis only at high temperature (60 °C). Their catalytic efficiency is much less than natural proteases. It leaves a great challenge to develop efficient and reusable nanoproteases with high protease activity at room temperature. Here, we have explored nanoproteases from eight low valent metal oxide nanoparticles. Among these nanoparticles, only perovskite LaCoO3 nanoparticles (LaCoO3 NPs) selectively hydrolyze bovine serum albumin (BSA) at X-Lys peptide bonds at 20 °C, producing fragments of 10–45 kDa, which are adapted to middle-down proteomics. LaCoO3 NPs have high stability and excellent reuse performance. LaCoO3 NPs also hydrolyze surfactant-denatured and heat-denatured BSA. Preliminary mechanism analysis indicates that the low valency of Co2+ and the strong Lewis acidity of La3+ played key roles in the high protease activity of LaCoO3 NPs. This work demonstrates that LaCoO3 NPs are a high promising nanoprotease to selectively hydrolyze insoluble and soluble proteins at 20 °C. More importantly, our findings highlight that manufacture of low valent metal ions-contained nanoparticle is an effectual strategy for fabricating high-efficiency nanoprotease.

Molecular characterization of a novel serine proteinase from Trichinella spiralis and its participation in larval invasion of gut epithelium.

A novel serine proteinase of Trichinells spiralis (TsSPc) has been identified in the excretion/secretion (ES) antigens, but its role in larval invasion is unclear. The aim of this study was to clone and express TsSPc, identify its biological and biochemical characteristics, and investigate its role on larval invasion of gut epithelium during T. spiralis infection. TsSPc has a functional domain of serine proteinase, and its tertiary structure consists of three amino acid residues (His88, Asp139 and Ser229) forming a pocket like functional domain. Recombinant TsSPc (rTsSPc) was expressed and purified. The rTsSPc has good immunogenicity. On Western blot analysis, rTsSPc was recognized by infection serum and anti-rTsSPc serum, natural TsSPc in crude and ES antigens was identified by anti-rTsSPc serum. The results of qPCR, Western blot and indirect immunofluorescence test (IIFT) showed that TsSPc was expressed at diverse stage worms, and mainly localized at cuticle, stichosome and intrauterine embryos of this nematode. The rTsSPc had enzymatic activity of native serine protease, which hydrolyzed the substrate BAEE, casein and collagen I. After site directed mutation of enzymatic active sites of TsSPc, its antigenicity did not change but the enzyme activity was fully lost. rTsSPc specifically bound to intestinal epithelium cells (IECs) and the binding sites were mainly localized in cell membrane and cytoplasm. rTsSPc accelerated larval invasion of IECs, whereas anti-rTsSPc antibodies and TsSPc-specific dsRNA obviously hindered larval invasion. TsSPc was a surface and secretory proteinase of the parasite, participated in larval invasion of gut epithelium, and may be considered as a candidate vaccine target molecule against Trichinella intrusion and infection.

Understanding the Molecular Basis for Enhanced Glutenase Activity of Actinidin using Structural Bioinformatics.

Management of gluten intolerance is currently possible only by consumption of a gluten-free diet (GFD) for a lifetime. The scientific community has been searching for alternatives to GFD, like the inclusion of natural proteases with meals or pre-treatment of gluten-containing foods with glutenases. Actinidin from kiwifruit has shown considerable promise in digesting immunogenic gliadin peptides compared to other plant-derived cysteine proteases. In this study, we aimed to understand the structural basis for the elevated protease action of actinidin against gliadin peptides by using an in silico approach. Docking experiments revealed key differences between the binding of gliadin peptide to actinidin and papain, which may be responsible for their differential digestive action. Sequence comparison of different plant cysteine proteases highlights amino acid residues surrounding the active site pocket of actinidin that are unique to this molecule and hence likely to contribute to its digestive properties.

Enzyme-sensitive soybean protein isolate/cinnamaldehyde antibacterial microcapsules triggered by blueberry proteases

In this study, enzyme-sensitive microcapsules using soybean protein isolate as the wall material, and cinnamaldehyde as an antimicrobial core material, were prepared by vacuum freeze-drying. The results showed that a 4% solution of soybean protein isolate with cinnamaldehyde added at a 4:1 (w/w) ratio provided an effective microcapsule formulation, with an embedding rate of 62.63 ± 0.95% and a particle size of 347.00 ± 31.18 nm. After lyze-drying, SPI-CA microcapsules showed uneven morphology, sag and agglomeration, but the microcapsules had small particle size and good dispersion stability. The 4%SPI/CA-4:1 microcapsules provided protection for cinnamaldehyde for 12 days. The release of cinnamaldehyde was slow from the 6th day, and the cumulative release was 79.08 ± 3.0% after 12 days. In addition, infrared spectroscopy, XRD and DSC prove that the hydration of soybean protein isolate, electrostatic repulsion charges, and the creation of cinnamaldehyde essential oil nanodroplets resulted in stable microcapsules. The microcapsules produced were successful in providing the controlled release of its antibacterial substance when exposed to natural protease concentrations triggered by fungal growth on blueberries. The release of cinnamaldehyde from SPia-Ca microcapsules was triggered by protease, which was secreted from blueberry by microbial contamination. The developed microcapsule has wide applications, and the results of this study provide new ideas for future food preservation technology development.

Proteolytic polymer: polyacrylamides functionalized with amino acids cleave bovine and human serum albumins

Polyacrylamides with various compositions of serine, aspartic acid, and histidine, which are the amino acids involved in the catalytic triad of natural serine protease chymotrypsin, were synthesized and their protein cleavage activity was investigated. SDS-PAGE analysis showed that some of the synthesized ternary copolymers showed cleavage activity against bovine and human serum albumins. Polyacrylamides incorporating a single type of amino acid were also able to cleave the protein substrates. These homopolymers exhibited unique cleavage profiles and pH and temperature sensitivities that differed from those of α-chymotrypsin. The results indicate the potential of polymers functionalized with amino acids as proteolytic artificial enzymes.

Thermoplastic molding of silk protein composite plastic toothbrush handles with on-demand degradability

Toothbrush handles made from synthetic polymers like polypropylene and polyethylene accumulate in the environment at an annual rate of 1 billion devices per year and without significant degradation, resulting in severe burdens particularly in marine environments. Herein, we report a new process to generate these plastic handles using natural, biopolymer-based components using a direct thermoplastic molding approach, that also offers on-demand degradation of the materials. The materials are prepared directly from whole silk cocoons that are thermoplastically transformed into biodegradable plastic parts for consumer needs, here in the form of toothbrush handles; either with silk alone or in combination with other natural sourced materials such as cellulose, chitosan and hydroxyapatite. We demonstrate the on-demand degradation of these bioplastics in different ways, using natural exogenous protease digestion either offered through soil exposure of the silk based plastics or via unique on-demand activation of proteases sequestered in the plastic materials to drive the degradation on-demand (hydration). The approaches described here demonstrate a new approach to bio-plastic formation and degradation from biopolymers that offers widespread options for future consumer materials with a focus on sustainability.