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Integrated bioprocessing strategies can facilitate ethanol production from both cellulose and hemicellulose fractions of lignocellulosic biomass. Consolidated bioprocessing (CBP) is an approach that combines enzyme production, biomass hydrolysis and sugar fermentation in a single step. However, technologies that propose the use of microorganisms together with solid biomass present the difficulty of the recovery and reuse of the biocatalyst, which can be overcome by cell immobilization. In this regard, this work applied immobilized cells of AC14 yeast, a recombinant yeast that secretes 7 hydrolytic enzymes, in the CBP process in a successful proof-of-concept for the enzyme access to the substrate polymers. The most appropriate cell load for CBP under the conditions studied with immobilized cells was selected among three optical densities (OD) 10, 55 and 100. These experiments were performed with free cells to ensure that the results were not biased by mass limitations effects. OD 10 achieved 100% of the sugar consumption and the higher specific production of enzymes, being selected for further studies. Diffusional effects were observed with immobilized cells under static conditions. However, mass transfer limitations were mitigated under agitation, with an 18.5% increase in substrate consumption rate (from 2.7 to 3.5 g/L/h), reaching the same substrate uptake rates as free cells. In addition, immobilized cells achieved 100% hydrolysis and consumption of all substrates offered within only 12 h. Overall, this is the first report of a successful application of immobilized yeast cells in CBP processes for bioethanol production, a promising technology that can be extended to other biorefinery bioproducts. [Display omitted] • Consolidated bioprocessing using 7 hydrolytic enzymes producer immobilized yeast. • Hydrolytic enzymes can diffuse through immobilization gel and access substrate. • Agitation improves mass transfer, increasing substrates consumption rate. • Immobilized yeast reached 100% hydrolysis and sugar conversion in only 12 h. [ABSTRACT FROM AUTHOR]

Ascorbic acid (AA) is a natural reducer that has been used as a prooxidant additive to improve dyes oxidation via Fenton processes (Fe2+/H 2 O 2 and Fe3+/H 2 O 2). In the present work, low concentration of AA (10 μmol L1) enhanced Bismarck Brown Y (BBY), Safranin T (ST), Rhodamine B (RB), and Reactive Black 5 (RB5) decolorizations in solution, mainly in reactions initially containing Fe3+ as a catalyst (Fe3+-reactions), e.g., Fe3+/H 2 O 2 decolorization of RB5 was increased from 54 % to 73 % after 60 min due to added AA. Decolorization increased from 0 up to 60–90 μmol L−1 of added reducer. At higher concentrations, AA did not improve decolorization. The dyes were decolorized below 10 % when incubated with 1 % tert-butanol, indicating that HO• radical is the main oxidant involved in the reactions. The kinetic analysis showed that the 1st- and 2nd-order kinetic models fitted well to both Fe2+- and Fe3+-reaction data. The BMG kinetic model also fitted well to the Fe2+-reactions (with and without AA), along with Fe3+/H 2 O 2 /AA. Based on these kinetic models, we verified that reaction rate constants were increased due to added AA. There was a decrease in activation energy (Ea) in decolorizing ST from added AA by varying the reaction temperatures. For example, Ea decreased from 111.6 to 81.8 kJ mol−1 for Fe3+-reactions and from 72.6 to only 69.3 kJ mol−1 for Fe2+-reactions. The AA/H 2 O 2 system was least effective in decolorizing ST since Ea was 130 kJ mol−1. In summary, ascorbic acid decreases the energy barrier to improve Fenton-based ST oxidation. [Display omitted] [ABSTRACT FROM AUTHOR]

Growing fossil raw material demand, especially for producing fuels, has caused serious damage to the environment, mainly in the form of greenhouse gas (GHG) emissions. To minimize non-renewable fuel use and impacts, biomasses can be used as substitutes to achieve a sustainable circular economy, especially in Brazil, which is one of the largest biomass producers in the world. Ethanol, biodiesel, and biogas/biomethane have been extensively studied as alternatives to fossil fuels, the two first being already produced at industrial scales and used as vehicle fuels in Brazil. Additionally, different biomasses have been used as raw materials to produce biofuels, and enzymatic catalysis have shown promising potential to achieve feasible and sustainable processes. Some main enzymes are amylases, which are used extensively in corn starch hydrolysis to produce first generation (1G) ethanol using a well-established process, while cellulases and xylanases are promising in hydrolyzing lignocellulosic materials for producing second generation (2G) ethanol. Lipases, on the other hand, are interesting catalysts for effectively converting triacylglycerols (TAGs), and free fatty acids (FFAs) from several vegetable oils or animal fats into biodiesel. This review comprehensively address biofuels production in Brazil, with a focus on the current status of industrial enzymes, exploring their characteristics, advantages, and discussing current technical challenges for their industrial application. [Display omitted] • Enzymatic catalysis is conventionally used for obtaining biofuels in Brazil. • Amylases and cellulases are used for industrial 1G and 2G ethanol production. • Lipases are promising catalysts in producing biodiesel. • Enzymes improves butanol, biogas, and hydrogen production from several feedstocks. • Improvements in enzymatic catalysis are continuously researched in Brazil. [ABSTRACT FROM AUTHOR]

Latex, a sap produced by cells called laticifers, occurs in plants of wide taxonomic diversity. Plants exude latex sap in response to physical damage. Questions about the function of latex or the underlying mechanisms persist, but a role in defense is likely. The presence of constitutive peptidases in latex sap in addition to inducible and de novo synthesized pathogenesis-related proteins (PR-proteins), raises the question about the role that each sap component plays to protect plants and how synergism occurs among sap proteins in the course of herbivory or infection. Here we discuss a variety of functions for laticifer and latex in plant defense. We propose that latex peptidases build the front line of defense against herbivores or pathogens. Laticifers are highly specialized cells forming a tube-like network structure throughout the plant body, occurring in phylogenetically unrelated groups. Laticifers produce and store latex that is released upon rupture of laticifers. Compounds preformed in the latex, such as peptidases, chitinases, and chitin-binding proteins, play important defensive roles against microbes and/or insects. Latex peptidases emerge as defensive molecules in several ways. Synergism and cooperative work involving chemically diversified latex compounds permits the plant to defend against natural enemies. [ABSTRACT FROM AUTHOR]

Caseins (α s1 -, α s2 , β-, and κ-caseins) form the major protein fraction of milk, irrespective of their origins. They are able to form well-ordered colloidal structures in association with colloidal calcium phosphate, named casein micelles. Chymosin-mediated milk coagulation takes place through loss of casein micelles' stability by hydrolyzing κ-caseins. This process is critical for the quality of cheese and other milk derivatives. Therefore, many microscopy techniques have been used to understand the structural aspects underlying the integrity of casein micelles during chymosin action. However, these technologies can be costly and laborious. In this study, atomic force microscopy (AFM) and dynamic light scattering (DLS) were used to study milk coagulation by chymosin. Following 15 min of chymosin action, the AFM images showed the start of the formation of casein micelle aggregates. After 30–45 min, the micelles continued aggregating, forming structures such as bunches of grapes. Finally, after 45–60 min, these structures formed large clusters of casein micelles. After 60 min, the zeta potential did not drop to zero when the milk clotted, but still had a negative value, suggesting that the κ-caseins on the micelle surface were not totally hydrolyzed. The results corroborate those previously described and suggest this tool as an alternative to study the milk-clotting process at the ultrastructural level induced by proteases. [ABSTRACT FROM AUTHOR]

Transitory allergies to cow milk proteins in infants or adults have become a public health problem. Although extensively or partially hydrolyzed cow milk protein formulas are available, these products are costly. Therefore, studies into innovative enzymes to digest cow milk proteins are needed. Danaus plexippus gut peptidases were purified and examined with regard to cow milk protein hydrolysis. The peptidases hydrolyzed caseins and whey proteins. However, after heat treatment, there was a significant improvement in β-lactoglobulin hydrolysis. The hydrolyzed cow milk proteins were not recognized by anti-casein antibodies and only reacted slightly with antibodies against whey proteins. This performance was better than that of partially hydrolyzed formulas and similar to that of an extensively hydrolyzed formula. These results suggest that D. plexippus gut peptidases are suitable and innovative enzymes to produce hypoallergenic cow milk protein formulas. [ABSTRACT FROM AUTHOR]

The latex of Calotropis procera has previously been reported to contain osmotin. This protein (CpOsm) inhibited phytopathogens and this was mechanistically characterized. Here, the time-course profile of CpOsm transcripts was examined in the salt-stressed cultivated callus of C. procera in order to better understand its role in the physiology of the plant. Stressed callus (80 mM NaCl) showed an unbalanced content of organic compounds (proline and total soluble sugar) and inorganic ions (Na + , Cl − , and K + ). Under salt treatment, the transcripts of CpOsm were detected after 12 h and slightly increased to a maximum at day seven, followed by reduction. Interestingly, CpOsm was not detected in the soluble protein fraction recovered from the salt-stressed callus as probed by electrophoresis, dot/Western blotting and mass spectrometry. The results suggested that (1) CpOsm is not constitutive in cultivated cells (laticifer-free tissues); (2) CpOsm transcripts appear under salt-stressed conditions; (3) the absence of CpOsm in the protein fractions of stressed cultivated cells indicated that salt-induced transcripts were not used for protein synthesis and this accounts to the belief that CpOsm may be a true laticifer protein in C. procera . More effort will be needed to unveil this process. In this study we show evidences that CpOsm gene is responsive to salt stress. However the corresponding protein is not produced in cultivated cells. Therefore, presently the hypothesis that CpOsm is involved in abiotic stress is not fully supported. [ABSTRACT FROM AUTHOR]

Studies have reported responses of plants to magnetism although the underlying mechanisms are not established. Here it is reported biochemical responses of seeds of Calotropis procera , a non-cultivated species, when exposed to static magnetic field (MF). Screening of the MF effect on seed germination was performed (0; 0.5; 1.0; 2.0 mT) and then the best response to field intensity (2 mT) was fixed in further analyses. Seeds were germinated for 5 days under 2 mT or seeds were exposed to 2 mT for 5 days and then germinated. Treatments were compared to seeds germinated without MF. MF reduced the time for seed germination (two days) and increased germination rate (65%–90%). This was accompanied by higher fresh and dry matter. Activity of catalase was not altered. Activity of POX increased and that of APX decreased in seeds after MF exposure, indicating oxidative unbalance. The increased contents of hydrogen peroxide and malondialdehyde were also indicators of oxidative stress. DNA integrity was preserved, while soluble protein and DNA quantity increased, compared to the control. Confocal analysis of seed tissues stained with propidium iodide suggested cellular proliferation stimulated by MF. Proteomics and bioinformatics analysis corroborated these data with up regulated protein involved in energetic and protein metabolism. These observations suggested increased cellular metabolism in seeds submitted to MF. It is concluded that MF accelerated the seed germination of Calotropis procera boosting seed metabolism. In this regard, MF might play a role of a mitogen-like inducer. • Many studies have demonstrated that Magnetic field (MF) affects plant metabolism and physiology. • MF exposure increased ratio of germination and induced faster germination of Calotropis procera seeds Faster germination. • It was accompanied by up regulation of proteins involved in protein and energetic metabolism and oxidative stress. • MF does not affect DNA integrity. • This study suggests that MF induces faster seed germination by increasing seed metabolism. [ABSTRACT FROM AUTHOR]

Background: The latex from the medicinal plant Calotropis procera is often used in folk medicine against infectious and inflammatory diseases.Purpose: In this study, we investigate a protein fraction with immunomodulatory properties, named LPPI, against experimental infections, in vitro and in vivo, with a virulent strain of Listeria monocytogenes.Study Design: LPPI was exposed to cultured macrophages or Swiss mice and then challenged with L. monocytogenes.Methods: Peritoneal macrophages were obtained from Swiss mice, and cultured in 96-well microplates. Soluble latex proteins (LP) were subjected to fractionation by ion-exchange chromatography. The major peak (LPPI) was added into wells at 10 or 100µg/ml. Albumin (100µg/ml) was used for comparison between protein treatments. After incubation for 1h at 5% CO2/ 37°C, the supernatant was discarded and 0.2ml of L. monocytogenes overnight culture was added in the wells. Following 4h and 24h infection, the cytokine mRNA expression was evaluated as well as the number of intracellular colony forming units. Swiss mice (n=16) were injected intraperitoneally (i.p.) with LPPI (5 and 10mg/kg) while the control mice received albumin (10mg/kg) or LP (10mg/kg). After 24h, all animal groups were challenged with L. monocytogenes (10(6) CFU/ ml), also by i.p. route.Results: LPPI was not toxic to uninfected macrophages (pMØ) and significantly increased mRNA expression of TNF-α, IL-6, IL-1β and iNOS. Following infection, cell viability was reduced by 50% in albumin-treated pMØ (control); but only 17% in pMØ treated with LPPI at 100µg/ml. In this case, LPPI increased expression of TNF-α and IL-6 whereas the number of bacterial colony-forming units was reduced 100-fold in comparison to control groups. Swiss mice pretreated with LPPI showed dose-dependent survival rates that reached 80%, while mice that received albumin died 1-3 days after infection. After 24h infection, leukocyte migration to the infectious foci was high in LPPI-treated mice whereas the number of viable bacteria in the peritoneal fluid, liver and bloodstream were significantly reduced.Conclusion: We conclude that LPPI present immunomodulatory properties that are beneficial for prevention of systemic bacterial infections caused by the intracellular bacteria L. monocytogenes. [ABSTRACT FROM AUTHOR]

Two new lectins were purified through affinity chromatography after crude extract preparation under high ionic strength. The hemagglutinating activity of these lectins from the seeds of the legumes Dioclea bicolor (DBL) and Deguelia scandens (DSL) was inhibited by galactose and glucose, respectively, and the molecular masses were estimated at 24 and 22 kDa (via SDS-PAGE), respectively. The alignment of internal peptides of DBL (MS/MS) with known protein sequences revealed similarity to other legume lectins. The N-terminal amino acid sequence of DSL also aligned with legume lectins. Cross-similarities among the two studied lectins were observed only after sequence permutation. More than a dozen lectins have been reported for the genus Dioclea but none that recognize galactose. DSL is the first lectin reported for the Deguelia genus in the tribe Millettieae. With the aid of bioinformatics tools and searches for genome/transcriptome information about closely related sequences, new lectin members of Millettieae were also identified. Electrophoresis profiling and amino acid sequence analysis suggested that DBL-Gal and DSL do not undergo post-transcriptional ConA-like circular permutation. Molecular modeling of the deduced amino acid sequences of the Millettieae lectins suggested that the overall folding of the monomeric structures of legume lectins is conserved. This and other recent studies highlight native plants of the Amazon as renewed sources of lectins. [ABSTRACT FROM AUTHOR]

Background: The osmotin from the medicinal plant Calotropis procera (CpOsm) has characteristics similar to adiponectin, a human protein with immunoregulatory actions.Purpose: This study aimed to investigate whether recombinant osmotin inclusion bodies from C. procera (IB/rCpOsm) produced in E. coli BL21(DE3) can prevent infection-induced inflammation. A virulent strain of Listeria monocytogenes was used as an infection model.Methods: Cells of E. coli BL21(DE3) carrying the plasmid pET303-CpOsm were used to express the recombinant osmotin, which accumulated at reasonable levels as inclusion bodies (IB/rCpOsm). IB/rCpOsm were purified from induced cells and SDS-polyacrylamide gel electrophoresis followed by mass spectrometry analyses confirmed the identity of the major protein band (23 kDa apparent molecular mass) as CpOsm. Peritoneal macrophages (pMØ) from Swiss mice were cultured with IB/rCpOsm (1 or 10 µg/ml) in 96-well plates and then infected with L. monocytogenes. IB/rCpOsm (0.1, 1 or 10 mg/kg) was also administered intravenously to Swiss mice, which were then infected intraperitoneally with L. monocytogenes.Results: Pretreatment of the pMØ with IB/rCpOsm significantly increased cell viability after infection and reduced the intracellular bacterial load. The infiltration of neutrophils into the peritoneal cavity of mice pretreated with IB/rCpOsm at 10 mg/kg (but not 0.1 and 1 mg/kg) was reduced after infection. In these mice, the bacterial load was high in the peritoneal fluid and the liver, but histological damage was discrete. The treatments with IB/rCpOsm at 10 mg/kg significantly increased the expression of the anti-inflammatory cytokine IL-10.Conclusion: This study shows that recombinant osmotin inclusion bodies from C. procera were bioactive and prompted anti-inflammatory actions at therapeutic dosages in the L. monocytogenes infection model. [ABSTRACT FROM AUTHOR]

Highlights: [•] A polyvinyl alcohol-based membrane was used as a delivery system for latex proteins (LP). [•] The biomembrane accelerated wound healing through faster neo-tissue formation. [•] This was accompanied by intensified fibroplasia and collagen deposition. [ABSTRACT FROM AUTHOR]

Abstract: The latex of Calotropis procera is a rich source of proteins that have anti-inflammatory, anti-nociceptive and selective cytotoxic and anti-tumorigenic properties. In this study, two distinct protocols were developed to culture C. procera in vitro in order to obtain active molecules. Soluble proteins of both callus and root cultures were extracted and tested for various activities found in the latex. Anti-inflammatory and anti-nociceptive proteins were present in callus and root extracts; however, these proteins did not possess cytotoxic or anti-tumorigenic activity. Larvicidal proteins were present, but they were not related to others that have been reported previously in latex. This study confirms that tissue culture of C. procera is able to produce therapeutic-grade proteins that have the potential to relieve inflammation and pain associated with inflammatory disorders. [Copyright &y& Elsevier]

Abstract: This study describes the digestive protease activity extracted from the gut of fifth-instar Monarch butterfly larvae, and its proteolytic activity on latex proteins of their host plant, Calotropis procera (the milkweed) and related non-host species from the milkweed family. Gut extracts digested azocasein, BANA and BApNA. Cysteine protease inhibitors such as E-64 and iodoacetamide inhibited proteolytic activity on azocasein; however, the serine protease inhibitors PMSF and leupeptin were more effective. Gut extracts promptly digested LP and were not affected by endogenous latex proteases. Gut extracts, however, did not digest LP from Cryptostegia grandiflora and only slightly digested LP from Plumeria rubra, two plant species that are not consumed by Monarch larvae. The protein profiles of latex proteins extracted from healthy and attacked plants were different. A protein identified as glycoside hydrolase was detected in increased concentrations in latex from damaged plants. Larvae fed on artificial diets containing 1% or 5% latex proteins were not adversely affected and gained weight faster than control larvae. These results provide new information on the resistance of Monarch larvae fed on C. procera and suggest that the ability of Monarch proteolytic enzymes to promptly digest LP can explain (at least in part) how these insects overcome the defensive proteins found in C. procera latex. [ABSTRACT FROM AUTHOR]

Abstract: Aim of the study: The latex of Calotropis procera has been used in the traditional medicinal system for the treatment of leprosy, ulcers, tumors, piles and diseases of liver, spleen, abdomen and toothache. It comprises of a non-dialyzable protein fraction (LP) that exhibits anti-inflammatory properties and a dialyzable fraction (DF) exhibiting pro-inflammatory properties. The present study was carried out to evaluate the effect of LP sub-fractions on neutrophil functions and nociception in rodent models and to elucidate the mediatory role of nitric oxide (NO). Material and methods: The LP was subjected to ion exchange chromatography and the effect of its three sub-fractions (LPPI, LPPII and LPPIII) thus obtained was evaluated on leukocyte functions in the rat peritonitis model and on nociception in the mouse model. Results: LP sub-fractions exhibit distinct protein profile and produce a significant decrease in the carrageenan and DF induced neutrophil influx and exhibit anti-nociceptive property. The LP and its sub-fractions produced a marked reduction in the number of rolling and adherent leukocytes in the mesenteric microvasculature as revealed by intravital microscopy. The anti-inflammatory effect of LPPI, the most potent anti-inflammatory fraction of LP, was accompanied by an increase in the serum levels of NO. Further, our study shows that NO is also involved in the inhibitory effect of LPPI on neutrophil influx. Conclusions: Our study shows that LP fraction of Calotropis procera comprises of three distinct sets of proteins exhibiting anti-inflammatory and anti-nociceptive properties of which LPPI was most potent in inhibiting neutrophil functions and its effects are mediated through NO production. [Copyright &y& Elsevier]

Background: The lectin from Cratylia argentea (CFL) is able to modulate the immune system response and is thus a potential phytotherapeutic substance.Hypothesis/purpose: In this study, we investigated the role of CFL on control of bacterial infection caused by Listeria monocytogenes, the causative agent of human listeriosis.Study Design: Swiss mice were infected with L. monocytogenes and then treated with CFL.Methods: Adult Swiss mice weighing with 30-40 g were infected intraperitoneally with a bacterial suspension (0.2 ml; 1 × 107 CFU/ml). After 30 min, the mice were treated with CFL intravenously at concentrations of 0.1 or 10 mg/kg. Control mice received phosphate-buffered saline (PBS). The animals were euthanized 24 h after infection.Results: We observed that i.v. administration of CFL to Swiss mice did not cause acute toxicity, and reduced the leukocyte counts in the bloodstream 24 h after infection with virulent L. monocytogenes. There was a reduction in the bacterial burden within peritoneal macrophages after infection in CFL-treated mice. Accordingly, the bacterial counts in the bloodstream, spleen and liver also decreased in comparison with the PBS group. Histological damage in the spleen and liver was lower in mice that received CFL treatment. In vitro antimicrobial assays demonstrated that CFL does not inhibit the growth of L. monocytogenes. The mRNA expression of the anti-inflammatory cytokine IL-10 was enhanced with CFL treatment after infection.Conclusion: The lectin from C. argentea (CFL) has immunomodulatory and anti-infective properties of pharmacological interest for control of infectious diseases. [ABSTRACT FROM AUTHOR]

Abstract: Latex-producing plants are widespread in different habitats. Usually these plants secrete an endogenous milk-like fluid in a network of laticifer cells in which sub-cellular organelles intensively synthesize proteins and secondary metabolites. The biological importance of latexes is still unclear and accumulated evidences of their physiological role are still limited. Here laticifer proteins (LP) from Calotropis procera were assayed for insecticidal action against different crop pests in attempt to give new insights for the biological role of latexes. Diets containing 4% LP affected survival (LD50 =4.61%) and decreased weight gain (ED50 =3.07%) of third instars Ceratitis capitata (Diptera: Tephritidae). Third instars Anticarsia gemmatalis (Lepidoptera: Noctuidae) fed on diets containing 0.1% (w/w) of LP showed reduced body mass while survival was reduced (LD50 =0.48%) only for insects grown on 0.5% LP-containing diets. Nonetheless, 1% LP was ineffective against third instars Spodoptera frugiperda (Lepidoptera: Noctuidae). Diets containing 1% LP slightly diminished survival of Dysdercus peruvianus (Hemiptera: Pyrrhocoridae) nymphs while 2% LP was more effective with consistent reduction in bodyweight (ED50 =1.4%) observed at the 14th day. Digestive enzymes of gut extracts of D. peruvianus were unable to breakdown LP. On the contrary, heat treated LP was capable of reducing by 50% proteolysis of gut extracts using BANA as substrate suggesting presence of inhibitory activity of cysteine proteinases. Adults of D. peruvianus were not affected when grown in diets containing 1% of LP. Laticifer proteins were shown to possess chitin-binding proteins and chitinolytic activity. Lectin activity was not detected. Occurrence of cysteine proteinase activity already reported in C. procera latex combined with the activities described here could explain, at least in part, the deleterious effects observed. [Copyright &y& Elsevier]

Abstract: Soluble proteins from the latex of Calotropis procera (LP) were investigated in vitro and in vivo for digestibility as the latex has previously been shown to produce considerable toxic effects on animals. The latex is also an important biologically active compound that displays antiinflammatory and antidiarrhea properties. The proteins were digested by the action of trypsin, pepsin or chemotrypsin as revealed by gel filtration and SDS–PAGE analysis. Furthermore, the full LP digestion was easily achieved by protease treatment. Rabbit polyclonal antibodies raised against LP failed to detect cross-reactive molecules in fecal material of experimental rats following 35 consecutive days of LP consumption in water. Similar patterns of electrophoresis were observed for the negligible amounts of protein observed in the fecal extracts of control and test animals. No death or toxic effects were observed among animals. Taken together these results suggest that harmful and toxic effects on animals of the latex from C. procera are present in its rubber and low molecular weight fractions rather than its protein content. [Copyright &y& Elsevier]

This study aimed to partially characterize the three main serine carboxypeptidases (SCP3, SCP20, and SCP47) from Nepenthes mirabilis. Furthermore, one peptidase (SCP3) was chosen for further heterologous expression in Escherichia coli Shuffle®T7. SCP3 also was characterized in terms of its allergenic potential using bioinformatics tools. SCP3, SCP20, and SCP47 showed very similar 3D structures and mechanistic features to other plant serine peptidases belonging to clan SC and family S10. Although SCP3 was obtained in its soluble form, using 1% ethanol during induction with 0.5 mM IPTG at 16 °C for 18 h, it did not show proteolytic activity by zymography or in vitro analysis. SCP3 presented a few allergenic peptides and several cleavage sites for digestive enzymes. This work describes additional features of these enzymes, opening new perspectives for further studies for characterization and analysis of heterologous expression, as well as their potential biotechnological applications. • Three serine carboxypeptidases (SCP3, SCP20, and SCP47) from N. mirabilis were partially characterized. • The sequences and 3D structures were similar to other SCPs belonging to clan SC and family S10. • Using E. coli Shuffle®T7, SCP3 was expressed with 0.5 mM IPTG at 16 °C after 18 h. • The best protocol to obtain soluble SCP3 was adding 1% ethanol during the expression. • Recombinant SCP3 did not exhibit proteolytic activity. [ABSTRACT FROM AUTHOR]

Glycoconjugates are the ubiquitous components of mammalian cells, mainly synthesized by covalent bonds of carbohydrates to other biomolecules such as proteins and lipids, with a wide range of potential applications in novel vaccines, therapeutic peptides and antibodies (Ab). Considering the emerging developments in glycoscience, renewable production of glycoconjugates is of importance and lignocellulosic biomass (LCB) is a potential source of carbohydrates to produce synthetic glycoconjugates in a sustainable pathway. In this review, recent advances in glycobiology aiming on glycoconjugates production is presented together with the recent and cutting-edge advances in the therapeutic properties and application of glycoconjugates, including therapeutic glycoproteins, glycosaminoglycans (GAGs), and nutraceuticals, emphasizing the integral role of glycosylation in their function and efficacy. Special emphasis is given towards the potential exploration of carbon neutral feedstocks, in which LCB has an emerging role. Techniques for extraction and recovery of mono- and oligosaccharides from LCB are critically discussed and influence of the heterogeneous nature of the feedstocks and different methods for recovery of these sugars in the development of the customized glycoconjugates is explored. Although reports on the use of LCB for the production of glycoconjugates are scarce, this review sets clear that the potential of LCB as a source for the production of valuable glycoconjugates cannot be underestimated and encourages that future research should focus on refining the existing methodologies and exploring new approaches to fully realize the potential of LCB in glycoconjugate production. [Display omitted] • Lignocellulosic biomass is a sustainable source for synthetic glycoconjugates. • Extraction method provides different monomers and oligomers for glycoconjugates synthesis. • Precursors purification is a challenge for glycoconjugates production from biomass. • Glycoconjugates have the potential to develop novel vaccines and therapeutic peptides. [ABSTRACT FROM AUTHOR]

Calotropis procera cysteine peptidases (CpCPs) have been used for reducing cow's milk allergenicity or as rennet in cheesemaking. Due to their residual presence in food products, the present study evaluated, for the first time, different protocols for their immobilization on different supports. Although the yield of immobilization on sulfopropyl-agarose (99%, at pH 7.0) was better than when using DEAE- and MANAE-agarose (40% and 15%, respectively), the derivatives had low recovered activity (4%). On the other hand, MANAE-agarose at pH 10.0 (200 mM buffer) exhibited the highest recovered enzymatic activity (~23%). Regarding the covalent immobilization, the peptidases immobilized on glyoxyl-agarose (glyoxyl-CpCPs) showed broader pH stability (pH 3.0–10.0), 60-fold more stable at 60 °C, and retained 70% of their initial activities after five reaction cycles, even though the immobilization has induced some structural changes analyzed by Fourier-Transform Infrared (FTIR) spectroscopy as well as altered some of enzyme kinetic parameters (V max , K m , K cat , and catalytic efficiency). In addition, this biocatalyst (glyoxyl-CpCPs) hydrolyzed the major cow's milk allergens (whey proteins) to a greater extent (65%) than the soluble enzymes (8%) and a commercial hypoallergenic formula (50%). [Display omitted] • Calotropis procera cysteine peptidases (CpCPs) were immobilized on different supports. • Glyoxyl-agarose was the best among all ionic and covalent supports. • The immobilization induced structural changes and altered the enzyme kinetics. • But the biocatalyst had better pH stability and was 60-fold more stable at 60 °C. • It reduced the milk protein antigenicity to a level lower than of a commercial formula. [ABSTRACT FROM AUTHOR]

• Antimicrobial peptides were generated from the hydrolysis of wheat gluten proteins. • The hydrolysates exhibited activity against the all six tested fungi. • The hydrolysates extended the shelf life of bread in three days. • 28 peptides were sequenced and then the best four peptides were synthesized. • The peptides were able to induce damage to the fungal plasma membrane. Antimicrobial peptides (AMPs) have been reported to be promising alternatives to chemical preservatives. Thus, this study aimed to characterise AMPs generated from the hydrolysis of wheat gluten proteins using latex peptidases of Calotropis procera , Cryptostegia grandiflora , and Carica papaya. The three hydrolysates (obtained after 16 h at 37 °C, using a 1: 25 enzyme: substrate ratio) inhibited the growth of Aspergillus niger , A. chevalieri , Trichoderma reesei , Pythium oligandrum , Penicillium sp., and Lasiodiplodia sp. by 60–90%, and delayed fungal growth on bread by 3 days when used at 0.3 g/kg. Moreover, the specific volume and expansion factor of bread were not affected by the hydrolysates. Of 28 peptides identified, four were synthesised and exhibited activity against Penicillium sp. Fluorescence and scanning electron microscopy suggested that the peptides damaged the fungal plasma membrane. Bioinformatics analysis showed that no peptide was toxic and that the antigenic ones had cleavage sites for trypsin or pepsin. [ABSTRACT FROM AUTHOR]

• A protease, named CpCP3, was purified and characterized from C. procera latex. • It hydrolyzed κ-casein and induced casein micelle aggregation similarly to chymosin. • It made cheeses with yield, protein, fat and ash contents equivalent to chymosin. • It had a very low allergenic and toxic potential. • The sensory analysis showed that cheeses made with CpCP3 had high acceptance index. This article reports the characterization and evaluation of the biotechnological potential of a cysteine protease purified from Calotropis procera (CpCP3). This enzyme was highly stable to different metal ions and was able to hydrolyze κ-casein similarly to bovine chymosin. Atomic force microscopy showed that the process of casein micelle aggregation induced by CpCP3 was similar to that caused by chymosin. The cheeses made using CpCP3 showed higher moisture content than those made with chymosin, but protein, fat, and ash were similar. The sensory analysis showed that cheeses made with CpCP3 had high acceptance index (>80%). In silico analysis predicted the presence of only two short allergenic peptides on the surface of CpCP3, which was highly susceptible to digestive enzymes and did not alter zebrafish embryos' morphology and development. Moreover, recombinant CpCP3 was expressed in Escherichia coli. All results support the biotechnological potential of CpCP3 as an alternative enzyme to chymosin. [ABSTRACT FROM AUTHOR]

Cysteine peptidases (EC 3.4.22) are the most abundant enzymes in latex fluids. However, their physiological functions are still poorly understood, mainly related to defense against phytopathogens. The present study reports the cDNA cloning and sequencing of five undescribed cysteine peptidases from Calotropis procera (Aiton) Dryand (Apocynaceae) as well as some in silico analyses. Of these, three cysteine peptidases (CpCP1, CpCP2, and CpCP3) were purified. Their enzymatic kinetics were determined and they were assayed for their efficacy in inhibiting the hyphal growth of phytopathogenic fungi. The mechanism of action was investigated by fluorescence and atomic force microscopy as well as by induction of reactive oxygen species (ROS). The deduced amino acid sequences showed similar biochemical characteristics and high sequence homology with several other papain-like cysteine peptidases. Three-dimensional models showed two typical cysteine peptidase domains (L and R domains), forming a "V-shaped" active site containing the catalytic triad (Cys, His, and Asn). Proteolysis of CpCP1 was higher at pH 7.0, whereas for CpCP2 and CpCP3 it was higher at 7.5. All peptidases exhibited optimum activity at 35 °C and followed Michaelis-Menten kinetics. However, the major difference among them was that CpCP1 exhibited highest V max , K m , K cat and catalytic efficiency. All peptidases were deleterious to the two fungi tested, with IC 50 of around 50 μg/mL. The peptidases promoted membrane permeabilization, morphological changes with leakage of cellular content, and induction of ROS in F. oxysporum spores. These results corroborate the hypothesis that latex cysteine peptidases play a role in defense against fungi. The latex peptidases promoted membrane permeabilization, morphological changes with leakage of cellular content, and induction of reactive oxygen species in fungal spores. Image 1 • The present study reports five undescribed cysteine peptidases from C. procera. • The sequences showed similar biochemical characteristics with other plant peptidases. • Three peptidases were purified and characterized. • All peptidases exhibited antifungal activity. • All peptidases induced ROS production and damaged membrane of fungal spores. [ABSTRACT FROM AUTHOR]

Highlights • Latex peptidases were able to perform total hydrolysis of caseins. • On the other hand, whey proteins were more resistant to proteolysis. • Heat pretreatment of the whey proteins enhanced the degree of hydrolysis. • In vivo tests showed that latex peptidases reduced antigenicity and allergenicity. • Results were similar to a commercial partially hydrolyzed formula. Abstract The present study evaluated four laticifer fluids as a novel source of peptidases capable of hydrolyzing proteins in cow's milk. The latex peptidases from Calotropis procera (CpLP), Cryptostegia grandiflora (CgLP), and Carica papaya (CapLP) were able to perform total hydrolysis of caseins after 30 min at pH 6.5, as confirmed by a significant reduction in the residual antigenicity. Casein hydrolysis by Plumeria rubra latex peptidases (PrLP) was negligible. Moreover, whey proteins were more resistant to proteolysis by latex peptidases; however, heat pretreatment of the whey proteins enhanced the degree of hydrolysis and reduced the residual antigenicity of the hydrolysates. The in vivo assays show that the cow's milk proteins hydrolysed by CgLP and CapLP exhibited no immune reactions in mice allergic to cow's milk, similar to a commercial partially hydrolysed formula. Thus, these peptidases are promising enzymes for the development of novel hypoallergenic formulas for children with a milk allergy. [ABSTRACT FROM AUTHOR]

Abstract Osmotin- and thaumatin-like proteins (OLPs and TLPs) have been associated with plant defense responses to different biotic stresses. In the present work, several in silico sequences from OLPs and TLPs were investigated by means of bioinformatics tools aiming to prospect for antimicrobial peptides. The peptide sequences chosen were further synthesized and characterized, and their activities and action mechanisms were assayed against some phytopathogenic fungi, bacteria and yeasts of clinical importance. From this survey approach, four peptide sequences (GDCKATSC, CPRALKVPGGCN, IVGQCPAKLKA, and CAADIVGQCPAKLK) were selected considering some chemical parameters commonly attributed to antimicrobial peptides. Antimicrobial assays showed that these peptides were unable to inhibit mycelial growth of phytopathogenic fungi and they did not affect bacterial cell growth. Nevertheless, significant inhibitory activity was found for CPRALKVPGGCN and CAADIVGQCPAKLK against Candida albicans and Saccharomyces cerevisiae. Fluorescence and scanning electron microscopy assays suggested that CAADIVGQCPAKLK did not damage the overall cell structure, or its activity was negligible on yeast membrane and cell wall integrity. However, it induced the production of reactive oxygen species (ROS) and apoptosis. Molecular docking analysis showed that CAADIVGQCPAKLK had strong affinity to interact with specific plasma membrane receptors of C. albicans and S. cerevisiae, which have been described as promoting the induction of apoptosis. The results indicate that CAADIVGQCPAKLK can be a valuable target for the development of a desired antimicrobial agent against the pathogen C. albicans. Graphical abstract Image 1 Highlights • Osmotin and thaumatin-like sequences were studied to prospect for antimicrobial peptides. • The CAADIVGQCPAKLK peptide had anticandidal activity. • The anticandidal peptide did not alter yeast membrane and cell wall integrity. • The peptide induced the production of reactive oxygen species and apoptosis. • Molecular docking suggested the peptide interaction with membrane receptor (PHO36). [ABSTRACT FROM AUTHOR]

Graphical abstract Abstract Phytomodulatory proteins from the latex of the medicinal plant Calotropis procera has been shown to be implicated in many pharmacological properties. However there is no current information about their activity on glucose metabolism, although the latex is used in folk medicine for treating diabetes. In this study the phytomodulatory proteins (LP) from C. procera latex were assessed on glycemic homeostasis. Control animals received a single intravenous dose (5 mg/kg) of LP or saline solution (CTL). Four hours after treatment, the animals were euthanized and their livers were excised for analysis by western blot and RT-PCR AMP-activated protein kinase (AMPK), phosphoenolpyruvate carboxykinase (PEPCK) and tumor necrosis factor alpha (TNF-α). In vivo tests of intraperitoneal tolerance to insulin, glucose and pyruvate were also performed, and the effect of LP administration on fed glycemia was studied followed by blood analysis to determine serum insulin levels. Treatment with LP reduced glycemia two hours after glucose administration (LP: 87.2 ± 3.70 mg/dL versus CTL: 115.6 ± 8.73 mg/dL). However, there was no change in insulin secretion (CTL: 14.16 ± 0.68 mUI/mL and LP: 14.96 ± 0.55 mUI/mL). LP improved the insulin sensitivity, represented by a superior glucose decay constant during an insulin tolerance test (kITT) (4.17 ± 0.94%/min) compared to the CTL group (0.82 ± 0.72%/min), and also improved glucose tolerance at 30 min (105.2 ± 12.4 mg/dL versus 154.2 ± 18.51 mg/dL), while it decreased hepatic glucose production at 15 and 30 min (LP: 75.5 ± 9.31 and 52.5 ± 12.05 mg/dL compared to the CTL: 79.0 ± 3.02 and 84.5 ± 7.49 mg/dL). Furthermore, there was a significant inhibition of gene expression of PEPCK (LP: 0.66 ± 0.06 UA and CTL: 1.14 ± 0.22 UA) and an increase of phosphorylated AMPK (LP: 1.342 ± 0.21 UA versus CTL: 0.402 ± 0.09 UA). These findings confirm the effect of LP on glycemic control and suggest LP may be useful in diabetes treatment. However, the pharmacological mechanism of LP in PEPCK modulation still needs more clarification. [ABSTRACT FROM AUTHOR]

The water-soluble protein fraction obtained from Plumeria pudica (LPPp) latex has previously been demonstrated to have anti-inflammatory and antinociceptive effects. In the present study, LPPp was tested for activity against diarrhea induced by castor oil, prostaglandin E 2 (PGE 2 ) or cholera toxin. Different doses of LPPp (10, 20 or 40 mg/kg) significantly inhibited the percentage of diarrheal stools (31.18%, 42.97% and 59.70%, respectively) induced by castor oil. This event was followed by significant reduction of both intestinal fluid accumulation (31.42%; LPPp 40 mg/kg) and intestinal transit (68.4%; LPPp 40 mg/kg). The pretreatment of animals with LPPp (40 mg/kg) prevented glutathione and malondialdehyde alterations induced by castor oil. The effects of LPPp against diarrhea induced by castor oil were lost when the fraction was submitted to protein denaturing treatment with heat. LPPp (40 mg/kg) also inhibited the average volume of intestinal fluid induced by PGE 2 (inhibition of 46.0%). Furthermore, LPPp (40 mg/kg) prevented intestinal fluid secretion accumulation (37.7%) and chloride ion concentration (50.2%) induced by cholera toxin. In parallel, colorimetric assays demonstrated that proteinases, chitinases and proteinase inhibitors were found in LPPp. Our data suggest that the antidiarrheal effect of LPPp is due to its protein content and is probably associated with its anti-inflammatory properties. [ABSTRACT FROM AUTHOR]

• Calotropis procera cysteine peptidases (CpCPs) were immobilized on glyoxyl-agarose. • Casein hydrolysis by glyoxyl-CpCPs was similar to soluble form and chymosin. • The casein micelle aggregation also was very similar to soluble form and chymosin. • Glyoxyl-CpCPs performed well after five reaction cycles. • Glyoxyl-CpCPs produced cheeses with characteristics similar to those using chymosin. Calotropis procera cysteine peptidases (CpCPs) have presented several potential biotechnological applications. Here, these enzymes were immobilized on glyoxyl-agarose (glyoxyl-CpCPs) with yields of 90–95 % and the recovered activities ranged from 10 % to 15 %, according to enzyme loadings (5, 10, 20, 40, and 50 mgBSAeq/g). Spectrophotometric assays and SDS-PAGE showed that the casein hydrolysis by glyoxyl-CpCPs was similar to soluble CpCPs. In addition, glyoxyl-CpCPs exhibited similar ratio of milk-clotting activity to proteolytic activity in comparison with soluble CpCPs and chymosin. Even after being stored for six months at 8 °C, the residual proteolytic activity of glyoxyl-CpCPs remained close to 100 %. Atomic force microscopy and dynamic light scattering techniques showed that the process of casein micelle aggregation after treatment with glyoxyl-CpCPs was very similar to its soluble form and chymosin. Glyoxyl-CpCPs performed well after five reaction cycles, producing cheeses with yield, moisture, protein, and fat similar to those produced with chymosin. [ABSTRACT FROM AUTHOR]

Calotropis procera latex protein (CpLP) is a popular anti-inflammatory and therefore we aimed to study its effects on inflammatory bone loss. Male Wistar rats were subjected to a ligature of molars. Groups of rats received intraperitoneally CpLP (0.3 mg/kg, 1 mg/kg, or 3 mg/kg) or saline (0.9% NaCl) one hour before ligature and then daily up to 11 days, compared to naïve. Gingiva was evaluated by myeloperoxidase activity and interleukin-1 beta (IL-1β) expression by ELISA. Bone resorption was evaluated in the region between the cement-enamel junction and the alveolar bone crest. The histology considered alveolar bone resorption and cementum integrity, leukocyte infiltration, and attachment level, followed by immunohistochemistry bone markers between 1st and 2nd molars. Systemically, the weight of the body and organs, and a leukogram were performed. The periodontitis significantly increased myeloperoxidase activity and the IL-1β level. The increased bone resorption was histologically corroborated by periodontal destruction, leukocyte influx, and attachment loss, as well as the increasing receptor activator of the nuclear factor-kappa B ligand (RANKL)/osteoprotegerin (OPG) ratio, and Tartrate-resistant acid phosphatase (TRAP)+ cells when compared to naïve. CpLP significantly reduced myeloperoxidase activity, level of IL-1β, alveolar bone resorption, periodontal destruction, leukocyte influx, and attachment loss. The CpLp also reduced the RANKL/OPG ratio and TRAP+ cells, when compared with the saline group, and did not affect the systemic parameters. CpLP exhibited a periodontal protective effect by reducing inflammation and restricting osteoclastic alveolar bone resorption in this rat model. [Display omitted] [ABSTRACT FROM AUTHOR]

Chitinases (EC 3.2.1.14) found in the latex of Calotropis procera (Ait) R. Br. were studied. The proteins were homogeneously obtained after two ion exchange chromatography steps. Most proteins were identified individually in 15 spots on 2-D gel electrophoresis with isoelectric points ranging from 4.6 to 6.0 and molecular masses extending from 27 to 30 kDa. Additionally, 66 kDa proteins were identified as chitinases in SDS-PAGE. Their identities were further confirmed by mass spectrometry (MS) analysis of the tryptic digests of each spot and MS analysis of the non-digested proteins. Positive reaction for Schiff's reagent suggested the proteins are glycosylated. The chitinases exhibited high catalytic activity toward to colloidal chitin at pH 5.0, and this activity underwent decay in the presence of increasing amounts of reducing agent dithiothreitol. Spore germination and hyphae growth of two phytopathogenic fungi were inhibited only marginally by the chitinases but were affected differently. This suggested a complex relationship might exist between the specificity of the proteins toward the fungal species. The chitinases showed potent insecticidal activity against the Bruchidae Callosobruchus maculatus , drastically reducing survival, larval weight and adult emergence. It is concluded that closely related chitinases are present in the latex of C. procera , and the first experimental evidence suggests these proteins are involved more efficiently in defence strategies against insects rather than fungi. [ABSTRACT FROM AUTHOR]

Latex fractions from Calotropis procera, Cryptostegia grandiflora, Plumeria rubra, and Himatanthus drasticus were assayed in order to prospect for new plant peptidases with milk-clotting activities, for use as rennet alternatives. Only C. procera and C. grandiflora latex fractions exhibited proteolytic and milk-clotting activities, which were not affected by high concentrations of NaCl and CaCl 2 . However, pre-incubation of both samples at 75 °C for 10 min eliminated completely their activities. Both proteolytic fractions were able to hydrolyze k-casein and to produce peptides of 16 kDa, a similar SDS-PAGE profile to commercial chymosin. RP-HPLC and mass spectrometry analyses of the k-casein peptides showed that the peptidases from C. procera or C. grandiflora hydrolyzed k-casein similar to commercial chymosin. The cheeses made with both latex peptidases exhibited yields, dry masses, and soluble proteins similar to cheeses prepared with commercial chymosin. In conclusion, C. procera and C. grandiflora latex peptidases with the ability to coagulate milk can be used as alternatives to commercial animal chymosin in the cheese manufacturing process. [ABSTRACT FROM AUTHOR]

Proteins that share similar primary sequences to the protein originally described in salt-stressed tobacco cells have been named osmotins. So far, only two osmotin-like proteins were purified and characterized of latex fluids. Osmotin from Carica papaya latex is an inducible protein lacking antifungal activity, whereas the Calotropis procera latex osmotin is a constitutive antifungal protein. To get additional insights into this subject, we investigated osmotins in latex fluids of five species. Two potential osmotin-like proteins in Cryptostegia grandiflora and Plumeria rubra latex were detected by immunological cross-reactivity with polyclonal antibodies produced against the C . procera latex osmotin (CpOsm) by ELISA, Dot Blot and Western Blot assays. Osmotin-like proteins were not detected in the latex of Thevetia peruviana , Himatanthus drasticus and healthy Carica papaya fruits. Later, the two new osmotin-like proteins were purified through immunoaffinity chromatography with anti-CpOsm immobilized antibodies. Worth noting the chromatographic efficiency allowed for the purification of the osmotin-like protein belonging to H . drasticus latex, which was not detectable by immunoassays. The identification of the purified proteins was confirmed after MS/MS analyses of their tryptic digests. It is concluded that the constitutive osmotin-like proteins reported here share structural similarities to CpOsm. However, unlike CpOsm, they did not exhibit antifungal activity against Fusarium solani and Colletotrichum gloeosporioides . These results suggest that osmotins of different latex sources may be involved in distinct physiological or defensive events. [ABSTRACT FROM AUTHOR]

A new peptide (1341 g/mol) from Jatropha curcas seeds was isolated. The linear sequence (APTLSGGSVPRDAD) was deduced by de novo peptide sequencing, and further used as scaffold for synthesis of linear (1342 g/mol) and cyclic (1324 g/mol) synthetic analogues. The full peptide sequence was identified as inserted in a putative conserved domain of late-embryogenesis proteins which produced a significant alignment hit (100% of identity and E -value of 1e−05) with a hypothetical protein JCGZ_12502 of J. curcas . Whereas in the linear peptide predominated the double charged ion state ( m / z 671.68), in the cyclic form was observed the mono charged ion state ( m / z of 1325.19) and an unusual MS/MS fragmentation pattern. The differences between the forms were discrete in terms of ionic mobility, retention time (reverse phase) and net charge as function of pH. Circular dichroism spectra presented an intense negative peak at 198 nm which is assigned for its disordered contents. A negative peak at 222 nm in the spectrum of the circular form suggested its structure was not as disordered as the linear form. The peptides were neither haemolytic nor cytotoxic and did not inhibit phytopathogenic fungi. Surprisingly, the circular but not the linear peptide increased the proteolytic activity of papain. [ABSTRACT FROM AUTHOR]