Technical-functional and surface properties of white common bean proteins (Phaseolus vulgaris L.): Effect of pH, protein concentration, and guar gum presence.

Legumes are abundant sources of proteins, and white common bean proteins play an important role in air-water interface properties. This study aims to investigate the technical-functional properties of white common bean protein isolate (BPI) as a function of pH, protein concentration, and guar gum (GG) presence. BPI physicochemical properties were analyzed in terms of solubility, zeta potential, and mean particle diameter at pH ranging from 2 to 9, in addition to water-holding capacity (WHC), oil-holding capacity (OHC), and thermogravimetric analysis. Protein dispersions were evaluated in terms of dynamic, interfacial, and foam-forming properties. BPI showed higher solubility (>80 %) at pH 2 and above 7. Zeta potential and mean diameter ranged from 15.43 to -34.08 mV and from 129.55 to 139.90 nm, respectively. BPI exhibited WHC and OHC of 1.37 and 4.97 g/g, respectively. Thermograms indicated decomposition temperature (295.81 °C) and mass loss (64.73 %). Flow curves indicated pseudoplastic behavior, with higher η100 values observed in treatments containing guar gum. The behavior was predominantly viscous (tg δ > 1) at lower frequencies, at all pH levels, shifting to predominantly elastic at higher frequencies. Equilibrium surface tension (γeq) ranged from 43.87 to 41.95 mN.m-1 and did not decrease with increasing protein concentration under all pH conditions. All treatments exhibited ϕ < 15°, indicating predominantly elastic surface films. Foaming properties were influenced by higher protein concentration and guar gum addition, and the potential formation of protein-polysaccharide complexes favored the kinetic stability of the system.

Chagasin from Trypanosoma cruzi as a molecular scaffold to express epitopes of TSA-1 as soluble recombinant chimeras.

Trypanosoma cruzi is the causative agent of Chagas disease, a global public health problem. New therapeutic drugs and biologics are needed. The TSA-1 recombinant protein of T. cruzi is one such promising antigen for developing a therapeutic vaccine. However, it is overexpressed in E. coli as inclusion bodies, requiring an additional refolding step. As an alternative, in this study, we propose the endogenous cysteine protease inhibitor chagasin as a molecular scaffold to generate chimeric proteins. These proteins will contain combinations of two of the five conserved epitopes (E1 to E5) of TSA-1 in the L4 and L6 chagasin loops. Twenty chimeras (Q1-Q20) were designed, and their solubility was predicted using bioinformatics tools. Nine chimeras with different degrees of solubility were selected and expressed in E. coli BL21 (DE3). Western blot assays with anti-6x-His and anti-chagasin antibodies confirmed the expression of soluble recombinant chimeras. Both theoretically and experimentally, the Q12 (E5-E3) chimera was the most soluble, and the Q20 (E4-E5) the most insoluble protein. Q4 (E5-E1) and Q8 (E5-E2) chimeras were classified as proteins with medium solubility that exhibited the highest yield in the soluble fraction. Notably, Q4 has a yield of 239 mg/L, well above the yield of recombinant chagasin (16.5 mg/L) expressed in a soluble form. The expression of the Q4 chimera was scaled up to a 7 L fermenter obtaining a yield of 490 mg/L. These data show that chagasin can serve as a molecular scaffold for the expression of TSA-1 epitopes in the form of soluble chimeras.

Isolation and evaluation of quinoa (Chenopodium quinoa Willd.) protein fractions. A nutritional and bio-functional approach to the globulin fraction.

This study evaluated the solubility profiles of quinoa grain proteins and applied a complete process for the isolation of its main protein fractions, namely: albumins, globulins, prolamins and glutelins, which corresponded to 26.96%, 41.3%, 1.7% and 23.16% of the total protein content, respectively. When these fractions were digested with pepsin followed by pancreatin, the degrees of hydrolysis achieved varied between 26.62% (for unheated globulin fraction) and 38.97% (for unheated glutelin), with casein reached 33.73% hydrolysis. After heating, the globulin hydrolysis degree increased to 34.7%, not significantly differing from casein. These results reflect its good susceptibility to hydrolysis by digestive enzymes, and this observation is reinforced with assays with pepsin, trypsin and chymotrypsin tested separately. Globulins, the largest protein fraction, showed promising results in additional assays regarding the amino acid profile, with limitation only for lysine in relation to the FAO standard, and the potential for releasing bioactive peptides after digestion. Although pepsin-digested globulin inhibited only 5% of ACE activity under the conditions tested, after 24h with the addition of pancreatin, the inhibition was 100%. Antioxidant activity (DPPH assay) also indicated very similar results, when hydrolysis with pepsin was inefficient in releasing antioxidant peptides, while hydrolysis by pancreatin led to 35 times greater results.

Foaming and Structural Studies on the Acidic Subunit of Amaranth 11S Globulin Modified with Antihypertensive Peptides as a Function of pH and Ionic Strength.

Some studies aimed at revealing the relationship between protein structure and their functional properties. However, the majority of these reports have been carried out using protein isolates. There are limited reports on the possible relationship between the functional properties and the structure of a purified protein. In this work the amaranth 11S globulin acidic subunit (AAC) and five mutations of the same protein that were modified in their variable regions with antihypertensive peptides (VYVYVYVY and RIPP), were analyzed at two ionic strength (2.9 and 17.6 g/L NaCl) and pH (3.0-7.0). Results revealed better solubility for the proteins mutated at the terminal ends (AACM.1 and AACM.4) and lower solubility for the protein inserted with RIPP peptide. Spectroscopy studies revealed an increase of ß-sheet structure at high salt concentration for all proteins. It was also observed that salt concentration acted as a modulator, which allowed a better foam features for all modified proteins limiting movement of side chains and reducing red-shifted displacement of λmax. All proteins showed foam capacity ranging from 76 to 93% although foam stability was twofold better for modified proteins than for AAC at high salt concentration. This study allowed better understanding about the structural changes that influence the foaming properties of engineered proteins.

Improved yield, stability, and cleavage reaction of a novel tobacco etch virus protease mutant.

The protease catalytic subunit of the nuclear inclusion protein A from tobacco etch virus (TEVp) is widely used to remove tags and fusion proteins from recombinant proteins. Some intrinsic drawbacks to its recombinant production have been studied for many years, such as low solubility, auto-proteolysis, and instability. Some point mutations have been incorporated in the amino acid protease sequence to improve its production. Here, a comprehensive review of each mutation reported so far has been made to incorporate them into a mutant called TEVp7M with a total of seven changes. This mutant with a His7tag at N-terminus was produced with remarkable purification yields (55 mg/L of culture) from the soluble fraction in a single step affinity purification. The stability of His7-TEVp7M was analyzed and compared with the single mutant TEVp S219V, making evident that His7-TEVp7M shows very constant thermal stability against pH variation, whereas TEVp S219V is highly sensitive to this change. The cleavage reaction was optimized by determining the amount of protease that could cleave a 100-fold excess substrate in the shortest possible time at 30 °C. Under these conditions, His7-TEVp7M was able to cleave His-tag in the buffers commonly used for affinity purification. Finally, a structural analysis of the mutations showed that four of them increased the polarity of the residues involved and, consequently, showed increased solubility of TEVp and fewer hydrophobic regions exposed to the solvent. Taken together, the seven changes studied in this work improved stability, solubility, and activity of TEVp producing enough protease to digest large amounts of tags or fusion proteins. KEY POINTS: • Production of excellent yields of a TEVp (TEVp7M) by incorporation of seven changes. • His-tag removal in an excess substrate in the common buffers used for purification. • Incorporated mutations improve polarity, stability, and activity of TEVp7M.

In vivo cleavage of solubility tags as a tool to enhance the levels of soluble recombinant proteins in Escherichia coli.

Recombinant proteins are generally fused with solubility enhancer tags to improve the folding and solubility of the target protein of interest. However, the fusion protein strategy usually requires expensive proteases to perform in vitro proteolysis and additional chromatographic steps to obtain tag-free recombinant proteins. Expression systems based on intracellular processing of solubility tags in Escherichia coli, through co-expression of a site-specific protease, simplify the recombinant protein purification process, and promote the screening of molecules that fail to remain soluble after tag removal. High yields of soluble target proteins have already been achieved using these protease co-expression systems. Herein, we review approaches for controlled intracellular processing systems tailored to produce soluble untagged proteins in E. coli. We discuss the different genetic systems available for intracellular processing of recombinant proteins regarding system design features, advantages, and limitations of the various strategies.

Alkaline hydrogen peroxide improves physical, chemical, and techno-functional properties of okara.

The objective was to evaluate the effect of modifying okara with alkaline hydrogen peroxide at different H2O2 concentrations and treatment temperatures on its soluble fiber content, water absorption and holding capacity, swelling capacity, and protein solubility in water. Multi-response optimization and characterization of physical, chemical, and techno-functional properties of unmodified and modified okara under optimal condition were performed. Treatment under optimal condition (2% H2O2 and 42 °C for 5 h) resulted in a 601% increase in soluble fiber content, a 26% increase in water absorption and holding capacity and swelling capacity, and a 609% increase in soluble protein. Scanning electron micrographs revealed that modified okara particles had a more fragmented structure and a rougher surface than control. Alkaline hydrogen peroxide treatment altered the color, chemical composition, and techno-functional properties of okara. The modification method has potential to add value to okara and contribute to the use of agro-industrial residues.

Magnetic fields: biomass potential of Spirulina sp. for food supplement.

This study evaluated the influence of the magnetic field on the chemical composition of Spirulina sp. LEB 18 and its digestibility and protein solubility. The highest protein digestibility of biomass was obtained at 30 °C and with 2.5 g L-1 NaNO3 (78.4%) in the medium, and the highest solubility was found in the cultivated biomass exposed to 60 mT, 30 °C and 2.5 g L-1 NaNO3 (89%, pH 6). MF application did not modify the protein concentration of biomass, but reduced the carbohydrate concentration by 69.1%, showing that the biomass obtained in the culture submitted to MF may be used as an ingredient in the development of protein supplements.

Single-Input Regulatory Cascade for in vivo Removal of the Solubility Tag in Fusion Recombinant Proteins Produced by Escherichia coli.

Solubility tags are commonly fused to target recombinant proteins to enhance their solubility and stability. In general, these protein tags must be removed to avoid misfolding of the partner protein and to allow for downstream applications. Nevertheless, in vitro tag removal increases process complexity and costs. Herein, we describe a synthetic biology-based strategy to permit in vivo removal of a solubility tag (EDA, KDPG aldolase), through co-expression of the fusion recombinant protein (EDA-EGFP) and the tag-cleaving protease (TEVp), in a controlled manner. Basically, the system uses three repressor proteins (LacI, cI434, and TetR) to regulate the expressions of EDA-EGFP and TEVp, in a regulatory cascade that culminates with the release of free soluble target protein (EGFP), following a single chemical induction by IPTG. The system worked consistently when all biological parts were cloned in a single plasmid, pSolubility(SOL)A (7.08 Kb, AmpR), and transformed in Escherichia coli Rosetta (DE3) or BL21(DE3) strains. Total soluble recombinant protein yield (EDA-EGFP + free EGFP) was ca. 272.0 ± 60.1 µg/mL of culture, following IMAC purification; free EGFP composed great part (average = 46.5%; maximum = 67.3%) of the total purified protein fraction and was easily separated from remaining fusion EDA-EGFP (53 KDa) through filtration using a 50 KDa cut-off centrifugal filter.

Surface patches on recombinant erythropoietin predict protein solubility: engineering proteins to minimise aggregation.

BACKGROUND: Protein solubility characteristics are important determinants of success for recombinant proteins in relation to expression, purification, storage and administration. Escherichia coli offers a cost-efficient expression system. An important limitation, whether for biophysical studies or industrial-scale production, is the formation of insoluble protein aggregates in the cytoplasm. Several strategies have been implemented to improve soluble expression, ranging from modification of culture conditions to inclusion of solubility-enhancing tags. RESULTS: Surface patch analysis has been applied to predict amino acid changes that can alter the solubility of expressed recombinant human erythropoietin (rHuEPO) in E. coli, a factor that has importance for both yield and subsequent downstream processing of recombinant proteins. A set of rHuEPO proteins (rHuEPO E13K, F48D, R150D, and F48D/R150D) was designed (from the framework of wild-type protein, rHuEPO WT, via amino acid mutations) that varied in terms of positively-charged patches. A variant predicted to promote aggregation (rHuEPO E13K) decreased solubility significantly compared to rHuEPO WT. In contrast, variants predicted to diminish aggregation (rHuEPO F48D, R150D, and F48D/R150D) increased solubility up to 60% in relation to rHuEPO WT. CONCLUSIONS: These findings are discussed in the wider context of biophysical calculations applied to the family of EPO orthologues, yielding a diverse range of calculated values. It is suggested that combining such calculations with naturally-occurring sequence variation, and 3D model generation, could lead to a valuable tool for protein solubility design.

Acid silage of tuna viscera: production, composition, quality and digestibility / Silagem ácida de vísceras de atum: produção, composição, qualidade e digestibilidade

Fish waste processed in the form of silage may constitute an alternative to the use of fishmeal (FM). In this study the composition and quality of the acid silage produced from tuna viscera (TV) were characterized and the digestibility of the nutrients of this product was determined for jundia Rhamdia quelen, using ytrium oxide as an inert marker and a completely randomized design. At the end of thirty days, 61.74% of the crude protein of TV was solubilized. Acid silage from TV presented good nutritional composition (high protein, good amino acid profile and essential fatty acids) and good microbiological quality. Crude protein digestibility was similar (88.52%) for TV and FM, but dry matter digestibility was higher (P<0.05) for (TV) (92.20%). Tuna silage presented as a high nutritional quality and nutrient digestibility for jundiá juveniles, R. quelen. Therefore, this novel ingredient has potential as an alternative protein source in aquafeeds.

Resíduos de pescado processados na forma de silagem podem se constituir em uma alternativa ao uso de farinha de peixe (FM). Neste estudo caracterizou-se a composição e qualidade da silagem ácida produzida a partir de vísceras de atum (TV) e determinou-se a digestibilidade dos nutrientes deste produto para jundiá Rhamdia quelen, utilizando-se o óxido de ítrio como marcador inerte em delineamento completamente casualizado. Ao final de trinta dias, 61. 74% da proteína bruta da TV estava solubilizada. A silagem ácida de TV apresentou boa composição nutricional (alta proteína, bom perfil de aminoácidos e de ácidos graxos essenciais) e qualidade microbiológica satisfatória. A digestibilidade da proteína bruta foi similar (88.52%) para TV e FM, mas a da matéria seca foi maior (P<0.05) para a TV (92.20%). A silagem de atum apresentou-se como um ingrediente proteico de alta qualidade nutritiva e digestiva para juvenis de jundiá, R. quelen. Portanto, este novo ingrediente tem potencial como fonte alternativa de proteína para rações de espécies aquícolas.

Acid silage of tuna viscera: production, composition, quality and digestibility / Silagem ácida de vísceras de atum: produção, composição, qualidade e digestibilidade

Fish waste processed in the form of silage may constitute an alternative to the use of fishmeal (FM). In this study the composition and quality of the acid silage produced from tuna viscera (TV) were characterized and the digestibility of the nutrients of this product was determined for jundia Rhamdia quelen, using ytrium oxide as an inert marker and a completely randomized design. At the end of thirty days, 61.74% of the crude protein of TV was solubilized. Acid silage from TV presented good nutritional composition (high protein, good amino acid profile and essential fatty acids) and good microbiological quality. Crude protein digestibility was similar (88.52%) for TV and FM, but dry matter digestibility was higher (P<0.05) for (TV) (92.20%). Tuna silage presented as a high nutritional quality and nutrient digestibility for jundiá juveniles, R. quelen. Therefore, this novel ingredient has potential as an alternative protein source in aquafeeds.(AU)

Resíduos de pescado processados na forma de silagem podem se constituir em uma alternativa ao uso de farinha de peixe (FM). Neste estudo caracterizou-se a composição e qualidade da silagem ácida produzida a partir de vísceras de atum (TV) e determinou-se a digestibilidade dos nutrientes deste produto para jundiá Rhamdia quelen, utilizando-se o óxido de ítrio como marcador inerte em delineamento completamente casualizado. Ao final de trinta dias, 61. 74% da proteína bruta da TV estava solubilizada. A silagem ácida de TV apresentou boa composição nutricional (alta proteína, bom perfil de aminoácidos e de ácidos graxos essenciais) e qualidade microbiológica satisfatória. A digestibilidade da proteína bruta foi similar (88.52%) para TV e FM, mas a da matéria seca foi maior (P<0.05) para a TV (92.20%). A silagem de atum apresentou-se como um ingrediente proteico de alta qualidade nutritiva e digestiva para juvenis de jundiá, R. quelen. Portanto, este novo ingrediente tem potencial como fonte alternativa de proteína para rações de espécies aquícolas.(AU)

Determinação da concentração de diferentes sistemas de solventes na solubilização de proteínas de análogo de carne / Determination of the concentration of different solvents systems in the protein solubilization of meat analogue

O uso de solventes específicos para extração de proteínas determina o tipo de reação química que ocorre entre os componentes proteicos, principalmente quando estes foram submetidos a tratamentos térmicos como a extrusão termoplástica, uma tecnologia de alta versatilidade, baixo custo, alta produtividade e que não gera efluentes. No entanto, é necessário o uso de concentrações adequadas dos solventes para maximizar a extração das proteínas. Neste trabalho foi avaliada a solubilização de proteínas de análogo de carne a base de isolado proteico de soja e glúten vital, submetidos ao processo de extrusão termoplástica a baixa (23%) e alta (60%) umidade. Os solventes utilizados foram: tampão fosfato (pH 7,5) de 10, 20, 40, 60, 80 e 100mM, dodecil sulfato de sódio de 1, 2, 3, 4 e 5%, -mercaptoetanol de 1, 2, 3 e 4%, Triton X-100 de 1, 2, 3 e 4% e ureia de 6, 7, 8, 9 e 10M. Todos os reagentes foram dissolvidos ou solubilizados em tampão fosfato 40mM (pH 7,5). Os resultados mostraram que as maiores extrações proteicas foram obtidas com o uso de 40mM de tampão fosfato, 2% de dodecil sulfato de sódio, 2% de -mercaptoetanol, 3% de Triton X-100 e 7M de ureia.(AU)

The use of specific solvents for protein extraction determines the type of chemical reaction, which occurs between the protein components, mainly when the protein was submitted to thermal treatment, such as thermoplastic extrusion, a technology with high versatility, low cost and high throughput and without effluent generation. However, it is necessary to use adequate solvents concentration in order to maximize the protein extraction. The aim of this research was to evaluate the protein solubilization of meat analogue based on isolate soy protein and vital gluten submitted to thermoplastic extrusion process at low moisture content (23%) and high moisture content (60%). The solvents used were: phosphate buffer (pH 7.5) at 10, 20, 40, 60, 80 and 100mM, sodium dodecyl sulphate at 1, 2, 3, 4 and 5%, -mercaptoethanol at 1, 2, 3 and 4%, Triton X-100 at 1, 2, 3 and 4% and urea at 6, 7, 8, 9 and 10M. All the chemical reagents were dissolved or solubilized in phosphate buffer 40mM (pH 7.5). The results showed that the highest protein extraction were obtained when phosphate buffer 40mM, sodium dodecil sulphate 2%, -mercaptoethanol 2%, Triton X-100 3% and urea 7M were used.(AU)

Determinação da concentração de diferentes sistemas de solventes na solubilização de proteínas de análogo de carne / Determination of the concentration of different solvents systems in the protein solubilization of meat analogue

O uso de solventes específicos para extração de proteínas determina o tipo de reação química que ocorre entre os componentes proteicos, principalmente quando estes foram submetidos a tratamentos térmicos como a extrusão termoplástica, uma tecnologia de alta versatilidade, baixo custo, alta produtividade e que não gera efluentes. No entanto, é necessário o uso de concentrações adequadas dos solventes para maximizar a extração das proteínas. Neste trabalho foi avaliada a solubilização de proteínas de análogo de carne a base de isolado proteico de soja e glúten vital, submetidos ao processo de extrusão termoplástica a baixa (23%) e alta (60%) umidade. Os solventes utilizados foram: tampão fosfato (pH 7,5) de 10, 20, 40, 60, 80 e 100mM, dodecil sulfato de sódio de 1, 2, 3, 4 e 5%, β-mercaptoetanol de 1, 2, 3 e 4%, Triton X-100 de 1, 2, 3 e 4% e ureia de 6, 7, 8, 9 e 10M. Todos os reagentes foram dissolvidos ou solubilizados em tampão fosfato 40mM (pH 7,5). Os resultados mostraram que as maiores extrações proteicas foram obtidas com o uso de 40mM de tampão fosfato, 2% de dodecil sulfato de sódio, 2% de β-mercaptoetanol, 3% de Triton X-100 e 7M de ureia.

The use of specific solvents for protein extraction determines the type of chemical reaction, which occurs between the protein components, mainly when the protein was submitted to thermal treatment, such as thermoplastic extrusion, a technology with high versatility, low cost and high throughput and without effluent generation. However, it is necessary to use adequate solvents concentration in order to maximize the protein extraction. The aim of this research was to evaluate the protein solubilization of meat analogue based on isolate soy protein and vital gluten submitted to thermoplastic extrusion process at low moisture content (23%) and high moisture content (60%). The solvents used were: phosphate buffer (pH 7.5) at 10, 20, 40, 60, 80 and 100mM, sodium dodecyl sulphate at 1, 2, 3, 4 and 5%, β-mercaptoethanol at 1, 2, 3 and 4%, Triton X-100 at 1, 2, 3 and 4% and urea at 6, 7, 8, 9 and 10M. All the chemical reagents were dissolved or solubilized in phosphate buffer 40mM (pH 7.5). The results showed that the highest protein extraction were obtained when phosphate buffer 40mM, sodium dodecil sulphate 2%, β-mercaptoethanol 2%, Triton X-100 3% and urea 7M were used.

Prediction of crude protein digestibility of animal by-product meals for dogs by the protein solubility in pepsin method.

Animal by-product meals have large variability in crude protein (CP) content and digestibility. In vivo digestibility procedures are precise but laborious, and in vitro methods could be an alternative to evaluate and classify these ingredients. The present study reports prediction equations to estimate the CP digestibility of meat and bone meal (MBM) and poultry by-product meal (PM) using the protein solubility in pepsin method (PSP). Total tract CP digestibility of eight MBM and eight PM samples was determined in dogs by the substitution method. A basal diet was formulated for dog maintenance, and sixteen diets were produced by mixing 70 % of the basal diet and 30 % of each tested meal. Six dogs per diet were used to determine ingredient digestibility. In addition, PSP of the MBM and PM samples was determined using three pepsin concentrations: 0·02, 0·002 and 0·0002 %. The CP content of MBM and PM ranged from 39 to 46 % and 57 to 69 %, respectively, and their mean CP digestibility by dogs was 76 (2·4) and 85 (2·6) %, respectively. The pepsin concentration with higher Pearson correlation coefficients with the in vivo results were 0·0002 % for MBM (r 0·380; P = 0·008) and 0·02 % for PM (r 0·482; P = 0·005). The relationship between the in vivo and in vitro results was better explained by the following equations: CP digestibility of MBM = 61·7 + 0·2644 × PSP at 0·0002 % (P = 0·008; R (2) 0·126); and CP digestibility of PM = 54·1 + 0·3833 × PSP at 0·02 % (P = 0·005; R (2) 0·216). Although significant, the coefficients of determination were low, indicating that the models were weak and need to be used with caution.