Recovery Of Soluble Proteins Research Articles

Manipulation Strategy to Increase Expression Level of Soluble Recombinant Protein Penicillin G Acylase (PGA) in Bacterial Host Escherichia coli: A Review Article

The strategy of producing PGA on a massive scale with high levels of soluble protein can be through recombinant genetic techniques and expressed in a certain host. E. coli is still a popular bacterial host to produce a recombinant protein which has advantages such as fast growth, low production cost, and high expression rate. Apart from its advantages, E. coli as a production host also has disadvantages including the expression of recombinant proteins often failing to form the proper folding conformation which makes the protein biologically inactive. Many strategies can be developed to overcome these problems, such as the selection of the host strain (E. coli HB101 & JM109), fusion protein to enhance the recovery of soluble protein (MBP & NusA), optimization of fermentation (low-temperature incubation), and optimization of the protein isolation process for the recovery of active PGA (Freeze-thawing method).

Recovery of soy whey protein from soy whey wastewater at various cavitation jet pretreatment time and their structural and emulsifying properties

Protein-polysaccharide composite is of great significance for the development of soluble protein recovery process. This study investigated the effects of cavitation jet (CJ) pretreatment at different time (0, 60, 120, 180, 240, 300 s) intervals on the recovery of soy whey protein (SWP) from soy whey wastewater using chitosan (CH). In addition, the structure and properties of the SWP/CH complexes were examined. The results showed that the recovery yield of SWP reached 84.44 % when the CJ pretreatment time was 180 s, and the EAI and ESI values of the SWP/CH complex increased from 32.39 m2/g and 21 min to 48.47 m2/g and 32 min, respectively. In the CJ pretreatment process, SWP promotes the recombination with chitosan through electrostatic interaction and hydrogen bond, while hydrophobic interaction is also involved. This study has guiding significance for CJ technology in the recovery and utilization of protein in industrial wastewater.

The effects of combined co-expression of groel/es and trigger factor chaperone towards Thermus thermophilus DNA polymerase enzyme expression on Escherichia coli BL21(DE3)

The Thermus thermophilus (Tth) DNA polymerase enzyme, originating from the thermophilic bacterium T. thermophilus, exhibits bifunctionally as both a DNA polymerase and a reverse transcriptase. When expressed in Escherichia coli, the T. thermophilus DNA polymerase enzyme frequently results in the formation of inclusion bodies. This occurrence is attributed to the rapid rate of protein expression in E. coli which surpasses the availability of chaperone proteins and reduced cytoplasmic conditions. The formation of inclusion bodies can reduce the recovery of soluble protein. A combination of GroEL/ES and trigger factor chaperone co-expression was used to overcome this deficiency. Thermus thermophilus gene DNA Pol/pG-Tf3 was transformed into the host E. coli BL21(DE3) and expressed with and without chaperone co-expression. Utilizing co-expression of the chaperone combination GroEL/ ES and trigger factor can reduce the formation of inclusion bodies or decrease the insoluble fraction in the expression of the T. thermophilus DNA polymerase enzyme, resulting in a co-expression total protein content of 3.9258 mg/ml.

Recovery of amino acids and peptides from pig bone soup using thermal pre‐treatment and enzymatic hydrolysis

Pig shoulder bones, by-products from the meat industry, are rich in protein and could be used to produce high amino acid products for food processing. In this study, after formulating the bone soup by heating it in water, we optimized the hydrolyzing process of the bone soup using three commercial enzymes (Flavourzyme, Protamex, and Alcalase). Alcalase hydrolysis gained the highest soluble protein recovery efficiency of 70.45% at the optimum condition, including hydrolysis temperature of 56°C, hydrolysis time of 138 min, and E/S ratio of 2.3%. The Alcalase hydrolysate achieved the peptide average molecular weight of 532 Da. In terms of nutritional value, the amino acid content gradually increased from the samples hydrolyzed with Alcalase, Flavourzyme, and Protamex (15.96, 16.60, and 20.71 mg/ml, respectively). However, the hydrolysate using Alcalase was the most favorable sample in sensory evaluation. Novelty Impact Statement Optimized soluble protein recovery of amino acids and peptides via enzymatic hydrolysis is essential to attain the highest possible nutritional value from raw materials like pig bone. Besides, thermal pretreatment and fat removal were implemented to identify soluble protein recovery efficiency (SPRE) precisely.

Nannochloropsis sp. Biorefinery: Recovery of Soluble Protein by Membrane Ultrafiltration/Diafiltration.

This work proposes a way to maximize the potential of a Nannochloropsis sp. biorefinery process, through membrane technology, producing an extract enriched in soluble proteins, free from the insoluble protein fraction, with a low lipid content and eliminating the colored chlorophyll-a. This procedure, following the principles of a circular economy approach, allows for the valorization of a stream from the biorefining of Nannochloropsis sp. that, otherwise, would be considered a residue without commercial value. The process proposed minimizes fouling phenomena at the membrane surface, making it possible to achieve high permeate fluxes, thus reducing the need for membrane cleaning and, therefore, contributing to an extended membrane lifetime. Supernatant obtained after centrifugation of a suspension of ruptured Nannochloropsis sp. cells was processed by ultrafiltration using a membrane with a cut-off of 100 kDa MWCO. Two different operating approaches were evaluated—controlled transmembrane pressure and controlled permeate flux—under concentration and diafiltration modes. Ultrafiltration operated in a diafiltration mode, under controlled permeate flux conditions, led to the highest soluble protein recovery (78%) with the highest constant permeate flux (12 L·m−2·h−1) and low membrane fouling.

Insight into IgG/IgE binding ability, in vitro digestibility and structural changes of shrimp (Litopenaeus vannamei) soluble extracts with thermal processing

The effects of six kinds of thermal processing on soluble protein recovery, potential allergenicity, in vitro digestibility and structural characteristics of shrimp soluble proteins were evaluated. Obtained results confirmed soluble protein recovery and IgG/IgE reactivity of shrimp soluble extracts were markedly suppressed by various thermal treatments with enhanced digestibility depended on the extent and type of heating applied, which correlated well with the structural alterations and modification. The maximum reduction of IgG/IgE-binding capacity and digestive stability were observed in the autoclaved shrimps because of unfolding of protein and hydrophobic residues exposed. Notably, tropomyosin (TM) and sarcoplasmic calcium-binding protein (SCP) were still IgG/IgE-reactive in various heat-processed shrimps, even higher IgG reactivity were found in heat-treated shrimps TM according to TM antiserum western-blotting and indirect ELISA results. Shrimp TM and SCP maintains its IgE/IgG-binding capacity after various cooking methods, thus most probably initiating allergic sensitization to both raw and cooked shrimps.

High-pressure biorefining of ora-pro-nobis (Pereskia aculeata)

Ora-pro-nobis (OPN) is an unconventional food plant used in some Brazilian regions in traditional culinary. A combination of high-pressure extraction processes was used, following the biorefinery concept, to value this underestimated raw material. A sequence of extractions was performed, with solvents of increasing polarity: supercritical fluid extraction (SFE-CO2), pressurized liquid extraction using ethanol (PLE-ethanol) and water (PLE-water). Soxhlet and buffer maceration were used for comparison. Extraction yield, antioxidant capacity, phenolics and soluble protein contents were evaluated. Samples recovered by water had better antioxidant capacity, while ethanol samples presented higher phenolic content. The process combination improved yield, antioxidant potential, phenolics and soluble protein recovery, especially combining SFE + PLE-ethanol + PLE-water. Overall, 13 phenolics were identified, mainly flavonoids and organic acids, known for its antioxidant and biological activities, confirming the extracts high value-added. Thus, downstream process combination for OPN leaves enabled the recovery of high value-added products following the biorefinery concept.

Recovery of soluble proteins from Chlorella vulgaris by bead-milling and microfiltration: Impact of the concentration and the physicochemical conditions during the cell disruption on the whole process

Chlorella vulgaris proteins show high potential for food applications. The challenge of biorefinery is to choose the best processes coupling which can ensure the integrity of the molecules and limit energy consumption. This study aimed at understanding the impact of biomass concentration and physicochemical conditions during cell disruption, on the whole process (bead milling (BM), centrifugation, microfiltration). The best scenario tested (BM: 60 g/LDW, 10 °C, lysate dilution: 30 g/LDW, pH 7, 20 °C before centrifugation and filtration of the supernatant at a volume reduction ratio VRR = 3) allowed a soluble protein recovery ηS = 12 % with a limited energy consumption of 10 kW h/g protein. Coupling BM at 90 g/LDW, pH 7, a dilution at 20 °C, with NaCl 0.1 M at 30 g/LDW and filtration at VRR = 20 would lead to ηS>25 %. The final soluble proteins contained no green pigment; their purity increased from 5 % in the raw biomass to 22 % in the permeate.

Soybean Protein Extraction by Alcalase and Flavourzyme, Combining Thermal Pretreatment for Enteral Feeding Product

Soybean is one of the essential ingredients when formulating a tube feeding formula. In this study, we initially focused on determining which enzyme is suitable for hydrolyzing soy and comparing the soy protein enzymatic hydrolysis of three different enzymes at the same enzyme content: Flavourzyme, Protamex, and Alcalase. The result showed that Flavourzyme attained the highest soluble protein recovery efficiency (SPRE). Secondly, the study determined the effect of thermal treatment conditions such as thermal treatment duration, and then it showed that when combining the thermal treatment and enzymatic hydrolysis, the yield reached (61.44 ± 0.22)%, which was much higher than only using enzymatic hydrolysis (52.57 ± 0.27)%. Next, optimizing the enzymatic hydrolysis (combining thermal treatment) using Flavourzyme and Alcalase, Flavourzyme achieved (62.47 ± 0.12)%, while Alcalase attained (41.32 ± 0.13)%. The soy hydrolyzate using Flavourzyme achieved an average molecular size of 3.19 kDa at the following optimizing conditions: enzyme concentration, 16.09 U·g−1; pH, 7.02; temperature, 45.8 °C; and beans/water ratio, 1:3. In contrast, when using Alcalase, the soy hydrolyzate achieved an average molecular size of 1.52 kDa at the following optimizing conditions: enzyme concentration, 28.01 U·g−1; pH, 7.2; temperature, 56.5 °C; beans/water ratio, 1:4.6. Soy protein hydrolyzate of suitable viscosity and particle size flow through the inhaler with branched-chain amino acids achieved a BCAA (Branched Amino Acid) ratio of 2:1:1 for Alcalase and 4:1:1 for Flavourzyme. Soybean hydrolyzate using both enzymes attained a high SPRE and was suitable for the digestive ability of patients recovering from surgery. Soy protein is divided into amino acids, di- and tri-amino acids, and peptides to create a soluble protein source that helps feed patients with a sonde tube easily. In addition, the molecular weight of peptides will reduce viscosity significantly when passing through a sonde tube, preventing tube congestion.

Protein composition and textural properties of inulin-enriched tofu produced by hydrothermal process

Tofu protein composition changes induced by a pilot plant processing method involving hydrothermal cooking, commercial rennet as coagulant and inulin as functional ingredient were assessed. Glycoproteins (γ-, β-conglycinin) were firmly bound to the matrix of the obtained gels. Extractable proteins of inulin-enriched tofu were with mainly basic 7S-globulin, and polypeptides of glycinin. Polypeptides A1,2,4 and B1,2,4 were found in higher concentrations in protein extracts of tofu than other 11S polypeptides. Tofu was characterized by high total (417.6–556.5 g/kg) and extractable soluble (155.3–367.5 g/kg) protein content, good protein extractability (37.2–68.6%), both total protein (52–69%) and soluble protein (50–76%) recovery. Increasing concentrations of inulin proportionally improved tofu hardness (6.23–8.98 N/cm2) contributing to the uniformity of gel microstructures. Inulin played an important role in incorporating β- and γ-conglycinins into the gel matrix and by increasing the concentration of inulin in soymilk before gelation, the content of extractable soluble proteins, protein extractability and soluble protein recovery of produced tofu were also proportionally increased. For all tested sensory attributes the grades were increased with increasing inulin content in the samples. Under the applied production method, protein composition and proportion of inulin had important roles in defining tofu quality.

Physicochemical Properties of Soy Protein Concentrate Treated with Ultrasound at Various Amplitudes

This research was conducted to investigate the influence of ultrasound amplitude on thephysicochemical properties of soy protein concentrate. Soy protein concentrates (SPC, Acron SM) were treatedwith a frequency of 20 kHz ultrasound and three different amplitudes of 50, 80, and 100% for 5 min. Untreated andultrasound-treated soy protein concentrate samples were evaluated in terms of recovery of soluble protein, particlesize, surface hydrophobicity, free sulfhydryl groups, turbidity and microstructure. The environmental scanningelectron microscope images of the treated and untreated soy protein concentrate samples were taken in order toanalyze the microstructure of the samples. The findings showed that the ultrasound treatment have a significanteffect on all physicochemical characteristics (p<0.05). All ultrasound treated samples showed significantly highersolubility compared to the untreated soy protein concentrates. In addition, the highest protein solubility wasdetermined for the samples treated with 100% amplitude. Ultrasound treatment reduced the size of all proteins.The sample which has the highest solubility also showed the lowest particle size compared to the others. Moreover,ultrasound treated (100% amplitude) soy protein concentrate was resulted with highest surface hydrophobicity andfree sulfhydryl groups. Microscope images of the soy protein concentrates showed a spherical morphology withparticle diameters which closely corresponding to the results obtained by dynamic light scattering. It was clearlyseen that increasing ultrasound amplitude enhance the functionality of soy protein concentrates.

Methodological bias associated with soluble protein recovery from soil

Proteins play a crucial role in many soil processes, however, standardised methods to extract soluble protein from soil are lacking. The aim of this study was to compare the ability of different extractants to quantify the recovery of soluble proteins from three soil types (Cambisol, Ferralsol and Histosol) with contrasting clay and organic matter contents. Known amounts of plant-derived 14C-labelled soluble proteins were incubated with soil and then extracted with solutions of contrasting pH, concentration and polarity. Protein recovery proved highly solvent and soil dependent (Histosol > Cambisol > Ferralsol) and no single extractant was capable of complete protein recovery. In comparison to deionised water (10–60% of the total protein recovered), maximal recovery was observed with NaOH (0.1 M; 61–80%) and Na-pyrophosphate (0.05 M, pH 7.0; 45–75% recovery). We conclude that the dependence of protein recovery on both extractant and soil type prevents direct comparison of studies using different recovery methods, particularly if no extraction controls are used. We present recommendations for a standard protein extraction protocol.

Expression and purification of the antigen binding fragment of the germline‐encoded precursor to the murine anti‐S1P metalloantibody in the baculovirus‐insect cell system

Sphinosine‐1‐phosphate (S1P) is a biologically active lipid that plays a signaling role in angiogenesis and in tissue remodeling after cardiac ischemia. In recent years, antibodies that target biologically active lipids have been studied as promising therapeutic agents. We determined the 1.9 Å x‐ray crystal structure of a humanized version of the murine anti‐S1P antibody, and reported the novel finding that it employs two bridging calcium ions in binding to its lipid antigen. In order to study the chemical properties of this novel metalloantibody, we are developing methods for recombinant expression and purification. Previously, germline‐encoded light and heavy immunoglobulin genes that served as precursors to the mature anti‐S1P antibody have been engineered into a plasmid vector for recombinant expression in E. coli. Recovery of soluble protein required that both light and heavy genes be directed to the periplasm of the Gram‐negative bacteria through implementation of PelB signal peptides on both genes in a coexpression plasmid. Unfortunately, the yield for the recombinant antibody was low and insufficient for further analyses. Curiously, the affinity chromatography purified protein partitions into two well resolved pools upon further purification by size exclusion chromatography. Sf9 insect cells infected with recombinant baculoviruses have been successfully used to express high yield secreted proteins. Selection of appropriate promoters and signal peptides has been shown to significantly affect protein yield. We employed the pFastBac Dual vector system in which one gene is expressed under the control of the AcNPV polyhedrin promoter while the other is under the control of the AcNPV P10 promoter and the gp64 signal peptide to express the histidine‐tagged heavy (VH and CH1 domains) and light chains of the metalloantibody precursor. The protein concentration is 12 mg/ml in 500 ml suspension insect cells. As part of our efforts to determine whether the incorporation of metals arises during affinity maturation of the antibody or if it is inherent to the germline encoded sequence and as a starting point to study the chemical potential of metalloantibodies, we used ICP‐MS to determine the Ca concentration in samples treated with EDTA. The concentration of Ca in the Fab antibody was significantly higher than the negative control antibody and the buffers. Our next step is to mutate residues that were previously shown to coordinate metals in the anti‐S1P antibody to introduce novel metal binding potential into an antibody scaffold.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

EXPLORATION OF THE FACTORS AFFECTING THE SOLUBLE PROTEIN EXTRACTION FROM CULTURED SNAKEHEAD FISH (Channa striata) MUSCLE

This study was conducted to identify the factors affecting the soluble protein extraction from cultured snakehead fish muscle, a protein having bioavailability and nutritional value. The study investigated the ratio of fish muscle to water (from 1:1 to 1:5 w/v) and salt (NaCl) concentration (from 0.05 M to 0.25 M) that are suitable for extracting soluble protein effectively. Research has applied a response surface methodology using a central composite design to optimize the effect of temperature and time extraction on soluble protein extraction efficiency. In addition, the study investigated the effect of pH of salt solution (pH 2 to pH 12) on the efficiency of soluble protein extraction from cultured snakehead fish muscle. As a result, the use of 0.15 M NaCl solution with 1:2 ratio of fish muscle to water gave high soluble protein extraction efficiency. The optimum extraction temperature and time were determined as 34.8 °C and 15.2 min respectively, and the appropriate pH value was also determined as pH 10. At the optimum extraction conditions, soluble protein recovery calculated by the amount of soluble protein extraction dividing total protein of fish muscle was 14.39 ± 0.18 %.

Recovery of soluble proteins from migratory locust (Locusta migratoria) and characterisation of their compositional and techno-functional properties

Edible insects emerged as an alternative source of high-quality proteins. Therefore, the effect of an extraction procedure for the recovery of migratory locust (Locusta migratoria) protein concentrate (MLPC) on the compositional characteristics and techno-functional properties was studied. The influence of pH value (2−10) and salt concentration (0, 1 and 3% w/v) on techno-functional properties was evaluated. Proteins were identified and characterized by RP-HPLC, SDS-PAGE and LC-MS/MS. The initial crude protein content of the whole locusts (65.9% on dry base) could be enhanced to 82.3% (MLPC). Solubility profiles of MLPC showed maximum solubility at pH9 (100%). Promising functionality comparable to egg white protein in terms of emulsifying activity at pH5, foamability at pH3 and 3% NaCl, and foam stability at pH9 were found. Consequently, MLPC offers a nutritious protein source with good functional properties at certain conditions, which could be used as food ingredient in a variety of food systems.

Enhanced dry anaerobic digestion of swine excreta after organic nitrogen being recovered as soluble proteins and amino acids using hydrothermal technology

This study investigated the feasibility of N recovery from swine excreta (SE) as value-added soluble proteins and amino acids using hydrothermal (HT) technology for improving dry anaerobic digestion (AD) performance. The effect of temperature on characteristics and transformation behaviors of N species in SE subjected to HT pretreatment at different temperatures (110–210 °C for holding 30 min) was investigated. After HT pretreatment at 150 °C, 58% of the total nitrogen (TN) content in SE became soluble, with 19.03%, 1.55% and 0.56% of TN being converted into soluble proteins, amino acids and urea, respectively. Temperatures higher than 170 °C favored the breakdown of these proteinaceous compounds, leading to accelerated formation of ammonia. After soluble proteins and amino acids being separated by water extraction, CH4 yield (322.13 cm3 g−1 of VSfed) from the solid residue pretreated at 150 °C was improved by 51% in comparison to that from raw excreta (213.70 cm3 g−1 of VSfed), achieving 3 days' shorter in effective CH4 production period. HT technology could be an effective pretreatment alternative for the recovery of soluble proteins and amino acids and subsequently enhanced dry AD of SE.

RELEASE BIOATIVE PEPTIDES FROM SOYBEAN BY OPTIMIZATION THE ENZYMATIC HYDROLYSIS BY ALCALASE AND PROTAMEX USING RESPONSE SURFACE METHODOLOGY

Bioactive peptides have been defined as specific protein fragments that have a positive impact on body functions and conditions and may ultimately influence health. The objective of this paper is to study the enzymatic hydrolysis process of soy protein to produce bioactive peptides. To study the action of Alcalase and Protamex on the proteins of soybean, the influence of the temperature, pH, substrate concentration, enzyme concentration and hydrolysis time on the soluble protein recovery of the soy proteins was evaluated. The soyprotein was hydrolyzed by two different enzymes. Response surface methodology was applied to optimize the hydrolysis capicity. The dried hydrolysate was low to medium molecular weight bioactive peptides. The results of amino acid analysis showed that the composition of amino acid of soy protein and its hydrolysate obtained under the optimized condition was considerably enriched in essential amino acids and ensure the nutrition and safety for human consumption.

Physicochemical Properties and ACE‐I Inhibitory Activity of Protein Hydrolysates from a Non‐Genetically Modified Soy Cultivar

AbstractArkansas‐grown non‐genetically modified soybean cultivar, R08‐4004, was selected to prepare a protein isolate, which was treated with Alcalase for limited enzymatic hydrolysis. The objective was to optimize the Alcalase hydrolysis condition to produce soy protein hydrolysate (SPH) with high protein yield, low bitterness, and clarity for beverage applications. The degree of hydrolysis ranged between 14 and 52 % during the study at varying incubation times using two different concentrations of Alcalase enzyme. Recovery of soluble protein, between 21 and 53 %, was achieved with a decrease in turbidity. There was an increase in surface hydrophobicity (S0) which is correlated with bitterness of SPH treated with 1.0 AU (3.2 µL/g) of Alcalase 2.4 L. The sodium dodecyl sulfate‐polyacrylamide gel electrophoresis analysis showed a distinct hydrolysis pattern in which 7S globulin and the two acidic sub‐units of 11S globulin were hydrolyzed extensively in comparison to the two basic sub‐units of 11S globulin. Limited enzymatic hydrolysis produced low molecular weight peptides <17 kDa. Among these SPHs, the one derived after 120 min incubation had the highest soluble protein yield (43 %), low S0 value (35.4), low turbidity (0.88), and highest angiotensin‐I converting enzyme (ACE‐I) inhibition activity (66.6 %). This hydrolysate has potential use as protein rich nutraceutical for developing many non‐genetically modified food product applications.

Production of a water-soluble fertilizer containing amino acids by solid-state fermentation of soybean meal and evaluation of its efficacy on the rapeseed growth

Soybean meal is a by-product of soybean oil extraction and contains approximately 44% protein. We performed solid-state fermentation by using Bacillus subtilis strain N-2 to produce a water-soluble fertilizer containing amino acids. Strain N-2 produced a high yield of protease, which transformed the proteins in soybean meal into peptide and free amino acids that were dissolved in the fermentation products. Based on the Plackett–Burman design, the initial pH of the fermentation substrate, number of days of fermentation, and the ratio of liquid to soybean meal exhibited significant effects on the recovery of proteins in the resulting water-soluble solution. According to the predicted results of the central composite design, the highest recovery of soluble proteins (99.072%) was achieved at the optimum conditions. Under these conditions, the resulting solution contained 50.42% small peptides and 7.9% poly-γ-glutamic acid (γ-PGA). The water-soluble fertilizer robustly increased the activity of the rapeseed root system, chlorophyll content, leaf area, shoot dry weight, root length, and root weight at a concentration of 0.25% (w/v). This methodology offers a value-added use of soybean meal.

Protein and water recovery from poultry processing wastewater integrating microfiltration, ultrafiltration and vacuum membrane distillation

A novel approach for the recovery of the proteins contained in the effluent formed during production of surimi-like material from mechanically recovered poultry meat was proposed, using an integrated membrane process comprising microfiltration, ultrafiltration and vacuum membrane distillation. Additionally, the possibility of using the integrated membrane process to achieve high-quality water for reuse was also investigated. Using cross-flow membrane techniques, it was found that the total recovery of soluble proteins as well as the average degree of concentration amounted to 84 % and 9.3, respectively. Moreover, using this process, the total recovery of water was 70 % and the percentage reduction in COD, TOEM and TSS in the permeate obtained using vacuum membrane distillation exceeded 99 %. As a result, the water recovered by the integrated membrane process could be reused in the manufacture of surimi-like material from mechanically recovered poultry meat.