Extraction Of Proteases Research Articles

Extraction, catalytic study and milk-clotting properties of proteases from Brassicaoleracea

Proteases were extracted and biochemically characterized from different Brassica oleracea varieties (B. oleracea var. palmifolia (black cabbage, BC), B. oleracea var. sabellica (curly kale, K), and B. oleracea var. botrytis (white cauliflower, WC)). Calf rennet (CR) and Cynara cardunculus crude extracts (CC) were used as reference samples. The catalytic study used two different substrates: the synthetic peptide Bz-Phe-Val-Arg-pNA (BPVA-pNA), which contains para-nitroaniline (pNA), and azocasein (AzC) as a macromolecular substrate. The effect of temperature and pH on the proteolytic activity were also evaluated. Brassica proteases were further proposed as milk coagulants, and their coagulating efficiencies were evaluated. Bovine casein was incubated with the plant extracts, and the relative breakdown products were observed through electrophoresis. BC, K, and WC contain proteases which hydrolyze the macromolecular substrate (AzC) and synthetic peptides with Arg in P-1 (BPVA-pNA). Therefore, among the three B. oleracea varieties and control extracts, K leaf extracts displayed greater catalytic efficiency against AzC. Brassica extracts showed a lesser milk clotting index but visible milk curd formation. More importantly, the protease activities of BC and K extracts exhibited great catalytic activity over a broad range of pH (3.6–9.0) and temperatures (40–70 °C) using AzC as a substrate. Current pH and thermal tolerance results indicate that B. oleracea var. palmifolia and B. oleracea var. sabellica proteases could have broad applications in food technology. Many plant resources, which could represent potential sources of efficient proteases, are still unexploited.

Extraction of collagenolytic proteases from Aspergillus heteromorphus URM 0269 in an aqueous two-phase system for application in collagen hydrolysis

Collagenolytic proteases produced by Aspergillus heteromorphus URM0269 were extracted using a PEG/sulfate aqueous two-phase system (ATPS). A 23 factorial design was performed to analyze the independent variables: PEG molar mass (MPEG), PEG concentration (CPEG), and sulfate concentration (Csulf). The extracted proteases were also evaluated for their optimum pH and stability at different pH levels (4.0 − 11.0) after 20 h of incubation. Collagen was extracted from mutton snapper (Lutjanus analis) skin using acetic acid (0.5 mol L−1). The enzyme was preferentially partitioned to the PEG-rich phase (K > 1), whose highest purification factor and recovery (PF = 6.256 and Y = 404.432%) were obtained under specific conditions: MPEG 8000 g.mol−1, CPEG 30%, Csulf 10%. The ATPS extraction provided an enzymatic activity range of pH 7.0 − 11.0, exhibiting greater stability compared to the crude extract. Approximately 80% of protease activity was maintained after 20 hours of incubation at all analyzed pH levels, except pH 11.0. Collagen extraction from L. analis skin yielded 8.056%, and both crude extract samples and ATPS-derived samples successfully hydrolyzed the extracted collagen, reaching peak hydrolysis after 36 hours of treatment. These findings demonstrate the feasibility of extracting highly purified and active proteases capable of hydrolyzing L. analis collagen.

Extraction of sulfated polysaccharide from Skipjack tuna (Katsuwonus pelamis) viscera using alcalase enzyme and rainbow trout (Oncorhynchus mykiss) visceral semi-purified alkaline proteases

Fisheries produce a considerable amount of by-products per ton of processed fish, which ranges from 350 to 600 kg. Currently, most of these by-products are used as fish and shrimp meal or as fertilizer, even though they contain significant amounts of quality proteins (enzymes), polysaccharides, and polyunsaturated fats. The present study deals with the extraction of crude alkaline proteases (AP) from rainbow trout (Oncorhynchus mykiss) viscera. The enzyme precipitated at 30–60% saturation displayed the highest yield, purity, and activity compared to other saturation ranges. The maximum activity of AP was obtained at 60 °C and pH 7. AP, ultrasound-AP, Alcalase, and ultrasound-Alcalase were used to isolate sulfated polysaccharides (SPs) from Skipjack tuna (Katsuwonus pelamis) viscera. The extraction yields showed no significant difference between all extracted SPs (p > 0.05). APU-Viscera-SP, AlU-Viscera-SP, and Al-Viscera-SP contained the most sugar (62.22%), sulfate (15.93%), uronic acid (8.84%), and protein (12.69%), respectively. Also, all SPs showed a mixed composition of monosaccharides, including xylose, mannose, rhamnose, GalA (galacturonic acid) and glucuronic acid (GlcA). AP-Viscera-SP, AlU-Viscera-SP, and AlU-Viscera-SP exhibited the highest antioxidant activity in DPPH, ABTS, and metal chelating tests. The highest inhibitor zone against L. monocytogenes, S. aureus, B. cereus, S. enterica, E. coli, and S. typhimurium were in APU-Viscera-SP, and AlU-Viscera-SP, respectively. Overall, the obtained results demonstrated that fish viscera could be a good source of alkaline protease. Besides, this obtained alkaline protease can also be used to extract sulfated polysaccharides with bioactive and functional properties from fish viscera byproducts.

Carangoides bartholomaei (Cuvier, 1833) stomach: a source of aspartic proteases for industrial and biotechnological applications.

The viscera and other residues from fish processing are commonly discarded by the fishing industry. These by-products can be a source of digestive enzymes with industrial and biotechnological potential. In this study, we aimed at the extraction, characterization, and application of acidic proteases from the stomach of Carangoides bartholomaei (Cuvier, 1833). A crude extract from the stomachs was obtained and submitted to a partial purification process by salting-out, which obtained a Purified Extract (PE) with a specific proteolytic activity of 54.0 U⋅mg-1. A purification of 1.9 fold and a yield of 41% were obtained. The PE presents two isoforms of acidic proteases and a maximum proteolytic activity at 45 °C and pH 2.0. The PE acidic proteolytic activity was stable in the pH range of 1.5 to 7.0 and temperature from 25 °C to 50 °C. Purified Extract kept 35% of its proteolytic activity at the presence of NaCl 15% (m/v) but was totally inhibited by pepstatin A. Purified Extract aspartic proteases presented high activity in the presence of heavy metals such as Cd2+, Hg2+, Pb2+, Al3+, and Cu2+. The utilization of PE as an enzymatic addictive in the collagen extraction from Nile tilapia scales has doubled the process yield. The results indicate the potential of these aspartic proteases for industrial and biotechnological applications.

Valorisation of viscera from fish processing for food industry utilizations

Fish viscera is a valuable source of functional materials for the food industry, such as protein, oil, enzymes, protein hydrolysate, peptones, sterols, producing biodiesel, and other oleochemical industries. The major components of fish viscera are oil and protein, and their quantity depends on the fish habitats. Viscera oil from fish contains appreciable amounts of omega-3 fatty acids. The protein of fish viscera is a raw material for protein concentrates, hydrolysate, and bioactive peptides. All are valuable ingredients in food processing and product formulation. Most viscera are fish digestive tracts and organs responsible for producing enzymes, and several enzymes are found in high activity, such as lipases and proteases. The extraction of lipases and proteases from fish viscera and their utilizations have been intensively studied. Currently, the isolation of each major component from fish viscera is conducted separately. Therefore, it is challenging to obtain all valuable components from fish viscera to have a zero-waste process. This article reviews the separation of major components of fish viscera by conventional and emerging technology and the proposed simultaneous and integrative separation of all valuable major components.

Extraction, partial purification and characterization of proteases from the red seaweed Gracilaria edulis with similar cleavage sites on κ-casein as calf rennet.

Enzymes currently used in cheesemaking have various drawbacks, and there is a continual need to find new coagulants. This study describes the extraction and biochemical characterization of two proteases from the red alga Gracilaria edulis. The proteases were extracted with phosphate buffer and partially purified by ammonium sulphate precipitation and dialysis. The enzymes exhibited optimum caseinolytic activity at 60°C and a pH range of 6-8. They showed a high ratio of milk-clotting over caseinolytic activity, indicating they had an excellent milk-clotting ability. The proteases were confirmed to be serine protease and metalloprotease with molecular weight (MW) of 44 and 108kDa. They exhibited high hydrolytic activity on κ-caseins, cleaving κ-casein at four main sites, one of which being the same as that of calf rennet, which is the first reported for an algal protease. The findings demonstrated that the proteases could potentially be used as a milk coagulant in cheesemaking.

Extraction of Proteases from Medicinal Plants and their Potential as Anti- Viral Targets

Medicinal plants especially leaves are used in traditional medicine for rapid anti-viral therapy against infectious diseases. Protease, a potential candidate in medicinal plants is not so for studied in leaves. So an attempt was made to determine the protease activity of various medicinal plants especially leaves. Buffers of different pH range were used for extraction of the leaves to identify the best buffer for extraction of protease. Firstly, protein from fresh plant leaves of these medicinal plants were determined and then evaluated its protease activity using crude enzyme of protein (leaves) against specific protein antigen i.e. Bovine serum albumin (BSA). Thereafter, exposure of these proteases (acid or basic) on virally infected human whole blood samples determined through flow cytometry. The results showed that protease at particular pH of PBS buffer range of these medicinal plant leaves on virally infected human whole blood samples showed anti-viral activity.

The impact of ingested potato type II inhibitors on the production of the major serine proteases in the gut of Helicoverpa armigera

The flowers of the ornamental tobacco produce high levels of a series of 6 kDa serine protease inhibitors (NaPIs) that are effective inhibitors of trypsins and chymotrypsins from lepidopteran species. These inhibitors have a negative impact on the growth and development of lepidopteran larvae and have a potential role in plant protection. Here we investigate the effect of NaPIs on the activity and levels of serine proteases in the gut of Helicoverpa armigera larvae and explore the adaptive mechanisms larvae employ to overcome the negative effects of NaPIs in the diet. Polyclonal antibodies were raised against a Helicoverpa punctigera trypsin that is a target for NaPIs and two H. punctigera chymotrypsins; one that is resistant and one that is susceptible to inhibition by NaPIs. The antibodies were used to optimize procedures for extraction of proteases for immunoblot analysis and to assess the effect of NaPIs on the relative levels of the proteases in the gut and frass. We discovered that consumption of NaPIs did not lead to over-production of trypsins or chymotrypsins but did result in excessive loss of proteases to the frass.

Extraction of fibrinolytic proteases from Streptomyces sp. DPUA1576 using PEG-phosphate aqueous two-phase systems

Extraction of fibrinolytic protease from Streptomyces sp. DPUA1576 fermentation broth was performed using polyethylene glycol (PEG) – phosphate salts aqueous two phase system (ATPS). The influence of pH, PEG molar mass, PEG and phosphate concentrations on fibrinolytic protease partition coefficient (K) were evaluated by 24 full factorial design. Fibrinolytic protease partitioned preferentially to the (PEG)-rich phase with the highest partition coefficient (K=37.00) obtained in the system 20% (w/w) PEG 1500 (g/mol)-14% (w/w) phosphate at pH 8.0. The system that allowed for the highest extraction consisted of 15% (w/w) PEG 3350-12% (w/w) phosphate, at pH 7.0. In these conditions, a purification factor of 1.51 was obtained for a partition coefficient of 6.41 with the fibrinolytic activity (FA) retained in the top phase. Obtained results confirm the interest of considering ATPS as an alternative extraction method for fibrinolytic protease from Streptomyces sp. DPUA1576.

Extraction and three-phase partitioning behavior of proteases from papaya peels

Abstract Dried papaya peels exhibited superior proteolytic activity to the fresh peels. An extraction with phosphate buffer pH 7.0 greatly maintained proteolytic activity when compared to water. SDS-PAGE verified that the extracted dried papaya peels held a wide range of proteins. To optimize the three-phase partitioning (TPP) for isolating the papaya peel proteases required a ratio of crude extract to t -butanol, the (NH 4 ) 2 SO 4 concentration and the TPP cycles. The ratio of the crude extract to t -butanol of 1.0:0.5 with the presence of 20% (NH 4 ) 2 SO 4 resulted in the highest proteolytic recovery at 253.5%, and 15.8-fold of purification in the bottom phase. The TPP was then optimized by adding up to 55% (NH 4 ) 2 SO 4 to the bottom phase of the first step. A purification of 10.1-fold with about 89.4% recovery was obtained. This study showed the TPP can be effectively employed for the extraction of proteases from papaya peels.

Liquid–liquid extraction of proteases from fermented broth by PEG/citrate aqueous two-phase system

This work deals with the use of an aqueous two-phase system (ATPS) of PEG/citrate to remove proteases from a Clostridium perfringens fermentation broth. To plan the experimental tests and evaluate the corresponding results, three successive experimental designs were employed, for which the PEG molar mass ( M PEG) and concentration ( C PEG), the citrate concentration ( C C) and the pH were selected as independent variables, while the purification factor (PF), the partition coefficient ( K), the activity yield ( Y) and the selectivity ( S) were selected as responses. PF of proteases in the top phase was shown to increase with increasing M PEG and decreasing C C, whereas a completely opposite trend was observed for K. On the other hand, Y was favored by simultaneous decreases in both these variables, while S decreased with increasing C C. Therefore, selecting a simultaneous increase in PF and Y as the most desirable result, the best performance of the system was obtained using M PEG = 10,000 g/mol, C PEG = 22% (w/w) and C C = 8.0% (w/w) at pH 8.5. Under these conditions, the activity yield was very high (131%) but the purification factor (4.2) and the selectivity (4.3) were lower than those ensured by more selective purification methods. According to these results, the ATPS seems to be an interesting alternative primary concentration/decontamination step for vaccine preparation from C. perfringens fermented broth.

Lipase and protease extraction from activated sludge

In the process of wastewater treatment hydrolysis of polymeric substances is the first and rate-limiting step. A closer study of the enzymes catalysing these reactions is essential for a better understanding of the microbial activity in the wastewater treatment process. Therefore, development of gentle and efficient enzyme extraction methods from environmental samples is very important. In this study we present a method for the extraction of lipases and proteases from activated sludge using the non-ionic detergent Triton X-100, EDTA, and cation exchange resin (CER), alone or in combination for the extraction of lipases and proteases from activated sludge. The sludge was continuously stirred in the presence of either buffer alone or in the presence of detergent and/or chelating agents. In all cases, a marked reduction in floc size was observed upon continuous stirring. However, no lipase activity and negligible protease activity was extracted in the presence of buffer alone, indicating that enzyme extraction was not due to shear force alone. The highest lipase activity was extracted using 0.1% Triton X-100 above which the activity was gradually decreasing. For proteases, the highest activity was obtained in the presence of 0.5% Triton X-100 and no decrease in activity was observed. Differences observed in the extraction efficiency of the two enzymes indicate the need for optimisation of the extraction process for the different enzymes or the extracellular polymeric substances from activated sludge.

Partial Purification and Characterization of Alkaline Thermostable Proteases of Jack Mackerel (Trachurus murphyi)

The objective of this research was to carry out the extraction, partial purification, characterization and inhibition of the alkaline thermostable proteases of jack mackerel white muscle (Trachurus murphyi). It was determined that the better extraction medium of proteolyt-ic enzymes was 60 mM bicarbonate buffer pH 9.5. The partial purification of protease was carried out applying a thermal shock at 50 °C for 10 min and gel filtration chromatography. Two peaks were collected with proteolytic activity and specific activity of 13.70 and 0.33 U'/mg, respectively. Frozen storage at −18 °C increased proteolytic activity of the extracts from 0.53 U′/ml to 7.1 U′/ml after 161 h. Inactivation constants ranged between 4.65 ×10−3 and 10.78 ×10−3 min−1 for a temperature of 40-65°C. The activation energy of denaturation was 31.85 KJ/ mol, the transition temperature and enthalpy were 59.01°C and 1.16 J/g, respectively. The protease was partially inactivated with 1.0 mM phenylmethylsulfonyl fluoride (PMSF) and 1 mM iodoacetamide (IA).

Extraction of harp seal gastric proteases and their immobilization on chitin

Gastric proteases from seal stomach were isolated following homogenization, extraction, centrifugation and ammonium sulfate precipitation/fractionation. The crude seal gastric proteases (SGP) were stable in acid conditions with optimum stability at pH 3.0, but were unstable in the alkaline range. The crude SGP possessed excellent temperature adaptability with relative activities of 70 and 90% at 5 and 25 °C, respectively. The activity of crude SGPs was retained up to about 40 min incubation at 70 °C. The isolated SGP were immobilized on glutaraldehyde-treated chitin. The immobilized SGP exhibited optimum performance at pH 2.0, were most stable at pH 4.0 and had a 90 h half-life in a column with continuous operation for haemoglobin hydrolysis at room temperature. The native SGP clotted milk rapidly at pH 5.8 to 6.6; however, immobilized SGP had a lower milk-clotting activity.

Extraction and Characterization of Midgut Proteases from Heliothis armigera and H. assulta (Lepidoptera: Noctuidae) and Their Inhibition by Tannic Acid

Crude preparations of larval midgut proteases from Heliothis armigera (Hübner) and H. assulta (Guenee) were made with double salt (ammonium sulfate) precipitation at 35% and then 75% saturation. Three proteases were resolved from each of these preparations with DEAE-cellulose chromatography. The elution profiles were similar for both preparations. These resolved proteases also had similar pH optima at pH 11, temperature optima between 55 and 60°C, and a wide pH stability range between pH 4 and pH 10. These proteases were illustrated to be serine proteases based on their inhibition properties toward specific protease inhibitors. Tannic acid had selective inhibitory effects on these proteases. The specificity of inhibition of tannic acid toward these proteases was found to correlate with previous estimates of its effect on digestibility of the larvae.

A modified procedure for simultaneous extraction and subsequent assay of calcium-dependent and lysosomal protease systems from a skeletal muscle biopsy.

An extraction and assay system was developed for quantifying endogenous muscle proteases from a single 5-g sample. A single extraction buffer was developed for simultaneous extraction of both calcium-dependent proteases (CDP) and cathepsins. Protease activity determined by the modified procedure was compared to standard procedures currently used in our laboratory. The successful use of the modified procedure on muscle biopsies was verified. Activities per gram of ovine longissimus muscle of CDP system components for 50-g standard and 5-g modified procedures were not different (P greater than .05) for CDP-I (1.16 vs 1.08), CDP-II (.89 vs 1.03), or CDP inhibitor (2.34 vs 2.32), respectively. Activities of cathepsins per gram of muscle for standard and modified procedures were higher (P less than .05) for the modified procedure (cathepsins B + L, 202.0 vs 309.8), but not different (P greater than .05) for cathepsin B (76.6 vs 98.8). Cystatin-like activity was not different (P greater than .05; 3.4 vs 3.2). To test the effect of location within the longissimus muscle on protease activities, 5 g of longissimus muscle was removed immediately postmortem from each of six locations from each side of three steer carcasses. Location within the longissimus muscle had no effect (P greater than .05) on the protease activities measured. Protease activities determined on bovine longissimus muscle biopsies with the modified procedure were similar to immediate postmortem activities. These data verify that the modified procedure was as able to quantify endogenous muscle proteases as the standard procedures and could be used on muscle biopsies. This procedure should be useful in studying the role of endogenous muscle proteases in muscle growth and postmortem proteolysis.

Extraction of Phosphatase, Urease, Proteases, Organic Carbon, and Nitrogen from Soil

Extraction of Phosphatase, Urease, Proteases, Organic Carbon, and Nitrogen from Soil

Extraction of Phosphatase, Urease, Proteases, Organic Carbon, and Nitrogen from Soil

Extraction of Phosphatase, Urease, Proteases, Organic Carbon, and Nitrogen from Soil

Extraction of Phosphatase, Urease, Proteases, Organic Carbon, and Nitrogen from Soil

AbstractExtractions of three soils at different incubation times demonstrate that 0.14M sodium pyrophosphate at pH 7.1 extracts phosphatase, urease, casein, and benzoylarginamide hydrolyzing proteases. Extraction yields of phosphatase and casein‐hydrolyzing proteases are remarkably high. The most efficient length of extraction depends both on the type of soil and on the selected enzyme.Correlation analyses of extracted enzymes and extracted C and organic N content generally show a significant correlation (p = 0.05 and 0.01).