Purification Of Protease Research Articles

Two-Step Preparation of Highly Pure, Soluble HIV Protease from Inclusion Bodies Recombinantly Expressed in Escherichia coli.

Heterologous expression of exogenous proteases in Escherichia coli often results in the formation of insoluble inclusion bodies. When sequestered into inclusion bodies, the functionality of the proteases is minimized. To be characterized structurally and functionally, however, proteases must be obtained in their native conformation. HIV protease is readily expressed as inclusion bodies, but must be recovered from the inclusion bodies. This protocol describes an efficient method for recovering HIV protease from inclusion bodies, as well as refolding and purifying the protein. HIV protease-containing inclusion bodies are treated with 8 M urea and purified via cation-exchange chromatography. Subsequent refolding by buffer exchange via dialysis and further purification by anion-exchange chromatography produces highly pure HIV protease that is functionally active. © 2020 by John Wiley & Sons, Inc. Basic Protocol: Recovery, refolding, and purification of HIV protease from inclusion bodies Support Protocol 1: Expression and extraction of inclusion bodies containing HIV protease expressed in Escherichia coli Support Protocol 2: Determination of the active site concentration of HIV protease via isothermal titration calorimetry.

Purification, characterization and optimization conditions of protease produced by Aspergillus brasiliensis strain BCW2

Proteases are a group of enzymes in high demand due to their wide and different biotechnological applications. However, the cost of production of proteases on industrial scale is high. Hence, this study was aimed at screening fungal strains having ability to produce protease using cheaper agricultural wastes, purifying and characterizing the protease produced. Fungi isolated were screened for protease production and identified using traditional and molecular means. Optimum conditions for protease production, purification and characterization were determined using standard methods. Proteolytic activity of the selected isolate BCW2 measured in zone of hydrolysis was 38 mm. The selected isolate shared 99% sequence homology with Aspergillus brasiliensis JC-A3. Hence, the isolate was tagged as Aspergillus brasiliensis BCW2. Production of protease by solid state fermentation was optimum at pH 9.0, temperature of 30 °C, 10% orange peel as carbon source, 2% yeast as nitrogen source, 60% moisture content and an incubation period of 72 h. The purified protease has a molecular weight of 68 KDa, a yield of 28% and 13.3 fold. The partially purified protease was stable between pH 4-6 and temperature of 30-40 °C, exhibited enhanced activity with Ca2+, and Tween-20 and had specificity to gelatin as substrate. The suitability of orange peel as a substrate for protease production by the strain Aspergillus brasiliensis BCW2 will reduce production cost. The use of orange peel as organic substrate is first being reported through this work. The purified enzyme has de-clotting and blood stains removal properties, making the enzyme open up to wider biotechnological applications. GenBank accession number for the strain A. brasiliensis BCW2 ITS rRNA gene sequence is MF599164.1.

Separation and partial purification of collagenolytic protease from peacock bass (Cichla ocellaris) using different protocol: Precipitation and partitioning approaches

Abstract This comparative study provides a useful protocol for the separation and partial purification of collagenolytic proteases obtained from peacock bass, by using precipitation and partitioning. In the precipitation process, 30% acetone (PF = 1.76; Y = 198%) and ethanol (PF = 2.56; Y = 272%) were used, while 30–60% fraction of (NH4)2SO4 was the most efficient in the separation process (Y = 201%; PF: 4.28).For the use of the three-phase partitioning (TPP), the sample was submitted to different proportions of t-butanol (1.0:0.5, 1.0:1.0, 1.0:1.5 and 1.0:2.0; v/v) and fixed proportion of (NH4)2SO4 (30%). The assay with the best Y (232%) of the collagenolytic protease in the interphase was in the ratio of 1.0:0.5, which also resulted in higher PF (5.07) of this same phase. The formation of the two-phase aqueous partitioning (ATPS) was obtained by mixing polyethylene glycol(1500, 4000 and 8000), according to a 24-factorial design. The highest values of PF (8.24) and Y (116%) were obtained at pH 8.0, 12.5% (w/w) PEG 8000, and 10.0% (w/w) phosphate. PEG-collagenolytic was physicochemically characterized and exhibited optimal state at a temperature of 55 °C and pH 7.5, which was activated by Ca2+ and inhibited by Zn2+; this activity was reduced when exposed to PMSF and TLCK. The PEG-collagenolytic showed three bands of protein, with molecular weights ranging from 10.0 to 60.3 kDa. The protease hydrolyzed native and fish skin collagen. Therefore, separation of the target molecule by precipitation and partitioningmay be a viable alternative. Furthermore, ATPS presented physicochemical characteristics compatible with the industrial interest.

Purification and characterization of extracellular alkaline protease from Streptomyces sp. LCJ12A isolated from Pichavaram mangroves

Purification and characterization of extracellular alkaline protease from Streptomyces sp. LCJ12A isolated from Pichavaram mangroves

Partial Purification and Characterization of Protease from Local Isolate of Beuveria bassiana

Partial Purification and Characterization of Protease from Local Isolate of Beuveria bassiana

Preparation and Application of a Decellularized Extracellular Matrix for Identification of ADAMTS Substrates.

Here we describe the use of a decellularized ECM produced in vitro by BALB/c 3T3 fibroblasts for the identification of ADAMTS substrates. Seeding of ADAMTS protease-producing HEK cells on top of the cell-free ECM followed by analysis of the conditioned medium by liquid chromatography tandem mass spectrometry (LC-MS/MS), allows for screening of ADAMTS substrates without prior purification of full-length protease.

Selective purification of protease from ginger and sodom apple by ultrasound assisted liquid-liquid microextraction using natural deep eutectic solvent

Ultrasonic assisted liquid-liquid microextraction (UA-LLME) of cysteine proteases from ginger and sodom apple were accomplished with natural deep eutectic solvents (NADES). Five imidazole-based NADES with carbohydrates were synthesised and their thermophysical properties were investigated. Selective partitioning of NADES assisted UA-LLME yielded more protease in NADES enriched top phase. Choice of phase forming compounds and their influence in the concentration of protease was evaluated by calculating the partition coefficient. The linearity of detection, LOD and LOQ for protease were found to be 10–50 μg/ml, 15 and 60 μg/ml respectively. Influential variables towards the extraction were optimised with response surface methodology and it was noted that maximum yield was achieved with 25% (v/v) of NADES, 15% (w/v) of source concentration and ultrasound temperature and time as 35 °C and 10 min respectively. The concentrated extract was purified by gel filtration chromatography was performed followed by affinity chromatography and purity fold with %yield of the enzyme was observed.

Получение высокоочищенных элюатов кислой протеазы из нативного раствора Aspergillus oryzae сорбционно-хроматографическим и мембранным методами

В работе представлены результаты экспериментов получения высокоочищенных элюатов кислой протеазы Aspergillus oryzae штамм 55 из нативного раствора на катионитах КУ-23, С-106, КБ-2. Установлено, что нативный раствор Aspergillus oryzae не однороден по белку и молекулярная массакислой протеазы составила 41кДа. Показано, что максимальный выход целевого продукта достигается на сульфокатионите КУ-23, и составляет 66% на стадии сорбции и 76 % на стадии десорбции. Очистка элюата кислой протеазы с использованием ультра- и диафильтрации позволяет увеличить удельную активность в 4.5 раза.

A novel metalloprotease from banana peel and its biochemical characterization

Protein cleaving enzymes, called proteases are one of the most commercially used enzymes possessing extensive applications in various industries. The present study was focused on screening, extraction, purification and characterization of protease from banana peel. Twelve different varieties of banana peels were screened for the protease activity. The maximum activity of the isolated enzyme was 230.4 CDU/mg from Nendran banana. The crude enzyme was purified up to 8.1 fold with the yield of 61.34%. The molecular weight of the purified fraction was found to be 40 kDa. The optimum conditions for maximum protease activity were achieved at 30 °C and pH 7. The Lineweaver-Burk plot exhibited the kinetic parameters of the enzyme such as Vmax and Km as 588.4 CDU/mg and 15.2 mg/ml respectively. The influence of different inhibitors, metal ions, organic solvents, detergents, oxidising agents and salinity were examined. The collagenolytic activity was tested with purified type I collagen as a substrate. The enzyme was tested for cell cytotoxicity, detection of apoptosis using fluorescent dyes and antitumor activity. The results demonstrated that protease from banana peel was of high significance and potential for usage in therapeutic for breaking down collagen/peptide bonds.

Isolation, Purification and Mass Production of Protease from Bacillus Subtilis

Isolation and identification of protease producing strains of bacteria were carried out from four different soil samples collected from various places in Tiruchirappalli. The organisms were tested by various biochemical tests, which led to their identification as Bacillus subtilis producing protease enzyme. These Bacillus subtilis could grow up to 60oC and in pH range of 6-9 with optimal growth temperature and pH of 55oC and 9.0 respectively. It was also optimized for carbon test and nitrogen test with optimal growth in dextrose and peptone respectively. Enzyme production was carried out in 1 litre of optimized media in the fermenter at 55oC for 48 hours at pH 9.0. Harvested protease product was purified by salt precipitation method. Finally the enzyme protease was purified by column chromatography. The protein was characterized using SDS-PAGE. This result showed that Bacillus subtilis is a good producer of extra cellular protease. The enzyme produced by B. subtilis is pH stable and thermo stable, which can be utilized in local detergent and leather industry.

Cloning, Expression and Purification of DegQ Protease, a Bacterial Analog to Pregnancy Related Serine Protease (HtrA3)

The High Temperature Requirement A (HtrA) family of enzymes are a diverse group of serine proteases, present in both prokarya and eukarya, and are generally responsible for the degradation of misfolded proteins. The dysregulation of HtrA3 in human somatic cells gives rise to various forms of cancer and preeclampsia. DegQ is a bacterial analog to HtrA3 found in the periplasm of E. coli and has the properties of an HtrA protease: it is a serine endopeptidase that works to protect the cell during high temperature by degrading specific damaged proteins. Currently, the interactions that dictate which specific substrates DegQ degrades are unknown. Insights into this interaction could lay the foundation for studying the interactions of the human HtrA3 protease. Previously, four N‐terminal truncated versions of DegQ were identified in prokarya, indicating an N‐terminal signaling sequence. Substrate cleavage in each of these variants has not been characterized, therefore, the goal of this study is to select for the active version to further characterize. Initial experiments utilized molecular cloning, protein expression and purification to obtain each variant. Affinity tags, such as the histidine‐tag, has greatly simplified the purification of recombinant proteins. However, the presence of such tags can interfere with a protein's biological function. To circumvent this problem, experiments were designed to obtain the protein without the use of a tag. To this end, four PCR forward primers were designed for the appropriate coding region using E. coli as template genomic DNA, each with an NdeI site engineered at the 5′ end. The reverse primer for each was the same, and designed with a stop codon followed by a SacI site at the 3′ end. The PCR product was then ligated into a linearized dephosphorylated pGEM vector using TA cloning. Orientation of the gene was determined through the use of restriction enzyme cuts. This vector and the pET24b expression vector were digested with NdeI and the gene was ligated into the pET24b vector. Orientation was again tested using SacI sites, and analyzed by gel electrophoresis. Based upon the theoretical mass distribution of the cleaved oligonucleotides from the gel electrophoresis, plasmids with positive results were purified for verification through sanger sequencing. Each pET24b‐DegQ vector was individually transformed into BL21(DE3) E. coli cells, grown in LB broth and expressed with 0.1 mM IPTG for 4 hours at room temperature. Protein was purified using a DEAE‐sephacel column, followed by an ammonium sulfate precipitation, and further purification on a G‐75‐sephadex size exclusion gel filtration column. Ultimately the proteases were purified to homogeneity without the use of an affinity tag for comparison of proteolytic activity.Support or Funding InformationMWSU Department of Chemistry MWSU Department of Biology MWSU PORTAL grantThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Purification of Protease from Aspergillus fumigatus

Purification of Protease from <i>Aspergillus fumigatus</i>

Molecular characterization of alkaline protease-coding gene from Bacillus licheniformis MK90 mutants with biofilm inhibitory activity

Background Enzymes are organic materials that accelerate biochemical processes without themselves undergoing change. They can be produced by plant, animal, fungi, and bacteria. Bacterial proteases are much favorable than any other sources, because bacteria can grow quickly and can be easily cultivated in laboratory. Objective To isolate and screen bacteria from soil samples for their ability to produce alkaline protease, and to improve the alkaline protease production followed by evaluation of its antimicrobials and antibiofilm activity. Materials and methods Sample collection was carried out from different locations in Egypt. Isolation of bacteria from soil samples was done using serial dilution method on skim milk agar. All isolated bacteria were screened for their ability to produce protease enzyme. The bacterial isolate showing maximum alkaline protease activity was identified using 16 S rRNA genetic identification. To induce mutations, ultraviolet (UV) irradiation was used. The most active mutant strains were selected for production, purification, and characterization of alkaline protease followed by evaluation of alkaline protease antimicrobial and antibiofilm activity. Results and conclusion Three UV mutants (MT2, MT4, and MT26) out of 48 displayed proteolytic activity more than other mutants and wild type (WT). Bacillus alkaline extracellular protease gene was genetically characterized through isolation of the genomic DNA of Bacillus licheniformis MK90 WT, and the best protease-producing UV mutants were followed by amplification, sequencing, and analyses. WT strain and best protease-producing mutants were compared at proteomic level through sodium dodecyl sulfate polyacrylamide gel electrophoresis for total cellular proteins. Then protease enzyme of WT and mutants was purified and characterized. This study reports that the B. licheniformis protease was active at an alkaline pH and wide range of temperatures (40–60°C), reflecting its potential application in detergent and laundry industries. On the contrary, the antibiofilm activity of the protease enzymes was evaluated toward four pathogenic bacterial strains, i.e., Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Bacillus subtilis, and the results showed that proteases from B. licheniformis MK90 may be useful for controlling biofilm formation by some pathogenic bacteria.

Purification and characterization of alkaline protease produced by Streptomyces flavogriseus and its application as a biocontrol agent for plant pathogens

Background A protease is an enzyme that helps proteolysis: protein catabolism by hydrolysis of peptide bonds. Proteases were the first enzymes to be commercialized, partly on the account of the history of availability and on account of need. Aim The aim of the present work is studying the properties of the partially purified enzyme and some application of the enzyme. Materials and methods In this study, alkaline protease was produced by Streptomyces flavogriseus using the shaking method. The enzyme was partially purified by ammonium sulfate precipitation. The most active fraction was evaluated on Sephadex G-100 column chromatography. The activity of the enzyme was determined at different temperatures and different pH values using various buffers. Results and conclusion In this study, alkaline protease produced by S. flavogriseus was partially purified by ammonium sulfate precipitation at 75% w/v saturation and pooled on Sephadex G-100 with a yield of 60.85% and specific activity of 85.14 U/mg protein. The enzyme was optimally active at 45°C in 0.5 mol/l potassium phosphate buffer (pH 8.0) after 30 min of incubation and was broadly stable at 45°C for 60 min and pH 6–8. The alkaline protease relative activity was increased in the presence Mn2+, Co2+, K+, Na+, Fe2+, and Mg2+ at concentration 1 mmol/l, respectively.

Partial Purification, Characterization, and Application of Extracellular Aspartic Protease from &lt;i&gt;Lactobacillus casei&lt;/i&gt; WSP in Producing the Bioactive Peptides with Antibacterial and Antioxidant Activity

Lactobacillus casei WSP-derived an aspartic protease was sequentially purified by using chromatography gel filtration sephadex G-50. It resulted in a 22.81-fold increase of specific activity (51.5 U/mg) with a final yield of 1.9%. The estimated molecular weight of the purified enzyme was 37 kDa and showed gelatinolytic activity in zymogram assay. The enzyme exhibited optimum activity at 40ºC and pH 6 with casein as the substrate. Enzyme activity was significantly inhibited by pepstatin A (0.5 mM and 1 mM), confirming that this enzyme is a group of aspartic proteases, while other inhibitors such as EDTA, PMSF and iodoacetic acid showed no inhibition effect on the activity of enzyme. The addition of metal ion to the enzyme decreased enzyme activity, indicating the proteolytic enzyme was metal ion- dependent. Denaturant such as DDT tended to increase caseinolytic activity. Furthermore, this enzyme was capable of generating the new peptides from skimmed milk with the size 8 kDa, 10 kDa and 15 kDa. These peptides have potential as antibacterial and antioxidant agents.

Purification and characterization of cysteine protease from miswak Salvadora persica

BackgroundGenerally, proteases in medicinal plants had different therapeutic effects such as anti-inflammatory effect; modulate the immune response and inhibitory effect toward tumor growth. In this study, protease was purified and characterized from miswak roots, as medicinal plant and natural toothbrush.ResultsPhysical and chemical characterization of cysteine protease P1 were studied such as pH optimum (6.5), optimum temperature (50 °C), thermal stability (50 °C) and Km (3.3 mg azocasein/ml). The enzyme digested some proteins in the order of caseine > haemoglobin > egg albumin >gelatin > bovine serum albumin. Hg2+ had strong inhibitory effect on enzyme activity compared with other metal ions. Kinetic of inhibition for determination the type of protease was studied. Iodoactamide and p-Hydroximercuribenzaoic acid (p-HMB) caused strong inhibitory effect on enzyme activity indicating the enzyme is cysteine protease.ConclusionsThe biochemical characterization of this enzyme will be display the suitable conditions for using of this enzyme in toothpaste in the future and the enzyme may be used in other applications.

Extraction and Purification of Protease from Nigella sativa for Its Potential Use in Celiac Disease

Celiac disease (CD) is a disease of the digestive system resulting from the intolerance of autoimmune system against gluten and gliadin proteins in wheat, barley, rye and some varieties of oats. In this work protease was isolated and partially purified from Nigella Sativa using ammonium sulphate, acetone and trichloroacetic acid. The purified protease was evaluated further for its possible use in celiac disease by its ability to hydrolyse both, gluten and gliadin. Data indicate that the highest purity of digestive protease enzymes was obtained by acetone 80% and 0.2M trichloroacetic acid (TCA). The enzyme purified by acetone exhibited the high value of Vmax (166.66 mg/min) and the low value of Km (0.133 mg/ml). When the obtained enzyme was tested to hydrolyse gluten and gliadin at two increasing concentrations of 1 and 2 mg/ml, almost similar efficacy was observed (28.46% and 32.02%; for gluten) and (22.7% and 16.84%; for gliadin), whereas Digestin, a positive control which contain Papain and Sanzyme 3500 and works by helping in the breakdown of proteins, produced higher efficacy at 2 mg/ml to hydrolyse gluten (38.67%) with 1 mg/ml (25.68%) whereas similar results were observed against gliadin at both concentrations (22.06% and 22.5%). This study show the efficacy of protease obtained from Nigella Sativa as a natural and economical source for the hydrolysis of gluten and gliadin, which is considered the primary reason for celiac disease.

Extraction, partial purification and characterization of alkaline protease from rainbow trout (Oncorhynchus Mykiss) viscera

In this study, crude alkaline proteases were recovered from rainbow trout (Oncorhynchus mykiss) viscera and partially purified by use of different saturation of ammonium sulfate. The enzyme exhibited highest yield, purity and activity when precipitated at a saturation of 40–60% compared to other ranges of saturation. Molecular weight for extracted protease was between 8-24 kDa. The protease had caseinolytic activity over a wide range of temperatures (30-55 °C) and pH (4-12). Soybean trypsin inhibitor and trypsin-chymotrypsin inhibitor strongly inhibited the enzyme activity but it was stable in the presence of surfactant, oxidizing reagents and organic solvents. The proteases had serine protease activity but no collagenase activity was detected. The current study showed that partially purified protease from the digestive tract of rainbow trout could be applicable in food and detergent industry because of its good activity over a wide temperature and pH range and its good thermal stability.

Process optimization for production and purification of novel fibrinolytic enzyme from Stenotrophomonas sp. KG-16-3

Intravascular thrombosis is a major cardiovascular complication responsible for high mortality worldwide. Existing thrombolytic agents are expensive and have various side effects. As a consequence, researchers continue to search for better thrombolytic agents. Fibrinolytic proteases especially those of microbial origin are considered as potential therapeutic candidates for thrombosis. The current study reports fibrinolytic protease from a bacterial isolate Stenotrophomonas sp. KG-16-3, as it exhibits high fibrinolytic activity on fibrin agarose plate. Studies on fibrinolytic protease from Stenotrophomonas sp. are lacking. So, a detailed study was conducted for the production and purification of fibrinolytic protease. Optimizing process parameters using the Design of Experiments method enhanced the yield by 1.5-fold. The fibrinolytic enzyme was purified by ammonium sulfate precipitation, ion-exchange and gel-filtration chromatography resulting in 7.1-fold purification and 16.7% yield with specific activity of 383.8 U/mg. The purified enzyme exhibited higher fibrinolytic activity than plasmin and had a molecular weight of 39 kDa. Optimal activity of the enzyme was observed at 50 °C and pH 10. The enzyme exhibited stability up to 60 °C, over pH 7–10 and in the presence of different metal ions and solvents. The activity of the enzyme was significantly reduced in the presence of phenylmethyl sulfonyl fluoride, iodoacetic acid and 1,10-phenanthroline, suggesting that the enzyme belonged to the serine–cysteine metalloprotease category. The present study is the first ever report on the Design of Experiments based optimization of fermentation conditions for the production of fibrinolytic protease from Stenotrophomonas sp.

Extraction, purification, and activity of protease from the leaves of Moringa oleifera

Background: Proteases cleave proteins, thereby providing essential amino acids for protein synthesis, and degrade misfolded and damaged proteins to maintain homeostasis. Proteases also serve as signaling molecules, therapeutic agents and find wide applications in biotechnology and pharmaceutical industry. Plant-derived proteases are suitable for many biomedical applications due to their easy availability and activity over a wide range of pH, temperature, and substrates. Moringa oleifera Lam (Moringaceae) is a very common food plant with medicinal property and geographically distributed in tropical countries. Here, we isolate proteases from the leaves of Moringa oleifera and characterize its enzymatic activity. Methods: Proteases were isolated from the aqueous leaf extract of Moringa oleifera by ammonium sulfate precipitation and purified by ion exchange chromatography. Subsequently, the enzyme kinetics was determined using casein as a substrate and calibrated over different pH and temperature range for maximal activity. Results: We obtained purified fraction of the protease having a molecular weight of 51 kDa. We observed that for the maximal caseinolytic activity of the protease, a pH of 8 and temperature of 37ºC was found to be most effective. Conclusion: The plant-derived proteolytic enzymes are finding increasing clinical and industrial applications. We could extract, purify and characterize the enzymatic activity of proteases from the leaves of Moringa oleifera. Further molecular characterization, substrate specificity and activity of the extracted protease are required for determining its suitability as a proteolytic enzyme for various applications.