Purification Of Proteases Research Articles

Expression, production and purification of proteinases from microbes

Screening and expression of protease producing 66 strains of different microbes were obtained from the various places in Chennai, Vellore, Tamilnadu, India. The isolates were positive on tyrosin caesin nitrate agar (1%) and thus are selected as protease producing strain. 11 of them belong to miscellaneous microbes. The microbial growth is revealed by the mycelial dry weight determination. Maximum growth is observed in the case of Bacillus (0.78 mg). Equally, the maximum growth is observed in Aspergillus sp. (0.064 mg), Fusarium sp. (0.62 mg) and Penicillium sp. (0.62 mg). Finally the enzyme protease was purified by column chromatography. The protein was characterized using SDS-PAGE. Maximum protein content is observed in the case of Alternaria sp. (0.902mg) and Penicillium sp. (0.624 mg). Maximum proteinase content is observed in Aspergillus sp. (0.866mg). This results showed that microbes under study is a good producer of extra cellular protease, which can be beneficial for industries. Keywords: Expression; production; purification; proteinases

Purification of alkaline protease from chicken intestine by aqueous two phase system of polyethylene glycol and sodium citrate.

An aqueous two phase system of polyethylene glycol (PEG) and salts was evaluated for separation and purification of alkaline proteases from chicken intestine. Among the different salts evaluated potassium phosphate and sodium citrate gave higher enzyme yield (73.5% and 69.7% respectively) and enzyme purification (5.3 and 7.4 fold) in PEG rich upper phase. Increase in concentration of sodium citrate in the system resulted in reduction in enzyme yield and enzyme purification factor, with 15% salt showing highest enzyme yield (59.8%) and purification (6.7 fold). Initial protein concentration in the system did not show any specific trend on the partition behavior of the enzyme. The temperature at which the system is incubated did not show any significant (p ≥ 0.05) effect on enzyme partition and purification. Increasing the PEG concentration in the system from 15 to 25% resulted in reduction in enzyme yield from 53.7 to 21.9% and enzyme purification from 5 fold to 1.4 fold in PEG rich upper phase. pH also had a significant (p < 0.05) effect on the partition of the enzyme to the upper phase with highest purification (3.4 fold) at pH9.0 and higher enzyme yield (46.2%) at pH10.

Purification and Characterization of Extracellular Proteases from a Marine Bacteriolytic Pseudoalteromonas sp. A1-J25a

Purification and Characterization of Extracellular Proteases from a Marine Bacteriolytic Pseudoalteromonas sp. A1-J25a

Recovery of Proteolytic and Collagenolytic Activities from Viscera By-products of Rayfish (Raja clavata)

The aim of this work was to study the recovery of proteolytic and collagenolytic activities from rayfish (Raja clavata) viscera wastes. Initially, different parts of the gastrointestinal tract by-products (stomach, duodenum section including pancreas, final intestine) were evaluated. The extracts from proximal intestine yielded the highest values of both enzymatic activities. Optimal conditions for protease activity quantification were established at pH = 6, T = 40 °C and incubation time ≤20 min. The mathematical equation used to model the joint effect of pH and temperature led to maximum activity at pH = 8.66 and 59.4 °C, respectively. Overcooled acetone was found to be best option for recovery of enzymatic activities in comparison with ethanol, PEG-4000, ammonium sulphate and ultrafiltration system. Finally, a simple and systematic protocol of partial purification and total recovery of proteases and collagenases was defined.

Isoenzyme characterization of proteases and amylases and partial purification of proteases from filamentous fungi causing biodeterioration of industrial paper

Biodeterioration is an undesirable process that can affect cultural heritage and economically important materials. Although several biotic and abiotic conditions can accelerate this process, microorganisms are perhaps its main promoters. Fungi are the most important microbial agents of biodeterioration of industrial paper stored in archives. The high genetic plasticity of these organisms allows them to adapt to different environments, using almost any class of materials as substrate. Fungi produce a wide array of enzymes, including cellulases, amylases, and proteases, which are responsible for their gross biodeterioration activity. Thirty-two morphotypes of filamentous fungi were isolated on different media from industrial paper at an advanced stage of biodeterioration. The isolates showed different degrees of cellulolytic, proteolytic, and amylolytic activities on plate assays. The highest proteolytic and amylolytic activities were selected for isoform characterization, which provided an indication of the biochemical diversity that allowed them to colonize these materials. Eladia sacculum was the morphotype selected for partial purification of basic proteases since it has three basic isoforms, simplifying the purification process. We obtained a protein of 35 kDa with a p I of 8.9.

Production, purification and characterisation of proteases from whey by some fungi

The results regarding the production of proteases fromAspergillus niger andAspergillus terreus revealed that different nitrogen complements induced different growth and enzyme productivity depending on the individual organism, nature of cosupplement and/or concentration. The two steps fermentation process increased the yield of fungal biomass and proteases. Proteases were isolated and purified fromA. niger andA. terreus with specific activity of 179.0 and 294.7 U mg−1 protein, respectively. The purification fold values were 28.9 and 81.9 from the two organisms in the same order. The Vmax values were 56 and 166 U ml−1 for acid and alkaline proteases fromA. niger. However, Vmax for alkaline protease fromA. terreus was 29 U ml−1. The three proteases expressed the same value of Hill coefficient. Abscisic acid inhibited activities of the three proteases particularly those produced by A.terreus, a phenomenon that completely reversed with gibberellic acid (GA3), more permanently with acid protease fromA. niger. The purified protease was hardly affected with chelating agents and SH-reagents whereas inhibition was dependent on the nature of agent concentration and/or the source of enzyme. Proteases from the testedAspergillus strains exhibited promising hydrolytic activities towards fibrinogen, fibrin and blood clot. Thrombolytic activity varied among organisms and/or substrates.

Purification and characterisation of Proteases A and D from Debaryomyces hansenii

The proteases A (PrA; EC. 3.4.23.25) and D (PrD; EC. 3.4.24.37) of Debaryomyces hansenii CECT 12487 were characterised after their isolation by fractionation with protamine sulfate followed by three chromatographic separations, which included two anion exchange and one gel filtration chromatographic steps. The whole procedures for PrA and PrD resulted in 1349 and 2560 purification-fold with a recovery yield of 1.4 and 1.3%, respectively. PrA was active at acidic–neutral pH with an optimum pH between 5.0 and 6.0. PrD was active at neutral–basic pH with an optimum pH between 7.0 and 8.0. The molecular mass of the native PrA was 55 kDa and (being) 42 kDa in denaturing conditions. Polyclonal-antibodies raised against PrA from Saccharomyces cerevisiae cross-reacted with the corresponding PrA from D. hansenii. PrD showed a native molecular mass of 68 kDa and 65 kDa in denaturing conditions. PrA was an aspartic protease effectively inhibited by pesptatin A while PrD was classified as a metallo protease inhibited by 1,10-phenantroline and affected by some divalent cations such as zinc, cadmium and magnesium. The homology of the PrA to the lisosomal cathepsin D suggests its possible participation in the ripening of fermented meat products.

Purification, primary structures and evolution of coagulant proteases from Deinagkistrodon actus venom

Deinagkistrodon (formerly Agkistrodon) actus (Taiwan) snake venom was found to contain at least seven closely related coagulant proteases. One of them, named actibin, was purified to homogeneity by means of four chromatographic steps. Actibin acted on fibrinogen to form fibrin clots with extremely high specific activity of 1630 NIH units/mg and preferentially released fibrinopeptide A. Actibin was an acidic glycoprotein (pI 3.4) with molecular weight of 41,000, which was reduced to 28,800 after deglycosylation with N-glycanase. The k cat/ K m values of actibin for hydrolysis of tosyl- l-arginine methyl ester and benzoyl- l-arginine p-nitroanilide were one-third to a half those for thrombin, reflecting a high potency of actibin in fibrinogen clotting. The amidase activities of actibin and its family proteases were inhibited by 3,4-dichloroisocoumarin, a serine protease inhibitor, indicating that actibin and its family proteases are serine proteases. Four cDNAs, named DaP1 and DaP7-DaP9, encoding D. actus coagulant proteases were cloned. All cDNAs contain an open reading frame of 780 bp coding for 260 amino acid residues, including a signal peptide of 24 amino acid residues. Their amino acid sequences predicted are highly homologous to one another with one to five amino acid substitutions. When four D. actus protease cDNAs were compared with the cDNAs coding for Trimeresurus flavoviridis and T. gramineus venom serine proteases, accelerated evolution was clearly observed. Similarity of the nucleotide sequences of four D. actus protease cDNAs with no synonymous and one to five nonsynonymous substitutions seems not to be in direct conformity with accelerated evolution. This possibly suggests that they have evolved to a similar direction to enhance their clotting activity rather than to produce other physiological activities.

Partial purification and comparison of precipitation techniques of proteolytic enzymes from trout ( Salmo gairdnerii) heads

Proteolytic enzymes have been detected and partially purified from trout ( Salmo gairdnerii) heads, which were preserved at −20 °C. Proteolytic enzymes, either in crude extract or in partial purified samples, were stable for 15 days with an optimum temperature of 55 °C. Proteolytic activity was very high in either alkaline or acidic pH regions. A particular ratio of cold acetone to crude extract (1.25:1) was found to be best for the partial purification of proteases, with a 99% recovery, compared with the partial purifications achieved using different cold acetone ratios or ammonium sulphate. This recovery was also confirmed via measurement of the particles (particle size analyser) contained either in crude extract or in the precipitated samples. The existence of mainly Zn–serine and possibly some Zn-acidic proteases was observed.

Partial purification and characterization of digestive trypsin-like proteases from the velvet bean caterpillar, Anticarsia gemmatalis

Trypsin-like proteases from the midgut of Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae) were purified on an aprotinin-agarose column equilibrated with 0.01 M Tris–HCl containing 5 mM CaCl 2 (pH 7.5). The yield was 66.7% with a purification factor of 107 and a final specific activity of 6.88 mM/min/mg protein with the substrate N-α-benzoyl- l-Arg- p-nitroanilide ( l-BApNA). The purified fraction showed three bands with proteolytic activity and molecular weights of 66,000, 71,000 and 91,000 (sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE)). Enzyme specificity assays were carried out using seven synthetic peptides containing 13 amino acid residues, but differing only on the 5th residue (K, R, Y, L, W or P). Peptide cleavage takes place only with amino acids K or R at the 5th position, which is typical of trypsin. The partially purified enzymes hydrolyzed casein and the synthetic trypsin substrates l-BApNA and N-α- p-tosyl- l-Arg methyl ester ( l-TAME). Higher activity was observed at pH 8.5 and 35 °C when using l-BApNA as substrate and at pH 8.0 and 30 °C when using l-TAME. Maximum enzyme activity against l-BApNA was obtained with 20 mM CaCl 2 in the reaction mixture. The partially purified enzymes showing trypsin activity were sensitive to inhibition by ethylenediaminetetraacetic acid (EDTA), phenylmethyl sulphonyl fluoride (PMSF), N-α-tosyl- l-lysine chloromethyl ketone (TLCK), benzamidine and aprotinin. Highest inhibition was obtained with TLCK and benzamidine. K M values obtained were 0.32 mM for l-BApNA and 52.5 μM for l-TAME.

Partial purification of proteases that are generated by processing of the Northern shrimp Pandalus borealis and which can tenderize beef

SummaryThe crude extracts obtained from the heads of Northern shrimps, Pandalus borealis, (adapted to cold), showed considerable collagenolytic activities. When tested for beef tenderization, resulted in an overdegradation of meat proteins, which was detected organoleptically. Subsequently, four fractions with proteolytic activity were partially purified from the crude extracts by hydroxyapatite followed by MonoQ or Superdex 200 column chromatography. Warner‐Bratzler shear force values of steaks treated with three protease fractions (Q, S2, S3) at 10 °C were significantly lower (P &lt; 0.01) than that of the control and the enzyme preparations were completely inactivated after mild heat treatment. These results suggest that the potential for Northern shrimp enzymes to be used in industrial processes, particularly in the food industry, is quite large, where working at lower temperatures to prevent undesirable chemical reactions is necessary.

Purification and characterization of serine proteases that exhibit caspase-like activity and are associated with programmed cell death in Avena sativa.

Victoria blight of Avena sativa (oat) is caused by the fungus Cochliobolus victoriae, which is pathogenic because of the production of the toxin victorin. The victorin-induced response in sensitive A. sativa has been characterized as a form of programmed cell death (PCD) and displays morphological and biochemical features similar to apoptosis, including chromatin condensation, DNA laddering, cell shrinkage, altered mitochondrial function, and ordered, substrate-specific proteolytic events. Victorin-induced proteolysis of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is shown to be prevented by caspase-specific and general protease inhibitors. Evidence is presented for a signaling cascade leading to Rubisco proteolysis that involves multiple proteases. Furthermore, two proteases that are apparently involved in the Rubisco proteolytic cascade were purified and characterized. These proteases exhibit caspase specificity and display amino acid sequences homologous to plant subtilisin-like Ser proteases. The proteases are constitutively present in an active form and are relocalized to the extracellular fluid after induction of PCD by either victorin or heat shock. The role of the enzymes as processive proteases involved in a signal cascade during the PCD response is discussed.

Purification and Characterization of Proteases from Bacillus amyloliquefaciens Isolated from Traditional Soybean Fermentation Starter

Bacillus amyloliquefaciens FSE-68 isolated from meju, a Korean soybean fermentation starter, was identified on the basis of biophysical tests and 16S rRNA gene sequence. A neutral metalloprotease (NPR68) and an alkaline serine-protease (APR68) were purified by ammonium sulfate precipitation and cation exchange chromatography and identified on the basis of their activities at different pH values and the selective protease inhibitors. The molecular weights of NPR68 and APR68 measured with ESI-MS were 32743 (+/- 0.8) and 27443 (+/- 0.5) Da, respectively. Against oxidized insulin chains, the NPR68 has a cleavage preference at the site where leucine is located as a P1' residue followed by phenylalanine, and the APR68 has broad specificity and favors leucine at the P1 site. These results indicate that the proteases are natural variants of subtilisin and bacillolysin.

Purification and characterization of collagenolytic proteases from the hepatopancreas of northern shrimp (Pandalus eous).

Three gelatinolytic proteases (A1, A2, and B) were purified using a synthetic substrate, DNP-Pro-Gln-Gly-Ile-Ala-Gly-Gln-d-Arg, from the hepatopancreas of Northern shrimp (Pandalus eous) by several chromatographic steps involving hydroxyapatite column chromatography, gel filtration on Superdex75, and ion-exchange chromatography on a MonoQ column. Collagenolytic proteases A2 and B, but not protease A1, were demonstrated to digest native porcine type I collagen at 25 degrees C and pH 7.5. Further characterizations of these two collagenolytic proteases showed that the pH optimum of enzyme A2 against DNP-peptide was found to be 11, whereas that of enzyme B was 8.5. The optimum temperature ranged between 40 and 45 degrees C for both enzymes, although enzyme B appeared to be thermally more stable than enzyme A2 at pH 7.5. Both enzymes were strongly inhibited by PMSF and antipain, which suggests that they belong to collagenolytic serine proteases.

Purification of soluble and membrane-bound proteases with substrate-analogous inhibitors by affinity chromatography

Specific modified substrate-analogous amino acids and peptides have been used as affinity ligands in the affinity chromatography of proteases. Alanine methyl ketone-Sepharose (AMK-Sepharose) is introduced as affinity support for the purification of a bacterial alanyl aminopeptidase (AAP) from a membrane protein extract and Arginine-Agarose as support for the preparation of a membrane-bound proteinase of myeloma cells (MP-1). Peptidyl methyl ketones as affinity ligands have been used to separate subtilisin enzymes and the cysteine proteases cathepsin B, L, and S. As a new type of ligands, spacer-bound peptidyl chloromethyl ketones are presented for a specific and oriented immobilization of proteinases. Oriented-immobilized cathepsin B was used to isolate antibodies against this enzyme.

Purification, characterization, and molecular modeling of pyrolysin and other extracellular thermostable serine proteases from hyperthermophilic microorganisms

Currently several serine proteases have been purified from hyperthermophiles with a variety of approaches and characterized at the enzyme and, in most cases also the gene level. Those that are genetically characterized all belong to the subtilisin-like family of serine proteases, also known as “subtilases.” Moreover, a variety of homologs of subtilases can be detected in the present databases of sequenced genomes of hyperthermophiles. The first of these serine proteases to be characterized at the enzyme and gene level has been pyrolysin from the hyperthermophilic archaeon Pyrococcus furiosus , which is the most thermostable protease to date and retains half of its activity after boiling for several hours. A characteristic feature of pyrolysin and other proteases is the fact that these enzymes are substrates of their proteolytic activity and degrade themselves in a process termed autoproteolysis. This has for a long time hampered the purification of pyrolysin and prevented its further characterization at the molecular level. This chapter discusses the procedures for the biochemical and genetic characterization of pyrolysin and other related thermostable serine proteases and the prediction of their properties by homology comparisons, database searches, and molecular modelling.

Purification and quantification of calcium-activated neutral proteases I and II and novel isoforms from cultured osteoblastic cells by ion-exchange fast protein liquid chromatography.

Purification and quantification of calcium-activated neutral proteases I and II and novel isoforms from cultured osteoblastic cells by ion-exchange fast protein liquid chromatography.

Protease activity induced by nicotine in human cells.

Nicotine has a wide range of biological effects, and proteases have been extensively studied for their biological roles in living creatures. The aim of this study is to determine whether nicotine can induce proteolytic protease activity in cultures of various human cell lines. Plasminogen activator-like fibrinolytic protease activity, using 125I-fibrin as substrate in the presence of plasminogen, was estimated in cells with and without nicotine treatment. Among 16 cell lines tested, APr-1 cells were found to have the highest induced protease activity. Partial purification of the proteases was carried out by high performance liquid chromatography gel filtration on TSKG2000SW. Protease inhibitor tests indicated that the proteases induced by nicotine are serine proteases.

Purification of mast cell proteases from murine skin.

Different subpopulations of mast cells are characterized by their abundant contents of either tryptase or in addition chymase. These two neutral proteases are found in mast cells and may thus hold a key to the understanding of mast cell dependent reactions. Such studies are however hampered by the lack of readily available supplies of chymase. We have therefore studied the simultaneous purification of both proteases from hairless moro hr/hr mouse skin, using a sequence of salt extractions and chromatographic separations. After three steps of extraction, a 13-fold purification with an 82% yield was obtained for tryptase and a 15-fold purification with a 90% yield for chymase. Further one step purification on conventional sephadex, sephacryl and octyl sepharose columns was unsatisfactory because of further protein contamination of the fractions. Heparin affinity chromatography caused a high loss of tryptase and residual protein contamination. Gradient elution on a benzamidine sepharose 6B column resulted however in a single, low yield (17.9%) tryptase peak and a broader, high yield (>90%) chymase peak, and a 34% yield high purity fraction. The proteases thus purified exhibited their typical inhibitor profile. On Western blot analysis and on autoradiography in the presence of the serine protease inhibitor diisopropylfluorophosphate (DFP), only one 28 kD molecule with chymase activity was identified, whereas a broad 32-38 kD band of tryptase monomers was noted. Taken together, these data show that, after salt extraction and a single benzamidine affinity chromatography step, both mast cell chymase and tryptase can be separated and in case of chymase also highly purified, allowing thus for the study of biological activities of this molecule.

Selective Inhibition, Separation, and Purification of Serine Proteases: A Strategy Based on a Photoremovable Inhibitor

Selective Inhibition, Separation, and Purification of Serine Proteases: A Strategy Based on a Photoremovable Inhibitor