Purification Of Protease Research Articles

Purification and characterization of alkaline, thermostable and organic solvent stable protease from a mutant of Bacillus sp.

Abstract An alkaline protease produced from wild Bacillus sp. with potential in many industrial applications and its hyper-protease producer mutant obtained through random mutagenesis was purified from the culture supernatant by using ethanol precipitation (0–70% saturation) and DEAE ion-exchange chromatography. The yield of the purified protease after purification in wild strain was found 7.5% with specific activity of 4729.0 U/mg and purification fold of 5.6. Under similar conditions the yield of purified protease in mutant strain was 8.8% with a specific activity of 6584.6 U/mg and purification fold of 7.8. The purified proteases fractions were found to be homogenous in SDS-PAGE in both the cases with a molecular weight of 17 kDa. Both mutant and wild strain showed optimum ativity at 65 °C and pH-9.0. Mutant strain retained 97% of activity even at pH-10.0 while wild strain showed decrease in activity (62%) at this pH. Both strain showed substrate specificity toward casein with Km and Vmax value (0.294 mg/ml, 3703.7 mg/ml/min and 0.167 mg/ml, 4761.9 mg/ml/min) in wild and mutant strain respectively. The protease activity in both wild and mutant showed inhibition in enzyme activity in the presence of EDTA (5 mM), thus confirming the presence of metal residues at the active site. In both wild and mutant strain Ca2+ (5 mM) showed stabilizing effect but presence of Cu2+ (5 mM) exhibited inhibitory effect on the activity of the purified protease. Purified protease of both strains showed stability in the presence of various detergents but decreased activity was noticed in the presence of NaCl. MALDI-TOF-MS analysis also confirmed that the purified protein was protease in wild and peptidase in mutant strain respectively.

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.

Purification and Characterization of Alkaline Protease from Bacillus sp. HD292

Proteolytic enzymes form a very significant group of enzymes which are used for a variety of purposes including as a detergent additive. In the present investigation, the protein hydrolyzing enzyme secreted by Bacillus sp. HD292 was purified and characterized. The purification of the proteolytic enzyme was undertaken by ammonium sulphate fractionation (20–50%) and gel filtration chromatography using Sephadex G-75 which resulted in 17.78 fold purification. On performing SDS-PAGE analysis, a single band of approx. 30 kD was observed. The purified protease displayed peak activity at pH 9.5 and 70 °C. The Km value of the enzyme was found to be 2.7 mg ml−1 with casein as the substrate. pBLAST analysis of the translated sequence resulting from the PCR product obtained from the conserved region of the gene, showed it to be similar to various intracellular and extracellular serine proteases. Also, the purified bacterial protease was found to retain its proteolytic activity at high pH as well as high temperature and was also observed to be quite compatible with the detergent Paras (R3 Organics).

Purification of Cold-adapted Protease from Janthinobacterium sp.

Purification of Cold-adapted Protease from Janthinobacterium sp.

Purification, characterization, molecular modeling and docking study of fish waste protease

In the present study, the alkaline protease has been extracted from the fish processing waste using ammonium sulphate fractionation followed by ion-exchange chromatography on sephadex G-25 and on DEAE column with a 4.0 fold increase in purification of yield 7.7%. The molecular weight of the purified protease was found to be 33 kDa as determined by SDS-PAGE. The optimum temperature was found to be 30 °C at pH 8. The activation energy (Ea) for casein hydrolysis and temperature quotient (Q10) was found to be 38.25 kJ/mol and 1.65, respectively. The kinetic constants km, Vmax, kcat, and kcat/km and thermodynamic parameters ΔH*, ΔS*, ΔG*, ΔG*E–S, and ΔG*E–T revealed high affinity of the fish protease for casein. Using CD spectroscopy it was found that the fish protease has 32.7% alpha-helical, 32.8% β-turn and 34.5% random coil. 3D structure of target protein was predicted by homology modeling. Ramachandran plot revealed that the total residues in favored, allowed and outlier regions are 96.6%, 2.3%, and 1.1% residues. The biological function of the modeled fish protease was predicted by COACH based on the I-TASSER model, suggests that the fish protease may be exploited as biocatalyst in various industrial applications and processes. AutoDock 4.2.6. was used to study the protein-ligand interactions, which may lead to the discovery of novel semisynthetic enzymes from renewable biowaste.

Purification and characterization of neutral protease from Aspergillus oryzae Y1 isolated from naturally fermented broad beans

The strain Y1, with a notably high production of neutral protease, was isolated from naturally fermented broad beans and subsequently identified as Aspergillus oryzae, through the analysis of its morphology characteristics and 18S rDNA sequence. Naturally fermented broad beans are the main raw material in Sichuan broad-bean sauce. The neutral protease from Aspergillus oryzae Y1 was purified using ammonium sulphate precipitation and DEAE-Sepharose Fast Flow chromatography, which resulted in a 10.0-fold increase in the specific activity (2264.3 U/mg) and a recovery rate of 21%. The estimated molecular mass of the purified protease was approximately 45 kDa. The optimal pH and temperature of the purified protease were 7.0 and 55 °C, respectively. The heat resistance of the purified protease was significantly higher than the commercial protease. The effect of metal ions on the activity of the purified protease approximated that of commercial neutral protease. Furthermore, the maximum hydrolysis rate (Vmax) and apparent Michaelis–Menten constant (Km) values of the purified protease were 256.4103 μg/mL min and 20.0769 mg/mL, respectively. The purified protease had a higher affinity for the substrate than the commercial neutral protease. All the results suggest that this neutral protease exhibits the potential for application in industry due to its good resistance to high temperatures and wide range of acids and bases.

Production and partial characterization of dehairing alkaline protease from Bacillus subtilis AKAL7 and Exiguobacterium indicum AKAL11 by using organic municipal solid wastes

Alkaline proteases have applications in numerous industries. In this study, we have isolated and screened proteolytic bacteria from poultry wastes mixed soil and identified two bacterial isolates as Bacillus subtilis AKAL7 and Exiguobacterium indicum AKAL11 based on 16S rDNA sequencing. Maximum level of protease production was achieved after 24 h of fermentation in a basal medium. The optimal temperature, initial pH of the media and agitation for alkaline protease production by these two isolates were 30 °C, pH 9.0 and 120 rpm, respectively. The both bacterial isolates produced maximum level of protease with 3.0% organic municipal solid wastes (OMSW) as the sole source of carbon and nitrogen under previously optimized fermentation conditions. In comparison with the shake flask, protease production increased about 2.5-fold in the bioreactor with reduction in fermentation period. The partial purification of protease resulted in a final 45.67 and 34.86-fold purified protease with a specific activity of 8335.34 and 9918.91 U/mg protein and a typical yield of 9.75 and 9.41% from B. subtilis and E. indicum, respectively. The optimum temperature and pH of the partially purified protease from the both sources was 40 °C and pH 9.0, respectively. Protease from the both isolates was stable at pH 7.0–12.0 and at temperatures up to 50 °C. The effects of protease inhibitors indicated that the protease from B. subtilis might be serine and cysteine type and from E. indicum might be cysteine type. Mg2+, K+ and Ca2+ stimulated but Zn2+, Hg2+, Co2+ and Fe3+ strongly inhibited the protease activity. The partially purified protease from B. subtilis substantially dehaired cow skin and decomposed gelatinous compound from X-ray film. Our study revealed that OMSW can be used as raw material for production of bacterial extracellular protease and alkaline protease from B. subtilis might be potential for industrial and biotechnological applications.

Purification and characterization of alkaline protease with novel properties from Bacillus cereus strain S8.

Proteases are the hydrolytic enzymes which hydrolyzes peptide bond between proteins with paramount applications in pharmaceutical and industrial sector. Therefore production of proteases with efficient characteristics of biotechnological interest from novel strain is significant. Hence, in this study, an alkaline serine protease produced by Bacillus cereus strain S8 (MTCC NO 11901) was purified and characterized. The alkaline protease was purified by ammonium sulfate precipitation (50%), ion exchange (DEAE-Cellulose) and gel filtration (Sephadex G-100) chromatographic techniques. As a result of this purification, a protein with specific activity of 300U/mg protein was obtained with purification fold 17.04 and recovery percentage of 34.6%. The molecular weight of the purified protease was determined using SDS-PAGE under non-reducing (71 kDa) and reducing conditions (35 kDa and 22 kDa). Zymogram analysis revealed that proteolytic activity was only associated with 22 kDa. These results indicate that existence of the enzyme as dimer in its native state. The molecular weight of the protease (22 kDa) was also determined by gel filtration (Sephadex G-200) chromatography and it was calculated as 21.8 kDa. The optimum activity of the protease was observed at pH 10.0 and temperature 70 °C with great stability towards pH and temperature with casein as a specific substrate. The enzyme was completely inhibited by PMSF and TLCK indicating that it is a serine protease of trypsin type. The enzyme exhibits a great stability towards organic solvents, oxidizing and bleaching agents and it is negatively influenced by Li2+ and Co2+ metal ions. The purified protein was further characterized by Matrix Assisted Laser Desorption Ionization/Mass Spectroscopy (MALDI/MS) analysis which reveals that total number of amino acids is 208 with isoelectric point 9.52.

Purification, biochemical, and molecular characterization of novel protease from Bacillus licheniformis strain K7A

A novel extracellular alkaline protease, called SAPHM, from Bacillus licheniformis strain K7A was purified by four steps procedure involving heat treatment (30 min at 70 °C) followed by ammonium sulfate precipitation (40–70%)-dialysis, UNO Q-12 FPLC, and ZORBAX PSM 300 HPLC, and submitted to biochemical characterization assays. The purified enzyme is a monomer of molecular mass of 30,325.12 Da. It was completely inhibited by phenylmethanesulfonyl fluoride (PMSF)and diiodopropyl fluorophosphates (DFP), which strongly suggested its belonging to the serine protease family. Its sequence of the 26 NH2-terminal residues showed high homology with those of Bacillus proteases. The purified enzyme was optimally active at pH 10 and temperature 70 °C. Its catalytic efficiency was higher than those of Alcalase and Thermolysin. SAPHM exhibited excellent stability to detergents and wash performance analysis revealed that it could remove blood-stains effectively. Data suggest also that SAPHM may be considered as potential candidate for future applications in non-aqueous peptide biocatalysis because it possesses an elevated organic solvent resistance. The sapHM gene encoding SAPHM was cloned, sequenced, and expressed in Escherichia coli strain BL21(DE3)pLysS. The biochemical properties of the extracellular purified recombinant enzyme (rSAPHM) were similar to those of native one. The deduced amino acid sequence showed strong homology with other Bacillus proteases. The highest sequence identity value (97%) was obtained with APRMP1 protease from Bacillus licheniformis strain MP1, with only 9 aa of difference.

Partial Purification and Characterization of Protease from Bacillus thuringiensis kurstaki HD-73 Upregulated by Limiting N2-Source

Partial Purification and Characterization of Protease from Bacillus thuringiensis kurstaki HD-73 Upregulated by Limiting N2-Source

Extraction and purification of protease from Aspergillus niger isolation

Extraction and purification of protease from Aspergillus niger isolation

Purification and Characterization of Microbial Protease Produced Extracellularly from Bacillus subtilis FBL-1

An ammonium sulfate precipitation of fermentation broth produced by Bacillus subtilis FBL-1 resulted in 2.9-fold increase of specific protease activity. An eluted protein fraction from the column chromatographies using DEAE-Cellulose and Sephadex G-75 had 94.2- and 94.9-fold higher specific protease activity, respectively. An SDS-PAGE revealed a band of purified protease at approximately 37.6 kDa. Although purified protease showed the highest activity at 45°C and pH 9.0, the activity remained stable in temperature range from 30 to 50°C and pH range from 7.0 to 9.0. Protease activity was activated by metal ions such as Ca2+, Mg2+, Mn2+, Fe2+, Ca2+ and K+, but 10 mM Fe3+ significantly inhibited enzyme activity (53%). Protease activity was inhibited by 2 mM EDTA as a metalloprotease inhibitor, but it showed good stability against surfactants and organic solvents. The preferred substrates for protease activity were found to be casein (100%) and soybean flour (71.6%).

Single step purification via magnetic nanoparticles of new broad pH active protease from Penicillium aurantiogriseum

A new set of applications can be achieved when using high stability proteases. Industrially, high costs can be related to production medium and purification process. Magnetic nanoparticles have been successfully used for rapid and scalable purification. In this work, azocasein were immobilized on magnetite nanoparticles and applied in a single step purification of protease produced by Penicillium aurantiogriseum using soybean flour medium, and the new purified enzyme was characterized. Glutaraldehyde activated nanoparticles were used in azocasein immobilization and then incubated with dialyzed 60–80% saline precipitation fraction of crude extract for purification. Adsorbents were washed 7 times (0.1 M NaCl solution) and eluted 3 times (1 M NaCl solution), these final elutions contained the purified protease. This protease was purified 55.68-fold, retaining 46% of its original activity. Presented approximately 40 kDa on SDS-PAGE and optimum activity at 45 °C and pH 9.0. Maintained over 60% of activity from pH 6.0 to 11.0. Kept more than 50% activity from 15 to 55 °C, did not lose any activity over 48 h at 25 °C. Inhibitors assay suggested a serine protease with aspartic residues on its active site. Results report a successful application of an alternative purification method and novel broad pH tolerant protease.

Purification and characterization of fibrinolytic protease from Bacillus amyloliquefaciens MCC2606 and analysis of fibrin degradation product by MS/MS

ABSTRACTA serine protease with preference for fibrin protein was purified and characterized from Bacillus amyloliquefaciens MCC2606, isolated from dosa batter. The protease was purified using ammonium sulfate precipitation, ion-exchange, and gel filtration chromatography. The degradation activity of the protease toward the fibrin was significantly higher compared with other protein substrates in the study. The molecular weight of the CFR15-protease was estimated to be 32 kDa based on SDS-PAGE. The purified enzyme exhibited both fibrinolytic and fibrinogenolytic activity. The optimum pH and temperature for the activity of the enzyme was found to be 10.5 and 45°C. A significant inhibition was seen with the protease inhibitors phenyl methyl sulphonyl fluoride and ethylene diamine tetra acetic acid and the activity of the enzyme was enhanced in presence of Mn2+. There was an observed increase in vitro activated partial thromboplastin time and prothrombin time of both time and dose dependent study. Among the four chains of fibrin, the β-chain of fibrin appears to be the primary component and site susceptible for CFR15-protease in early action as indicated by MS/MS analysis of initial degradation products. These results indicated that the CFR15-protease have the potential to be an effective fibrinolytic agent.

Cloning, Expression and Purification of Trypsin-like Protease of the Periodontal Pathogen Tannerella forsythia ATCC 43037

Tannerella forsythia, a bacterial species frequently associated with the pathogenesis of periodontal disease and its proteases are implicated as virulence factors, is known to possess trypsin-like activity. Here, we characterize a trypsin-like protease of T. forsythia referred to as tlp. Full length (without a signal peptide) recombinant tlp (102 kDa). The evidence suggests that the enzyme is a serine protease since it was strongly inhibited by Pefabloc, Tosyl-L-Lysyl-chloromethane hydrochloride (TLCK) and Leupeptin. The ability to degrade elastin, fibrinogen and the antimicrobial peptide LL-37 may contribute to the pathogenicity of periodontitis.

Submerged fermentation, purification, and characterization of L-asparaginase from Streptomyces sp. isolated from soil

Purification and characterization of extracellular alkaline protease from Streptomyces sp. LCJ12A isolated from Pichavaram mangrovesM. Parthasarathy, J. Joel Gnanadoss

Characterization of a robust serine protease from Bacillus subtilis K-1.

Process apt microbial proteases due to their wide range industrial applications have become the focus of intense scientific research during recent years. Considering the hostile process milieu, the proteases intended for application must be robust enough to withstand the extremes of temperature and pH, and presence of organic solvents and other potential enzyme inhibitors. Current study presents the characterization of a robust protease from a previously isolated bacterium Bacillus subtilis K-1 (BSK-1). Purification of BSK-1 protease (5.21-fold) was achieved to homogeneity by salt (ammonium sulfate) precipitation, and ion-exchange (diethyl-aminoethyl-sephadex) and size exclusion chromatography (Sephadex G-100). Molecular weight of BSK-1 protease was determined by SDS-PAGE analysis (42 kDa). Though the optimum temperature and pH for BSK-1 protease activity was 50 °C and 10, respectively, but, the protease exhibited remarkable activity and stability over elevated temperatures (60-80 °C) and a broad pH range (pH 7-11). Protease showed resistance towards several organic solvents/other potential enzyme inhibitors. Drastic activity loss in presence of phenylmethylsulfonyl fluoride indicated that the enzyme is a serine protease. Kinetic parameters (Km and Vmax ) for BSK-1 protease were found to be 0.14 mg ml-1 and 1176 mg min-1 , respectively. Putative amino acid sequence of BSK-1 protease (derived from nucleotide sequence of protease gene) suggested that the enzyme belonged to peptidases S8/S53 super family with multidomain of S8. BSK-1 protease being stable under harsh conditions may serve a model system for understanding the molecular basis of stability, and may help designing novel proteases that are suitable for industrial applications.

Partial purification and characterization of serine protease produced through fermentation of organic municipal solid wastes by Serratia marcescens A3 and Pseudomonas putida A2.

Proteolytic bacteria isolated from municipal solid wastes (MSW) were identified as Serratia marcescens A3 and Pseudomonas putida A2 based on 16S rDNA sequencing. Protease produced through fermentation of organic MSW by these bacteria under some optimized physicochemical parameters was partially purified and characterized. The estimated molecular mass of the partially purified protease from S. marcescens and P. putida was approximately 25 and 38 kDa, respectively. Protease from both sources showed low Km 0.3 and 0.5 mg ml−1 and high Vmax 333 and 500 µmole min−1 at 40 °C, and thermodynamics analysis suggested formation of ordered enzyme-substrate (E-S) complexes. The activation energy (Ea) and temperature quotient (Q10) of protease from S. marcescens and P. putida were 16.2 and 19.9 kJ/mol, and 1.4 and 1.3 at temperature range from 20 to 40 °C, respectively. Protease of the both bacterial isolates was serine and cysteine type. The protease retained approximately 97% of activity in the presence of sodium dodecyl sulphate. It was observed that the purified protease of S. marcescens could remove blood stains from white cotton cloth and degrade chicken flesh remarkably. Our study revealed that organic MSW can be used as raw materials for bacterial protease production and the protease produced by S. marcescens A3 might be potential for applications.

Isolation, partial purification, biochemical characterization and detergent compatibility of alkaline protease produced by Bacillus subtilis, Alcaligenes faecalis and Pseudomonas aeruginosa obtained from sea water samples.

In the current study, bacteria isolated from sea water samples of Murdeshwar, Karnataka, were screened for the production of alkaline protease by culturing them onto skim milk agar media. Of the isolated bacteria, Bacillus subtilis, Pseudomonas aeruginosa and Alcaligenes faecalis showed distinct zones of hydrolysis due to enzyme production. They were each inoculated into enzyme production media under submerged fermentation conditions at 37 °C for 48 h with a constant agitation of 120 rpm. Partial purification of alkaline protease was carried out by isoelectric precipitation. Enzyme activity was determined under varying conditions of pH, incubation temperature, different substrates, carbon and nitrogen sources and salt concentrations using sigma’s universal protease activity assay. Enzyme immobilization was carried out using 2% Sodium alginate and 0.1 M ice cold CaCl2 and its activity under varying pH, temperature conditions and detergent compatibility was assayed. Efficacy of enzyme in stain removal was tested and haemolysis was observed within of 60 s which resulted in removal of the stain. Among the three organisms, enzyme from Bacillus subtilis showed highest activity in all cases indicating that it was the most ideal organism for enzyme production.

Purification, characterization and application of novel alkaline protease from new Bacillus cereus UV-15 mutant

The alkaline protease produced by Bacillus cereus UV-15 mutant was purified by precipitation with ammonium sulphate and gel filtration through sephadex G-100. The enzyme has shown to have a molecular weight of 29kDa by SDS polyacrylamide gel electrophoresis. The extracted protease enzyme was purified by 16.64 fold through ammonium sulphate precipitation and chromatography separation in Sephadex G-100. The purified protease had a specific activity of 2915 (U/mg). The zymogram also revealed a clear hydrolytic zone due to proteolytic activity, which coincided with the band obtained with SDS–PAGE. The enzyme was remained active and stable at pH 8-11, with an optimum at pH 10.0. The protease was stable in the temperature ranging from 40°C to 60°C, but gradually decreased at temperature 70°C. The optimum temperature for protease activity was determined at 60°C. The enzyme showed stability towards non-ionic and anionic surfactants, and oxidizing agents. At 1% concentration of Tween-20 and Tween-80, the enzyme retained 78% and 94% relative activity respectively. Alkaline protease retained 95% activity toward 0.5% concentration of the anionic detergent SDS. The enzyme showed compatibility at 50°C with commercial detergents such as Ariel, Surf excel, Rin, wheel, Tide and Nirma. In the presence of Ariel and Rin the enzyme retained about 72 and 75% of the original activity respectively. The supplementation of the enzyme in detergents could improve the cleansing performance towards the blood stains and suggested to be used as a detergent additive. The enzyme also removed goat hide hairs completely after 15 hr of incubation. These characteristics may make the enzyme suitable for several industrial applications, especially in leather industries.