Purification Of Protease Research Articles

Purification, immobilization, and characterization of protease from local Bacillus subtilis M‐11

AbstractProtease from Bacillus subtilis M‐11 was produced and purified using ammonium sulfate precipitation, gel filtration chromatography with 15.8‐fold. The purified enzyme showed a single band on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, and it was determined that its molecular weight was 32 kDa. The purified enzyme was immobilized on polysulfone membrane containing Silica gel‐3 aminopropyltriethoxysilane (Si‐APTS) by physical adsorption. The optimal immobilization conditions (pH, temperatures, and incubation time) were investigated. The properties of immobilized protease were determined and compared with free enzyme. The investigations on immobilized enzyme indicated that the optimum pH was shifted 0.5 units to alkaline pH (pH 8.0–8.5) after immobilization with physical adsorption. Immobilization improved pH stability of enzyme between pH 7.5 and 8.5. Immobilized enzyme sustained 77.3% of its activity after usage of 10 times. However, there is no change on optimum temperature (50 °C) after immobilization.All these results indicated that polysulfone membrane containing Si‐APTS could be a proper support for protease immobilization, and immobilized protease by using physical adsorption could be used for industrial applications efficiently. © 2015 Curtin University of Technology and John Wiley & Sons, Ltd.

Production, purification and characterization of alkaline protease by ascidian associated Bacillus subtilis GA CAS8 using agricultural wastes

Abstract In the present study, protease producing Bacillus subtilis GA CAS8 was isolated from marine ascidian Phallusia arabica and identified by 16 S rRNA gene sequence. Optimization of culture condition for protease production using agricultural wastes had done by response surface methodology. The statistical optimization studies revealed that the protease production was higher when the groundnut oil cake and cabbage leaf were used as substrate. The protease activity was optimum at 50 °C and pH 9 and it retained more than 60% of activity, even at pH 12 and the molecular weight of the purified protease was 41 kDa. The activity of the purified protease was stimulated by Mg2+ and Ca2+ and it was resistant to Tween 20, Tween 40 and SDS. The protease activity was strongly inhibited in the presence of EDTA. The remarkable properties such as temperature and pH stability found with this enzyme assure that it could be a potential candidate for industrial applications.

Extraction, Purification and Characterization of Protease from Aspergillus Niger Isolated from Yam Peels

Protease was obtained from Aspergillus niger isolated from yam peels; a food waste, purified and characterized. Purification was achieved using ion exchange DEAE column and gel filtration (Sephadex G-200) chromatography. Effects of temperature; pH and production time on protease production were investigated. Also, physicochemical characteristics of the purified enzyme were investigated. The optimum production of protease was at temperature, pH and time of 37oC, 7.0 and 42 hrs respectively. The results showed that purified protease had more specific enzymatic activity than crude samples from Aspergillus Niger. whereas the specific activity of crude enzyme was 0.51 (U/mg), while the purified enzyme had an improved specific activity of 8.51 (U/mg).Optimum temperature and pH values of the purified protease were found to be 50°C and 10.0, respectively. pH stability of the enzyme ranged from 3.0- 12.0. At 3.0 and 10.0 it retained 70% and 60% of its activity after 5 hrs of incubation. Temperature stability ranged between 30oC and 90oC but most stable at 50oC retaining 94% of its activity after 1 h of incubation. The enzyme exhibited maximum activity on casein, among other protein substrates. EDTA, Cu2+, Fe2+, Mg 2+, and Ca2+ inhibited its activity while Na+ enhanced it. The enzyme was purified 16.60-fold, had a yield of 10.96 and the apparent molecular weight was 46.90 kDa. The study revealed that protease from A. niger can be exploited for protein conversion biotechnologies.

Characterization and Purification of Protease Extracted from Two Maturity Stages of ‘Noni’ (Morinda citrifolia L.) fruit

Protease was extracted from two maturity stages of noni fruits (Morinda citrifolia L.), unripe (stage 1) and ripe (stage 5). The crude extract was partially purified by acetone precipitation method followed by dialysis, gel filtration chromatography and freeze drying. Protein concentrations, proteolytic activity, molecular weight distribution, pH stability, temperature stability and storage efficiency of the resulting protease were evaluated. The unripe and ripe noni fruit contains 0.65 and 0.35% protein, respectively. Molecular weight of the proteases from both stages ranged approximately between 3 to 28 kDa based on the SDS-PAGE results. The optimum activity were at pH 7s and 6, temperatures of 40 and 50°C, respectively for proteases obtained from the unripe and ripe fruit. Analysis from the freeze dried protease indicated that protease from ripe noni fruits had higher protein concentration and specific activity compared to those from unripe fruit. However, it is more sensitive to pH and temperature and less stable during storage as it shows lower proteolytic activity compared to protease from unripe fruit. Based on its high proteolytic activity reaching up to 70.31 U/mg and storage stability (30% lost of activity), noni fruit could be an alternative source of plant protease.

Isolation, purification and characterization of extracellular protease produced by marine-derived endophytic fungus Xylaria psidii KT30

Isolation, purification and characterization of extracellular protease produced by marine-derived endophytic fungus Xylaria psidii KT30

Purification of protease from Coriandrum sativum using ion-exchange chromatography and gel filtration method

In the past decades, the interest towards plant proteases has increased significantly. Plant proteases are widely used in environment field, food and medicine industries. Proteases such as bromelain, papain and ficin are used in various areas such as in biosensors for detection of heavy metals, meat tenderization, brewing, cancer treatment, milk-clotting, viral disorders and digestion. In this study, protease from coriander leaf (Coriandrum sativum) was evaluated for protease activity using a Bradford-protease-casein assay system. This enzyme was purified through anion exchanger using DEAE-Cellulose column and gel filtration using Agilent ZORBAX column. Its molecular weight was around 55 kDa. The specific activity of the purified protein is 45.0 units/mg protein, total activity is 2745.0 units, yield 33.2% and fold purification is 2.8. Further investigation on gene sequence should be performed in order to identify the type of protease involved.

Partial Purification and Characterization of Extracellular Protease from <i>Pedicoccus acidilactici</i>

Microbial proteases have wide industrial applications and proteases of the lactic acid bacteria (LAB) have received special attention because of their importance in the food and dairy industry. Of all the LAB, the proteolytic system of the pediococci is the least studied. Therefore, this study was aimed at characterizing and purifying the protease produced by Pediococcus acidilactici grown in de Man, Rogosa and Sharpe (MRS) broth. Casein concentration of 2% (w/v) and 2.5 ml of the crude enzyme were optimal for the activity of the protease. The crude protease had temperature and pH optima of 28 oC and 4.0 respectively thus indicating that the enzyme is a mesophilic and acidic protease. Purification of the enzyme by gel filtration chromatography on Sephadex G75 following ammonium sulphate precipitation gave 2.26 fold increase in purification with specific activity of 46.13 units/mg protein while purification on Sephadex CM50 resulted in reduced purification fold (1.24 - 1.59) with specific activity ranging between 25.39 - 32.54 units/mg protein. The protease showed a characteristic band on SDS-PAGE and the molecular weight was estimated between 45 and 66 kDa. The potential applications of the protease are discussed.Keywords: Protease, lactic acid bacteria, Pediococcus acidilactici, enzyme purification.

Purification and characterization of secretory serine protease from necrotrophic oomycete, Pythium myriotylum Dreschler

Progressive increase in extracellular proteolytic activity with respect to growth was detected in cultures of necrotrophic oomycete Pythium myriotylum Dreschler with maximum activity detected at stationary phase of growth. The secretory protease from P. myriotylum designated, spPm1 was purified to homogeneity giving a single band of 47kDa molecular mass on non-reducing SDS-PAGE and exhibiting caseinolytic activity in the zymogram. Under reducing conditions, an additional band of 27.0kDa molecular size observed was subjected to matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) analysis. Resulting peptide identified as an autolytic product and generated under reducing conditions on SDS-PAGE, showed homology to domains of oomycete effector molecules. spPm1 retained proteolytic activity over broad pH (5.0-12.0) and temperature (10-80°C) ranges with optimal pH and temperature at 8.0and 60°C respectively. spPm1 was identified as a serine protease following experiments with inhibitors specific to various protease groups. spPm1 displayed high stability to surfactants, organic solvents, oxidizing agents and to metal-ion chelator, EDTA. Kinetic parameters, Km and Vmax were determined as 0.04mM and 7.52Umin(-1)mg(-1) respectively. Preferential hydrolysis of synthetic fluorogenic substrate, SAAPPPA (N-succinyl-L-alanyl-alanyl-proline-phenylalanine-p-nitroanilide) confirmed spPm1 as belonging to subtilisin serine protease family. The excellent stability of spPm1 protease characterized from P. myriotylum is discussed with respect to its potential industrial applications.

Optimization of fibrinolytic protease production from Bacillus subtilis I-2 using agro-residues

The aim of this work was to study the production of fibrinolytic protease by Bacillus subtilis I-2 on agricultural residues. Molasses substantially enhanced (63%) protease production (652.32 U/mL) than control (398.64 U/mL). Soybean meal supported maximum protease production (797.28 U/mL), followed by malt extract (770.1 U/mL), cotton cake (761.04 U/mL), gelatin (742.92 U/mL) and beef extract (724.8 U/mL). Based on the Plackett-Burman designed experiments, incubation time, soybean meal, mustard cake and molasses were identified as the significant fermentation parameters. Ammonium sulfate precipitation and DEAE sephadex chromatography resulted 4.8-fold purification of protease. Zymography showed the presence of three iso-forms in the partially purified protease preparation, which was confirmed by the SDS-PAGE analysis (42, 48, 60 kDa). Protease exhibited maximum activity at 50oC and at pH 8.0. Significant stability was observed at 30-50oC and at pH 7.0-10.0. Mg2+, Zn2+, Co2+, Ca2+, Mn2+ and Cu2+,EGTA, EDTA and aprotinin severely decreased the enzyme activity.

Unveiling the potential of metal-tolerant fungi for efficient enzyme production

The present study first time reports the utilization of metal tolerant fungi as a potential source for efficient enzyme production. The purification and characterization of alkaline protease from an indigenous zinc-metal tolerant fungal isolate Aspergillus flavus NJP08 has been demonstrated. The specific activity of enzyme was determined as 89.1Umg−1 which is found to be the highest among the reported Aspergillus flavus isolates so far. The protease was purified 55.87 fold up to homogeneity and identified as “alkaline protease” with a molecular mass of 33kDa. The N-terminal sequence was GLTTQKSAPWGLG which showed high similarity with other reported proteases of genus Aspergillus. Physico-chemical characterization of enzyme revealed an estimated half-life of >20h with aliphatic and GRAVY index values of 79.65 and −0.161 respectively, depicting high thermo-stability and secretory nature of protease. The protease was active within the temperature range of 25–50°C with an optimum temperature of 50°C and was stable in the pH range of 6.0–11.0. The enzyme was activated by Ca2+ and Fe2+ ions, partially inhibited by Cu2+ ions and strongly inhibited by PMSF. High enzyme stability in presence of various detergents further strengthens enzyme applicability in industrial applications.

Production, optimization and partial purification of protease from Bacillus subtilis

Proteases have characteristics of biotechnological interest and have thus become important industrial enzymes. We report the production of thermostable protease and its characterization in Bacillus species, which is a thermotolerant bacterium; Bacillus subtilis is widely used for isolating protease enzyme. Gelatin was used as the substrate in nutrient agar medium for screening and showed the maximum zone of activity (22mm) after overnight incubation and addition of the indicator. Under submerged fermentation conditions, a high level of protease production was found at 45°C after 36h at pH 10, with continuous agitation (180rpm). The presence of galactose and peptone in the medium enhanced enzyme production by 0.5% when compared with other carbon and nitrogen sources. Thus, such additions can augment protease production and their application in various industries.

ISOLATION, PARTIAL PURIFICATION AND CHARACTERIZATION OF ALKALINE SERINE PROTEASE FROM SEEDS OF CUCUMIS MELO VAR AGRESTIS

The present study shows the isolation, partial purification and characterization of alkaline protease from the seeds of Cucumis melo var agrestis, by a four step purification process. Its molecular weight was estimated to be 54KDa. Enzyme showed maximum activity at p H 9.0 and optimum temperature at40 o Cwith casein as substrate. The enzyme exhibited homogeneity as attested by a single protein band on both native PAGE and SDS PAGE. It is a monomeric enzyme and nonglycoprotein in nature. The km value of enzyme for casein as determined by double reciprocal plot was 2.5 mg/ml. It was strongly inhibited by PMSF but not by EDTA. The results indicate that the alkaline protease is a serine protease, similar to cucumisin from sarcocarp of melon fruit.

Environmental chitinous materials as adsorbents for one-step purification of protease and chitosanase

We describe the use of chitinous materials as adsorbents for purification of protease and chitosanase from bromelain solution and chitosanase from culture supernatants of three bacterial strains: Serratia marcescens TKU011, Bacillus cereus TKU022 and Acinetobacter calcoaceticus TKU024. The best adsorption results were observed when crude shrimp shell chitin (CSSC) and 750-nm chitin nanoparticles (CNP) were used. The optimum temperatures for protease adsorption from bromelain solution (22.9 mg/mL) by CSSC (0.1 g) and by 750-nm CNP (0.1 g) were 4 and 25 °C, respectively. The purification folds of bromelain by CSSC and 750-nm CNP were 5.2 and 4.5, respectively. For purification of protease from culture supernatants of TKU011, 750-nm CNP was 4.0-fold better than CSSC. However, CSSC exhibited purification folds of 2.9 and 3.3 for the chitosanases from TKU022 and TKU024, respectively. The adsorbed chitinolytic enzymes TKU015 chitinase (30 kDa) and TKU024 chitosanase (27 kDa) exhibited high purity by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Thus, CSSC and 750-nm CNP indicate potential for use as tools for one-step purification of bacterial chitinolytic enzymes from culture supernatants.

Statistical analysis of production of protease and esterase by a newly isolated Lysinibacillus fusiformis AU01: purification and application of protease in sub-culturing cell lines

A bacterial strain producing both a protease and an esterase was isolated from the seed of Tamarindus indica. The isolate was identified as Lysinibacillus fusiformis AU01 by phylogenetic analysis of the 16S rDNA sequence. The isolate produced an extracellular protease and an intracellular esterase simultaneously under the same culture conditions. Statistical methods, i.e., Plackett-Burman followed by response surface methodology (RSM), were applied to optimize media components and culture conditions in 50 mL shake flask cultures. Culturing in shake flasks with the optimized medium resulted in a 6-fold and a 3.5-fold increase in protease and esterase production, respectively, when compared to nutrient medium. The productivity of the protease and esterase was increased to 75 U/mL and 370 U/mL when cultivated under controlled conditions in a 3-L bioreactor. The extracellular protease was purified to 34.6 fold with 38.8 % recovery and the molecular mass of the enzyme was found to be 48 kDa. The partial amino acid sequence of the protease was determined by MALDI-TOF-MS analysis. The optimum temperature and pH for protease activity were found to be pH 9.0 and 40 °C. The inhibition of purified protease by EDTA and PMSF confirmed that the enzyme belonged to the family of serine metalloproteases. The enzyme was found to be stable in the presence of some hydrophobic and hydrophilic solvents. The ability of AU01 protease to dissociate monolayer cells for sub-culturing adherent cell lines was also investigated.

Purification and characterization of detergent-compatible protease from Aspergillus terreus gr.

The possibility of using Aspergillus terreus protease in detergent formulations was investigated. Sodium dodecyl sulfate (SDS) and native polyacrylamide gel electrophoresis indicated that the purified alkaline protease (148.9 U/mg) is a monomeric enzyme with a molecular mass of 16 ± 1 kDa. This was confirmed by liquid chromatography–mass spectrometry. The active enzyme degraded the co-polymerized gelatin. The protease demonstrated excellent stability at pH range 8.0–12.0 with optimum at pH 11.0. It was almost 100 % stable at 50 °C for 24 h, enhanced by Ca2+ and Mg2+, but inhibited by Hg2+, and strongly inhibited by phenylmethyl sulfonyl fluoride. It showed maximum activity against casein followed by gelatin; its Vmax was 12.8 U/ml with its corresponding KM of 5.4 mg/ml. The proteolytic activity was activated by Tween-80, Triton-100 and SDS, and remained unaltered in the presence of H2O2 and NaClO. The enzyme exhibited higher storage stability at 4, 28 and −20 °C. It was stable and compatible to the desired level in the local detergents. The addition of the protease to the Super wheel improved its blood stain removal. The isolated protease can thus be a choice option in detergent industry.

Purification and characterization of intracellular serine protease produced in mycelia during fruit-body growth stage of Hypsizygus marmoreus

Purification and characterization of intracellular serine protease produced in mycelia during fruit-body growth stage of Hypsizygus marmoreus

Extraction and Partial Purification of Protease from Bilimbi (Averrhoa bilimbi L.)

Unripe and ripe bilimbi (Averrhoa bilimbi L.) were ground and the extracted juices were partially purified by ammonium sulfate precipitation at the concentrations of 40 and 60% (w/v). The collected proteases were analysed for pH, temperature stability, storage stability, molecular weight distribution, protein concentration and protein content. Protein content of bilimbi fruit was 0.89 g. Protease activity of both the unripe and ripe fruit were optimum at pH 4 and 40°C when the juice were purified at 40 and 60% ammonium sulfate precipitation. A decreased in protease activity was observed during the seven days of storage at 4°C. Molecular weight distribution indicated that the proteases protein bands fall between IO to 220 kDa. Protein bands were observed at 25, 50 and 160 kDa in both the unripe and ripe bilimbi proteases purified with 40% ammonium sulfate, however, the bands were more intense in those from unripe bilimbi. No protein bands were seen in proteases purified with 60% ammonium sulfate. Protein concentration was higher for proteases extracted with 40% ammonium sulfate at both ripening stages. Thus, purification using 40% ammonium sulfate precipitation could be a successful method to partially purify proteases from bilimbi especially from the unripe stage.

Extraction and Partial Purification of Protease from Bilimbi (Averrhoa bilimbi L.)

Unripe and ripe bilimbi (Averrhoa bilimbi L.) were ground and the extracted juices were partially purified by ammonium sulfate precipitation at the concentrations of 40 and 60% (w/v). The collected proteases were analysed for pH, temperature stability, storage stability, molecular weight distribution, protein concentration and protein content. Protein content of bilimbi fruit was 0.89 g. Protease activity ofboth the unripe and ripe fruit were optimum at pH 4 and 40°C when the juice were purified at 40 and 60% ammonium sulfate precipitation. A decreased in protease activity was observed during the seven days of storage at 4°C. Molecular weight distribution indicated that the proteases protein bands fall between IO to 220 kDa. Protein bands were observed at 25, 50 and 160 kDa in both the unripe and ripe bilimbi proteases purified with 40% ammonium sulfate, however, the bands were more intense in those from unripe bilimbi. No protein bands were seen in proteases purified with 60% ammonium sulfate. Protein concentration was higher for proteases extracted with 40% ammonium sulfate at both ripening stages. Thus, purification using 40% ammonium sulfate precipitation could be a successful method to partially purify proteases from bilimbi especially from the unripe stage.

Purification and Characterization of Heat-Tolerant Protease Produced by Bacillus polyfermenticus SCD

A protease produced by Bacillus polyfermenticus SCD was purified and characterized as a new detergent material. The protease was purified from supernatant produced by B. polyfermenticus SCD, by ammonium sulfate precipitation, ion-exchange chromatography on a DEAE-Sephadex A-50, and finally gel filtration chromatography on Sephadex G-50. The molecular mass of this enzyme was 44 kDa based on SDS-PAGE. The optimum temperature and pH were 50°C and pH 8.0. The ranges of its stability to the pH and temperature were 7.0 to 9.0 and under 40°C, respectively. The enzyme was highly stable in the presence of the surfactants like Triton X-100 (0.1%), showing a 2-fold increase in its proteolytic activity. However, the enzyme was slightly inhibited by the chelating agent EDTA (1 mM). The enzyme has a maximum activity at 50°C and the activity can be increased by surfactants such as Triton X-100 and Tween 80.

Partial Purification and Characterization of Extracellular Protease of Proteus vulgaris and its Inhibition by the Volatile Oil of Syzygium samarangense

Background: The spread of drug resistant pathogens is one of the most serious threats to successful treatment of microbial infections. However, in the recent time, unrelenting effort to explore the medicinal importance of plants has become a major concern to scientists because of the less toxic and reliable phytopharmacoactive components of these plants. Purpose: The present study was carried out to evaluate the growth inhibitory effect of the volatile oil of Syzygium samarangense leaves against Proteus vulgaris and to examine the mode of inhibition of this oil on partially purified extracellular protease of this pathogen. Procedures: The volatile oil was extracted by hydrodistillation from air-dried leaves of Syzygium samarangense. The inhibitory effect of the oil was tested against the growth of Proteus vulgaris under favourable conditions using spectrophotometric method in nutrient broth. Similarly, the mode of inhibition against partially purified and characterized extracellular protease of this nosocomial opportunistic enteric pathogen was determined from Lineweaver Burke plot. The activity of this protease was also assessed based on the effect of different chloride salt solutions. Findings: The volatile oil of Syzygium samarangense inhibited Proteus vulgaris with IC50 of 0.42%v/v. The enzyme had optimum activity at pH 7.5 and 45°C. This enzyme was relatively stable at pH range of 7.0 - 8.0. The activity of this enzyme was moderately activated by K + but most inhibited by Co 2+ . Double reciprocal plot of this enzyme showed a competitive inhibition by the volatile oil with Vmax of 8.33 x 10 3 µmol/min and the K m in the absence and presence of volatile oil were 0.23mg/ml and 1.25mg/ml respectively. Highest percentage yield was 35.9 while the highest purification fold was 4.49. Conclusion: This study has shown that the relative stability of the protease between pH 7.0 – 8.0 confirmed one of the reasons why this pathogen is responsible for most of the urinary tract infections. The volatile oil extracted from the leaves of Syzygium samarangense possessed antimicrobial activity and its inhibition on the extracellular protease of Proteus vulgaris may just be