Separation Of Proteases Research Articles

Extraction of protease from Aspergillus tamarii URM 4634 in aqueous two-phase system under continuous and discontinuous process

Aqueous two-phase systems have been studied for almost a century to separate biomolecules in harmless conditions. Proteases produced by Aspergillus tamarii URM 4634 were extracted in polyethylene glycol (PEG)/phosphate aqueous two-phase system under discontinuous and continuous (perforated rotative discs column) process. On the discontinuous process, it was evaluated the effect of operational conditions (PEG molar mass and its concentration, phosphate concentration and pH) over the partition coefficient, activity yield and purification factor. Protease partitioned to PEG-phase with partition coefficients up to 55.73. The best process parameters were 17.5% of PEG, with molar mass 8000 g·mol−1, 15% of phosphate salt at pH 6, with 113.15% of activity yield and purification factor of 2.62. Under continuous extraction, hold up data showed that 57.1% of the discontinuous phase was available for protein extraction. Further, separation achieved 90.0% of efficiency. The yields surpassed 100% in almost all runs, and the best purification factor was 1.84, with both flows of 2 mL·min−1. Thus, the best operational conditions reached an activity yield of 95.3% and 90.0% of separation efficiency. Hence, aqueous two-phase system PEG/phosphate extraction is an efficient process for separation of proteases produced by Aspergillus tamarii URM 4634, under continuous extraction likewise under discontinuous process.

Silver-Stained Fibrin Zymography: Separation of Proteases and Activity Detection Using a Single Substrate-Containing Gel.

Silver-stained fibrin zymography for separation of protease bands and activity detection using a single substrate gel was designed. The method takes advantage of the nano-scale sensitivity of both zymography and silver staining. After sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) in a gel containing fibrin (protease substrate), the gel was incubated in enzyme reaction buffer and the zymogram gel was silver-stained. Bands with protease activity were stained with silver in clear areas where the protein substrate had been degraded. The molecular sizes of proteases were accurately determined.

Comparison of four purification methods to purify cysteine protease from Asian pear fruit (Pyrus pyrifolia)

Abstract Pear fruit is one of the most popular fruits, extensively exploited for its protease activity commercially in the food, cosmetic, and pharmacological industries. The aim of this study was to compare four different purification methods for protease purification from an Asian pear cultivar. Pear proteases were purified by ethanol precipitation, two phase partitioning, or ion exchange chromatography (DEAE Sepharose alone or followed by Mono Q). Purified proteases from each method were further evaluated for purification efficiency by SDS-PAGE analysis and enzyme activity with succinlyated casein substrate. All methods produced two protease bands with molecular weights of 36 kDa and 38 kDa. Among the methods, 15% PEG 1000–20% MgSO4 system and DEAE followed by Mono Q chromatography showed efficient separation of protease with high recovery of enzymatic activity (91–98%). DEAE followed by Mono Q chromatography achieved the highest purification fold (15). These result suggested that a two phase partitioning technique is effective as a single purification step for pear protease.

Optimization of Aqueous Two-Phase Extraction of Protease Produced from Bacillus licheniformis NCIM 2042 Using Response Surface Methodology

An aqueous two-phase system (ATPS) composed of polyethylene glycol (PEG) 4000 and potassium phosphate was explored for the separation of protease produced from Bacillus licheniformis NCIM 2042. The various experimental parameters viz. potassium phosphate, PEG, and NaCl concentration as well as system pH were optimized for higher extraction yield using central composite rotatable design (CCRD) of response surface methodology (RSM). The RSM predicted model delivered 86.80% yield with 1.89 purification factor and 11.65 partition coefficient of protease with ATPS comprising of potassium phosphate 22.36% (w/w), PEG 4000 15.93% (w/w), pH (10.06), and NaCl (1.08 mM). The experimental results of optimized conditions showed partition coefficient of 11.95 with 2.2 purification factor and %yield of 89.75 of protease and close resemblance of both the results validated the predicted model. SDS PAGE analysis showed a protein bands for partially purified protease.

Silver-stained fibrin zymography: separation of proteases and activity detection using a single substrate-containing gel

A new zymogram method, silver-stained fibrin zymography, for separation of protease bands and activity detection using a single substrate gel, was developed. The method takes advantage of the nanoscale sensitivity of both zymography and silver staining. After SDS-PAGE in a gel containing fibrin, the gel was incubated in enzyme reaction buffer and the zymogram was silver-stained. Bands with protease activity were stained with silver in clear areas where the protein substrate had been degraded. The molecular sizes of proteases were accurately determined. Furthermore, proteases of high molecular weight were clearly and sharply resolved.

Separation of protease from yellowfin tuna spleen extract by ultrafiltration: Effect of hydrodynamics and gas sparging on flux enhancement and selectivity

The pretreated yellowfin tuna spleen extract was used as feed in ultrafiltration for recovery of protease. The combined impact of crossflow rate and gas sparging on critical flux, limiting flux and selectivity was studied by a total recycle mode using a hollow fiber membrane with molecular weight cutoff 30 kDa. The critical flux varied from 28.8 to 44.2 L/m 2 h and limiting flux varied from 34.3 to 52.4 L/m 2 h while crossflow rate increased from 17.55 to 69.98 L/h without gas sparging. A low gas injection factor of 0.15 could improve critical and limiting flux significantly comparing to that without gas sparging. Higher gas injection factors varied from 0.30 to 0.61 did not give remarkable improvement of both critical and limiting flux. The benefit of increasing crossflow rate to enhance critical and limiting flux was great when gas sparging did not applied. Selectivity of ultrafiltration process was increased with increasing permeate flux at subcritical condition and critical flux condition. It became insensitive to the flux and crossflow rate at limiting flux condition when gas sparging was not applied. Gas sparging gave negative effect on soluble protein and peptide transmission and resulted in the decay of selectivity at subcritical condition and critical flux condition. The selectivity at limiting flux condition with gas sparging was not sensitive to gas injection factor.

Separation of proteases from yellowfin tuna spleen by ultrafiltration

Separation of protease, trypsin and chymotrypsin from yellowfin tuna spleen extract by ultrafiltration (UF) using regenerated cellulose membranes with molecular weight cut off (MWCO) 30 and 100 kDa was studied. The 100 kDa membrane had a higher transmission of enzymes than that of the 30 kDa membrane. The enzyme transmission varied from 0.01 to 0.18 and from 0.6 to 0.8 for the 30 kDa membrane and 100 kDa membrane, respectively. The protein transmission was about 0.8 for both membranes. Increasing cross-flow rate and transmembrane pressure (TMP) increased permeate flux. The limiting fluxes at cross-flow rate 120, 240 and 360 L/h for the 30 kDa membrane were 17.3, 43.9 and 54.7 L/m 2 h, respectively and the limiting fluxes at the same flow rate for 100 kDa membrane were 34.1, 51.1 and 68.4 L/m 2 h, respectively. The separation of these proteases was achieved using the 30 kDa membrane. The purities of proteases were increased more than ten times at TMP 1.5 bar and cross-flow rate 360 L/h by diafiltration using 30 kDa membrane.

Separation of proteases: old and new approaches.

All methods of protein separations can be applied to proteases. Some emphasis is put in this review on a powerful technique specific to proteases purification: cyclic peptide antibiotics may be seen as general affinity ligands for proteases. Also, some examples of affinity chromatography of proteases on ligands with narrower specificity are given. The special interest of hydrophobic interaction chromatography for proteases purification is discussed. The merits of immobilized dye chromatography for proteases purification and the interest in empirically screening many immobilized dyes, as well as several eluents are discussed.

Extracellular Proteases of Mucor pusillus

Mucor pusillus was grown in different media for a period of 92 h, and the media were investigated for both milk-clotting and protease activities. It was observed that the ratio of extracellular milk-clotting activity to protease activity was the highest for 3% corn steep liquor containing 1% glucose as the source of carbon. Variation of both milk-clotting and protease activities was studied during the growth of the organism in the medium stated above. Separation of protease was carried out by ion-exchange chromatography at pH 8.0. Fractions collected were assayed for both activities simultaneously. The findings suggested that, instead of only one major acid protease, as reported by previous workers, two major acid proteases were produced. One of them had significant rennin-like activity, and the other lacked it. The former could be assumed to be the enzyme reported and studied by previous workers. The existence of two proteases was further confirmed by the appearance of two protease activity bands on polyacrylamide gels after electrophoresis. An attempt was made to separate the rennin-like enzyme from nonspecific protease activity by ammonium sulfate fractionation followed by ion-exchange chromatography at pH 6.0. The results indicated that the nonspecific protease activity due to the enzyme that lacked rennin action was substantially removed by the ammonium sulfate fractionation.