Purification Of Proteases Research Articles

Maltodextrin-modified hyperoxic graphene oxide as potential matrix for one-step purification and immobilization of MBP-tagged proteins

One-step purification and immobilization directly from crude extracts is a development trend to obtain high purity and performance target proteins due to the simple operation and high efficiency. Maltose binding protein (MBP)-tagged proteins usually need to be purified in biological experiments. It is very necessary to develop a low-cost, environmentally friendly and one-step purification and immobilization matrix. In this study, maltodextrin-modified hyperoxic graphene oxide (MHGO) was prepared using hyperoxic graphene oxide (HGO) and maltodextrin, which was characterized by scanning electron microscopy (SEM), (powder X-ray diffraction) PXRD and Fourier transform infrared spectroscopy (FTIR). MHGO was then used as a one-step purification and immobilization matrix to treat the crude extract and obtain MlrA@MHGO with 84.2 % purity of MlrA. The loading capacity of MHGO for MlrA was 379 ± 26 mg g−1 MHGO. Furthermore, the activity recovery of MlrA using the one-step strategy with MHGO as the matrix was found to be 26.5 times that of the two-step strategy. The Kcat value of MlrA@MHGO was 1.84 times that of free MlrA. Compared with free MlrA, MlrA@MHGO demonstrated enhanced stability, including half-life, thermal stability and pH stability, which were significantly improved than free MlrA. Based on these findings, we proposed a new paradigm for the purification and immobilization of MlrA using MHGO can be an effective matrix for the one-step purification and immobilization of MBP-tagged proteases.

Systematic review: How to obtain Fibrinolytic Enzymes from Fungi

Fibrinolytic complications such as stroke and peripheral occlusive disease due to vascular occlusion are significant causes of poor prognosis and death. Given the ability of mushroom fibrinolytic enzymes to dissolve blood clots and prevent side effects, their use as a potential thrombolytic therapy has attracted great interest. Serine protease, metalloprotease, and serine metalloprotease are protease enzymes that can affect the fibrinolytic mechanism by dissolving blood clots and increasing blood flow. This systematic review aimed to disclose all information regarding the production, purification, and characterization of fibrinolytic proteases from fungi. Searches were conducted on online media databases, including Google and journal sites (NCBI, PubMed, and others, using the keywords "(Fibrinolytic Enzymes) OR (Fibrinolytic Proteases) AND (Fungi or Mushrooms)" from 2012-2023. This systematic review observed reviews from the purification and characterization steps, indicating the need for attention to the manufacturing process and enzymatic applications. The production of these enzymes by fungi is associated with developments in pharmaceutical applications.

Isolation and characterization of Bacillus sp. IMV B-7883 proteases

The representatives of Bacillus are some of the best protease producers studied so far since they exhibit broad substrate specificity, significant activity, stability, simple downstream purification, short period of fermentation and low cost. Earlier, we showed that Bacillus sp. IMV B-7883 strain synthesizes an extracellular proteases, which exhibit elastolytic and fibrinogenolytic activity. The aim of the work was to isolate and purify these enzymes from the culture liquid of the Bacillus sp. IMV B-7883 strain, as well as to study their properties. Isolation and purification of proteases was carried out by precipitation of the culture liquid with ammonium sulfate, gel permeation and ion exchange chromatography and rechromatography on Sepharose 6B. As a result, proteases with elastolytic and fibrinogenolytic activity with a molecular weight of 23 and 20 kDa respectively were isolated with elastase activity increased by 63.6 and fibrinogenolytic activity by 44.1 times. The enzyme with elastase activity had a pH-optimum of 7.0 and hydrolyzed only elastin, while the enzyme with fibrinogenolytic activity was an alkaline protease with a pH-optimum of 8.0 and in addition to fibrinogen, showed specificity for fibrin and, in trace amounts, for collagen. Keywords: Bacillus sp. IMV B-7883, elastase, fibrinogenase, pH optimum, substrate specificity

Cloning, sequencing, expression, and purification of aspartic proteases isolated from two human Demodex species

Aspartic proteases (ASPs) are important hydrolases for parasitic invasion of host tissues or cells. This was the first study on Demodex ASP. First, the complete coding sequence (CDS) was amplified, cloned and sequenced. Then, the protein physical and chemical properties was analysed. Finally, the recombinant plasmid, expression and purification system was established. Results showed that the lengths of CDS of Demodex folliculorum and D. brevis were 1161 and 1173 bp, respectively. The molecular weight of the protein was approximately 40 KDa. It contained an aspartic acid residue, a substrate-binding site and signal peptide, yet lacked a transmembrane domain and was located in the membrane or extracellular matrix. The phylogenetic and conserved motif analyses showed that D. folliculorum and D. brevis clustered separately and then formed a single branch, which finally clustered with other Acariformes species. The prokaryotic expression systems for recombinant ASP with His-tag (rASP-His) and GST-tag (rASP-GST) were constructed. The inclusion bodies of rASP-His were renaturated by gradient urea and purified using NI beads, while those of rASP-GST were renaturated by sarkosyl and Triton X-100 and purified using GST beads. Conclusively, the prokaryotic expression and purification system of Demodex rASP was successfully established for further pathogenic mechanism research.

Production, optimization and partial purification of bacterial and fungal proteases for animal skin dehairing: A sustainable development in leather-making process

Crude microbial protease was produced from Bacillus subtilis KM5B and Aspergillus flavus KMAF1 after their 16S/18S rDNA molecular identification with 99.59 % and 98.99 % respectively through solid state fermentation in a medium containing hair, feathers and agro wastes. The crude protease after optimization had an optimum production with Bacillus subtilis of 35 mm, enzyme activity of 123.2 U/mL at 35 °C, pH 8.0 for 48 h while A. flavus recorded 18 mm and an enzyme activity of 122.1 U/ml at 30C, pH 7.0 for 120 h. The percentage protease recovery for the B. subtilis and A. flavus were 71.5 % and 94.8 %, fold purification at 1.5 and 2.0, total protein was 107.90 mg and 150.60 mg, enzyme activity was 125.06 U/ml and 122.01 U/ml, and specific activity was 1.159 U/mg and 0.81 U/mg respectively. Therefore, protease from the two organisms have potential to dehair animal skins in tanneries industries to improve the quality of leather without environmental pollution.

Two-Step Purification and Partial Characterization of Keratinolytic Proteases from Feather Meal Bioconversion by Bacillus sp. P45

This study aimed to purify and partially characterize a keratinolytic protease produced by Bacillus sp. P45 through bioconversion of feather meal. Crude protease extract was purified using a sequence of an aqueous two-phase system (ATPS) in large volume systems (10, 50, and 500 g) to increase obtaining purified enzyme, followed by a diafiltration (DF) step. Purified protease was characterized in terms of protein profile analysis by SDS-PAGE, optimum temperature and pH, thermal deactivation kinetics at different temperatures and pH, and performance in the presence of several salts (NaCl, CaCl2, MnCl2, CaO, C8H5KO4, MgSO4, CuSO4, ZnSO4, and FeCl3) and organic solvents (acetone, ethanol, methanol, acetic acid, diethyl ether, and formaldehyde). ATPS with high capacities resulted in purer protease extract without compromising purity and yields, reaching a purification factor up to 2.6-fold and 6.7-fold in first and second ATPS, respectively, and 4.0-fold in the DF process. Recoveries were up to 79% in both ATPS and reached 84.3% after the DF step. The electrophoretic analysis demonstrated a 25–28 kDa band related to keratinolytic protease. The purified protease’s optimum temperature and pH were 55 °C and 7.5, respectively. The deactivation energy (Ed) value was 118.0 kJ/mol, while D (decimal reduction time) and z (temperature interval required to reduce the D value in one log cycle) values ranged from 6.7 to 237.3 min and from 13.6 to 18.8 °C, respectively. Salts such as CaCl2, CaO, C8H5KO4, and MgSO4 increased the protease activity, while all organic solvents caused its decrease. The results are useful for future studies about ATPS scale-up for enzyme purification and protease application in different industrial processes.

Enhanced Biosynthesis and Purification of Proteases from Bacillus sp. AI-5 by SmF: A Green Approach for Degradation of Peptide Bonds in Complex Proteins

Proteases are theprotein degrading commercial enzymes with considerable importance for many industrial applications such as paper, leather, food industries as well as used in detergent formulation, toxic waste removal, pharmaceutical and drilling for oil. The focus of the current study was to isolate the novel protease producing bacterial sp. from environment, to optimize the submerged fermentation parameters in order to design the best supportive media for maximum enzyme yield and to purify the enzyme to increase its utilization in various industries, most importantly the pharmaceutical industry.Five pure cultures were isolated from soil of coastal area, Karachi and the bacterial strain that was proved to be the most potent producer of protease was selected for research purposeand named as Bacillus sp AI-5 after series of morphological and biochemical tests. The fermentation conditions and media composition were optimized using the starter medium that was selected among the three previously reported media for protease production. It was found that protease production from Bacillus sp AI-5 reached to maximum when media was supplemented with 0.4 gm % casein as carbon source, the combination of 0.5 gm % yeast extract and 0.5 gm % peptone as nitrogen source and 0.05 gm CaCl₂ as inducer and stabilizer of proteases. The optimum pH and temperature for maximum production of protease were found to be pH 5 and 45 and#176;C respectively after 24 hours of incubation. The protease of the Bacillus sp. AI-5 was purified to homogeneity by salt fractionation method using 60 gm % ammonium sulfate, dialysis and Sephadex G-100 Gel filtration chromatography. The specific activity of purified protease was found to be 322.25 U/mg with 13.80 fold purification as compared to crude enzyme. The purified enzyme gave a single band on Sodiumdodecyl sulfate Polyacrylamide gel electrophoresis (SDS-PAGE)corresponding to a molecular weight of 66 kDa.

Purification, Characterization, and Application of Alkaline Protease Enzyme from a Locally Isolated Bacillus cereus Strain

Among the microbial enzymes protease and amylase are the most valuable enzymes which have been has diversified applications and used extensively because of their capabilities in the degradation of organic wastes, application in biofuels, agricultural, pharmaceuticals, chemical and biotechnological industries. The aim of the current research work was the purification, characterization and application of alkaline proteases extracted from Bacillus cereus AUST-7. Various concentrations of ammonium sulphate were applied for enzyme precipitation. Sephadex-G 100 was used in FPLC system for separation of protease from other proteins. SDS-PAGE was used to measure the molecular weight of required alkaline protease. Relative activities were determined against different pH, temperature, and incubation period to measure the enzymes activity. Stability of pH, temperature and various metal ions and inhibiter were also studied. Purified enzymes were applied on the goat skin to explore the dehairing efficacy. A 6.5 purification fold and 1163.50 U/mg of specific activity were obtained at 70% saturation and 35. 91 purification fold and 8902 U/mg of specific activity were observed after FPLC separation. The 35 kDa molecular size of protease enzyme was exhibited on the SDS-PAGE. The purified enzyme was stable at pH 10, temperature 55 °C and 35 min of incubation period. The purified enzyme was found to be stable at pH 8–11, thermo-stability at 50 °C and phenyl methyl sulphonyl fluoride (PMSF) and di-isopropyl fluorophosphates (DFP) inhibited the enzyme activity. The enzyme has good potential as dehairing agent in leather industries.

Purification, biochemical characterization and fibrinolytic potential of proteases produced by bacteria of the genus Bacillus: a systematic literature review.

Thrombosis is a hematological disorder characterized by the formation of intravascular thrombi, which contributes to the development of cardiovascular diseases. Fibrinolytic enzymes are proteases that promote the hydrolysis of fibrin, promoting the dissolution of thrombi, contributing to the maintenance of adequate blood flow. The characterization of new effective, safe and low-cost fibrinolytic agents is an important strategy for the prevention and treatment of thrombosis. However, the development of new fibrinolytics requires the use of complex methodologies for purification, physicochemical characterization and evaluation of the action potential and toxicity of these enzymes. In this context, microbial enzymes produced by bacteria of the Bacillus genus are promising and widely researched sources to produce new fibrinolytics, with high thrombolytic potential and reduced toxicity. Thus, this review aims to provide a current and comprehensive understanding of the different Bacillus species used for the production of fibrinolytic proteases, highlighting the purification techniques, biochemical characteristics, enzymatic activity and toxicological evaluations used.

Partial purification, characterization and application of thermoalkaliphilic proteases from Priestia endophytica, Lysinibacillus cresolivorans and Bacillus subtilis isolated from desert soil

Partial purification, characterization and application of thermoalkaliphilic proteases from Priestia endophytica, Lysinibacillus cresolivorans and Bacillus subtilis isolated from desert soil

Production, Optimization, and Partial Purification of Alkali-Thermotolerant Proteases from Newly Isolated Bacillus subtilis S1 and Bacillus amyloliquefaciens KSM12

Proteases that can remain active under extreme conditions such as high temperature, pH, and salt concentration are widely applicable in the commercial sector. The majority of the proteases are rendered useless under harsh conditions in industries. Therefore, there is a need to search for new proteases that can tolerate and function in harsh conditions, thus improving their commercial value. In this study, 142 bacterial isolates were isolated from diverse alkaline soil habitats. The two highest protease-producing bacterial isolates were identified as Bacillus subtilis S1 and Bacillus amyloliquefaciens KSM12, respectively, based on 16S rRNA sequencing. Optimal protease production was detected at pH 8, 37 °C, 48 h, 5% (w/v) NaCl for Bacillus subtilis S1 (99.8 U/mL) and pH 9, 37 °C, 72 h, 10% (w/v) NaCl for Bacillus amyloliquefaciens KSM12 (94.6 U/mL). The molecular weight of these partially purified proteases was then assessed on SDS-PAGE (17 kDa for Bacillus subtilis S1 and 65 kDa for Bacillus amyloliquefaciens KSM12), respectively. The maximum protease activity for Bacillus subtilis S1 was detected at pH 8, 40 °C, and for Bacillus amyloliquefaciens KSM12 at pH 9, 60 °C. These results suggest that the proteases secreted by Bacillus subtilis S1 and Bacillus amyloliquefaciens KSM12 are suitable for industries working in a highly alkaline environment.

Systematic analysis on the obtaining of fibrinolytic fungi enzymes

Fibrinolytic proteases operate directly on fibrin clot and are able to maintain blood flow. Fungi show up as viable sources for obtaining this enzyme. The purpose of this systematic review is to is to unveil all the information concerning production, purification and characterization of fibrinolytic proteases by fungi. The search was conducted in ScienceDirect, PubMed and Scopus databases, using as keywords “(Fibrinolytic enzyme) OR (Fibrinolytic protease) AND (Fungal or Fungus or Fungi)”. Delimiting period of 10 years (2011-2021). The results obtained were filtered by selection criteria, and review articles or articles outside the scope of this work were excluded. The articles were evaluated and scored (0-10) according to pre-established criteria. None of the studies obtained the score 10, however the study with the highest score (9) presented relevant data in all criteria analyzed, obtaining fibrinolytic enzyme from Xylaria curta. Among the 21 selected articles, 12 different genera appear and Submerged Fermentation and purification of Serino-proteases were more described. This work also observed a greater representation of purification and characterization steps, indicating the need for attention to cultivation process and enzymatic application. It is clear that the production of these enzymes by fungi is pertinent towards the high recovery observed even after purification and its tendency for pharmaceutical application.

Partial purification and biochemical characterization of proteases of common spice seeds of Carom (Trachyspermum ammi), Coriander (Coriandrum sativum), Black cardamom (Amomum subulatum) and Green cardamom (Elettaria cardamomum)

Partial purification and biochemical characterization of proteases of common spice seeds of Carom (Trachyspermum ammi), Coriander (Coriandrum sativum), Black cardamom (Amomum subulatum) and Green cardamom (Elettaria cardamomum)

Purification of His-Tagged Proteases from the Apoplast of Agroinfiltrated N. benthamiana.

Protein expression in plants by agroinfiltration and subsequent purification is increasingly used for the biochemical characterization of plant proteins. In this chapter we describe the purification of secreted, His-tagged proteases from the apoplast of agroinfiltrated Nicotiana benthamiana using immobilized metal affinity chromatography (IMAC). We show quality checks for the purified protease and discuss potential problems and ways to circumvent them. As a proof of concept, we produce and purify tomato immune protease Pip1 and demonstrate that the protein is active after purification.

Protease Produced by Endophytic Fungi: A Systematic Review.

The purpose of this systematic review was to identify the available literature of production, purification, and characterization of proteases by endophytic fungi. There are few complete studies that entirely exhibit the production, characterization, and purification of proteases from endophytic fungi. This study followed the PRISMA, and the search was conducted on five databases: PubMed, PMC, Science Direct, Scopus Articles, and Web of Science up until 18 May 2021, with no time or language restrictions. The methodology of the selected studies was evaluated using GRADE. Protease production, optimization, purification, and characterization were the main evaluated outcomes. Of the 5540 initially gathered studies, 15 met the inclusion criteria after a two-step selection process. Only two studies optimized the protease production using statistical design and two reported enzyme purification and characterization. The genus Penicillium and Aspergillus were the most cited among the eleven different genera of endophytic fungi evaluated in the selected articles. Six studies proved the ability of some endophytic fungi to produce fibrinolytic proteases, demonstrating that endophytic fungi can be exploited for the further production of agents used in thrombolytic therapy. However, further characterization and physicochemical studies are required to evaluate the real potential of endophytic fungi as sources of industrial enzymes.

Production, Optimization, Purification, Kinetic analysis and applications of alkaline proteases produced from Bacillus subtilis through solid state fermentation of agricultural byproducts

Proteases have gained more commercial value to date due to multiple applications in different industrial sectors. Current research was aimed to use the cheaper agricultural waste for optimal protease production. Maximum level of protease production was achieved at 37 °C, incubation period of 24 h, pH 9.0, inoculum size 3%, 1.5 g sucrose as a carbon source and 30% moisture content by using solid-state fermentation. Among the various inorganic and organic nitrogen sources, ammonium nitrate and yeast extract tremendously increased the production of protease. Among metal ions and surfactants tested, Ca2+ and Tween 40 showed the optimal protease production. The purification of protease was carried out by ammonium salt precipitation followed by sephadex G-100 gel filtration chromatography. The purification resulted in 1.3 fold of purified protease with a specific activity of 51.5 and a total yield of 37.5 %. Molecular weight of purified protease was predicted upon SDS-PAGE with a single band of ~36 kDa. The protease was quite stable over a temperature range of 35-45 °C and pH 7-9 with maximal activity at 40 °C and pH 9. The kinetic parameters Vmax (maximum rate) and Km (Michaelis-Menten constant) were calculated as 0.307 U/g and 11.2 mg/mL, respectively. The alkaline protease significantly de-hair the goat skin and successfully removed the animal blood stain from cotton cloth.

Extraction, partial purification and characterization of proteases from the red seaweed Gracilaria edulis with similar cleavage sites on κ-casein as calf rennet.

Enzymes currently used in cheesemaking have various drawbacks, and there is a continual need to find new coagulants. This study describes the extraction and biochemical characterization of two proteases from the red alga Gracilaria edulis. The proteases were extracted with phosphate buffer and partially purified by ammonium sulphate precipitation and dialysis. The enzymes exhibited optimum caseinolytic activity at 60°C and a pH range of 6-8. They showed a high ratio of milk-clotting over caseinolytic activity, indicating they had an excellent milk-clotting ability. The proteases were confirmed to be serine protease and metalloprotease with molecular weight (MW) of 44 and 108kDa. They exhibited high hydrolytic activity on κ-caseins, cleaving κ-casein at four main sites, one of which being the same as that of calf rennet, which is the first reported for an algal protease. The findings demonstrated that the proteases could potentially be used as a milk coagulant in cheesemaking.

Reduce, Reuse and Recycle in Protein Chromatography: Development of an Affinity Adsorbent from Waste Paper and Its Application for the Purification of Proteases from Fish By-Products.

In the present study, we report the development of a cellulose-based affinity adsorbent and its application for the purification of proteases from fish by-products. The affinity adsorbent was synthesized using cellulose microfibers as the matrix, isolated from recycled newspapers using the acid precipitation method. As an affinity ligand, the triazine dye Cibacron Blue 3GA (CB3GA) was used and immobilized directly onto the cellulose microfibers. Absorption equilibrium studies and frontal affinity chromatography were employed to evaluate the chromatographic performance of the adsorbent using as model proteins bovine serum albumin (BSA) and lysozyme (LYS). Absorption equilibrium studies suggest that the adsorption of both proteins obeys the Langmuir isotherm model. The kinetics of adsorption obey the pseudo-second-order model. The affinity adsorbent was applied for the development of a purification procedure for proteases from Sparus aurata by-products (stomach and pancreas). A single-step purification protocol for trypsin and chymotrypsin was developed and optimized. The protocol afforded enzymes with high yields suitable for technical and industrial purposes.

Facile Identification and Isolation of Protease Using SDS-PAGE and Zymography

Purification and identification of novel proteases is critical in the development of new therapeutic drugs, such as those for ischemic stroke. However, it's a time-consuming and expensive job to purify enzymes from natural sources. Furthermore, even if the process is completed, the enzyme could be a known protease. Zymography is a protease-confirming assay, but its molecular weight is hard to be accurately measured due to background noise and the broad zone of lysis. Moreover, a sample in a zymography gel cannot be used for protein identification because of the staining substrate in the gel. However, a protease in an unstained SDS-PAGE gel is not damaged by staining buffer and its proteolytic potential will be maintained and be identified, even though these bands are invisible. In this research, to take advantage of both zymography and unstained SDS-PAGE, each band in an unstained SDS-PAGE gel was cut and transferred to a zymography gel. Through this method, bands on the SDS-PAGE gel and the accompanying electroblotted membrane could be used to confirm the target proteolytic activity. Screened samples could be used for protease identification and to check for novelty.

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.