Extracellular Alkaline Serine Protease Research Articles

Purification and characterization of a high molecular weight serine protease from Microbacterium paraoxydans sp. SKS10.

Alkaline proteases from microbial sources have been found suitable for diverse industrial applications, with serine proteases being the most common enzymes used in the detergent industry. In the present study, we have purified and characterized an extracellular alkaline serine protease from Microbacterium paraoxydans sp. SKS10. The protease was purified using ammonium sulfate precipitation followed by different chromatography techniques (fold purification 6.919). Km and Vmax for the protease were determined to be 0.183mg/mL and 4.904U/mL, respectively. This enzyme is a thermostable high molecular weight (∼109.4kDa) protease which has maximal activity at 60°C, and above pH 10. Inhibitor assays revealed the enzyme to be a serine protease whose activity increased by 2.5-fold in the presence of EDTA. This enzyme remained active in the presence of various metal salts and organic solvents and was compatible with commercially available laundry detergents highlighting its potential for use in the detergent industry.

Characterization of the Proteolytic Activity of a Halophilic Aspergillus reticulatus Strain SK1-1 Isolated from a Solar Saltern.

Salterns are hypersaline environments that are inhabited by diverse halophilic microorganisms, including fungi. In this study, we isolated a fungal strain SK1-1 from a saltern in the Republic of Korea, which was identified as Asperillus reticulatus. This is the first reported saline-environment-derived A. reticulatus that belongs to the Aspergillus penicillioides clade and encompasses xerophilic fungi. SK1-1 was halophilic, obligately requiring NaCl for growth, with a maximum radial growth of 6%–9% (w/v) NaCl. To facilitate the biotechnological application of halophilic fungi, we screened the SK1-1 strain for proteolytic activity. Proteases have widespread applications in food processing, detergents, textiles, and waste treatment, and halophilic proteases can enable protein degradation in high salt environments. We assessed the proteolytic activity of the extracellular crude enzyme of SK1-1 using azocasein as a substrate. The crude protease exhibited maximum activity at 40–50 °C, pH 9.5–10.5, and in the absence of NaCl. It was also able to retain up to 69% of its maximum activity until 7% NaCl. Protease inhibitor assays showed complete inhibition of the proteolytic activity of crude enzymes by Pefabloc® SC. Our data suggest that the halophilic A. reticulatus strain SK1-1 produces an extracellular alkaline serine protease.

Production of Extracellular Alkaline Serine Protease from Pediococcus acidilactici NCDC 252: Isolation, Purification, Physicochemical and Catalytic Characterization

Proteases are the most important industrial enzymes capable of hydrolyzing proteins into small peptides and amino acids and used commercially in food, textile and pharmaceutical industries. An alkaline serine protease from Pediococcus acidilactici NCDC 252 was purified using a simple two step purification procedure i.e. ammonium sulphate precipitation and gel filtration chromatography with purification fold of 5.9, a yield and specific activity of 11.8% and 23.3 U mg/ml respectively. Purified enzyme worked optimally at pH 8.5 and 37 °C. The enzyme was stable in the pH range of 6.5–9.5 and up to 50 °C. The Km for BAPNA was 38 µM and Vmax was 10.6 U/mg. Purified enzyme exhibited highest hydrolyzing activity for BAPNA. Inhibition of purified enzyme by soybean trypsin inhibitor, benzamide and β-ME suggested the enzyme to be alkaline serine protease with involvement of cysteine residues either in binding or regulation. In-silico characterization of alkaline serine protease revealed the presence of Ser, His and Asp residues at its catalytic site. This shows the resemblance of alkaline protease under study with other studied serine proteases.

Purification and biochemical characterization of a novel thermostable serine alkaline protease from Aeribacillus pallidus C10: a potential additive for detergents

An extracellular thermostable alkaline serine protease enzyme from Aeribacillus pallidus C10 (GenBank No: KC333049), was purified 4.85 and 17. 32-fold with a yield of 26.9 and 19.56%, respectively, through DE52 anion exchange and Probond affinity chromatography. The molecular mass of the enzyme was determined through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with approximately 38.35 kDa. The enzyme exhibited optimum activity at pH 9 and at temperature 60 °C. It was determined that the enzyme had remained stable at the range of pH 7.0–10.0, and that it had preserved more than 80% of its activity at a broad temperature range (20–80 °C). The enzyme activity was found to retain more than 70% and 55% in the presence of organic solvents and commercial detergents, respectively. In addition, it was observed that the enzyme activity had increased in the presence of 5% SDS. KM and Vmax values were calculated as 0.197 mg/mL and 7.29 μmol.mL−1.min−1, respectively.

Cloning, heterologous expression and structural characterization of an alkaline serine protease from sea water haloalkaliphilic bacterium

Cloning and functional attributes of a serine protease gene from haloalkaliphilic bacteria are described. The protease gene of ∼1,600 bp amplified from the genomic DNA was cloned into TA vector followed by the sub-cloning into pUC19 for expression. Growth of the organism and gene expression was studied at 30 and 37 °C in the presence of 0.5–2.0 mM IPTG. Sequencing of the gene and homology search of the sequence revealed that the gene encoded an extracellular alkaline serine protease belonging to superfamily subtilisin-like hydrolases. The amino acid sequence alignment resulted from the BLAST search of the subtilisin exhibited high sequence homology with the Bacillus subtilis ssp. subtilis strain 168 and subtilisins of other Bacillus sp., B. subtilis and B. mojavensis. The deduced amino acid sequence exhibited a mature protease of a 419 amino acid, single-chained monomeric peptide with the large number of the positively charged amino acids suggesting its hydrophilic nature.

Purification and characterization of a novel alkaline serine protease secreted by Vibrio metschnikovii

A novel extracellular alkaline serine protease secreted by Vibrio metschnikovii (V. metschnikovii) ATCC700040 cells was purified by three chromatographic steps and characterized in terms of enzymatic kinetics and substrate specificity. The purified enzyme (named AKP-Vm) was composed of a single polypeptide with an apparent molecular weight of 50 kDa on 12% SDS-polyacrylamide gel in the presence of CuCl₂. The optimal temperature and the pH for the enzyme were found to be 37˚C and 9.5, respectively. However, the enzyme activity was inhibited by inhibitors such as PMSF and aprotinin. AKP-Vm could hydrolyze a peptide bond at the carboxyl side of the arginine residue, as revealed by its amidolytic activity toward a chromogenic substrate, Boc-Val-Pro-Arg-pNA. The kinetic parameters of the enzyme were as follows: KM=0.91 mM, kcat=0.8 sec⁻¹ and kcat/KM=0.88 mM⁻¹sec⁻¹. AKP-Vm protease could cleave various blood coagulation-associated proteins, including fibrinogen, prothrombin and thrombin. In particular, the enzyme showed powerful fibrinogenolytic and fibrinolytic activities, as it could cleave all major chains of fibrinogen and also digest cross-linked fibrin. The results obtained suggest that AKP-Vm is a novel alkaline serine protease that can actively cleave fibrinogen and cross-linked fibrin.

Characterization of an extracellular alkaline serine protease from marine Engyodontium album BTMFS10

An alkaline protease from marine Engyodontium album was characterized for its physicochemical properties towards evaluation of its suitability for potential industrial applications. Molecular mass of the enzyme by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) analysis was calculated as 28.6 kDa. Isoelectric focusing yielded pI of 3-4. Enzyme inhibition by phenylmethylsulfonyl fluoride (PMSF) and aprotinin confirmed the serine protease nature of the enzyme. K (m), V (max), and K (cat) of the enzyme were 4.727 x 10⁻² mg/ml, 394.68 U, and 4.2175 x 10⁻² s⁻¹, respectively. Enzyme was noted to be active over a broad range of pH (6-12) and temperature (15-65 °C), with maximum activity at pH 11 and 60 °C. CaCl₂ (1 mM), starch (1%), and sucrose (1%) imparted thermal stability at 65 °C. Hg²⁺, Cu²⁺, Fe³⁺, Zn²⁺, Cd⁺, and Al³⁺ inhibited enzyme activity, while 1 mM Co²⁺ enhanced enzyme activity. Reducing agents enhanced enzyme activity at lower concentrations. The enzyme showed considerable storage stability, and retained its activity in the presence of hydrocarbons, natural oils, surfactants, and most of the organic solvents tested. Results indicate that the marine protease holds potential for use in the detergent industry and for varied applications.

Preliminary studies on partial reduction of Colletotrichum acutatum infection by proteinase inhibitors extracted from apple skin

Colletotrichum acutatum, a fungal pathogen that causes soft rot in fruit, produced protease when grown on casein or apple cell wall, as revealed by a clear zone around the well filled with C. acutatum medium in a radial diffusion assay. A protease-inhibiting protein (PI) was also extracted from healthy stored apple, cultivar Cripps Pink, and its activity was tested in vitro and in vivo against a protease produced by C. acutatum. In in vitro trials the inhibition rate determined by radial diffusion assay was over 41% after 24 h, while in inoculated fruit the inhibition ranged from 23.5% to 45% after 5 days at 20 °C. The protease inhibitor extracted from healthy apple skin was a heat-denaturable protein since the halo produced by protein extracted from C. acutatum and added to boiled protein extracted from healthy apple skin tissue was 338.7 mm 2, significantly higher than the halo produced by protein extracted from C. acutatum diluted with fresh protein extracted from healthy tissue (220.7 mm 2). Protein secreted by C. acutatum grown in induced buffer media was tested by polyacrylamide gel electrophoresis. SDS-PAGE of crude enzyme extract revealed the presence of various protein bands that could be ascribed by their molecular mass as putative aspartic proteinase, extracellular alkaline proteinase and serine protease. Genomic analyses are, however, in progress to identify the proteins involved in Colletotrichum patogenicity. More investigations are required to identify the nature of the substance responsible for C. acutatum inhibition in apple and to evaluate the possibility of manipulating the protease inhibitor levels in fruit to reduce soft rot caused by C. acutatum.

Purification and characterization of the cold-active alkaline protease from marine cold-adaptive Penicillium chrysogenum FS010

An extracellular cold-active alkaline serine protease from Penicillium chrysogenum FS010 has been purified. The purification procedure involved: ammonium sulfate precipitation, DEAE ion-exchange chromatography and sephadex G-100 gel chromatography. SDS-PAGE of the purified enzyme indicated a molecular weight of 41,000 +/- 1,000 Da. The protease is stable in a pH range of 7.0-9.0 and has a maximum activity at pH 9.0. Compared with other industrial proteases, the enzyme shows a high hydrolytic activities at lower temperatures and a high sensitivity at a temperature over 50 degrees C. The isoelectric point of the enzyme is approximate to 6.0. Enzymatic activity is enhanced by the addition of divalent cations such as Mg(2+) and Ca(2+) and inhibited by addition of Cu(2+)and Co(2+). PMSF and DFP are its specific inhibitors. The application of the cold-active alkaline protease is extremely extensive, and widely used in detergents, feed, food, leather and many other industries.

Roles of LuxR in regulating extracellular alkaline serine protease A, extracellular polysaccharide and mobility ofVibrio alginolyticus

In marine Vibrio species, the Vibrio harveyi-type LuxR protein, a key player in a quorum-sensing system, controls the expression of various genes. In this study, the luxR homologue in Vibrio alginolyticus was identified and named luxR(val), whose expression was greatly induced by the increase of cell number. The luxR(val) in-frame deletion mutant showed a significant downregulation of total extracellular protease activity, and especially caused a 70% decrease in the transcript levels of extracellular alkaline serine protease A (proA), which was an important virulent factor of V. alginolyticus. Complementation in trans with luxR(val) could restore the expression of proA to the level of the wild-type strain. Deletion of the luxR(val) gene also resulted in changes of colony morphology, extracellular polysaccharide production and mobility. Therefore, another member of the V. harveyi-type LuxR regulator family has been characterized in V. alginolyticus.

Purification and properties of an alkaline protease of Aspergillus clavatus

An extracellular alkaline serine protease has been purified from a strain of Aspergillus clavatus, to apparent homogeneity, by ammonium sulfate precipitation and chromatography on Sephadex G-75. Its molar mass, estimated by SDS-PAGE, was 35 kDa. Maximum protease activity was observed at pH 9.5 and 40°C. The enzyme was active between pH 6.0 and 11.0 and was found to be unstable up to 50°C. Calcium at 5 mM increased its thermal stability. The protease was strongly inhibited by PMSF and chymostatin as well as by SDS, Tween 80 and carbonate ion. Substrate specificity was observed with N-p-Tos-Gly-Pro-Arg-p-nitroanilide and N-Suc-Ala-Ala-Ala-p-nitroanilide being active substates. Parts of the amino acid sequence were up to 81% homologous with those of several fungal alkaline serine proteases.

Purification and characterization of an extracellular serine protease from the nematode-trapping fungus Dactylella shizishanna

To evaluate the production of an extracellular serine protease by Dactylella shizishanna and its potential as a pathogenesis factor. An extracellular alkaline serine protease (Ds1) was purified and characterized from the nematode-trapping fungus D. shizishanna using cation-exchange chromatography and hydrophobic interaction chromatography. The molecular mass of the protease was approximately 35 kDa estimated by SDS-PAGE. The optimum activity of Ds1 was at pH 10 and 55 degrees C (over 30 min). The purified protease could degrade purified cuticle of Penagrellus redivivus and a broad range of protein substrates. The purified protease was highly sensitive to phenylmethyl sulfonyl fluoride (PMSF) (0.1 mmol l(-1)), indicating it belonged to the serine protease family. The N-terminal amino acid residues of Ds1 are AEQTDSTWGL and showed a high homology with Aozl and PII, two serine proteases purified from the nematode-trapping fungus Arthrobotrys oligospora. Nematicidal activity of D. shizishanna was partly related to its ability to produce extracellular serine protease. In this report, we purified a new serine protease from D. shizishanna and provided a good foundation for future research on infection mechanism.

Purification and characterization of extracellular alkaline serine protease from Stenotrophomonas maltophilia strain S-1

The present study was conducted by screening soil bacteria in an attempt to isolate a bacterium that produced extracellular alkaline protease, and for purification and characterization of the protease. Soil bacteria were screened by growth on casein as the sole carbon source. Characterization of a strain isolated from soil of Abashiri, Japan indicated a taxonomic affiliation to Stenotrophomonas maltophilia, and was named S-1 strain. The purified S-1 protease, designed S. maltophilia Protease-1 (SmP-1), exhibited an optimal pH of 12.0, optimal reaction temperature of 50 degrees C and a molecular mass of approximately 40 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The cleavage sites of the oxidized-insulin B chain by SmP-1 were identified as Leu6-Cys7, Cys7-Gly8, Tyr16-Leu17 and Leu17-Val18. The N-terminal amino acid sequence of the purified alkaline protease was determined as NH2-SASAPMVSGVAALVLE. A novel extracellular alkaline serine protease was isolated from S. maltophilia strain S-1. The optimal pH of the proteolytic activity was pH 12.0. The extremely high optimal pH and heat stability of the alkaline serine protease SmP-1 might make it widely applicable to food and other industries.

Purification and characterization of a protease from solid state cultures of Aspergillus parasiticus

An extracellular alkaline serine protease was produced by Aspergillus parasiticus by solid state fermentation. The protease was purified and characterized. A 200-fold purified protease was obtained by acetone precipitation, ion exchange and gel filtration chromatography (FPLC). The molecular weight of the protease was 23 kDa as estimated by SDS-PAGE. This enzyme was stable over a wide range of pH (6–10) and temperature (4–40 °C) and showed maximum activity at pH 8.0 and 40 °C. The enzyme remained active in the presence of oxidizing, reducing agents and also stable in various detergents. Several heavy metals such as Hg +2, Co +2 and Sn +2 had no effect on protease activity. According to the inhibition profiles obtained with the serine protease inhibitor, PMSF, it was confirmed that purified protease belongs to the serine protease family.

Purification and characterization of an extracellular alkaline serine protease with dehairing function from Bacillus pumilus.

An extracellular alkaline serine protease (called DHAP), produced by a Bacillus pumilus strain, demonstrates significant dehairing function. This protease is purified by hydrophobic interaction chromatography, ion exchange, and gel filtration. DHAP had a pI of 9.0 and a molecular weight of approximately 32,000 Dalton. It shows maximal activity at pH 10 and with a temperature of 55 degrees C; the enzyme activity can be completely inhibited by phenylmethylsulfonyl fluoride (PMSF) and diisopropyl fluorophosphates (DFP). The first 20 amino acid residues of the purified DHAP have been determined with a sequence of AQTVPYGIPQIKAPAVHAQG. Alignment of this sequence with other alkaline protease demonstrates its high homology with protease from another B. pumilus strain.

Purification and characterization of an oxidation-stable, thiol-dependent serine alkaline protease from Bacillus mojavensis

An extracellular, thiol-dependent and oxidation-stable alkaline serine protease (molecular mass 30 kDa on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE)) from Bacillus mojavensis was purified to homogeneity with 17-fold purification as unbound fractions using a single step anion exchange chromatography on fast flow Q-sepharose column pre-equilibrated with buffer of pH 10.5. The N-terminal sequence of first 15 amino acids of purified protease showed 91% similarity with other subtilisins and alkaline proteases. The enzyme exhibited pH and temperature optima of 10.5 and 60 °C, respectively, and was stable between a wide pH range of 7.0 and 11.5 for 48 h. The half-lives of protease at 60, 65, and 70 °C were 150, 15 and 7 min, respectively. Various stabilizers and additives stabilized protease for more than 4 h at 60 °C, and 45 min at 65 °C. Specific protease inhibitors, such as phenylmethyl sulfonyl fluoride (PMSF), bestatin, chymostatin, iodoacetic acid, and N-bromosuccinimide completely inhibited the enzyme activity, whereas, the enzyme activity was increased up to more than two-fold in presence of 2-mercaptoethanol, glutathione, and dithiothreitol, suggesting it to be a thiol-dependent serine protease. Among metal ions, Cu 2+ and Mn 2+ ions increased enzyme activity up to 36%. The enzyme was also stable towards several commercially available laboratory bleaches (H 2O 2, sodium perborate), surfactants (tweens, Triton X-100, sodium choleate), and other commercial detergents used in laundries. The protease hydrolyzed several native proteinacious substrates, such as gelatin, elastin, albumin, haemoglobin, and skim milk, thus making it suitable for its use as an effective detergent additive. Wash performance analysis of enzyme revealed that it could effectively remove a variety of stains, such as blood, beetle and grass most effectively at 60 °C.

Purification and characterization of an extracellular alkaline serine protease from Aspergillus terreus (IJIRA 6.2).

An extracellular alkaline serine protease has been purified from Aspergillus terreus (IJIRA 6.2). The purification procedure involved chromatography on DEAE-Sephadex A25, phosphocellulose, hydroxyapatite, casein-Sepharose, gel filtration on Sephacryl-S-300 and by glycerol density gradient centrifugation. The enzyme was further purified to apparent homogeneity through a combination of electrophoresis in polyacrylamide gel containing 0.1% sodium dodecyl sulfate (SDS) with or without protease substrate (gelatin) and subsequent regeneration of its activity in situ by removal of SDS. The active enzyme was visualized in a zymogram or on the basis of protease activity exhibited on an X-ray film. The protein in the unstained segment of the gel was electroeluted. The eluted protein with protease activity exhibited a molecular mass of 37,000-daltons on electrophoresis in SDS-polyacrylamide gel. A sedimentation coefficient of 3.2S was obtained by glycerol density gradient contrifugation. Maximum activity of protease was observed at pH 8.5 and at 37 degrees C. Purified protease was active between pH 5.5 and 9.5 and was found to be stable up to 60 degrees C. With Na-caseinate, the K(m) of the purified protease was found to be 0.055 mM. Antipain, phenylmethane sulfonyl fluoride, and chymostatin served as non-competitive inhibitors. Substrate specificity was determined by using a synthetic chromogenic peptide containing N-P-Tosyl-Gly-Pro-Arg-p-nitroanilide. Results showed that the protease cleaved the peptide on the -COOH end of arginine residue.

Characterization of an alkaline serine protease from an alkaline-resistant Pseudomonas sp.: Cloning and expression of the protease gene in Escherichia coli

A gene, aprP, encoding an extracellular alkaline serine protease from a newly isolated Pseudomonas sp. KFCC 10818 was cloned and characterized. Nucleotide sequence analysis revealed an open reading frame of 1,266 nucleotides which could encode a polypeptide comprised of 422 amino acids. The C-terminal 283 residues showed an overall sequence homology with the subtilisin-type serine proteases. When expressed in E. coli, the alkaline protease, AprP, was released to the culture medium. The purified AprP was most active at pH 11. The k Cat/K m value of this enzyme was 9.2 × 103 S−1mM−1, which is much higher than those of subtilisins.

Cloning and characterization of the gene encoding an extracellular alkaline serine protease from Vibrio metschnikovii strain RH530

The gene vapT, encoding VapT, one of the extracellular sodium dodecyl sulfate (SDS)-resistant alkaline serine proteases (Serp) from the Gram - Vibrio metschnikovii strain RH530 has been cloned in Escherichia coli. The recombinant E. coli produced a protease which co-migrated with VapT on gelatin polyacrylamide gels. The nucleotide (nt) sequence of the cloned vapT revealed a single open reading frame of 1641 bp encoding 547 amino acids (aa) (58 961 Da). Upon analysis of the N-terminal aa sequence, VapT was shown to be processed properly in recombinant E. coli and to consist of 428 aa (45 626 Da). The deduced aa sequence of VapT showed significant sequence homology to subtilisin Carlsberg from Bacillus licheniformis, particularly in the regions containing active site residues and calcium-binding sites. VapT had an intervening region of approx. 149 aa between the His and Ser residues of the active site, as compared with other Serp.

Purification and characterization of alkaline serine protease from an alkalophilic Streptomyces sp.

SAP, an extracellular alkaline serine protease produced by Streptomyces sp. YSA-130, was purified to homogeneity by CM-Sephadex column chromatography and crystallization. The enzyme was a monomeric protein with a molecular weight of 19,000 as estimated by SDS-PAGE and gel filtration. The amino acid composition and amino-terminal sequence of SAP were similar to those of other bacterial serine proteases, i.e., Streptomyces griseus proteases A and B, Lysobacter enzymogenes alpha-lytic protease and Nocardiopsis dassonvillei subsp. prasina OPC-210 alkaline serine protease NDP-I. The optimum temperature and pH for the enzyme activity were 60 degrees C and 11.5. The enzyme was stable up 50 degrees C, and between pHs 4 and 12. The activity was inhibited by Ag+, Hg2+, Co2+, sodium dodecyl sulfate. N-bromosuccinimide, diisopropyl phosphorofluoridate (DFP), 2,3-butanedione, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), iodoacetate, N-ethylmaleimide (NEM), phenylmethanesulfonyl fluoride (PMSF), and phenylglyoxal.