Molecular characterization and growth optimization of halo-tolerant protease producing <Emphasis Type="Italic">Bacillus Subtilis</Emphasis> Strain BLK-1.5 isolated from salt mines of Karak,Pakistan

Microbial proteolytic enzyme is one of the most important industrial enzymes that hydrolyze proteins. The applications of proteases under harsh industrial conditions like alkalinity, salinity, and temperature make them inactive and unstable. This suggests need for search for novel microbial sources for protease production having diverse properties. For this purpose, 54 bacterial strains were isolated from different salt mines of Karak, Pakistan and were investigated for their proteolytic activity on skim milk agar plates. The strain which showed maximum protease activity was characterized by 16S rRNA gene sequence analysis. Furthermore, growth and protease production was optimized for the characterized bacteria under different physical factors, i.e., pH, temperature and salinity. The isolate BLK-1.5 exhibited strong protease production and was identified as Bacillus subtilis based on biochemical characteristics and 16S rRNA gene sequence analysis. Maximum production of protease was recorded at pH 10, 37 °C and 7 % (w/v) NaCl. Molecular weight of proteases was estimated 38 kDa and its optimum activity was observed at pH 10, 50 °C and 2 % (w/v) NaCl. In conclusion, the protease produced by halo-tolerant Bacillus subtilis strain BLK-1.5 has diverse characteristics and could be useful in various industrial applications.     

A thermostable collagenolytic protease with a very large molecular mass produced by thermophilic Bacillus sp. strain MO-1

A collagenolytic protease was purified to homogeneity from thermophilic Bacillus sp. strain MO-1. The protease from strain MO-1 showed high activity toward type I and IV collagens and gelatin. However, peptide substrates (4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-Arg and 2-furylacryloyl-Leu-Gly-Pro-Ala) for collagenases were inert as substrates. The collagenolytic protease cleaved oxidized insulin B-chain at 11 sites and degraded type I and IV collagens into anonymous small pieces, suggesting that the protease digests collagens at multiple sites. The collagenolytic protease was far more thermostable than a mesophilic Clostridium histolyticum collagenase. The collagenolytic protease possesses two salient features: (1) it has a very large molecular mass, 210 kDa, and consists of two, identical 105-kDa subunits; (2) it belongs to a serine protease group. The high molecular mass is unique among serine proteases but common for collagenases. The features of the enzyme from strain MO-1 suggest that it is a new collagenolytic protease which is distinct from previously reported collagenases and serine proteases.     

Purification and characterization of manganese-dependent alkaline serine protease from Bacillus pumilus TMS55

The purification and characterization of a Mn2+-dependent alkaline serine protease produced by Bacillus pumilus TMS55 were investigated. The enzyme was purified in three steps: concentrating the crude enzyme using ammonium sulfate precipitation, followed by gel filtration and cation-exchange chromatography. The purified protease had a molecular mass of approximately 35 kDa, was highly active over a broad pH range of 7.0 to 12.0, and remained stable over a pH range of 7.5 to 11.5. The optimum temperature for the enzyme activity was found to be 60 degreesC. PMSF and AEBSF (1 mM) significantly inhibited the protease activity, indicating that the protease is a serine protease. Mn2+ ions enhanced the activity and stability of the enzyme. In addition, the purified protease remained stable with oxidants (H2O2, 2%) and organic solvents (25%), such as benzene, hexane, and toluene. Therefore, these characteristics of the protease and its dehairing ability indicate its potential for a wide range of commercial 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 and characterization of two extracellular alkaline proteases from a newly isolated obligate alkalophilic Bacillus sphaericus

Two novel extracellular serine proteases were purified to homogeneity from the cell-free culture filtrate of an obligate alkalophilic Bacillus sphaericus by a combination of ultrafiltration, ammonium sulfate precipitation and chromatographic methods. The enzymes showed similar substrate specificities, but differed in hydrophobicity and molecular mass. Protease A was a monomeric protease with a relative molecular mass (M _r) of 28.7 kDa, whereas protease B, with a M _r of 68.0 kDa, apparently consisted of smaller subunits. The purified protease A had a specific activity on hemoglobin of 5.1 U/mg protein compared to 40.9 U/mg protein in the case of protease B. Both proteases were most active on SAAPF-pNa, a substrate for chymotrypsin-like serine proteases. However, the K _m values of these two proteases on SAAPF-pNa were higher than that for α-chymotrypsin, indicating a lower affinity of proteases A and B for this substrate compared to chymotrypsin. Unlike other Bacillus serine proteases, neither protease A nor B stained with Coomasie blue R-250, even with loading of a large amount of protein, and they stained poorly with the silver staining method. However, NH₂-terminal amino acid sequencing of protease B revealed a high similarity with subtilisin Carlsberg (67% homology). Almost total inhibition of both proteases by PMSF, but very little/no inhibition by trypsin and chymotrypsin inhibitors (TPCK and TLCK) or thiol reagents (PCMB and iodoacetic acid), further supported the view that the enzyme belonged to the serine protease family. Journal of Industrial Microbiology & Biotechnology (2001) 26, 387–393. Received 05 November 2000/ Accepted in revised form 23 April 2001     

Purification and characterization of an extracellular protease from Penicillium chrysogenum Pg222 active against meat proteins

An extracellular protease from Penicillium chrysogenum (Pg222) isolated from dry-cured ham has been purified. The purification procedure involved several steps: ammonium sulfate precipitation, ion-exchange chromatography, filtration, and separation by high-performance liquid chromatography. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and gel filtration, the purified fraction showed a molecular mass of about 35 kDa. The hydrolytic properties of the purified enzyme (EPg222) on extracted pork myofibrillar proteins under several conditions were evaluated by SDS-PAGE. EPg222 showed activity in the range of 10 to 60 degrees C in temperature, 0 to 3 M NaCl, and pH 5 to 7, with maximum activity at pH 6, 45 degrees C, and 0.25 M NaCl. Under these conditions the enzyme was most active against tropomyosin, actin, and myosin. EPg222 showed collagenolytic activity but did not hydrolyze myoglobin. EPg222 showed higher activity than other proteolytic enzymes like papain, trypsin, and Aspergillus oryzae protease. The N-terminal amino acid sequence was determined and was found to be Glu-Asn-Pro-Leu-Gln-Pro-Asn-Ala-Pro-Ser-Trp. This partial amino acid sequence revealed a 55% homology with serine proteases from Penicillium citrinum. The activity of this novel protease may be of interest in ripening and generating the flavor of dry-cured meat products.     

Purification and partial characterisation of a novel trypsin-like cysteine protease from Metarhizium anisopliae

Abstract Trypsin-like enzymes from the entomopathogenic fungus Metarhizium anisopliae have been characterised. Two proteases with tryptic activity were purified by narrow range isoelectric focussing and affinity chromatography. One of these proteases, with an isoelectric point of 5.4 and a molecular mass of 28.8 kDa is a 'classical' trypsin belonging to the serine protease class. The other protease, with an isoelectric point of 4.6 and a molecular mass of 26.7 kDa, demonstrates trypsin-like specificity but, on the basis of inhibition and activation studies, belongs to the cysteine protease family and as such is the first fungal protease to be found of this type. The amino acid composition, kinetic constants and activity against proteinaceous substrates, including locust cuticle have been determined.     

Purification and characterization of an alkaline serine-protease produced by a new isolated Aspergillus clavatus ES1

An extracellular bleach stable protease from the fungus Aspergillus clavatus ES1, isolated from wastewater, was purified and characterized. The protease of ES1 strain was purified to homogeneity using acetone precipitation, Sephadex G-100 gel filtration and CM-Sepharose ion exchange chromatography, with a 7.5-fold increase in specific activity and 29% recovery. The molecular mass was estimated to be 32 kDa on SDS-PAGE. The optimum pH and temperature for the proteolytic activity were pH 8.5 and 50 °C, respectively. The enzyme was stable in the pH range of 7.0–9.0. The protease was activated by divalent cations such as Ca²⁺ and Mg²⁺.The alkaline protease showed extreme stability towards non-ionic surfactants (5% Tween 80 and 5% Triton X-100). In addition, the enzyme was relatively stable towards oxidizing agents, retaining more than 71 and 53% of its initial activity after 1 h incubation in the presence of 1 and 2% (w/v) sodium perborate, respectively.The N-terminal sequence of the first 15 amino acids of the purified alkaline protease of A. clavatus ES1 showed high similarity with other fungal alkaline proteases. The activity was totally lost in the presence of PMSF, suggesting that the purified enzyme is a serine-protease.     

Purification and biochemical characterization of a novel cysteine protease of Entamoeba histolytica.

Cysteine proteases are important virulence factors of Entamoeba histolytica, the causative agent of amoebiasis. A novel cysteine protease from parasite extracts was purified 15-fold by a procedure including concanavalin A-Sepharose, hydroxylapatite and DEAE-Sepharose chromatography. The purification resulted in the obtainment of an homogeneous protein with a molecular mass of 66 kDa on native PAGE. In 10% SDS/PAGE, three bands of 60, 54 and 50 kDa were evident. Each of the three specific mouse antisera raised against these proteins showed cross-reactivity with the three bands obtained from the purified eluate. The N-terminal sequencing of the first 10 amino acids from the three proteins showed 100% identity. These results support the hypothesis of a common precursor for the 60, 54 and 50-kDa proteins. Protease activity of the purified enzyme was demonstrated by electrophoresis in a gelatine-acrylamide copolymerized gel. Its activity was quantified by cleaving a synthetic fluorogenic peptide substrate such as N-carbobenzyloxy-arginyl-arginyl-7-amido-4-methylcoumarin. The optimum pH for the protease activity was 6.5; however, enzymatic activity was observed between pH 5 and pH 7.5. Typical of cysteine proteases, the enzyme was inhibited by 4-[(2S, 3S)-carboxyoxiran-2-ylcarbonyl-L-leucylamido]butylg uanidine and iodoacetamide, and activated by free sulfhydryl groups. The cellular location of the enzyme was examined on trophozoites before and after contact with red blood cells using indirect immunofluorescence and cellular fractionation. The 60-kDa cysteine protease translocated to the amoebic surface upon the interaction of trophozoites with red blood cells. This result provided evidence for participation of the 60-kDa protease in erythrophagocytosis.     

A neutral protease from Bacillus nematocida, another potential virulence factor in the infection against nematodes

A neutral protease (npr) (designated Bae16) toxic to nematodes was purified to homogeneity from the strain Bacillus nematocida. The purified protease showed a molecular mass of approximately 40 kDa and displayed optimal activity at 55°C, pH 6.5. Bioassay experiments demonstrated that this purified protease could destroy the nematode cuticle and its hydrolytic substrates included gelatin and collagen. The gene encoding Bae16 was cloned, and the deduced amino acid sequence showed 94% sequence identity with npr gene from B. amyloliquefaciens, but had low similarity (13–43%) with the previously reported virulence serine proteases from fungi or bacteria, which reflected their differences. Recombinant mature Bae16 (rm-Bae16) was expressed in Escherichia coli BL21 using pET30 vector system, and its nematicidal activity confirmed that Bae16 could be involved in the infection process. Our present study revealed that the npr besides the known alkaline serine protease could serve as a potential virulence factor in the infection against nematodes, furthermore, the two proteases with different characteristics produced by the same strain co-ordinated efforts to kill nematodes. These data helped to understand the interaction between this bacterial pathogen and its host.Qiuhong Niu, Xiaowei Huang have contributed equally to this work.     

Production, purification and characterization of thermophilic lipase from Bacillus sp. THL027

A low-cost medium, MGRS, has been developed for growth and lipase production from Bacillus THL027 at 65 degrees C and pH 7.0. MGRS was composed of 2% (v/v) buffer solution (7.3% (w/v) Na(2)HPO(4), 3.2% (w/v) KH(2)PO(4), pH 7.2), 40 microg ml(-1) FeSO(4) and 40 microg ml(-1) MgSO(4), 0.1% (w/v) (NH(4))(2)SO(4) supplemented with 3% NaCl, 0.1% glucose, 1.0% rice bran oil and 0.5% (w/v) rice bran. The lipase was purified 2.6-fold to apparent homogeneity by ultrafiltration and gel filtration chromatography. Its molecular mass was 69 kDa. The purified enzyme was characterized for its general physical properties.     

Purification and characterization of a 33 kDa serine protease from Acanthamoeba lugdunensis KA/E2 isolated from a Korean keratitis patient

In order to evaluate the possible roles of secretory proteases in the pathogenesis of amoebic keratitis, we purified and characterized a serine protease secreted by Acanthamoeba lugdunensis KA/E2, isolated from a Korean keratitis patient. The ammonium sulfate-precipitated culture supernatant of the isolate was purified by sequential chromatography on CM-Sepharose, Sephacryl S-200, and mono Q-anion exchange column. The purified 33 kDa protease had a pH optimum of 8.5 and a temperature optimum of 55 degrees C. Phenylmethylsulfonylfluoride and 4-(2- Aminoethyl)-benzenesulfonyl-fluoride, both serine protease specific inhibitors, inhibited almost completely the activity of the 33 kDa protease whereas other classes of inhibitors did not affect its activity. The 33 kDa enzyme degraded various extracellular matrix proteins and serum proteins. Our results strongly suggest that the 33 kDa serine protease secreted from this keratopathogenic Acanthamoeba play important roles in the pathogenesis of amoebic keratitis, such as in corneal tissue invasion, immune evasion and nutrient uptake.     

A novel nonionic surfactant- and solvent-stable alkaline serine protease from <Emphasis Type="Italic">Serratia</Emphasis> sp. SYBC H with duckweed as nitrogen source: production,purification, characteristics and application

A novel nonionic surfactant- and hydrophilic solvent-stable alkaline serine protease was purified from the culture supernatant of Serratia sp. SYBC H with duckweed as nitrogen source. The molecular mass of the purified protease is about 59 kDa as assayed via SDS-PAGE. The protease is highly active over the pH range between 5.0 and 11.0, with the maximum activity at pH 8.0. It is also fairly active over the temperature range between 30 and 80°C, with the maximum activity at 40°C. The protease activity was substantially stimulated by Mn²⁺ and Na⁺ (5 mM), up to 837.9 and 134.5% at 40°C, respectively. In addition, Mn²⁺ enhanced the thermostability of the protease significantly at 60°C. Over 90% of its initial activity remained even after incubating for 60 min at 40°C in 50% (v/v) hydrophilic organic solvents such as DMF, DMSO, acetone and MeOH. The protease retained 81.7, 83.6 and 76.2% of its initial activity in the presence of nonionic surfactants 20% (v/v) Tween 80, 25% (v/v) glycerol and Triton X-100, respectively. The protease is strongly inhibited by PMSF, suggesting that it is a serine protease. Washing experiments revealed that the protease has an excellent ability to remove blood stains.     

Purification and Characterization of an Extracellular Protease from Penicillium chrysogenum Pg222 Active against Meat Proteins

An extracellular protease from Penicillium chrysogenum (Pg222) isolated from dry-cured ham has been purified. The purification procedure involved several steps: ammonium sulfate precipitation, ion-exchange chromatography, filtration, and separation by high-performance liquid chromatography. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and gel filtration, the purified fraction showed a molecular mass of about 35 kDa. The hydrolytic properties of the purified enzyme (EPg222) on extracted pork myofibrillar proteins under several conditions were evaluated by SDS-PAGE. EPg222 showed activity in the range of 10 to 60°C in temperature, 0 to 3 M NaCl, and pH 5 to 7, with maximum activity at pH 6, 45°C, and 0.25 M NaCl. Under these conditions the enzyme was most active against tropomyosin, actin, and myosin. EPg222 showed collagenolytic activity but did not hydrolyze myoglobin. EPg222 showed higher activity than other proteolytic enzymes like papain, trypsin, and Aspergillus oryzae protease. The N-terminal amino acid sequence was determined and was found to be Glu-Asn-Pro-Leu-Gln-Pro-Asn-Ala-Pro-Ser-Trp. This partial amino acid sequence revealed a 55% homology with serine proteases from Penicillium citrinum. The activity of this novel protease may be of interest in ripening and generating the flavor of dry-cured meat products.     

Isolation and characterization of novel inducible serine protease inhibitors from larval hemolymph of the greater wax moth Galleria mellonella.

Three inducible serine protease inhibitors (ISPI-1, 2, 3) have been purified from larval hemolymph of greater wax moth larvae, Galleria mellonella, and characterized at a molecular level. These inhibitors were synthesized after larvae were injected with a yeast polysaccharide, zymosan preparation. ISPI-1,2,3 were active against various serine proteases including trypsin and toxic proteases released by the entomopathogenic fungus Metarhizium anisopliae. Precipitation by trichloroacetic acid and heat, followed by FPLC and HPLC separation steps were used for purification of the protease inhibitors from cell-free hemolymph samples. The molecular masses of purified proteins were determined by MS to be 9.2 kDa (ISPI-1), 6.3 kDa (ISPI-2) and 8.2 kDa (ISPI-3) with isoelectric points ranging between 7.2 and 8.3. The N-terminal amino-acid sequences of ISPI-1 and ISPI-3 are not similar to other known proteins, whereas that of ISPI-2 exhibits extensive similarity to known Kunitz-type protease inhibitors.     

A novel alkaline protease from wild edible mushroom Termitomyces albuminosus

A protease with a molecular mass of 30 kDa and the N-terminal sequence of GLQTNAPWGLARSS, was isolated from fresh fruiting bodies of the wild edible mushroom Termitomyces albuminosus. The purification protocol included ion exchange chromatography on DEAE-cellulose, Q-Sepharose, SP-Sepharose and FPLC-gel filtration on Superdex 75. The protein was unadsorbed on DEAE-cellulose and Q-Sepharose, but adsorbed on SP-Sepharose. The optimal pH and temperature of the purified enzyme were 10.6 and 60 °C, respectively. The enzyme was stable in the presence of 2 % (v/v) Tween 80 and 4 M urea. More than 80 % of the enzyme activity was retained in 2 % (v/v) Triton X 100, 54 % in 10 mM EDTA and 31 % in 2 % (w/v) SDS. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), but not inhibited by dithiothreitol (DTT), pepstatin or lima bean trypsin inhibitor suggesting that it was a serine protease but not a trypsin-like one. The protease was inhibited by Hg(2+), Cu(2+), and Fe(3+) ions. The K(m) and V(max) values of the purified enzyme for casein were 8.26 mg ? ml(-1) and 0.668 mg ? ml(-1) ? min(-1), respectively.     

Purification and characterization of a thermostable, haloalkaliphilic extracellular serine protease from the extreme halophilic archaeon Halogeometricum borinquense strain TSS101

A novel haloalkaliphilic, thermostable serine protease was purified from the extreme halophilic archaeon, Halogeometricum borinquense strain TSS101. The protease was isolated from a stationary phase culture, purified 116-fold with 18% yield and characterized biochemically. The molecular mass of the purified enzyme was estimated to be 86 kDa. The enzyme showed the highest activity at 60 degrees C and pH 10.0 in 20% NaCl. The enzyme had high activity over the pH range from 6.0 to 10.0. Enzymatic activity was strongly inhibited by 1 mM phenyl methylsulfonyl fluoride, but activity was increased 59% by 0.1% cetyltrimethylammonium bromide. The enzyme exhibited relatively high thermal stability, retaining 80% of its activity after 1 h at 90 degrees C. Thermostability increased in the presence of Ca2+. The stability of the enzyme was maintained in 10% sucrose and in the absence of NaCl.     

Purification and characterization of organic solvent stable protease from Bacillus licheniformis RSP-09-37

A protease was purified from the cell-free supernatant of Bacillus licheniformis RSP-09-37, a mutant from a thermophilic bacterial strain, B. licheniformis RSP-09, using affinity chromatography with alpha-casein agarose resin. The protease was purified 85-fold to electrophoretic homogeneity. The apparent molecular mass of purified protease was 55 kDa using gel filtration in high-performance liquid chromatography, which is in agreement with the results obtained from sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting a monomeric nature of the protein. The purified protease revealed temperature optima of 50 degrees C and pH optima of 10.0 and was classified as serine protease based on its complete inhibition with phenyl methyl sulfonyl fluoride. The purified protease exhibited tolerance to both detergents and organic solvent. The synthetic activity of the protease was tested using the transesterification reaction between N-acetyl-L-phenylalanine-ethyl ester and n-propanol in organic solvents varying in their log P values and the kinetic parameters of the enzyme in these organic solvents were studied. The enzyme has potential to be employed for synthetic reactions and in detergent formulations.     

Expression, purification, and characterization of a thermophilic neutral protease from Bacillus stearothermophilus in Bacillus subtilis

The gene coding for a thermophilic neutral protease from Bacillus stearothermophilus was expressed in Bacillus subtilis DB104, under the control of the sacB gene promoter. This was followed by either the native signal peptide sequence of this protease or the signal peptide sequence of the sacB gene. The protease was purified 3.8-fold, with a specific activity of 16530 U mg-1. As analyzed by SDS-PAGE, the molecular mass of the expressed protease was about 35 kDa, and the optimal temperature and pH of the protease were 65℃ and 7.5, respectively. Moreover, it still had about 80% activity after 1 h reaction at 65℃.     

Subtilisin like protease secreted in Bacillus pumilus KMM 62 on different growth stages

A protease secreted in Bacillus pumilus KMM 62 culture liquid on different growth stages was isolated using ion-exchange chromatography. On the basis of pattern of specific chromogenic substrates hydrolysis and inhibitory analysis the protease was classified as subtilisin like serine protease. The molecular weight ofprotease is 31 kDa. Proteolytic activity towards Z-Ala-Ala-Leu-pNa substrate was maximal at pH 8-8.5. The optimal temperature for proteolytic activity was observed at a temperature of 30 degrees C, and the protein was stable within the pH range of 7.5-10.0. Bacillus pumilus KMM 62 subtilisin like serine protease was shown to have thrombolytic activity.