PURIFICATION AND CHARACTERIZATION OF A PROTEINASE FROM THE PROBIOTIC Lactobacillus rhamnosus OXY

A proteinase produced by the human gastrointestinal isolate Lactobacillus rhamnosus strain OXY was identified and characterized. The prtR2 gene coding for proteinase activity was detected in the examined strain. The PCR primers used were constructed on the basis of the sequence of the prtR2 proteinase gene from Lactobacillus rhamnosus GG. The enzyme was purified by fast protein liquid chromatography (FPLC) using CM-Sepharose Fast Flow and Sephacryl S-300 columns. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the enzyme had a relatively low molecular mass of 60 kD. Protease activity was observed at a pH range from 6.5 to 7.5 with optimum k (cat)/K (m) values at pH 7.0 and 40°C. Maximum proteolytic activity (59?U mL(-1)) was achieved after 48?hr of cultivation. The activity of the enzyme was inhibited only by irreversible inhibitors specific for serine proteinases (PMSF and 3,4-dichloro-isocumarine), suggesting that the enzyme was a serine proteinase. Proteinase activity was increased by Ca(2+) and Mg(2+), and inhibited by Cu(2+), Zn(2+), Cd(2+), and Fe(2+.).     

An alkaline protease from Bacillus circulans BM15, newly isolated from a mangrove station: characterization and application in laundry detergent formulations

An investigation on the properties of an alkaline protease secreted by Bacillus circulans BM15 strain isolated from a mangrove sediment sample was carried out in order to characterize the enzyme and to test its potency as a detergent additive. The protease was purified to apparent homogeneity by ammonium sulphate precipitation and was a 30-kDa protease as shown by SDS-PAGE and its proteolytic activity was detected by casein zymography. It had optimum activity at pH 7, was stable at alkaline pH range (7 to 11), had optimum temperature of activity 40°C and was stable up to a temperature of 55°C after incubation for one hour. Hg²⁺, Zn²⁺, Co²⁺, and Cu²⁺completely inhibited the enzyme activity, while Ca²⁺, Mg²⁺, K⁺ and Fe³⁺ were enhancing the same. The serine protease inhibitor PMSF and metal chelator EDTA inhibited the activity of this protease while the classic metalloprotease inhibitor 1, 10 phenanthroline did not show inhibition. The enzyme was stable in SDS, Triton-X-100 and H₂ O₂ as well as in various commercial detergents after incubation for one hour. The extracellular production of the enzyme, the pH and temperature stability and stability in presence of oxidants, surfactants and commercial detergents suggest its possible use as a detergent additive.     

PURIFICATION AND PROPERTIES OF DETERGENT-COMPATIBLE EXTRACELLULAR ALKALINE PROTEASE FROM Scopulariopsis spp.

A fungal alkaline protease of Scopulariopsis spp. was purified to homogeneity with a recovery of 32.2% and 138.1 U/mg specific activity on lectin-agarose column. The apparent molecular mass was 15 ± 1 kD by sodium dodecyl sulfate polyacryalamide gel electrophoresis (SDS-PAGE). It was a homogenous monomeric glycoprotein as shown by a single band and confirmed by native PAGE and gelatin zymography. The enzyme was active and stable over pH range 8.0–12.0 with optimum activity at pH 9.0. The maximum activity was recorded at 50°C and remained unaltered at 50°C for 24 hr. The enzyme was stimulated by Co²⁺ and Mn²⁺ at 10 mM but was unaffected by Ba²⁺, Mg²⁺, Cu²⁺, Na⁺, K⁺, and Fe²⁺. Ca²⁺ and Fe³⁺ moderately reduced the activity (～18%); however, a reduction of about 40% was seen for Zn²⁺ and Hg²⁺. The enzyme activity was completely inhibited by 5 mM phenylmethylsulfonyl fluoride (PMSF) and partially by N-bromosuccinimide (NBS) and tocylchloride methylketone (TLCK). The serine, tryptophan, and histidine may therefore be at or near the active site of the enzyme. The protease was more active against gelatin compared to casein, fibrinogen, egg albumin, and bovine serum albumin (BSA). With casein as substrate, Km and Vmax were 4.3 mg/mL and 15.9 U/mL, respectively. An activation was observed with sodium dodecyl sulfate (SDS), Tween-80, and Triton X-100 at 2% (v/v); however, H₂O₂ and NaClO did not affect the protease activity. Storage stability was better for all the temperatures tested (?20, 4, and 28 ± 2°C) with a retention of more than 85% of initial activity after 40 days. The protease retained more than 50% activity after 24 hr of incubation at 28, 60, and 90°C in the presence (0.7%, w/v) of commercial enzymatic and nonenzymatic detergents. The Super Wheel–enzyme solution was able to completely remove blood staining, differing from the detergent solution alone. The stability at alkaline pH and high temperatures, broad substrate specificity, stability in the presence of surfactants and oxidizing and bleaching agents, and excellent compatibility with detergents clearly suggested the use of the enzyme in detergent formulations.     

Isolation and characterization of β-galactosidase fromLactobacillus crispatus

β-Galactosidase was isolated from the cell-free extracts ofLactobacillus crispatus strain ATCC 33820 and the effects of temperature, pH, sugars and monovalent and divalent cations on the activity of the enzyme were examined.L. crispatus produced the maximum amount of enzyme when grown in MRS medium containing galactose (as carbon source) at 37°C and pH 6.5 for 2 d, addition of glucose repressing enzyme production. Addition of lactose to the growth medium containing galactose inhibited the enzyme synthesis. The enzyme was active between 20 and 60°C and in the pH range of 4–9. However, the optimum enzyme activity was at 45°C and pH 6.5. The enzyme was stable up to 45°C when incubated at various temperatures for 15 min at pH 6.5. When the enzyme was exposed to various pH values at 45°C for 1 h, it retained the original activity over the pH range of 6.0–7.0. Presence of divalent cations, such as Fe²⁺ and Mn²⁺, in the reaction mixture increased enzyme activity, whereas Zn²⁺ was inhibitory. TheK _m was 1.16 mmol/L for 2-nitrophenyl-β-d-galactopyranose and 14.2 mmol/L for lactose.     

Purification and properties of a fibrinogenase from the venom of Naja nigricollis

A fibrinogenolytic proteinase from the venom of Naja nigricollis was purified by chromatography on Bio-Rex 70 and Phenyl-Sepharose. The purified enzyme, designated proteinase F1, was homogeneous by the criterion of SDS-polyacrylamide gel electrophoresis, and consisted of a single chain with a molecular weight of 58 000. Purified proteinase F1 had approximately 15-fold more proteinase activity than the crude venom, based on its ability to inactive α₂-macroglobulin. The enzyme acted on only the Aα-chain of fibrinogen and left the Bβ- and γ-chains intact. The pH optimum for this fibrinogenolytic activity was in the range of pH 8 to 10. In addition to its activity on fibrinogen, proteinase F1 was active on α₂-macroglobulin and fibronectin, but did not degrade casein, hemoglobin or bovine serum albumin. The enzyme was not inhibited by inhibitors of serine proteinases, cysteine proteinases or acid proteinases, but only by the metalloproteinase inhibitor, EDTA. The inhibition by EDTA could be prevented by Zn²⁺, but not by Ca²⁺ or Mg²⁺.     

Characterization,Gene Cloning,and Sequencing of a Fungal Phytase,PhyA, From Penicillium oxalicum PJ3

A phytase from Penicillium oxalicum PJ3, PhyA, was purified near to homogeneity with 427-fold increase in specific phytase activity by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatographies. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and zymogram analysis of the purified enzyme indicated an estimated molecular mass of 65 kD. The optimal pH and temperature of the purified enzyme were pH 4.5 and 55°C, respectively. The enzyme activity was strongly inhibited by Ca²⁺, Cu²⁺, Zn²⁺, and phenylmethylsulfonyl fluoride (PMSF). The K_m value for sodium phytate was 0.545 mM with a V_max of 600 U/mg of protein. The phyA gene was cloned, and it contains an open reading frame of 1,383 with a single intron (118 bp), and encodes a protein of 461 amino acids.     

A Review of: “HPLC of Biological Macromolecules (Methods and Applications) K.M. Goodring F.E. Regnier,Eds Marcel Dekker,New York, 1990; hardbound, 676 pages, $150”

An ionically unbound and thermostable polyphenol oxidase (PPO) was extracted from the leaf of Musa paradisiaca. The enzyme was purified 2.54-fold with a total yield of 9.5% by ammonium sulfate precipitation followed by Sephadex G-100 gel filtration chromatography. The purified enzyme exhibited a clear single band on native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS) PAGE. It was found to be monomeric protein with molecular mass of about 40 kD. The zymographic study using crude extract as enzyme source showed a very clear band around 40 kD and a faint band at around 15 kD, which might be isozymes. The enzyme was optimally active at pH 7.0 and 50°C temperature. The enzyme was active in wide range of pH (4.0–9.0) and temperature (30–90°C). From the thermal inactivation studies in the range 60–75°C, the half-life (t_1/2) values of the enzyme ranged from 17 to 77 min. The inactivation energy (Ea) value of PPO was estimated to be 91.3 kJ mol^?1. It showed higher specificity with catechol (K_m = 8 mM) as compared to 4-methylcatechol (K_m = 10 mM). Among metal ions and reagents tested, Cu²⁺, Fe²⁺, Hg²⁺, Mn²⁺, Ni²⁺, protocatechuic acid, and ferrulic acid enhanced the enzyme activity, while K⁺, Na⁺, Co²⁺, kojic acid, ascorbic acid, ethylenediamine tetraacetic acid (EDTA), sodium azide, β-mercaptoethanol, and L-cysteine inhibited the activity of the enzyme.     

Partial Purification and Characterization of Chromate Reductase of a Novel Ochrobactrum sp. Strain Cr-B4

Hexavalent chromium contamination is a serious problem due to its high toxicity and carcinogenic effects on the biological systems. The enzymatic reduction of toxic Cr(VI) to the less toxic Cr(III) is an efficient technology for detoxification of Cr(VI)-contaminated industrial effluents. In this regard, a chromate reductase enzyme from a novel Ochrobactrum sp. strain Cr-B4, having the ability to detoxify Cr(VI) contaminated sites, has been partially purified and characterized. The molecular mass of this chromate reductase was found to be 31.53 kD, with a specific activity 14.26 U/mg without any addition of electron donors. The temperature and pH optima for chromate reductase activity were 40°C and 8.0, respectively. The activation energy (E_a) for the chromate reductase was found to be 34.7 kJ/mol up to 40°C and the activation energy for its deactivation (E_d) was found to be 79.6 kJ/mol over a temperature range of 50–80°C. The frequency factor for activation of chromate reductase was found to be 566.79 s^?1, and for deactivation of chromate reductase it was found to be 265.66 × 10³ s^?1. The reductase activity of this enzyme was affected by the presence of various heavy metals and complexing agents, some of which (ethylenediamine tetraacetic acid [EDTA], mercaptoethanol, NaN₃, Pb²⁺, Ni²⁺, Zn²⁺, and Cd²⁺) inhibited the enzyme activity, while metals like Cu²⁺ and Fe³⁺ significantly enhanced the reductase activity. The enzyme followed Michaelis–Menten kinetics with K_m of 104.29 µM and a V_max of 4.64 µM/min/mg.     

Purification and Characterization of Alkaline Serine Proteinase from Photosynthetic Bacterium,Rubrivivax gelatinosus KDDS1

In order to reduce the protein content of wastewater, photosynthetic bacteria producing proteinases were screened from wastewater of various sources and stocked in culture. An isolated strain, KDDS1, was identified as Rubrivivax gelatinosus, a purple nonsulfur bacterium that secretes proteinase under micro-aerobic conditions under light at 35°C. Molecular weight of the purified enzyme was estimated to be 32.5 kDa. The enzyme showed the highest activity at 45°C and pH 9.6, and the activity was completely inhibited by phenylmethyl sulfonyl fluoride (PMSF), but not by EDTA. The amino-terminal 24 amino acid sequence of the enzyme showed about 50% identity to those of serine proteinases from Pseudoalteromonas piscicida strain O-7 and Burkholderia pseudomallei. Thus, the enzyme from Rvi. gelatinosus KDDS1 was thought to be a serine-type proteinase. This was the first serine proteinase characterized from photosynthetic bacteria.     

Protective Effect of Some Anions on the Heat-Denaturation of Ovotransferrin

An alkaline serine-proteinase from Bacillus sp. PN51 isolated from bat feces collected in Phang Nga, Thailand, was purified and characterized. The molecular mass was estimated to be 35.0 kDa. The N-terminal 25 amino acid sequence was about 70% identical with that of Natrialba magadii halolysin-like extracellular serine protease. The enzyme showed the highest proteinase activity at 60 °C at pH 10.0. The activity was strongly inhibited by PMSF and chymostatin. The proteinase activity was not affected by the presence of 2% urea, 2% H₂O₂, 12% SDS, 15% triton X-100, or 15% tween 80. The proteinase preferred Met, Leu, Phe, and Tyr residues at the P₁ position, in descending order. The k _cat, K _m and k _cat/K _m values for Z-Val-Lys-Met-MCA were 16.8±0.14 min^?1, 5.1±0.28 μM, and 3.3±0.28 μM^?1 min^?1 respectively. This is the first report of an alkaline serine-proteinase with extremely high stability against detergents such as SDS.     

Isolation and general characterization of a heat-stable proteinase from Pseudomonas fluorescens AFT 36

An extracellular proteinase from Pseudomonas fluorescens, strain AFT 36, was isolated to homogeneity by chromatography on DEAE-cellulose and Sephadex G-150; a 230-fold increase in specific activity with a recovery of 53% was obtained. The enzyme was optimally active at pH 6.5 and 45°C; activity declined rapidly at higher temperatures but significant activity persisted down to 4°C. Activity was strongly inhibited by 10^?3 M EDTA and was partially restored by addition of Zn²⁺, Ca²⁺ or Co²⁺. The K_m values on methylated casein and sodium caseinate were 18.2 and 7.1 mg/ml, respectively. The enzyme was very labile in phosphate buffer and in a milk salts buffer at 55°C but was very stable in the latter at more than 80°C.     

Purification and Characterization of a Thermostable Alkaline Protease from Alkalophilic Thermoactinomyces sp. H8682

Protease secreted into the culture medium by alkalophilic Thermoactinomyces sp. HS682 was purified to an electrophoretically homogeneous state through only two chromatograhies using Butyl-Toyopearl 650M and SP-Toyopearl 650S columns. The purified enzyme has an apparent relative molecular mass of 25, 000 according to gel filtration on a Sephadex G-75 column and SDS-PAGE and an isoelectric point above 11.0.

Its proteolytic activity was inhibited by active-site inhibitors of serine protease, DFP and PMSF, and metal ions, Cu²⁺ and Hg²⁺. The enzyme was stable toward some detergents, sodium perborate, sodium triphosphate, sodium-n-dodecylbenzenesulfonate, and sodium dodecyl sulfate, at a concentration of 0.1% and pH 11.5 and 37°C for 60 min. The optimum pH was pH 11.5–13.0 at 37°C and the optimum temperature was 70°C at pH 11.5. Calcium divalent cation raised the pH and heat stabilities of the enzyme. In the presence of 5 mM CaCl₂, it showed maximum proteolytic activity at 80°C and stability from pH 4–12.5 at 60°C and below 75°C at pH 11.5. The stabilization by Ca²⁺ was observed in secondary conformation deduced from the circular dichroic spectrum of the enzyme. The protease hydrolyzed the ester bond of benzoyl leucine ester well. The amino acid terminal sequence of the enzyme showed high homology with those of Microbiol serine protease, although alanine of the NH₂-terminal amino acid was deleted.     

Extracellular proteinase fromLactobacillus murinus

Lactobacillus murinus CNRZ 313 produced an extracellular proteinase irrespective of the Ca²⁺ content in the culture medium. Proteinase activity was optimal at 37 °C and pH 7.5 in phosphate buffer (0.2 mol/L). It was stimulated by Mg²⁺ and Mn²⁺ and was inhibited by Zn²⁺. Ca²⁺ did not affect the enzymic activity but the proteinase liberated in the presence of this ion is more stable. The enzyme was purified to homogeneity from cell-free culture medium.     

Purification and characterization of an extracellular haloalkaline serine protease from the moderately halophilic bacterium, <Emphasis Type="Italic">Bacillus iranensis (</Emphasis>X5B)

This study reports the purification and characterization of an extracellular haloalkaline serine protease from the moderately halophilic bacterium, Bacillus iranensis, strain X5B. The enzyme was purified to homogeneity by acetone precipitation, ultrafiltration and carboxymethyl (CM) cation exchange chromatography, respectively. The purified protease was a monomeric enzyme with a relative molecular mass of 48–50 kDa and it was inhibited by PMSF indicating that it is a serine-protease. The optimum pH, temperature and NaCl concentration were 9.5, 35 °C and 0.98 M, respectively. The enzyme showed a significant tolerance to salt and alkaline pH. It retained approximately 50 % of activity at 2.5 M NaCl and about 70 % of activity at highly alkaline pH of 11.0; therefore, it was a moderately halophilic and also can be activated by metals, especially by Ca²⁺. The specific activity of the purified protease was measured to be 425.23 μmol of tyrosine/min per mg of protein using casein as a substrate. The apparent K _m and V _max values were 0.126 mM and 0.523 mM/min, respectively and the accurate value of k _cat was obtained as 3.284 × 10^?2 s^?1. These special and important characteristics make this serine protease as valuable tool for industrial applications.     

Purification and characterization of extracellular inulinase from a marine yeast <Emphasis Type="Italic">Pichia guilliermondii</Emphasis> and inulin hydrolysis by the purified inulinase

The extracellular inulinase of the marine yeast Pichia guilliermondii strain 1 was purified to homogeneity resulting in a 7.2-fold increase in specific inulinase activity. The molecular mass of the purified enzyme was estimated to be 50.0 kDa. The optimal pH and temperature for the purified enzyme were 6.0 and 60°C, respectively. The enzyme was activated by Mn²⁺, Ca²⁺, K⁺, Li⁺, Na⁺, Fe³⁺, Fe²⁺, Cu²⁺, and Co²⁺, but Mg²⁺, Hg²⁺, and Ag⁺ inhibited activity. The enzyme was strongly inhibited by phenylmethanesulphonyl fluoride (PMSF), iodoacetic acid, EDTA, and 1, 10-phenanthroline. The K _m and V _max values of the purified inulinase for inulin were 21.1 mg/mL and 0.08 mg/min, respectively. A large number of monosaccharides were detected after the hydrolysis of inulin. The deduced protein sequence from the cloned P. guilliermondii strain 1 inulinase gene contained the consensus motifs R-D-P-K-V-F-W-H and W-M-N-D-P-N-G, which are conserved among the inulinases from other microorganisms.     

PURIFICATION AND CHARACTERIZATION OF MUTANASE PRODUCED BY Paenibacillus curdlanolyticus MP-1

Mutanases are enzymes that catalyze hydrolysis of α-1,3-glucosidic bonds in various α-glucans. One of such glucans, mutan, which is synthesized by cariogenic streptococci, is a major virulence factor for induction of dental caries. This means that mutan-degrading enzymes have potential in caries prophylaxis. In this study, we report the purification, characterization, and partial amino acid sequence of extracellular mutanase produced by the MP-1 strain of Paenibacillus curdlanolyticus, bacterium isolated from soil. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme showed a single protein band of molecular mass 134 kD, while native gel filtration chromatography confirmed that the enzyme was a monomer of 142 kD. Mutanase showed a pH optimum in the range from pH 5.5 to 6.5 and a temperature optimum around 40–45°C. It was thermostable up to 45°C, and retained 50% activity after 1 hr at 50°C. The enzyme was fully stable at a pH range of 4 to 10. The enzyme activity was stimulated by the addition of Tween 20, Tween 80, and Ca²⁺, but it was significantly inhibited by Hg²⁺, Ag⁺, and Fe²⁺, and also by p-chloromercuribenzoate, iodoacetamide, and ethylenediamine tetraacetic acid (EDTA). Mutanase preparation preferentially catalyzed the hydrolysis of various streptococcal mutans and fungal α-1,3-glucans. It also showed binding activity to insoluble α-1,3-glucans. The N-terminal amino acid sequence was NH₂-Ala-Gly-Gly-Thr-Asn-Leu-Ala-Leu-Gly-Lys-Asn-Val-Thr-Ala-Ser-Gly-Gln. This sequence indicated an analogy of the enzyme to α-1,3-glucanases from other Paenibacillus and Bacillus species.     

Characterization of an extracellular alkaline serine protease from marine <Emphasis Type="Italic">Engyodontium album</Emphasis> 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 × 10⁻² mg/ml, 394.68 U, and 4.2175 × 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.     

An extracellular glucoamylase produced by endophytic fungus EF6

A strain of endophytic fungus EF6 isolated from Thai medicinal plants was found to produce higher levels of extracellular glucoamylase. This strain produced glucoamylase of culture filtrate when grown on 1% soluble starch. The enzyme was purified and characterized. Purification steps involved (NH₄)₂SO₄ precipitation, anion exchange, and gel filtration chromatography. Final purification fold was 14.49 and the yield obtained was 9.15%. The enzyme is monomeric with a molecular mass of 62.2 kDa as estimated by SDS-PAGE, and with a molecular mass of 62.031 kDa estimated by MALDI-TOF spectrometry. The temperature for maximum activity was 60°C. After 30 min for incubation, glucoamylase was found to be stable lower than 50°C. The activity decrease rapidly when residual activity was retained about 45% at 55°C. The pH optimum of the enzyme activity was 6.0, and it was stable over a pH range of 4.0–7.0 at 50°C. The activity of glucoamylase was stimulated by Ca²⁺, Co²⁺, Mg²⁺, Mn²⁺, glycerol, DMSO, DTT and EDTA, and strongly inhibited by Hg²⁺. Various types of starch were test, soluble starch proved to be the best substrate for digestion process. The enzyme catalyzes the hydrolysis of soluble starch and maltose as the substrate, the enzyme had K _m values of 2.63, and 1.88 mg/ml and V _max, values of 1.25, and 2.54 U/min/mg protein, and V _max/K _m values of 0.48 and 1.35, respectively. The internal amino acid sequences of endophytic fungus EF6 glucoamylase; RALAN HKQVV DSFRS have similarity to the sequence of the glucoamylase purified form Thermomyces lanuginosus. From all results indicated that this enzyme is a glucoamylase (1,4-α-D-glucan glucanohydrolase).     

Phylogenetic analysis of alkaline proteinase producing fluorescent pseudomonads associated with green gram (<Emphasis Type="Italic">Vigna radiata</Emphasis> L.) rhizosphere

Fifty fluorescent pseudomonads were isolated from rhizospheric soil of green gram from nearby area of Kaziranga, Assam, India and assayed for their extracellular proteinase production. Out of these isolates, 20 were found to be prominent in proteinase production. Genetic diversity of the 20 isolates were analyzed through BOX-PCR fingerprinting and 16S rDNA-RFLP along with three reference strains, viz., Pseudomonas fluorescens (NCIM2099^T), Pseudomonas aureofaciens (NCIM2026^T), and Pseudomonas aeruginosa (MTCC2582^T). BOX-PCR produced two distinct clusters at 56% similarity coefficient and seven distinct BOX profiles. 16S rDNA-RFLP with three tetra-cutters restriction enzymes (HaeIII, AluI, and MspI) revealed two major clusters A and B; cluster A contained only single isolate FPS9 while the rest of 22 isolates belonged to the cluster B. Based on phenotypic characters and 16S rDNA sequence similarity, all the eight highly proteinase-producing strains were affiliated with P. aeruginosa. The proteinase was extracted from two most prominent strains (KFP1 and KFP2), purified by a three-step process involving (NH₄)₂SO₄ precipitation, gel filtration, and ion exchange chromatography. The enzyme had an optimal pH of 8.0 and exhibit highest activity at 60°C and 37°C by KFP1 and KFP2 respectively. The specific activities were recorded as 75,050 (for KFP1) and 81,320 U/mg (for KFP2). The purified enzyme was migrated as a single band on native and SDS-PAGE with a molecular mass of 32 kDa. Zn²⁺, Cu²⁺, and Ni²⁺ ion inhibited the enzyme activity. Enzyme activity was also inhibited by EDTA established as their metallo-proteinase nature.