Purification and characterization of a nitroreductase from the soil bacterium Streptomyces mirabilis

A NADH-dependent nitroreductase from an efficient nitro-reducing soil bacterium, Streptomyces mirabilis DUT001, was isolated and characterized. The enzyme was purified to near homogeneity using ammonium sulfate precipitation, ion exchange chromatography, and gel filtration chromatography. The native enzyme was estimated by gel filtration to have a molecular weight of 68 kDa, and its subunit molecular weight determined by SDS-PAGE was about 34 kDa, which indicated this enzyme was a dimer. Polycyclic nitroaromatic compounds were preferred substrates for this enzyme. The purified enzyme exhibited maximum activity at pH 7.5 and 40 °C. The addition of various chemicals such as reducing agents, metal ions, and chelating agents, had effects on enzyme activity. Mg²⁺, Ca²⁺, Sr²⁺, and 1% (w/v) Triton X-100 increased activity. However, Hg²⁺, Co²⁺, Ni²⁺, Cu²⁺, and SDS reduced activity. The maximum reaction rate (V_max) was 64 μM min^?1 mg^?1 enzyme and the apparent Michaelis–Menten constants (K_m) for 4-nitro-1,8-naphthalic anhydride and NADH were 276 and 29 μM, respectively. Menadione, bimethylenebis, sodium benzoate, and antimycin A were inhibitors of the purified nitroreductase with apparent inhibition constants (K_is) of 20, 36, 44 and 80 μM, respectively.     

A novel fibrinolytic protease from Streptomyces sp. CS684

A fibrinolytic protease (FP84) was purified from Streptomyces sp. CS684, with the aim of isolating economically viable enzyme from a microbial source. SDS-PAGE and fibrin zymography of the purified enzyme showed a single protein band of approximately 35 kDa. Maximal activity was at 45 °C and pH 7–8, and the enzyme was stable between pH 6 and 9 and below 40 °C. It exhibited fibrinolytic activity, which is stronger than that of plasmin. FP84 hydrolyzed Bβ-chains of fibrinogen, but did not cleave Aα- and γ-chains. K_m, V_max and K_cat values for azocasein were 4.2 mg ml⁻¹, 305.8 μg min⁻¹ mg⁻¹ and 188.7 s⁻¹, respectively. The activity was suppressed by Co²⁺, Zn²⁺, Cu²⁺ and Fe²⁺, but slightly enhanced by Ca²⁺ and Mg⁺². Additionally, the activity was slightly inhibited by aprotinin and PMSF, but significantly inhibited by pefabloc, EDTA and EGTA. The first 15 amino acids of N-terminal sequence were GTQENPPSSGLDDID. They are highly similar to those of serine proteases from various Streptomyces strains, but different with known fibrinolytic enzymes. These results suggest that FP84 is a novel serine metalloprotease with potential application in thrombolytic therapy.     

Purification and characterization of SDG-β-d-glucosidase hydrolyzing secoisolariciresinol diglucoside to secoisolariciresinol from Aspergillus oryzae

The SDG-β-d-glucosidase that hydrolyzes the glucopyranoside bond of secoisolariciresinol diglucoside (SDG) to release secoisolariciresinol (SECO) was isolated from Aspergillus oryzae 39 strain and the enzyme was purified and characterized. The enzyme was purified to one spot in SDS polyacrylamide gel electrophoresis, and its molecular weight was about 64.9 kDa. The optimum temperature of the SDG-β-d-glucosidase was 40 °C, and the optimum pH was 5.0. The SDG-β-d-glucosidase was stable at less than 65 °C, and pH 4.0–6.0. Ca²⁺, K⁺, Mg²⁺ and Na⁺ ions have no significant effect on enzyme activity, Zn²⁺ and Cu²⁺ ions have weakly effect on enzyme activity, but Fe³⁺ ion inhibits enzyme activity strongly. The K_m value of SDG-β-d-glucosidase was 0.14 mM for SDG.     

Production of β-fructofuranosidases by Aspergillus niveus using agroindustrial residues as carbon sources: Characterization of an intracellular enzyme accumulated in the presence of glucose

The production of β-fructofuranosidases by Aspergillus niveus, cultivated under submerged fermentation using agroindustrial residues, was investigated. The highest productivity of β-fructofuranosidases was obtained in Khanna medium supplemented with sugar cane bagasse as carbon source. Glucose enhanced the production of the intracellular enzyme, whereas that of the extracellular one was decreased. The intracellular β-fructofuranosidase was a trimeric protein of approximately 141 kDa (gel filtration) with 53.5% carbohydrate content, composed of 57 kDa monomers (SDS-PAGE). The optimum temperature and optimum pH were 60 °C and 4.5, respectively. The purified enzyme showed good thermal stability and exhibited a half-life of 53 min at 60 °C. β-Fructofuranosidase activity was slightly activated by Cu²⁺, Mn²⁺, Mg²⁺, and Na⁺ at 1 mM concentration. The enzyme hydrolyzed sucrose, raffinose, and inulin, with K_d values of 5.78 mM, 5.74 mM, and 1.74 mM, respectively.     

Purification and biochemical properties of an extracellular acid phytase produced by the Saccharomyces cerevisiae CY strain

An extracellular acid phytase was purified to homogeneity from the culture supernatant of the Saccharomyces cerevisiae CY strain by ultrafiltration, DEAE-Sepharose column chromatography, and Sephacryl S-300 gel filtration. The molecular weight of the purified enzyme was estimated to be 630 kDa by gel filtration. Removing the sugar chain by endoglycosidase H digestion revealed that the molecular mass of the protein decreased to 446 kDa by gel filtration and gave a band of 55 kDa by SDS-PAGE. The purified enzyme was most active at pH 3.6 and 40 °C and was fairly stable from pH 2.5 to 5.0. The phytase displayed broad substrate specificity and had a K_m value of 0.66 mM (sodium phytate, pH 3.6, 40 °C). The phytase activity was completely inhibited by Fe³⁺ and Hg²⁺, and strongly inhibited (maximum of 91%) by Ba²⁺, Co²⁺, Cu⁺, Cu²⁺, Fe²⁺, Mg²⁺, and Sn²⁺ at 5 mM concentrations.     

Characterization of an extracellular thermophilic alkaline esterase produced by Bacillus subtilis DR8806

This work is a report of the characterization of an alkaline lipolytic enzyme isolated from Bacillus subtilis DR8806. The extracellular extract was concentrated using ammonium sulfate, and ultrafiltration. The active enzyme was purified by Q-sepharose ion exchange chromatography. The molecular mass of the enzyme was estimated to be 60.25 kDa based on SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). The optimum pH and temperature of this enzyme were observed to be 8.0 and 50 °C, respectively. The enzyme exhibited a half-life of 72 min at its optimum temperature. It was stable in the presence of metal ions (10 mM) such as Ca²⁺, K⁺ and Na⁺, whereas Cu²⁺, Fe²⁺, Zn²⁺, Mn²⁺, Co²⁺, Mg²⁺ and Hg²⁺ were found to have inhibitory effects. However, the enzyme activity was not affected significantly by 1% Triton X-100. The study of substrate specificity showed that the purified enzyme has a preferential specificity for small ester of p-nitrophenyl acetate (C₂), and it was the most efficiently hydrolyzed substrate as compared to the other esters. The kinetic parameters showed that the enzyme has K_m of 4.2 mM and V_max of 151 μmol min⁻¹ mg⁻¹ for p-nitrophenyl acetate. The hydrolysis rates of the fluorescence substrates were increased in the presence of the purified enzyme. Regarding the features of the enzyme, it may be utilized as a novel candidate for industrial applications.     

Purification and characterization of a thermostable α-galactosidase with transglycosylation activity from Aspergillus parasiticus MTCC-2796

An extracellular thermostable α-galactosidase from Aspergillus parasiticus MTCC-2796 was purified 16.59-fold by precipitation with acetone, followed by sequential column chromatography with DEAE-Sephadex A-50 and Sephadex G-100. The purified enzyme was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It was found to be a monomeric protein with a molecular weight of about 67.5 kDa. The purified enzyme showed optimum activity against o-nitrophenyl-α-d-galactopyranoside (oNPG) at pH 5.0 and a temperature of 50 °C. The enzyme was thermostable, showing complete activity even after heating at 65 °C for 30 min. The enzyme showed strict substrate specificity for α-galactosides and hydrolyzed oNPG (K_m = 0.83 mM), melibiose (K_m = 2.48 mM) and raffinose (K_m = 5.83 mM). Among metal ions and reagents tested, Ca²⁺ and K⁺ enhanced the enzymatic activity, but Mg²⁺, Mn²⁺, ethylenediaminetetraacetic acid (EDTA) and 2-mercaptoethanol showed no effect, while Ag⁺, Hg²⁺ and Co²⁺ strongly inhibited the activity of the enzyme. The enzyme catalyzed the transglycosylation reaction for the synthesis of melibiose.     

Purification and characterization of piceid-β-d-glucosidase from Aspergillus oryzae

The piceid-β-d-glucosidase that hydrolyzes the β-d-glucopyranoside bond of piceid to release resveratrol was isolated from Aspergillus oryzae sp.100 strain, and the enzyme was purified and characterized. The enzyme was purified to one spot in SDS polyacrylamide gel electrophoresis, and its molecular weight was about 77 kDa. The optimum temperature of the piceid-β-d-glucosidase was 60 °C, and the optimum pH was 5.0. The piceid-β-d-glucosidase was stable at less than 60 °C, and pH 4.0–5.0. Ca²⁺, Mg²⁺ and Zn²⁺ ions have no significant effect on enzyme activity, but Cu²⁺ ion inhibits enzyme activity strongly. The K_m value was 0.74 mM and the V_max value was 323 nkat mg⁻¹ for piceid.     

Purification and characterization of an extracellular antifungal chitinase from Penicillium ochrochloron MTCC 517 and its application in protoplast formation

A highly chitinolytic strain Penicillium ochrochloron MTCC 517 was procured from MTCC, Chandigarh, India. Culture medium supplemented with 1% chitin was found to be suitable for maximum production of chitinase. Purification of extracellular chitinase was done from the culture medium by organic solvent precipitation and DEAE-cellulose column chromatography. The chitinase was purified 6.92-fold with 29.9% yield. Molecular mass of purified chitinase was found to be 64 kDa by SDS-PAGE. The chitinase showed optimum temperature 40 °C and pH 7.0. The enzyme activity was completely inhibited by Hg²⁺, Zn²⁺, K⁺ and NH₄⁺. The enzyme kinetic study of purified chitinase revealed the following characteristics, such as apparent K_m 1.3 mg ml^?1, V_max 5.523 × 10^?5 moles l^?1 min^?1 and K_cat 2.37 s^?1 and catalytic efficiency 1.82 s^?1 M^?1. The enzyme hydrolyzed colloidal chitin, glycol chitin, chitosan, glycol chitosan, N,N′-diacetylchitobiose, p-nitrophenyl N-acetyl-β-d-glucosaminide and 4-methylumbelliferyl N-acetyl-β-d-glucosaminide. The chitinase of P. ochrochloron MTCC 517 is an exoenzyme, which gives N-acetylglucosamine as the main hydrolyzate after hydrolysis of colloidal chitin. Protoplasts with high regeneration capacity were obtained from Aspergillus niger using chitinase from P. ochrochloron MTCC 517. Since it also showed antifungal activity, P. ochrochloron MTCC 517 seems to be a promising biocontrol agent.     

A thermoalkaliphilic halotolerant esterase from Rhodococcus sp. LKE-028 (MTCC 5562): Enzyme purification and characterization

A newly isolated Rhodococcus sp. LKE-028 (MTCC 5562) from soil samples of Gangotri region of Uttarakhand Himalayan produced a thermostable esterase. The enzyme was purified to homogeneity with purification fold 62.8 and specific activity 861.2 U mg^?1 proteins along with 26.7% recovery. Molecular mass of the purified enzyme was 38 kDa and values of K_m and V_max were 525 nM and 1666.7 U mg^?1 proteins, respectively. The esterase was active over a broad range of temperature (40–100 °C) and pH (7.0–12.0). The esterase was most active at pH 11.0. The optimum temperature of enzyme activity was 70 °C and the enzyme was completely stable after 3 h pre-incubation at 60 °C. Metal ions like Ca²⁺, Mg²⁺ and Co²⁺ stimulated enzyme activities. Purified esterase remarkably retained its activity with 10 M NaCl. Enzyme activity was slightly increased in presence of non-polar detergents (Tween 20, Tween 80 and Triton X 100), and compatible with oxidizing agents (H₂O₂) and reducing agents (β-mercaptoethanol). Activities of the enzyme was stimulated in presence of organic solvents like DMSO, benzene, toluene, methanol, ethyl alcohol, acetone, isoamyl alcohol after 10 days long incubation. The enzyme retained over 75% activity in presence of proteinase K. Besides hyperthermostability and halotolerancy the novelty of this enzyme is its resistance against protease.     

Purification and characterization of a new carbonyl reductase from Leifsonia xyli HS0904 involved in stereoselective reduction of 3,5-bis(trifluoromethyl) acetophenone

Leifsonia xyli HS0904 can stereoselectively catalyze the bioreduction of 3,5-bis(trifluoromethyl) acetophenone (BTAP) to its corresponding alcohol, which is a valuable chiral intermediate in the pharmaceuticals. In this study, a new carbonyl reductase derived from L. xyli HS0904 was purified and its biochemical properties were determined in detail. The carbonyl reductase was purified by 530-fold with a specific activity of 13.2 U mg⁻¹ and found to be a homodimer with a molecular mass of 49 kDa, in which the subunit molecular-weight was about 24 kDa. The purified enzyme exhibited a maximum enzyme activity at 34 °C and pH 7.2, and retained over 90% of its initial activity at 4 °C and pH 7.0 for 24 h. The addition of various additives, such as Ca²⁺, Mg²⁺, Mn²⁺, l-cysteine, l-glutathione, urea, PEG 1000 and PEG 4000, could enhance the enzyme activity. The maximal reaction rate (V_max) and apparent Michaelis–Menten constant (K_m) of the purified carbonyl reductase for BTAP and NADH were confirmed as 33.9 U mg⁻¹, 0.383 mM and 69.9 U mg⁻¹, 0.412 mM, respectively. Furthermore, this enzyme was found to have a broad spectrum of substrate specificity and can asymmetrically catalyze the reduction of a variety of ketones and keto esters.     

Application of chitosan beads immobilized Rhodococcus sp. NCIM 2891 cholesterol oxidase for cholestenone production

The cell-bound cholesterol oxidase from the Rhodococcus sp. NCIM 2891 was purified three fold by diethylaminoethyl–sepharose chromatography. The estimated molecular mass (SDS-PAGE) and K_m of the enzyme were ∼55.0 kDa and 151 μM, respectively. The purified cholesterol oxidase was immobilized on chitosan beads by glutaraldehyde cross-linking reaction and immobilization was confirmed by Fourier transform infrared spectroscopy, scanning electron microscopy and energy dispersive X-ray analysis. The optimum temperature (45 °C, 5 min) for activity of the enzyme was increased by 5 °C after immobilization. Both the free and immobilized cholesterol oxidases were found to be stable in many organic solvents except for acetone. Fe²⁺ and Pb²⁺ at 0.1 mM of each acted as inhibitors, while Ag⁺, Ca²⁺, Ni²⁺ and Zn²⁺ activated the enzyme at similar concentration. The biotransformation of cholesterol (3.75 mM) with the cholesterol oxidase immobilized beads (3.50 U) leads to ∼88% millimolar yield of cholestenone in a reaction time of 9 h at 25 °C. The immobilized enzyme retains ∼67% activity even after 12 successive batches of operation. The biotransformation method thus, shows a great promise for the production of pharmaceutically important cholestenone.     

Purification and physicochemical properties of polygalacturonase from Aspergillus niger MTCC 3323

Polygalacturonases are the pectinolytic enzymes that catalyze the hydrolytic cleavage of the polygalacturonic acid chain. In the present study, polygalacturonase from Aspergillus niger (MTCC 3323) was purified. The enzyme precipitated with 60% ethanol resulted in 1.68-fold purification. The enzyme was purified to 6.52-fold by Sephacryl S-200 gel-filtration chromatography. On SDS–PAGE analysis, enzyme was found to be a heterodimer of 34 and 69 kDa subunit. Homogeneity of the enzyme was checked by NATIVE-PAGE and its molecular weight was found to be 106 kDa. The purified enzyme showed maximum activity in the presence of polygalacturonic acid at temperature of 45 °C, pH of 4.8, reaction time of 15 min. The enzyme was stable within the pH range of 4.0–5.5 for 1 h. At 4 °C it retained 50% activity after 108 h but at room temperature it lost its 50% activity after 3 h. The addition of Mn²⁺, K⁺, Zn²⁺, Ca²⁺ and Al³⁺ inhibited the enzyme activity; it increased in the presence of Mg²⁺ and Cu²⁺ ions. Enzyme activity was increased on increasing the substrate concentration from 0.1% to 0.5%. The K_m and V_max values of the enzyme were found to be 0.083 mg/ml and 18.21 μmol/ml/min. The enzyme was used for guava juice extraction and clarification. The recovery of juice of enzymatically treated pulp increased from 6% to 23%. Addition of purified enzyme increased the %T₆₅₀ from 2.5 to 20.4 and °Brix from 1.9 to 4.8. The pH of the enzyme treated juice decreased from 4.5 to 3.02.     

Homologous expression,purification and characterization of a novel high-alkaline and thermal stable lipase from Burkholderia cepacia ATCC 25416

The lipase secreted by Burkholderia cepacia ATCC 25416 was particularly attractive in detergent and leather industry due to its specific characteristics of high alkaline and thermal stability. The lipase gene (lipA), lipase chaperone gene (lipB), and native promoter upstream of lipA were cloned. The lipA was composed of 1095 bp, corresponding to 364 amino acid residues. The lipB located immediately downstream of lipA was composed of 1035 bp, corresponding to 344 amino acid residues. The lipase operon was inserted into broad host vector pBBRMCS1 and electroporated into original strain. The homologous expression of recombinant strain showed a significant increase in the lipase activity. LipA was purified by three-step procedure of ammonium sulfate precipitation, phenyl-sepharose FF and DEAE-sepharose FF. SDS-PAGE showed the molecular mass of the lipase was 33 kDa. The enzyme optimal temperature and pH were 60 °C and 11.0, respectively. The enzyme was stable at 30–70 °C. After incubated in 70 °C for 1 h, enzyme remained 72% of its maximal activity. The enzyme exhibited a good stability at pH 9.0–11.5. The lipase preferentially hydrolyzed medium-chain fatty acid esters. The enzyme was strongly activated by Mg²⁺, Ca²⁺, Cu²⁺, Zn²⁺, Co²⁺, and apparently inhibited by PMSF, EDTA and also DTT with SDS. The enzyme was compatible with various ionic and non-ionic surfactants as well as oxidant H₂O₂. The enzyme had good stability in the low- and non-polar solvents.     

Purification of a laccase from fungus combs in the nest of Odontotermes formosanus

A new enzyme was isolated from the fungus combs in the nest of Odontotermes formosanus and identified as a laccase. The single laccase was purified with a purification factor of 16.83 by ammonium sulphate precipitation and anion exchange chromatography, to a specific activity of 211.11 U mg⁻¹. Its molecular mass was 65 kDa. The optimum pH value and temperature were 4.0 °C and 10 °C with ABTS as the substrate, respectively. The enzyme activity stabilized at temperatures between 10 °C and 30 °C and decreased rapidly when the temperature was above 30 °C. The V_max and K_m values were 3.62 μmol min⁻¹ mg⁻¹ and 119.52 μM, respectively. Ethanol concentration affected laccase activity, inhibiting 60% of enzyme activity at a concentration of 70%. Metal ions of Mg²⁺, Ba²⁺ and Fe²⁺ showed inhibition on enzyme activity of 17.2%, 5.3% and 9.4%, respectively, with the increase of metal ions concentration from 1 mM to 5 mM. Especially Fe²⁺ strongly inhibited enzyme activity up to 89% inhibition at a concentration of 1 mM.     

Purification of a thermostable laccase from Leucaena leucocephala using a copper alginate entrapment approach and the application of the laccase in dye decolorization

Laccase from a tree legume, Leucaena leucocephala, was purified to homogeneity using a quick two-step procedure: alginate bead entrapment and celite adsorption chromatography. Laccase was purified 110.6-fold with an overall recovery of 51.0% and a specific activity of 58.5 units/mg. The purified laccase was found to be a heterodimer (∼220 kDa), containing two subunits of 100 and 120 kDa. The affinity of laccase was found to be highest for catechol and lowest for hydroquinone, however, highest K_cat and K_cat/K_m were obtained for hydroquinone. Purified laccase exhibited pH and temperature optima of 7.0 and 80 °C, respectively. Mn²⁺, Cd²⁺, Fe²⁺, Cu²⁺ and Na⁺ activated laccase while Ca²⁺ treatment increased laccase activity up to 3 mM, beyond which it inhibited laccase. Co²⁺, Hg²⁺, DTT, SDS and EDTA showed an inhibition of laccase activity. The Leucaena laccase was found to be fairly tolerant to organic solvents; upon exposure for 1 h individually to 50% (v/v) each of ethanol, DMF, DMSO and benzene, more than 50% of the activity was retained, while in the presence of 50% (v/v) each of methanol, isopropanol and chloroform, a 40% residual activity was observed. The purified laccase efficiently decolorized synthetic dyes such as indigocarmine and congo red in the absence of any redox mediator.     

Calcium dependent,alkaline detergent-stable trypsin from the viscera of Goby (Zosterisessor ophiocephalus): Purification and characterization

An alkaline calcium dependent trypsin from the viscera of Goby (Zosterisessor ophiocephalus) was purified to homogeneity with a 16-fold increase in specific activity and 20% recovery. The purified trypsin appeared as a single band on sodium dodecyl sulphate-polyacrylamide gel (SDS-PAGE) and native-PAGE. The enzyme had an estimated molecular weight of 23.2 kDa.The optimum pH was 9.0, and the enzyme was extremely stable in various pH buffers between pH 7.0 and 11.0. The optimum temperature for enzyme activity was 60 °C, and the activity and stability of trypsin was highly dependent on the presence of calcium ion. At 60 °C, Ca²⁺ (5 mM) stimulated the protease activity by 220%. The trypsin kinetic constants, K_m and k_cat, were 0.312 mM and 2.03 s^?1.The enzyme showed high stability towards non-ionic surfactants and oxidizing agent. In addition, the enzyme showed excellent stability and compatibility with some commercial solid and liquid detergents.     

Biochemical properties of a β-mannanase and a β-xylanase produced by Ceriporiopsis subvermispora during biopulping conditions

One mannanase and one of the three xylanases produced by Ceriporiopsis subvermispora grown on Pinus taeda wood chips were characterized. A combination of ion exchange chromatography and SDS-PAGE data revealed the existence of a high-molecular-weight mannanase of 150 kDa that was active against galactoglucomannan and xylan. Its activity was optimal at pH 4.5. The K_m value with galactoglucomannan as substrate was 0.50 mg ml^?1. One xylanase with molecular mass of 79 kDa was also purified and characterized. Its activity was optimal at 60 °C and pH 8.0. Its K_m value with birchwood xylan as substrate was 1.65 mg ml^?1. Both the mannanase and the 79 kDa xylanase displayed relatively high activity on carboxymethyl cellulose. The sensitivity of the xylanase and mannanase to various salts was evaluated. None of the tested salts inhibited the xylanase, but Mn⁺², Fe⁺³, and Cu⁺² were strong inhibitors for the mannanase.     

Arginase from Bacillus thuringiensis SK 20.001: Purification,characteristics, and implications for l-ornithine biosynthesis

An l-ornithine high producing strain Bacillus thuringiensis SK20.001 was screened by our laboratory. An intracellular arginase used to biosynthesize l-ornithine from the strain was purified and characterized. The final specific arginase activity was 589.2 units/mg, with 70.1 fold enrichment and 22.4% recovery. The molecular weight of the enzyme was approximately 33,000 Da as evaluated by SDS-PAGE and 191,000 Da as determined by gel filtration. The enzyme had an optimum pH of 10.0 and an optimum temperature of 40 °C. It was stable from pH 8.0–12.0 and <50 °C without Mn²⁺. The presence of Mn²⁺ and Ni²⁺ had strong effects on the enzyme activity, and Mn²⁺ significantly increased the thermal stability of the enzyme. The arginase was slightly inhibited by Ca²⁺, Fe²⁺ and Zn²⁺. Trp, Asp, Glu, Tyr, and Arg residues were directly involved in the arginase activity evaluated by chemical modifications. The K_m and V_max for l-arginine were estimated to be 15.6 mM and 538.9 μmol/min/mg. The biosynthesis yield of l-ornithine was 72.7 g/L with the enzyme.     

Enzymatic properties of a novel highly active and chelator resistant protease from a Pseudomonas aeruginosa PD100

Pseudomonas aeruginosa PD100 capable of producing an extracellular protease was isolated from the soil collected from local area (garbage site) from Shivage market in Pune, India. The purified protease showed a single band on native and SDS-PAGE with a molecular weight of 36 kDa on SDS-PAGE. The optimum pH value and temperature range were found to be 8 and 55–60 °C, respectively. The enzyme exhibited broad range of substrate specificity with higher activity for collagen. The enzyme was inhibited with low concentration of Ag²⁺, Ni²⁺, and Cu²⁺. β-Mercaptoethanol was able to inactivate the enzyme at 2.5 mM, suggesting that disulfide bond(s) play a critical role in the enzyme activity. Studies with inhibitors showed that different classes of protease inhibitors, known to inhibit specific proteases, could not inhibit the activity of this protease. Amino acid modification studies data and pK_a values showed that Cys, His and Trp were involved in the protease activity. P. aeruginosa PD100 produces one form of protease with some different properties as compared to other reported proteases from P. aeruginosa strains. With respect to properties of the purified protease such as pH optimum, temperature stability with capability to degrade different proteins, high stability in the presences of detergents and chemicals, and metal ions independency, suggesting that it has great potential for different applications.