The mechanism of myeloperoxidase-dependent chlorination of monochlorodimedon

Chlorination of monochlorodimedon is routinely used to measure the production of hypochlorous acid catalysed by myeloperoxidase from H2O2 and Cl-. We have found that the myeloperoxidase/H2O2/Cl- system, at pH 7.8, catalysed the loss of monochlorodimedon with a rapid burst phase followed by a much slower steady-state phase. The loss of monochlorodimedon in the absence of Cl- was only 10% of the steady-state rate in the presence of Cl-, which indicates that the major reaction of monochlorodimedon was with hypochlorous acid. During the steady-state reaction, myeloperoxidase was present as 100% compound II, which cannot participate directly in hypochlorous acid formation. Monochlorodimedon was necessary for formation of compound II, since it was not formed in the presence of methionine. Both the amount of hypochlorous acid formed during the burst phase, and the steady-state rate of hypochlorous acid production, increased with increasing concentrations of myeloperoxidase and with decreasing concentrations of monochlorodimedon. Inhibition by monochlorodimedon was competitive with Cl-. From these results, and the ability of myeloperoxidase to slowly peroxidase monochlorodimedon in the absence of Cl-, we propose that the reaction of monochlorodimedon with the myeloperoxidase/H2O2/Cl- system involves a major pathway due to hypochlorous acid-dependent chlorination and a minor peroxidative pathway. Only a small fraction of compound I needs to react with monochlorodimedon instead of Cl- at each enzyme cycle, for compound II to rapidly accumulate. Monochlorodimedon, therefore, cannot be regarded as an inert detector of hypochlorous acid production by myeloperoxidase, but acts to limit the chlorinating activity of the enzyme. In the presence of reducing species that act like monochlorodimedon, the activity of myeloperoxidase would depend on the rate of turnover of compound II. Components of human serum promoted the conversion of ferric-myeloperoxidase to compound II in the presence of H2O2. We suggest, therefore, that in vivo the rate of turnover of compound II may determine the rate of myeloperoxidase-dependent production of hypochlorous acid by stimulated neutrophils.     

Purification and characterization of alcohol oxidase from <Emphasis Type="Italic">Paecilomyces variotii</Emphasis> isolated as a formaldehyde-resistant fungus

Paecilomyces variotii IRI017 was isolated as a formaldehyde-resistant fungus from wastewater containing formaldehyde. The fungus grew in a medium containing 0.5% formaldehyde and had consumed formaldehyde completely after 5 days. Alcohol oxidase was purified from the fungus grown on methanol. A 20-fold purification was achieved with a yield of 44%. The molecular mass of the purified enzyme was estimated to be 73 and 450 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography, respectively, suggesting that the enzyme consists of six identical subunits. The N-terminal amino acid sequence of the subunit was TIPDEVDIII. The enzyme showed an absorption spectrum typical of a flavoprotein and had a noncovalently bound flavin different from FAD, FMN, and riboflavin. The pH optimum of the enzyme activity was pH 6–10. The enzyme was stable in the pH range of pH 5–10. The enzyme retained full activity after incubation at 50°C for 30 min. The enzyme oxidized not only methanol but also lower primary alcohols and formaldehyde. The K _m values for methanol, ethanol, and formaldehyde were 1.9, 3.8, and 4.9 mmol l⁻¹, respectively.     

Purification and characterization of a mesophilic organic solvent tolerant lipase produced by Acinetobacter sp. K5b4

The extracellular lipase produced by Acinetobacter sp. K5b4 was purified to homogeneity using ultrafiltration (cutoff 30?KDa) followed by gel filtration chromatography on Sephadex G-50. The enzyme was purified to homogeneity with an apparent molecular mass of 133?KDa by SDS-PAGE. This purification resulted on 10.24 fold with 18.3% recovery. The K_m and V_max of purified enzyme when using pNPL hydrolysis were 4.0?mM and 73.53?nmol/ml/min, respectively. The pure enzyme was greatly stimulated in the presence of 20, 40 and 60% (v/v) methanol, DMSO and acetone whereas, ethanol, acetonitrile and propanol decreased the enzyme activity. Maximum enzyme activity was achieved at pH 7.0 and incubation temperature of 27?°C. The enzyme was stable within a pH range of 6.5 to 7 at 27?°C for 1?h. The enzyme activity was enhanced up to 36% by KCl, BaCl₂, MgCl₂ and CaCl₂ while obviously inhibited (10–20%) by CoCl₂, ZnCl₂, MnCl₂ and CuCl₂. No inhibitory effects were observed with 1.0 and 5.0?mM of 2-mercaptoethanol and EDTA. Similarly, SDS at 1.0?mM does not affect the enzyme activity while high reduction (80%) was observed at 5.0?mM SDS concentration. The enzyme was active against p-nitrophenyl esters of C8, C12 and C16 with highest preference to the medium carbon chain p-nitrophenyl caprylate (C8). The fact that the enzyme displays distinct stability in the presence of methanol, DMSO and acetone suggests that this lipase is suitable as biocatalyst in organic synthesis where such hydrophilic organic solvents are used as a reaction media.     

Effect of organic solvents on a chloroperoxidase biotransformation

The effect of organic solvents on the chlorination activity of chloroperoxidase (CPO) was identified for use in biotransformations with CPO. CPO was found to chlorinate monochlorodimedon (MCD) in the presence of organic solvents with log P values less than 0. The relative rates of chlorination with chloride ion in the presence of H₂O₂, buffer and 2.5–20% of either dimethyl sulfoxide, N,N-dimethyl formamide, methanol or acetonitrile, were in the range of 10–58% of that in buffer (pH 2.8) at the same reactant concentrations. The presence of such organic solvents was found to alter CPO catalysis by altering the protein conformation and the local environment at the active site. CPO did not display chlorination activity in the presence of organic solvents which had log P values greater than 0.     

Burkholderia cepacia lipase is a promising biocatalyst for biofuel production

Lipases resistant to inhibition and denaturation by methanol are valuable tools for biotechnological applications, in particular for biofuel production. Microbial lipases have attracted a great deal of interest because of their stability at high concentrations of organic solvents. Burkholderia cepacia lipase (BCL) is tested here for robustness towards methanol in terms of conformational stability and catalytic activity in transesterification assays. This lipase turns out to be even more tolerant than the homologous and better characterized enzyme from Burkholderia glumae. BCL unfolding transition, as monitored by far‐UV circular dichroism (CD) and intrinsic fluorescence, displays a T_m above 60°C in the presence of 50% methanol. The protein unfolds at low pH, and the organic solvent affects the nature of the denatured state under acidic conditions. The protein performs well in transesterification assays upon prolonged incubations at high methanol concentrations. BCL is highly tolerant to methanol and displays particularly high conformational stability under conditions employed for transesterification reactions. These features depict BCL as a promising enzyme for biofuel industry.     

Partial purification and kinetic properties of cytoplasmic malate dehydrogenase from ovine liver Echinococcus granulosus protoscolices

Cytoplasmic malate dehydrogenase from ovine liver Echinococcus granulosus protoscolices was purified 22-fold by QAE- and SP-Sephadex chromatography. The pH optimum of the enzyme was 8.0 in either Tris-HCl or barbital buffer. The κ_m values of oxaloacetate and NADH were 0.400 ± 0.018 and 0.410 ± 0.038 mM, respectively. The enzyme lost about 90% of its activity when heated for 2 min at 65°C. A 61.4% inhibition of the enzyme was noted at 4 mM concentration of diethyl pyrocarbonate. A 3 mM concentration of fructose 1,6-diphosphate inhibited the enzyme by 76.5%. The inhibition was non-competitive with respect to NADH with a κ_i value of 0.85 mM. A 75% inhibition of the enzyme was noted at 1 mM concentration of mebendazole that inhibited the enzyme upon competing with NADH with a κ_i value of 0.176 mM. A 2-mM concentration of citrate almost doubled the enzyme activity. The enzyme was inhibited at high concentrations of either substrate. The enzyme was not inhibited by p-hydroxymercuribenzoate or fumarate. The enzyme was absolutely specific for NADH as a cofactor. The properties of this enzyme are compared with those of the enzyme from the host liver, the cyst fluid and some other animal sources. The results are discussed in terms of the differences among the properties of the host liver, the cyst fluid and the protoscolices enzymes. The biochemical basis for the use of mebendazole in the treatment of echinococcosis is also elucidated.     

pH-dependent modulation of alkaline phosphatase activity in Serratia marcescens

Serratia marcescens is an opportunistic pathogen responsible for causing nosocomial infections, corneal ulcer, necrotizing fasciitis, cellulites, and brain abscess. Alkaline phosphatase (APase) is believed to play an important role in the survival of several intracellular pathogens and their adaptation. We have studied the effect of low phosphate concentration and acid pH on the APase activities of S. marcescens. In a low phosphate medium, some strains of S. marcescens synthesize two different types of APases, a constitutive (CAPase) and an inducible (IAPase). Both the CAPase and IAPase isoenzymes completely lost their enzyme activities at pH 2.3, within 10 min of incubation at 0°C. Acid-treated IAPase isoenzymes I, II, III, and IV solutions when adjusted to pH 7.8 showed recovery of 70%, 52%, 72%, and 60% of the lost activities, respectively. When the pH of the CAPase reaction mixture was raised to pH 7.8, the enzyme activity regained only 5% of its initial activity. Variations in protein concentration also affected the pH-dependent reversible changes of the IAPase activity. The higher the protein concentration, the faster the inactivation of enzyme activity observed at acidic pH at 0°C. Conversely, the lower the protein concentration, the higher the rate of reactivation of enzyme activity observed for IAPase at alkaline pH. Protein interaction studies revealed a lack of similarity between CAPase and IAPase, suggesting separate genetic origin of these potentially virulent genes of S. marcescens. Received: 4 December 2001 / Accepted: 7 January 2002     

Characterisitics and Gene Cloning of Phospholipase D of the Psychrophile,Shewanella sp.

Phospholipase D, with a molecular mass of 64 kDa, was purified from the psychrophile, Shewanella sp. The enzyme showed maximal activity at pH 7.8 and 40 °C in the presence of the Ca²⁺-ion, and its activity at 10 °C was 6.5% of maximum. The enzyme exhibited high activity to the non-micelle form of phosphatidylcholine in an aqueous solution containing water miscible alcohols such as methanol, ethanol, iso-propanol, and n-propanol. Nucleotide sequencing of the enzyme gene yielded a deduced amino acid sequence, which showed 36.2% identity to that of Streptomyces chromofuscus phopsholipase D alone. The low sequence similarity to other phopsholipase D enzymes suggests that the purified enzyme might be a novel phospholipase D.     

Oxidation of Methanol by the Yeast,Pichia pastoris. Purification and Properties of the Alcohol Oxidase

The enzymes of methanol oxidation were investigated in a new yeast strain, Pichia pastoris IFP 206, with high yield (0.42 g cell per g of methanol). The following enzymes were detected in cell free extracts of P. pastoris: alcohol oxidase, catalase, formaldehyde and formate dehydrogenases. The alcohol oxidase was purified from cell free extracts of P. pastoris containing high amount of the enzyme (33%) with a good yield (55%). The preparation was homogenous by immunochemical methods. The enzyme had a molecular weight of 675,000 and was composed of eight identical subunits of M.W. 80,000. Each subunit contained one FAD. The N-terminal sequence was found to be: Ala-Ile-Pro-Glu-Glu-Phe-Asp-Ile-Leu-Val-Leu-Gly-The protein had 65 free ?SH groups per molecule. The optimum temperature for the enzyme activity was 37°C and the activation energy was 11.1 kcal/mol. Optimum pH was 7.5 and the enzyme activity was unstable at acidic pH. The apparent Km for methanol were 1.4 and 3.1 mm at oxygen concentrations of 0.19 and 0.93 mm. Similarly, the apparent Kms for oxygen were 0.40 and 1.0 mm at methanol concentrations of 1, 10 and 100 mm. The enzyme oxidized primary alcohols with short carbon chains like ethanol and propanol. Inhibition of enzyme activity by hydrogen peroxide was a consequence of the oxidation of essential ?SH groups. The inhibition was reversed by reducing agents.     

Production of cellulases by Thermomucor indicae-seudaticae: characterization of a thermophilic β-glucosidase

Abstract

The current study evaluated the production and characterization of β-glucosidase by the thermophilic fungus Thermomucor indicae-seudaticae in solid-state fermentation of wheat bran. Isolated fungi have significant amounts of β-glucosidase, an enzyme that may be applied to different industrial processes, such as the production of fuels, food, and other chemical compounds. Maximal enzyme activity occurred in pH 3.5–4.5 and at 70?°C. The enzyme exhibited high thermostability, for 1?h, up to 60?°C, and good tolerance to glucose (10?mM) and ethanol (10%). The optimization of fermentative parameters on the production of β-glucosidase was carried out by evaluating the best supplementary nutrient source, pH of nutrient solution, initial substrate moisture and fermentation temperature. The optimization of the above fermentation parameters increased enzyme activity by 120.0%. The highest enzymatic activity (164.0?U/g) occurred with wheat bran containing 70% initial moisture, supplemented with 1.0% (NH₄)₂SO₄ solution at pH 5.5–6.0 and fungus incubated at 40?°C. A more detailed study of β-glucosidase suggested that Sulfur is an important component of the main amino acid present in this enzyme. The enhancer of the enzyme activity occurred when the fungus was grown on wheat bran supplemented with a sulfur-containing solution. In fact, increasing the concentration of sulfur in the solution increased its activity.     

Effect of several factors on soluble lipase-mediated biodiesel preparation in the biphasic aqueous-oil systems

Biodiesel is increasingly perceived as an important component of solutions to the important current issues of fossil fuel shortages and environmental pollution. Utilization of soluble lipase offers an alternative approach to lipase-catalyzed biodiesel production using immobilized enzyme or whole-cell catalysis. Soluble lipase NS81020, produced by submerged fermentation of genetically modified Aspergillus oryzae microorganism, was first proposed here as the catalyst of biodiesel preparation with oleic acid in the biphasic aqueous-oil systems. The effect factors such as enzyme concentration, water content, temperature, molar ratio of methanol to oil, stirring rate and pH of buffer solution on the esterification rate were investigated systematically. The reaction time could be shortened with the increasing of enzyme concentration as long as the maximum enzyme absorptive capacity on the interface in the biphasic aqueous-oil systems was not achieved. The optimal water content in the biphasic aqueous-oil systems was 10 wt% by oleic acid weight. The reaction rate was enhanced with the increasing molar ratio of methanol to oil, the increasing stirring rate or the decreasing temperature. Although soluble lipase NS81020 had lower activity at pH 10.55, hydroxyl ion conduced to restrain hydrolysis of methyl ester and facilitated the reaction toward the methyl ester formation.     

Inhibition of hyaluronan uptake in lymphatic tissue by chondroitin sulphate proteoglycan.

Stimulated neutrophils discharge large quantities of superoxide (O2.-), which dismutates to form H2O2. In combination with Cl-, H2O2 is converted into the potent oxidant hypochlorous acid (HOCl) by the haem enzyme myeloperoxidase. We have used an H2O2 electrode to monitor H2O2 uptake by myeloperoxidase, and have shown that in the presence of Cl- this accurately represents production of HOCl. Monochlorodimedon, which is routinely used to assay production of HOCl, inhibited H2O2 uptake by 95%. This result confirms that monochlorodimedon inhibits myeloperoxidase, and that the monochlorodimedon assay grossly underestimates the activity of myeloperoxidase. With 10 microM-H2O2 and 100 mM-Cl-, myeloperoxidase had a neutral pH optimum. Increasing the H2O2 concentration to 100 microM lowered the pH optimum to pH 6.5. Above the pH optimum there was a burst of H2O2 uptake that rapidly declined due to accumulation of Compound II. High concentrations of H2O2 inhibited myeloperoxidase and promoted the formation of Compound II. These effects of H2O2 were decreased at higher concentrations of Cl-. We propose that H2O2 competes with Cl- for Compound I and reduces it to Compound II, thereby inhibiting myeloperoxidase. Above pH 6.5, O2.- generated by xanthine oxidase and acetaldehyde prevented H2O2 from inhibiting myeloperoxidase, increasing the initial rate of H2O2 uptake. O2.- allowed myeloperoxidase to function optimally with 100 microM-H2O2 at pH 7.0. This occurred because, as previously demonstrated, O2.- prevents Compound II from accumulating by reducing it to ferric myeloperoxidase. In contrast, at pH 6.0, where Compound II did not accumulate, O2.- retarded the uptake of H2O2. We propose that by generating O2.- neutrophils prevent H2O2 and other one-electron donors from inhibiting myeloperoxidase, and ensure that this enzyme functions optimally at neutral pH.     

Immobilization imparts stability to watermelon urease to work in water miscible organic media

The behaviour of alginate immobilized and soluble watermelon (Citrullus vulgaris) urease in water miscible organic solvents like, acetonitrile, dimethylformamide (DMF), ethanol, methanol, and propanol is described. The organic solvents exhibited a concentration dependent inhibitory effect on both the immobilized and the soluble urease in the presence of urea. Pretreatment of soluble enzyme preparations with organic solvents in the absence of substrate for 10 min at 30°C led to rapid loss in the activity, while similar pretreatment of immobilized urease with 50% (v/v) of ethanol, propanol, and acetonitrile was ineffective. Time-dependent inactivation of immobilized urease, both in the presence and in the absence of urea, revealed stability for longer duration of time even at very high concentration of organic solvents. The soluble enzyme, on the other hand, was rapidly inactivated even at fairly lower concentrations. The results suggest that the immobilization of watermelon urease in calcium alginate make it suitable for its application in organic media. the observations are discussed.     

Methanol-Assimilation durch ein acidophiles Bacterium der Gattung Acetobacter

Generally, methylotrophic bacteria grow optimally in a pH range between 6 and 7.2. The assimilation of methanol can take place via several pathways. Acetobacter methanolicus preiers an acidic pH range for growth, the pH optimum is about 4, and it uses the FBP variant for methanol assimilation. The latter is interesting from a regulatory point of view because phosphofructokinase disappears during growth on glucose, which is assimilated via the hexosemonophosphate pathway. Since Entner-Doudoroff enzymes and phosphoketolase are absent in A. `ethanolicus as well as in non-methylotrophic Acetobacter and Gluconobacter species phosphofructokinase becomes a key enzyme of the assimilation of methanol. Although A. methanolicus uses the hexulosephosphate pathway the growth yield on methanol is smaller than with other “hexulosephosphate pathway bacteria” e. g. with obligate methanol assimilating bacteria. At first sight it may appear that the acidic optimum pH is responsible for the smaller growth yield and the discrepancy between the experimental and predicted values. The relationship between the dependence on and the protection from, high external proton concentration on the one hand and the causes of the low growth yield on the other are discussed. Accordingly, A. methanolicus and another heterotrophic acidophiles seem to be acidoresistant above all, their machinery guaranteeing the protection from the high proton concentration is responsible for the acidophily and the low growth efficiency is caused by a simple respiratory chain.     

The alkaline adenosine triphosphatase activity of 30S dynein after modification of the SH groups. Possible involvement of some of the most reactive SH groups.

An apparent 'triphasic' alteration of 30S dynein ATPase activity was produced by treatment with various amounts of NEM when the modification and susequent ATPase assay were carried out at pH 7.4 and pH 10-10.2, respectively. The Mg-ATPase activity was markedly inhibited by modification of the most reactive SH groups with 10 microM NEM, although the same treatment had no significant effect on the activity when assayed at neutral pH. Increasing the NEM concentration to 0.3 mM largely restored the enzyme activity, but a further increase in NEM concentration inhibited the enzyme activity again. This unusual response of 30S dynein ATPase at pH 10-10.2 was accounted for by the results of Arrhenius plots of the enzyme activity at pH 10.1; the enzyme protein modified with not more than 10 microM NEM was not stable under the assay conditions (pH 10-10.2 at 25 degrees C), whereas modification with 0.3 mM NEM stabilized 30S dynein against the assay conditions. The possible significance of the 10 microM NEM-induced inhibition of the 30S dynein alkaline ATPase activity is discussed in connection with the participation of SH groups of 30S dynein in the enzyme activity.     

Bioconversion of methanol to formaldehyde. II. By purified methanol oxidase from modified yeast, Hansenula polymorpha

Modified methylotrophic yeast Hansenula polymorpha (HP A16) that was obtained by repressing leucine oxotrophic yeast; a wild type of Hansenula polymorpha CB4732 was used in this study. The yeast is grown with methanol, which is used as a sole carbon source, using various methanol concentrations and temperatures, and methanol oxidase (MOX) which is a key enzyme of methanol metabolism; production is maximized. Whole yeast cells were cultivated under optimized inoculation conditions; they were separated into two portions. One portion of these cells was directly used in bioconversion of methanol to formaldehyde. The second portion of the free cells was broken into pieces and a crude enzyme extract was obtained. The MOX enzyme in this extract was purified via salt precipitation, dialysis, and chromatographic methods. The purified MOX enzyme of yeast (HP A16) oxidized the methanol to formaldehyde. Optimization of bioconversion conditions was studied to reach maximum activity of enzyme. The optimum temperature and pH were found to be 35 degrees C and pH 8.0 in boric acid/NaOH buffer, and it was stable over the pH range of 6-9, at the 20 degrees C 15 min. A suitable reaction period was found as 50 min. The enzyme indicated low carbon primary alcohols (C2 to C4), as well as methanol. Initially, MOX activity increased with the increase of methanol concentration, but enzyme activity decreased. The apparent Km and Vmax values for methanol substrate of HP A16 MOX were 0.25 mM and 30 U/mg, respectively. The purified MOX enzyme was applied onto sodium dodecyl sulphate-polyacrylamide gel electrophoresis; molecular weight of the enzyme was calculated to be about 670 kDa. Each MOX enzyme is composed of eight identical subunits, each of whose molecular weight is around 82 kDa and which contain eight moles of FAD as the prosthetic group, and the pI of the natural enzyme is found to be 6.4. The purified MOX enzyme was used in the bioconversion of methanol to formaldehyde as a catalyst; this conversion was compared to the conversion percentages of whole cells in our previous article in terms of catalytic performances.     

Improved production of polygalacturonate lyase by combining a pH and online methanol control strategy in a two-stage induction phase with a shift in the transition phase

Polygalacturonate lyase is a kind of enzyme that is abundantly used in the textile industry for cotton scouring. Previously, we reconstructed the polygalacturonate lyase gene in Pichia pastoris for the expression of this enzyme. To enhance the production of polygalacturonate lyase (PGL), a combined strategy was formulated by combining online methanol control and two-stage pH control strategies. For the two-stage pH control strategy during the growth phase, the pH was controlled at 5.5, and in the induction phase different pH levels were investigated for the optimum enzyme production. During the online methanol control strategy, the different levels of methanol (v/v) were investigated for the best enzyme production at pH 5.5. These two strategies were combined together for enhanced PGL productivity, and the induction phase was divided into two stages in which methanol concentrations were maintained at different levels online. The transition phase was introduced during the induction phase instead of introducing it after the growth phase. The two-stage combination strategy was formulated on the bases of methanol consumption of cells, optimal pH, cell viability and the production of polygalacturonate lyase by P. pastoris. By using this strategy, the production was doubled compared with common conditions, and the highest polygalacturonate lyase activity reached 1,631 U/ml. This strategy proved to be very useful for the enhancement of polygalacturonate lyase production by achieving higher cell viability, alcohol oxidase activity and phosphate-related compounds of the cells during the induction phase.     

Inversion of sucrose with β- -fructofuranosidase (invertase) immobilized on bead DEAHP-cellulose: Batch process

The inversion of sucrose with β- -fructofuranosidase (EC 3.2.1.26) immobilized by an ionic bond on bead cellulose containing weak basic N,N-diethylamino-2-hydroxypropyl groups has been investigated. The immobilized enzyme is strongly bound at an ionic strength up to 0.1 M in the pH range 3–6. The amount adsorbed is proportional to porosity and to the exchange capacity of the ion exchange cellulose, reaching values up to 200 mg/g dry carrier, with an activity in 10% sucrose solution at 30°C, pH 5, >8000 μmol min⁻¹ g⁻¹. The inversion of sucrose with immobilized β- -fructofuranosidase was carried out in a stirred reactor. The dependence of activity on pH (3–7), temperature (0–70°C) and concentration of the substrate (2–64 wt%) were determined, and the inversion was compared with that obtained using non-immobilized enzyme under similar conditions. The rate of inversion at low substrate concentration (2–19 wt%) was described by Michaelis-Menten kinetics.     

Production and partial characterization of chitinase from a halotolerant <Emphasis Type="Italic">Planococcus rifitoensis</Emphasis> strain M2-26

This paper is the first to investigate the production and partial characterization of the chitinase enzyme from a moderately halophilic bacterium Planococcus rifitoensis strain M2-26, earlier isolated from a shallow salt lake in Tunisia. The impact of salt, salinity concentration, pH, carbon and nitrogen sources on chitinase production and activity have been determined. This is the first report on a high salt-tolerant chitinase from P. rifitoensis, since it was active at high salinity (from 5 to 30% NaCl) as well as in the absence of salt. This enzyme showed optimal activity at 70°C and retained up to 82 and 66% of its original activity at 80 or 90°C, respectively. The activity of the enzyme was also shown over a wide pH range (from 5 to 11). For characterization of the enzyme activity, the chitinase secreted in the culture supernatant was partially purified. The preliminary study of the concentrated dialysed supernatant on native PAGE showed at least three chitinases produced by strain M2-26, with highest activity approximately at 65 kDa. Thus, the thermo-tolerant and high salt-tolerant chitinases produced by P. rifitoensis strain M2-26 could be useful for application in diverse areas such as biotechnology and agro-industry.     

Fibrinolysis and anticoagulant potential of a metallo protease produced by <Emphasis Type="Italic">Bacillus subtilis</Emphasis> K42

In this study, a potent fibrinolytic enzyme-producing bacterium was isolated from soybean flour and identified as Bacillus subtilis K42 and assayed in vitro for its thrombolytic potential. The molecular weight of the purified enzyme was 20.5 kDa and purification increased its specific activity 390-fold with a recovery of 14%. Maximal activity was attained at a temperature of 40°C (stable up to 65°C) and pH of 9.4 (range: 6.5–10.5). The enzyme retained up to 80% of its original activity after pre-incubation for a month at 4°C with organic solvents such as diethyl ether (DE), toluene (TO), acetonitrile (AN), butanol (BU), ethyl acetate (EA), ethanol (ET), acetone (AC), methanol (ME), isopropanol (IP), diisopropyl fluorophosphate (DFP), tosyl-lysyl-chloromethylketose (TLCK), tosyl-phenylalanyl chloromethylketose (TPCK), phenylmethylsulfonylfluoride (PMSF) and soybean trypsin inhibitor (SBTI). Aprotinin had little effect on this activity. The presence of ethylene diaminetetraacetic acid (EDTA), a metal-chelating agent and two metallo protease inhibitors, 2,2′-bipyridine and o-phenanthroline, repressed the enzymatic activity significantly. This, however, could be restored by adding Co²⁺ to the medium. The clotting time of human blood serum in the presence of this enzyme reached a relative PTT of 241.7% with a 3.4-fold increase, suggesting that this enzyme could be an effective antithrombotic agent.