Structural stability and functional analysis of L-asparaginase from <Emphasis Type="Italic">Pyrococcus furiosus</Emphasis>

We report studies on an L-asparaginase from Pyrococcus furiosus, cloned and expressed in Escherichia coli and purified to homogeneity. Protein stability and enzyme kinetic parameters were determined. The enzyme was found to be thermostable, natively dimeric, and glutaminase-free, with optimum activity at pH 9.0. It showed a K _m of 12 mM and a substrate inhibition profile above 20 mM L-asparagine. Urea could not induce unfolding and enzyme inactivation; however, with guanidine hydrochloride (GdnCl) a two-state unfolding pattern was observed. Reduced activity and an altered near-UV-CD signal for protein at low GdnCl concentration (1 M) suggested tertiary structural changes at the enzyme active site. A homology three-dimensional model was developed and the structural information was combined with activity and stability data to give functional clues about the asparaginase.     

Optimization,purification, and characterization of L-asparaginase from Actinomycetales bacterium BkSoiiA

Actinobacteria are promising source of a wide range of important enzymes, some of which are produced in industrial scale, with others yet to be harnessed. L-Asparaginase is used as an antineoplastic agent. The present work deals with the production and optimization of L-asparaginase from Actinomycetales bacterium BkSoiiA using submerged fermentation in M9 medium. Production optimization resulted in a modified M9 medium with yeast extract and fructose as carbon and nitrogen sources, respectively, at pH 8.0, incubated for 120 hr at 30 ± 2°C. The crude enzyme was purified to near homogeneity by ammonium sulfate precipitation following dialysis, ion-exchange column chromatography, and finally gel filtration. The sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) revealed an apparent molecular weight of 57 kD. The enzyme was purified 95.06-fold and showed a final specific activity of 204.37 U/mg with 3.49% yield. The purified enzyme showed maximum activity at a pH 10.0 and was stable at pH 7.0 to 9.0. The enzyme was activated by Mn²⁺ and strongly inhibited by Ba²⁺. All these preliminary characterization suggests that the L-asparaginase from the source may be a tool useful to pharmaceutical industries after further research.     

CHEMICAL MODIFICATION OF L-ASPARAGINASE FROM Cladosporium sp. FOR IMPROVED ACTIVITY AND THERMAL STABILITY

L-Asparaginase (ASNase), an antileukemia enzyme, is facing problems with antigenicity in the blood. Modification of L-asparaginase from Cladosporium sp. was tried to obtain improved stability and improved functionality. In our experiment, modification of the enzyme was tried with bovine serum albumin, ovalbumin by crosslinking using glutaraldehyde, N-bromosuccinimide, and mono-methoxy polyethylene glycol. Modified enzymes were studied for activity, temperature stability, rate constants (k_d), and protection to proteolytic digestion. Modification with ovalbumin resulted in improved enzyme activity that was 10-fold higher compared to native enzyme, while modification with bovine serum albumin through glutaraldehyde cross-linking resulted in high stability of L-asparaginase that was 8.5- and 7.62-fold more compared to native enzyme at 28°C and 37°C by the end of 24 hr. These effects were dependent on the quantity of conjugate formed. Modification also markedly prolonged L-asparaginase half-life and serum stability. N-Bromosuccinimide-modified ASNase presented greater stability with prolonged in vitro half-life of 144 hr to proteolytic digestion relative to unmodified enzyme (93 h). The present work could be seen as producing a modified L-asparaginase with improved activity and stability and can be a potential source for developing therapeutic agents for cancer treatment.     

Indicator dye based screening of glutaminase free L-asparaginase producer and kinetic evaluation of enzyme production process

Abstract

Several soil isolates from 1 g of soil sample were isolated and screened for the production of L-asparaginase. Primary screening was performed using rapid plate assay; dye indicator studies were conducted, and phenol red with 0.005% concentration was found to be optimum. The secondary screening was carried out using the Nesslerization method. The bacteria screened for L-asparaginase production with no glutaminase activity was identified as Bacillus subtilis. Crude L-asparaginase enzyme was partially purified 1.57 folds of purity and 110 U/mg of specific activity. The glutaminase-free L-asparaginase activity was also confirmed using LC-MS analysis. The presence of mass peaks at 147.0 in the reaction mixture suggested an absence of glutaminase activity. An optimized medium obtained comprised of Dextrose 1.5 g/L, K₂HPO₄ 1.2 g/L, L-asparagine 15 g/L, and Tryptone 5 g/L. The highest L-asparaginase activity was observed at 6.0 pH and 30 °C. Kinetic parameters associated with biomass and L-asparaginase production were also studied. The computed values were µ_m 0.104 h^?1, X_m 6g/L P₀ 1.7U/mL P_m 8.2 U/mL Y_X/S 4 g-cell/g-glucose µP_m 0.35 h^?1 q_p 5.46 U/g/h Y_P/x 13.6667 U/g-cell. The novel bacterial isolates showed promise as a potential glutaminase-free L-asparaginase producer, which can prove to be of industrial applications.     

Isolation and screening of endophytes from the rhizomes of some Zingiberaceae plants for L-asparaginase production

Endophytes are described as microorganisms that colonize the internal tissues of healthy plants without causing any disease. Endophytes isolated from medicinal plants have been attracting considerable attention due to their high biodiversity and their predicted potential to produce a plethora of novel compounds. In this study, an attempt was made to isolate endophytes from rhizomes of five medicinal plants of Zingiberaceae family, and to screen the endophytes for L-asparaginase activity. In total, 50 endophytes (14 bacteria, 22 actinomycetes, and 14 fungi) were isolated from Alpinia galanga, Curcuma amada, Curcuma longa, Hedychium coronarium, and Zingiber officinale; of these, 31 endophytes evidenced positive for L-asparaginase production. All the L-asparaginase-positive isolates showed L-asparaginase activity in the range of 54.17–155.93 U/mL in unoptimized medium. An endophytic fungus isolated from Curcuma amada, identified as Talaromyces pinophilus, was used for further experiments involving studies on the effect of certain nutritional and nonnutritional factors on L-asparaginase production in submerged fermentation. Talaromyces pinophilus initially gave an enzyme activity of 108.95 U/mL, but gradually reduced to 80 U/mL due to strain degeneration. Perhaps this is the first report ever on the production of L-asparaginase from endophytes isolated from medicinal plants of Zingiberaceae family.     

Studies on pH and thermal stability of novel purified L-asparaginase from <Emphasis Type="Italic">Pectobacterium carotovorum</Emphasis> MTCC 1428

Glutaminase free L-asparaginase is known to be an excellent anticancer agent. In the present study, the combined effect of pH and temperature on the performance of purified novel L-asparaginase from Pectobacterium carotovorum MTCC 1428 was studied under assay conditions using response surface methodology (RSM). Deactivation studies and thermodynamic parameters of this therapeutically important enzyme were also investigated. The optimum pH and temperature of the purified L-asparaginase were found to be 8.49 and 39.3°C, respectively. The minimum deactivation rate constant (k _d ) and maximum half life (t _1/2) were found to be 0.041 min⁻¹ and 16.9 h, respectively at pH of 8.6 and 40°C. Thermodynamic parameters (ΔG, ΔH, ΔS, and activation energies) were also evaluated for purified L-asparaginase. The probable mechanism of deactivation of purified L-asparaginase was explained to an extent on the basis of deactivation studies and thermodynamic parameters.     

Estimation of Kinetic Parameters for Bioremediation of Cr(VI) from Wastewater Using Pseudomonas taiwanensis,an Isolated Strain from Enriched Mixed Culture

An aerobic mixed culture collected in the form of activated sludge was enriched for Cr(VI) reduction. An indigenous microorganism was isolated from the enriched aerobic mixed culture and identified as Pseudomonas taiwanensis. Bioremediation studies were carried out for treating Cr(VI)-contaminated wastewater using the indigenous microorganism. The kinetic studies were carried out for initial Cr(VI) concentrations ranging from 20 to 200 mg L^?1. The maximum consumption of Cr(VI) obtained was 108.3 mg L^?1 for an initial Cr(VI) concentration of 150 mg L^?1 at a solution pH of 7.0. The effect of nutrient dosage and pH were studied to get their optimum values. The same isolated bacterial strain was also used to treat Cr(VI)-contaminated industrial wastewater collected from a local plating industry. Various growth kinetic models, such as Monod, Powell, Haldane, Luong, and Edward models, were fitted with the obtained experimental data. The obtained results for different growth kinetic models indicate that the growth kinetics of Pseudomonas taiwanensis for bioremediation of Cr(VI) can be better understood by the Luong model (R² = .913). The rate kinetic analysis was performed using zero-order and three-half-order kinetic models. The three-half-order kinetic model was found to be suitable for the present bioremediation study.     

Gene cloning and characterization of recombinant L-Asparaginase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strain R5

L-asparaginase gene from Bacillus subtilis strain R5 (Asn-R5), comprising 990 nucleotides corresponding to a polypeptide of 329 amino acids, was cloned and expressed in Escherichia coli. Recombinant Asn-R5 was produced in soluble and active form exhibiting a specific activity of 223 μmol min^?1 mg^?1. The optimal temperature and pH for L-asparaginase activity of Asn-R5 were 35 °C and 9.0, respectively. Asn-R5 displayed a 50% activity with D-asparagine and 2% with L-glutamine compared to 100% with L-asparagine. No activity could be detected when D-glutamine was used as substrate. Half-life of the enzyme was 180 min at 35 °C and 40 min at 50 °C. There was no effect of metal ions and EDTA on the activity indicating that Asn-R5 enzyme activity is not metal ion dependent. The K_m and V_max values were 2.4 mM and 265 μmol min^?1 mg^?1, respectively. Activation energy for reaction catalyzed by Asn-R5 was 28 kJ mol^?1. High L-asparaginase activity and thermostability of recombinant Asn-R5 may be beneficial for industrial production and application.     

L-asparaginase production by <Emphasis Type="Italic">Streptomyces albidoflavus</Emphasis>

Attempts were made to optimize the cultural conditions for the production of L-asparaginase by Streptomyces albidoflavus under submerged fermentations. Enhanced level of L-asparaginase was found in culture medium supplemented with maltose as carbon source. Yeast extract (2%) was served as good nitrogen source for the production of L-asparaginase. The optimum pH for enzyme production was 7.5 and temperature was 35°C. The release of L-asparaginase from the cells of S. albidoflavus was high when strain was treated with cell disrupting agents like EDTA and lysozyme. The enzyme produced by the strain was purifi ed by ammonium sulfate, Sephadex G-100 and CM-Sephadex C-50 gel fi ltration and the molecular weight was apparently determined as 112 kDa.     

Purification and characterization of L-asparaginase with anti-lymphoma activity from Vibrio succinogenes.

Homogeneols L-asparaginase with anti-lymphoma activity was prepared from Vibrio succinogenes, an anaerobic bacterium from the bovine rumen. An overall yield of pure L-asparaginase of 40 to 45% and a specific activity of 200 +/- 2 IU per mg of protein was obtained. The pure enzyme can be stored at -20 degrees for at least 3 months with no loss of activity. The isoelectric point of the L-asparaginase is 8.74. No carbohydrate, phosphorus, tryptophan, disulfide, or sulfhydryl groups were detected. The enzyme has a molecular weight of 146,000 and a subunit weight of approximately 37,000. The Km of the enzyme for L-asparagine is 4.78 X 10(-5) M and the pH optimum of the L-asparaginase reaction is 7.3. D-Asparagine was hydrolyzed at 6.5% of the rate found with the L isomer. L-Glutamine and a variety of other amides were not hydrolyzed at significant rates; the activity of the enzyme for L-glutamine was 130- to 600-fold less than that of other therapeutically effective L-asparaginases of bacterial origin. The L-asparaginase from V. succinogenes is immunologically distinct from the L-asparaginase (EC-2) of Escherichia coli.     

L-Asparaginase activity in Leptosphaeria michotii. Isolation and properties of two forms of the enzyme

The properties of L-asparaginase (EC 3.5.1.1) in Leptosphaeria michotii (West) Sacc., which has previously been shown to have an activity rhythm, were analyzed. Two forms of L-asparaginase were isolated from acetic acid and ammonium sulfate fractionations followed by DEAE-Sephacel chromatography. The activity of L-asparaginase changed rhythmically with the same period as that of crude extracts, but the rhythms of the two enzyme forms were out of phase. The two asparaginase forms differed in their isoelectric points and the substrate concentrations for attaining half-maximal velocity; non-Michaelis-Menten kinetics for hydrolysis of L-asparagine were observed. Analyses of asparaginase form II by polyacrylamide gel electrophoresis showed that four proteins, irrespective of the phase of the activity rhythm at which the enzyme was extracted, could be detected: asparaginase oligomer (M_r 130 000 to 140 000), its dimer, an aggregate (M_r 500 000 to 600 000) having a low asparaginase activity, and a protein (M_r 60 000) without asparaginase activity; the same proteins were found in asparaginase form I. These results indicate that L. michotii asparaginase could be implicated in a protein complex.     

PRODUCTION AND OPTIMIZATION OF L-GLUTAMINASE ENZYME FROM Hypocrea jecorina PURE CULTURE

L-Glutaminase (L-glutamine amidohydrolase, EC 3.5.1.2) is the important enzyme that catalyzes the deamination of L-glutamine to L-glutamic acid and ammonium ions. Recently, L-glutaminase has received much attention with respect to its therapeutic and industrial applications. It acts as a potent antileukemic agent and shows flavor-enhancing capacity in the production of fermented foods. Glutaminase activity is widely distributed in plants, animal tissues, and microorganisms, including bacteria, yeasts, and fungi. This study presents microbial production of glutaminase enzyme from Hypocrea jecorina pure culture and determination of optimum conditions and calculation of kinetic parameters of the produced enzyme. The optimum values were determined by using sa Nesslerization reaction for our produced glutaminase enzyme. The optimum pH value was determined as 8.0 and optimum temperature as 50°C for the glutaminase enzyme. The Km and Vmax values, the kinetic parameters, of enzyme produced from Hypocrea jecorina, pure culture were determined as 0.491 mM for Km and 13.86 U/L for Vmax by plotted Lineweaver–Burk graphing, respectively. The glutaminase enzyme from H. jecorina microorganism has very high thermal and storage stability.     

Effect of chemical and physical parameters on the production of L-asparaginase from a newly isolated Serratia marcescens SK-07

Aims: The objective of this study is to optimize the levels of carbon and nitrogen sources of the medium in shake flask experiments and evaluate the effect of pH and dissolved oxygen (DO) on the production of l ‐asparaginase from a newly isolated Serratia marcescens SK‐07 in a batch bioreactor. Methods and Results: Central composite rotatable design (CCRD) was applied to optimize the levels of carbon and nitrogen sources of the medium in shake flask experiments. The optimal levels of l ‐asparagine, glucose, yeast extract and peptone were found to be 4·93, 3·81, 3·65 and 1·47 g l^?1, respectively, and maximal l ‐asparaginase production of 25·02 U mg^?1 was obtained under these conditions. Among the carbon sources tested, l ‐asparagine was identified to be the most favourable carbon source for enhanced production of l ‐asparaginase. The maximum l ‐asparaginase production of 29·89 U mg^?1 was achieved in a batch bioreactor at initial pH of 6·5 (uncontrolled) and DO level of 40% in the culture. Conclusions: We have isolated, screened and identified the potential micro‐organism, S. marcescens, for the production of l ‐asparaginase. An overall 5·55‐fold increase in the production was achieved under optimal levels of carbon and nitrogen sources, DO level and at initial pH of 6·5 (uncontrolled). Significance and Impact of the Study: The experiments illustrate the importance of statistical method for optimization of carbon and nitrogen sources and study the effect of physical process parameters on the production of l ‐asparaginase in shake flask and bioreactor, respectively. This study would be helpful for bioprocess development of bacterial l ‐asparaginase production.     

Production,purification, characterization,antioxidant and antiproliferative activities of extracellular L-asparaginase produced by Fusarium equiseti AHMF4

L-Asparaginase is an antileukemic agent that depletes L-asparagine “an important nutrient for cancer cells” through the hydrolysis of L-asparagine into L-aspartic acid and ammonia leading to leukemia cell starvation and apoptosis in susceptible leukemic cell populations. Moreover currently, bacterial L-asparaginase has been limited by problems of lower productivity, stability, selectivity and a number of toxicities along with the resistance towards bacterial L-asparaginase. Then the current work aimed to provide pure L-asparaginase with in-vitro efficacy against various human carcinomas without adverse effects related to current L-asparaginase formulations. Submerged fermentation (SMF) bioprocess was applied and improved to maximize L-asparaginase production from Fusarium equiseti AHMF4 as alternative sources of bacteria. The enzyme production in SMF was maximized to reach 40.78 U mL⁻¹ at the 7th day of fermentation with initial pH 7.0, incubation temperature 30 °C, 1.0% glucose as carbon source, 0.2% asparagine as nitrogen source, 0.1% alanine as amino acid supplement and 0.1% KH₂PO₄. The purification of AHMF4 L-asparaginase yielded 2.67-fold purification and 48% recovery with final specific activity of 488.1 U mg⁻¹ of protein. Purified L-asparaginase was characterized as serine protease enzyme with molecular weight of 45.7 kDa beside stability at neutral pH and between 20 and 40 °C. Interestingly, purified L-asparaginase showed promising DPPH radical scavenging activity (IC₅₀ 69.12 μg mL⁻¹) and anti-proliferative activity against cervical epitheloid carcinoma (Hela), epidermoid larynx carcinoma (Hep-2), hepatocellular carcinoma (HepG-2), Colorectal carcinoma (HCT-116), and breast adenocarcinoma (MCF-7) with IC₅₀ equal to 2.0, 5.0, 12.40, 8.26 and 22.8 μg mL⁻¹, respectively. The enzyme showed higher activity, selectivity and anti-proliferative activity against cancerous cells along with tiny cytotoxicity toward normal cells (WI-38) which indicates that it has selective toxicity and it could be applied as a less toxic alternative to the current formulations.     

Engineering of Helicobacter pylori L-Asparaginase: Characterization of Two Functionally Distinct Groups of Mutants

Bacterial L-asparaginases have been used as anti-cancer drugs for over 4 decades though presenting, along with their therapeutic efficacy, several side effects due to their bacterial origin and, seemingly, to their secondary glutaminase activity. Helicobacter pylori type II L-asparaginase possesses interesting features, among which a reduced catalytic efficiency for L-GLN, compared to the drugs presently used in therapy. In the present study, we describe some enzyme variants with catalytic and in vitro cytotoxic activities different from the wild type enzyme. Particularly, replacements on catalytic threonines (T16D and T95E) deplete the enzyme of both its catalytic activities, once more underlining the essential role of such residues. One serendipitous mutant, M121C/T169M, had a preserved efficiency vs L-asparagine but was completely unable to carry out L-glutamine hydrolysis. Interestingly, this variant did not exert any cytotoxic effect on HL-60 cells. The M121C and T169M single mutants had reduced catalytic activities (nearly 2.5- to 4-fold vs wild type enzyme, respectively). Mutant Q63E, endowed with a similar catalytic efficiency versus asparagine and halved glutaminase efficiency with respect to the wild type enzyme, was able to exert a cytotoxic effect comparable to, or higher than, the one of the wild type enzyme when similar asparaginase units were used. These findings may be relevant to determine the role of glutaminase activity of L-asparaginase in the anti-proliferative effect of the drug and to shed light on how to engineer the best asparaginase/glutaminase combination for an ever improved, patients-tailored therapy.     

Purification and characterization of cyclodextrin β-glucanotransferase from novel alkalophilic bacilli

The discovery of novel bacterial cyclodextrin glucanotransferase (CGTase) enzyme could provide advantages in terms of its production and relative activity. In this study, eight bacterial strains isolated from soils of a biodiversity-rich vegetation in Egypt based on their hydrolyzing activity of starch, were screened for CGTase activity, where the most active strain was identified as Bacillus lehensis. Optimization process revealed that the using of rice starch (25 %) and a mixture of peptone/yeast extract (1 %) at pH 10.5 and 37 °C for 24 h improved the bacterial growth and enzyme activity. The bacterial CGTase was successively purified by acetone precipitation, gel filtration chromatography in a Sephadex G-100 column and ion exchange chromatography in a DEAE-cellulose column. The specific activity of the CGTase was increased approximately 274-fold, from 0.21 U/mg protein in crude broth to 57.7 U/mg protein after applying the DEAE-cellulose column chromatography. SDS-PAGE showed that the purified CGTase was homogeneous with a molecular weight of 74.1 kDa. Characterization of the enzyme exhibited optimum pH and temperature of 7 and 60 °C, respectively. CGTase relative activity was strongly inhibited by Mg²⁺, Zn²⁺, Al³⁺ and K⁺, while it was slightly enhanced by 5 and 9 % with Cu²⁺ and Fe²⁺ metal ions, respectively.     

PURIFICATION AND PHYSICOKINETIC CHARACTERIZATION OF A GLUCONOLACTONE INHIBITION-INSENSITIVE β-GLUCOSIDASE FROM Andrographis paniculata NEES. LEAF

A gluconolactone inhibition-insensitive β-glucosidase from Andrographis paniculata (Acanthaceae) leaves has been isolated, homogeneity purified, and characterized for its physicokinetic properties. The purified enzyme appeared to be a monomeric structure with native molecular weight about 60 kD. The enzyme exhibited optimum pH 5.5 and pI 4.0, meso-thermostability and high temperature optimum (55°C) for catalytic activity, with activation energy of 6.8 kcal Mol^?1. The substrate saturation kinetics studies of the enzyme revealed a Michaelis–Menten constant (K_m) of 0.25 mM for pNPG and catalytic efficiency (K_cat/K_m) of 52,400 M ^?1 s^?1, respectively. Substrate specificity of the enzyme was restricted to β-linked gluco-, manno- and fuco-conjugates. The gluconolactone inhibition insensitivity was evident from its very low inhibition at millimolar inhibitor concentrations. Interestingly, the enzyme showed geraniol transglucosylating activity with pNPG as glucosyl donor but not with cellobiose. The catalytic activity of the enzyme has been reported to be novel with respect to its activity and preferences from a medicinal plant resource.     

Partial Purification and Characterization of A Novel Histidine Decarboxylase from Enterobacter aerogenes DL-1

Histidine decarboxylase (HDC) from Enterobacter aerogenes DL-1 was purified in a three-step procedure involving ammonium sulfate precipitation, Sephadex G-100, and DEAE-Sepharose column chromatography. The partially purified enzyme showed a single protein band of 52.4 kD on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH for HDC activity was 6.5, and the enzyme was stable between pH 4 and 8. Enterobacter aerogenes HDC had optimal activity at 40°C and retained most of its activity between 4 and 50°C. HDC activity was reduced in the presence of numerous tested compounds. Particularly with SDS, it significantly (p < 0.01) inhibited enzyme activity. Conversely, Ca²⁺ and Mn²⁺ showed prominent activation effects (p < 0.01) with activity increasing to 117.20% and 123.42%, respectively. The Lineweaver–Burk plot showed that K _m and V _max values of the enzyme for L-histidine were 0.21 mM and 71.39 µmol/min, respectively. In comparison with most HDCs from other microorganisms and animals, HDC from E. aerogenes DL-1 displayed higher affinity and greater reaction velocity toward L-histidine.     

Bacterial aerosols in an urban nursery school in Gliwice,Poland: a case study

This work presents the results of the study of airborne bacteria in a kindergarten in Gliwice, Upper Silesia, Poland. In this study, the samples of bioaerosols were collected using six-stage Andersen cascade impactor (with aerodynamic cutoff diameters 7.0, 4.7, 3.3, 2.1, 1.1, and 0.65 μm). The level of culturable bacterial aerosols indoors was about 3000 CFU m^?3—six to eight times higher than outdoors. In the classrooms, respirable bacterial particles, <4.7 µm, contributed up to 85 % of the total number of culturable bacteria, increasing the possible adverse health effects due to their inhalation. The identification of the bacterial species showing the dominance of gram-positive cocci in the indoor environment and non-sporing gram-positive rods in the outdoor air indicates that most of the bacteria present in the studied kindergarten are human origin. Using the obtained data, the nursery school exposure dose (NSED) of bioaerosols was estimated for the children and personnel of this kindergarten (nursery school). The highest value of NSED was obtained for younger children (930 CFU kg^?1) compared to older children (about 600 CFU kg^?1) and to the kindergarten staff (about 300 CFU kg^?1). This result suggests the elevated risk of adverse health effects in younger children exposed to the bioaerosols in the kindergarten, including infections.