Approximate Molecular Weight Research Articles

Isolation and Purification of a Cyclooxygenase-2 from the Blood of a Patient Suffering from Rheumatoid Arthritis and Studying the Effect of Natural Products of the Soapwort on the Activity of Purified Enzyme

In this paper to isolate and study the properties of the cyclooxygenase-2 (EC: 1.14.99.1) enzyme in the blood of a patient suffering from rheumatoid arthritis and study the effect of natural products of the Soapwort on the activity of purified enzyme. The study involves taking 30 ml of blood from an adult woman 40 years old, who suffers from rheumatoid arthritis disease for 13 years. Serum is separated and subjected to a series of purification processes including: precipitation by ammonium sulfate, filtration by centrifugation radiator, dialysis in presence of ammonium bicarbonate, separation using the technology of ion exchange, lipholization and then estimating approximate molecular weight of the enzyme using gel filtration technique and sodium dodecyl sulfate (SDS)-page polyacrylamide gel electrophoresis. The study also includes isolating the natural products of Soapwort plant and study the effect of isolated natural products on the activity of the purified enzyme. The result of the study indicates that cycloxygenase-2 has an approximate molecular weight of 71.5 kDa and that the extracted oil of the Soapwort has a negative impact on the activity of the enzyme (r= -0.824; P=0.006), while flavonoids and Saponin have no such impact (r= -0.565; P=0.113; r= -0.634; P=0.067 respectively).

Isolation and Characterization of Midgut Lectin From Aedes aegypti (L.) (Diptera: Culicidae)

The present investigation deals with the isolation and characterization of a lectin from Aedes aegypti (Ae aegypti) female mid gut extract that agglutinates various mammalian red blood cells (RBCs) such as human three groups A, B, and O (RH+), mouse, rat, guinea-pig, sheep and goat erythrocytes. The highest activity of both crude and isolated mid gut lectins were detected against sheep RBCs. Using (NH4)2 SO4 fractionation, ion-exchange and mannose-CNBr-Sepharose 6B affinity chromatography techniques, Ae. aegypti midgut lectin (Aelec) was purified to homogeneity.Isoelectric focusing (IEF) and reducing SDS/PAGE revealed that the isolated mid gut lectin had isoelectric point (PI) of 5.90, and subunits approximate molecular weights of 35.50 and 27.35 KDa. The hemagglutination (HA) of lectins were Ca2+ - independent and heat-resistant. The sugar specificity of the purified Aelec was strongly inhibited by D (+)-mannose and raffinose, followed by D (+) glucose. N-acetyl-D-manosamine and N-acetyl-D-glucosamine were moderate inhibitors. None of the lectins were inhibited by the disaccharides such as galactose, lactose, trehalose (IC50 up to 200 mM or fetuin up to 1%) but the glycosubstances mucin and laminarin were strong inhibitors up to very low concentrations (0.030 - 0.003%).

Identification and characterization of a cathepsin L-like cysteine protease from Rhipicephalus (Boophilus) annulatus.

The tick Rhipicephalus (Boophilus) annulatus is one of the most important ectoparasites of bovines and is responsible for the transmission of different pathogens such as Babesia and Anaplasma. Cysteine proteases are involved in several host-tick interactions including invasion of host tissues, immune evasion, pathogen transmission, embryogenesis and blood digestion. In this study, the gene encoding R. annulatus cathepsin L-like enzyme (RaCL1) was cloned into pTZ57R/T vector, sequenced and analyzed using bioinformatics approaches. The nucleotide length of RaCL1 was 999 bp. Bioinformatics analysis showed 332 amino acids with an approximate molecular weight of 36.3 kDa which contained a signal peptide sequence (18 amino acids), pro-region (97 amino acids) and mature enzyme (217 amino acids). Multiple sequence alignment of the RaCL1 revealed high similarity to cathepsin L-like cysteine proteases from other tick species such as Rhipicephalus (Boophilus) microplus and Amblyomma variegatum. Based on bioinformatics analyses, results of this work suggest that RaCL1 can be a suitable candidate for the development of vaccine against R. annulatus.

Bioflocculant production from untreated corn stover using Cellulosimicrobium cellulans L804 isolate and its application to harvesting microalgae.

BackgroundMicroalgae are widely studied for biofuel production. Nevertheless, harvesting step of biomass is still a critical challenge. Bioflocculants have been applied in numerous applications including the low-cost harvest of microalgae. A major bottleneck for commercial application of bioflocculant is its high production cost. Lignocellulosic substrates are abundantly available. Hence, the hydrolyzates of rice stover and corn stover have been used as carbon source to produce the bioflocculant in previous studies. However, the hydrolyzates of biomass required the neutralization of pH before the downstream fermentation processes, and the toxic by-products produced during hydrolysis process inhibited the microbial activities in the subsequent fermentation processes and contaminated the bioflocculant product. Therefore, strains that can secrete plant cell-wall-degrading enzymes and simultaneously produce bioflocculants through directly degrading the lignocellulosic biomasses are of academic and practical interests.ResultsA lignocellulose-degrading strain Cellulosimicrobium cellulans L804 was isolated in this study, which can produce the bioflocculant MBF-L804 using untreated biomasses, such as corn stover, corn cob, potato residues, and peanut shell. The effects of culture conditions including initial pH, carbon source, and nitrogen source on MBF-L804 production were analyzed. The results showed that over 80 % flocculating activity was achieved when the corn stover, corn cob, potato residues, and peanut shell were used as carbon sources and 4.75 g/L of MBF-L804 was achieved under the optimized condition: 20 g/L dry corn stover as carbon source, 3 g/L yeast extract as nitrogen source, pH 8.2. The bioflocculant MBF-L804 contained 68.6 % polysaccharides and 28.0 % proteins. The Gel permeation chromatography analysis indicated that the approximate molecular weight (MW) of MBF-L804 was 229 kDa. The feasibility of harvesting microalgae Chlamydomonas reinhardtii and Chlorella minutissima using MBF-L804 was evaluated. The highest flocculating efficiencies for C. reinhardtii and C. minutissima were 99.04 and 93.83 %, respectively.ConclusionsThis study shows for the first time that C. cellulans L804 can directly convert corn stover, corn cob, potato residues and peanut shell into the bioflocculants, which can be used to effectively harvest microalgae.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0354-4) contains supplementary material, which is available to authorized users.

Cloning and characterization of lipoxygenase gene from germinating seedlings of green gram (Vigna radiata L.)

Plant LOXs play a significant role in plant physiological processes such as growth, senescence, defence and stress-related responses. In the present study a full length lipoxygenase cDNA (VrLOX) has been cloned from green gram germinating seedlings using 5′- and 3′-RACE PCR method and nomenclatured VrLOX. The ORF (2601) encodes 867 amino acid residues with an approximate molecular weight of 97.43 kDa and an isoelectric point of 5.93. The VrLOX comprises of a putative PLAT domain and an iron-binding catalytic domain, and the sequence displayed a significant identity to known plant LOXs deposited in Protein Data Bank. Bioinformatics analysis demonstrated that this gene is most probably a LOX putative protein of type 13-LOX, and shared highest identity (91 %) with the Glycine max LOX-10. Enzymatic assay showed LOX activity was more on day 3 at pH 6.5 and higher MDA levels were observed on day 5 after germination. Semi-quantitative RT-PCR analysis indicated highest VrLOX mRNA expression on day 3 after germination, and could be induced by methyl jasmonate (MeJA). The 3-dimensional structure of the VrLOX was elucidated using in silico approach and molecular dynamics simulations.

Assessing the antimicrobial activities of Ocins.

The generation of a zone of inhibition on a solid substrate indicates the bioactivity of antimicrobial peptides such as bacteriocin and enterocin. The indicator strain plays a significant role in bacteriocin assays. Other characteristics of bacteriocins, such as their dispersal ability and the different zymogram components, also affect bacteriocin assays. However, universal well diffusion assays for antimicrobials, irrespective of their ability to diffuse (bacteriocin and enterocin), do not exist. The ability of different zymography components to generate non-specific activities have rarely been explored in the literature. The purpose of the present work was to evaluate the impact of major factors (diffusion and rate of diffusion) in a solid substrate bioassay, and to document the adverse effects of sodium dodecyl sulfate in zymograms used to estimate the approximate molecular weight of bacteriocins.

Cytolytic and systemic toxic effects induced by the aqueous extract of the fire coral Millepora alcicornis collected in the Mexican Caribbean and detection of two types of cytolisins.

BackgroundMillepora alcicornis is a branching hydrocoral common throughout the Caribbean Sea. Like other members of this genus, this species is capable of inducing skin eruptions and blisters with severe pain after contact. In the present study, we investigated the toxicity of the M. alcicornis aqueous extract on several animal models. Considering that some cnidarian hemolysins have been associated to local tissue damage, since they also induce lysis of other cell types, we also made a partial characterization of the hemolytic activity of M. alcicornis aqueous extract. This information is important for understanding the defense mechanisms of the “fire corals”.MethodsThe effects of pH, temperature, and some divalent cations on the hemolytic activity of the extract were assayed, followed by a zymogram analysis to detect the cytolysins and determine their approximate molecular weight. The toxicity of the aqueous extract was assayed in mice, by intravenous administration, and histopathological changes on several tissues were analyzed by light microscopy. The toxicity of the extract was also tested in Artemia salina nauplii, and the damages caused on the crustaceans were analyzed by transmission and scanning electron microscopy.ResultsThe hemolytic activity of the hydrocoral extract was enhanced in the presence of Ca2+ (≥2 mM), Mg2+ (≥6 mM), and Ba2+ (≥0.1 mM); however, it was reduced in the presence of Cu2+ (≥0.1 mM), Zn2+ (≥6 mM), and EDTA (≥0.34 mM). Differences in the pH did not affect the hemolytic activity, but it was temperature-sensitive, since preincubation at ≥ 50 °C sharply reduced hemolysis. The zymogram showed the presence of two types of hemolysins: ~ 28–30 kDa proteins with phospholipase A2 activity and ~ 200 kDa proteins that do not elicit enzymatic activity. The aqueous extract of this cnidarian was lethal to mice (LD50 = 17 μg protein/g), and induced kidney, liver, and lung damages. Under denaturing conditions, the aqueous extract completely lost its toxic and hemolytic activities.ConclusionsThe results showed that the M. alcicornis aqueous extract contains two types of thermolabile hemolysins: proteins of approximately 28–30 kDa with PLA2 activity, while the others are larger proteins of approximately 200 kDa, which do not possess PLA2 activity. Those thermolabile cytolysins, which are stable to pH changes and whose activity is calcium dependent, are capable of inducing damage in lung, kidney and liver tissues, resulting in a slow death of mice. M. alcicornis cytolysins also provoke tissue dissociation in Artemia salina nauplii that might be attributed to pore forming mechanisms.

Alkaline thermostable pectinase enzyme from Aspergillus niger strain MCAS2 isolated from Manaslu Conservation Area, Gorkha, Nepal.

Pectinase enzymes are one of the commercially important enzymes having great potential in various industries especially in food industry. Pectinases accounts for 25 % of global food enzymes produced and their market is increasing day by day. Therefore, the exploration of microorganism with novel characteristics has always been the focus of the research. Microorganism dwelling in unique habitat may possess unique characteristics. As such, a pectinase producing fungus Aspergillus niger strain MCAS2 was isolated from soil of Manaslu Conservation Area (MCA), Gorkha, Nepal. The optimum production of pectinase enzyme was observed at 48 h of fermentation. The pectinase enzyme was partially purified by cold acetone treatment followed by Sephadex G-75 gel filtration chromatography. The partially purified enzyme exhibited maximum activity 60 U/mg which was almost 8.5-fold higher than the crude pectinase. The approximate molecular weight of the enzyme was found to be 66 kDa as observed from SDS-PAGE. The pectinase enzyme was active at broad range of temperature (30–70 °C) and pH (6.2–9.2). Optimum temperature and pH of the pectinase enzyme were 50 °C and 8.2 respectively. The enzyme was stable up to 70 °C and about 82 % of pectinase activity was still observed at 100 °C. The thermostable and alkaline nature of this pectinase can meet the demand of various industrial processes like paper and pulp industry, in textile industry, fruit juice industry, plant tissue maceration and wastewater treatment. In addition, the effect of different metal ions on pectinase activity was also studied.

Molecular cloning, characterization and three-dimensional structure prediction of Lipoxygenase from Finger millet [Eleusine coracana (L.) Gaertn.] germinating seedlings

Lipoxygenases (LOXs) are functionally diverse class of non-heme iron containing dioxygenases. They involve in multiple physiological processes in plants and animals. In current study a full length cDNA of LOX has been cloned from finger millet germinating seedlings using PCR and RACE PCR methods. The ORF (2661 bp) encodes 887 amino acid residues, with an approximate molecular weight of 100.8 kDa and an isoelectric point of 5.90. The sequence exhibited significant similarity to already known LOXs. It comprises of a putative PLAT domain and an iron-binding catalytic domain. Sequence analysis demonstrated that this LOX is most probably a type 9-LOX, and shared highest identity (85 %) with the Setaria italica 9-LOX. TargetP predicted EcLOX as a cytoplasmic protein. It has an optimum pH of about 6.4 and increased lipid peroxidation levels were observed on day 5 of germination. Semi-quantitative RT-PCR analysis showed that EcLOX gene expression was preferentially higher on day 4 after germination, and in growing leaf and root tissues. Its expression could be induced by Methyl Jasmonate (MeJA), but not by Salicylic acid (SA). The 3-dimensional structure of the EcLOX was modeled using in silico approach and Molecular dynamics (MD) simulations were performed. Further, to understand the protein active site, docking studies were performed with substrates and inhibitors. In summary, EcLOX protein was characterized using various molecular, biochemical and in silico methods which improves our understanding of these fascinating enzymes.

Heterologous expression and production of Trichoderma reesei cellobiohydrolase II in Pichia pastoris and the application in the enzymatic hydrolysis of corn stover and rice straw

The cellobiohydrolase II (CBH II) gene cbh2 from Trichoderma reesei was cloned and its codons were optimized in accordance with the codon usage frequencies of the host Pichia pastoris. The AOX1 strong promoter inducible by methanol was employed to efficiently express the foreign gene cbh2 in P. pastoris. It was found that 5.84 ± 0.42 U cm−3 CBH II was obtained at 96 h using the synthetic cbh2 gene whose codons were optimized, 2.02-fold higher than using the native cbh2 gene (2.89 ± 0.32 U cm−3), indicating that the codon optimization strategy was an effective approach to enhance the heterologous expression of CBH II in P. pastoris. The product of recombinant P. pastoris CBH II had an approximate molecular weight 58 kDa. Its optimal pH and temperature were 5.0 and 50 °C, respectively. The recombinant CBH II was used to enhance the yields of the enzymatic hydrolysis of the corn stover and rice straw pretreated with sodium hydroxide by improving the exo-exo-synergism between CBH II and CBH I in T. reesei cellulase. The yields 94.7% and 83.3% were achieved in the enzymatic hydrolysis of corn stover and rice straw pretreated by sodium hydroxide, respectively.

Purification and biochemical characterization of a novel β-fructofuranosidase from Penicillium oxalicum with transfructosylating activity producing neokestose

Neokestose is a novel fructooligosaccharide (FOS) exhibiting greater prebiotic effects and chemical stability than commercial FOSs. In this study, a neokestose-producing β-fructofuranosidase was purified from Penicillium oxalicum GXU20. The enzyme is a glycoprotein with an approximate 111kDa molecular weight, and it has an N-linked carbohydrate composition that accounts for approximately 38% of its total mass. Optimal enzymatic activity occurred at pH 5.5 and 60°C. The enzyme remained stable over a wide pH range (2–9.5). Metal ions and chemical reagents had no significant effect on its enzymatic activity, with the exception of silver (Ag+). The enzyme could hydrolyze fructosyl-(2-1)-linked carbohydrates. Using sucrose as a substrate, the Km and Vmax values for a transfer reaction were 163.9±10.3mmol/L and 800.1±19.8μmol/(minmg), respectively, whereas the Km, Vmax, and Ki values for a hydrolysis reaction with substrate inhibition were 48.3±1.7mmol/L, 1631.3±28.2μmol/(minmg), and 162.6±4.9mmol/L, respectively. In the catalysis of 500g/L sucrose, the maximum concentration of neokestose and total FOS were 94.2g/L and 224.7g/L, respectively. Finally, the gene encoding the β-fructofuranosidase was cloned, analyzed, and functionally expressed in Pichia pastoris.

Isolation and characterization of a 17-kDa FKBP-type peptidyl-prolyl cis/trans isomerase from Vibrio anguillarum

Peptidyl-prolyl cis/trans isomerase (PPIase) catalyzes the isomerization of peptide bonds to achieve conformational changes in native folded proteins. An FKBP-type PPIase with an approximate molecular weight of 17kDa was isolated from Vibrio anguillarum O1 and named VaFKBP17. To investigate its biochemical properties, the ppi gene from V. anguillarum O1 was isolated and overexpressed in Escherichia coli. A protease-coupled assay for isomerization activity, using Succinyl-Ala-Phe-Pro-Phe-p nitroanilide as substrate, indicated that the activity of VaFKBP17 was highest at low temperature (5°C) and alkaline conditions (pH 10). The immunosuppressant FK506 inhibited the isomerization activity of VaFKBP17. The chaperone activity of VaFKBP17 was assessed using a citrate synthase thermal aggregation activity assay. To evaluate its ability to catalyze protein refolding, the effect of VaFKBP17 on inclusion bodies was investigated during a dilution process. In this assay, VaFKBP17 was able to assist protein refolding. These results provide evidence that VaFKBP17 possesses chaperone-like activity. The structural homology of VaFKBP17 relative to other known bacterial FKBPs was also examined.

What Is The New Tumor Marker For Detection Of Different Kidney Tumors? Modern Study To Isolation, Purification, And Characterization Of Galactose Binding Lectin From Sera Of Patients With Kidney Cancer

The present study was designed to investigate lectins in sera of patients with kidney tumors, in addition to non tumoral kidney disease patients. Fifty five patients of malignant kidney tumors were enrolled in addition to 23 patients of benign kidney tumors, and 18 patients of non tumoral kidney diseases used as control groups, in addition to 46 healthy individuals were also investigated. The age of patients and healthy individuals were 10-90 years. The measurement of total serum proteins revealed significant (p < 0.001) decrease in patients of malignant tumors when compared with those of benign, non tumoral diseases, and healthy individuals. The conditions of the hemagglutination assay of serum lectin activity were optimized. They were Tris buffer of 20 mM and pH 7.4, 60 mM CaCl2, 800 µg of defatted serum, 30 ˚C for serum samples, 60 minutes for serum samples, and human blood of group A+ suspension with 1.4 optical density. The measurement of the specific hemagglutination activity of lectins demonstrated significant (p < 0.001) elevation in patients of malignant tumors when compared with those of other patients and healthy individuals. Lectin activity was pointed out to be significantly (r = 0.767 at p < 0.0005) positively correlated with stage of malignancies. The cutoff value of the specific hemagglutination activity was found to equal 6 SHU for discriminatory malignant kidney tumors. Serum lectins activity were indicated to be inhibited by galactose, mannose, lactose, and N-acetyl galactosamine. Purification of lectin from sera of patients with malignant kidney tumors by affinity chromatography with the use of silver stain revealed galactose binding lectin (GalBL). The purified folds and the yield was 132 with 22.1. The polyacrylamide gel electrophoresis (PAGE) of purified lectin demonstrated one band consisted lectin activity. The approximate molecular weight of GalBL was determined and found to be 98.40. Purified lectin was characterized through the assessment of the capability to agglutinate RBCs, inhibition by EDTA, pH dependency, thermal dependent, and carbohydrate contents. GalBL was observed to be calcium independence lectins. These results suggest that the diagnosis of the specific hemagglutination activity of lectin is a promising biomarker for discrimination of malignant kidney tumor patients and the purified lectin could be introduced in the field of biomarkers.

Structural and functional analysis of two universal stress proteins YdaA and YnaF from Salmonella typhimurium: possible roles in microbial stress tolerance.

In many organisms "Universal Stress Proteins" (USPs) are induced in response to a variety of environmental stresses. Here we report the structures of two USPs, YnaF and YdaA from Salmonella typhimurium determined at 1.8Å and 2.4Å resolutions, respectively. YnaF consists of a single USP domain and forms a tetrameric organization stabilized by interactions mediated through chloride ions. YdaA is a larger protein consisting of two tandem USP domains. Two protomers of YdaA associate to form a structure similar to the YnaF tetramer. YdaA showed ATPase activity and an ATP binding motif G-2X-G-9X-G(S/T/N) was found in its C-terminal domain. The residues corresponding to this motif were not conserved in YnaF although YnaF could bind ATP. However, unlike YdaA, YnaF did not hydrolyse ATP in vitro. Disruption of interactions mediated through chloride ions by selected mutations converted YnaF into an ATPase. Residues that might be important for ATP hydrolysis could be identified by comparing the active sites of native and mutant structures. Only the C-terminal domain of YdaA appears to be involved in ATP hydrolysis. The structurally similar N-terminal domain was found to bind a zinc ion near the segment equivalent to the phosphate binding loop of the C-terminal domain. Mass spectrometric analysis showed that YdaA might bind a ligand of approximate molecular weight 800daltons. Structural comparisons suggest that the ligand, probably related to an intermediate in lipid A biosynthesis, might bind at a site close to the zinc ion. Therefore, the N-terminal domain of YdaA binds zinc and might play a role in lipid metabolism. Thus, USPs appear to perform several distinct functions such as ATP hydrolysis, altering membrane properties and chloride sensing.

Alkaline and thermostable polygalacturonase from Streptomyces halstedii ATCC 10897 with applications in waste waters

Abstract Pectin degrading enzymes with polygalacturonase (PG) activity hydrolyze α-(1,4) glycosidic bonds of polysaccharides present in higher plants. In the current study one hundred bacterial strains were screened for extracellular PG activity using an inductive culture medium. Optimization of fermentation conditions for Streptomyces halstedii ATCC 10897 was conducted using experimental designs. The maximum enzymatic activity obtained was 3.489 U/mL and 98.0% of viscosity reduction after 12 h of fermentation using soy peptone as unique source of carbon and nitrogen. PG from S. halstedii ATCC 10897 showed high thermal stability, an approximate molecular weight of 48 kDa and its optimum conditions for catalytic reaction were 50 °C and pH 12.0. This study reveals that alkaline PG is a useful enzyme for depectinization in alkaline pulping mill and papermaking waste waters.

Detection of serum anti-melanocyte antibodies and identification of related antigens in patients with vitiligo.

We detected autoantibodies against melanocytes in serum samples obtained from 50 patients, including 4 with HBV, with vitiligo and identified the associated membrane antigens. Heat shock protein 70 (HSP70) and anti-tyrosinase-related protein 1 (TRP-1) antibody levels were analyzed. The associated antigens in normal human melanocyte were identified by immunofluorescence. Autoantibodies against melanocyte membrane and cytoplasmic proteins were detected by western blot. Membrane antigens with higher frequencies were identified by protein mass spectrometry. The HSP70 and anti-TRP-1 antibody levels (N = 70; 10 with HBV) were detected by ELISA. The specific antigens were detected in melanocyte cytoplasm and membrane (40/50; 80% incidence; western blot). The autoantibodies reacted with several membrane antigens with approximate molecular weights (Mr) of 86,000, 75,000, 60,000, 52,000, and 44,000 (strip positive rates: 36, 58, 22, 2, and 2%, respectively). Thirty percent of the patients showed the presence of cytoplasmic antigens (Mr: 110,000, 90,000, 75,000, 50,000, and 400,000; strip positive rates: 12, 4, 12, 10, and 2%, respectively). Fifteen and 5% of the healthy subjects showed positive expression of membrane and cytoplasmic antigens, respectively. Protein mass spectrometry predicted membrane proteins with Mr of 86,000 and 75,000 and 60,000 to be Lamin A /C and Vimentin X1, respective. High titers of anti-TRP-1 antibody were detected and showed positive correlation with HSP70 (r = 0. 927, P < 0. 01). This study identified a novel membrane antigen associated with vitiligo, which might assist future investigations into autoimmune pathogenesis of vitiligo and formation of autoantibodies. HBV infection was correlated to vitiligo.

Isolation, Partial Purification and Characterization of L-Asparaginase from Hedgehog Serum

The need to further explore other sources of therapy for leukemias prompted this current work. It’s already known that L-asparaginase possess a lot of potentials in the treatment of cancers of the white blood cell but the sources of these enzyme and its properties determines the efficacy of this drug. Generally, for an asparaginase to be ideally suited for use in anti-tumour therapy, it had to satisfy a variety of criteria. The organism that is selected should produce the asparaginase in high quantity or yield, and it should be capable of being isolated from simple mammalian sources. The procedures developed for purification of the enzyme should be as rapid and simplified as possible. The purified enzyme should have long term stability on storage, maximal activity at a physiological pH, and a Km for substrate below the concentration of the substrate in the blood. Therefore the aim of this present research was to partially purify and characterize this enzyme from Hedgehog serum in order to compare its anti-leukemic potentials with those from documented literature for possible future medical application. L-asparaginase was isolated and partially purified from Hedgehog serum which is also known to be a reservoir of many other useful proteins including anti venoms using a four step profile of ammonium sulphate fractionation, dialysis, and ion exchange and gel filtration chromatography. The enzyme gave an overall yield of 77.58%, optimum pH and temperature of 7.8 and 39oC respectively, and a Km of 0.0125 mM. Gel filtration gave an approximate molecular weight of 139,000 Da, while SDS PAGE indicated a subunit molecular weight of 36,000 and 34,600 Da respectively. The enzyme also catalyses the hydrolysis of glutamine though slightly. Mg2+ and Zn2+ serve as activators for Hedgehog serum L-asparaginase, while inhibition from heavy ions like Hg2+ was also observed. This work shows that mammalian sources of this enzyme could be more preferable for treatment of asparagine dependent tumours in time to come.

Production and Purification of Cutinase from &amp;lt;i&amp;gt;Fusarium oxysporum&amp;lt;/i&amp;gt; Using Modified Growth Media and a Specific Cutinase Substrate

Cutinases are hydrolytic enzymes used by phytopathogenic fungi to gain entry into plants by breaking down the cuticular barrier of higher plants. Cutinase displayed hydrolytic activity not only towards cutin, the main component of the plant cuticle, but also towards a variety of soluble synthetic esters, insoluble triglycerides and polyesters. Therefore, cutinase was evaluated for use in the chemical, food and cotton bio-scouring industry and for synthetic fibers modification. This research examined the production and purification of extracellular cutinase from the phytopathogenic fungus Fusarium oxysporum. The addition of apple cutin or its hydrolysate to the fungus growth medium resulted in an enhanced secretion of cutinase into the extracellular fluid. Testing 1-hexadecanol as an alternative to natural cutin to induce cutinase production resulted in a high process yield under modified growth conditions. Cutinase enzyme production was followed by an optimized purification method for enzyme preparation using high-performance liquid chromatography and high-specificity 4-nitrophenyl (16-methyl sulfide ester) hexadecanoate (pNMSEH) cutinase substrate. Electrophoresis sodiumdodecyl sulfate-polyacrylamide and isoelectric focusing gels enabled the final separation and identification of the protein. The purified cutinase had an approximate molecular weight of 20 kDa and an isoelectric point of 4.7. The method presented here could be modified and used for cutinase production and purification in other microorganisms that exhibit cutinolytic activity.

Optimization of enzymatic hydrolysis of tilapia (Oreochromis Spp.) scale gelatine

Gelatine hydrolysate is a type of gelatine that undergoes a controlled hydrolysis treatment to further break apart the gelatine or collagen molecules. In this study, gelatine hydrolysate was produced from commercial tilapia scale gelatine via controlled enzymatic hydrolysis. Commercial Alcalase 2.4 L, a protease enzyme was used to breakdown the peptide chains present in the gelatine. Optimization of hydrolysis conditions (temperature, time and enzyme to substrate ratio) was conducted by utilizing response surface methodology (RSM). Results showed that a hydrolysis temperature of 57.6 °C together with a hydrolysis time of 80 min and enzyme to substrate ratio of 1.20 % (v/w) were the optimum conditions to obtain the highest degree of hydrolysis (10.91 %). The freeze-dried gelatine hydrolysate was characterized with respect to chemical composition:approximate composition, viscosity and molecular weight. The gelatine hydrolysate produced contained a high content of protein (85.26 %); thus, it may serve as a potential protein source for human needs.

Prolonged Acetaminophen-Protein Adduct Elimination During Renal Failure, Lack of Adduct Removal by Hemodiafiltration, and Urinary Adduct Concentrations After Acetaminophen Overdose.

Elevated concentrations of serum acetaminophen-protein adducts, measured as protein-derived acetaminophen-cysteine (APAP-CYS), have been used to support a diagnosis of APAP-induced liver injury when histories and APAP levels are unhelpful. Adducts have been reported to undergo first-order elimination, with a terminal half-life of about 1.6 days. We wondered whether renal failure would affect APAP-CYS elimination half-life and whether continuous venovenous hemodiafiltration (CVVHDF), commonly used in liver failure patients, would remove adducts to lower their serum concentrations. Terminal elimination half-lives of serum APAP-CYS were compared between subjects with and without renal failure in a prospective cohort study of 168 adults who had ingested excessive doses of APAP. APAP-CYS concentrations were measured in plasma ultrafiltrate during CVVHDF at times of elevated serum adduct concentrations. Paired samples of urine and serum APAP-CYS concentrations were examined to help understand the potential importance of urinary elimination of serum adducts. APAP-CYS elimination half-life was longer in 15 renal failure subjects than in 28 subjects with normal renal function (41.3 ± 2.2 h versus 26.8 ± 1.1 h [mean ± SEM], respectively, p < 0.001). CVVHDF failed to remove detectable amounts of APAP-CYS in any of the nine subjects studied. Sixty-eight percent of 557 urine samples from 168 subjects contained no detectable APAP-CYS, despite levels in serum up to 16.99 μM. Terminal elimination half-life of serum APAP-CYS was prolonged in patients with renal failure for reasons unrelated to renal urinary adduct elimination, and consideration of prolonged elimination needs to be considered if attempting back-extrapolation of adduct concentrations. CVVHDF did not remove detectable APAP-CYS, suggesting approximate APAP-protein adduct molecular weights ≥ 50,000 Da. The presence of urinary APAP-CYS in the minority of instances was most compatible with renal adduct production and protein shedding into urine rather than elimination of serum adducts.