Isolation and characterization of DM40 and DM43, two snake venom metalloproteinase inhibitors from Didelphis marsupialis serum

From Didelphis marsupialis serum, two antihemorrhagic proteins were isolated by DEAE-Sephacel, Phenyl-Sepharose and Superdex 200 and characterized. Their masses by mass spectrometry were 40318 AMU for DM40 and 42373 and 43010 AMU for DM43, indicating the presence of isoforms for the last. Molecular masses of 44.8 and 47.3 were obtained by SDS-PAGE, respectively for DM40 and DM43. Both inhibitors showed isoelectric points lower than 3.5 and glycosylation percentages varying from 20.5 to 29.0%, as estimated by chemical deglycosylation and amino acid analysis. N-terminal sequences of the first 17 residues of DM40 and DM43 were identical except for the exchange of R9 for P9. Both were homologous to oprin, a similar inhibitor from Didelphis virginiana serum. No evidence of complex formation between DM40 and DM43 was observed either by native PAGE or gel filtration chromatography. In addition to the antihemorrhagic activity, DM40 and DM43 inhibited the hydrolysis of casein, fibrinogen and fibronectin by Bothrops jararaca venom. DM43 also showed antilethal, antiedematogenic and antihyperalgesic activities. None of the inhibitors showed enzymatic activity on casein. Both proteins formed stable complexes with jararhagin and inhibited its hemorrhagic effect as well as the enzymatic activity of this toxin on fluorogenic substrate.     

Using mass spectrometry to explore the neglected glycan moieties of the antiophidic proteins DM43 and DM64

The resistance of the opossum Didelphis aurita to Bothrops snake venoms is attributed to the opossum's antihemorrhagic (DM43) and antimyotoxic (DM64) acidic serum glycoproteins. The aim of this study was to characterize the N-glycosylation sites of these antiophidic proteins and to determine whether their glycans influence the biological activity measured by in vitro assays. Our experimental pipeline included the sequential enzymatic digestion of the inhibitors with two different proteinases (trypsin and endoproteinase Asp-N) and eventually with trypsin, peptide-N-glycosidase F (PNGase F) and endoproteinase Asp-N, used in that order. All of the peptide and protein samples were analyzed by MALDI-TOF/TOF MS. The results experimentally confirmed the putative N-glycosylation sites of DM43 (Asn23, Asn156, Asn160, and Asn175) and DM64 (Asn46, Asn179, Asn183, and Asn379). Following treatments with specific glycosidases, complex-type oligosaccharides containing galactose and sialic acid could be assigned to both proteins. The removal of these monosaccharide units by exoglycosidase digestion did not measurably affect the inhibitory activity. In contrast, partially deglycosylated DM43 treated with PNGase F under nondenaturing conditions was half as effective as native DM43. In conclusion, we have demonstrated that the contribution of the carbohydrate portion of these potentially therapeutic molecules, for their mechanism of action, should not be overlooked.     

Functional analysis of DM64, an antimyotoxic protein with immunoglobulin-like structure from Didelphis marsupialis serum.

Bothrops snake venoms are known to induce local tissue damage such as hemorrhage and myonecrosis. The opossum Didelphis marsupialis is resistant to these snake venoms and has natural venom inhibitors in its plasma. The aim of this work was to clone and study the chemical, physicochemical and biological properties of DM64, an antimyotoxic protein from opossum serum. DM64 is an acidic protein showing 15% glycosylation and with a molecular mass of 63 659 Da when analysed by MALDI-TOF MS. It was cloned and the amino acid sequence was found to be homologous to DM43, a metalloproteinase inhibitor from D. marsupialis serum, and to human alpha1B-glycoprotein, indicating the presence of five immunoglobulin-like domains. DM64 neutralized both the in vivo myotoxicity and the in vitro cytotoxicity of myotoxins I (mt-I/Asp49) and II (mt-II/Lys49) from Bothrops asper venom. The inhibitor formed noncovalent complexes with both toxins, but did not inhibit the PLA2 activity of mt-I. Accordingly, DM64 did not neutralize the anticoagulant effect of mt-I nor its intracerebroventricular lethality, effects that depend on its enzymatic activity, and which demonstrate the dissociation between the catalytic and toxic activities of this Asp49 myotoxic PLA2. Furthermore, despite its similarity with metalloproteinase inhibitors, DM64 presented no antihemorrhagic activity against Bothrops jararaca or Bothrops asper crude venoms, and did not inhibit the fibrinogenolytic activity of jararhagin or bothrolysin. This is the first report of a myotoxin inhibitor with an immunoglobulin-like structure isolated and characterized from animal blood.     

Characterization of digestive gland esterase-lipase activity of juvenile redclaw crayfish Cherax quadricarinatus

Investigation of esterase-lipase activity in the digestive gland of redclaw crayfish Cherax quadricarinatus showed that the optimum enzyme activity occurred between 35 and 40 degrees C, with 100 mM NaCl at pH 8.5. Heavy metals completely inhibited and calcium ions partially inhibited enzyme activity. The enzyme activity diminished as the length of the fatty acid chain of substrates increased. Molecular masses for four isozymes were 43, 46, 63 and 118 kDa, respectively, as determined by PAGE.     

Purification and Characterization of a New Weak Hemorrhagic Metalloproteinase BmHF-1 from <Emphasis Type="Italic">Bothrops marajoensis</Emphasis> Snake Venom

BmHF-1, from the venom of Bothrops marajoensis, was purified by Sephadex G-75 and HPLC-RP on μ-Bondapak C-18 column chromatography. It presented a molecular mass of 27162.36 Da determined by MALDI-TOF MS. BmHF-1 had a sequence of 238 residues of amino acids. The multiple alignment of its amino acid sequence and those of other snake venom metalloproteinases showed high structural similarity, mainly among P–I class. The enzyme initially cleaves the Aα-chain of fibrinogen, followed by the Bβ-chain, and shows no effects on the γ-chain. BmHF-1 had, caseinolytic and weakly hemorrhagic activities, which were inhibited by EDTA. In contrast, PMSF did not affect these activities. The caseinolytic activity of BmHF-1 had a pH optimum of 8.0 and was stable in solution up to 40 °C; activity was completely lost at ≥70 °C. The proteolytic activity was also inhibited by sDa (opossum sera) and Da2-1, Da2-II, antihemorrhagic factors isolated from the opossum sera of Didelphis albiventris. BmHF-1 presents weak hemorrhagic activity, with a MHD of 41.14 μg and it induces dose-dependent edema. We could concluded that, despite its weak hemorrhagic activity, BmHF-1 contributes to local tissue damage by inducing edema, releasing pharmacologically active mediators from protein precursors due to its enzymatic action.     

Isolation and purification of Mucor circinelloides intracellular chitosanolytic enzymes

This study aimed at isolation, purification and characterization of a chitosanase from Mucor circinelloides mycelium. The latter contains also a mycelium-bound lipase and lipids. The chitosanase and lipase were extracted from defatted M. circinelloides mycelium with a detergent and purified through a two-step procedure comprising chromatography on bacitracin–CNBr-Sepharose 4B and gel filtration on Sephadex G-100. Purification degree of the chitosanase (endo-type enzyme) and lipase was 23 and 12, respectively. These enzymes were optimally active at pH of 5.5–6.0 (chitosanase) and 7.2 (lipase in olive oil hydrolysis) and at 37 °C. Both purified enzymes were activated by Ca²⁺ and Mg²⁺ ions. The preferred substrates of chitosanase were chitosan preparations with a high degree of deacetylation. This enzyme showed no activity for colloidal chitin, Na-CMC and starch. SDS–PAGE of both purified enzymes showed two bands with molecular masses of 42 and 43 kDa. Our results suggest that M. circinelloides synthesizes an oligomeric (bifunctional) lipase which also efficiently depolymerizes chitosan.     

Two detergent stable alkaline serine-proteases from Bacillus mojavensis A21: Purification,characterization and potential application as a laundry detergent additive

Two detergent stable alkaline serine-proteases (BM1 and BM2) from Bacillus mojavensis A21 were purified. The molecular weights of BM1 and BM2 enzymes determined by SDS–PAGE were approximately 29,000 Da and 15,500 Da, respectively. The optimum pH values of BM1 and BM2 proteases were shown to be 8.0–10.0 and 10.0, respectively. Both enzymes exhibited maximal activity at 60 °C, using casein as a substrate.     

Effect of compressed fluids treatment on β-galactosidase activity and stability

This study aimed at assessing the influence of different pressurized fluids treatment on the enzymatic activity and stability of a lyophilized β-galactosidase. The effects of system pressure, exposure time and depressurization rate, using propane, n-butane, carbon dioxide and liquefied petroleum gas on the enzymatic activity were evaluated. The β-galactosidase activity changed significantly depending on the experimental conditions investigated, allowing the selection of the proper compressed fluid for advantageous application of this biocatalyst in enzymatic reactions. The residual activity ranged from 32.1 to 93.8?% after treatment. The storage stability of the enzyme after high-pressure pre-treatment was also monitored, and results showed that the biocatalyst activity presents strong dependence of the fluid used in the pretreatment. The activity gradually decreases over the time for the enzyme treated with LGP and propane, while the enzyme treated with n-butane maintained 96?% of its initial activity until 120?days. For CO₂, there was a reduction of around 40?% in the initial activity 90?days of storage. The enzyme treated with n-butane also showed a better thermostability in terms of enzymatic half-life.     

Comparative Characteristics of Human and Porcine Staphylococci and Their Differentiation in Burn Xenografting Procedures

Staphylococcus epidermidis from porcine skin differed from human cutaneous S. epidermidis in that the former strains were principally of the Baird-Parker biotype III group. The porcine-type strains were more proteolytic on casein and gelatin than were human strains, which were primarily of biotype II. Porcine strains were also elastolytic. Using supernatant fluids of broth cultures, the biotype II strains, but not the type III strains, were lipolytic in action on triolein. Both types of staphylococci were similar in enzymatic activities on Tween 80, egg yolk, and tributyrin. Elastase activity was not found in broth supernatant fluid of these bacteria. The porcine strains were retarded or inhibited from growing in media at pH 5.5. Action on casein agar followed by demonstration of elastase activity were used as markers to detect the porcine S. epidermidis strains in xenografts and on human burn wound grafting sites.     

Isolation and purification ofβ-galactosidase ofLactobacillus murinus CNRZ 313

β-Galactosidase has been isolated inLactobacillus murinus CNRZ 313, and its properties have been studied. The enzyme was purified 292-fold by chromatography on Ultrogel ACA 34, DEAE-Sephadex A-50 columns, and by affinity chromatography in agarose-p-aminophenyl-β-d-thiogalactoside. The purified extract exhibited a single band following polyacrylamide gel electrophoresis (PAGE). Molecular weight was estimated to be 170,000 on an Ultrogel ACA 34 column. Maximum enzymatic activity was observed at 45°C and pH 7 in 50 mM phosphate buffer. The Km for ONPG and ONPG + 20 mM of lactose were 480 μM and 870 μM, respectively. The effect of different salts on the enzymatic activity was studied. An inhibitory effect was observed when using 10 mM CaCl₂, and a stimulating effect when using 10 mM MgCl₂. The latter protected the enzyme from thermal denaturation. Among the protective agents belonging to the sulfhydryl group that was tested, mercaptoethanol and dithiotreitol acted as activators, while glutathione and cysteine acted as inhibitors, of the enzymatic activity.p-β-Galactosidase activity was not observed.     

Two new β-glucosidases from ethanol-fermenting fungus Mucor circinelloides NBRC 4572: enzyme purification, functional characterization, and molecular cloning of the gene

Two β-glucosidases (BGLs 1 and 2) were purified to homogeneity from the extracellular enzyme preparations of the ethanol-fermenting Mucor circinelloides NBRC 4572 statically grown on rice straw. BGLs 1 and 2 are monomeric glycoproteins whose apparent molecular masses (Ms) are around 78 kDa, which decreased by approximately 10 kDa upon enzymatic deglycosylation. Both BGLs showed similar enzyme characteristics in optimal temperature and pH, stability, and inhibitors. They were active against a wide range of aryl-β-glucosides and β-linked glucose oligosaccharides. Their amino acid sequences shared 81 % identity and exhibited less than 60 % identity with the known family-3 BGLs. Considering properties such as reduced inhibition by ethanol, glucose, and cellobiose, low transglucosylation activity, wider substrate range, less binding affinity to lignocellulosic materials, and abundant expression, BGL1 is likely to be more suitable for bioethanol production than BGL2 via simultaneous saccharification and fermentation of rice straw with M. circinelloides.     

Purification and properties of glucose-1-phosphate uridylyltransferase fromNeurospora crassa

A method was developed to assay glucose-1-phosphate uridylyltransferase and 2-acetamido-2-deoxyglucose-1-phosphate uridylyltransferase by separation and quantitation of the corresponding sugar nucleotides by HPLC. Glucose-1-phosphate uridylyltransferase (GPUT) fromNeurospora crassa was purified by a method involving ion-exchange, gel filtration, adsorption, and affinity chromatographic procedures. The enzyme was stable until the last step of purification, after which it became extremely labile, apparently due to disaggregation. With the purified enzyme, kinetic properties of GPUT were determined. Polyacrylamide gel electrophoresis (PAGE) of the purified enzyme under nondenaturing conditions showed a single band which contained all the enzymatic activity. Denaturation of the enzyme with sodium dodecyl sulfate followed by PAGE resolved the single band into four polypeptides of different molecular masses. The minimal molecular mass of the enzyme was calculated to be 537,000 Da. This value was similar to that calculated by sucrose density sedimentation, 580,000 Da, but different from that estimated by gel filtration, 1,600,000 Da. It is proposed that the native enzyme is a trimer which may be disaggregated. By electron microscopy of negatively stained samples, the enzyme appeared in the form of rosettes 10 nm in diameter.     

Purification and Characterization of an Endoglucanase from Pseudomonas solanacearum

Comparative analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of culture supernatants of a virulent Pseudomonas solanacearum strain and of a spontaneous avirulent mutant derived from it was performed. The results show that the levels of two major polypeptides with molecular masses of 43 and 25 kilodaltons (kDa) were markedly reduced in the spent culture medium of the avirulent mutant. In addition, enzyme assays showed that the level of carboxymethyl cellulase (endoglucanase) activity in the culture supernatants of the avirulent mutant was reduced over 25-fold, whereas polygalacturonase activities in both strains were nearly identical. Purification of the endoglucanase from the spent culture medium of the virulent P. solanacearum strain by adsorption to phosphocellulose, salt elution, and gel-filtration chromatography yielded a >95% pure preparation of the 43-kDa polypeptide. The kinetic and enzymatic properties of the purified endoglucanase were subsequently analyzed. Antibody prepared against the purified 43-kDa endoglucanase was used to demonstrate its production by several strains of P. solanacearum races 1 and 2.     

Kinetics of trypsin-catalyzed hydrolysis determined by isothermal titration calorimetry

Isothermal titration calorimetry (ITC) was applied to determine enzymatic activity and inhibition. We measured the Michaelis–Menten kinetics for trypsin-catalyzed hydrolysis of two substrates, casein (an insoluble macromolecule substrate) and Nα-benzoyl-dl-arginine β-naphthylamide (a small substrate), and estimated the thermodynamic parameters in the temperature range from 20 to 37 °C. The inhibitory activities of reversible (small molecule benzamidine) and irreversible (small molecule phenylmethanesulfonyl fluoride and macromolecule α1-antitrypsin) inhibitors of trypsin were also determined. We showed the usefulness of ITC for fast and direct measurement of inhibition constants and half-maximal inhibitory concentrations and for predictions of the mechanism of inhibition. ITC kinetic assays could be an easy and straightforward way to estimate Michaelis–Menten constants and the effectiveness of inhibitors as well as to predict the inhibition mechanism. ITC efficiency was found to be similar to that of classical spectrophotometric enzymatic assays.     

Partial Biochemical Characterization of a Metalloproteinase from the Bloodstream Forms of Trypanosoma brucei brucei Parasites

Metalloproteinases (MMP) belong to the family of cation dependent endopeptidases that degrade matrices at physiological pH and to cleave extracellular matrix proteins. They play an important role in diverse physiological and pathological processes; not only there diverse types of MMP differ in structure and functionally, but also their enzymatic activity is regulated at multiple levels. Trying to shed some light over the processes that govern the pathology of African Trypanosomiasis, the aim of the present study was to examine the proteolytic activity of the crude trypanosome protein extract obtained from the bloodstream forms of Trypanosoma brucei brucei parasites. We hereby report the partial biochemical characterization of a neutral Trypanosoma brucei-metalloproteinase that displays marked proteolytic activities on gelatin and casein, with a molecular mass of approximately 40 kDa, whose activity is strongly dependent of pH and temperature. Furthermore, we show that this activity can be inhibited by classical MMP inhibitors such as EDTA, EGTA, phenantroline, and also by tetracycline and derivatives. This study has a relevant role in the search for new therapeutical targets, for the use of metalloproteinases inhibitors as treatment strategies, or as enhancement to trypanocidal drugs used in the treatment of the disease.     

X-Prolyl-Dipeptidyl Aminopeptidase of Lactobacillus delbrueckii subsp. bulgaricus: Characterization of the Enzyme and Isolation of Deficient Mutants

Lactobacillus delbrueckii subsp. bulgaricus CNRZ 397 is able to hydrolyze X-proline-para-nitroanilides and X-proline-β-naphthylamides (X for alanyl- or glycyl-). A single metal-independent cytoplasmic enzyme with a molecular weight estimated to be 82,000 is responsible for these activities and was named X-prolyl-dipeptidyl aminopeptidase (X-Pro-DPAP). Isolation and analysis of mutants totally deficient for X-Pro-DPAP activity showed that a total lack of this enzyme induces (i) a decrease in the growth rate; (ii) an increase in cell wall proteinase activity; (iii) the loss of three cell wall proteins with respective molecular masses of 16, 40, and 52 kilodaltons; and (iv) enhancement of a cell wall protein with a molecular mass of 150 kilodaltons. The involvement of X-Pro-DPAP in casein catabolism is discussed.     

Aggregation properties of cold-active lipase produced by a psychrotolerant strain of Pseudomonas palleroniana (GBPI_508)

Abstract

The present study aims to exploit microbial potential from colder region to produce lipase enzyme stable at low temperatures. A newly isolated bacterium GBPI_508 from Himalayan environment, was investigated for the production of cold-active lipase emphasizing on its aggregation properties. Plate based assays followed by quantitative production of enzyme was estimated under different culture conditions. Further characterization of partially purified enzyme was done for molecular weight determination and activity and stability under varying conditions of pH, temperature, and in presence of organic solvents, inhibitors, and metal ions. The psychrotolerant bacterium was identified as Pseudomonas palleroniana following 16S rRNA gene sequencing. Maximum lipase production by GBPI_508 was recorded in 7?days at 25?°C utilizing yeast extract as nitrogen source and olive oil as substrate in the lipase production medium. Triton X-100 (1%) in the medium as emulsifier significantly enhanced the lipase production. Lipase produced by bacterium showed aggregation which was confirmed by dynamic light scattering and native PAGE. SDS-PAGE followed by zymogram analysis of partially purified enzyme showed two active bands of ～50?kDa and ～54?kDa. Optimum activity of partially purified enzymatic preparation was recorded at 40?°C while the activity remained nearly consistent from pH 7.0 to 12.0, whereas, maximum stability was recorded at pH values 7.0 and 11.0 at 25?°C. Interestingly, lipase in the partially purified fraction retained 60% enzyme activity at 10?°C. Medium chain pNP ester (C10) was the most preferred substrate for the lipase of GBPI_508. The lipase possessed >50% residual activity when incubated with different organic solvents (25% v/v) except toluene and dichloromethane which inhibited the activity below 50%. Partially purified enzyme was also stable in the presence of metal ions and inhibitors. The study suggests applicability of GBPI_508 lipase in low temperature conditions such as cold-active detergent formulations and cold bioremediation.     

Partial Purification of Proteinase K Inhibitors from Liquid-Cultured Mycelia of the White Rot Basidiomycete Trametes versicolor

Novel protease inhibitors were isolated from liquid-cultured mycelia of the white rot fungus Trametes versicolor. Two bands of antiproteinase K activity, TvPI-A and TvPI-B, were detected in the crude cell extract by native polyacrylamide gel electrophoresis (PAGE). Proteins corresponding to TvPI-A were purified by heat treatment, anion-exchange chromatography, and gel filtration. Sodium dodecyl sulfate (SDS)-PAGE demonstrated the presence of three proteins with molecular masses of 14.5, 16.6, and 20 kDa, respectively. T. versicolor protease inhibitors suppressed the activity of proteinase K and, to a smaller extent, of Carlsberg subtilisin, whereas trypsin and chymotrypsins were not inhibited. The inhibitors were acidic proteins and showed remarkable heat stability. To our knowledge, this is the first report about proteinase K inhibitors from fungi.     

Purification and characterisation of monoamine oxidase from Avena sativa

FAD-containing monoamine oxidase (MAO; EC 1.4.3.4) oxidises monoamines to their corresponding aldehydes, H₂O₂, and NH₃. It has been purified to homogeneity in mammals, but to our knowledge, there have been no reports of the enzyme in plants. MAO activity was detected in Avena sativa seedlings during germination using benzylamine as substrate. The enzyme was purified to homogeneity (as assessed by native PAGE) by Sephadex G-25, DEAE Sephacel, hydroxyapatite, Mono Q, and TSK-GEL column chromatographies. The molecular mass estimated by gel filtration using the TSK-GEL column was 220?kDa. SDS-PAGE yielded four distinct protein bands of 78, 58, 55, and 32?kDa molecular masses. The pI value of the enzyme was 6.3. The enzyme showed high substrate specificity for an endogenous amine, phenethylamine, which was oxidised to phenylacetaldehde, but not for ethylamine, propylamine, butylamine, pentylamine, dopamine, serotonin, tryptamine, or tyramine. The K _m values for benzylamine and phenethylamine were 2.7?×?10^?4 and 7.1?×?10^?4?M, respectively. Enzyme activity was not inhibited by pargyline, clorgyline, semicarbazide, or Na-diethyldithiocarbamate. Benzaldehyde, the product of benzylamine oxidation, exhibited strong competitive inhibition of enzyme activity with a Ki of 3???M. FAD was identified by ODS-column chromatography as an enzyme cofactor. The enzyme contained 2?mol of FAD per 220,000?g of enzyme.