Activity Of Enzymatic Extracts Research Articles

Enzymatic activity of extracts from higher fungi for manufacturing fermented dairy products

The present study investigates the enzymatic activity and chemical composition of extracts obtained from the substrate mycelium of higher fungi. The investigated object is the biomass of fungi Piptoporus betulinus (substrate mycelium) gathered after solid-phase cultivation on natural substrate. The extracts were obtained using distilled water (pH=7.0), acetate (pH=4.7) and phosphate (pH=7.4) buffers, and Mcllvaine buffer (pH=4.0). Milk-clotting, proteolytic, cellulosolytic and lipolytic activity, as well as protein content, were determined in both aqueous or buffer extracts. As a result, the values of cellulosolytic (3.75–3.90 units/g), lipolytic (40.00–44.24 units/g) and milk-clotting (65.80–66.60 units/mL) activity of the substrate mycelium was determined. These values differ slightly in the extracts prepared on distilled water and buffers. Moreover, the concentration of protein substances in the native aqueous extract from the substrate mycelium of P. betulinus was 14.50 mg/mL. The values of proteolytic activity varied from 0.22 to 0.78 units/mL. Distilled water was found to be the most effective solvent for achieving high values of milk-clotting activity. Extract purification by microfiltration or with bentonite leads to a significant decrease in protein concentration (up to 5.90 mg/mL), cellulose(up to 1.40 units/g), lipo(up to 5.30 units/g), and proteolytic (up to 0.11 units/mL) activity, and an increase in milk-clotting activity values (up to 285.80 units/mL). The bentonite sorbs cellulosolytic, lipolytic, and non-specific proteolytic enzymes, resulting in a noticeable increase in the value of milk-clotting activity. In the aqueous extract of P. betulinus fungi, a high ratio of milk-clotting to proteolytic activity was found with a value of 2598.20. This may lead to an increased yield and improvement of the organoleptic properties of cheese and its storage period.

Cannonball jellyfish digestion: an insight into the lipolytic enzymes of the digestive system.

The digestive system and metabolism of the cannonball jellyfish Stomolophus sp. 2 are not well-known. The digestion study was critical to explain its ecology and bloom success. Different enzymes are involved in food digestion, which hydrolyze carbohydrates, proteins, and lipids. This study detected lipolytic activity in enzymatic extracts from gastric pouches of Stomolophus sp. 2 collected in the summer of 2013 at Bahía de Kino, Sonora, México (28°47′47″N 111°57′25″W). Lipase/esterase activity showed optimal pH at 11.0 and 50–60 °C with a half-life (t1/2) of 33 min at 55 °C, whereas halotolerance of this activity was recorded from 0-4 M NaCl. Metal ions Ca2+ and Mn2+ did not affect the activity, but Mg2+ decreased it 14.2% ± 3.15, while chelating agents as ethylenediaminetetraacetic acid reduced the activity 8.55% ± 2.13. Inhibition of lipase/esterase activity with tetrahydrolipstatin and paraoxon-ethyl decreased the activity 18.2% ± 2.3, and 62.80% ± 0.74, respectively, whereas phenylmethanesulfonyl fluoride (a protease inhibitor) did not affect it. The enzyme displayed a higher specificity for short-chain triglycerides, but triolein, coconut oil, olive oil, and fish oil were hydrolyzed. For the first time, phospholipase activity from the gastric pouch of Stomolophus sp. 2 was detected using L-α-phosphatidylethanolamine from chicken egg yolk as a substrate. These results suggest that Stomolophus sp. 2 hydrolyze several kinds of lipids, and lipolytic enzymes are active at alkaline pH under different saline conditions, which may be essential to digest different preys.

Network of proteins, enzymes and genes linked to biomass degradation shared by Trichoderma species

Understanding relationships between genes responsible for enzymatic hydrolysis of cellulose and synergistic reactions is fundamental for improving biomass biodegradation technologies. To reveal synergistic reactions, the transcriptome, exoproteome, and enzymatic activities of extracts from Trichoderma harzianum, Trichoderma reesei and Trichoderma atroviride under biodegradation conditions were examined. This work revealed co-regulatory networks across carbohydrate-active enzyme (CAZy) genes and secreted proteins in extracts. A set of 80 proteins and respective genes that might correspond to a common system for biodegradation from the studied species were evaluated to elucidate new co-regulated genes. Differences such as one unique base pair between fungal genomes might influence enzyme-substrate binding sites and alter fungal gene expression responses, explaining the enzymatic activities specific to each species observed in the corresponding extracts. These differences are also responsible for the different architectures observed in the co-expression networks.

Influence of the ripening stage and the lyophilization of wild cardoon flowers on their chemical composition, enzymatic activities of extracts and technological properties of cheese curds

The selection of an appropriate Cynara cardunculus flowers batch is a preliminary step to produce rennet with better clotting properties. For this reason, we proposed to study the influence of the ripening stage and the lyophilization of cardoon flowers on their chemical composition, enzymatic activities of extracts, and technological properties of cheese curds. Results of flowers composition have shown that lyophilized flowers harvested at the middle of ripening stage (A) could be employed to produce mainly proteins or milk-clotting proteases. To confirm this, enzymatic activities of extracts and technological properties of curds were assessed. The experimental findings revealed that flowers lyophilization seems to be an efficient way to produce rennet with better clotting properties, leading to higher yield, moisture, and texture parameters of curd. These findings allowed us to select lyophilized flowers (A) for further cheese making process.

Use of biotechnology for creation of structured food

The influence of enzymatic complex of plant material on physical and chemical properties of pectin substances and structuring of fruit and vegetable raw materials is determined. It is analyzed an activity of enzymatic extracts of plant material by the action on apple pectin, and structure-forming properties of vegetable puree with addition of enzymatically treated apple pectin. It is obtained structured product with enhanced functional properties

Enzymatic Activity of Allergenic House Dust and Storage Mite Extracts

Proteases are involved in the pathogenicity of allergy, increasing epithelial permeability and acting as adjuvants. Enzymatic activity is therefore important for the allergenicity of an extract and also affects its stability and safety. However, the enzymatic activity of extracts is not usually evaluated. The objective of this study was to evaluate the enzymatic activity of the most allergenic mite extracts and to investigate their allergenic properties. Extracts from nine allergenic mite species (Dermatophagoides pteronyssinus, Dermatophagoides farinae Hughes, Euroglyphus maynei, Lepidoglyphus destructor, Tyrophagus putrescentiae (Schrank), Glycyphagus domesticus (DeGeer), Acarus siro L., Chortoglyphus arcuatus, and Blomia tropicalis) were characterized. Protein and allergen profiles were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western-blot, respectively. Gelatinolytic activity was evaluated with a zymogram and the activity of other enzymes (cysteine, serine proteases, and esterases) was evaluated individually or with the API-ZYM system. The main differences in protease activity were found between house dust mites and storage mites. House dust mites presented higher cysteine protease activity while storage mites presented higher serine protease activity. These differences are in line with their trophic specialization. A wide range of different activities was found in all the extracts analyzed, reflecting the fact that the extracts preserve the activity of many enzymes, this being necessary for a correct diagnosis. However, enzymes may act as adjuvants and, therefore, could lead to undesirable effects in immunotherapies, making this activity not suitable for treatment products. Modified extracts with lower enzymatic activity could be more appropriate for immunotherapy.

Effect of pH and Temperature on the Activity of Enzymatic Extracts from Pineapple Peel

This study was carried out to characterize a crude extract from pineapple peel after precipitation by three methods with the aim of obtaining an enzymatic extract from agro-industrial waste. The characterization of these extracts involved the determination of both protein content and specific protease activities. The effects of pH and temperature on specific protease activity and on the stability of the extracts were also evaluated. The optimal values of specific activity for the crude extract (CE) were pH 6.0 (5.76 U mg−1 protein) and 7.0 (5.71 U mg−1 protein) and a temperature of 70 °C (16 U mg−1 protein). The average values for the relative specific activity were 17.4% (pH 3.0 to 9.0) and 42.7% (at 30, 50, and 70 °C). The ethanolic extract had the highest specific activity (10.7 U mg−1 protein) in comparison to the best results obtained for the isoelectric precipitation (7.7 U mg−1 protein) and the ammonium sulfate precipitation (4.7 U mg−1 protein). Moreover, the ethanolic extract was more stable than the CE, retaining 60.9% and 53.7% of the initial specific activity during the evaluation of the stability at different pH and temperature values, respectively. The optimal values of pH and temperature were almost the same for the crude and the ethanolic extracts. In addition, the ethanolic extract was more stable than the CE in the experimental conditions tested in this work.

Proteolytic activity in enzymatic extracts from Carica papaya L. cv. Maradol harvest by-products

Proteolytic activity and the cysteine protease profile were determined for enzymatic extracts (EE) from Carica papaya L. cv. Maradol harvest by-products (stems, unripe fruit, petioles and leaves). The proportion of each by-product type in the sampled plantation was calculated. Polypeptide bands were identified by SDS–PAGE for each EE and molecular weight calculated for the cysteine proteases. Leaf and fruit tissue had the highest protein contents of the by-products. Leaf tissue also produced the highest total EE yield. All the SDS–PAGE gels for the EE’s exhibited an approximately 23 kDa band probably corresponding to papain. The zymography profiles of the EE’s were similar, with bands at approximately >202.8, 76.8, 55.4 and 46.5 kDa. The fruit EE had the highest specific proteolytic activity and the leaf EE the lowest. Fruit and stem by-products are the most promising for proteolytic enzyme extraction.

Enzymatic Activity in Body and Fecal Extracts of the Storage Mite Chortoglyphus arcuatus

Background:Chortoglyphus arcuatus has been described in many countries. Many allergens are potent enzymes, which may promote a Th2 immune response. The aim of this study was to evaluate the enzymatic activity of body and fecal extracts of C. arcuatus. Material and Methods: Feces and bodies of full-grown C. arcuatus cultures were separated by sieving, extracted in PBS, dialyzed and lyophilized. The antigenic profile of both extracts was determined by SDS-PAGE. Immunoblot experiments were conducted using a pool of sera from allergic individuals residing in Galicia, a region of Spain, where this species is abundant. The enzymatic activity of the extracts was evaluated by the zymogram technique. Serine and cysteine protease activity was measured using in vitro methods. The API Zym® system was used to determine the enzymatic properties of the extracts. Results: The antigenic profile showed that the body extract contained more and better defined bands than the fecal extract. Allergens were detected in both extracts in a molecular weight range between 14 and 100 kDa. Gelatinolytic gels confirmed that fecal extracts contain more hydrolytic enzymatic activity than body extracts. Serine protease activity in fecal extracts was higher than in body extracts (5.98 vs. 2.701 IU of trypsin/mg of freeze-dried material). No cysteine protease activity was detected. Conclusion:C. arcuatus extracts contain several allergens and proteins with high enzymatic activity, especially in the feces. Some of these allergens may be enzymes. Fecal extracts have more enzymatic activity than body extracts.

Enzymatic Activity in Extracts of Allergy-Causing Astigmatid Mites

Enzymatic Activity in Extracts of Allergy-Causing Astigmatid Mites

Enzymatic Activity in Extracts of Allergy-Causing Astigmatid Mites

Many of the previously characterized allergens of house dust mites are known to be proteases, and this enzymatic activity is thought to contribute to their allergenicity. Other astigmatid mites, including stored-product mites and the ectoparasitic itch mite, Sarcoptes scabiei De Geer, are also known to be allergenic, but little or nothing is known about their enzymatic activities. The purpose of this study was to characterize the enzymatic activities present in extracts of the parasitic itch mite and from eight other species of free-living astigmatid mites. Extracts were prepared from one parasitic mite (S. scabiei), five stored-product mites (Chortoglyphus arcuatus (Troupeau), Lepidoglyphus destructor (Schrank), Blomia tropicalis Bronswijk, Cock, Oshima, Tyrophagus putrescentiae (Schrank), and Acarus siro L.), and three house dust mites [Dermatophagoidesfarinae Hughes, Dermatophagoides pteronyssinus (Troussart), and Euroglyphus maynei (Cooreman) ]. ApiZym strips were used to screen for the presence of 19 individual enzyme activities. Digestion of nine other substrates was evaluated by spectrophotometric or electrophoretic methods. All mite extracts exhibited some form of phosphatase, esterase, aminopeptidase, and glycosidase activity, although their substrate specificities varied considerably. Itch mite extract did not possess detectable serine peptidase activity nor was it able to hydrolyze gelatin or casein, whereas all other mite extracts exhibited these activities. Storage mite extracts possessed enzymes capable of degrading the widest range of substrates, whereas itch mite extract had the most limited proteolytic capacity. Extracts of nine species of allergy-causing astigmatid mites contain wide and diverse repertoires of enzymatic activities. These catalytic activities may be important contributors to the induction and manifestation of inflammatory and immune responses to mites in patients.

A potential role for beta-carotene in avian embryonic development.

Vitamin A is essential for vertebrate embryonic development; dietary carotenoids are the primary source of vitamin A since animals cannot synthesize it de novo. To study the role of beta-carotene during embryonic development, we analyzed in chick embryos the expression of beta,beta-carotene 15,15'-oxygenase (beta-oxy) which cleaves beta-carotene to produce two molecules of retinal. Beta-oxy transcripts were detected in one-and-a-half- to five-day-old embryo homogenates and in situ hybridization in five-day-old embryos, revealing their presence in tissues including the central nervous system, lungs, limbs, and cardiovascular system. Moreover, we detected beta-oxy enzymatic activity in extracts from five-day-old embryos as well as small amounts of beta-carotene in the egg yolk. These results indicate that beta-oxy is present during early developmental stages, raising the possibility that yolk-stored beta-carotene is utilized as a source of vitamin A. Thus, our results suggest that beta-carotene could play an important role in early avian embryonic development as a local source of vitamin A in specific tissues.

Involvement of multiple copper/topa quinone-containing and flavin-containing amine oxidases and NAD(P) + aldehyde dehydrogenases in amine degradation by filamentous fungi

Eighty-five different fungal strains were screened for the presence of amine oxidase after it was induced by n-butylamine, methylamine or spermine. Amine oxidase activity was detected in all Aspergillus, Penicillium, and most of Monascus strains, as well as in Pullularia, Doratomyces, Armillaria, Fusarium, Gibberella, Sporotricus, Trichophyton, Beauveria, Caldariomyces, Helicostylum, and Trichoderma, but not in Mucor, Neurospora, Syncephalis, Keratinomyces, Eurotium, Zygorhynchus, or Cladosporium. By electrophoresis of native proteins with amine oxidase activity staining, two to three different enzymes were found in most of the strains. Predominant among these were copper-containing amine oxidases (EC 1.4.3.6), which were detected by Western blotting with polyclonal antibody against copper amine oxidase from Aspergillus niger AKU 3302. On SDS-PAGE, most of the enzymes showed bands corresponding to a molecular mass of about 75 kDa. Enzymes that did not react with the antibody against copper amine oxidase did not react either with the antibody against flavin-containing monoamine oxidase from Micrococcus luteus. However, the enzymatic activities of extracts that contained non-copper amine oxidases was substantially decreased by pargyline, an inhibitor specific to flavin-containing amine oxidases. Therefore, these enzymes are thought to belong to the class of flavin-containing amine oxidases (EC 1.4.3.4.). Besides amine oxidase activity, increased aldehyde dehydrogenase activity was found in selected strains after induction with amines, from which it is supposed that amines are degraded via aldehydes to corresponding carboxylic acids. Two alternate metabolic pathways are suggested for methylamine and for larger aromatic amines.

Bombyx mori nuclear polyhedrosis virus-based vectors for expressing passenger genes in silkmoth cells under viral or cellular promoter control

The polyhedrin gene of the nuclear polyhedrosis virus of the silkmoth Bombyx mori (BmNPV) has been subjected to deletion mutagenesis. A number of clones containing partially deleted polyhedrin genes were characterized and four clones containing limited deletions of the 5' -untranslated or 5' -flanking sequences of the gene were further analyzed with respect to polyhedrin promoter activity. The functional characterization of the deletion mutants was achieved through the insertion of a chloramphenicol acetyl transferase (CAT) gene (cat) into each deletion junction. The resultant cat constructs were introduced into the genome of BmNPV through homologous recombination and the effect of each deletion on the activity of the polyhedrin promoter was evaluated by measurements of CAT enzymatic activity in extracts of tissue culture cells infected with the corresponding recombinant BmNPVs as well as by primer extension assays. Removal of the entire leader region and eleven adjacent residues of the 5'-flanking sequences of the polyhedrin gene results in a dramatic decrease in promoter activity, which, however, remains detectable through CAT activity measurements. Elimination of an additional 30 nucleotides (nt) of the upstream sequences results in the complete inactivation of the polyhedrin promoter. The functional characterization of a deletion mutant lacking 41 nt of the 5'-flanking sequences has demonstrated that no functions necessary for viral infectivity, replication or assembly are disrupted by this deletion, since the corresponding recombinant viruses propagate in the cells with the same kinetics and to the same extent as wild-type BmNPV. As a result of the deletion mutagenesis, two classes of transfer vectors have become available. The first class can be used for introducing into the viral genome foreign nucleotide sequences under polyhedrin promoter control, while the second one can be used for obtaining recombinant viruses harboring foreign genetic material in an environment which is devoid of polyhedrin promoter activity.

Introduction of UAG, UAA, and UGA nonsense mutations at a specific site in the Escherichia coli chloramphenicol acetyltransferase gene: use in measurement of amber, ochre, and opal suppression in mammalian cells.

We have used oligonucleotide-directed site-specific mutagenesis to convert serine codon 27 of the Escherichia coli chloramphenicol acetyltransferase (cat) gene to UAG, UAA, and UGA nonsense codons. The mutant cat genes, under transcriptional control of the Rous sarcoma virus long terminal repeat, were then introduced into mammalian cells by DNA transfection along with UAG, UAA, and UGA suppressor tRNA genes derived from a human serine tRNA. Assay for CAT enzymatic activity in extracts from such cells allowed us to detect and quantitate nonsense suppression in monkey CV-1 cells and mouse NIH3T3 cells. Using such an assay, we provide the first direct evidence that an opal suppressor tRNA gene is functional in mammalian cells. The pattern of suppression of the three cat nonsense mutations in bacteria suggests that the serine at position 27 of CAT can be replaced by a wide variety of amino acids without loss of enzymatic activity. Thus, these mutant cat genes should be generally useful for the quantitation of suppressor activity of suppressor tRNA genes introduced into cells and possibly for the detection of naturally occurring nonsense suppressors.

Introduction of UAG, UAA, and UGA nonsense mutations at a specific site in the Escherichia coli chloramphenicol acetyltransferase gene: use in measurement of amber, ochre, and opal suppression in mammalian cells.

We have used oligonucleotide-directed site-specific mutagenesis to convert serine codon 27 of the Escherichia coli chloramphenicol acetyltransferase (cat) gene to UAG, UAA, and UGA nonsense codons. The mutant cat genes, under transcriptional control of the Rous sarcoma virus long terminal repeat, were then introduced into mammalian cells by DNA transfection along with UAG, UAA, and UGA suppressor tRNA genes derived from a human serine tRNA. Assay for CAT enzymatic activity in extracts from such cells allowed us to detect and quantitate nonsense suppression in monkey CV-1 cells and mouse NIH3T3 cells. Using such an assay, we provide the first direct evidence that an opal suppressor tRNA gene is functional in mammalian cells. The pattern of suppression of the three cat nonsense mutations in bacteria suggests that the serine at position 27 of CAT can be replaced by a wide variety of amino acids without loss of enzymatic activity. Thus, these mutant cat genes should be generally useful for the quantitation of suppressor activity of suppressor tRNA genes introduced into cells and possibly for the detection of naturally occurring nonsense suppressors.

Studies by NMR of in vitro spontaneous oscillations of activity in enzymatic extracts

NMR has been used to study slow spontaneous oscillations previously observed for the activity of enzymes extracted from plant leaves and maintained under constant conditions. The randomly modulated period of these oscillations has a mean value of several hours. This phenomenon is interpreted in terms of synchronous transition among several conformational states of biomolecules.

The proteolytic enzyme system of skin. VII. Effect of immune reactions.

SummaryPassive cutaneous anaphylaxis was induced in the rat using the egg albumin-rabbit anti-egg albumin system. Enzymatic activities of extracts of skin exposed to this immune reaction were assayed with casein, N-acetyl-L-tyrosine ethyl ester, and L-tyrosine ethyl ester as substrates. No difference existed between these extracts and those of control skin extracts. Purified Proteinase C was maximally inactivated by mixing the enzyme with its specific inhibitor. The inactivated enzyme solution was added to preformed antigen-antibody aggregates and no activation effect was observed. Similarly, no activation effect was found when the immune reaction was allowed to proceed in vitro in the presence of inactivated Proteinase C.

The inactivation of insulin by liver slices of the rat.

THE demonstration that extracts of rat liver contain an insulininactivating system (insulina.se) (Mirsky and Broh-Kahn, 1949) and an inhibitor of this system (insulinase-inhibitor (Mirsky, Simkin and Broh-Kahn, 1950) raises the possibility that the rate of insulin inactivation within the intact cell is determined by the balance of activity resulting from the mobilization of either of the two systems. Since the assay of the enzymatic activity of extracts may not represent the “working” activity of the cell prior to disintegration by homogenization, it was deemed essential to investigate the inactivation of insulin by relatively intact cells such as comprise surviving slices of liver. METHODS Fed male rats of from 150 to 200 gm in weight were decapitated and as much blood as possible was drained from the carcass of each animal. The liver was removed and placed in cold phosphate buffer, pH 7.4. Slices of approximately 1 cm2 in size were prepared with a Stadie-Riggs apparatus (Studie and Riggs, 1944).