Academic literature on the topic 'Enzyme activity determination'

The use of enzymes on industrial scale saves a lot of energy and avoids pollution, thus holding a promise for green and economically sustainable alternative strategies in industrial transformations. Generally, the fungi Aspergillus niger secretes enzymes which can be used in different industries. Thus, coming up with these enzymes in large amounts will definitely result in reduced costs encountered in importing them for industrial use. This study focussed on isolation and activity determination of an enzyme phosphatase secreted by Aspergillus niger. This enzyme can be of great importance in molecular biology industries, particularly for recombinant DNA technology. For this study, pure cultures of Aspergillus niger were used. Aspergillus niger was resuscitated on potato dextrose agar and then subcultured in Adam’s medium, a medium specific for the production of phosphatase. Cells were centrifuged and the filtrate was collected whilst the residue was discarded. The filtrate was expected to contain the crude enzyme phosphatase since Aspergillus niger secretes the extracellular enzyme into the medium. Disodium phenyl phosphate was used as a substrate for the determination of the phosphatase activity. The enzyme activity was determined spectrophotometrically by reading absorbance of phenol formed in the presence of enzyme and the substrate. The concentration of phenol liberated was then used to calculate the enzyme activity expressed in King Armstrong Units (KAU). Further work on enzyme activity determination was done by varying enzyme and substrate concentrations. Results showed that the isolated alkaline phosphatase had activity of 4.0 KAU and 4.5 KAU at 25 ºC and 37 ºC respectively. Acidic phosphatase had activity of 5 KAU and 7 KAU at 25 ºC and 37 ºC respectively. Rate of activity increased upon increasing enzyme concentration and substrate. Thus, Aspergillus niger produces the enzyme phosphatase, however, there is need to induce the production of these enzymes for industrial use.

"α-mannosidase is a hydrolytic enzyme that cleaves α-glycosidic bonds of mannopyranoside derivatives. In this study, the enzyme activity and the specific activity of α-mannosidase have been determined using the p-nitrophenyl-alpha-Dmannopyranoside hydrolysis assay."

"α-mannosidase is a hydrolytic enzyme that cleaves α-glycosidic bonds of mannopyranoside derivatives. In this study, the enzyme activity and the specific activity of α-mannosidase have been determined using the p-nitrophenyl-alpha-Dmannopyranoside hydrolysis assay."

Traditionally, the dependence of enzyme activity on temperature has been described by a model consisting of two processes: the catalytic reaction defined by ΔGDaggercat, and irreversible inactivation defined by ΔGDaggerinact. However, such a model does not account for the observed temperature-dependent behaviour of enzymes, and a new model has been developed and validated. This model (the Equilibrium Model) describes a new mechanism by which enzymes lose activity at high temperatures, by including an inactive form of the enzyme (Einact) that is in reversible equilibrium with the active form (Eact); it is the inactive form that undergoes irreversible thermal inactivation to the thermally denatured state. This equilibrium is described by an equilibrium constant whose temperature-dependence is characterized in terms of the enthalpy of the equilibrium, ΔHeq, and a new thermal parameter, Teq, which is the temperature at which the concentrations of Eact and Einact are equal; Teq may therefore be regarded as the thermal equivalent of Km. Characterization of an enzyme with respect to its temperature-dependent behaviour must therefore include a determination of these intrinsic properties. The Equilibrium Model has major implications for enzymology, biotechnology and understanding the evolution of enzymes. The present study presents a new direct data-fitting method based on fitting progress curves directly to the Equilibrium Model, and assesses the robustness of this procedure and the effect of assay data on the accurate determination of Teq and its associated parameters. It also describes simpler experimental methods for their determination than have been previously available, including those required for the application of the Equilibrium Model to non-ideal enzyme reactions.

Background: In order to organize and give a better understanding of the existing population of protease activity units together with their respective methods of enzymatic activity assessment, there is a need of their clear classification system. Results and Conclusion: The following system has been proposed: Enzyme Centered Units (ECU) equivalent to Enzyme Process Unit notation; Protein Centered Units (PCU) equivalent to Protein Process Unit notation; Legal Authority and Enzyme Centered Units (LAECU) equivalent to Enzyme Centered Units system additionally related to a legal authority or an organization. The suitable ways for the mutual conversion of commonly used units and their conversion into the standard SI units have been included. A convenient gravity/spectrophotometer test of proteolytic activity with the use of three protein types has also been proposed. The test gives high degree of confidence of the experimental determination for a wide spectrum of protease activity in samples of plant origin. The whole paper allows both theoretical and practical orientation in the range of different proteolytic activity units as well as in the methods of their determination.

Abstract Pancreatin is a biotechnological product containing an enzyme complex, obtained from porcine pancreas, that is employed in treating pancreatic diseases. Experiments regarding the stability of the pharmaceutical formulation containing pancreatin were performed using standard binary mixtures with 6 excipients in a 1:1 ratio (m/m) and a commercial formulation. To accomplish these goals, samples were stored for 1, 3 and 6 months at 40 ± 1 °C and 75 ± 5 % relative humidity (RH) and 40 ± 1 °C and 0 % RH. Stress testing was also performed. All samples were analyzed to evaluate the α-amylase, lipase and protease activities through UV/Vis spectrophotometry. The results revealed that the excipient proprieties and the storage conditions affected enzyme stability. Humidity was a strong influencing factor in the reduction of α-amylase and protease activities. Stress testing indicated that pH 9.0 and UV light did not induce substantial alterations in enzyme activity.

In this study, three subspecies of Apis mellifera L. (A. m. caucasica, A. m. carnica, and A. m. syriaca) from different climatic regions were evaluated electrophoretically at ontogenetic level by means of four enzymes, namely Phosphoglucomutase (PGM), Hexokinase (HK), Phosphoglucose isomerase (PGI) and Glukose-6-phosphate dehydrogenase (G6PD). It is determined that only Pgm and Hk loci were polymorphic. Allele and genotype frequencies at Pgm locus changes seasonally whereas Hk locus does not exhibit seasonal variation. Within the scope of this study we investigated at which developmental stage shifting to heterozygotes prior to winter occurs. It is found that there is a seasonal fluctuation throughout the year in Pgm genotype frequencies at each developmental stage studied and correlated with enzyme activity and glycogen content. As the studied enzymes have crucial v roles in insect energy metabolism, results of this study provided further information about the relationship between carbohydrate metabolism and enzyme polymorphism of honey bees.

碩士
大同大學
生物工程學系(所)
102
The characteristics of the currently available enzymes for rice-cooking are investigated. These properties included heat-resistance, abilities to cut amylose and to produce mono-, di-, tri-, and tetrasaccharides. The function of rice-cooking was also evaluated. First, the ability of the enzyme to produce low-molecular-weight saccharides from starch was determined. Results indicated that the rice soaked in water for 15 minutes without addition of enzyme yielded mono- and disaccharides alone. However, the rice soaked in water in the presence of the enzyme yielded not only more amounts of mono- and disaccharides but also tri- and tetrasaccharides compared to that without enzyme. The increase of mono- and disaccharides enhanced the sweetness of rice. The production of tri- and tetra-saccharides resulted from the cleavage of starch by the enzyme. Therefore, the molecular weights of starch and the viscosity of rice should decrease. The enzyme for rice-cooking posses the following properties: (1) the enzyme activity increased with the increase of temperature in the range of 30~35°C, (2) Inactivation of the enzyme occurred at 60°C, (3) Increase of enzyme activity by ~10% when temperature increased by 10°C, (4) The linear correlation declined as the enzyme reaction was prolonged. Two enzymes, A and B, possessed different heat-resistance. Enzyme A lost 4.5~33% of activity for every increase of 10°C, whereas enzyme B was very stable at the temperature ranged from 30° to 50°C. Although different enzymes for rice-cooking possessed varied heat resistance, these enzymes can be used for improving digestibility, texture and sweetness of cooked rice.

碩士
中山醫學院
生物化學研究所
87
Part 1: The aim of the present study was to determine the level of eryth-rocyte carbonic anhydrase isoenzyme of G6PD deficiency patient in Taiwan . Carbonic anhydrase (CA) is a zinc-containing enzyme, it reversibly catalyzes the hydration of carbon dioxide to bicarbonate and hydrogen ions. CA II is believed to be a chloride/bicarbonate exchanger in red blood cells, and also directly coupled to band 3 protein. Human erythrocyte CA I and CAII were measured in normal subjects (n=30) and G6PD deficiency patients (n=30), by using Western blot assay (with 12.5 % SDS-PAGE- 0.8 % bisacrylamide). In the groups of G6PD deficiency patients, there was a tendency for CA I / CA II ratio (mean= 2.3) to be lower than normal subjects (mean= 5.2)(P<0.001). The band 3 protein were resol-ved by 8 % SDS-PAGE, normal subjects was also significantly higher than the G6PD deficiency patients. We may speculate that in G6PD deficiency might be to a change in CA II and band 3 protein expression and stability. The cytoskeletal proteins which bind spectrin, and that this change makes the affected protein more susceptible to oxidative damage, band 3 protein loss and increased levels of CAII in G6PD deficiency patients may lead to hemolytic anemia. Detailed mechanism is unclear. Part 2: A rapid automated system for assessing the activities of superoxide dismutase (SOD), total antioxidant status (TAS), and glutathione peroxidase (GSHPx) in blood was developed using a set of commercial kits and the Beckman Synchrom CX-5 automatic analyser. Reproducibility data for assaying SOD, GSHPx, and TAS by our proposed procedure were excellent (CVs < 6.0 %). We then recruited a total of 188 apparently healthy individuals and their antioxidative status were assessed by our proposed method. We found that female (n=90) had a significant higher SOD (1082±261 unit/g Hb) and GSH-Px (91±16 unit/g Hb) than its male counterparts (n=98)[ SOD: 989±196 unit/g Hb; GSH-Px: 79±11 unit/g Hb] (P < 0.01). GSH-Px (R = 0.26) and TAS (R = -0.38) was correlated with age. Interestingly, we also found that alcoholics and motor cyclists seemed to have lower GSH-Px (P < 0.05). In contrast, we did not find any significant discrepancy in SOD, GSH-Px and TAS between smokers and non- smokers.

Bones are multifunctional passive organs of movement that supports soft tissue and directly attached muscles. They also protect internal organs and are a reserve of calcium, phosphorus and magnesium. Each bone is covered with periosteum, and the adjacent bone surfaces are covered by articular cartilage. Histologically, the bone is an organ composed of many different tissues. The main component is bone tissue (cortical and spongy) composed of a set of bone cells and intercellular substance (mineral and organic), it also contains fat, hematopoietic (bone marrow) and cartilaginous tissue. Bones are a tissue that even in adult life retains the ability to change shape and structure depending on changes in their mechanical and hormonal environment, as well as self-renewal and repair capabilities. This process is called bone turnover. The basic processes of bone turnover are: • bone modeling (incessantly changes in bone shape during individual growth) following resorption and tissue formation at various locations (e.g. bone marrow formation) to increase mass and skeletal morphology. This process occurs in the bones of growing individuals and stops after reaching puberty • bone remodeling (processes involve in maintaining bone tissue by resorbing and replacing old bone tissue with new tissue in the same place, e.g. repairing micro fractures). It is a process involving the removal and internal remodeling of existing bone and is responsible for maintaining tissue mass and architecture of mature bones. Bone turnover is regulated by two types of transformation: • osteoclastogenesis, i.e. formation of cells responsible for bone resorption • osteoblastogenesis, i.e. formation of cells responsible for bone formation (bone matrix synthesis and mineralization) Bone maturity can be defined as the completion of basic structural development and mineralization leading to maximum mass and optimal mechanical strength. The highest rate of increase in pig bone mass is observed in the first twelve weeks after birth. This period of growth is considered crucial for optimizing the growth of the skeleton of pigs, because the degree of bone mineralization in later life stages (adulthood) depends largely on the amount of bone minerals accumulated in the early stages of their growth. The development of the technique allows to determine the condition of the skeletal system (or individual bones) in living animals by methods used in human medicine, or after their slaughter. For in vivo determination of bone properties, Abstract 10 double energy X-ray absorptiometry or computed tomography scanning techniques are used. Both methods allow the quantification of mineral content and bone mineral density. The most important property from a practical point of view is the bone’s bending strength, which is directly determined by the maximum bending force. The most important factors affecting bone strength are: • age (growth period), • gender and the associated hormonal balance, • genotype and modification of genes responsible for bone growth • chemical composition of the body (protein and fat content, and the proportion between these components), • physical activity and related bone load, • nutritional factors: – protein intake influencing synthesis of organic matrix of bone, – content of minerals in the feed (CA, P, Zn, Ca/P, Mg, Mn, Na, Cl, K, Cu ratio) influencing synthesis of the inorganic matrix of bone, – mineral/protein ratio in the diet (Ca/protein, P/protein, Zn/protein) – feed energy concentration, – energy source (content of saturated fatty acids - SFA, content of polyun saturated fatty acids - PUFA, in particular ALA, EPA, DPA, DHA), – feed additives, in particular: enzymes (e.g. phytase releasing of minerals bounded in phytin complexes), probiotics and prebiotics (e.g. inulin improving the function of the digestive tract by increasing absorption of nutrients), – vitamin content that regulate metabolism and biochemical changes occurring in bone tissue (e.g. vitamin D3, B6, C and K). This study was based on the results of research experiments from available literature, and studies on growing pigs carried out at the Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences. The tests were performed in total on 300 pigs of Duroc, Pietrain, Puławska breeds, line 990 and hybrids (Great White × Duroc, Great White × Landrace), PIC pigs, slaughtered at different body weight during the growth period from 15 to 130 kg. Bones for biomechanical tests were collected after slaughter from each pig. Their length, mass and volume were determined. Based on these measurements, the specific weight (density, g/cm3) was calculated. Then each bone was cut in the middle of the shaft and the outer and inner diameters were measured both horizontally and vertically. Based on these measurements, the following indicators were calculated: • cortical thickness, • cortical surface, • cortical index. Abstract 11 Bone strength was tested by a three-point bending test. The obtained data enabled the determination of: • bending force (the magnitude of the maximum force at which disintegration and disruption of bone structure occurs), • strength (the amount of maximum force needed to break/crack of bone), • stiffness (quotient of the force acting on the bone and the amount of displacement occurring under the influence of this force). Investigation of changes in physical and biomechanical features of bones during growth was performed on pigs of the synthetic 990 line growing from 15 to 130 kg body weight. The animals were slaughtered successively at a body weight of 15, 30, 40, 50, 70, 90, 110 and 130 kg. After slaughter, the following bones were separated from the right half-carcass: humerus, 3rd and 4th metatarsal bone, femur, tibia and fibula as well as 3rd and 4th metatarsal bone. The features of bones were determined using methods described in the methodology. Describing bone growth with the Gompertz equation, it was found that the earliest slowdown of bone growth curve was observed for metacarpal and metatarsal bones. This means that these bones matured the most quickly. The established data also indicate that the rib is the slowest maturing bone. The femur, humerus, tibia and fibula were between the values of these features for the metatarsal, metacarpal and rib bones. The rate of increase in bone mass and length differed significantly between the examined bones, but in all cases it was lower (coefficient b <1) than the growth rate of the whole body of the animal. The fastest growth rate was estimated for the rib mass (coefficient b = 0.93). Among the long bones, the humerus (coefficient b = 0.81) was characterized by the fastest rate of weight gain, however femur the smallest (coefficient b = 0.71). The lowest rate of bone mass increase was observed in the foot bones, with the metacarpal bones having a slightly higher value of coefficient b than the metatarsal bones (0.67 vs 0.62). The third bone had a lower growth rate than the fourth bone, regardless of whether they were metatarsal or metacarpal. The value of the bending force increased as the animals grew. Regardless of the growth point tested, the highest values were observed for the humerus, tibia and femur, smaller for the metatarsal and metacarpal bone, and the lowest for the fibula and rib. The rate of change in the value of this indicator increased at a similar rate as the body weight changes of the animals in the case of the fibula and the fourth metacarpal bone (b value = 0.98), and more slowly in the case of the metatarsal bone, the third metacarpal bone, and the tibia bone (values of the b ratio 0.81–0.85), and the slowest femur, humerus and rib (value of b = 0.60–0.66). Bone stiffness increased as animals grew. Regardless of the growth point tested, the highest values were observed for the humerus, tibia and femur, smaller for the metatarsal and metacarpal bone, and the lowest for the fibula and rib. Abstract 12 The rate of change in the value of this indicator changed at a faster rate than the increase in weight of pigs in the case of metacarpal and metatarsal bones (coefficient b = 1.01–1.22), slightly slower in the case of fibula (coefficient b = 0.92), definitely slower in the case of the tibia (b = 0.73), ribs (b = 0.66), femur (b = 0.59) and humerus (b = 0.50). Bone strength increased as animals grew. Regardless of the growth point tested, bone strength was as follows femur > tibia > humerus > 4 metacarpal> 3 metacarpal> 3 metatarsal > 4 metatarsal > rib> fibula. The rate of increase in strength of all examined bones was greater than the rate of weight gain of pigs (value of the coefficient b = 2.04–3.26). As the animals grew, the bone density increased. However, the growth rate of this indicator for the majority of bones was slower than the rate of weight gain (the value of the coefficient b ranged from 0.37 – humerus to 0.84 – fibula). The exception was the rib, whose density increased at a similar pace increasing the body weight of animals (value of the coefficient b = 0.97). The study on the influence of the breed and the feeding intensity on bone characteristics (physical and biomechanical) was performed on pigs of the breeds Duroc, Pietrain, and synthetic 990 during a growth period of 15 to 70 kg body weight. Animals were fed ad libitum or dosed system. After slaughter at a body weight of 70 kg, three bones were taken from the right half-carcass: femur, three metatarsal, and three metacarpal and subjected to the determinations described in the methodology. The weight of bones of animals fed aa libitum was significantly lower than in pigs fed restrictively All bones of Duroc breed were significantly heavier and longer than Pietrain and 990 pig bones. The average values of bending force for the examined bones took the following order: III metatarsal bone (63.5 kg) <III metacarpal bone (77.9 kg) <femur (271.5 kg). The feeding system and breed of pigs had no significant effect on the value of this indicator. The average values of the bones strength took the following order: III metatarsal bone (92.6 kg) <III metacarpal (107.2 kg) <femur (353.1 kg). Feeding intensity and breed of animals had no significant effect on the value of this feature of the bones tested. The average bone density took the following order: femur (1.23 g/cm3) <III metatarsal bone (1.26 g/cm3) <III metacarpal bone (1.34 g / cm3). The density of bones of animals fed aa libitum was higher (P<0.01) than in animals fed with a dosing system. The density of examined bones within the breeds took the following order: Pietrain race> line 990> Duroc race. The differences between the “extreme” breeds were: 7.2% (III metatarsal bone), 8.3% (III metacarpal bone), 8.4% (femur). Abstract 13 The average bone stiffness took the following order: III metatarsal bone (35.1 kg/mm) <III metacarpus (41.5 kg/mm) <femur (60.5 kg/mm). This indicator did not differ between the groups of pigs fed at different intensity, except for the metacarpal bone, which was more stiffer in pigs fed aa libitum (P<0.05). The femur of animals fed ad libitum showed a tendency (P<0.09) to be more stiffer and a force of 4.5 kg required for its displacement by 1 mm. Breed differences in stiffness were found for the femur (P <0.05) and III metacarpal bone (P <0.05). For femur, the highest value of this indicator was found in Pietrain pigs (64.5 kg/mm), lower in pigs of 990 line (61.6 kg/mm) and the lowest in Duroc pigs (55.3 kg/mm). In turn, the 3rd metacarpal bone of Duroc and Pietrain pigs had similar stiffness (39.0 and 40.0 kg/mm respectively) and was smaller than that of line 990 pigs (45.4 kg/mm). The thickness of the cortical bone layer took the following order: III metatarsal bone (2.25 mm) <III metacarpal bone (2.41 mm) <femur (5.12 mm). The feeding system did not affect this indicator. Breed differences (P <0.05) for this trait were found only for the femur bone: Duroc (5.42 mm)> line 990 (5.13 mm)> Pietrain (4.81 mm). The cross sectional area of the examined bones was arranged in the following order: III metatarsal bone (84 mm2) <III metacarpal bone (90 mm2) <femur (286 mm2). The feeding system had no effect on the value of this bone trait, with the exception of the femur, which in animals fed the dosing system was 4.7% higher (P<0.05) than in pigs fed ad libitum. Breed differences (P<0.01) in the coross sectional area were found only in femur and III metatarsal bone. The value of this indicator was the highest in Duroc pigs, lower in 990 animals and the lowest in Pietrain pigs. The cortical index of individual bones was in the following order: III metatarsal bone (31.86) <III metacarpal bone (33.86) <femur (44.75). However, its value did not significantly depend on the intensity of feeding or the breed of pigs.

We attempted to study the immune response in M. rosenbergii by melanization reaction produced by plasma phenoloxidase (PO) activity. The substrate affinity of the PO enzyme was determined using different phenolic substrates, and it was found that the diphenols were only oxidized. The enzyme was characterized as catechol oxidase type of PO and L-3,4 dihydroxyphenylalanine (L-DOPA) showed the highest substrate affinity to the enzyme. The biochemical parameters that determined optimum enzyme activity were found to be 2.5 mM L-DOPA at an absorbance of 470 nm, 10 mM Tris–HCl buffer at pH 7.5, temperature at 25°C, and 15 min incubation. Kinetic characteristics of plasma were studied from the M. rosenbergii. The hemocyanin was isolated by gel filtration chromatographic technique using Sephadex G-100. The M. rosenbergii hemocyanin (MrHC) showed only one band with a molecular weight of 325 kDa on native polyacrylamide gel electrophoresis (PAGE) when stained with Coomassie Brilliant Blue (CBB) and bathocuproine sulfonic acid. The reduction of MrHC protein in SDS-PAGE displayed three subunits with a molecular weight of 74, 76, and 78 kDa, respectively. Determination of optimal condition for PO activity of plasma has also been attempted. The plasma optimal condition taken for the MrHC was tested for its ability to oxidize diphenols such as L-DOPA was shown only PO activity. These results showed that in the presence of PO and peroxidase inhibitors, phenylthiourea (PTU) and tropolone respectively have decreased plasma and MrHC PO activity. This indicates that hemocyanin triggers innate immunity probably through one of its subunits that function as the active moiety.

Flavonoids are a major class of secondary metabolites that comprises more than 6000 compounds that have been identified. They are biosynthesized via the phenylpropanoid metabolic pathway that involves groups of enzymes such as isomerases, hydroxylases, and reductases that greatly affect the determination of the flavonoid skeleton. For example, transferase enzymes responsible for the modification of sugar result in changes in the physiological activity of the flavonoids and changes in their physical properties, such as solubility, reactivity, and interaction with cellular target molecules, which affect their pharmacodynamics and pharmacokinetic properties. In addition, flavonoids have diverse biological activities such as antioxidants, anticancer, and antiviral in managing Alzheimer’s disease. However, most marine flavonoids are still incompletely discovered because marine flavonoid biosynthesis is produced and possesses unique substitutions that are not commonly found in terrestrial bioactive compounds. The current chapter will illustrate the importance of flavonoids’ role in metabolism and the main difference between marine and terrestrial flavonoids.

In drug-delivery systems containing nano-drug structures, targeting the tumorous tissue by anthraquinone molecules with high biological activity, and reaching and destroying tumors by their tumor-killing effect reveals remarkable results for the treatment of tumors. The various biological activities of anthraquinones and their derivatives depend on molecular conformation; hence, their intra-cell interaction mechanisms including deoxyribonucleic acid (DNA), ribonucleic acid (RNA), enzymes, and hormones. Computer-based drug design plays an important role in the design of drugs and the determination of goals for them. Molecular docking has been widely used in structure-based drug design. The effects of anthraquinone analogues in tumor cells as a result of their interaction with DNA strand has increased the number of studies done on them, and they have been shown to have a wide range of applications in chemistry, medicine, pharmacy, materials, and especially in the field of biomolecules.

In recent years, the application of evolutionary methods for protein engineering has created tremendous optimism regarding our ability to generate proteins with tailored functional properties such as ligand binding, improved stability, allostery, and catalytic activity. The power of directed protein evolution lies in its simplicity : First, a gene encoding a polypeptide is subjected to mutagenesis, and the resulting ensemble of mutated genes is expressed in a suitable cellular host. Second, the population of expressed proteins is subjected to a screening process. Often, multiple rounds of screening are required to isolate the rare clones within the population that can satisfy the functional screen. Third, DNA is isolated from the enriched clones and subjected to additional rounds of mutagenesis and screening under increasingly stringent conditions. This iterative process is repeated several times until either little functional improvement is observed between sequential rounds or proteins that satisfy the chosen criteria have been generated. There is a plethora of methods for generating an ensemble of mutated genes. Specifically, sequence diversity can be created by random mutagenesis, typically accomplished using error-prone polymerase chain reaction techniques ; by homologous in vitro recombination ; or by nonhomologous recombination. The latter involves two families of methods collectively known as incremental truncation for the creation of hybrid enzymes and sequence-homology independent protein recombination. Regardless of the means for generating sequence diversity, the next and by far the more technically challenging step in directed evolution is the screening of the resulting library of protein-expressing cells to isolate those that are expressing a protein variant that exhibits the desired function. It is fair to say that evolutionary protein design has been hampered by limitations in screening technologies. The quantitative determination of protein function for each and every clone in a library in a high-throughput fashion is a difficult and technically demanding task. In broad terms, there are four general strategies suitable for the screening of combinatorial protein libraries: phage display; biological assays that include selections and assays that use reporter enzymes [e.g., two-hybrid-like techniques for detecting interacting proteins ]; single-well assays using high-density microtiter well plates; and flow cytometry (FC) methods. Each of these methods has a different set of advantages and shortcomings.

Factor XIII (FXIIl) of blood coagulation is a zymogen which is converted into an active transglutaminase during the clotting process. Earlier methods used for its determination are cumbersome, laborious, and not suitable for routine laboratory measurements.Most recently we have designed a new simple UV-kinetic assay for the determination of FXIII in the plasma (Muszbek et al., Clin. Chem., 3JL, 35, 1985). The assay is performed on def:j.b-rinated plasma in which FXIII is activated by thrombin and Ca2+. Acetylateddephosphorylated (AD)β-casein and ethylamine are used as substrates and the ammonia released during thereaction is continuously monitored by a NADPH dependent indicator reaction at 340 nm. As the enzymatically active a subunit of FXIII is also present in platelets and monocytes/macrophages we attempted to adapt the above method for the measurement of cellular FXIII activity. Experiments were carried out on Lubrol extract of washed sonicated platelets. It was found that the small amount of fibrinogen present in platelets does not need to be removed and in the blank hirudin used for preventing activation of plasma FXIII should be replaced by EGTA. The concentration of substrates and activators were optimized. The methodwas found linear at least up-to 40 U/l enzyme activity. It had a good reproducibility (optimal conditionvariance was less than 3%) and correlated well with the most commonly used fluorescent amine (dansylcadaverine) incorporation assay. The method was adapted to a centrifugal fast analyser (Baker, Centrifichem). In addition to congenital FXIII deficiency the determination of FXIII in platelets by this new methodmight have a diagnostic importance in haemopoietic diseases with diminished or accelerated platelet production.

Lipoxygenase activities were estimated in platelet subcellular fractions as well as in intact platelets obtained from normal subjects and patients with deficient platelet lipoxygenase activities (<mean - 2SD of normal activities). From a washed platelet suspension (intact platelets), subcellular fractions including 12,000 x g supernatant of sonicated platelets (F-I), 105,000 x g supernatant (cytosol, F-II) and sediment (microsomal fraction, F-III) of F-I were prepared by differential centrifugation at 4°C. The enzyme activity was studied by the determination of 12-hy-droxyeicosatetraenoic acid (HETE; ng) produced by the reaction of 100μM arachidonic acid with 108 platelets or the subcellular fraction derived from them at pH 7.4 for 5 min at 37°C in the presence or absence of 2.5 mM CaCl2 and/or 2 mM ATP by the use of reversed-phase high-performance liquid chromatography. In experiments with subcellular fractions, reduced glutathione was added to the reaction mixture. In normal subjects, HETE production by intact platelets, F-I, F-II and F-III was 1,162.4±203.3, 1,029.7±403.8, 368.8±175.8 and 194.4±73.4 (M±SD, n=9), respectively, and was not significantly affected by the addition of CaCl2 and/or ATP. HETE produced by 12,000 x g sediment of sonicated platelets was negligible (<1 % of the product by intact platelets). One of the 7 patients showed no detectable lipoxygenase activity both in intact platelets and in any subcellular fractions, while, the other 6 patients showed reduced lipoxygenase activities in all subcellular fractions as well as in intact platelets: HETE produced by intact platelets, F-I, F-II and F-III was 78.8± 112.3, 59.1± 36.3, 37.0± 18.9 and 17.7±15.8 (n=6), respectively. The addition of CaCl2 significantly increased HETE production only by the patient’s F-I (p< 0.02),while ATP showed no significant effect in any experiments.Thus, it was shown that lipoxygenaseactivities were not fully exhibited in intact platelets with the deficient enzyme activities and that F-I could produce more HETE than intact platelets especially in the presence of CaCl2 only in the case of such patient’s platelets.

В статье приводятся данные изучения цитокинового профиля (определение уровней содержания интерлейкинов: IL-4, IL-10, IL-18 и γ-интерферона (γ-IFN) методом твердофазного иммуноферментного анализа у17 спортсменов - биатлонистов высшей спортивной квалификации и 17 здоровых добровольцев со средней физической активностью. Полученные результаты подтверждают значительную вариабельность содержания иммунорегуляторных цитокинов в сыворотке крови здоровых людей. Повышение уровней провоспалительных цитокинов у спортсменов свидетельствует о напряженности иммунного ответа при интенсивных физических нагрузках. The article presents the data of the study of the cytokine profile (determination of the levels of interleukins: IL-4, IL-10, IL-18 and γ-interferon (γ-IFN) by enzyme-linked immunosorbent assay in 17 athletes - biathletes of the highest sports qualification and 17 healthy volunteers with moderate physical activity. The results confirm significant variability in the content of immunoregulatory cytokines in the blood serum of healthy people. An increase in the levels of pro-inflammatory cytokines in athletes indicates the immunity tension during high-intensity exercise.

Microvascular thrombosis is considered an important pathogenetic factor in renal failure associated with OJ. Plasma levels of PA-I, measured by an amidolytic assay in 29 patients with OJ, were significantly increased as compared with 20 control patients (X ± SEM: 26.5 ± 3.5 vs 6.0 ± 1 units/ml; p < 0.001). Fibrin autography of plasma supplemented with tissue plasminogen activator (t-PA,50 IU/ml) revealed that in jaundiced samples most of the added activator migrated with an apparent Mr of 100 Kd, corresponding to t-PA-PA-I complex, whereas in control samples virtually all t-PA migrated as free enzyme. Rabbits made icteric by bile duct ligation showed an early and progressive increase in plasma PA-I activity (3- and 6-fold higher than the preoperative levels on the 3rd and 10th day respectively) indicating that OJ itself may cause a rise in circulating PA-I. Venous stasis (10 min) in jaundiced patients (n=4) was neither associated with an increase in blood fibrinolytic activity nor with a decrease in PA-I activity. t-PA antigen determination showed that activator release was impaired in 3 out of 4 patients. In controls (n=4), venous occlusion induced a rise in both fibrinolytic activity and t-PA antigen and a reduction in PA-I activity. Finally, bile duct recanalization in patients subjected to surgery (n=7) was accompanied by a decrease in plasma PA-I activity which paralleled the decrease in seric bilirubin levels. It is concluded that OJ, independently of the primary disease, may induce a marked increase in circulating PA-I and perhaps an impairment of t-PA release. Defective fibrinolysis may well contribute to microvascular thrombosis and renal failure associated with OJ.

Fresh pig heart tissues were homogenized and deli-pidized with cold acetone. The dry acetone powder was extracted with 0.45 M potassium acetate pH 4.5, the extract was fractionated with ammonium sulfate to 60% saturation, successively followed by four chromatography steps: chromatography on CM-Sepharose CL-6B; gel filtration on Sephadex G-100; Fibrin-Sepharose affinity chromatography and chromatography on DEAE-Sepharose CL-6B. The fibrin plate method was used for the tissue plasminogen activator (t-PA) activity determination. The porcine t-PA purified was proved to be homogeneous either by SDS gel electrophoresis or by high performance liguid chromatography with a molecular weight of 67,000. Using the human melanoma t-PA antigen and its rabbit antiserum, the double immunodiffusion and immu-noelectrophoretic tests demonstrated that the porcine t-PA possessed a cross-reaction with the human t-PA. During the purification procedure, it was found that the porcine t-PA was contaminated and reversibly bound with the cell fibronectin. The binding ability depends on salt concentration. They could be separated from each other on Sepharose G-100 gel filtration at high salt concentration, this reversible binding was further confirmed by mixing these two purified components monitored on high performance liquid chromatography. The fibronectin-bound t-PA retained its ability to activate plasminogen, indicating that these two proteins may exist in a form of complex in the in vivo tissue. Some monoclonal antibodies against the porcine t-PA were obtained, being used to screen what domain of the enzyme is responsible for binding the cell fibronectin.

The new drug pro-urokinase, a proenzyme of urokinase (scu-PA), seems to have advantages in comparison with other fibrinolytic agents. Properties like higher fibrin specifity, non-systemic activity and lower antigenity may lead to a lower rate of complications. In a pilot study 10 patients with acute myocardial infarction have been treated under angiographical control with pro-urokinase (3-9 millions IU) by i.v. application. In case of no perfusion a further administration of streptokinase was carried on. The blood samples were obtained at therapy begin and after 5, 10, 30, 60 and 120 minutes. The therapy monitoring was performed by determination of established haemostasis parameters, like fibrinogen, fibrin(ogen)-split products (FSP), a2-antiplasmin. Plasminogen and batroxobin-time. Furthermore, the diagnostic relevance of new laboratory tests for fibrinolysis, D-Dimer and thrombin-anti thrombin Ill-complex (TAT) has been investigated considering some typical follow-ups. D-Dimer were determined by latex agglutination test and TAT by enzyme immunoassay.Generally the application of pro-urokinase in contrast to streptokinase results in minimal changes of the classic fibrinolysis parameters like fibrinogen, FSP, batroxobin-time etc. demonstrating no systemic lysis. The appearance of plasmic degradation products of cross-linked fibrin (D-Dimer) is a specific indi-cater of the release of thrombotic material. Other non-specific degradation products (fibrinogenolysis) were detected by the measurement of FSP. In some cases in which perfusion ocurred an increase of TAT followed by a rapid decrease was observed. This indicates a higher thromboplastic activity which may originate from the infarcted area producing TAT complex formation.

Native one-chain t-PA is cleaved by plasmin or by trypsin after the Arg in the sequence -Gln-Phe-Arg-Ile-Lys-. Variants of one-chain t-PA where the -Arg- was replaced by a His (Arg to His) or by a Lys (Arg to Lys) or by a Thr (Arg to Thr) were made through genetic modification. The three mutants and the wild type were expressed in animal cells and purified in the one-chain form by affinity chromatography as was t-PA from Bowes melanoma cells. In contrast to wild type and melanoma t-PA the mutants reacted poorly with polyclonal antibodies raised against the peptide -Gln-Pro-Gln-Phe-Arg-Ile-Lys--Gly-Gly- indicating mutation in the sequence. Of these proteins only the Arg to Thr mutant was resistant to plasmin cleavage as evidenced by SDS-PAGE. t-PA antigen values (ELISA) and fibrinolytic activity values (fibrin clot lysis assay) yielded the following specific activities expressed in IU/|μg: 810 (Arg to His), 640 (Arg to Lys), 290 (Arg to Thr), 810 (wild type) and 660 (melanoma t-PA). The amidolytic activities for the one-chain proteins against D-Ile-Pro-Arg-pNA at pH 9.0 and 37°C, expressed in mOD per minute at 1 M-g/mL of enzyme were: 15.8 (Arg to His), 13.6 (Arg to Lys), 8.3 (Arg to Thr), 10.0 (wild type), 9.6 (melanoma t-PA) as compared to 55.2 for two-chain melanoma t-PA.All mutants including the uncleavable Arg to Thr mutant could be used in determination of PAI activity in plasma samples. Only one-chain t-PA reacts selectively with PAI 1. Thus, use of the Arg to Thr mutant represents a theoretical advantage in PAI 1 activity determination since preparations of this mutant most likely is free of contaminating two-chain t-PA.The plasminogen activation rate as measured in a coupled assay in the presence and absence of fibrin at 0.5 jiM plasminogen and 37°C was measured and the stimulation factor calculated. This was about 950 fold for the Arg to Thr mutant wich was considerably higher than that of melanoma one chain t-PA and the other mutants wich all were about 550 fold. The stimulation factor for melanoma two-chain t-PA was in the same experiment about 120 fold. The extra fibrin sensitivity of the Arg to Thr mutant resulted in improved soluble fibrin assay according to Wiman and Renby Thromb. Haemostas, (1986) 55:189-193.In conclusion: the use of a plasmin insensitive protein-engineered mutant of t-PA gives advantages in assays for PAI 1 and soluble fibrin.

Abstract Controlling bacteria in Oil & Gas operations is essential for flow assurance and avoiding unhealthy environments developing in the systems. The resistance of bacteria grows stronger as they manage to survive environments, In gaming terms, the bacteria is "leveling up" every time it faces and overcomes adversities, thus getting more resilient. Once bacteria are present in medias of Oil & Gas, they will already be bacteria with high survival skills and with very complex development and growth path. Knowing the total content of bacteria can provide confident decision-making on aspects such as HSE, system integrity, biocide selection, and dose frequency. As anaerobe bacteria are very harsh on metal structures, they can cause corrosion and damage and shortened lifetime on flow structures and pipelines. Ideally, bacteria are monitored closely with short testing times to ensure rapid decision-making and initiation of mitigating actions. However, the traditional methods for detecting and enumerating bacteria in Oil & Gas operations are cumbersome and time-consuming, with analysis times of 24 hours to 28 days, making them inefficient in an operational environment that requires rapid decision-making to stay in control. Tools that address these limitations are thus in high demand. In this study, we will describe how a field portable technology for fluorometric determination of a hydrolase activity can be used to identify and monitor bacterial contamination in an offshore operation in near real-time.The study demonstrates how the use of the enzyme method allowed the platform chemical engineer to quickly identify a bacterial contamination issue, related to a failed biocide injection pump and allowed the platform manager to rapidly initiate relevant mitigation of the problem.

Polyphenol oxidases (PPOs) participate in the preparation of many plant products on the one hand and cause considerable losses during processing of plant products on the other hand. However, the physiological functions of plant PPO were still a subject of controversy at the onset of the project. Preliminary observations that suggested involvement of PPOs in resistance to herbivores and pathogens held great promise for application in agriculture but required elucidation of PPO's function if modulation of PPO expression is to be considered for improving plant protection or storage and processing of plant products. Suggestions on a possible role of PPO in various aspects of chloroplast metabolism were also relevant in this context. The characterization of plant PPO genes opened a way for achieving these goals. We reasoned that "understanding PPO targeting and routing, designing ways to manipulate its expression and assessing the effects of such modifications will enable determination of the true properties of the enzyme and open the way for controlling its activity". The objective of the project was to "obtain an insight into the function and biological significance of PPOs" by examining possible function(s) of PPO in photosynthesis and plant-pest interactions using transgenic tomato plants; extending our understanding of PPO routing and assembly and the mechanism of its thylakoid translocation; preparing recombinant PPOs for use in import studies, determination of the genuine properties of PPOs and understanding its assembly and determining the effect of PPO's absence on chloroplast performance. Results obtained during work on the project made it necessary to abandon some minor objectives and devote the effort to more promising topics. Such changes are mentioned in the 'Body of the report' which is arranged according to the objectives of the original proposal. The complex expression pattern of tomato PPO gene family was determined. Individual members of the family are differentially expressed in various parts of the plant and subjected to developmentally regulated turnover. Some members are differentially regulated also by pathogens, wounding and chemical wound signals. Wounding systemically induces PPO activity and level in potato. Only tissues that are developmentally competent to express PPO are capable of responding to the systemic wounding signal by increased accumulation of PPO mRNA. Down regulation of PPO genes causes hyper susceptibility to leaf pathogens in tomato while over expression regulation of PPO expression in tomato plants is their apparent increased tolerance to drought. Both the enhanced disease resistance conferred by PPO over expression and the increased stress tolerance due to down regulation can be used in the engineering of improved crop plants. Photosynthesis rate and variable fluorescence measurements in wild type, and PPO-null and over expressing transgenic tomato lines suggest that PPO does not enable plants to cope better with stressful high light intensities or reactive oxygen species. Rather high levels of the enzyme aggravate the damage caused under such conditions. Our work suggests that PPO's primary role is in defending plants against pathogens and herbivores. Jasmonate and ethylene, and apparently also salicylate, signals involved in responses to wounding and defense against herbivores and pathogens, enhance markedly and specifically the competence of chloroplasts to import and process pPPO. The interaction of the precursor with thylakoid membranes is primarily affected. The routing of PPO shows other unusual properties: stromal processing occurs in two sites, resulting in intermediates that are translocated across thylakoids by two different mechanisms - a DpH- and a Sec-dependent one. It is suggested that the dual pattern of processing and routing constitutes a'fail safe' mechanism, reflecting the need for a rapid and flexible response to defense challenges. Many of the observations described above should be taken into consideration when manipulation of PPO expression is contemplated for use in crop improvement.