Studies on the enzyme α-glucan phosphorylase

ac-qlncnn phosphorylsse is en ilportent enzyme in glycoiysis. It is the first used knows to exhibit ellosteric properties and lance its inhibition end ectivetion have significant effect on the rete ot qlycolysis. The thesis deals with 11 detailed study of the structure. inhibition and control or this snlrlls from rabbit uncle and troll e merino eninelo ‘the thesis is divided into two parts. Port 1 deals with studies on rabbit uncle glycogen phospherylese. After e review of the relevant literetnre (Chapter 1) the inhibition and chancel sodiiicetion studies on rabbit ensyle ere discussed in chepters 2 to 5. Chapter 6. gives the methods used for the study

“Studies on extracellular enzyme production during growth of Pleurotus sp. on lignocellulosic agriwaste and the utilization of spent mushroom substrate”

Commercially, Pleurotus spp. of mushroom are cultivated in bags. After mushroom cultivation, spent substrate remains as residual material. Proper recycling of spent substrate is beneficial for our economy. Spent substrate can be utilized for various other value added purposes through the proper knowledge of its components. Composition of various components depends on the activity of extracellular enzymes in the spent substrate. The present study was conducted to know the enzyme profile of some major extracellular enzymes - cellulase, hemicellulase (xylanase), pectinase and ligninase (lignin peroxidase and laccase) and to estimate cellulose, hemicellulose, pectin and lignin in the substrate. The use of spent substrate as a source of fibre and ethanol, and in the biodegradation of phenol by Pleurotus spp. was also investigated

Purification and Characterization of an Anti-cancer Enzyme Produced by Marine Vibrio Costicola Under A Novel Solid State Fermentation Process

L - Glutaminase, a therapeutically and industrially important enzyme, was produced from marine Vibrio costicola by a novel solid state fermentation process using polystyrene beads as inert support. The new fermentation system offered several advantages over the conventional systems, such as the yield of leachate with minimum viscosity and high specific activity for the target product besides facilitating the easy estimation of biomass. The enzyme thus produced was purified and characterised. It was active at physiological pH, showed high substrate specificity towards L - glutamine and had a Km value of 7.4 x 10-2 M. It also exhibited high salt and temperature tolerance indicating good scope for its industrial and therapeutic applications

Effects of antioxidant enzyme inhibitors on free radical levels in mice and possible clinical applications for decreasing ROS damage in the cochlea

This paper examines an experiment to determine if impairment of antioxident protective agents resulted in elevated ROS levels in mice.

Shifts in rhizosphere microbial communities and enzyme activity of Poa alpina across an alpine chronosequence

This study quantifies the influence of Poa alpina on the soil microbial community in primary succession of alpine ecosystems, and whether these effects are controlled by the successional stage. Four successional sites representative of four stages of grassland development (initial, 4 years (non-vegetated); pioneer, 20 years; transition, 75 years; mature, 9500 years old) on the Rotmoos glacier foreland, Austria, were sampled. The size, composition and activity of the microbial community in the rhizosphere and bulk soil were characterized using the chloroform-fumigation extraction procedure, phospholipid fatty acid (PLFA) analysis and measurements of the enzymes beta-glucosidase, beta-xylosidase, N-acetyl-beta-glucosaminidase, leucine aminopeptidase, acid phosphatase and sulfatase. The interplay between the host plant and the successional stage was quantified using principal component (PCA) and multidimensional scaling analyses. Correlation analyses were applied to evaluate the relationship between soil factors (C-org, N-t, C/N ratio, pH, ammonium, phosphorus, potassium) and microbial properties in the bulk soil. In the pioneer stage microbial colonization of the rhizosphere of P. alpina was dependent on the reservoir of microbial species in the bulk soil. As a consequence, the rhizosphere and bulk soil were similar in microbial biomass (ninhydrin-reactive nitrogen (NHR-N)), community composition (PLFA), and enzyme activity. In the transition and mature grassland stage, more benign soil conditions stimulated microbial growth (NHR-N, total amount of PLFA, bacterial PLFA, Gram-positive bacteria, Gram-negative bacteria), and microbial diversity (Shannon index H) in the rhizosphere either directly or indirectly through enhanced carbon allocation. In the same period, the rhizosphere microflora shifted from a G(-) to a more G(+), and from a fungal to a more bacteria-dominated community. Rhizosphere beta-xylosidase, N-acetyl-beta-glucosaminidase, and sulfatase activity peaked in the mature grassland soil, whereas rhizosphere leucine aminopeptidase, beta-glucosidase, and phosphatase activity were highest in the transition stage, probably because of enhanced carbon and nutrient allocation into the rhizosphere due to better growth conditions. Soil organic matter appeared to be the most important driver of microbial colonization in the bulk soil. The decrease in soil pH and soil C/N ratio mediated the shifts in the soil microbial community composition (bacPLFA, bacPLFA/fungPLFA, G(-), G(+)/G(-)). The activities of beta-glucosidase, beta-xylosidase and phosphatase were related to soil ammonium and phosphorus, indicating that higher decomposition rates enhanced the nutrient availability in the bulk soil. We conclude that the major determinants of the microllora vary along the successional gradient: in the pioneer stage the rhizosphere microflora was primarily determined by the harsh soil environment; under more favourable environmental conditions, however, the host plant selected for a specific microbial community that was related to the dynamic interplay between soil properties and carbon supply. (C) 2004 Elsevier Ltd. All rights reserved.

Shifts in rhizosphere microbial communities and enzyme activity of Poa alpina across an alpine chronosequence

This study quantifies the influence of Poa alpina on the soil microbial community in primary succession of alpine ecosystems, and whether these effects are controlled by the successional stage. Four successional sites representative of four stages of grassland development (initial, 4 years (non-vegetated); pioneer, 20 years; transition, 75 years; mature, 9500 years old) on the Rotmoos glacier foreland, Austria, were sampled. The size, composition and activity of the microbial community in the rhizosphere and bulk soil were characterized using the chloroform-fumigation extraction procedure, phospholipid fatty acid (PLFA) analysis and measurements of the enzymes beta-glucosidase, beta-xylosidase, N-acetyl-beta-glucosaminidase, leucine aminopeptidase, acid phosphatase and sulfatase. The interplay between the host plant and the successional stage was quantified using principal component (PCA) and multidimensional scaling analyses. Correlation analyses were applied to evaluate the relationship between soil factors (C-org, N-t, C/N ratio, pH, ammonium, phosphorus, potassium) and microbial properties in the bulk soil. In the pioneer stage microbial colonization of the rhizosphere of P. alpina was dependent on the reservoir of microbial species in the bulk soil. As a consequence, the rhizosphere and bulk soil were similar in microbial biomass (ninhydrin-reactive nitrogen (NHR-N)), community composition (PLFA), and enzyme activity. In the transition and mature grassland stage, more benign soil conditions stimulated microbial growth (NHR-N, total amount of PLFA, bacterial PLFA, Gram-positive bacteria, Gram-negative bacteria), and microbial diversity (Shannon index H) in the rhizosphere either directly or indirectly through enhanced carbon allocation. In the same period, the rhizosphere microflora shifted from a G(-) to a more G(+), and from a fungal to a more bacteria-dominated community. Rhizosphere beta-xylosidase, N-acetyl-beta-glucosaminidase, and sulfatase activity peaked in the mature grassland soil, whereas rhizosphere leucine aminopeptidase, beta-glucosidase, and phosphatase activity were highest in the transition stage, probably because of enhanced carbon and nutrient allocation into the rhizosphere due to better growth conditions. Soil organic matter appeared to be the most important driver of microbial colonization in the bulk soil. The decrease in soil pH and soil C/N ratio mediated the shifts in the soil microbial community composition (bacPLFA, bacPLFA/fungPLFA, G(-), G(+)/G(-)). The activities of beta-glucosidase, beta-xylosidase and phosphatase were related to soil ammonium and phosphorus, indicating that higher decomposition rates enhanced the nutrient availability in the bulk soil. We conclude that the major determinants of the microllora vary along the successional gradient: in the pioneer stage the rhizosphere microflora was primarily determined by the harsh soil environment; under more favourable environmental conditions, however, the host plant selected for a specific microbial community that was related to the dynamic interplay between soil properties and carbon supply. (C) 2004 Elsevier Ltd. All rights reserved.

Converting the nanodomains of a diblock-copolymer thin film from spheres to cylinders with an external electric field

We investigate the ability of an applied electric field to convert the morphology of a diblock-copolymer thin film from a monolayer of spherical domains embedded in the matrix to cylindrical domains that penetrate through the matrix. As expected, the applied field increases the relative stability of cylindrical domains, while simultaneously reducing the energy barrier that impedes the transition to cylinders. The effectiveness of the field is enhanced by a large dielectric contrast between the two block-copolymer components, particularly when the low-dielectric contrast component forms the matrix. Furthermore, the energy barrier is minimized by selecting sphere-forming diblock copolymers that are as compositionally symmetric as possible. Our calculations, which are the most quantitatively reliable to date, are performed using a numerically precise spectral algorithm based on self-consistent-field theory supplemented with an exact treatment for linear dielectric materials.

Influence of exogenous fibrolytic enzyme level and incubation pH on the in vitro ruminal fermentation of alfalfa stems

A series of in vitro experiments was carried out to examine the impact of enzyme application rate and incubation medium pH on the rate and extent of fermentation of alfalfa stems. In Experiment 1, a commercial enzyme product (Liquicell 2500, Specialty Enzyme and Biochemicals, Fresno, CA, USA) was added to alfalfa stems at six levels: 0, 0.51, 1.02, 2.55, 5.1, and 25.5 mu l/g (control and L1-L5, respectively) to forage DM in a completely randomized design, with a factorial arrangement of treatments. Rate and extent of fermentation and apparent organic matter degradation (OMD) were determined in vitro, using a gas production technique. Addition of enzyme linearly increased (P < 0.01) gas production for up to 12 h (68.9, 70.9, 67.6, 67.9, 71.9, and 74.9 ml/g OM for control, L1-L5, respectively) and OMD for up to 19 h incubation (0.425, 0.444, 0.433, 0.446, 0.443, and 0.451 for control, L1-L5, respectively), but no increases (P > 0.05) were detected thereafter. In Experiment 2, the effect of the same enzyme as used previously (added at 0.51 mu l/g forage DM, directly into the incubation medium), and buffer pH were examined using the ANKOM system, in a completely randomized design. Incubation medium pH was altered using 1 M citric acid, in order to obtain target initial pH values of 6.8 (control, no citric acid added), 6.2, 5.8, and 5.4. Actual initial pH values achieved were 6.72, 6.50, 6.20, and 5.72. Lowering the pH decreased (P < 0.01) dry matter disappearance (DMD) at 18 h incubation (0.339, 0.341, 0.314, and 0.291 for 6.72, 6.50, 6.20, and 5.72, respectively), whereas enzyme addition increased (P < 0.05) DMD at 24 h (0.363 versus 0.387 for control and enzyme-treated, respectively). Addition of enzyme increased (P < 0.05) neutral detergent fibre (NDF), acid detergent fibre (ADF), and hemicellulose (HC) degradation at pH 6.50 (0.077 versus 0.117; 0.020 versus 0.051; 0.217 versus 0.270 for control and enzyme-treated NDF, ADF and hemicellulose degradation, respectively) and 6.72 (0.091 versus 0.134; 0.041 versus 0.079; 0.205 versus 0.261 for control and enzyme-treated NDF, ADF and HC degradation, respectively). It is concluded that the positive effects of this enzyme product were independent of the pre-treatment period, but pH influenced the responses to enzyme supplementation. Under the conditions of this experiment, exogenous fibrolytic enzymes seemed to work better at close to neutrality ruminal pH conditions. (C) 2006 Elsevier B.V. All rights reserved.

In vitro evaluation of fibrolytic enzymes as additives for maize (Zea mays L.) silage - I. Effects of ensiling temperature, enzyme source and addition level

A series of experiments was completed to investigate the impact of addition of enzymes at ensiling on in vitro rumen degradation of maize silage. Two commercial products, Depot 40 (D, Biocatalysts Ltd., Pontypridd, UK) and Liquicell 2500 (L, Specialty Enzymes and Biochemicals, Fresno, CA, USA), were used. In experiment 1, the pH optima over a pH range 4.0-6.8 and the stability of D and L under changing pH (4.0, 5.6, 6.8) and temperature (15 and 39 degreesC) conditions were determined. In experiment 2, D and L were applied at three levels to whole crop maize at ensiling, using triplicate 0.5 kg capacity laboratory minisilos. A completely randomized design with a factorial arrangement of treatments was used. One set of treatments was stored at room temperature, whereas another set was stored at 40 degreesC during the first 3 weeks of fermentation, and then stored at room temperature. Silages were opened after 120 days. Results from experiment I indicated that the xylanase activity of both products showed an optimal pH of about 5.6, but the response differed according to the enzyme, whereas the endoglucanase activity was inversely related to pH. Both products retained at least 70% of their xylanase activity after 48 h incubation at 15 or 39 degreesC. In experiment 2, enzymes reduced (P < 0.05) silage pH, regardless of storage temperature and enzyme level. Depol 40 reduced (P < 0.05) the starch contents of the silages, due to its high alpha-amylase activity. This effect was more noticeable in the silages stored at room temperature. Addition of L reduced (P < 0.05) neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents. In vitro rumen degradation, assessed using the Reading Pressure Technique (RPT), showed that L increased (P < 0.05) the initial 6 h gas production (GP) and organic matter degradability (OMD), but did not affect (P > 0.05) the final extent of OMD, indicating that this preparation acted on the rumen degradable material. In contrast, silages treated with D had reduced (P < 0.05) rates of gas production and OMD. These enzymes, regardless of ensiling temperature, can be effective in improving the nutritive quality of maize silage when applied at ensiling. However, the biochemical properties of enzymes (i.e., enzymic activities, optimum pH) may have a crucial role in dictating the nature of the responses. (C) 2003 Elsevier B.V. All rights reserved.

Effect of prolonged intravenous glucose and essential amino acid infusion on nitrogen balance, muscle protein degradation and ubiquitin-conjugating enzyme gene expression in calves

Background: Intravenous infusions of glucose and amino acids increase both nitrogen balance and muscle accretion. We hypothesised that co-infusion of glucose ( to stimulate insulin) and essential amino acids (EAA) would act additively to improve nitrogen balance by decreasing muscle protein degradation in association with alterations in muscle expression of components of the ubiquitin-proteasome proteolytic pathway. Methods: We examined the effect of a 5 day intravenous infusions of saline, glucose, EAA and glucose + EAA, on urinary nitrogen excretion and muscle protein degradation. We carried out the study in 6 restrained calves since ruminants offer the advantage that muscle protein degradation can be assessed by excretion of 3 methyl-histidine and multiple muscle biopsies can be taken from the same animal. On the final day of infusion blood samples were taken for hormone and metabolite measurement and muscle biopsies for expression of ubiquitin, the 14-kDa E2 ubiquitin conjugating enzyme, and proteasome sub-units C2 and C8. Results: On day 5 of glucose infusion, plasma glucose, insulin and IGF-1 concentrations were increased while urea nitrogen excretion and myofibrillar protein degradation was decreased. Co-infusion of glucose + EAA prevented the loss of urinary nitrogen observed with EAA infusions alone and enhanced the increase in plasma IGF-1 concentration but there was no synergistic effect of glucose + EAA on the decrease in myofibrillar protein degradation. Muscle mRNA expression of the ubiquitin conjugating enzyme, 14-kDa E2 and proteasome sub-unit C2 were significantly decreased, after glucose but not amino acid infusions, and there was no further response to the combined infusions of glucose + EAA. Conclusion: Prolonged glucose infusion decreases myofibrillar protein degradation, prevents the excretion of infused EAA, and acts additively with EAA to increase plasma IGF-1 and improve net nitrogen balance. There was no evidence of synergistic effects between glucose + EAA infusion on muscle protein degradation or expression of components of the ubiquitin-proteasome proteolytic pathway.

Effect of method of application of a fibrolytic enzyme product on digestive processes and milk production in Holstein-Friesian cows

Four multiparous cows with cannulas in the rumen and proximal duodenum were used in early lactation in a 4 x 4 Latin square experiment to investigate the effect of method of application of a fibrolytic enzyme product on digestive processes and milk production. The cows were given ad libitum a total mixed ration (TMR) composed of 57% (dry matter basis) forage (3:1 corn silage:grass silage) and 43% concentrates. The TMR contained (g/kg dry matter): 274 neutral detergent fiber, 295 starch, 180 crude protein. Treatments were TMR alone or TMR with the enzyme product added (2 kg/1000 kg TMR dry matter) either sprayed on the TMR 1 h before the morning feed (TMR-E), sprayed only on the concentrate the day before feeding (Concs-E), or infused into the rumen for 14 h/d (Rumen-E). There Was no significant effect on either feed intake or milk yield but both were highest on TMR-E. Rumen digestibility of dry matter, organic matter, and starch was unaffected by the enzyme. Digestibility of NDF was lowest on TMR-E in the rumen but highest postruminally. Total Tract digestibility was highest on TMR-E for dry matter, organic matter, and starch but treatment differences were nonsignificant for neutral detergent fiber: Corn silage stover retention time in the rumen was reduced by all enzyme treatments but postruminal transit time vas increased so the decline in total tract retention. time with enzymes was not significant. It is suggested that the tendency for enzymes to reduce particle retention time in the rumen may, by reducing the time available for fibrolysis to occur, at least partly explain the variability in the reported responses to enzyme treatment.

Budgetary implications of, and motives for, converting to organic farming: Case study farm business evidence from Great Britain

This paper explores the financial implications of converting to organic farming in Great Britain through a case study of farmers considering conversion in 2002. Most study farmers were motivated to convert for financial, not ideological or life-style reasons; organic meat production was the most common planned enterprise, although those choosing to produce milk, vegetables and cereals were also studied in depth. At the time of study, organic beef and sheep meat production was particularly profitable. It was found that, in these product sectors, a large improvement in Family Farm Income would result if organic production was introduced on the case study farms. With few exceptions, a fall in Family Farm Income during the conversion period would not be an obstacle to farmers changing to organic methods. Fixed cost changes would also not deter conversion but expensive investment in new livestock and appropriate buildings would be required by some of those businesses studied. These findings are, however, dependent upon the price premia assumptions used and, whilst these premia have dropped slightly since the time of study, this would lessen the financial shortfall during the conversion period. There is also the possibility that reversion to conventional agricultural production might occur, perhaps at a faster rate than the original conversion process that was taking place around the turn of the century.

Evaluation of a nucleoprotein-based enzyme-linked immunosorbent assay for the detection of antibodies against infectious bronchitis virus

As an immunogen of the coronavirus, the nucleoprotein (N) is a potential antigen for the serological monitoring of infectious bronchitis virus (IBV). In this report, recombinant N protein from the Beaudette strain of IBV was produced and purified from Escherichia coli as well as Sf9 ( insect) cells, and used for the coating of enzyme-linked immunosorbent assay ( ELISA) plates. The N protein produced in Sf9 cells was phosphorylated whereas N protein from E. coli was not. Our data indicated that N protein purified from E. coli was more sensitive to anti-IBV serum than the protein from Sf9 cells. The recombinant N protein did not react with the antisera to other avian pathogens, implying that it was specific in the recognition of IBV antibodies. In addition, the data from the detection of field samples and IBV strains indicated that using the recombinant protein as coating antigen could achieve an equivalent performance to an ELISA kit based on infected material extracts as a source of antigen(s). ELISAs based on recombinant proteins are safe ( no live virus), clean ( only virus antigens are present), specific ( single proteins can be used) and rapid ( to respond to new viral strains and strains that cannot necessarily be easily cultured).