Increase In Total Enzyme Activity Research Articles

Bioproduction of quercetin using recombinant thermostable glycosidases from Dictyoglomus thermophilum

Quercetin is an essential ingredient in functional foods and nutritional supplements, as well as a promising therapeutic reagent. Also, the green technique to produce quercetin via rutin biotransformation is attractive. Genes encoding two thermostable glycosidases from Dictyoglomus thermophilum were cloned and expressed in Escherichia coli, which were applied in rutin biotransformation to produce highly pure quercetin at a high temperature. The production of biocatalysts were scaled up in a 5-L bioreactor, yielding a several-fold increase in total enzyme activity and a quercetin production of 14.22 ± 0.26 g/L from 30 g/L of rutin. Feeding strategies were optimized to boost biomass and enzyme production, achieving an activity of 104,801.80 ± 161.99 U/L for rhamnosidase and 12,637.23 ± 17.94 U/L for glucosidase, and a quercetin yield of 20.24 ± 0.27 g/L from the complete conversion of rutin. This study proposes a promising approach for producing high-quality quercetin in an industrial setting.Graphical

Efficient extracellular recombinant production and purification of a Bacillus cyclodextrin glucanotransferase in Escherichia coli

BackgroundCyclodextrin glucanotransferases (CGTases) catalyze the synthesis of cyclodextrins, cyclic oligosaccharides composed of glucose monomers that find applications in the pharmaceutical, food, and cosmetic industries. An economic application of these industrially important enzymes requires their efficient production and recovery. In this study, the effect of Sec-type signal peptides on the recombinant expression of a CGTase derived from Bacillus sp. G825-6 was investigated in Escherichia coli BL21(DE3) using a codon-adapted gene. In addition, a novel purification method for the CGTase using starch adsorption was developed.ResultsExpression vectors encoding N-terminal PelB, DacD, and the native Bacillus sp. G825-6 CGTase signal peptides (SP) were constructed for the recombinant CGTase. With the DacD SP derived from E. coli, a 3.9- and 3.1-fold increase in total enzyme activity was obtained compared to using the PelB and the native CGTase SP, respectively. DacD enabled a 7.3-fold increase of activity in the extracellular fraction after induction for 24 h compared to the native CGTase SP. After induction for 48 h, 75% of the total activity was detected in the extracellular fraction. By a batch wise adsorption to starch, the extracellular produced CGTase could be purified to homogeneity with a yield of 46.5% and a specific activity of 1637 U/mg.ConclusionsThe signal peptide DacD promoted the high-level heterologous extracellular expression of a recombinant CGTase from Bacillus sp. G825-6 with a pET20b(+) vector in E. coli BL21(DE3). A protocol based on starch adsorption enabled a fast and efficient purification of the recombinant enzyme.

Body and intestinal growth of broiler chicks on a commercial starter diet. 2. Development and characteristics of intestinal enzymes

1. Investigations were conducted into the development of intestinal enzyme function in broiler chickens on a commercial starter diet. The differences between intestinal regions and localisation of enzymes on the villus were assessed. 2. The specific activity of maltase, sucrase, aminopeptidase N (APN) and alkaline phosphatase (AP) at all intestinal sites decreased with age. There were also variations between intestinal sites although this variation depended on age. The specific activity of maltase was higher than that of the other enzymes examined, regardless of age and intestinal site. The total activities of the enzymes also increased with age at all intestinal sites. 3. Results of the localisation of enzymes on the crypt : villus axis showed that activity was expressed over a large proportion of the villus. There was an increase in the total villus activity of α -glu cosidase (AG), APN and AP with age. Activity per unit villus surface area was similar between ages, except for jejunal AP. At hatch enzyme activity was expressed over 44.1, 55.8 and 63.3% of villus height in the duodenum, jejunum and ileum, respectively. At 21 d of age, corresponding values were 68.7, 65.6 and 77.2%. The point of peak activity from the crypt : villus junction increased with age. In the jejunum, most enterocytes were capable of secreting active enzymes within 1 h of formation. Cells maintained their secretory capabilities until they were more than 60 h old in the case of AG. 4. Although the specific activities of the enzymes were maximal at hatch, the digestive capacity of older birds may be sustained by an increase in total enzyme activity brought about by increased surface area. The pattern of enzyme activity along the gastrointestional tract (GIT) and crypt : villus axis is similar to that reported for some mammalian species.

Repression of the acetyl-CoA carboxylase gene in ovine adipose tissue during lactation: the role of insulin responsiveness.

We have investigated the mechanisms whereby lipogenesis is markedly suppressed in adipose tissue depots of lactating sheep. Expression of the gene encoding acetyl-CoA carboxylase (ACC), the flux-determining enzyme of the lipogenic pathway, is reduced approximately threefold in both omental and subcutaneous adipose tissue depots during late pregnancy and remains so into lactation when compared with non-pregnant, non-lactating animals. By comparison, total ACC enzyme activity in these adipose depots is suppressed approximately 25- to 30-fold in lactation. Culture of explants from the subcutaneous depot of lactating sheep with insulin plus dexamethasone for 72 h resulted in an approximately sevenfold increase in ACC mRNA, a fivefold increase in total enzyme activity and a marked increase in the proportion of the enzyme in the active state when compared with explants cultured with no added hormones for the same period. However, there was a lag of between 32 and 48 h before marked induction of any of these parameters by insulin plus dexamethasone was observed. Induction of the alpha-tubulin gene paralleled that of the ACC gene, suggesting that cytoskeletal rearrangements are associated with the aquisition of sensitivity to insulin plus dexamethasone. These results demonstrate that the reduction in lipogenic capacity in ovine adipose tissue during lactation is related to repression of the ACC gene, both at the level of steady-state mRNA abundance and possibly at translation, as well as to suppression of the mechanisms that regulate the proportion of ACC in the active state, and these are further related to the marked insensitivity of these parameters to insulin plus dexamethasone in vitro.

Effects of pyridostigmine on acetylcholinesterase in different muscles of the mouse.

1. Pyridostigmine bromide was administered subcutaneously in mice, in a dose of 4.0 or 2.0 mu moles/kg, and the activity of the predominant (G1, G4 and A12) molecular forms of acetylcholinesterase were examined in diaphragm, extensor digitorum longus (EDL), and soleus muscles at 3 h, 6 h, 24 h and 5 days. 2. In diaphragm, no effect was apparent after the low dose, but after the high dose there was a reduction in activity of the functional A12 form at 24 h, followed by an increase which had overshot the control level at 5 days. 3. In the fast EDL, after the low dose, all three molecular forms were decreased at 3 h but had returned to normal by 6 h. This effect was not apparent after the high dose. 4. In the slow soleus the low dose caused a significant increase in total enzyme activity at 5 days, but the high dose caused significant increases in all molecular forms at 3 hours. 5. Thus pyridostigmine had delayed effects on the levels of acetylcholinesterase. The three muscles displayed different sensitivities to the drug, but the changes were consistent with initial inhibition of the activity leading to down-regulation of the enzyme followed by up-regulation, which could overshoot the normal levels.

Human alcohol dehydrogenase isoenzyme activity in the sera of non-alcoholic liver cirrhotic patients.

The activities of class I and II alcohol dehydrogenase isoenzymes were examined in the sera of patients with non-alcoholic liver cirrhosis using a fluorometric method. The analysis of these results shows a statistically significant increase (2,5-times) in the activity of class I alcohol dehydrogenase, and no marked differences in the activity of class II in cirrhotic and control patients. The observed increase in total enzyme activity measured using a photometric method was not very high but confirmed the elevation of class I isoenzyme activity. Activities of both classes of alcohol dehydrogenase isoenzymes have a good correlation with aspartate aminotransferase. Class II isoenzyme activity additionally correlates with alkaline phosphatase. These results suggest that serum activity of class I alcohol dehydrogenase is a better indicator of liver cell destruction during non-alcoholic cirrhosis than total enzyme activity, and is comparable with the value of aspartate aminotransferase.

Activity of microbial peptidases, oxidases, and esterases in lake waters of varying trophic status.

The range and activities of microbial enzymes present in lake water were assessed directly in cells concentrated by tangential flow filtration. A total of 108 enzymes were assayed in this study, which included tests for 60 peptidases, 20 oxidases, and 10 esterases, and 18 miscellaneous tests. In general, no trends in the range of enzymes were associated with trophic status of the lakes. However, one lake that was hypereutrophic had a greater range of enzymes than the other lakes tested. An increase in total enzyme activity (activity/mL) was recorded with an increase in trophic status of the water. The relationship between the physical and chemical attributes of each lake and microbial enzyme activities was investigated by principal component analysis. Quantitative changes between lakes in 11 of the 21 variables were shown to be closely related to changes in the enzyme activities of the lakes; total organic carbon, particulate carbon, particulate nitrogen, pH, and chlorophyll a showed the closest relationships.

Expression of activities of two 20 alpha-hydroxysteroid dehydrogenase isozymes in rat corpora lutea.

The rat ovary contains two isozymes of 20 alpha-hydroxysteroid dehydrogenase (HSD-1 and HSD-2). In this study, the expression of activity of each isozyme was investigated in ovaries that contained a single generation of corpora lutea during pseudopregnancy. This condition was induced by cervical stimulation in rats that had been rendered anovulatory by housing them in a continuously lit environment. The total activity of cytosolic 20 alpha-HSD was lower in the ovaries of these pseudopregnant rats than in ovaries containing multiple generations of corpora lutea. In normal pseudopregnancy, HSD-1 activity was low on days 5 and 9 and increased markedly on day 15, whereas HSD-2 was lower than HSD-1 and did not vary throughout pseudopregnancy. However, on days 5 and 9 of continuous-light pseudopregnancy, low activity of HSD-1 only was detected; by day 15, HSD-1 activity had increased sixfold and HSD-2 activity could be detected. Immunohistochemical methods using a specific antibody recognizing both HSD-1 and HSD-2 revealed that the number of 20 alpha-HSD-positive luteal cells increased by day 15. Thus, the increase in total enzyme activity and appearance of HSD-2 activity observed at late pseudopregnancy was accompanied by an increase in the number of 20 alpha-HSD-positive luteal cells.

The developmental regulation of l-ornithine decarboxylase in Dictyostelium discoideum and its induction by cAMP

By the use of an in vivo assay, ornithine decarboxylase ( l-ornithine carboxy-lyase, EC 4.1.1.17) is shown to be developmentally regulated in Dictyostelium discoideum. High levels of cAMP can induce ornithine decarboxylase activity in preaggregative cells kept in shaking suspension, under similar conditions as where other markers for development can also be induced. This induction by cAMP is solely dependent on the total amount of cAMP to which the cells have been exposed, and not on the manner of cAMP addition. Induction of ornithine decarboxylase activity, when measured in vitro, is caused by both an increase in total enzyme activity and by a proportional increase in activity of the high-affinity form for the cofactor pyridoxal phosphate. When measured in vivo, an additional regulatory mechanism seems to be involved. Kinetic studies with the competitive inhibitor putrescine suggest that in cAMP-stimulated cells the low affinity form of the enzyme may also be active in vivo.

Effects of exercise on riboflavin requirements: biological validation in weight reducing women

The present study was designed to evaluate our previous estimates for riboflavin requirement, 0.96 mg/1000 kcal during nonexercise and 1.16 mg/1000 kcal during exercise in overweight women. Two groups of 6 weight reducing women consumed either 1.16 mg riboflavin/1000 kcal (HR) or 0.96 mg/1000 kcal (MR). The study was two, 3 period by 2 treatment (exercise or nonexercise) crossover designs, one design at each level of riboflavin. Erythrocyte glutathione reductase activity coefficients (AC) significantly increased in both groups from 1.16 +/- .02 to 1.20 +/- .03 in group HR and from 1.31 +/- .04 to 1.36 +/- .02 in the MR group during nonexercise and exercise, respectively. ACs increased in the HR group due to an increase in total enzyme activity while ACs increased in the MR group due to a decrease in basal enzyme activity reflecting decreased flavin availability. There were no differences in aerobic capacity, weight loss, nor change in lean body mass between the two groups. Thus, 0.96 mg/1000 kcal was not adequate during either nonexercise or exercise periods while the 1.16 mg/1000 kcal was adequate.

Regulation of transglutaminase activity in Chinese hamster ovary cells

We have investigated the regulation of transglutamine activity (ϵ-(γ-glutamyl)lysine crosslinking enzme) in Chinese hamster ovary cells in culture. We report that transglutaminase activity increases several-fold in CHO cells at maximum density in suspension culture. This increase cannot be explained by the presence of the soluble regulators of the enzyme activity or the appearance of a new enzyme activity with a different affinity for substrate, but appears to be due to an increase in total enzyme activity. Treatment of CHO cells at low cell density with 8-bromo cyclic AMP results in a small increase (20–70%) in transglutaminase activity. By studying CHO mutants which have altered or absent cyclic-AMP-dependent protein kinases, we have demonstrated that the effect of cyclic AMP on transglutaminase activity at low cell density is mediated by cyclic-AMP-dependent protein kinase. However, the protein kinase mutants show normal increases in transglutaminase activity at high cell density, indicating that cyclic AMP-dependent protein kinase does not mediate density-dependent changes in transglutaminase activity.

The induction of collagenase and acid phosphatase by thyroxine in resorbing tadpole gills in vitro

AbstractExplants of tadpole gills were cultured in Hanks balanced salt solution or in Hanks supplemented with thyroid hormone. Gills in control media maintain their integrity in vitro while those treated with thyroxine undergo resorption. The activity of collagenase in the media increases in parallel with thyroxine induced resorption and is accompanied by a decrease in tissue collagen levels. T4 induced gill resorption is also associated with an increase in the specific activity of acid phosphatase. However, this increase is not the result of a significant increase in total enzyme activity but rather the result of a differential loss in total protein concentration in thyroxine treated gills as compared to control.

Lipoprotein lipase of cultured mesenchymal rat heart cells: I. Synthesis, secretion and releasability by heparin

Cell suspensions prepared from rat hearts were separated by replating into F 1, F 2 and M cultures, and cultured for 3–11 days. Lipoprotein lipase activity was highest in the F 1 cultures which consisted mainly of non-beating, mesenchymal cells. The enzyme activity was released into the medium only after addition of heparin. The release occurred by an initial rapid phase and a continuous slow phase. Both the rapid and the slow release of enzyme activity by heparin were inhibited by about 70% after a 4 h pretreatment with colchicine. Thus, it seems that the vesicular transport is responsible for the translocation of lipoprotein lipase to the cell surface also during the slow process of release. The residual activity in the colchicine treated cultures was higher than in the controls indicating that no inhibition of enzyme synthesis occurred. The slow phase of enzyme release continued also after removal of heparin from the medium but was reduced markedly when protein synthesis was inhibited by cycloheximide. Thus the increase in total enzyme activity encountered after exposure to heparin resulted from stimulation of new enzyme synthesis. The half-time of lipoprotein lipase in the F 1 cultures was 35 min and full restoration of enzyme activity was found 60 min after complete removal of cycloheximide from the system. These data indicate that the culture system can be used to study regulation of new enzyme synthesis and its turnover.

Activities of glycolytic enzymes during the early stages of differentiation in the cellular slime mold Dictyostelium discoideum

Activities of fructose diphosphatase (EC 3.1.3.11) and the enzymes in the Embden-Meyerhof glycolytic sequence from hexokinase (EC 2.7.1.1) through lactate dehydrogenase (EC 1.1.1.27) were estimated in extracts of starved myxamoebae and cells at the preculmination stage of Dictyostelium discoideum. The activities, in terms of μmoles of substrate or product/min per 100 mg soluble protein at 22°, ranged from 0.1 or less and 0.4 for fructose diphosphatase and hexokinase, resp., to 35 to 49 for glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) and phosphoglycerate kinase (EC 2.7.2.3), resp., in starved myxamoebae. Activities tended to increase slightly on progression to the preculmination stage, but in no case did the increase exceed a factor of 3, and in some instances (aldolase (EC 4.1.2.7) for example) a slight decline occured. Allowing the possible exception of pyruvate kinase (EC 2.7.1.40) the least active enzyme in the direction of gluconeogenesis was fructose diphosphatase, and there was no detectable increase in its activity during differentiation. It is concluded that any great acceleration in the rate of gluconeogenesis via glycolysis during culmination is brought about by changing substrate levels or by intracellular relocalization rather than by an increase in total enzyme activity. Comparison of the profile of glycolytic enzyme activities in the slime mold with those of liver, brain, and ascites tumor (literature values) revealed that the slime mold profile resembled that of liver most closely, but differed from all in its low lactate dehydrogenase activity.