Apparent Enzyme Activity Research Articles

An anti-CRISPR that pulls apart a CRISPR-Cas complex.

In biological systems, the activities of macromolecular complexes must sometimes be turned off. Thus, a wide variety of protein inhibitors has evolved for this purpose. These inhibitors function through diverse mechanisms, including steric blocking of crucial interactions, enzymatic modification of key residues or substrates, and perturbation of post-translational modifications1. Anti-CRISPRs-proteins that block the activity of CRISPR-Cas systems-are one of the largest groups of inhibitors described, with more than 90 families that function through diverse mechanisms2-4. Here, we characterize the anti-CRISPR AcrIF25, and weshow that it inhibits the type I-F CRISPR-Cas system by pulling apart the fully assembled effector complex. AcrIF25 binds to the predominant CRISPR RNA-binding components of this complex, comprising six Cas7 subunits, and strips them from the RNA. Structural and biochemical studies indicate that AcrIF25 removes one Cas7 subunit at a time, starting at one end of the complex. Notably, this feat is achieved with no apparent enzymatic activity. To our knowledge, AcrIF25 is the first example of a protein that disassembles a large and stable macromolecular complex in the absence of an external energy source. As such, AcrIF25 establishes a paradigm for macromolecular complex inhibitors that may be used for biotechnological applications.

Characterisation of Variants of Cyclic di-GMP Turnover Proteins Associated with Semi-Constitutive rdar Morphotype Expression in Commensal and Uropathogenic Escherichia coli Strains.

Expression of rdar (red, dry, and rough) colony morphology-based biofilm formation in Escherichia coli is highly variable. To investigate the molecular mechanisms of semi-constitutive rdar morphotype formation, we compared their cyclic di-GMP turnover protein content and variability to the highly regulated, temperature-dependent morphotype of the historical and modern ST10 isolates E. coli MG1655 and Fec10, respectively. Subsequently, we assessed the effects of cyclic di-GMP turnover protein variants of the EAL phosphodiesterases YcgG and YjcC and the horizontally transferred diguanylate cyclase DgcX on biofilm formation and motility. The two YcgG variants with truncations of the N-terminal CSS signaling domain were oppositely effective in targeting downregulation of rdar biofilm formation compared to the full-length reference protein. Expression of the C-terminal truncated variants YjcCFec67 and YjcCTob1 showed highly diminished apparent phosphodiesterase activity compared to the reference YjcCMG1655. For YjcCFec101, substitution of the C-terminus led to an apparently inactive enzyme. Overexpression of the diguanylate cyclase DgcX contributed to upregulation of cellulose biosynthesis but not to elevated expression of the major biofilm regulator csgD in the "classical" rdar-expressing commensal strain E. coli Fec10. Thus, the c-di-GMP regulating network is highly complex with protein variants displaying substantially different apparent enzymatic activities.

Effects of curcumin on the growth performance, apparent nutrient digestibility, intestinal morphology, digestive enzyme activity, and antioxidant capacity of meat rabbits

The purpose of this study was to investigate the effect of curcumin on the growth performance, slaughter performance, apparent nutrient digestibility, intestinal morphology, and digestive enzyme activity of meat rabbits. One hundred and sixty healthy weaned meat rabbits were randomly divided into four groups (forty replicates per group and one rabbit per replicate). The control group was fed a basal diet and the test groups (T1, T2 and T3) were fed diets supplemented with 50, 100 and 150 g/t curcumin in the basal diet, respectively. The study lasted for 28 days, including a pre-feeding period of 7 days and a formal trial period of 21 days. The results showed that diets supplemented with curcumin significantly increased rabbits’ body weight gain and average daily feed intake; Apparent digestibility of energy, crude protein, crude fat, crude fibre, NDF and ADF; Jejunal villus height, the ratio of villus height to crypt depth; The activities of amylase and lipase in jejunal and caecal digesta and cellulase in caecal digesta; Caecal mucosal thickness, eviscerated dressing percentage, and dorsal and abdominal hair thickness (p < .05). Diets supplemented with curcumin significantly decreased rabbits’ feed to gain ratio, jejunal crypt depth and thickness of the intestinal wall (p < .05). In conclusion, curcumin is a kind of valuable feed additive, which can improve the nutrient digestibility, growth performance, slaughter ratio and fur development of meat rabbits by improving digestive tract development and digestive enzyme activity. The appropriate dosage in the meat rabbit diet is 100 g/t. HIGHLIGHTS Dietary supplementation of curcumin improved the growth performance, gastrointestinal digestive enzyme activities and slaughter performance of meat rabbits. Dietary supplementation of curcumin increased the antioxidant capacity of meat rabbits. Curcumin could be used as a new kind of feed additive for meat rabbits.

Mapping the proximity interactome of ATG9A reveals unexpected dynamics of ULK1 complex proteins

ABSTRACT ATG9A is essential for macroautophagy/autophagy and considered to be one of the earliest ATG (autophagy related) proteins recruited to sites of autophagosome biogenesis. Recent data suggest ATG9A vesicles may even form the lipid seed of the autophagosome. However, ATG9A regulation is still poorly understood, which is likely at least partly due to challenges inherent to studying an intracellular transmembrane protein with no apparent enzymatic activity. To help overcome these challenges, we used BioID and quantitative LC-MS/MS to map the proximity interactome of ATG9A, which included entire protein complexes involved in protein trafficking, and proteins implicated in autophagy but previously lacking any physical link to core autophagy machinery. We also unexpectedly found an ATG9A interaction with an ULK1-independent ATG13-ATG101 dimer that promotes autophagy in fed cells.

Coated tannin supplementation improves growth performance, nutrients digestibility, and intestinal function in weaned piglets.

To explore the effect of coated tannin (CT) on the growth performance, nutrients digestibility, and intestinal function in weaned piglets, a total of 180 piglets Duroc × Landrace × Yorkshire (28 d old) weighing about 8.6 kg were randomly allotted to three treatments: 1) Con: basal diet (contains ZnSO4); 2) Tan: basal diet + 0.15% CT; and 3) ZnO: basal diet + ZnO (Zn content is 1,600 mg/kg). The results showed that 0.15% CT could highly increase the average daily gain and average daily feed intake of weaned piglets compared with the control group, especially decreasing diarrhea incidence significantly (P < 0.05). Compared with the control group, crude protein apparent digestibility and digestive enzyme activity of the piglets fed with 0.15% CT were enhanced obviously (P < 0.05). Meanwhile, the intestinal villi and microvilli arranged more densely, while the content of serum diamine oxidase was decreased, and the protein expressions of zonula occludens-1 (ZO-1) and claudin-1 were significantly upregulated (P < 0.05). In addition, CT altered the structure of intestinal microbiota and augmented some butyrate-producing bacteria such as Ruminococcaceae and Megasphaera. PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) analysis also showed that the abundances of pathways related to butyrate metabolism and tryptophan metabolism were increased; however, the function of lipopolysaccharide biosynthesis proteins was significantly decreased. The results demonstrated that 0.15% CT could improve growth performance, digestibility, and intestinal function of weaned piglets, and it had the potential to replace ZnO applied to farming.

Growth performance, apparent digestibility coefficients and digestive enzyme activities of Pacific white shrimp, Penaeus vannamei fed low fishmeal diets supplemented with DL‐methionine and phytase

An eight-week growth trial and a four-week digestibility trial was undertaken to assess the impact of DL-methionine and phytase supplementation on growth performances, apparent digestibility coefficients (ADC), phosphorus retention and digestive enzyme activities of Penaeus vannamei. Five treatment groups in three replicates fed with a control diet (fishmeal 200 g/kg), and four low fishmeal (50 g/kg) diets with soy protein concentrate-soybean meal protein viz., SPC1 (without supplementation), SPC 2 (DL-methionine), SPC 3 (Phytase) and SPC4 (DL-methionine and phytase). SPC 2 had significantly improved (p < .05) the weight gain (5.79 ± 0.12), FCR (1.53 ± 0.06) and SGR (3.21 ± 0.06) than other low fishmeal diets, but not significantly different to that of SPC 4 and control. SPC 2 also showed significantly higher protease activity. P retention was significantly higher in SPC 2 than SPC 1, SPC 3 and control, however, not significantly different from SPC 4. Faecal phosphorus load was significantly reduced in SPC 2 than SPC 1 and control. Protein digestibility was significantly higher in SPC 2 and SPC 4, whilst phosphorus digestibility was significantly higher in SPC 4. Thus, it is concluded that the supplementation of DL-methionine is essential for the overall better performances of P. vannamei in low fishmeal diets and phytase for improving phosphorus digestibility.

Impact of the size effect on enzymatic electrochemical detection based on metal-organic frameworks

Metal-organic frameworks (MOFs) hold a great promise as immobilization carriers for enzymes and other biomolecules, owing to their enhanced stability, selectivity and controllability. However, enzyme-MOF complexes usually lead to a decrease in the apparent enzyme activity and apparent substrate affinity as a result of the constrained structure of the enzyme and the mass transfer limitation of the substrate, respectively. These results consequently impede the applications of enzyme-MOF complexes in biocatalysis and biosensing. In this study, zeolitic imidazolate framework-8 (ZIF-8) was synthesized to immobilize cytochrome c (Cyt c) via a one-step co-precipitation process under mild conditions. By adjusting the molar ratio of precursors, enzyme-MOF composites with different sizes from 100 nm to 1.3 μm were prepared. The decreased size of the prepared MOFs generated an increase in substrate affinity (with an over 50% decrease in the Michaelis constant Km) and a 6.4-fold improvement in the apparent enzyme activity with a 6.26-fold increase in the enzymatic electrochemical detection sensitivity compared with native Cyt c. The enzyme-MOF composites were coated on a screen-printed electrode for the sensitive and fast detection of H2O2, which is the most common representative of reactive oxygen species in cellular environments, showing the potential for the construction of efficient biosensors with applications in biomedicine.

Urinary steroid profiles: comparison of spot and 24-hour collections

Urinary steroid profiling is commonly used in clinical routine for the diagnosis of steroid-related diseases through the analysis of absolute steroid excretion values as well as apparent enzyme activities based on catalysed product-to-precursor ratios. To compensate for diurnal fluctuations in steroid concentrations, 24 hour collections are preferred yet impractical and sometimes not feasible. We therefore measured 40 steroid metabolites by GC-MS in 24 hour and spot urine samples of healthy volunteers and systematically evaluated to which extent 24 hour urine collections can be replaced by spot urine collections for diagnostic purposes. Whereas most steroid concentrations show poor correlation between 24 hour and spot urine collection, apparent enzyme activities show better correlation and defects in steroidogenic enzymes such as SRD5A2, CYP17A1, CYP21A2 and CYP11B1. We confirmed our findings in patient samples from our clinic.

Peroxidase Encapsulated in Peroxidase Mimics via in situ Assembly with Enhanced Catalytic Performance

AbstractEnzymatic catalysis is of great importance due to its high catalytic activity and high selectivity. However, intrinsic fragile property of enzyme limits its application in a wide range, where inactive inorganic materials have proven promising in enhancing the stability of the enzyme in industry. Here, Prussian blue nanoparticles (PB) as peroxidase mimics were adopted for encapsulation of peroxidase, Cytochrome c (Cyt c). Prussian blue/Cytochrome c (PB@Cyt c) composite developed via in‐situ assembly exhibited a 2.6‐fold higher apparent enzymatic activity than the sum activity of equivalent Cyt c and PB due to synergistic effect. Mechanism investigation showed that interaction between irons ions from PB and carbonyl group and nitrogen from enzyme led to a favorable catalytic conformation of Cyt c. Moreover, PB shielded the encapsulated Cyt c against harsh conditions (e. g. high temperature and organic solvents). This new efficient hybrid catalyst would open opportunities for wide applications in biosensors and biocatalysis.

Effects of dietary chitosan on growth rate, small intestinal morphology, nutrients apparent utilization and digestive enzyme activities of growing Huoyan geese

Dietary chitosan (CS) supplementation could improve the growth rate, small intestinal morphology, nutrients apparent digestibility and digestive enzyme activities in pigs, broiler chickens, rats and fish, whereas no data has been reported about the effect of CS on the growing Huoyan geese. Therefore, this study was designed to investigate the effects of CS on growth rate, small intestinal morphology, nutrients apparent utilization and digestive enzyme activities of growing Huoyan geese. Three hundred and twenty (28 days of age, gender balance) Huoyan geese were randomly divided into control, CS100, CS200 and CS400 groups (based on BW) with 20 geese per pen and 4 replicates pen per group, and the feeding experiment lasted for 4 weeks. The 4 diets contained 0, 100, 200 and 400 mg CS per kg feed, respectively. The results showed that CS200 groups had higher average daily gain, final BW, apparent utilization of DM and CP, and lower feed/gain ratio compared with the control group (P < 0.05). Meanwhile, CS100 and CS200 groups had higher villus height, villus height/crypt depth ratio and lower crypt depth in duodenum and jejunum than those in the control group (P < 0.05). The geese in CS100 and CS200 groups had higher villus height, villus height/crypt depth ratio and lower crypt depth of ileum compared with those in control and CS400 groups (P < 0.05). In addition, compared with the control group, CS200 group has higher trypsin activities and lower lipase activities in duodenal, jejunal and ileal contents (P < 0.05). The results suggested that addition of 200 mg/kg CS had positive effects on growth rate, small intestinal morphology, nutrients apparent utilization and digestive enzyme activities of growing Huoyan geese.

Engineering a nicotinamide mononucleotide redox cofactor system for biocatalysis.

Biological production of chemicals often requires the use of cellular cofactors, such as nicotinamide adenine dinucleotide phosphate (NADP+). These cofactors are expensive to use in vitro and difficult to control in vivo. We demonstrate the development of a noncanonical redox cofactor system based on nicotinamide mononucleotide (NMN+). The key enzyme in the system is a computationally designed glucose dehydrogenase with a 107-fold cofactor specificity switch toward NMN+ over NADP+ based on apparent enzymatic activity. We demonstrate that this system can be used to support diverse redox chemistries in vitro with high total turnover number (~39,000), to channel reducing power in Escherichia coli whole cells specifically from glucose to a pharmaceutical intermediate, levodione, and to sustain the high metabolic flux required for the central carbon metabolism to support growth. Overall, this work demonstrates efficient use of a noncanonical cofactor in biocatalysis and metabolic pathway design.

N-3 essential fatty acids in Nile tilapia, Oreochromis niloticus: Bioconverting LNA to DHA is relatively efficient and the LC-PUFA biosynthetic pathway is substrate limited in juvenile fish

This study aimed to gain a better understanding of the long-chain (≥C20) polyunsaturated fatty acid (LC-PUFA) biosynthetic metabolism, with a specific focus on the metabolic fate of linolenic acid (18:3n-3, LNA) and linoleic acid (18:2n-6, LA) and the relationship between these two substrates in the process of LC-PUFA biosynthesis in juvenile Nile tilapia, Oreochromis niloticus. Fish were fed four isoproteic (37.5%) and isolipidic (6.2%) diets formulated with fixed levels of LA (0.60%) and gradual levels of LNA (0.10, 0.63, 1.56 and 2.04% of dry weight, respectively) for 10 weeks. The whole-body fatty acid balance method was used to evaluate fish in vivo fatty acid metabolism. The results showed that most of dietary LNA was bioconverted to LC-PUFA when tilapia receiving not enough LNA for optimal growth. In contrast, when fish receiving sufficient dietary LNA, most of the LNA was β-oxidized, with only <10% LNA being bioconverted. The in vivo apparent △-5/-6 desaturase and elongase activities on n-3 and n-6 PUFA, respectively, exhibited a significant linear increase and decrease with the increasing dietary LNA levels, clearly indicating that for all of the enzyme activities, n-3 PUFA is a preferred substrate and the LC-PUFA biosynthetic pathway is substrate limited in Nile tilapia. Dietary higher inclusion of LNA could block or at least slow down n-6 LC-PUFA biosynthesis from LA, indicating that a direct substrate competition for accessing the Δ-6 desaturase and elongase exists between these two substrates. Furthermore, Nile tilapia were relatively efficient in bioconversion from LNA to DHA, and the DHA biosynthesis from EPA may be more direct or faster than the production of EPA from LNA. Despite of the active bioconversion of LNA up to DHA, this metabolic effort was still insufficient to compensate for the significant reduced dietary intake of n-3 LC-PUFA. No significant difference was observed in the gene expression of fatty acyl desaturase (fads2) and fatty acid elongase (elovl5) among all groups, which suggests that the total in vivo apparent enzyme activities are directly and positively affected by substrate availability rather than the extent of the enzyme gene expression.

7-Ketocholesterol impairs phagocytosis and efferocytosis via dysregulation of phosphatidylinositol 4,5-bisphosphate.

The plasma membrane is inhomogeneously organized containing both highly ordered and disordered nanodomains. 7-Ketocholesterol (7KC), an oxysterol formed from the nonenzymatic oxidation of cholesterol, is a potent disruptor of membrane order. Importantly, 7KC is a component of oxidized low-density lipoprotein and accumulates in macrophage and foam cells found in arterial plaques. Using a murine macrophage cell line, J774, we report that both IgG-mediated and phosphatidylserine-mediated phagocytic pathways are inhibited by the accumulation of 7KC. Examination of the well-studied Fcγ receptor pathway revealed that the cell surface receptor abundance and ligand binding are unaltered while downstream signaling and activation of spleen tyrosine kinase is not affected. However, while the recruitment of phospholipase Cγ1 was unaffected its apparent enzymatic activity was compromised resulting in sustained phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2 ] levels and polymerized actin at the base of the phagocytic cup. Additionally, we found that 7KC prevented the activation of PLCβ downstream of the P2Y6 G-protein coupled receptor and that 7KC impaired PLCγ activity in response to a direct elevation of cytosolic calcium induced by ionomycin. Finally, we demonstrate that 7KC partly attenuates the activity of rapamycin recruitable constitutively active PLCβ3. Together, our results demonstrate that the accumulation of 7KC impairs macrophage function by altering PtdIns(4,5)P2 catabolism and, thus, impairing actin depolymerization required for the completion of phagocytosis.

Effects of dietary biofloc on growth, digestibility, protein turnover and energy budget of sea cucumber Apostichopus japonicus (Selenka)

Bioflocs are not only rich in various bioactive compounds but also proven to be sustainable protein sources of aquafeed. A 60-day experiment was conducted to evaluate the feasibility and capacity of dietary replacement of macroalgae Sargassum thunbergii with biofloc meal as an ingredient in the diet of Apostichopus japonicus. During the experiment, the sea cucumbers of initial wet weight ∼25.18 g were fed seven diets contained graded biofloc levels of 0%, 5%, 10%, 20%, 30%, 45% and 60% (referred as group B0, B5, B10, B20, B30, B45 and B60, respectively) in a recirculating aquaculture system. Each group contained 150 sea cucumbers that were equally allocated into 3 cylinder aquaria. The results showed that dietary supplementation levels of 20% and 30% biofloc significantly enhanced specific growth rate (SGR), food conversion efficiency (FCE) and protein efficiency ratio (PER) of sea cucumber. Based on the second-order polynomial regression models of SGR and FCE, it was concluded that 27.74−30.75% were optimal dietary replacement levels of biofloc. Apparent digestibility coefficients and digestive enzyme activities all showed remarkable trends of the first increase and then decrease as dietary biofloc level increased with relatively higher values in diet B30, which might have stimulated the digestion and absorption of nutrients to maintain faster growth of sea cucumber. There were significant differences in parameters estimated by time-based turnover model of δ15 N between the experimental diets, and the sea cucumbers in diets B30 and B45 exhibited faster nitrogen turnover rates. The average formula of energy allocation was 100C = 3.8G + 65.4F + 3.3U + 27.5R in the present study. The fluctuation in energy for growth and metabolism suggested that dietary biofloc modified their energy allocation strategy by balancing the energy accumulation of growth and energy consumption of feces, excretion and respiration. In conclusion, ca. 30% replacement of S. thunbergii with biofloc powder could improve growth performance of A. japonicus, by enhancing feeding utilization and digestibility, accelerating protein turnover and optimizing energy budget of sea cucumber.

Immunostimulating and Growth Promoting Activity of Dietary Levamisole on Cirrhinus mrigala Fingerlings

The effects of supplementing diets with levamisole on growth, immune response and resistance against pathogenic Aeromonas hydrophila were determined. Fish were assigned to three treatments with different level of levamisole(125, 250 and 500 mg kg¯¹ diet) and the control diet was prepared without levamisole. Cirrhinus mrigala were fed at 4% of their body mass for 60 days. Growth performance, enzyme activities and immunological parameters were analyzed among groups. The total erythrocyte count, total leucocyte count, phagocytic activity and NBT reduction were significantly (P<0.05) enhanced in levamisole treated group when compared to control. The relative percentage survival, A. hydrophila post-challenge was significantly higher in fishes fed on 250 mg kg¯¹ of levamisole supplemented diet. The same dose also resulted in significantly high specific growth rate (SGR), feed conversion ratio (FCR), apparent protein digestibility (APD) and elevated intestinal enzyme activities compared to other groups. Thus, administration of 250 mg of levamisole kg¯¹diet is recommended for enhancement of immunity, growth and survival of C. mrigala hence acting as a potent immunostimulant agent.

Growth of Pseudomonas weihenstephanensis, Pseudomonas proteolytica and Pseudomonas sp. in raw milk: Impact of residual heat-stable enzyme activity on stability of UHT milk during shelf-life

One of the reasons for spoilage of UHT milk during shelf-life is the presence of residual proteolytic activity produced from Pseudomonas spp. during storage of raw milk. The aim of this study was to describe the product defects occurring in indirectly heated UHT milk during shelf-life, and to establish a correlation between proteolytic activity and onset of product spoilage. UHT milk was produced from raw milk incubated with different Pseudomonas strains, and examined over four months during storage at 20 °C. Inactivation kinetics of the peptidases were determined. In UHT milk, product defects occurred in the order: bitterness – particles – creaming – sediment – gelation in all the samples containing peptidases (apparent enzyme activity ≥ 0.03 pkat mL−1). A linear correlation was found between proteolytic activity and onset of product defects, apart from onset of gelation.

Born from pre-eclamptic pregnancies predisposes infants to altered cortisol metabolism in the first postnatal year.

Pre-eclampsia leads to disturbed fetal organ development, including metabolic syndrome, attributed to altered pituitary-adrenal feedback loop. We measured cortisol metabolites in infants born from pre-eclamptic and normotensive women and hypothesised that glucocorticoid exposure would be exaggerated in the former. Twenty-four hour urine was collected from infants at months 3 and 12. Cortisol metabolites and apparent enzyme activities were analysed by gas chromatography-mass spectrometry. From 3 to 12 months, excretion of THS, THF and pregnandiol had risen in both groups; THF also rose in the pre-eclamptic group. No difference was observed with respect to timing of the visit or to hypertensive status for THE or total F metabolites (P>0.05). All apparent enzymes activities, except 17α-hydroxylase, were lower in infants at 12 compared to 3 months in the normotensive group. In the pre-eclamptic group, only 11β-HSD activities were lower at 12 months.17α-hydroxylase and 11β-HSD activities of tetrahydro metabolites were higher in the pre-eclamptic group at 3 months (P<0.05). 11β-hydroxylase activity increased in the pre-eclamptic group at 12 months. Cortisol excretion, determined by increased 11β-hydroxylase, compensates for high 11β-HSD-dependent cortisol degradation at 3 months and at 12 months counterbalances the reduced cortisol substrate availability in infants born from pre-eclamptic mothers.

Enhancing the resolution of (R,S)-2-octanol catalyzed by magnetic cross-linked lipase aggregates using an alternating magnetic field

Magnetic cross-linked lipase aggregates (MCLEAs) were used as a biocatalyst to catalyze the resolution of (R,S)-2-octanol under an alternating magnetic field. The magnetic field resulted in enhanced apparent enzymatic activity. The effects of varying the magnetic field parameters showed that the magnetic field frequency played a more dominant role than field strength on the resolution of (R,S)-2-octanol. Using the optimal conditions, the MCLEAs were highly stable under the alternating magnetic field and were suitable for multiple reuse cycles for the resolution of (R,S)-2-octanol.

Probing the Enzymatic Activity of Alkaline Phosphatase within Quantum Dot Bioconjugates

Enzymes provide the critical means by which to catalyze almost all biological reactions in a controlled manner. Methods to harness and exploit their properties are of strong current interest to the growing field of biotechnology. In contrast to depending upon recombinant genetic approaches, a growing body of evidence suggests that apparent enzymatic activity can be enhanced when located at a nanoparticle interface. We use semiconductor quantum dots (QDs) as a well-defined and easily bioconjugated nanoparticle along with Escherichia coli-derived alkaline phosphatase (AP) as a prototypical enzyme to seek evidence for this process in a de novo model system. We began by first assessing whether the relatively large dimeric AP protein (∼100 kDa) can be assembled onto two differentially sized green and red CdSe/ZnS core/shell QDs in a ratiometric and structurally controlled manner; such assembly is necessary to minimize heterogeneity within the bioconjugate and provide intimate control over the experimental format. For this, analysis is undertaken using both structural modeling and physicochemical characterization techniques including dynamic light scattering and agarose gel electrophoresis; these all provide strong supporting evidence for controlled AP attachment to the QDs. The enzymatic activity of AP-QD bioconjugates assembled on the different QDs and displaying variable AP:QD ratios was then assayed against equivalent amounts of freely diffusing enzyme controls in both conventional excess substrate formats and a varying enzyme–fixed substrate format that is more amenable in general to concentration-limited nanoparticle conjugates. The resulting experimental data were then analyzed in the context of the Michaelis–Menten model and compared. The results show a general equivalency between the two assay formats while also providing evidence for an increase in apparent AP activity of ca. 25% when attached to the QDs. Some discussion is provided on the underlying mechanisms that may contribute to the enhanced activity along with the implications of this work toward future research.

Effects of dietary carbohydrate source on growth performance, diet digestibility and liver glucose enzyme activity in blunt snout bream, Megalobrama amblycephala

Abstract A 3-month feeding trial was conducted to investigate the effects of dietary carbohydrate source on the growth performance, apparent digestibility coefficients and liver glucose enzyme activity in juvenile blunt snout bream. Six experimental diets were formulated to contain 33% wheat starch, corn starch, dextrin, maltose, glucose or cellulose. At the end of the experiment, the best final body weight (18.4 g), specific growth rate (SGR, 2.46%/day), feed conversion ratio (FCR, 1.41 g feed/g gain) and protein efficiency ratio (PER, 2.22 g gain/protein) were found in the group fed with the dextrin diet, while the poorest values were observed in the group fed with the cellulose diet among the treatments. Fish fed with the glucose (1.55 g feed/g gain) or maltose (1.53 g feed/g gain) diet showed significantly higher FCR than those fed with the dextrin diet (1.41 g feed/g gain). Dietary cellulose caused the highest feed intake (2.91 g/fish/day) and FCR (1.87 g feed/g gain) among the treatments. The apparent digestibility coefficient (ADC) of dry matter was significantly lower in fish fed with the cellulose diet than those fed with other diets. ADC of protein was independent of dietary carbohydrate source. The highest ADC of energy was observed in fish fed with diets containing glucose, maltose or dextrin; followed by wheat starch, corn starch and cellulose. Carbohydrate source did not affect whole body moisture, protein and ash contents, and plasma total protein and cholesterol contents. Lipid contents of whole body and plasma triglyceride contents increased with increasing carbohydrate complexities. Plasma glucose contents and liver enzyme activities were affected by sampling time and dietary carbohydrate source. Fish fed with the dextrin diet showed the highest liver glucokinase (GK), pyruvate kinase (PK) and glucose-6-phosphate dehydrogenase (G6PDH) activities, and the lowest glucose-6-phosphatase (G6Pase) activity among all the groups. Significantly higher G6Pase activity was observed in fish fed with the glucose diet than those fed with the dextrin or wheat starch diet at 2 h postprandial. These results indicated that dextrin was the best dietary carbohydrate source for juvenile blunt snout bream, based on growth performance, feed utilization, ADC and liver glucose enzyme activities.