A Molecular Modelling Approach for Designing a Novel Semisynthetic Metalloenzyme Based on Thermolysin

Current computational chemistry tools were used to solve the problem of screening for the best conformation of potential protein-ligand-metal complex in designing a novel semisynthetic metalloenzyme. The computational tools used were Computational Atlas Topography of Protein (CASTp), a sophisticated molecular modeling environment InsightII, a conventional drug-docking algorithm Autodock 3.05 and a schematic diagram for protein-ligand interactions for a given PDB file LIGPLOT. Overall 48 protein pockets on the thermolysin structure were measured using CASTp and the four biggest pockets based on their number of residues and surface area were identified to be suitables site for the modification. Ten different sizes and multifunctional groups of chemical ligands were studied for their thermodynamic valuation using the AutoDock 3.05 program. For further modification, phosphoethanolamaine (PSE), phenylalanine (PHE), phenylacetic acid (PAC) and phenanthroline (PHN) were chosen as they possessed the lowest docking energy of -8.49, -8.34, -7.33 and -7.06 kcal/mol, respectively. Non-covalent interactions included hydrogen bonding and hydrophobic interaction between the ligands and the thermolysin were determined using CASTp. The result showed that larger ligands with multifunctional groups such as PSE and PHE showed higher number interactions compared to the smaller ligands. In terms of specific pockets for the modification, different protein-ligand complexes showed different suitable pockets; complex of thermolysin and PSE ligand at pocket 45, complex of thermolysin and PAC ligand at pocket 48 and both complexes of thermolysin with PHE and PHN ligands at pocket 45, respectively. To verify the final metal ion orientation, three procedures were conducted to narrow down the number of possible conformations for the modification. From four tested metal ions (Ca2+, Mg2+, Fe2+ and Zn2+ ), Ca2+ was identified to be the most favorable metal ion for the modification. It had orientated within an allowed geometry in all tested protein ligand complexes. Meanwhile, both Mg2+ and Fe2+ were identified as favorable metal ions in KEI-PSE and KEI-PAC complexes, respectively. Zn2+ however, showed non favorable docking in all tested complexes due to improper parameterized file for zinc ion in AutoDock

The prosegment catalyzes native folding of Plasmodium falciparum plasmepsin II

Plasmepsin II is a malarial pepsin-like aspartic protease produced as a zymogen containing an N-terminal prosegment domain that is removed during activation. Despite structural similarities between active plasmepsin II and pepsin, their prosegments adopt different conformations in the respective zymogens. In contrast to pepsinogen, the proplasmepsin II prosegment is 80 residues longer, contains a transmembrane region and is non-essential for recombinant expression in an active form, thus calling into question the prosegment's precise function. The present study examines the role of the prosegment in the folding mechanism of plasmepsin II. Both a shorter (residues 77–124) and a longer (residues 65–124) prosegment catalyze plasmepsin II folding at rates more than four orders of magnitude faster compared to folding without prosegment. Native plasmepsin II is kinetically trapped and requires the prosegment both to catalyze folding and to shift the folding equilibrium towards the native conformation. Thus, despite low sequence identity and distinct zymogen conformations, the folding landscapes of plasmepsin II and pepsin, both with and without prosegment, are qualitatively identical. These results imply a conserved and unusual feature of the pepsin-like protease topology that necessitates prosegment-assisted folding

Fatigue onset through oxidative stress, dehydration and lactic acid accumulation and its in vivo study using experimental animals

Among athletes, endurance is one of the key elements to victory. In addition to training, athletes normally used supplement to prevent fatigue during the event. With prolonged and intense activity, our body started to experience decrease in muscle performance due to several factors such as oxidative stress, dehydration and accumulation of lactic acid in the body fluids. The free radicals generated during intense exercise will expose the cells to oxidative damages. In the event of dehydration, there will be significant losses of water and functional electrolytes during intense exercise which affected the body fluid balance. Fatigue will also occur during reduced oxygen in aerobic metabolism which later caused accumulation of lactic acid in the muscle. This will change the pH balance toward more acidic and caused the muscles to lose contractile efficiency. In addition, fatigue can also be studied using rats as model organism. Results from this activity can be useful to analyse cellular metabolism and physiology effects of the tested rats toward physical exercise. Therefore, this review aims to discuss the causes of fatigue through oxidative stress, dehydration and lactic acid accumulation. In addition, the effectiveness of using rats as a model system in measuring fatigue is also included in illustrating examples on fatigue assessment in vivo

Ergogenic attributes of young and mature coconut (Cocos nucifera L.) water based on physical properties, sugars and electrolytes contents

The present work aimed to compare the main ergogenic attributes of two commercialized stages (young and mature) of coconut water (CW) obtained from four coconut varieties. The changes of electrolytes and sugars in CW upon maturation were quantified by inductively coupled plasma mass spectrophotometer and high-performance liquid chromatography, respectively. Based on the electrolyte profiling, potassium yielded the highest amount (ranging from 237.41 to 361.20 mg/100 mL) followed by sodium, magnesium, calcium, iron, manganese, copper, selenium, and zinc across all the maturity stages tested. For sugars, there were lower amounts of fructose and glucose, but a higher amount of sucrose with the maturation of the fruits. In conclusion, the amount of beneficial nutrients in the form of sugars and minerals was higher than that of young CW, and the ergogenic attributes of mature CW especially from MATAG variety (M-MATAG) were the best to be exploited further in the development of natural energy drinks

Different maturities and varieties of coconut (Cocos nucifera L.) flesh as fat replacers in reduced-fat meatballs

Meatball is a popular meat product in Malaysia but it has high-fat content that could often bring health problems such as non-communicable diseases. This study aimed to produce replaced-fat meatballs using coconut flesh with three different varieties (i.e. MAWA, MYD, and Pandan) at two levels of maturities (young and matured). Meatballs with animal fat were used as control. All samples were analysed for the proximate composition, cooking yield, water holding capacity (WHC), texture and sensory properties. Two-way ANOVA was conducted to determine the interaction between the maturity and variety of the coconut for all parameters with post-hoc analysis to observe the significant difference (p<0.05). The maturity and variety of coconuts showed significant interactions (p<0.05) in affecting most of the meatball properties. Young coconuts were superior to mature coconut with better cooking yield (p<0.05), texture and sensory properties. Meatballs with young Pandan had the highest moisture content (p<0.05) (71.78%), low fat (3.56%) (p<0.05), and overall sensory acceptance. Meatballs with young MYD had low fat content (3.50%) (p<0.05), high WHC (52.11) (p<0.05), high redness (6.09) (p<0.05) with a high score of the colour (7.13) taste (7.17), texture (7.30) and overall acceptability (7.03) for the sensory properties. Since both young Pandan and young MYD managed to reduce the fat content in the meatballs as compared to the control sample and other treatments while maintaining the quality characteristics of the meatballs, both can be concluded to have potential as the fat replacers in the production of reduced-fat meatballs

Design of novel semisynethetic metalloenzyme from thermolysin

Initial applications of biocatalysis involved the used of naturally occurring enzyme. With new challenges in green chemical reaction, biocatalyst that shed the light is metalloenzyme,which function as enzyme and contain metal that are tightly attached and always isolated with the protein[1]. In recent years, enzyme engineering has proven to be an invaluable tool for elucidating biocatalytic mechanisms as well as producing enzymes for industrial purposes.Approaches developed for in vivo chemical modification and in silico computational methods promise to increase the scope and have already been used successfully to alter existing protein so that they have better stability and functionality [2]. This task might be good to address in designing a new biocatalyst with improved properties

Metabolomics approach to investigate the ergogenic effect of Morinda citrifolia L. leaf extract on obese Sprague Dawley rats

Introduction: Natural products are obtaining much acceptance as ergogenic aid, not only among athletes but also among the general population including people with excess body fat. Under normal circumstances, an obese person will have the desire and ability to exercise reduced; mainly because they are easily fatigued. Thus, they need to boost their energy production so that they can be more active and healthier. Objective: In this present work, Morinda citrifolia L. leaf extract (MLE) which is believed to possess ergogenic property, was evaluated on its effect on an obese animal model using 1 H-NMR based metabolomics. Material and methods: Rats were fed with high fat diet (HFD) for 12 weeks for obese development. Once this was achieved, all the rats underwent endurance exercise (forced swimming test) every 2 weeks for 8 weeks together with treatment. The time to exhaustion was recorded for each rat. Three different dosages of MLE: 50 mg/kg, 100 mg/kg and 200 mg/kg of body weight were used together with two positive controls: 5 mg/kg caffeine and 100 mg/kg green tea. Blood was collected before and after treatments for metabolomics study. Results: Findings showed that feeding the rats at a dose of 200 mg/kg body weight MLE significantly prolonged the exhaustive swimming time of the rats, and altered the metabolites present in their serum. Discriminating metabolites involved were the product of various metabolic pathways, including carbohydrate, lipids metabolism and energy metabolism. Treatment with 200 mg/kg body weight MLE resulted in significant improvement in the metabolic perturbations where the proximity of the obese exercised treated group to that of normal exercised group in the partial least squares discriminant analysis score plot was observed. Conclusion: The present work demonstrated ergogenic property of MLE based on the improved metabolic perturbation in exercised obese rats

Ergogenic property of Morinda citrifolia L. leaf extract affects energy metabolism in obese Sprague Dawley rats

Ergogenic property is the ability to enhance capacity for physical activities through efficient production of energy and is potentially beneficial in weight management for the obese. In this study, ergogenic property of Morinda citrifolia leaf's extract (MCL) was evaluated using AMP-activated protein kinase (AMPK) activity and high fat diet-induced obese rats. Findings from the study showed that MCL demonstrated ergogenic activity via enhancement of AMPK activity using L6 skeletal muscle cell line. Interestingly, the result also revealed that rats treated with the intermediate dosage of MCL experienced the lowest % weight gain. The rats fed the highest dose of 200 mg/kg BW MCL demonstrated the longest swimming time of approximately three times that of green tea and caffeine-fed rats. The highest dose fed rats were also found to have lower glucose and lactate levels, suggesting that energy metabolism was more effective in these rats. In addition, lactate dehydrogenase and creatinine kinase activities, the muscle injury indicators, were found to be the lowest in rats fed the highest MCL dose. The same effect was not seen in rats fed either caffeine or green tea, indicating that MCL treatment is may be protective of the rats' muscles. It was also shown that MCL consisted of various flavonoids with epicatechin, catechin, and quercetin that may be responsible for the effects measured. In conclusion, improvements were seen in rats fed MCL in terms of weight management, endurance capacity, energy metabolism, and muscle injury parameters. PRACTICAL APPLICATIONS: Results of the study revealed that Morinda citrifolia leaf has great potential to be used as functional ingredient in the development of designer food/drink as ergogenic aid for both obese and non-obese individuals. Morinda citrifolia leaf could help in the weight management of obese people and enhance endurance capacity and energy metabolism in active individuals

Potential use of rosemary extract to increase the quality and safety of rendered chicken fat

A commercial rosemary extract was evaluated for antioxidant effectiveness in rendered chicken fat and antibacterial activity against selected foodborne pathogens. Four treatments were used in the experiment comprising of 25 and 50 ppm rosemary extract (RE), 200 ppm butylated hydroxyanisole (BHA; commercial antioxidant as positive control), and a negative control without antioxidant. All treatments were subjected to oxidative stability analysis tests including free fatty acid (FFA), peroxide value (PV), p-Anisidine value (AV), and total oxidation value (TOTOX), which were analysed weekly for a total period of seven weeks. In addition, RE was tested for antibacterial activity against Bacillus cereus, Escherichia coli, Klebsiella pneumoniae, Listeria monocytogenes, Salmonella enterica, and Staphylococcus aureus using disc diffusion assay (DDA). The results showed that the addition of 50 ppm of RE yielded the most potent antioxidant effect as evidenced by the lowest amounts for negative oxidation indicators (i.e., FFA, PV, AV, and TOTOX) as compared to the other treatments. BHA showed significant antioxidant effect between treatments for FFA and PV, and 25 ppm RE showed significant antioxidant effect between treatments for FFA only. DDA resulted in inhibition zones against all the tested bacteria ranging from 7.7 ± 0.6 mm (S. enterica; weakest inhibition) to 11.8 ± 0.4 mm (K. pneumonia; strongest inhibition). The findings showed that rosemary extract can be used as a natural antioxidant that effectively delays oxidation and exhibits antibacterial properties