Nowadays dengue virus infection (DENV) is one of the major health complications in the world. Although DENV is an old and common disease, unfortunately, until now, there are no specific relevant treatments available for it. This study, therefore, aimed to design, as well as synthesize selective peptide inhibitors, and investigate their activity by in-vitro NS2B/NS3 protease inhibition assay. The design of the peptide ligands was based on studying the interactions with the dengue NS2B/NS3 protease using the computational docking technique in the MOE and AutoDock (version 4.2) software. To this end, the researchers designed 26 linear pentapeptides based on previous studies. It was revealed that two linear pentapeptides (i.e., GKRRK and KRRRK) are the best potential inhibitors. Furthermore, based on the findings of the two independent docking programs, the peptide GKRRK was synthesized by solid-phase peptide synthesis and its structure was confirmed. The in-vitro protease inhibitor study was conducted for these two peptides to examine their activity against the dengue virus using a protin in as a control. It was found that the designed potential peptides possess interesting inhibition against the NS2B/NS3 protease. Additionally, the findings showed that the peptide GKRRK had the highest percentage of inhibition (71.11%) at 100 µM with the IC50 of 48.87 µM; therefore, this linear peptide could serve as a good inhibitor for the DENV.
In the current study, we used molecular screening and simulation approaches to target I7L protease from monkeypox virus (mpox) from the Traditional Chinese Medicines (TCM) database. Using molecular screening, only four hits TCM27763, TCM33057, TCM34450 and TCM31564 demonstrated better pharmacological potential than TTP6171 (control). Binding of these molecules targeted Trp168, Asn171, Arg196, Cys237, Ser240, Trp242, Glu325, Ser326, and Cys328 residues and may affect the function of I7L protease in in vitro assay. Moreover, molecular simulation revealed stable dynamics, tighter structural packing and less flexible behaviour for all the complexes. We further reported that the average hydrogen bonds in TCM27763, TCM33057, TCM34450 and TCM31564I7L complexes remained higher than the control drug. Finally, the BF energy results revealed -62.60 ± 0.65 for the controlI7L complex, for the TCM27763I7L complex -71.92 ± 0.70 kcal/mol, for the TCM33057I7L complex the BF energy was -70.94 ± 0.70 kcal/mol, for the TCM34450I7L the BF energy was -69.94 ± 0.85 kcal/mol while for the TCM31564I7L complex the BF energy was calculated to be -69.16 ± 0.80 kcal/mol. Although, we used stateoftheart computational methods, these are theoretical insights that need further experimental validation.
The cell-free extract of locally isolated Rhodococcus UKMP-5M strain was used as an alternative to develop greener and cost effective cyanide removal technology. The present study aims to assess the viability of the cell-free extract to detoxify high concentrations of cyanide which is measured through the monitoring of protein concentration and specific cyanide-degrading activity. When cyanide-grown cells were subjected to grinding in liquid nitrogen which is relatively an inexpressive and fast cell disruption method, highest cyanide-degrading activity of 0.63 mM min(-1) mg(-1) protein was obtained in comparison to enzymatic lysis and agitation with fine glass beads. The cell-free extracts managed to degrade 80% of 20 mM KCN within 80 min and the rate of cyanide consumption increased linearly as the concentration of protein was raised. In both cases, the addition of co-factor was not required which proved to be advantageous economically. The successful formation of ammonia and formate as endproducts indicated that the degradation of cyanide by Rhodococcus UKMP-5M proceeded via the activity of cyanidase and the resulting non-toxic products are safe for disposal into the environment. Further verification with SDS-PAGE revealed that the molecular weight of the active enzyme was estimated to be 38 kDa, which is consistent with previously reported cyanidases. Thus, the utilization of cell-free extracts as an alternative to live microbial in cyanide degradation offers numerous advantageous such as the potential to tolerate and degrade higher concentration of cyanide and total reduction in the overall cost of operation since the requirement for nutrient support is irrelevant.
We report the degradation of quorum sensing N-acylhomoserine lactone molecules by a bacterium isolated from a Malaysian marine water sample. MALDI-TOF and phylogenetic analysis indicated this isolate BM1 clustered closely to Labrenzia sp. The quorum quenching activity of this isolate was confirmed by using a series of bioassays and rapid resolution liquid chromatography analysis. Labrenzia sp. degraded a wide range of N-acylhomoserine lactones namely N-(3-hexanoyl)-L-homoserine lactone (C6-HSL), N-(3-oxohexanoyl)-L-homoserine lactone (3-oxo-C6-HSL) and N-(3-hydroxyhexanoyl)-L-homoserine lactone (3-hydroxy-C6-HSL). Re-lactonisation bioassays confirmed Labrenzia sp. BM1 degraded these signalling molecules efficiently via lactonase activity. To the best of our knowledge, this is the first documentation of a Labrenzia sp. capable of degrading N-acylhomoserine lactones and confirmation of its lactonase-based mechanism of action.
Two microbial isolates from a Malaysian shoreline were found to be capable of degrading N-acylhomoserine lactones. Both Matrix Assisted Laser Desorption Ionization-Time of Flight-Mass Spectrometry and 18S rDNA phylogenetic analyses confirmed that these isolates are Rhodotorula mucilaginosa. Quorum quenching activities were detected by a series of bioassays and rapid resolution liquid chromatography analysis. The isolates were able to degrade various quorum sensing molecules namely N-hexanoyl-L-homoserine lactone (C6-HSL), N-(3-oxo-hexanoyl)-L-homoserine lactone (3-oxo-C6-HSL) and N-(3-hydroxyhexanoyl)-L-homoserine lactone (3-hydroxy-C6-HSL). Using a relactonisation assay to verify the quorum quenching mechanism, it is confirmed that Rh. mucilaginosa degrades the quorum sensing molecules via lactonase activity. To the best of our knowledge, this is the first documentation of the fact that Rh. mucilaginosa has activity against a broad range of AHLs namely C6-HSL, 3-oxo-C6-HSL and 3-hydroxy-C6-HSL.
Heavy metals pollution has become a great threat to the world. Since instrumental methods are expensive and need skilled technician, a simple and fast method is needed to determine the presence of heavy metals in the environment. In this study, an inhibitive enzyme assay for heavy metals has been developed using crude proteases from Coriandrum sativum. In this assay, casein was used as a substrate and Coomassie dye was used to denote the completion of casein hydrolysis. In the absence of inhibitors, casein was hydrolysed and the solution became brown, while in the presence of metal ions such as Hg²⁺ and Zn²⁺, the hydrolysis of casein was inhibited and the solution remained blue. Both Hg²⁺ and Zn²⁺ exhibited one-phase binding curve with IC₅₀ values of 3.217 mg/L and 0.727 mg/L, respectively. The limits of detection (LOD) and limits of quantitation (LOQ) for Hg were 0.241 and 0.802 mg/L, respectively, while the LOD and LOQ for Zn were 0.228 and 0.761 mg/L, respectively. The enzyme exhibited broad pH ranges for activity. The crude proteases extracted from Coriandrum sativum showed good potential for the development of a rapid, sensitive, and economic inhibitive assay for the biomonitoring of Hg²⁺ and Zn²⁺ in the aquatic environments.
Keratinous wastes have increasingly become a problem and accumulate in the environment mainly in the form of feathers, generated mainly from a large number of poultry industries. As keratins are very difficult to degrade by general proteases, they pose a major environmental problem. Therefore, microorganisms which would effectively degrade keratins are needed for recycling such wastes. A geophilic dermatophyte, Microsporum fulvum IBRL SD3 which was isolated from a soil sample collected from a chicken feather dumping site using a baiting technique, was capable to produce keratinase significantly. The crude keratinase was able to degrade whole chicken feathers effectively. The end product of the degradation was protein that contained essential amino acids and may have potential application in animal feed production. Thus, M. fulvum could be a novel organism to produce keratinase for chicken feathers degradation.
Polyhydroxyalkanoates (PHAs) are hydrophobic biodegradable thermoplastics that have received considerable attention in biomedical applications due to their biocompatibility, mechanical properties, and biodegradability. In this study, the degradation rate was regulated by optimizing the interaction of parameters that influence the enzymatic degradation of P(3HB) film using response surface methodology (RSM). The RSM model was experimentally validated yielding a maximum 21 % weight loss, which represents onefold increment in percentage weight loss in comparison with the conventional method. By using the optimized condition, the enzymatic degradation by an extracellular PHA depolymerase from Acidovorax sp. DP5 was studied at 37 °C and pH 9.0 on different types of PHA films with various monomer compositions. Surface modification of scaffold was employed using enzymatic technique to create highly porous scaffold with a large surface to volume ratio, which makes them attractive as potential tissue scaffold in biomedical field. Scanning electron microscopy revealed that the surface of salt-leached films was more porous compared with the solvent-cast films, and hence, increased the degradation rate of salt-leached films. Apparently, enzymatic degradation behaviors of PHA films were determined by several factors such as monomer composition, crystallinity, molecular weight, porosity, and roughness of the surface. The hydrophilicity and water uptake of degraded salt-leached film of P(3HB-co-70%4HB) were enhanced by incorporating chitosan or alginate. Salt-leached technique followed by partial enzymatic degradation would enhance the cell attachment and suitable for biomedical as a scaffold.
By-products from different animal sources are currently being utilised for beneficial purposes. Chicken processing plants all over the world generate large amount of solid by-products in form of heads, legs, bones, viscera and feather. These wastes are often processed into livestock feed, fertilizers and pet foods or totally discarded. Inappropriate disposal of these wastes causes environmental pollution, diseases and loss of useful biological resources like protein, enzymes and lipids. Utilisation methods that make use of these biological components for producing value added products rather than the direct use of the actual waste material might be another viable option for dealing with these wastes. This line of thought has consequently led to researches on these wastes as sources of protein hydrolysates, enzymes and polyunsaturated fatty acids. Due to the multi-applications of protein hydrolysates in various branches of science and industry, and the large body of literature reporting the conversion of animal wastes to hydrolysates, a large section of this review was devoted to this subject. Thus, this review reports the known functional and bioactive properties of hydrolysates derived from chicken by-products as well their utilisation as source of peptone in microbiological media. Methods of producing these hydrolysates including their microbiological safety are discussed. Based on the few references available in the literature, the potential of some chicken by-product as sources of proteases and polyunsaturated fatty acids are pointed out along with some other future applications.
The ability of immobilized cell cultures of Aspergillus niger FETL FT3 to produce extracellular tannase was investigated. The production of enzyme was increased by entrapping the fungus in scouring mesh cubes compared to free cells. Using optimized parameters of six scouring mesh cubes and inoculum size of 1 × 10(6) spores/mL, the tannase production of 3.98 U/mL was obtained from the immobilized cells compared to free cells (2.81 U/mL). It was about 41.64% increment. The immobilized cultures exhibited significant tannase production stability of two repeated runs.
The protease activity of different isolates of dermatophytes representing different ecological groups namely geophilic, zoopahilic and anthropophilic, in their vegetative and sporulation growth phases were compared. Unlike their geophilic and zoophilic counterparts, all the isolates of anthropophilic dermatophytes viz. Trichophyton rubrum, T. mentagrophytes, T. tonsurans, T. violaceum and Epidermophyton floccosum recorded reduced protease activity during artificially induced sporulation phase in comparison to their vegetative growth phase. Even among the anthropophilic group, a classical moderation of protease activity was recorded in Trichyphyton rubrum which also correlates to its clinical manifestation. This enzyme moderation could also be an evolutionary adaptation of the anthropization of these species.
Two experiments were conducted to study the effects of beta-glucanase produced by transformed Lactobacillus strains on the intestinal characteristics and feed passage rate of broiler chickens fed barley-based diets. Supplementation of transformed Lactobacillus strains to the diet of chickens significantly (P < 0.05) reduced the intestinal fluid viscosity by 21 to 46% compared with chickens fed an unsupplemented diet or a diet supplemented with parental Lactobacillus strains. The relative weights of pancreas, liver, duodenum, jejunum, ileum, ceca, and colon were reduced (P < 0.05) by 6 to 27%, and the relative length of duodenum, jejunum, ileum, and ceca was reduced (P < 0.05) by 8 to 15%. Histological examination of the intestinal tissues showed that the jejunal villus height of chickens fed a diet supplemented with transformed Lactobacillus strains was significantly (P < 0.05) higher than that of chickens fed other dietary treatments. The transformed Lactobacillus strains were found to reduce (P < 0.05) the time of feed passage rate by 2.2 h. Supplementation of transformed Lactobacillus strains to the diet improved the intestinal characteristics and feed, passage rate of the chickens.
Loss of heterozygosity (LOH) on several loci and mutations on PTEN tumor suppressor gene (10q23.3) occur frequently in sporadic gliomas. We have performed polymerase chain reaction (PCR)-LOH analysis using microsatellite markers and single-stranded conformational polymorphism (SSCP) analysis to determine the incidence of allelic losses on chromosome 10q, 9p, 17p and 13q and mutations of exons 5, 6 and 8 of the PTEN gene in malignant gliomas. Twelve of 23 (52.2%) malignant glioma cases showed allelic losses whereas 7 of 23, (30.4%) samples showed aberrant band patterns and mutations of the PTEN gene. Four of these cases showed LOH on 10q23 and mutations of the PTEN gene. The data on LOH indicated the involvement of different genes in gliomagenesis whereas mutations of the PTEN gene indicated the role of PTEN tumor suppressor gene in the progression of glioma in Malay population.
1. Toxicity evaluations of DDT, lindane, abate and carbaryl were carried out in the larvae of two wild Aedes aegypti strains from Kuala Lumpur and Klang. The Kuala Lumpur strain was more susceptible to the insecticides than the Klang strain. 2. The lethal toxicity time was also determined. The insecticides were found to take a longer time to exert their effect in the Klang strain as compared to the Kuala Lumpur strain. 3. Carboxylesterase activity was determined to be higher in the Kuala Lumpur strain, but glutathione transferase activities were higher in the Klang strain.
An abnormal, fast-moving 5'-nucleotide phosphodiesterase isozyme was found in 90.0% of 20 Malaysian patients with primary hepatoma and in 23.5% of 391 Malaysian patients with various malignant diseases; it was also discovered in 42.9% of 14 Malaysian and American patients with clinically active hepatitis B infection; in 16.7% of 18 healthy American blood bank donors who were positive for hepatitis B surface antigen (HBsAg); in 13.9% of 287 healthy Malaysian blood bank donors, some positive for HBsAg; and in none of 160 healthy American donors who were negative for HBsAg. A correlation of this abnormal isozyme with hepatoma and with infectious hepatitis B is clearly evident.
Calmodulin, an activator protein in most calcium-dependent processes, was isolated to apparent homogeneity from the femurs of 1-day old chicks using phenyl-Sepharose and high performance liquid chromatography. The purified calmodulin was found to produce a 6-fold increase in the activity of alkaline phosphatase isolated from the same source. A Ca2+ concentration of 10(-5) M was required for the activation. Purification of alkaline phosphatase involved acetone precipitation, DEAE-Sephacel and Sephadex G-200 column chromatography. The enzyme was purified to 540-fold and had a specific activity of 10.75 U/mg protein.
Conventional refolding methods are associated with low yields due to misfolding and high aggregation rates or very dilute proteins. In this study, we describe the optimization of the conventional methods of reverse dilution and affinity chromatography for obtaining high yields of a cysteine rich recombinant glycoside hydrolase family 19 chitinase from Streptomyces griseus HUT6037 (SgChiC). SgChiC is a potential biocontrol agent and a reference enzyme in the study and development of chitinases for various applications. The overexpression of SgChiC was previously achieved by periplasmic localization from where it was extracted by osmotic shock and then purified by hydroxyapatite column chromatography. In the present study, the successful refolding and recovery of recombinant SgChiC (r-SgChiC) from inclusion bodies (IB) by reverse dilution and column chromatography methods is respectively described. Approximately 8 mg of r-SgChiC was obtained from each method with specific activities of 28 and 52 U/mg respectively. These yields are comparable to that obtained from a 1 L culture volume of the same protein isolated from the periplasmic space of E. coli BL21 (DE3) as described in previous studies. The higher yields obtained are attributed to the successful suppression of aggregation by a stepwise reduction of denaturant from high, to intermediate, and finally to low concentrations. These methods are straight forward, requiring the use of fewer refolding agents compared with previously described refolding methods. They can be applied to the refolding of other cysteine rich proteins expressed as inclusion bodies to obtain high yields of actively folded proteins. This is the first report on the recovery of actively folded SgChiC from inclusion bodies.
The effects of beta-glucanase expressed by transformed Lactobacillus strains on growth performance, apparent digestibilities of dry matter and crude protein, and apparent metabolisable energy were studied. Two hundred and forty 1-d-old chicks (Avian-43) were randomly divided into three dietary treatment groups and fed with the following diets: (i) basal diet (control) (BD); (ii) basal diet with parental Lactobacillus strains (BDP) and (iii) basal diet with transformed Lactobacillus strains (BDT). At 21 d of age, the body weight, body weight gain and feed conversion ratio of the BDT-fed chickens were significantly improved. At 14 and 21 d of age, the proportions of dry matter in the duodenum, jejunum, ileum, caeca and excreta of chickens given the BDT diet were significantly higher than those of chickens given the BD and BDP diets. Apparent metabolisable energy, digestibilities of crude protein and dry matter were also significantly improved (by 3.5, 5.6 and 3.5%, respectively) by the BDT diet. These results showed that the transformed Lactobacillus strains improved digestibility as well as enhanced the growth performance of chickens.
Kadazans, the largest indigenous group in Sabah, northern Borneo, were surveyed for glyoxalase I, phosphoglucomutase I, red cell acid phosphatase, esterase D, adenosine deaminase, soluble glutamate pyruvate transaminase, soluble glutamate oxaloacetate transaminase, 6-phosphogluconate dehydrogenase, uridine monophosphate kinase, adenylate kinase, peptidase B and D, superoxide dismutase, C5, group specific component, haptoglobin and transferrin. Kadazans were found to be polymorphic for GLO I, PGM I, RCAP, esterase D, ADA, s-Gpt, 6PGD, UMPK, Gc, C5, haptoglobin and peptidase B. Rare variants were found for transferrin and peptidase D. No variant was found for s-Got, SOD and AK.
A Rhodococcus sp. UKMP-5M isolate was shown to detoxify cyanide successfully, suggesting the presence of an intrinsic property in the bacterium which required no prior cyanide exposure for induction of this property. However, in order to promote growth, Rhodococcus sp. UKMP-5M was fully acclimatized to cyanide after 7 successive subcultures in 0.1 mM KCN for 30 days. To further shorten the lag phase and simultaneously increase the tolerance towards higher cyanide concentrations, the bacterium was induced with various nitrile compounds sharing a similar degradatory pathway to cyanide. Acetonitrile emerged as the most favored inducer and the induced cells were able to degrade 0.1 mM KCN almost completely within 18 h. With the addition of subsequent aliquots of 0.1 mM KCN a shorter period for complete removal of cyanide was required, which proved to be advantageous economically. Both resting cells and crude enzyme of Rhodococcus sp. UKMP-5M were able to biodegrade cyanide to ammonia and formate without the formation of formamide, implying the identification of a simple hydrolytic cyanide degradation pathway involving the enzyme cyanidase. Further verification with SDS-PAGE revealed that the molecular weight of the active enzyme was estimated to be 38 kDa, which is consistent with previously reported cyanidases. Since the recent advancement in the application of biological methods in treating cyanide-bearing wastewater has been promising, the discovery of this new bacterium will add value by diversifying the existing microbial populations capable of cyanide detoxification.