The effect of the recently developed graphene nanoflakes (GNFs) on the polymerase chain reaction (PCR) has been investigated in this paper. The rationale behind the use of GNFs is their unique physical and thermal properties. Experiments show that GNFs can enhance the thermal conductivity of base fluids and results also revealed that GNFs are a potential enhancer of PCR efficiency; moreover, the PCR enhancements are strongly dependent on GNF concentration. It was found that GNFs yield DNA product equivalent to positive control with up to 65% reduction in the PCR cycles. It was also observed that the PCR yield is dependent on the GNF size, wherein the surface area increases and augments thermal conductivity. Computational fluid dynamics (CFD) simulations were performed to analyze the heat transfer through the PCR tube model in the presence and absence of GNFs. The results suggest that the superior thermal conductivity effect of GNFs may be the main cause of the PCR enhancement.
Molecular dynamics simulation was used to study the dynamic differences between native Aspergillus niger PhyA phytase and a mutant with 20 % greater thermostability. Atomic root mean square deviation, radius of gyration, and number of hydrogen bonds and salt bridges are examined to determine thermostability factors. The results suggest that, among secondary structure elements, loops have the most impact on the thermal stability of A. niger phytase. In addition, the location rather than the number of hydrogen bonds is found to have an important contribution to thermostability. The results also show that salt bridges may have stabilizing or destabilizing effect on the enzyme and influence its thermostability accordingly.
Bromelain, a cysteine protease with various therapeutic and industrial applications, was expressed in Escherichia coli, BL21-AI clone, under different cultivation conditions (post-induction temperature, L-arabinose concentration and post-induction period). The optimized conditions by response surface methodology using face centered central composite design were 0.2% (w/v) L-arabinose, 8 hr and 25°C. The analysis of variance coupled with larger value of R2 (0.989) showed that the quadratic model used for the prediction was highly significant (p < 0.05). Under the optimized conditions, the model produced bromelain activity of 9.2 U/mg while validation experiments gave bromelain activity of 9.6 ± 0.02 U/mg at 0.15% (w/v) L-arabinose, 8 hr and 27°C. This study had innovatively developed cultivation conditions for better production of recombinant bromelain in shake flask culture.
This study enhanced the production of thermostable organic solvent-tolerant (TS-OST) lipase by locally isolated thermotolerant Rhizopus sp. strain using solid-state fermentation (SSF) of palm kernel cake (PKC). The optimum conditions were achieved using a series of statistical approaches. The cultivation parameters, which include fermentation time, moisture content, temperature, pH, inoculum size, various carbon and nitrogen sources, as well as other supplements, were initially screened by the definitive screening design, and one-factor-at-a-time using PKC as the basal medium. Three significant factors (olive oil concentration, pH, and inoculum size) were further optimized using face-centred central composite design. The results indicated a successful and significant improvement of lipase activity by almost two-fold compared to the initial screening production. The findings showed that the optimal conditions were 2% (v/w) inoculum size, 2% (v/w) olive oil, 0.6% (w/w) peptone, 2% (v/w) ethanol, 70% moisture content at initial pH 10.0 and 45 °C within 72 h of fermentation. Process optimization resulted in maximum lipase activity of 58.63 U/gram dry solids (gds). The analysis of variance showed that the statistical model was significant (p value <0.0001) and reliable with a high value of R2 (0.98) and adjusted R2 (0.96). This indicates a better correlation between the actual and predicted responses of lipase production. By considering this study, the low-cost PKC through SSF appears to be promising in the utilization of agro-industrial waste for TS-OST lipase production. This is because satisfactory enzyme activity could be attained that promises industrial applications.
Fish by-product oil and lemon oil have potential applications as active ingredients in many industries, including cosmetics, pharmaceuticals and food. However, the physicochemical properties, especially the poor stability, compromised the usage. Generally, nanoemulsions were used as an approach to stabilize the oils. This study employed an ultrasonication method to form oil-in-water nanoemulsion of lemon and fish by-product oils (NE-FLO). The formulation is produced at a fixed amount of 2 wt% fish by-product oil, 8 wt% lemon oil, 10 wt% surfactant, 27.7 wt% co-surfactants and 42 min of ultrasonication time. The size, polydispersity index (PDI) and zeta potential obtained were 44.40 nm, 0.077, and -5.02 mV, respectively. The biological properties, including antioxidant, antibacterial, cell cytotoxicity, and anti-inflammatory, showed outstanding performance. The antioxidant activity is comparable without any significant difference with ascorbic acid as standard and is superior to pure lemon oil. NE-FLO successfully inhibits seven Gram-positive and seven Gram-negative bacterial strains. NE-FLO's anti-inflammatory activity is 99.72%, comparable to nordihydroguaiaretic acid (NDGA) as the standard. At a high concentration of 10,000 µg·mL-1, NE-FLO is non-toxic to normal skin cells. These findings demonstrate that the NE-FLO produced in this study has significant potential for usage in various industries.
Over two hundred bacteria were isolated from the halosphere, rhizosphere and endophyte of Malaysian maize plantation and screened for phytases activity. Thirty isolates with high detectable phytase activity were chosen for media optimization study and species identification. Ten types of bacterial phytase producers have been discovered in this study, which provides opportunity for characterization of new phytase(s) and various commercial and environmental applications. The majority of the bacterial isolates with high detectable phytase activity were of endophyte origin and 1.6% of the total isolates showed phytase activity of more than 1 U/ml. Most of the strains produced extra-cellular phytase and Staphylococcus lentus ASUIA 279 showed the highest phytase activity of 1.913 U/ml. All 30 species used in media optimization study exhibit favorable enzyme production when 1% rice bran was included in the growth media.
Growing cells on microcarriers may have overcome the limitation of conventional cell culture system. However, the surface functionality of certain polymeric microcarriers for effective cell attachment and growth remains a challenge. Polycaprolactone (PCL), a biodegradable polymer has received considerable attention due to its good mechanical properties and degradation rate. The drawback is the non-polar hydrocarbon moiety which makes it not readily suitable for cell attachment. This report concerns the modification of PCL microcarrier surface (introduction of functional oxygen groups) using ultraviolet irradiation and ozone (UV/O3) system and investigation of the effects of ozone concentration, the amount of PCL and exposure time; where the optimum conditions were found to be at 60,110.52 ppm, 5.5 g PCL and 60 min, respectively. The optimum concentration of carboxyl group (COOH) absorbed on the surface was 1495.92 nmol/g and the amount of gelatin immobilized was 320 ± 0.9 µg/g on UV/O3 treated microcarriers as compared to the untreated (26.83 ± 3 µg/g) microcarriers. The absorption of functional oxygen groups on the surface and the immobilized gelatin was confirmed with the attenuated total reflectance Fourier transformed infrared spectroscopy (ATR-FTIR) and the enhancement of hydrophilicity of the surface was confirmed using water contact angle measurement which decreased (86.93°-49.34°) after UV/O3 treatment and subsequently after immobilization of gelatin. The attachment and growth kinetics for HaCaT skin keratinocyte cells showed that adhesion occurred much more rapidly for oxidized surfaces and gelatin immobilized surface as compared to untreated PCL.
A panel of six monoclonal antibodies (mAbs) against the nucleocapsid (NP) protein of Newcastle disease virus (NDV) was produced by immunization of Balb/c mice with purified recombinant NP protein. Western Blot analysis showed that all the mAbs recognized linearized NP epitopes. Three different NP antigenic sites were identified using deleted truncated NP mutants purified from Escherichia coli. One of the antigenic sites was located at the C-terminal end (residues 441 to 489) of the NP protein. Two other antigenic sites were located within the N-terminal end (residues 26-121 and 122-375). This study demonstrates that the N- and C-terminal ends of the NP proteins are responsible in eliciting immune response, thus it is most likely that these ends are exposed on the NP.
The aim of this study was to develop gelatin coated polystyrene (PS) microcarriers with good cell adhesion and proliferation properties. PS microspheres, prepared using oil-in water (o/w) solvent evaporation method, were loaded with oxygen containing functional groups using an ultraviolet/ozone (UVO3) system. Using water-soluble carbodiimide chemistry, gelatin was subsequently immobilized on UVO3 treated PS microspheres. The amount of immobilized gelatin was found to be directly proportional to the surface carboxyl (COOH) concentration on PS microspheres. Face Centered Central Composite Design (FCCD) was employed to optimize the process conditions of UVO3 treatment to maximize the surface COOH concentration on PS microspheres for allowing higher gelatin immobilization. Statistical results revealed that, the optimized process conditions were ozone flow rate of ∼64,603 ppm, exposure time of ∼60 minutes and sample amount of 5.05 g. Under these conditions, the surface COOH concentration on PS microspheres was ∼1,505 nmol/g with the corresponding amount of immobilized gelatin was ∼2,725 µg/g. Characterization analyses strongly suggest that the optimized UVO3 treatment and successive gelatin immobilization have successfully improved surface wettability and dispersion stability of PS microspheres. Moreover, gelatin coated PS microcarriers were also proven as able to support the growth of CHO-K1 cells in high cell density culture.
Microencapsulation is a promising approach in drug delivery to protect the drug from degradation and allow controlled release of the drug in the body. Fucoxanthin-loaded microsphere (F-LM) was fabricated by two step w/o/w double emulsion solvent evaporation method with poly (L-lactic-co-glycolic acid) (PLGA) as carrier. The effect of four types of surfactants (PVA, Tween-20, Span-20 and SDS), homogenization speed, and concentration of PLGA polymer and surfactant (PVA), respectively, on particle size and morphology of F-LM were investigated. Among the surfactants tested, PVA showed the best results with smallest particle size (9.18 µm) and a smooth spherical surface. Increasing the homogenization speed resulted in a smaller mean F-LM particle size [d(0.50)] from 17.12 to 9.18 µm. Best particle size results and good morphology were attained at homogenization speed of 20 500 rpm. Meanwhile, increased PLGA concentration from 1.5 to 11.0 (% w/v) resulted in increased F-LM particle size. The mean particle size [d(0.5)] of F-LM increased from 3.93 to 11.88 µm. At 6.0 (% w/v) PLGA, F-LM showed the best structure and external morphology. Finally, increasing PVA concentration from 0.5 to 3.5 (% w/v) resulted in decreased particle size from 9.18 to 4.86 µm. Fucoxanthin characterization before and after microencapsulation was carried out to assess the success of the microencapsulation procedure. Thermo gravimetry analysis (TGA), glass transition (Tg) temperature of F-LM and fucoxanthin measured using DSC, ATR-FTIR and XRD indicated that fucoxanthin was successfully encapsulated into the PLGA matrix, while maintaining the structural and chemical integrity of fucoxanthin.
Aquilaria malaccensis has been traditionally used to treat several medical disorders including inflammation. However, the traditional claims of this plant as an anti-inflammatory agent has not been substantially evaluated using modern scientific techniques. The main objective of this study was to evaluate the anti-inflammatory effect of Aquilaria malacensis leaf extract (ALEX-M) and potentiate its activity through nano-encapsulation. The extract-loaded nanocapsules were fabricated using water-in-oil-in-water (w/o/w) emulsion method and characterized via multiple techniques including DLS, TEM, FTIR, and TGA. The toxicity and the anti-inflammatory activity of ALEX-M and the extract-loaded nanocapsules (ALEX-M-PNCs) were evaluated in-vitro on RAW 264.7 macrophages and in-vivo on zebrafish embryos. The nanocapsules demonstrated spherical shape with mean particle diameter of 167.13 ± 1.24 nm, narrow size distribution (PDI = 0.29 ± 0.01), and high encapsulation efficiency (87.36 ± 1.81%). ALEX-M demonstrated high viability at high concentrations in RAW 264.7 cells and zebrafish embryos, however, ALEX-M-PNCs showed relatively higher cytotoxicity. Both free and nanoencapsulated extract expressed anti-inflammatory effects through significant reduction of the pro-inflammatory mediator nitric oxide (NO) production in LPS/IFNγ-stimulated RAW 264.7 macrophages and zebrafish embryos in a concentration-dependent manner. The findings highlight that ALEX-M can be recognized as a potential anti-inflammatory agent, and its anti-inflammatory activity can be potentiated by nano-encapsulation. Further studies are warranted toward investigation of the mechanistic and immunomodulatory roles of ALEX-M.