Yeast producing alcohol dehydrogenase 1 (YADH 1) enzyme has been used as a biocatalyst for the synthesis of an optically active flavouring compound known as citronellol. However, the slow growth of yeast (Saccharomyces cerevisiae) has deterred the progress of biotransformation. The main purpose of this work is to clone the genes producing YADH1 enzyme from yeast into a faster growing bacteria, Escherichia coli. Initially, the sequence of the gene encoding this protein has been identified in the S. cerevisiae Genome Databases (SGD). The so-called Yadh1 gene sequence is located from coordinate 159548 to 160594 on chromosome XV of yeast. Based on this information, two primer sequences (Forward and Reverse) were constructed. Each of these primers will bind to either end of the Yadh1 gene. The Yadh1 gene was then amplified using Polymerase Chain Reaction (PCR) technique. The amplified Yadh 1 gene was successfully cloned into a cloning vector, TOPO TA plasmid. This plasmid also contains a gene which confers resistance to ampicillin. This recombinant
plasmid was then inserted into Escherichia coli TOP 10 using heat shock protocol at 42oC. Finally, the cloned bacteria containing the recombinant TOPO TA plasmid harbouring Yadh1 gene was able to grow on Luria Bertani (LB) media supplied with antibiotic.
Current ethanol production processes utilizing crops such as sugar cane and corn starch have been well established over the decade. Other crop such as cassava is a potential candidate in producing ethanol. However, thermal processes are required to hydrolyze starch for the production of fermentable sugars. The processes are energy intensive and could lead to undesirable by-products generation. In this work, the hydrolysis of cassava starch is studied following an experimental design as a statistical problem solving approach. Central composite design (CCD) is used in order to select the most important variables from the simultaneous study on the effect and influence of operating conditions of bioreactor utilized, namely, pH, temperature and substrate concentration, as well as to optimize the process of cassava starch hydrolysis. From the results obtained, it can be concluded that the cassava starch hydrolysis is enhanced by pH and temperature. Model validations show good agreement between experimental results and the predicted responses.
In this study polymer electrolytes composed of poly(methyl methacrylate) (PMMA) as a host polymer and ethylene carbonate (EC) as a plasticizer complexed with different lithium salts, i.e. lithium tetrafluoroborate (LiBF4) and lithium triflate (LiCF3SO3) were prepared by the solution casting technique. The conductivities of the films were characterized by impedance spectroscopy. At room temperature, the highest conductivities were 4.07 × 10–7S cm–1 and 3.40 × 10–5 S cm–1 achieved, respectively from the films containing 30 wt% LiBF4 in the PMMA-EC-LiBF4 system and 35 wt% LiCF3SO3 in the PMMA-EC-LiCF3SO3 system. The conductivity-temperature dependence of the films seemed to obey the Arrhenius equation in which the ion transport in these materials was thermally assisted. Scanning electron microscopy analysis showed that the surface of PMMA-EC-LiCF3SO3 film was smooth and homogeneous, hence lithium ions could traverse through the PMMA-EC-LiCF3SO3 film more easily compared to the PMMA-EC-LiBF4 film. X-Ray diffraction studies revealed that complexation had occurred and the complexes formed were amorphous.
Elevated temperature affects marine benthic algae by reducing growth and limits the transport of electron or carbon fixation which may reduce the ability of the cell to use light. This resulting excess light energy may cause photoinhibition. In this study, the photosystem II of the benthic microalgal communities from Casey, eastern Antarctic were relatively unaffected by significant changes in temperatures up to 8ºC, along with high PAR level (450 μmol photons m–2 s–1). Similarly, the community was able to photosynthesize as the temperature was reduced to –5ºC. Recovery from saturating and photoinhibiting irradiances was not significantly influenced by temperatures at both –5ºC and 8ºC. These responses were consistent with those recorded by past experiments on Antarctic benthic diatoms and temperate diatoms which showed that climate change did not have a significant impact on the ability of benthic microalgae to recover from photoinhibitory temperature stress.
Chlorella is one of the common microalgae found in a wide range of habitats, including Antarctica. Chlorella UMACC 234 is an interesting isolate in the collection of Antarctic microalgae in the University of Malaya algae culture collection (UMACC) as it grows well at temperatures much higher than the ambience. The alga was isolated from snow samples collected from Casey, Antarctica. This study investigates the influence of nitrogen source on the growth, biochemical composition and fatty acid profile of Chlorella UMACC 234. The cultures were grown in Bold’s Basal Medium with 3.0 mM NaNO3, NH4Cl or urea. The cultures grown on NaNO3 attained the highest specific growth rate (μ = 0.43 day–1) while the specific growth rates of those grown on NH4Cl and urea were not significantly different (p > 0.05). The urea-grown cells produced the highest amounts of lipids (25.7% dry weight) and proteins (52.5% dry weight) compared to those grown on other nitrogen sources. The cell numbers attained by the cultures grown at NaNO3 levels between 0.3 and 3.0 mM were similar but decreased markedly at 9.0 mM NaNO3. The fatty acids of Chlorella UMACC 234 were dominated by saturated fatty acids, especially 16:0 and 18:0. The percentage of polyunsaturated fatty acids was very low, especially in cells grown on urea (0.9% total fatty acids). Characterisation of the growth and biochemical composition of this Antarctic Chlorella is important to our studies on the relationship of Chorella isolates from tropical, temperate and polar regions, especially in terms of phylogeny and stress adaptation.
Acanthamoeba is a free-living protozoa which causes serious ocular problem. Acanthamoeba keratitis is becoming more prevalent amongst contact lens wearers and it can cause loss of vision and blindness if not treated properly. The objective of this research is to determine the effectiveness of gentamicin against six Acanthamoeba spp. isolates, of which three were clinical isolates (HS 6, HKL 95, HTH 73) and three environmental isolates (SMAL 7, SMAL 8, TTT 9). Cyst suspension from the chosen isolates were exposed to gentamicin. After 48 hours of incubation at temperature of 30°C and 37oC, each mixture was filtered and filtration membrane was put onto non-nutrient agar laid with Escherichia coli. The agar plates were incubated for three days at 30oC and 37oC and the plates were examined daily until day 14 to look for the presence of Acanthamoeba trophozoites under inverted microscope. The presence of trophozoites indicated the ineffectiveness of gentamicin. Gentamicin was found to be effective against Acanthamoeba cysts from all the test strains at both incubation temperatures. The minimum cysticidal concentration (MCC) mean value of gentamicin was 0.193 mg/mL at 30oC and 0.229 mg/mL at 37oC. So, we concluded that gentamicin has cysticidal potential towards Acanthamoeba.
The β-1,6-glucanases are ubiquitous enzymes which appear to be implicated in the morphogenesis and have the ability to become virulence factor in plant-fungal symbiotic interaction. To our knowledge, no report on ß-1,6-glucanases purification from Trichoderma longibrachiatum has been made, although it has been proven to have a significant effect as a biocontrol agent for several diseases. Therefore, the aim of this study was to purify β-1,6- glucanase from T. longibrachiatum T28, with an assessment on the physicochemical properties and substrate specificity. β-1,3-glucanase enzyme, from the culture filtrate of T. longibrachiatum T28, was successively purified through precipitation with 80% acetone, followed by anionexchange chromatography on Neobar AQ and chromatofocusing on a Mono P HR 5/20 column. (One β-1,6-glucanase) band at 42kDa in size was purified, as shown by the SDS-PAGE. The physicochemical evaluation showed an optimum pH of 5 and optimum temperature of 50°C for enzyme activity with an ability to maintain 100% enzyme stability. Enzyme activity was slightly reduced by 10-20% in the presence of 20 mM of Zn2+, Ca2+, Co2+, Mg2+, Cu2+, Mn2+ and Fe2+. The highest β-1,6-glucanase hydrolysis activity was obtained on pustulan due to the similarity of β-glucosidic bonds followed by laminarin, glucan and cellulose. Therefore, it can be concluded that the characterization of ß-1,6-glucanase secreted by T. longibrachiatum in term of molecular weight, responsed to selected physicochemical factors and the substrate specificity are approximately identical to other Trichoderma sp.
An antimicrobial (AM) Active Packaging can be made by incorporating and immobilizing suitable AM agents into food package matrices and applying a bio switch concept. A starchbased film was prepared and incorporated with an antimicrobial agent, i.e. lysozyme with EDTA as a chelating agent. This film was then inoculated with the bacteria Escherichia coli and Bacillus subtilis to carry out the microbial contamination study. The inhibition of both E. coli and B. subtilis by the AM film was clearly observed as a clear zone formation in the culture agar test. The film appearance showed that lysozymes could give a better inhibition to the growth of E. coli and to B. subtilis, at a satisfying inhibition rate. From the broth test, the decreased in the optical densities were found to be 65.83% and 91.30%, suggesting an effective growth inhibition of E. coli and B. subtilis, respectively. Physically, the film which was incorporated with lysozymes was found to be slightly different from the control film. The moisture content of the film, with lysozymes, was found to be below 10.5%, as compared to the control, after 24 hours of formation in the storage at ambient temperature.
This study was carried out to observe thermotolerance ability of Acanthamoeba spp. A total of 32 Acanthamoeba spp. isolates obtained from water taps, sinks, swimming pools and sea water were used. Trophozoites of Acanthamoeba spp. were inoculated onto non-nutrient agar (NNA) seeded with heat-killed Escherichia coli using aseptic technique and incubated for 14 days at 30°C to obtain the cyst. The cysts were subcultured onto new agar plates for thermotolerance test at 37°C and 42°C. The plates were observed until 96 hours after incubation for excystation of Acanthamoeba before being declared negative. Overall, 81.25% of samples were able to excyst at 37°C while 37.5% were able to excyst at 42°C. Thermotolerant Acanthamoeba is associated with high pathogenicity potential.
Climate change is a product of human actions. The extreme events such as flash floods, droughts, heat waves, earthquakes, volcano eruptions and tsunamis seen in the world today are the result of indiscriminate human intrusion into the environment. Vulnerable countries and populations are the most affected by these climatic events. This places a burden on the resources of these countries. The Kyoto Protocol is a milestone in environmental management and the impetus created by it must be maintained by carrying out the much needed research into appropriate mitigating measures that will alleviate the climate
change impact globally. A paradigm shift is needed in addressing the associated risks on human health to assess socioeconomic determinants and the related impacts on disease burden. Some wealthy nations emphasize economic benefits and downplay sustainability goals, health and equality. However the rising cost of energy is beginning to influence their outlook towards this issue. The implications on economics, human health and wellbeing are implicit. In order to strike a balance between disadvantaged and privileged nations, many
international agencies are spearheading various research agenda to improve adaptation programmes on effects of changing climatic conditions on health. Malaysia too has such programmes initiated under its 5-year development plans.
Limnonectes kuhlii and Limnonectes leporinus are two of the Bornean fanged frogs (without advertisement call) which are widely distributed, thus thought to exhibit different evolutionary lineages and the existence of genetically cryptic species. Yet, the two species are still under study especially at the molecular level. Hence, cytochrome c oxidase I (COI) of mitochondrial gene was used to investigate suitable parameters for DNA amplification using the Polymerase Chain Reaction (PCR) method. Three PCR programmes (varied in the temperatures and period of each PCR step) were employed to identify the most efficient parameters in amplifying PCR products for both species. From the three programmes, Programme B (Initial denaturation: 96°C for 5 min; denaturation: 95°C for 45 sec; annealing: 48-53°C for 1 min 30 sec; extension: 72°C for 1 min 30 sec; final extension: 72°C for 10 min, 30 cycles) showed the highest percentage (53%) of optimal PCR products. The other two programmes showed non-specific products or “primer-dimers”. The results also suggest that the annealing temperature of 52°C, 0.025-0.05 units/µl of 1.5mM Taq polymerase, 0.04 mM of
dNTPs mix and optimal concentrations of magnesium in 50 µl of reaction mixture were sufficient enough to amplify high quality PCR products for both species. However, using Programme B, the re-amplification of the PCR products yielded “primer-dimer”. In addition, a ‘Hot-Start’ PCR method was also applied and mostly yielded in an optimal PCR amplification. Nevertheless, further research on the second amplification of the two species should be conducted to determine the causes of the primer-dimer production.
HCFCs, in addition to destroying the ozone layer, have been recognized as a contributing factor that increases global warming. It is widely used as working fluid in window air-conditioning system, where capillary tube serves as an expansion device. Literature reports have shown that no single refrigerant can solve the problem of ozone layer depletion and global warming. Refrigerant HC290/HC600a/HFC407C mixture, an eco-friendly refrigerant, has been recognized as an alternative to HCFC22. The objective of this study is to, for cost effectiveness, develop an empirical correlation to predict the refrigerant HC290/ HC600a/HFC407C mixture mass flow rate using statistical experimental design approach. A review of relevant literature shows that refrigerant’s mass flow rate depends on condensing temperature, degree of subcooling, inner diameter and length of capillary tube. The relationship between the mass flow rate and the four independent variables was established as an empirical mathematical correlation using central composite design (CCD), a response surface methodology (RSM). This empirical correlation was examined using analysis of variance (ANOVA) of 5% level of significance. The results of these analysis showed that the correlation fitted well with the experimental data yielding an average and standard deviation of 1.05% and 2.62%, respectively. The validity of the present correlation was further assessed by comparing it with published empirical correlation in literature and the result showed that the present correlation is consistent.
Dicofol, an extensively used organochlorine pesticide and a recommended Stockholm convention persistent organic pollutant (POP) candidate is well known for its endocrine disruptive properties. The sonochemical degradation of Dicofol in aqueous media has been investigated using a 20-kHz probe type sonicator with power inputs from 150 to 450 W. The degradation rate was determined as a function of concentration of Dicofol, solution pH, bulk phase temperature, ultrasonic power density and H2O2 addition. At optimum operating conditions, the pseudo-first-order degradation rate constant (k) was determined to be 0.032 min-1 and the extent of degradation was found to be 86% within 60 min of ultrasound treatment. High performance liquid chromatography (HPLC) and Gas chromatography coupled with mass spectroscopy (GC-MS) analysis indicated the presence of degraded products. The obtained results of Dicofol degradation and control experiments in the presence of H2O2 and radical scavenger test suggest thermal decomposition along with radical attack at bubble-vapor interface to be the dominant degradation pathway. Sonochemical treatment is effective and promising for successful removal of harmful pesticides such as Dicofol and superior removal efficiency for other POPs is expected in the near future with the successful implementation of ultrasound-based wastewater treatment.
Identifying the consequences of tropical forest degradation is essential to mitigate its effects upon forest fauna. Large forest-dwelling mammals are often highly sensitive to environmental perturbation through processes such as fragmentation, simplification of habitat structure, and abiotic changes including increased temperatures where the canopy is cleared. Whilst previous work has focused upon species richness and rarity in logged forest, few look at spatial and temporal behavioural responses to forest degradation. Using camera traps, we explored the relationships between diel activity, behavioural expression, habitat use and ambient temperature to understand how the wild free-ranging Bornean banteng (Bos javanicus lowi) respond to logging and regeneration. Three secondary forests in Sabah, Malaysian Borneo were studied, varying in the time since last logging (6-23 years). A combination of generalised linear mixed models and generalised linear models were constructed using >36,000 trap-nights. Temperature had no significant effect on activity, however it varied markedly between forests, with the period of intense heat shortening as forest regeneration increased over the years. Bantengs regulated activity, with a reduction during the wet season in the most degraded forest (z = -2.6, Std. Error = 0.13, p = 0.01), and reductions during midday hours in forest with limited regeneration, however after >20 years of regrowth, activity was more consistent throughout the day. Foraging and use of open canopy areas dominated the activity budget when regeneration was limited. As regeneration advanced, this was replaced by greater investment in travelling and using a closed canopy. Forest degradation modifies the ambient temperature, and positively influences flooding and habitat availability during the wet season. Retention of a mosaic of mature forest patches within commercial forests could minimise these effects and also provide refuge, which is key to heat dissipation and the prevention of thermal stress, whilst retention of degraded forest could provide forage.
The in situ polymorphic forms and thermal transitions of refined, bleached and deodorized palm oil (RBDPO), palm stearin (RBDPS) and palm kernel oil (RBDPKO) were investigated using coupled X-ray diffraction (XRD) and differential scanning calorimetry (DSC). Results indicated that the DSC onset crystallisation temperature of RBDPO was at 22.6°C, with a single reflection at 4.2Å started to appear from 23.4 to 17.1°C, and were followed by two prominent exothermic peaks at 20.1°C and 8.5°C respectively. Further cooling to -40°C leads to the further formation of a β'polymorph. Upon heating, a of β'→βtransformation was observed between 32.1 to 40.8°C, before the sample was completely melted at 43.0°C. The crystallization onset temperature of RBDPS was 44.1°C, with the appearance of the α polymorph at the same temperature as the appearance of the first sharp DSC exothermic peak. This quickly changed from α→β´ in the range 25 to 21.7°C, along with the formation of a small β peak at -40°C. Upon heating, a small XRD peak for the β polymorph was observed between 32.2 to 36.0°C, becoming a mixture of (β´+ β) between 44.0 to 52.5°C. Only the β polymorph survived further heating to 59.8°C. For RBDPKO, the crystallization onset temperature was 11.6°C, with the formation of a single sharp exothermic peak at 6.5°C corresponding to the β' polymorphic form until the temperature reached -40°C. No transformation of the polymorphic form was observed during the melting process of RBDPKO, before being completely melted at 33.2°C. This work has demonstrated the detailed dynamics of polymorphic transformations of PKO and PS, two commercially important hardstocks used widely by industry and will contribute to a greater understanding of their crystallization and melting dynamics.
A new cloud point methodology was successfully used for the extraction of carcinogenic pesticides in milk samples as a prior step to their determination by spectrophotometry. In this work, non-ionic silicone surfactant, also known as 3-(3-hydroxypropyl-heptatrimethylxyloxane), was chosen as a green extraction solvent because of its structure and properties. The effect of different parameters, such as the type of surfactant, concentration and volume of surfactant, pH, salt, temperature, incubation time and water content on the cloud point extraction of carcinogenic pesticides such as atrazine and propazine, was studied in detail and a set of optimum conditions was established. A good correlation coefficient (R2 ) in the range of 0.991-0.997 for all calibration curves was obtained. The limit of detection was 1.06 µg l-1 (atrazine) and 1.22 µg l-1 (propazine), and the limit of quantitation was 3.54 µg l-1 (atrazine) and 4.07 µg l-1 (propazine). Satisfactory recoveries in the range of 81-108% were determined in milk samples at 5 and 1000 µg l-1, respectively, with low relative standard deviation, n = 3 of 0.301-7.45% in milk matrices. The proposed method is very convenient, rapid, cost-effective and environmentally friendly for food analysis.
Kenaf seed oil has been suggested to be used as nutritious edible oil due to its unique fatty acid composition and nutritional value. The objective of this study was to optimize the bleaching parameters of the chemical refining process for kenaf seed oil, namely concentration of bleaching earth (0.5 to 2.5% w/w), temperature (30 to 110 °C) and time (5 to 65 min) based on the responses of total oxidation value (TOTOX) and color reduction using response surface methodology. The results indicated that the corresponding response surface models were highly statistical significant (P < 0.0001) and sufficient to describe and predict TOTOX value and color reduction with R2 of 0.9713 and 0.9388, respectively. The optimal parameters in the bleaching stage of kenaf seed oil were: 1.5% w/w of the concentration of bleaching earth, temperature of 70 °C, and time of 40 min. These optimum parameters produced bleached kenaf seed oil with TOTOX value of 8.09 and color reduction of 32.95%. There were no significant differences (P > 0.05) between experimental and predicted values, indicating the adequacy of the fitted models.
In vivo and in vitro depolymerizations of intracellular medium-chain-length poly-3-hydroxyalkanoates (mcl-PHA) in Pseudomonas putida Bet001 grown on lauric acid was studied. Both processes were studied under optimum conditions for mcl-PHA depolymerization viz. 0.2 M Tris-HCl buffer, pH 9, ionic strength (I) = 0.2 M at 30°C. For in vitro depolymerization studies, cell-free system was obtained from lysing bacterial cells suspension by ultrasonication at optimum conditions (frequency 37 kHz, 30% of power output, <25°C for 120 min). The comparison between in vivo and in vitro depolymerizations of intracellular mcl-PHA was made. In vitro depolymerization showed lower depolymerization rate but higher yield compared to in vivo depolymerization. The monomer liberation rate reflected the mol% distribution of the initial polymer subunit composition, and the resulting direct individual products of depolymerization were identical for both in vivo and in vitro processes. It points to exo-type reaction for both processes, and potential biological route to chiral molecules.
Uncontrolled disposal of feathers from the poultry industry and slaughterhouses is environmentally undesirable. The feathers are composed of approximately 90% of keratin which is an important ingredient of cosmetics, shampoos and hair treatment creams. This study aimed to determine the optimum conditions for the extraction of keratin from chicken feathers. The extraction of keratin using various reducing agents was studied using statistical experimental design. In the extraction process, pH, temperature, ratio of reducing agents, mass of chicken feathers and incubation time were analyzed. The keratin in the total extracted protein was purified by size exclusion chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and further characterized using amino acids profile analysis. The surface morphology and chemical composition were studied by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR) analysis. Sodium sulfide (Na2S) yielded 84.5% of keratin as compared to sodium hydroxide (43.8), urea mixture (50.6), mixture of sodium dodecyl sulfate (SDS) and sodium bisulfite (18.3) and a mixture of Na2S and sodium hydroxide (41.5%) under optimized conditions. The optimum yield of keratin was achieved at 80.9 °C in 9.5 h with 0.05 M sodium sulfide using response surface methodology (RSM). Among the five parameters screened, pH was found not to be significant because the p value was greater than 0.05.