Photocatalytic and Antibacterial Studies on Poly(Hydroxybutyrate-co-Hydroxyhexanoate) / Titanium Dioxide Composites

Bacteria and viruses causes food poisoning outbreaks. To prevent this, antimicrobial films can be used as packaging material or coatings on food processing surfaces. Titanium dioxide (TiO2) irradiated with ultraviolet light produces free radicals that can destroy organic contaminants and bacteria. U.S. Food and Drug Administration and the U.S. Pharmacopeia approves TiO2 as a colorant. Polyhydroxybutyrate is a bio-based polymer. Blends of this polymer are being studied for implants and drug delivery. TiO2 immobilized to blends of this polymer may be suitable in food processing. Poly(hydroxybutyrate-co-hydroxyhexanoate) and titanium oxide (PHB-HH/TiO2) composite films were irradiated under fluorescent and blacklight lamps. The results show that they can be activated by both lamps. However, the photocatalytic activity is higher in blacklight. The films were irradiated in the presence of Escherichia coli and Staphylococcus aureus. Both had a 0 log count when a 3% PHB-HH/TiO2 composite film was exposed to blacklight for 5 h. Exposure to fluorescent light showed some antibacterial activity. The photocatalytic activity of the films was enough to inhibit bacterial growth when exposed to fluorescent lamps. PHB-HH/TiO2 composite films have photocatalytic and antibacterial properties when exposed to fluorescent and blacklight lamps. The films can be used in the food industry

Optimized Conditions for the Production of Hydroxamic Fatty Acids from Coconut Oil by a Lipase from Rhizopus delemar

Alipase from Rhizopus delemar was used as a biocatalyst for the synthesis of hydroxamic fatty acids (HFA) by reacting hydroxylamine with coconut oil, methyl laurate and lauric acid. The effects of various reaction parameters such as amount of hydroxylamine-HCl, incubation time, temperature, and amount of enzyme were evaluated to achieve high product yield.The reaction products were monitored by Thin Layer Chromatography using hexane: diethyl ether: acetic acid (20:80:1 v/v/v) as solvent system. Identification of the products was done by Gas-Liquid Chromatography. Analysis revealed a mixture of HFA produced using coconut oil as substrate: 30.33% myristylhydroxamic acid (MHA), 24.17% olelyhydroxamic acid (OHA) and 45.50% laurylhydroxamic acid (LHA). On the other hand, methyl laurate and lauric acid yielded only LHA.Purification of the product was done by crystallization in hexane at 14 °C. Identification of the functional groups present was determined by Infrared (IR) spectroscopy

Downregulation of RWA genes in hybrid aspen affects xylan acetylation and wood saccharification

High acetylation of angiosperm wood hinders its conversion to sugars by glycoside hydrolases, subsequent ethanol fermentation and (hence) its use for biofuel production. We studied the REDUCED WALL ACETYLATION (RWA) gene family of the hardwood model Populus to evaluate its potential for improving saccharification. The family has two clades, AB and CD, containing two genes each. All four genes are expressed in developing wood but only RWA-A and -B are activated by master switches of the secondary cell wall PtNST1 and PtMYB21. Histochemical analysis of promoter:: GUS lines in hybrid aspen (Populus tremula x tremuloides) showed activation of RWA-A and -B promoters in the secondary wall formation zone, while RWA-C and -D promoter activity was diffuse. Ectopic downregulation of either clade reduced wood xylan and xyloglucan acetylation. Suppressing both clades simultaneously using the wood-specific promoter reduced wood acetylation by 25% and decreased acetylation at position 2 of Xylp in the dimethyl sulfoxide-extracted xylan. This did not affect plant growth but decreased xylose and increased glucose contents in the noncellulosic monosaccharide fraction, and increased glucose and xylose yields of wood enzymatic hydrolysis without pretreatment. Both RWA clades regulate wood xylan acetylation in aspen and are promising targets to improve wood saccharification.Peer reviewe

IMMOBILIZATION OF INVERTASE ON CARBOXYMETHYLCELLULOSE PREPARED FROM NATA DE COCO FOR THE INVERSION OF SUCROSE

Nata de coco, the cellulose produced by Acetobacter aceti with coconut water as substrate, was used as starting material in the synthesis of carboxymethylcellulose after treatment with NaOH and monochloroacetic acid. The product, referred to as carboxymethyl-“nata” (CMN), had a degree of substitution of 0.76, a higher value than those previously reported. This was used in the immobilization of invertase via ionic interaction and adsorptive forces, which produced a viscous colloidal suspension. Agar was incorporated to facilitate pellet formation. Interactions between the agar and the CMN-invertase may have resulted from ionic interactions as well as H-bonding. The immobilized enzyme retained 71% of its initial activity and exhibited optimum pH of 4.5 and an optimum temperature of 55°C. It was more sensitive to pH and temperature changes. The Michaelis constant, Km, was 107.43 mM for the immobilized enzyme, and 71.42 mM for the free enzyme. The Vmax values were 89.28 µmole min-1 and 82.64 µmole min for the free and immobilized enzyme, respectively. Statistical analyses showed that V values did not vary significantly. The higher K of the immobilized enzyme may be attributed to diffusional effects, steric hindrance and conformational modifications of the enzyme. The immobilized enzyme has potential for further applications because of its stability with storage, repeated and continuous use

IMMOBILIZATION OF INVERTASE ON CARBOXYMETHYLCELLULOSE PREPARED FROM NATA DE COCO FOR THE INVERSION OF SUCROSE

Nata de coco, the cellulose produced by Acetobacter aceti with coconut water as substrate, was used as starting material in the synthesis of carboxymethylcellulose after treatment with NaOH and monochloroacetic acid. The product, referred to as carboxymethyl-“nata” (CMN), had a degree of substitution of 0.76, a higher value than those previously reported. This was used in the immobilization of invertase via ionic interaction and adsorptive forces, which produced a viscous colloidal suspension. Agar was incorporated to facilitate pellet formation. Interactions between the agar and the CMN-invertase may have resulted from ionic interactions as well as H-bonding. The immobilized enzyme retained 71% of its initial activity and exhibited optimum pH of 4.5 and an optimum temperature of 55°C. It was more sensitive to pH and temperature changes. The Michaelis constant, Km, was 107.43 mM for the immobilized enzyme, and 71.42 mM for the free enzyme. The Vmax values were 89.28 µmole min-1 and 82.64 µmole min for the free and immobilized enzyme, respectively. Statistical analyses showed that V values did not vary significantly. The higher K of the immobilized enzyme may be attributed to diffusional effects, steric hindrance and conformational modifications of the enzyme. The immobilized enzyme has potential for further applications because of its stability with storage, repeated and continuous use

Capillary electrophoresis with laser induced fluorescence for DNA damage analysis

Due to the advancements in organic chemistry, millions of new compound are created everyday. Some of these compounds when absorbed by the body can be activated by cytochromes p450 and react with DNA. Therefore, these compounds have to be assessed for possible genotoxicity before its release to the general public. Abasic sites are excellent biomarkers to quantify damage from reactions of DNA with genotoxic compounds and/or their metabolites. Abasic sites may occur in very small amounts. Therefore, a quick and sensitive analytical method like capillary electrophoresis with laser induced fluorescence (CE-LW) is required to measure them.^ In the first part of this thesis, a method to detect abasic sites was developed using CE-LIF. In this method a fluorescent tag that reacts specifically to abasic sites is incubated DNA. Even after ethanol precipitation and extensive washing excess unreacted probe is still present with DNA. This is probably due to intercalation of the fluorescent tag with the double stranded DNA. The unreacted tagged was however separated from tag attached to the abasic site by capillary electrophoresis. Thus the tagged and untagged probe elided as 2 different peaks in the electropherogram. Using this method we were able to get a limit of detection of 20 attomoles abasic sites.^ Damage to cellular DNA results in cell death or cancer. Of the 2 consequences, it is the latter which is more deadly. Cancer results when damage occurs at specific points within the DNA. In the second part of this thesis, we developed a method to determine sequence specificity of DNA damage using CE-LIF array. This is done by determining the exact location of the abasic site which was formed by the removal of the damaged base. A 5\u27 fluoropore tagged DNA fragment of known sequence was incubated with styrene oxide. Heating the DNA fragment under optimized conditions releases the adenine and guanine adducts while leaving the unmodified guanines and adenines intact. This procedure leaves a piperidine cleavable abasic site at the point where the damaged nucleobase existed. Using correction tools we were able to measure the exact size of the fragments which helped us determine the exact location of the damaged point.

Capillary electrophoresis with laser induced fluorescence for DNA damage analysis

Due to the advancements in organic chemistry, millions of new compound are created everyday. Some of these compounds when absorbed by the body can be activated by cytochromes p450 and react with DNA. Therefore, these compounds have to be assessed for possible genotoxicity before its release to the general public. Abasic sites are excellent biomarkers to quantify damage from reactions of DNA with genotoxic compounds and/or their metabolites. Abasic sites may occur in very small amounts. Therefore, a quick and sensitive analytical method like capillary electrophoresis with laser induced fluorescence (CE-LW) is required to measure them.^ In the first part of this thesis, a method to detect abasic sites was developed using CE-LIF. In this method a fluorescent tag that reacts specifically to abasic sites is incubated DNA. Even after ethanol precipitation and extensive washing excess unreacted probe is still present with DNA. This is probably due to intercalation of the fluorescent tag with the double stranded DNA. The unreacted tagged was however separated from tag attached to the abasic site by capillary electrophoresis. Thus the tagged and untagged probe elided as 2 different peaks in the electropherogram. Using this method we were able to get a limit of detection of 20 attomoles abasic sites.^ Damage to cellular DNA results in cell death or cancer. Of the 2 consequences, it is the latter which is more deadly. Cancer results when damage occurs at specific points within the DNA. In the second part of this thesis, we developed a method to determine sequence specificity of DNA damage using CE-LIF array. This is done by determining the exact location of the abasic site which was formed by the removal of the damaged base. A 5\u27 fluoropore tagged DNA fragment of known sequence was incubated with styrene oxide. Heating the DNA fragment under optimized conditions releases the adenine and guanine adducts while leaving the unmodified guanines and adenines intact. This procedure leaves a piperidine cleavable abasic site at the point where the damaged nucleobase existed. Using correction tools we were able to measure the exact size of the fragments which helped us determine the exact location of the damaged point.

Detection of abasic sites with capillary electrophoresis with laser induced flourescence

Damaged bases and abasic sites are two of the major DNA lesions caused by free radicals. These damaged bases could further be removed by specific enzymes leaving an abasic site in the DNA making the site a very good marker for detection of specific DNA damage. Right now the most widely used method for abasic site detection is an ELISA like method in which the DNA with abasic sites is tagged with biotin, adsorbed on a surface and then attached with streptavidin-HRP. This method is susceptible to non specific binding, uses more sample than our method (150 µl vs 15 µl) and takes 1 or 2 days to get results. A more rapid method which we are developing is Capillary Electrophoresis with Laser Induced Flourescence (CE-LIF) which takes only about 5 hrs. Our method uses a Fluorescent Aldehyde Reactive Probe to tag the abasic sites. The tagged DNA was then separated by capillary electrophoresis from the excess probe that was not completely removed by ethanol precipitation. Our results show that CE-LIF could be used as an alternative to the ELISA like method of detecting abasic sites

Detection of labeled abasic sites in damaged DNA by capillary electrophoresis with laser-induced fluorescence

Removal of nucleobases from the DNA backbone leads to the formation of abasic sites. The rate of abasic site formation is significantly increased for chemically damaged nucleobases. Thus, abasic sites serve as general biomarkers for the quantification of DNA damage. Herein, we show that capillary electrophoresis with laser-induced fluorescence (CE-LIF) can be used to detect the amount of abasic sites with very high sensitivity. For proof of concept, DNA was incubated with methylmethane sulfonate (MMS) and the damaged bases were removed by incubation at 80 degrees C. The resulting abasic sites were then tagged with a fluorescent aldehyde-reactive probe (FARP). The DNA was precipitated with ethanol, and then analyzed by CE-LIF. CE-LIF and HPLC analysis shows that the fluorescently tagged DNA (DNA-FARP) had a peak area directly proportional to the amount of N-7 methyl guanines. The CE-LIF method had a detection limit of 1.2 abasic sites per 1,000,000 bases or ca. 20 attomoles of abasic sites. This provides a general method for detecting DNA damage that is not only faster but also has comparable or better sensitivity than the alternative ELISA-like method