A SIMPLE METHOD OF DNA EXTRACTION OF MYCOBACTERIUM TUBERCULOSIS FROM SPUTUM CULTURES FOR SEQUENCING ANALYSIS.

BACKGROUND: Concern has been raised about DNA extraction from Mycobacterium tuberculosis due to its complex procedure. This study demonstrates a simple and fast DNA extraction method of mycobacterial genome to subsequent molecular investigation, such as Polymerization Chain Reaction (PCR) amplification, with species-specific primers and sequencing. MATERIALS AND METHODS: Total DNA was isolated from M. tuberculosis cultured by using boil method. DNA was evaluated via measures of DNA quantity and quality (absorbance at 230, 260 and 280 nm), DNA integrity (electrophoresis). Molecular tests were tested namely PCR and sequencing. CONCLUSIONS: The quality of DNA obtained is acceptable for PCR and sequencing analysis. These findings demonstrate that the method used is inexpensive and suitable for minimum infrastructure facilities.

Laccase oxidation and removal of toxicants released during combustion processes.

This study reports for the first time the ability of laccases adsorbed on cellulose acetate to eliminate toxicants released during combustion processes. Laccases directly oxidized and eliminated more than 40% w/v of 14 mM of 1,4-dihydroxybenzene (hydroquinone); 2-methyl-1,4-benzenediol (methylhydroquinone); 1,4-dihydroxy-2,3,5-trimethylbenzene (trimethylhydroquinone); 3-methylphenol (m-cresol); 4-methylphenol (p-cresol); 2-methylphenol (o-cresol); 1,3-benzenediol (resorcinol); 1,2-dihydroxybenzene (catechol); 3,4-dihydroxytoluene (4-methylcatechol) and 2-naphthylamine. Further, laccase oxidized 2-naphthylamine, hydroquinone, catechol, methylhydroquinone and methylcatechol were also able to in turn mediate the elimination of >90% w/v of toxicants which are per-se non-laccase substrates such as 3-aminobiphenyl; 4-aminobiphenyl; benz[a]anthracene; 3-(1-nitrosopyrrolidin-2-yl) pyridine (NNN); formaldehyde; 4-(methyl-nitrosamino-1-(3-pyridyl)-1-butanone (NNK); 2-butenal (crotonaldehyde); nitric oxide and vinyl cyanide (acrylonitrile). These studies demonstrate the potential of laccase immobilized on solid supports to remove many structurally different toxicants released during combustion processes. This system has great potential application for in situ removal of toxicants in the manufacturing, food processing and food service industries.

Synthesis of multifunctional bioresponsive polymers for the management of chronic wounds.

Novel multifunctional bioresponsive gelatin and alginate based hydrogels with in-built antioxidant regenerating system and antimicrobial properties were successfully synthesized. These hydrogels are based on the versatile reactions catalyzed by cellobiose dehydrogenase (CDH). CDH uses cellobiose and cello-oligosacharides as electron donors to reduce oxidized phenolic antioxidants, quinones, or molecular oxygen to H2O2 (a well-known antimicrobial agent). The antioxidant regenerating system consisting of CDH and cellobiose increased the ability of catechol to quench nitric oxide (NO), superoxide (O2⁻) and hydroxyl radicals (OH•) in solution and when incorporated into hydrogels. The CDH loaded into the hydrogels free of oxidized phenolic antioxidants and quinones reduced molecular to H2O2 resulting in the complete inhibition of the growth of Stapylococcus aeureus, Bacillus subtilis, Pseudomonas putida, Escherichia coli and Cellulomonasmicrobium cellulans. This study therefore presents a new concept for synthesizing multifunctional bioresponsive chronic wound dressing polymers with in-built continuous antioxidant system able to continuously quench [reactive oxygen species (ROS) and reactive nitrogen species (RNOS)], and antimicrobial properties able to prevent microbial colonization of wound.

An antioxidant regenerating system for continuous quenching of free radicals in chronic wounds.

A novel antioxidant regenerating system consisting of cellobiose dehydrogenase (CDH), cellobiose, and phenolic antioxidants with potential application for continuous quenching of free radical species in chronic wounds was developed. This antioxidant regenerating system, continuously quenched in situ produced ·NO, O(2)(·-) and OH· radicals and the produced oxidized phenolic antioxidants were regenerated back to their original parent compounds by CDH using cellobiose as electron donor. This system therefore prevented the accumulation of oxidized phenolic antioxidants. Interestingly, this study also challenges the relevance of using total antioxidant capacities values of plant crude extracts obtained using biologically none relevant radical species like (2,2-diphenyl-1-picrylhydrazyl (DPPH)), Trolox Equivalent Antioxidant Capacity (TEAC), etc. when applied as medicinal remedies. This is because methoxylated phenolic antioxidants like sinapic acid, ferulic acid; 2,6-dimethoxyphenol readily donate their electrons to these radicals (DPPH, TEAC, etc.), thereby greatly influencing the total antioxidant values although this study showed that they are not at all effective in quenching O(2)(·-) radicals and again are not the most effective quenchers of NO and OH radicals as demonstrated during this study.

Cellulose oxidation and bleaching processes based on recombinant Myriococcum thermophilum cellobiose dehydrogenase.

Myriococcum thermophilum cellobiose dehydrogenase (MtCDH) was expressed in Pichia pastoris using the pPICZαA expression vector under the control of methanol inducible AOX promoter. The purified recombinant MtCDH with a specific activity of 3.1 Umg(-1) was characterized to obtain kinetic constants for various carbohydrate substrates. Additionally, the C1 oxidation of the reducing ends of cellobiose, cellotetraose and maltotriose by MtCDH was verified by HPLC-MS. MtCDH was employed to oxidize several different cellulose-based materials by production of hydrogen peroxide. Based on the obtained results a one-pot enzymatic scouring/bleaching process for cotton fabrics was developed using pectinases as scouring agent and MtCDH to produce H(2)O(2) for bleaching. An average increase in whiteness (Berger) ΔE of 26 and an average 95% increase in wettability were observed in all MtCDH treated fabrics. In addition, MtCDH oxidized typical colored cotton flavonoids (morin, rutin, isoquercitrin).

Enzymatic synthesis of lignin-siloxane hybrid functional polymers.

This study combines the properties of siloxanes and lignin polymers to produce hybrid functional polymers that can be used as adhesives, coating materials, and/or multifunctionalized thin-coating films. Lignin-silica hybrid copolymers were synthesized by using a sol-gel process. Laccases from Trametes hirsuta were used to oxidize lignosulphonates to enhance their reactivity towards siloxanes and then were incorporated into siloxane precursors undergoing a sol-gel process. In vitro copolymerization studies using pure lignin monomers with aminosilanes or ethoxytrimethylsilane and analysis by ²9Si NMR spectroscopy revealed hybrid products. Except for kraft lignin, an increase in lignin concentration positively affected the tensile strength in all samples. Similarly, the viscosity generally increased in all samples with increasing lignin concentration and also affected the curing time.

Cellular and plasma antioxidant activity assay using tetramethoxy azobismethylene quinone.

The kinetics of the reduction of enzymatically generated tetramethoxy azobismethylene quinone (TMAMQ), a newly developed antioxidant activity assay method, by pure cellular and plasma antioxidants was studied. Further, the potential application of TMAMQ to the estimation of the antioxidant activity of clinical serum samples was investigated. The highest reduction rate (k) was obtained with ascorbic acid (1.11x10(-2)microM(-1) s(-1)) and glutathione showed the lowest (2.94x10(-5)microM(-1) s(-1)). Comparing TMAMQ and the commercially available antioxidant method Total Antioxidant Capacity clearly shows a similar trend, although the values differ. This study also shows that TMAMQ is highly sensitive (only a minute plasma sample was required) and reproducible, and the reaction proceeds until steady state (until all antioxidants have reacted). TMAMQ is very stable in acetonitrile (>3months), making it a highly flexible method because it can be easily adapted for analysis of just a single sample or for high-throughput analysis. This has direct implications on reducing costs and experimental steps. TMAMQ is therefore a highly promising antioxidant activity assay method for cellular and plasma antioxidant activity assay.

Reactivity of long chain alkylamines to lignin moieties: implications on hydrophobicity of lignocellulose materials.

Enzymatic processes provide new perspectives for modification of lignocellulose materials. In the current study, laccase catalyzed coupling of long chain alkylamines to lignin model molecules and lignocellulose was investigated. Up to two molecules of dodecylamine (DA) and dihexylamine (DHA) were successfully coupled with lignin monomers (guaiacol, catechol and ferulic acid) while coupling onto complex lignin model compounds (syringylglycerol beta-guaiacyl ether, guaiacylglycerol beta-guaiacyl ether and dibenzodioxocin) yielded 1:1 coupling products. Surface analysis of beech veneers enzymatically grafted with DA showed an increase in nitrogen content of 3.18% compared to 0.71% in laccase only treated controls while the O/C ratio decreased from 0.52 to 0.46. Concomitantly the grafting of DHA or DA onto beech veneers resulted in a 53.8% and 84.2% increase in hydrophobicity, respectively when compared to simple adsorption. Therefore, laccase-mediated grafting of long chain alkylamines onto lignocellulose materials can be potentially exploited for improving their hydrophobicity.

Laccase catalyzed covalent coupling of fluorophenols increases lignocellulose surface hydrophobicity.

This work presents for the first time the mechanistic evidence of a laccase-catalyzed method of covalently grafting hydrophobicity enhancing fluorophenols onto Fagus sylvatica veneers. Coupling of fluorophenols onto complex lignin model compounds guaiacylglycerol beta-guaiacyl ether and syringylglycerol beta-guaiacyl ether was demonstrated by LC-MS and NMR. Laccase-mediated coupling increased binding of 4-[4-(trifluoromethyl)phenoxy]phenol (4,4-F3MPP) and 4-(trifluoromethoxy)phenol (4-F3MP) to veneers by 77.1% and 39.2%, respectively. XPS studies showed that laccase-catalyzed grafting of fluorophenols resulted in a fluorine content of 6.39% for 4,4-F3MPP, 3.01% for 4-F3MP and 0.26% for 4-fluoro-2-methylphenol (4,2-FMP). Grafting of the fluorophenols 4,2-FMP, 4-F3MP and 4,4-F3MPP led to a 9.6%, 28.6% and 65.5% increase in hydrophobicity, respectively, when compared to treatments with the respective fluorophenols in the absence of laccase, in good agreement with XPS data.