Oscar F. Sánchez

Degradation of chlorophenols by sequential biological-advanced oxidative process using Trametes pubescens and TiO2/UV

The degradation of 2-chlorophenol (2-CP), 2,4-dichlorophenol (2,4-DCP), 2,4,6-trichlorophenol (2,4,6-TCP) and pentachlorophenol (PCP) via biological, advanced oxidative process (AOP) and sequential biological-AOP was investigated in this work. The white-rot fungus Trametes pubescens was used for the biodegradation of chlorophenols, while in AOP TiO(2)/UV was used. In the biological degradation, the effect of glucose as a cofactor was also evaluated. The highest degradations were obtained when the reaction medium was supplemented with glucose, ranging from 94.6% to 37.8%, with degradation activity for 2-CP>2,4-DCP>PCP>2,4,6-TCP. During the AOP the removal initial rate increased in the following order 2-CP>2,4,6-TCP>2,4-DCP>PCP, and the obtained degradation range from 82.0% to 24.0%. When biological removal process, supplemented with glucose, was followed for an AOP process, 100% degradation was obtained for all the chlorophenols tested. These results suggest that the white-rot fungi T. pubescens could be used for the degradation of xenobiotic compounds, and its use with an advanced oxidative process, in a sequential mode, may be considered to obtain a complete removal of them.

Evaluation of toxicity and degradation of a chlorophenol mixture by the laccase produced by Trametes pubescens

In this study, the biodegradation of a mixture of 2-chlorophenol (2-CP), 2,4-dichlorophenol (2,4-DCP), 2,4,6-trichlorophenol (2,4,6-TCP) and pentachlorophenol (PCP) using the laccase produced by the white-rot fungus Trametes pubescens CBS 696.94 was evaluated. Two laccase isoenzymes with molecular weights of about 60 and 120 kDa were identified in the enzymatic crude extract. The highest laccase activity with syringaldazine was observed with pH 6.0 and 60°C, while with 2,2-azino-bis(3-ethylbenzothiazoline-6) sulphonic acid the highest activity was observed between 50 and 60°C and 3.0-4.0 pH. A biodegradation of 100%, 99%, 82.1% and 41.1% for 2-CP, 2,4-DCP, 2,4,6-TCP and PCP, respectively, was observed after 4h of reaction. The reduction in chlorophenols concentration allowed 90% reduction in mixture toxicity. In summary, these results show the feasibility of a laccase enzymatic crude extract from T. pubescens for the reduction of concentration and toxicity of chlorophenols.

Impact of enzyme replacement therapy and hematopoietic stem cell transplantation in patients with Morquio A syndrome

Patients with mucopolysaccharidosis IVA (MPS IVA) can present with systemic skeletal dysplasia, leading to a need for multiple orthopedic surgical procedures, and often become wheelchair bound in their teenage years. Studies on patients with MPS IVA treated by enzyme replacement therapy (ERT) showed a sharp reduction on urinary keratan sulfate, but only modest improvement based on a 6-minute walk test and no significant improvement on a 3-minute climb-up test and lung function test compared with the placebo group, at least in the short-term. Surgical remnants from ERT-treated patients did not show reduction of storage materials in chondrocytes. The impact of ERT on bone lesions in patients with MPS IVA remains limited. ERT seems to be enhanced in a mouse model of MPS IVA by a novel form of the enzyme tagged with a bone-targeting moiety. The tagged enzyme remained in the circulation much longer than untagged native enzyme and was delivered to and retained in bone. Three-month-old MPS IVA mice treated with 23 weekly infusions of tagged enzyme showed marked clearance of the storage materials in bone, bone marrow, and heart valves. When treatment was initiated at birth, reduction of storage materials in tissues was even greater. These findings indicate that specific targeting of the enzyme to bone at an early stage may improve efficacy of ERT for MPS IVA. Recombinant N-acetylgalactosamine-6-sulfate sulfatase (GALNS) in Escherichia coli BL21 (DE3) (erGALNS) and in the methylotrophic yeast Pichia pastoris (prGALNS) has been produced as an alternative to the conventional production in Chinese hamster ovary cells. Recombinant GALNS produced in microorganisms may help to reduce the high cost of ERT and the introduction of modifications to enhance targeting. Although only a limited number of patients with MPS IVA have been treated with hematopoietic stem cell transplantation (HSCT), beneficial effects have been reported. A wheelchair-bound patient with a severe form of MPS IVA was treated with HSCT at 15 years of age and followed up for 10 years. Radiographs showed that the figures of major and minor trochanter appeared. Loud snoring and apnea disappeared. In all, 1 year after bone marrow transplantation, bone mineral density at L2–L4 was increased from 0.372 g/cm2 to 0.548 g/cm2 and was maintained at a level of 0.48±0.054 for the following 9 years. Pulmonary vital capacity increased approximately 20% from a baseline of 1.08 L to around 1.31 L over the first 2 years and was maintained thereafter. Activity of daily living was improved similar to the normal control group. After bilateral osteotomies, a patient can walk over 400 m using hip–knee–ankle–foot orthoses. This long-term observation of a patient shows that this treatment can produce clinical improvements although bone deformity remained unchanged. In conclusion, ERT is a therapeutic option for MPS IVA patients, and there are some indications that HSCT may be an alternative to treat this disease. However, as neither seems to be a curative therapy, at least for the skeletal dysplasia in MPS IVA patients, new approaches are investigated to enhance efficacy and reduce costs to benefit MPS IVA patients.

Production of Trametes pubescens Laccase under Submerged and Semi-Solid Culture Conditions on Agro-Industrial Wastes

Laccases are copper-containing enzymes involved in the degradation of lignocellulosic materials and used in the treatment of phenol-containing wastewater. In this study we investigated the effect of culture conditions, i.e. submerged or semi-solid, and copper supplementation on laccase production by Trametes pubescens grown on coffee husk, soybean pod husk, or cedar sawdust. The highest specific laccase activity was achieved when the culture was conducted under submerged conditions supplemented with copper (5 mM), and using coffee husk as substrate. The crude extracts presented two laccase isoforms with molecular mass of 120 (Lac1) and 60 kDa (Lac2). Regardless of the substrate, enzymatic crude extract and purified fractions behaved similarly at different temperatures and pHs, most of them presented the maximum activity at 55 °C and a pH range between 2 and 3. In addition, they showed similar stability and electro-chemical properties. At optimal culture conditions laccase activity was 7.69±0.28 U mg-1 of protein for the crude extract, and 0.08±0.001 and 2.86±0.05 U mg-1 of protein for Lac1 and Lac2, respectively. In summary, these results show the potential of coffee husk as an important and economical growth medium to produce laccase, offering a new alternative use for this common agro-industrial byproduct.

Delignification Process of Agro-Industrial Wastes an Alternative to Obtain Fermentable Carbohydrates for Producing Fuel

Fossil fuels, mainly petroleum, coal, and natural gas, were the main energy sources for most industries during the 20th century, and are still the most important feedstocks to produce energy in the world. “Currently, the world energy market worth around 1.5 trillion dollars is dominated by fossil fuels” (Goldemberg, 2006). However, these sources are not longer regarded sustainable, and their availability is much lower. Shafiee and Topal (2009) predicted that oil, coal and gas reserves will last around 35, 107 and 37 years for petroleum, coal and natural gas, respectively. In addition their combustion involve environmental issues such as global warming due to greenhouse gas emissions (Naik et al., 2010). Therefore, the interest for questing sustainable and environmental energy sources has risen in the last two decades; giving origin to the production of fuels from renewable feedstocks, such as biomass. These feedstocks are commonly divided in three categories: wood, residues from agricultural, industrial or domestic origin, and energy crops from dedicated farming (Bringezu et al., 2007). The use of these renewable resources to produce fuel has created two different generations in biofuel production. The first biofuel generation is based on grain or food sources, and is constituted mainly by ethanol, fatty acid methyl ester (FAME), and pure plant oil (PPO) (Bringezu et al., 2007). However, environmental impacts, energy efficiency, and eutrophycation have limited the production of these first generation biofuels. In addition, land competition of energy crops with food crops has arisen a fuel-versus-food debate exacerbated by the increase of food prices, particularly those of maize, wheat, sugar beet, cassava, sweet sorghum, sugarcane, oilseed rape, soybean and oil palm (Borjesson & Tufvesson, 2011; Vries et al., 2010). For example, the United States Department of Energy reported that in 2005 the bioethanol production reached about 15 billion litters for which nearly 36 MMt of maize, 13% of total US maize crops, were used. These values were estimated to double in 2010 by the Renewable Fuel Association (Cassman & Liska, 2007). The second generation of biofuels is produced from non-grain and non-food sources such as lignocellulosic sources and algae biomass (Naik et al., 2010; Simmons et al., 2008).

Recent Trends in Fructooligosaccharides Production

Prebiotics are food ingredients that promote host health beneficially due to their effect over the growth and activity of probiotic bacterial species. Prebiotic properties have been demonstrated for inulin-type fructans, galactoolicosaccharides and lactulose. Fructooligosaccharides (FOS), considered as inulin-type fructans, represent an important source of prebiotic compounds that are widely used as an ingredient in functional foods. FOS are produced by the action of fructosyltransferase from many plants, fungi and bacteria, and they are mainly composed of 1-kestose, nystose, and 1-beta-fructofuranosyl nystose. Among them, 1-kestose has better therapeutic properties than those with a high polymeric degree (GF(n > 4)). FOS exhibited properties than those with a high polymeric degree (GF(n>4)). FOS exhibited properties such as low caloric values, non-cariogenic properties, decrease levels of lipids and cholesterol, help gut absorption of ions, and stimulate the bifidobacteria growth in the human colon. This review presents a summary of the patents related with FOS production by industrial sucrose biotransformation or the use of recombinant fructosyltransferase enzymes. Also, a brief description of recent FOS applications will be discussed.

Computational analysis of the fructosyltransferase enzymes in plants, fungi and bacteria.

Fructosyltransferases (FTases) are enzymes produced by plants, fungi, and bacteria, which are responsible for the synthesis of fructooligosaccharides. In this study, we conducted a computational analysis of reported sequences for FTase from a diverse source of organisms, such as plants, fungi, and bacteria. Ninety-one proteins sequences were obtained; all belonging to the glycoside hydrolase 32 (GH32) and 68 (GH68) families. The sequences were grouped in seven clades, five for plants, one for fungi, and one for bacteria. Our findings suggest that FTases from fungi and bacteria likely evolved from dicotyledonous FTases. The analysis of catalytic domains A, D and E, which contain the amino acids involved in the catalytic binding site, allowed the identification of clade-specific conserved characteristics. The analysis of sequence motifs involved in donor/acceptor molecule affinity showed that additional sequences could be responsible for donor/acceptor molecule affinity. The correlation of this large set of FTases allowed to identify additional features that might be used for the identification and classification of new FTases, and to improve the understanding of these valuable enzymes.

Human recombinant lysosomal enzymes produced in microorganisms

Lysosomal storage diseases (LSDs) are caused by accumulation of partially degraded substrates within the lysosome, as a result of a function loss of a lysosomal protein. Recombinant lysosomal proteins are usually produced in mammalian cells, based on their capacity to carry out post-translational modifications similar to those observed in human native proteins. However, during the last years, a growing number of studies have shown the possibility to produce active forms of lysosomal proteins in other expression systems, such as plants and microorganisms. In this paper, we review the production and characterization of human lysosomal proteins, deficient in several LSDs, which have been produced in microorganisms. For this purpose, Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, and Ogataea minuta have been used as expression systems. The recombinant lysosomal proteins expressed in these hosts have shown similar substrate specificities, and temperature and pH stability profiles to those produced in mammalian cells. In addition, pre-clinical results have shown that recombinant lysosomal enzymes produced in microorganisms can be taken-up by cells and reduce the substrate accumulated within the lysosome. Recently, metabolic engineering in yeasts has allowed the production of lysosomal enzymes with tailored N-glycosylations, while progresses in E. coli N-glycosylations offer a potential platform to improve the production of these recombinant lysosomal enzymes. In summary, microorganisms represent convenient platform for the production of recombinant lysosomal proteins for biochemical and physicochemical characterization, as well as for the development of ERT for LSD.

Native and heterologous production of bacteriocins from gram-positive microorganisms.

In nature, microorganisms can present several mechanisms for setting intercommunication and defense. One of these mechanisms is related to the production of bacteriocins, which are peptides with antimicrobial activity. Bacteriocins can be found in Gram-positive and Gram-negative bacteria. Nevertheless, bacteriocins produced by Gram-positive bacteria are of particular interest due to the industrial use of several strains that belong to this group, especially lactic acid bacteria (LAB), which have the status of generally recognized as safe (GRAS) microorganisms. In this work, we will review recent tendencies in the field of invention and state of art related to bacteriocin production by Gram-positive microorganism. Hundred-eight patents related to Gram-positive bacteriocin producers have been disclosed since 1965, from which 57% are related bacteriocins derived from Lactococcus, Lactobacillus, Streptococcus, and Pediococcus strains. Surprisingly, patents regarding heterologous bacteriocins production were mainly presented just in the last decade. Although the major application of bacteriocins is concerned to food industry to control spoilage and foodborne bacteria, during the last years bacteriocin applications have been displacing to the diagnosis and treatment of cancer, and plant disease resistance and growth promotion.

A sensitive protein-based sensor for quantifying histone acetylation levels.

H3K14ac (acetylation of lysine 14 of histone H3) is one of the most important epigenetic modifications in cells. Aberrant changes in H3K14ac are commonly found in various types of cancers and neurological disorders. Current detection approaches for histone modifications, however, require either tedious sample pre-treatments or lack the quantitative accuracy required for biochemical and biomedical applications. In this study, we engineered a protein sensor using the amino acid sequences derived from the bromodomain of human polybromo-1 (PB1). The protein sensor was conjugated to a fluorescent dye for sensitive detection of H3K14ac. Different detection conditions, such as additive concentrations and probe concentrations, were optimally selected by balancing signal strength (I(Rel)) and signal-to-noise ratio (SNR). The protein sensor was verified using histone H3 peptides containing different H3K14 acetylation levels. The detection signal was found to be linearly dependent on acetylation levels of H3K14 ranging from 5% to 100%. The designed platform can be used for screening epigienetic drugs regulating H3K14 acetylation levels as well as monitoring H3K14 acetylation level of circulating nucleosomes for disease progression.