Development of Thermophilic Tailor-Made Enzyme Mixtures for the Bioconversion of Agricultural and Forest Residues

Even though the main components of all lignocellulosic feedstocks include cellulose, hemicellulose, as well as the protective lignin matrix, there are some differences in structure, such as in hardwoods and softwoods, which may influence the degradability of the materials. Under this view, various types of biomass might require a minimal set of enzymes that has to be tailor-made. Partially defined complex mixtures that are currently commercially used are not adapted to efficiently degrade different materials, so novel enzyme mixtures have to be customized. Development of these cocktails requires better knowledge about the specific activities involved, in order to optimize hydrolysis. The role of filamentous fungus Myceliophthora thermophila and its complete enzymatic repertoire for the bioconversion of complex carbohydrates has been widely proven. In this study, four core cellulases (MtCBH7, MtCBH6, MtEG5 and MtEG7), in the presence of other four accessory enzymes (mannanase, lytic polyssacharide monooxygenase MtGH61, xylanase, MtFae1a) and β-glucosidase MtBGL3, were tested as a 9-component cocktail against one model substrate (phosphoric acid swollen cellulose) and four hydrothermally pretreated natural substrates (wheat straw as an agricultural waste, birch and spruce biomass, as forest residues). Synergistic interactions among different enzymes were determined using a suitable design of experiments methodology. The results suggest that for the hydrolysis of the pure substrate (PASC), high proportions of MtEG7 are needed for efficient yields. MtCBH7 and MtEG7 are enzymes of major importance during the hydrolysis of pretreated wheat straw, while MtCBH7 plays a crucial role in case of spruce. Cellobiohydrolases MtCBH6 and MtCBH7 act in combination and are key enzymes for the hydrolysis of the hardwood (birch). Optimum combinations were predicted from suitable statistical models which were able to further increase hydrolysis yields, suggesting that tailor-made enzyme mixtures targeted towards a particular residual biomass can help maximize hydrolysis yields. The present work demonstrates the change from ‘one cocktail for all’ to ‘tailor-made cocktails’ that are needed for the efficient saccharification of targeted feed stocks prior to the production of biobased products through the biorefinery concept

Doppler tissue imaging unmasks right ventricular function abnormalities in HIV-infected patients

Background: We sought to investigate right ventricular (RV) function with Doppler tissue imaging (DTI) in human immunodeficiency virus (HIV)-infected patients receiving highly-active antiretroviral treatment, without any heart-related symptoms. Methods: We studied 38 asymptomatic HIV patients (aged 44.5 ± 9.2 years, 22 of them men) and 25 age-matched and sex-matched controls. All subjects underwent conventional and DTI estimation of left ventricular (LV) systolic and diastolic function, measuring peak systolic and diastolic myocardial velocities at the mitral annulus (Sm, Em, Am). Two-dimensional (2-D) echocardiographic study of the right ventricle (RV) was performed from the four-chamber view, and RV end-diastolic dimensions were measured. DTI recordings from the RV free wall at the tricuspid annulus were used to determine systolic (SmRV) and diastolic function (EmRV and AmRV). Results: HIV-infected patients compared to controls exhibited significantly lower peak systolic velocities at the septal-SmIVS (7.9 ± 1.3 vs 9.1 ± 1.4 cm/s, p = 0.002) and lateral mitral annulus - SmLAT (9.8 ± 1.7 vs 11.2 ± 1.3 cm/s, p = 0.025); no difference was observed regarding conventional 2-D examination of LV systolic and diastolic function and DTI-derived Em and Am. No significant difference occurred between HIV patients and controls regarding RV end-diastolic dimensions and pulmonary artery systolic pressure. However, SmRV (13.8 ± 1.6 vs 14.9 ± 2.2 cm/s, p = 0.040), EmRV (11.6 ± 3 vs 13.5 ± 2.6 cm/s, p = 0.028) and AmRV (10.9 ± 2.5 vs 13.8 ± 4 cm/s, p = 0.003) were significantly reduced in HIV patients as compared to controls. Conclusions: DTI unmasks subtle and otherwise undetectable abnormalities of the longitudinal LV systolic function and both RV systolic and diastolic function, in asymptomatic HIV patients receiving highly-active antiretroviral treatment. (Cardiol J 2010; 17, 6: 587-593

Fine-Tuned Enzymatic Hydrolysis of Organosolv Pretreated Forest Materials for the Efficient Production of Cellobiose

Non-digestible oligosaccharides (NDOs) are likely prebiotic candidates that have been related to the prevention of intestinal infections and other disorders for both humans and animals. Lignocellulosic biomass is the largest carbon source in the biosphere, therefore cello-oligosacharides (COS), especially cellobiose, are potentially the most widely available choice of NDOs. Production of COS and cellobiose with enzymes offers numerous benefits over acid-catalyzed processes, as it is milder, environmentally friendly and produces fewer by-products. Cellobiohydrolases (CBHs) and a class of endoglucanases (EGs), namely processive EGs, are key enzymes for the production of COS, as they have higher preference toward glycosidic bonds near the end of cellulose chains and are able to release soluble products. In this work, we describe the heterologous expression and characterization of two CBHs from the filamentous fungus Thermothelomyces thermophila, as well as their synergism with proccessive EGs for cellobiose release from organosolv pretreated spruce and birch. The properties, inhibition kinetics and substrate specific activities for each enzyme are described in detail. The results show that a combination of EGs belonging to Glycosyl hydrolase families 5, 6, and 9, with a CBHI and CBHII in appropriate proportions, can enhance the production of COS from forest materials, underpinning the potential of these biocatalysts in the production of NDOs

DataSheet1_Fine-Tuned Enzymatic Hydrolysis of Organosolv Pretreated Forest Materials for the Efficient Production of Cellobiose.DOCX

<p>Non-digestible oligosaccharides (NDOs) are likely prebiotic candidates that have been related to the prevention of intestinal infections and other disorders for both humans and animals. Lignocellulosic biomass is the largest carbon source in the biosphere, therefore cello-oligosacharides (COS), especially cellobiose, are potentially the most widely available choice of NDOs. Production of COS and cellobiose with enzymes offers numerous benefits over acid-catalyzed processes, as it is milder, environmentally friendly and produces fewer by-products. Cellobiohydrolases (CBHs) and a class of endoglucanases (EGs), namely processive EGs, are key enzymes for the production of COS, as they have higher preference toward glycosidic bonds near the end of cellulose chains and are able to release soluble products. In this work, we describe the heterologous expression and characterization of two CBHs from the filamentous fungus Thermothelomyces thermophila, as well as their synergism with proccessive EGs for cellobiose release from organosolv pretreated spruce and birch. The properties, inhibition kinetics and substrate specific activities for each enzyme are described in detail. The results show that a combination of EGs belonging to Glycosyl hydrolase families 5, 6, and 9, with a CBHI and CBHII in appropriate proportions, can enhance the production of COS from forest materials, underpinning the potential of these biocatalysts in the production of NDOs.</p