Nei Pereira

Optimized Expression of a Thermostable Xylanase from Thermomyces lanuginosus in Pichia pastoris

ABSTRACT Highly efficient production of a Thermomyces lanuginosus IOC-4145 β-1,4-xylanase was achieved in Pichia pastoris under the control of the AOX1 promoter. P. pastoris colonies expressing recombinant xylanase were selected by enzymatic activity plate assay, and their ability to secrete high levels of the enzyme was evaluated in small-scale cultures. Furthermore, an optimization of enzyme production was carried out with a 23 factorial design. The influence of initial cell density, methanol, and yeast nitrogen base concentration was evaluated, and initial cell density was found to be the most important parameter. A time course profile of recombinant xylanase production in 1-liter flasks with the optimized conditions was performed and 148 mg of xylanase per liter was achieved. Native and recombinant xylanases were purified by gel filtration and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, circular dichroism spectroscopy, matrix-assisted laser desorption ionization-time of flight-mass spectrometry and physicochemical behavior. Three recombinant protein species of 21.9, 22.1, and 22.3 kDa were detected in the mass spectrum due to variability in the amino terminus. The optimum temperature, thermostability, and circular dichroic spectra of the recombinant and native xylanases were identical. For both enzymes, the optimum temperature was 75°C, and they retained 60% of their original activity after 80 min at 70°C or 40 min at 80°C. The high level of fully active recombinant xylanase obtained in P. pastoris makes this expression system attractive for fermentor growth and industrial applications.

Production of biosurfactants from Pseudomonas aeruginosa PA 1 isolated in oil environments

The potential production of rhamnolipid-type biosurfactants is assessed based on the development of a fermentative process with a strain of Pseudomonas aeruginosa PA1, which was isolated from oil production wastewater in the Northeast of Brazil. These production of molecules using different carbon (n-hexadecane, paraffinic oil, glycerol and babassu oil) and nitrogen sources (NaNO3, (NH4)2SO4 and CH4N2O) was studied. The best results were obtained when using glycerol as substrate. A C/N ratio of 60/1 and use of sodium nitrate as nitrogen source resulted in higher production of the rhamnolipid, expressed by rhamnose (3.16 g/L) and by the yield in relation to biomass (Yp/x = 0.70 g/g). Additionally, physical-chemical characteristics of the spent broth with and without cells were studied, providing a low critical micelle concentration of 19 mg/L and toxicity values of 13 and 13.8 mg/L using two test organisms, the micro crustacean Daphnia similis and the bacterium Vibrio fisheri (Microtox), respectively.

Enzymatic hydrolysis optimization to ethanol production by simultaneous saccharification and fermentation

There is tremendous interest in using agro-industrial wastes, such as cellulignin, as starting materials for the production of fuels and chemicals. Cellulignin are the solids, which result from the acid hydrolysis of the sugarcane bagasse. The objective of this work was to optimize the enzymatic hydrolysis of the cellulose fraction of cellulignin, and to study its fermentation to ethanol using Saccharomyces cerevisiae. Cellulose conversion was optimized using response surface methods with pH, enzyme loading, solid percentage, and temperature as factor variables. The optimum conditions that maximized the conversion of cellulose to glucose, calculated from the initial dried weight of pretreated cellulignin, (43°C, 2%, and 24.4 FPU/g of pretreated cellulignin) such as the glucose concentration (47°C, 10%, and 25.6 FPU/g of pretreated cellulignin) were found. The desirability function was used to find conditions that optimize both, conversion to glucose and glucose concentration (47°C, 10%, and 25.9 FPU/g of pretreated cellulignin). The resulting enzymatic hydrolyzate was fermented yielding a final ethanol concentration of 30.0 g/L, in only 10 h, and reaching a volumetric productivity of 3.0 g/L·h, which is close to the values obtained in the conventional ethanol fermentation of sugar cane juice (5.0–8.0 g/L·h) in Brazil.

Evaluation of Different Carbon and Nitrogen Sources in Production of Rhamnolipids by a Strain of Pseudomonas aeruginosa

Culture conditions involving variations in carbon and nitrogen sources and different C:N ratios were examined with the aim of increasing productivity in the process of rhamnolipid synthesis by Pseudomonas aeruginosa. In addition to the differences in productivity, the use of different carbon sources resulted in several proportions related to the types of rhamnolipids synthesized (monorhamnolipids and dirhamnolipids). Furthermore, the variation in nutrients, mainly the nitrogen source, resulted in different amounts of virulence factors, as phenazines and extracellular proteins. The data point out a new concern in the choice of substrate to be used for rhamnolipid production by P. aeruginosa: toxic byproducts.

PRODUÇÃO, PROPRIEDADES E APLICAÇÃO DE CELULASES NA HIDRÓLISE DE RESÍDUOS AGROINDUSTRIAIS

PRODUCTION, PROPERTIES AND APPLICATION OF CELLULASES IN THE HYDROLYSIS OF AGROINDUSTRIAL RESIDUES. Cellulases have been intensively studied in the past few years, due to the interests in biofuels production from lignocellulosic materials, since they permit maintaining mild conditions during the conversion process. These enzymes can be produced by a broad variety of naturally occurring microorganisms, such as from genera Aspergillus, Trichoderma, Penicillium and Humicola. Targeting the increasing of expression levels, molecular biology tools have been used for heterologous genes insertion in host cells, e. g., Pichia pastoris and Escherichia coli. Enzymes from fungal cellulolytic complex usually act best at pH between 4 and 5 under temperatures from 40 to 60 °C and can be used for either sequential (SHF) or simultaneous (SSF) hydrolysis together alcoholic fermentation. In this review, the main raw materials for production of cellulases are identified, as well as the state of art of enzymes’ properties, production and main applications.

Use of Corncob for Endoxylanase Production by Thermophilic Fungus Thermomyces lanuginosus IOC-4145

The production of cellulase-free end oxylanase by the thermophilic fungus Thermomyces lanuginosus was investigated insemisolid fermentation and liquid fermentation. Different process variables were investigated in semisolid fermentation, employing corncobas the carbon source. The best results were with the following conditions: grain size=4.5 mm, solid:liquid ratio=1:2, and inoculum size=20% (v/v). Corncob, xylan, and xylose were the best inducers for endoxylanase production. Additionally, organic nitrogen sources were necessary for the production of high endoxylanase activities. The crude enzyme had optimum activity at pH 6.0 and 75°C, displaying a high thermostability. The apparent K25 and Vmax were 1.77 mg of xylan/mL and 21.5 U/mg of protein, respectively.

Production of ethanol 3G from Kappaphycus alvarezii: evaluation of different process strategies.

This study evaluated the potential of Kappaphycus alvarezii as feedstock for ethanol production, i.e. ethanol 3G. First, aquatic biomass was subjected to a diluted acid pretreatment. This acid pretreatment generated two streams--a galactose-containing liquid fraction and a cellulose-containing solid fraction, which were investigated to determine their fermentability with the following strategies: a single-stream process (simultaneous saccharification and co-fermentation (SSCF) of both fractions altogether), which achieved 64.3 g L(-1) of ethanol, and a two-stream process (fractions were fermented separately), which resulted in 38 g L(-1) of ethanol from the liquid fraction and 53.0 g L(-1) from the simultaneous saccharification and fermentation (SSF) of the solid fraction. Based on the average fermentable carbohydrate concentration, it was possible to obtain 105 L of ethanol per ton of dry seaweed. These preliminaries results indicate that the use of the macro-algae K. alvarezii has a good potential feedstock for bioethanol production.

Joint X‐ray crystallographic and molecular dynamics study of cellobiohydrolase I from Trichoderma harzianum: deciphering the structural features of cellobiohydrolase catalytic activity

Aiming to contribute toward the characterization of new, biotechnologically relevant cellulolytic enzymes, we report here the first crystal structure of the catalytic core domain of Cel7A (cellobiohydrolase I) from the filamentous fungus Trichoderma harzianum IOC 3844. Our structural studies and molecular dynamics simulations show that the flexibility of Tyr260, in comparison with Tyr247 from the homologous Trichoderma reesei Cel7A, is enhanced as a result of the short side‐chains of adjacent Val216 and Ala384 residues and creates an additional gap at the side face of the catalytic tunnel. T. harzianum cellobiohydrolase I also has a shortened loop at the entrance of the cellulose‐binding tunnel, which has been described to interact with the substrate in T. reesei Cel7A. These structural features might explain why T. harzianum Cel7A displays higher kcat and Km values, and lower product inhibition on both glucoside and lactoside substrates, compared with T. reesei Cel7A.

Usefulness of a real-time PCR platform for G+C content and DNA-DNA hybridization estimations in vibrios

The aim of this study was to evaluate the utility of a real-time PCR platform to estimate the DNA G+C content (mol%) and DNA-DNA hybridization (DDH) values in the genus Vibrio. In total, nine vibrio strains were used to determine the relationship between genomic DNA G+C content and T(m) (°C). The T(m) and HPLC datasets fit a linear regression curve with a significant correlation coefficient, corroborating that this methodology has a high correlation with the standard methodology based on HPLC (R(2) = 0.94). Analysis of 31 pairs of vibrios provided a wide range of ΔT(m) values, varying between 0.72 and 12.5 °C. Pairs corresponding to strains of the same species or strains from sister species showed the lowest ΔT(m) values. For instance, the ΔT(m) of the sister species Vibrio harveyi LMG 4044(T) and Vibrio campbellii LMG 11216(T) was 5.2 °C, whereas the ΔT(m) of Vibrio coralliilyticus LMG 20984(T) and Vibrio neptunius LMG 20536(T) was 8.75 °C. The mean ΔT(m) values corresponding to pairs of strains with DDH values lower than 60 % or higher than 80 % were, respectively, 8.29 and 2.21 °C (significant difference, P<0.01). The high correlation between DDH values obtained in previous studies and the ΔT(m) values (R(2) = 0.7344) indicates that the fluorimetric methodology is a reliable alternative for the estimation of both DNA G+C content and ΔT(m) in vibrios. We suggest that strains of the same Vibrio species will have less than 4 °C ΔT(m). The use of a real-time PCR platform represents a valuable alternative for the development of the taxonomy of vibrios.

Application of xylanase from Thermomyces lanuginosus IOC-4145 for enzymatic hydrolysis of corncob and sugarcane bagasse.

Xylanases have significant current and potential uses for several industries including paper and pulp, food, and biofuel. For the biofuel industry, xylanases can be used to aid in the conversion of lignocellulose to fermentable sugars (e.g., xylose). We investigated the thermophilic fungus Thermomyces lanuginosus was yielded for xylanase production and found that the highest activity (850 U/mL) was yielded after 96 h of semisolid fermentation. The enzyme was used for hydrolyzing agricultural residues with and without pretreatment. Such residues were characterized in relation to the maximum xylose content by total acid hydrolysis. The highest xylose yields realized by enzymatic hydrolysis were 24 and 52%, achieved by using 3000 U/g (dried material) of sugarcane bagasse and corncob, respectively, which received both alkali and thermal pretreatment.