Ricardo Sposina Sobral Teixeira

Amino acids interference on the quantification of reducing sugars by the 3,5-dinitrosalicylic acid assay mislead carbohydrase activity measurements

This study evaluated the interference of the amino acids tryptophan, cysteine, histidine, tyrosine, hydroxyproline, leucine, proline, serine, glycine, valine, glutamic acid, phenylalanine, and methionine on the measurement of reducing sugars using a phenol-free 3,5-dinitrosalicylic acid (DNS) reagent. It was found that in reaction mixtures containing 20mM of either tryptophan, cysteine, histidine, tyrosine, or hydroxyproline the measurement of 3.7 mM glucose was overestimated by 76%, 50%, 35%, 18%, and 10%, respectively. The amino acids valine, glutamic acid, and phenylalanine did not affect the DNS reaction, while methionine decreased the color development by 5%. The measurement of glucose, xylose, arabinose, and cellobiose at the 3.7-12.4 mM range in the presence of 20 mM cysteine resulted in an overestimated concentration of 34.8-50%. Enzymatic assays for measuring xylanolytic and filter paper activity (FPAse) were conducted in the presence of 20-60 mM cysteine, and compared to cysteine-free assays. In the presence of cysteine, the measured xylanase activity increased threefold and the FPAse activity increased twofold due to the overestimation of the reducing sugar concentrations in the assays. The interference from cysteine was reduced to a maximum of 8.6% when a DNS reagent containing phenol was used.

Continuous pretreatment of sugarcane bagasse at high loading in an ionic liquid using a twin-screw extruder

Ionic liquids (ILs) are innovative and effective solvents for pretreating lignocellulose biomasses because they bring about a noticeable increase in the enzymatic saccharification of these materials. However, the reported solid loadings in ILs of approximately 5.0 wt% reflect their large consumption per gram of biomass and hinder their use in biomass pretreatment. In the present study, a twin-screw extruder with high shearing force was used as a pretreatment reactor to process sugarcane bagasse at high loadings in the IL 1-ethyl-3-methylimidazolium acetate. This procedure allowed effective pretreatment of biomasses at loadings as high as 25 wt% for 8 min at 140 °C, resulting in glucose yields of more than 90% after 24 h of enzymatic saccharification of the pretreated material. This glucose yield was comparable to that of a 4.8 wt% bagasse loading that was pretreated for 120 min at 120 °C in a stirring reactor and enzymatically hydrolyzed under the same conditions. A higher bagasse loading, 50 wt%, also afforded a high glucose yield of 76.4%. Characterization of the pretreated materials by examining the surface morphology at the nanoscopic scale, measuring the specific surface area (SSA), and analyzing the degree of crystallinity showed that the use of the extruder as a pretreatment reactor significantly decreased the crystallinity and increased the SSA by more than 100-fold. In addition, the extrusion process can be conducted continuously and is appropriate for industrial-scale biomass processing, allowing higher reactant concentrations and throughputs, higher mixing rates, and more uniform products compared to batch processes.

Efficient production of lignocellulolytic enzymes xylanase, β-xylosidase, ferulic acid esterase and β-glucosidase by the mutant strain Aspergillus awamori 2B.361 U2/1.

The production of xylanase, β-xylosidase, ferulic acid esterase and β-glucosidase by Aspergillus awamori 2B.361 U2/1, a hyper producer of glucoamylase and pectinase, was evaluated using selected conditions regarding nitrogen nutrition. Submerged cultivations were carried out at 30 °C and 200 rpm in growth media containing 30 g wheat bran/L as main carbon source and either yeast extract, ammonium sulfate, sodium nitrate or urea, as nitrogen sources; in all cases it was used a fixed molar carbon to molar nitrogen concentration of 10.3. The use of poor nitrogen sources favored the accumulation of xylanase, β-xylosidase and ferulic acid esterase to a peak concentrations of 44,880; 640 and 118 U/L, respectively, for sodium nitrate and of 34,580, 685 and 170 U/L, respectively, for urea. However, the highest β-glucosidase accumulation of 10,470 U/L was observed when the rich organic nitrogen source yeast extract was used. The maxima accumulation of filter paper activity, xylanase, β-xylosidase, ferulic acid esterase and β-glucosidase by A. awamori 2B.361 U2/1 was compared to that produced by Trichoderma reesei Rut-C30. The level of β-glucosidase was over 17-fold higher for the Aspergillus strain, whereas the levels of xylanase and β-xylosidase were over 2-fold higher. This strain also produced ferulic acid esterase (170 U/L), which was not detected in the T. reesei culture.

Extraction and Application of Laccases from Shimeji Mushrooms (Pleurotus ostreatus) Residues in Decolourisation of Reactive Dyes and a Comparative Study Using Commercial Laccase from Aspergillus oryzae

Oxidases are able to degrade organic pollutants; however, high costs associated with biocatalysts production still hinder their use in environmental biocatalysis. Our study compared the action of a commercial laccase from Aspergillus oryzae and a rich extract from Pleurotus ostreatus cultivation residues in decolourisation of reactive dyes: Drimaren Blue X-3LR (DMBLR), Drimaren Blue X-BLN (DMBBLN), Drimaren Rubinol X-3LR (DMR), and Drimaren Blue C-R (RBBR). The colour removal was evaluated by considering dye concentration, reaction time, absence or presence of the mediator ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), and the source of laccase. The presence of ABTS was essential for decolourisation of DMR (80–90%, 1 h) and RBBR (80–90%, 24 h) with both laccases. The use of ABTS was not necessary in reactions containing DMBLR (85–97%, 1 h) and DMBBLN (63–84%, 24 h). The decolourisation of DMBBLN by commercial laccase showed levels near 60% while the crude extract presented 80% in 24 h.

Optimized atrazine degradation by Pleurotus ostreatus INCQS 40310: an alternative for impact reduction of herbicides used in sugarcane crops

The herbicide atrazine (2-chloro-4-ethylamine-6-isopropylamine-s-triazine) is extensively used for weed control in sugarcane crops. The application of fungi for the biodegradation of xenobiotics has been studied with promising results. Therefore, atrazine degradation mediated by Pleurotus ostreatus INCQS 40310 was evaluated, and the involvement of ligninolytic enzymes along with the degradation process was also investigated. To promote high degradation percentages and rates, a fractional factorial experimental design was first used to determine the most significant medium components for atrazine degradation. This strategy improved atrazine degradation from 39.0% to 71.0% after 15 days, with the formation of different metabolites. Afterward, a 32 full factorial design was performed using the variables selected in the first part of this study. The salts FeSO4 and MnSO4 showed significant influence in the percentages and the rates of atrazine degradation. The medium optimization resulted in 90.3% and 94.5% of atrazine degradation after 10 days and 15 days, respectively. Although laccase activity was measured during the degradation process, it was not possible to correlate laccase activity with atrazine degradation. The results demonstrated the efficiency of P. ostreatus INCQS 40310 for atrazine degradation, thus demonstrating the potential of this fungus as a bioremediation agent.

Combining biomass wet disk milling and endoglucanase/β-glucosidase hydrolysis for the production of cellulose nanocrystals.

Cellulose nanocrystals (CNCs), a biomaterial with high added value, were obtained from pure cellulose, Eucalyptus holocellulose, unbleached Kraft pulp, and sugarcane bagasse, by fibrillating these biomass substrates using wet disk milling (WDM) followed by enzymatic hydrolysis using endoglucanase/β-glucosidase. The hydrolysis experiments were conducted using the commercial enzyme OptimashBG or a blend of Pyrococcus horikoshii endoglucanase and Pyrococcus furiosus β-glucosidase. The fibrillated materials and CNCs were analyzed by X-ray diffraction, atomic force microscopy, scanning electron microscopy, and the specific surface area (SSA) was measured. WDM resulted in the formation of long and twisted microfibers of 1000-5000 nm in length and 4-35 nm in diameter, which were hydrolyzed into shorter and straighter CNCs of 500-1500 nm in length and 4-12 nm in diameter, with high cellulose crystallinity. Therefore, the CNCs aspect ratio was successfully adjusted by endoglucanases under mild reaction conditions, relative to the reported acidic hydrolysis method.

Use of cellobiohydrolase-free cellulase blends for the hydrolysis of microcrystalline cellulose and sugarcane bagasse pretreated by either ball milling or ionic liquid [Emim][Ac]

This study investigated the requirement of cellobiohydrolases (CBH) for saccharification of microcrystalline cellulose and sugarcane bagasse pretreated either by ball milling (BM) or by ionic liquid (IL) [Emim][Ac]. Hydrolysis was done using CBH-free blends of Pyrococcus horikoshii endoglucanase (EG) plus Pyrococcus furiosus β-glucosidase (EGPh/BGPf) or Optimash™ BG while Acremonium Cellulase was used as control. IL-pretreated substrates were hydrolyzed more effectively by CBH-free enzymes than were the BM-pretreated substrates. IL-treatment decreased the crystallinity and increased the specific surface area (SSA), whereas BM-treatment decreased the crystallinity without increasing the SSA. The hydrolysis of IL-treated cellulose by EGPh/BGPf showed a saccharification rate of 3.92 g/Lh and a glucose yield of 81% within 9h. These results indicate the efficiency of CBH-free enzymes for the hydrolysis of IL-treated substrates.

Purification and characterization of asparaginase II from Saccharomyces cerevisiae cloned in Pichia pastoris: a study on a possible antileukemic drug

Bacterial asparaginase obtained from Escherichia coli and Erwinia chrysanthemi are used as medicine to treat acute lymphocytic leukemia and non-Hodgkin lymphoma. Despite the therapeutic properties of such enzymes there have been reports on adverse reactions, eventually so severe as to impede some patients of using the medicine. Besides, the only drug Brazil used to import is no longer produced. Considering these two factors our proposition is to produce non-bacterial asparaginase.

Productive Chain of Biofuels and Industrial Biocatalysis: Two Important Opportunities for Brazilian Sustainable Development

Abstract The integration between biofuels and chemicals production from biomass stimulates the transition to the inevitable bioeconomy era; this era can be achieved by implementing new technologies in existing industrial units where waste streams and by-products can be used as a renewable source of raw materials for the production of commodities and other value-added chemicals. This synergistic approach requires less capital investment, creates new business and job opportunities, expands the market and reduces the environmental impact caused by the operation of industrial plants. This chapter depicts the current situation of the two main biofuels in Brazil, ethanol and biodiesel, and introduces the discussion of opportunities and bottlenecks in the exploitation of lignocellulosic and oleaginous materials, focusing on the important role of enzymatic and microbial processes to support a sustainable industry.

Chlorine-Free Biomass Processing: Enzymatic Alternatives for Bleaching and Hydrolysis of Lignocellulosic Materials

This chapter presents biocatalytic alternatives for the processing of lignocellulosic materials with chlorine compounds in the pulp and paper industry and in the hydrolysis of lignocellulosic materials for the production of biomass sugar syrups and, ultimately, biofuels and chemicals. The advantages and disadvantages of the current chemical processes, their effects on the organisms and the environment, and the options for biocatalytic routes are discussed with the aim of contributing to the shift toward innovative and sustainable industrial processes. This shift is not an easy task due to economic reasons and to the impact on the operation logistics of the chemical industry worldwide. However, this investment is a necessary step for the future, when green processes will improve production and be required by the ever-growing restrictive regulations for the industrial sector.