Characterization and optimization of ArtinM lectin expression in Escherichia coli

Background ArtinM is a D-mannose-specific lectin from Artocarpus integrifolia seeds that induces neutrophil migration and activation, degranulation of mast cells, acceleration of wound healing, induction of interleukin-12 production by macrophages and dendritic cells, and protective T helper 1 immune response against Leishmania major, Leishmania amazonensis and Paracoccidioides brasiliensis infections. Considering the important biological properties of ArtinM and its therapeutic applicability, this study was designed to produce high-level expression of active recombinant ArtinM (rArtinM) in Escherichia coli system. Results The ArtinM coding region was inserted in pET29a(+) vector and expressed in E. coli BL21(DE3)-Codon Plus-RP. The conditions for overexpression of soluble ArtinM were optimized testing different parameters: temperatures (20, 25, 30 or 37°C) and shaking speeds (130, 200 or 220 rpm) during induction, concentrations of the induction agent IPTG (0.01-4 mM) and periods of induction (1-19 h). BL21-CodonPlus(DE3)-RP cells induced under the optimized conditions (incubation at 20°C, at a shaking speed of 130 rpm, induction with 0.4 mM IPTG for 19 h) resulted in the accumulation of large amounts of soluble rArtinM. The culture provided 22.4 mg/L of rArtinM, which activity was determined by its one-step purification through affinity chromatography on immobilized D-mannose and glycoarray analysis. Gel filtration showed that rArtinM is monomeric, contrasting with the tetrameric form of the plant native protein (jArtinM). The analysis of intact rArtinM by mass spectrometry revealed a 16,099.5 Da molecular mass, and the peptide mass fingerprint and esi-cid-ms/ms of amino acid sequences of peptides from a tryptic digest covered 41% of the total ArtinM amino acid sequence. In addition, circular dichroism and fluorescence spectroscopy of rArtinM indicated that its global fold comprises β-sheet structure. Conclusions Overall, the optimized process to express rArtinM in E. coli provided high amounts of soluble, correctly folded and active recombinant protein, compatible with large scale production of the lectin.

Treatment of rice straw hemicellulosic hydrolysates with advanced oxidative processes: a new and promising detoxification method to improve the bioconversion process

Abstract Background The use of lignocellulosic constituents in biotechnological processes requires a selective separation of the main fractions (cellulose, hemicellulose and lignin). During diluted acid hydrolysis for hemicellulose extraction, several toxic compounds are formed by the degradation of sugars and lignin, which have ability to inhibit microbial metabolism. Thus, the use of a detoxification step represents an important aspect to be considered for the improvement of fermentation processes from hydrolysates. In this paper, we evaluated the application of Advanced Oxidative Processes (AOPs) for the detoxification of rice straw hemicellulosic hydrolysate with the goal of improving ethanol bioproduction by Pichia stipitis yeast. Aiming to reduce the toxicity of the hemicellulosic hydrolysate, different treatment conditions were analyzed. The treatments were carried out according to a Taguchi L16 orthogonal array to evaluate the influence of Fe+2, H2O2, UV, O3 and pH on the concentration of aromatic compounds and the fermentative process. Results The results showed that the AOPs were able to remove aromatic compounds (furan and phenolic compounds derived from lignin) without affecting the sugar concentration in the hydrolysate. Ozonation in alkaline medium (pH 8) in the presence of H2O2 (treatment A3) or UV radiation (treatment A5) were the most effective for hydrolysate detoxification and had a positive effect on increasing the yeast fermentability of rice straw hemicellulose hydrolysate. Under these conditions, the higher removal of total phenols (above 40%), low molecular weight phenolic compounds (above 95%) and furans (above 52%) were observed. In addition, the ethanol volumetric productivity by P. stipitis was increased in approximately twice in relation the untreated hydrolysate. Conclusion These results demonstrate that AOPs are a promising methods to reduce toxicity and improve the fermentability of lignocellulosic hydrolysates.

A new method for decision making in multi-objective optimization problems

Many engineering sectors are challenged by multi-objective optimization problems. Even if the idea behind these problems is simple and well established, the implementation of any procedure to solve them is not a trivial task. The use of evolutionary algorithms to find candidate solutions is widespread. Usually they supply a discrete picture of the non-dominated solutions, a Pareto set. Although it is very interesting to know the non-dominated solutions, an additional criterion is needed to select one solution to be deployed. To better support the design process, this paper presents a new method of solving non-linear multi-objective optimization problems by adding a control function that will guide the optimization process over the Pareto set that does not need to be found explicitly. The proposed methodology differs from the classical methods that combine the objective functions in a single scale, and is based on a unique run of non-linear single-objective optimizers.

Multiobjective engineering design optimization problems: a sensitivity analysis approach

This paper proposes two new approaches for the sensitivity analysis of multiobjective design optimization problems whose performance functions are highly susceptible to small variations in the design variables and/or design environment parameters. In both methods, the less sensitive design alternatives are preferred over others during the multiobjective optimization process. While taking the first approach, the designer chooses the design variable and/or parameter that causes uncertainties. The designer then associates a robustness index with each design alternative and adds each index as an objective function in the optimization problem. For the second approach, the designer must know, a priori, the interval of variation in the design variables or in the design environment parameters, because the designer will be accepting the interval of variation in the objective functions. The second method does not require any law of probability distribution of uncontrollable variations. Finally, the authors give two illustrative examples to highlight the contributions of the paper.

Optimization and control of a continuous polymerization reactor

This work studies the optimization and control of a styrene polymerization reactor. The proposed strategy deals with the case where, because of market conditions and equipment deterioration, the optimal operating point of the continuous reactor is modified significantly along the operation time and the control system has to search for this optimum point, besides keeping the reactor system stable at any possible point. The approach considered here consists of three layers: the Real Time Optimization (RTO), the Model Predictive Control (MPC) and a Target Calculation (TC) that coordinates the communication between the two other layers and guarantees the stability of the whole structure. The proposed algorithm is simulated with the phenomenological model of a styrene polymerization reactor, which has been widely used as a benchmark for process control. The complete optimization structure for the styrene process including disturbances rejection is developed. The simulation results show the robustness of the proposed strategy and the capability to deal with disturbances while the economic objective is optimized.

'Synthetic lipase' production from a newly isolated Sporidiobolus pararoseus strain by submerged fermentation

The lipase produced by a newly isolate Sporidiobolus pararoseus strain has potential catalysis ability for esterification reactions. In order to improve its synthetic activity, this work aimed at optimizing 'synthetic lipase' production by submerged fermentation of a conventional media based on peptone, yeast extract, NaCl and olive oil using experimental design technique. According to the results obtained in the first experimental design (2(4-1)), yeast extract and NaCl concentrations were tested to further optimization by response surface methodology. The maximum 'synthetic lipase' activity obtained was 26.9 U/mL in the optimized media (5.0, 6.8, 7.0 and 1.0% (wt/v) of peptone, yeast extract, NaCl and olive oil, respectively), representing a 6.36-fold increase compared to the initial medium. The time course of 'synthetic lipase' production in the optimized condition was evaluated in terms of synthetic activity, protease activity, biomass and total carbon and the maximum synthetic activity was observed during the stationary phase of growth.

Towards the stabilization of the low density elements in topology optimization with large deformation

This work addresses the treatment of lower density regions of structures undergoing large deformations during the design process by the topology optimization method (TOM) based on the finite element method. During the design process the nonlinear elastic behavior of the structure is based on exact kinematics. The material model applied in the TOM is based on the solid isotropic microstructure with penalization approach. No void elements are deleted and all internal forces of the nodes surrounding the void elements are considered during the nonlinear equilibrium solution. The distribution of design variables is solved through the method of moving asymptotes, in which the sensitivity of the objective function is obtained directly. In addition, a continuation function and a nonlinear projection function are invoked to obtain a checkerboard free and mesh independent design. 2D examples with both plane strain and plane stress conditions hypothesis are presented and compared. The problem of instability is overcome by adopting a polyconvex constitutive model in conjunction with a suggested relaxation function to stabilize the excessive distorted elements. The exact tangent stiffness matrix is used. The optimal topology results are compared to the results obtained by using the classical Saint Venant–Kirchhoff constitutive law, and strong differences are found.

Environmental assessment of residues generated after consecutive acid-base pretreatment of sugarcane bagasse by advanced oxidative process

Abstract Background Biofuels produced from sugarcane bagasse (SB) have shown promising results as a suitable alternative of gasoline. Biofuels provide unique, strategic, environmental and socio-economic benefits. However, production of biofuels from SB has negative impact on environment due to the use of harsh chemicals during pretreatment. Consecutive sulfuric acid-sodium hydroxide pretreatment of SB is an effective process which eventually ameliorates the accessibility of cellulase towards cellulose for the sugars production. Alkaline hydrolysate of SB is black liquor containing high amount of dissolved lignin. Results This work evaluates the environmental impact of residues generated during the consecutive acid-base pretreatment of SB. Advanced oxidative process (AOP) was used based on photo-Fenton reaction mechanism (Fenton Reagent/UV). Experiments were performed in batch mode following factorial design L9 (Taguchi orthogonal array design of experiments), considering the three operation variables: temperature (°C), pH, Fenton Reagent (Fe2+/H2O2) + ultraviolet. Reduction of total phenolics (TP) and total organic carbon (TOC) were responsive variables. Among the tested conditions, experiment 7 (temperature, 35°C; pH, 2.5; Fenton reagent, 144 ml H2O2+153 ml Fe2+; UV, 16W) revealed the maximum reduction in TP (98.65%) and TOC (95.73%). Parameters such as chemical oxygen demand (COD), biochemical oxygen demand (BOD), BOD/COD ratio, color intensity and turbidity also showed a significant change in AOP mediated lignin solution than the native alkaline hydrolysate. Conclusion AOP based on Fenton Reagent/UV reaction mechanism showed efficient removal of TP and TOC from sugarcane bagasse alkaline hydrolysate (lignin solution). To the best of our knowledge, this is the first report on statistical optimization of the removal of TP and TOC from sugarcane bagasse alkaline hydrolysate employing Fenton reagent mediated AOP process.