Enrichments of gene replacement events by Agrobacterium-mediated recombinase-mediated cassette exchange

We report recombinase-mediated cassette exchange (RMCE), which can permit integration of transgenes into pre-defined chromosomal loci with no co-expressed marker gene by using Agrobacterium-mediated transformation. Transgenic tobacco plants which have a single copy of negative marker genes (codA) at target loci in heterozygous and homozygous conditions were used for gene exchange by the RMCE method. By negative selection, we were able to obtain five heterozygous and four homozygous transgenic plants in which the genes were exchanged from 64 leaf segments of heterozygous and homozygous target plants, respectively. Except for one transgenic plant with an extra copy, the other eight plants had only a single copy of marker-free transgenes, and no footprint of random integrated copies was detected in half of the eight plants. The RMCE re-transformation frequencies were calculated as 6.25 % per explant and were approximately the same as the average percentage of intact single-copy transformation events for standard tobacco Agrobacterium-mediated transformation

THREE METHODS TO EVALUATE THE USE OF EVAPORATIVE COOLING FOR HUMAN THERMAL COMFORT

This paper presents three methods that can be used as reference for efficientuse of evaporative cooling systems, applying it, latter, to several Braziliancities, characterized by different climates. Initially it presents the basicprinciples of direct and indirect evaporative cooling and defines theeffectiveness of the systems. Afterwards, it presents three methods thatallows to determinate where the systems are more efficient. It concludesthat evaporative cooling systems have a very large potential to propitiatethermal comfort and can still be used as an alternative to conventionalsystems in regions where the design wet bulb temperature is under 24ºC

A MATHEMATICAL MODEL FOR DIRECT EVAPORATIVE COOLING AIR CONDITIONING SYSTEM

Air conditioning systems are responsible for increasing men's work efficiency as well for his comfort, mainly in the warm periods of the year. Currently, the most used system is the mechanical vapor compression system. However, in many cases, evaporative cooling system can be an economical alternative to replace the conventional system, under several conditions, or as a pre-cooler in the conventional systems. It leads to a reduction in the operational cost, comparing with systems using only mechanical refrigeration. Evaporative cooling operates using induced processes of heat and mass transfer, where water and air are the working fluids. It consists in water evaporation, induced by the passage of an air flow, thus decreasing the air temperature. This paper presents the basic principles of the evaporative cooling process for human thermal comfort, the principles of operation for the direct evaporative cooling system and the mathematical development of the equations of thermal exchanges, allowing the determination of the effectiveness of saturation. It also presents some results of experimental tests in a direct evaporative cooler that take place in the Air Conditioning Laboratory at the University of Taubaté Mechanical Engineering Department, and the experimental results are used to determinate the convective heat transfer coefficient and to compare with the mathematical model

Sistemas aquosos bifásicos uma ferramenta sustentável para a extração de ácido clavulânico a partir de diferentes fontes

O ácido clavulânico (AC) é um inibidor de β-lactamases que tem vindo a ser largamente utilizado na área médica. Embora seja de extrema importância, o desenvolvimento de processos alternativos de produção e purificação é ainda insignificante, sendo fundamental o estudo de técnicas de extração mais biocompatíveis, como os Sistemas Aquosos Bifásicos (SABs). Assim, este trabalho objetivou o estudo de Sistemas Aquosos Bifásicos baseados em polímeros como uma ferramenta alternativa para a extração de AC. Foram testados dois SPAB compostos por Polietileno Glicol (PEG) com massa molecular (M) de 4000 g/mol e Poliacrilato de Sódio de 8000 g/mol, nos quais foi alterado o eletrólito indutor da formação de fases, em particular, sulfato de sódio (Na2SO4,) e cloreto de sódio (NaCl). Ademais, este trabalho visou também avaliar a eficiência de extração do AC, bem como compreender o efeito dos contaminantes no processo de migração. Para tal, foi avaliada a extração do AC a partir de três fontes distintas: solução pura (99,9%); solução comercial (60%); diretamente a partir do sobrenadante de um meio fermentando de Streptomyces clavuligerus. Os resultados obtidos demonstraram que independentemente da fonte inicial do AC, ambos os SABs poliméricos promoveram uma partição preferencial do AC para a fase rica em PEG, sendo o coeficiente de partição maior nos sistemas com Na2SO4 do que com NaCl. Após identificar a grande capacidade de partição de AC, o SAB com PEG/NaPA/Na2SO4 foi também utilizado para avaliar a partição de proteínas presente no meio fermentado, sendo também obtida uma preferencial partição destas para a fase rica em PEG. Assim, apesar da baixa capacidade de purificação de AC frente a proteínas contaminantes, os SABs estudados demonstraram que podem ser uma técnica alternativa sustentável e bastante econômica para uma etapa inicial de clarificação/concentração de bioprodutos a partir de caldos fermentados

Development of suported ionic liquids for the purification of antileukemic drugs

L-asparaginase (LA) is an antileukemic biopharmaceutical of current high-cost. LA is produced via fermentation and its purification usually comprises precipitation, liquid- liquid extraction and chromatography techniques [1]. This work aims to develop sustainable technologies to purify LA. Functionalized nanomaterials, namely supported ionic liquids (SILs), are used as cost-effective purification techniques for the target enzyme. Initially, the synthesis and modification of SILs was performed. Different SILs were obtained and used for the purification of LA. Commercial LA was used for the first purification tests, in order to understand the behavior of the enzyme in contact with the nanomaterial. Experimental conditions, such as pH, and material/LA ratio, contact time were optimized. LA activity was quantified by Nessler reaction [2]. The first results reveal a total adsorption of LA by the SILs with a recovered activity reaching 90% depending on the SILs functionalization/ treatment. The modified SILs are shown to be very promising nanomaterials for the purification of LA. The LA was easily attached to SILs by adsorption under mild conditions. SILs supports can be a real alternative for a single step immobilization/purification of LA.publishe

Production and characterization of recombinant Aliivibrio fischeri L-Asparaginase with low L-Glutamine affinity: a potential antileukemic drug obtained by genetic engineering

L-Asparaginase has been successfully applied in the treatment of lymphoid malignancies. Some limitations in the use of the commercial preparations of this drug include several side effects that may be correlated to L-Glutaminase activity, as immunosuppressive effects (Castro et al., 2021). The objective of this study was to evaluate the characteristics of a novel engineered Aliivibrio fischeri L-asparaginase type II expressed by Bacillus subtilis. Cultivations were carried out in shaken flasks at 30 ºC, 200 rpm, 24 h, using Luria-Bertani medium. Intracellular enzyme was recovered by sonication and enzymatic activities were evaluated by Nessler colorimetric method (Mashburn; Wriston, 1963). Recovered enzymatic extracts achieved L-Asparaginase activity up to 1.43 U.mL-1 at optimum pH 7.5. Substrate affinity was much higher for L-Asparagine than for L-Glutamine (Km= 1.226 mmol.L-1 and Km= 28.584 mmol.L-1, respectively), which indicate the potential application of the recombinant enzyme as biopharmaceutical.publishe

L-Asparaginase

L-Asparaginase (ASNase, EC 3.5.1.1) is a tetrameric aminohydrolase enzyme that catalyses the hydrolysis of the amino acid L-Asparagine into ammonia and L-aspartic acid. ASNase is present in different organisms such as bacteria, fungi, plant tissues and algae. ASNase is used in the pharmaceutical field as an anticancer drug for the treatment of acute lymphoblastic leukemia (ALL) and other malignant diseases such as Hodgkin’s disease. In the food sector, ASNase is used to prevent the formation of acrylamide, a toxic compound formed when starch-rich foods are cooked at temperatures above 100 °C. ASNase can also be used as a biosensor for the detection of L-asparagine levels.publishe