RESUMO
The purpose of this study was to isolate, purify and optimize the production conditions of an organic solvent tolerant and thermostable lipase from Acinetobacter sp. AU07 isolated from distillery waste. The lipase production was optimized by response surface methodology, and a maximum production of 14.5 U/mL was observed at 30 ºC and pH 7, using a 0.5% (v/v) inoculum, 2% (v/v) castor oil (inducer), and agitation 150 rpm. The optimized conditions from the shake flask experiments were validated in a 3 L lab scale bioreactor, and the lipase production increased to 48 U/mL. The enzyme was purified by ammonium sulfate precipitation and ion exchange chromatography and the overall yield was 36%. SDS-PAGE indicated a molecular weight of 45 kDa for the purified protein, and Matrix assisted laser desorption/ionization time of flight analysis of the purified lipase showed sequence similarity with GDSL family of lipases. The optimum temperature and pH for activity of the enzyme was found to be 50 ºC and 8.0, respectively. The lipase was completely inhibited by phenylmethylsulfonyl fluoride but minimal inhibition was observed when incubated with ethylenediaminetetraacetic acid and dithiothreitol. The enzyme was stable in the presence of non-polar hydrophobic solvents. Detergents like SDS inhibited enzyme activity; however, there was minimal loss of enzyme activity when incubated with hydrogen peroxide, Tween 80 and Triton X-100. The kinetic constants (Km and Vmax) revealed that the hydrolytic activity of the lipase was specific to moderate chain fatty acid esters. The Vmax, Km and Vmax/Km ratio of the enzyme were 16.98 U/mg, 0.51 mM, and 33.29, respectively when 4-nitrophenyl palmitate was used as a substrate.(AU)
Assuntos
Lipase/análise , Lipase/isolamento & purificação , Solventes/isolamento & purificação , Acinetobacter/imunologia , Acinetobacter/fisiologiaRESUMO
Background Cyclodextrin glucanotransferase (CGTase) from Amphibacillus sp. NPST-10 was covalently immobilized onto amino-functionalized magnetic double mesoporous core-shell silica nanospheres (mag@d-SiO2@m-SiO2-NH2), and the properties of the immobilized enzyme were investigated. The synthesis process of the nanospheres included preparing core magnetic magnetite (Fe3O4) nanoparticles, coating the Fe3O4 with a dense silica layer, followed by further coating with functionalized or non-functionalized mesoporous silica shell. The structure of the synthesized nanospheres was characterized using TEM, XRD, and FT-IR analyses. CGTase was immobilized onto the functionalized and non-functionalized nanospheres by covalent attachment and physical adsorption. Results The results indicated that the enzyme immobilization by covalent attachment onto the activated mag@d-SiO2@m-SiO2-NH2, prepared using anionic surfactant, showed highest immobilization yield (98.1%), loading efficiency (96.2%), and loading capacity 58 µg protein [CGTase]/mg [nanoparticles]) which were among the highest yields reported so far for CGTase. Compared with the free enzyme, the immobilized CGTase demonstrated a shift in the optimal temperature from 50°C to 50-55°C, and showed a significant enhancement in the enzyme thermal stability. The optimum pH values for the activity of the free and immobilized CGTase were pH 8 and pH 8.5, respectively, and there was a significant improvement in pH stability of the immobilized enzyme. Moreover, the immobilized CGTase exhibited good operational stability, retaining 56% of the initial activity after reutilizations of ten successive cycles. Conclusion The enhancement of CGTase properties upon immobilization suggested that the applied nano-structured carriers and immobilization protocol are promising approach for industrial bioprocess for production of cyclodextrins using immobilized CGTase.
Assuntos
Bacillaceae/enzimologia , Enzimas Imobilizadas , Glucosiltransferases/isolamento & purificação , Glucosiltransferases/metabolismo , Solventes/isolamento & purificação , Temperatura , Porosidade , Dióxido de Silício , Ciclodextrinas , Nanosferas , Glucosiltransferases/biossíntese , Concentração de Íons de HidrogênioRESUMO
Owing to limitations of pump-and-treat, several technologies are being investigated for groundwater treatment. One of the most promising is the treatment of contaminants through the use of reactive barriers installed in situ, especially in the case of aquifers contaminated with chlorinated solvents. This work presents results of batch and column tests with metallic iron and some chlorinated solvents (1,2-DCA, 1,1,2-TCA and TCE). Such tests provided means to evaluate the degradation rates of these compounds and their byproducts. It is concluded that the reductive dechlorination with metallic iron can have different results, depending on the type of contaminant. Some contaminants may not present any degradation, or they have a half-life time so high that the use of the reactive barrier technology may not be practical. Furthermore, the formation of chlorinated byproducts, eventually more toxic than the original contaminant and that are not degradable using this same technology, emphasises that the treatment of aquifers should be sequential.
Assuntos
Água Doce/análise , Água Doce/química , Modelos Químicos , Poluentes da Água/isolamento & purificação , Poluição da Água/prevenção & controle , Biodegradação Ambiental , Hidrocarbonetos Clorados/isolamento & purificação , Ferro/química , Cinética , Solventes/isolamento & purificaçãoRESUMO
This study presents the design, monitoring and remediation strategy for a soil vapor extraction (SVE) system installed at an industrial site contaminated with a mixture of organic compounds, in particular 1,2 dichloroethane, carbon tetrachloride and chloroform. The unsaturated zone at the site is very deep and presents near ideal conditions for SVE, with the contaminated portion of the porous media characterized by alluvial deposits of a well graded sand followed by a capillary barrier at 28m of depth, which limited DNAPL downward migration. The soil cleanup time and the optimal removal rates of mass, considering different values of vacuum/discharge applied in the SVE system, have been estimated through the use of numerical sensitivity analysis. Results of the pilot-scale testing activities allowed for the calibration of flow and contaminated mass transport models and the determination, in a preliminary phase, of the infiltrated mass of different compounds.