Juliana Vaz Bevilaqua

Biodiesel Fuel Production by the Transesterification Reaction of Soybean Oil Using Immobilized Lipase

The enzymatic alcoholysis of soybean oil with methanol and ethanol was investigated using a commercial, immobilized lipase (Lipozyme RMIM). The effect of alcohol (methanol or ethanol), enzyme concentration, molar ratio of alcohol to soybean oil, solvent, and temperature on biodiesel production was determined. The best conditions were obtained in a solvent-free system with ethanol/oil molar ratio of 3.0, temperature of 50°C, and enzyme concentration of 7.0% (w/w). Three-step batch ethanolysis was most effective for the production of biodiesel. Ethyl esters yield was about 60% after 4 h of reaction.

Phenol removal through combined biological and enzymatic treatments

This work studies the use of biological and combined biological/enzymatic treatments in phenol degradation. The systems studied were conventional batch aerobic biological followed or preceded by enzymatic treatment. Tyrosinase extracted from the mushroom Agaricus bispora was employed. Biological treatment efficiently degraded effluents containing up to 420 mg.L-1 of phenol, removing 97% of the COD and 99% of the phenol in 48-hour batches. Alterations in phenol concentration intake reduced treatment efficiency significantly. Enzymatic polishing of biotreated effluent removed up to 75% of the remaining phenol in a four-hour reaction with 46 U.mL-1 of tyrosinase and 50 mg.L-1 of chitosan (used as coagulant). Enzymatic pretreatment with 20 U.mL-1 of tyrosinase reduced the phenol concentration by 25 % after 2 hours of reaction, although initial COD increased up to 58%. The subsequent biological treatment of that enzymatic pretreated effluent reduced COD to 151 mgO2.L-1 and phenol concentration to 1 mg.L-1 in 24-hours batches.

Characterization of Gordonia sp. strain F.5.25.8 capable of dibenzothiophene desulfurization and carbazole utilization

A dibenzothiophene (DBT)-degrading bacterial strain able to utilize carbazole as the only source of nitrogen was identified as Gordonia sp. F.5.25.8 due to its 16S rRNA gene sequence and phenotypic characteristics. Gas chromatography (GC) and GC–mass spectroscopy analyses showed that strain F.5.25.8 transformed DBT into 2-hydroxybiphenyl (2-HBP). This strain was also able to grow using various organic sulfur or nitrogen compounds as the sole sulfur or nitrogen sources. Resting-cell studies indicated that desulfurization occurs either in cell-associated or in cell-free extracts of F.5.25.8. The biological responses of F.5.25.8 to a series of mutagens and environmental agents were also characterized. The results revealed that this strain is highly tolerant to DNA damage and also refractory to induced mutagenesis. Strain F.5.25.8 was also characterized genetically. Results showed that genes involved in desulfurization (dsz) are located in the chromosome, and PCR amplification was observed with primers dszA and dszB designed based on Rhodococcus genes. However, no amplification product was observed with the primer based on dszC.

Immobilization of Yarrowia lipolytica Lipase—a Comparison of Stability of Physical Adsorption and Covalent Attachment Techniques

Lipase immobilization offers unique advantages in terms of better process control, enhanced stability, predictable decay rates and improved economics. This work evaluated the immobilization of a highly active Yarrowia lipolytica lipase (YLL) by physical adsorption and covalent attachment. The enzyme was adsorbed on octyl–agarose and octadecyl–sepabeads supports by hydrophobic adsorption at low ionic strength and on MANAE–agarose support by ionic adsorption. CNBr–agarose was used as support for the covalent attachment immobilization. Immobilization yields of 71, 90 and 97% were obtained when Y. lipolytica lipase was immobilized into octyl–agarose, octadecyl–sepabeads and MANAE–agarose, respectively. However, the activity retention was lower (34% for octyl–agarose, 50% for octadecyl–sepabeads and 61% for MANAE–agarose), indicating that the immobilized lipase lost activity during immobilization procedures. Furthermore, immobilization by covalent attachment led to complete enzyme inactivation. Thermal deactivation was studied at a temperature range from 25 to 45°C and pH varying from 5.0 to 9.0 and revealed that the hydrophobic adsorption on octadecyl–sepabeads produced an appreciable stabilization of the biocatalyst. The octadecyl–sepabeads biocatalyst was almost tenfold more stable than free lipase, and its thermal deactivation profile was also modified. On the other hand, the Y. lipolytica lipase immobilized on octyl–agarose and MANAE–agarose supports presented low stability, even less than the free enzyme.

Separation and Immobilization of Lipase from Penicillium simplicissimum by Selective Adsorption on Hydrophobic Supports

Lipases are an enzyme class of a great importance as biocatalysts applied to organic chemistry. However, it is still necessary to search for new enzymes with special characteristics such as good stability towards high temperatures, organic solvents, and high stereoselectivity presence. The present work’s aim was to immobilize the lipases pool produced by Penicillium simplissicimum, a filamentous fungi strain isolated from Brazilian babassu cake residue. P. simplissicimum lipases were separated into three different fractions using selective adsorption method on different hydrophobic supports (butyl-, phenyl-, and octyl-agarose) at low ionic strength. After immobilization, it was observed that these fractions’ hyperactivation is in the range of 131% to 1133%. This phenomenon probably occurs due to enzyme open form stabilization when immobilized onto hydrophobic supports. Those fractions showed different thermal stability, specificity, and enantioselectivity towards some substrates. Enantiomeric ratio for the hydrolysis of (R,S) 2-O-butyryl-2-phenylacetic acid ranged from 1 to 7.9 for different immobilized P. simplissicimum lipase fractions. Asymmetry factor for diethyl 2-phenylmalonate hydrolysis ranged from 11.8 to 16.4 according to the immobilized P. simplissicimum lipase fractions. Those results showed that sequential adsorption methodology was an efficient strategy to obtain new biocatalysts with different enantioselectivity degrees, thermostability, and specificity prepared with a crude extract produced by a simple and low-cost technology.

Estolides Synthesis Catalyzed by Immobilized Lipases

Estolides are vegetable-oil-based lubricants obtained from oleic acid or any source of hydroxy fatty acids. In this work, the estolides synthesis from oleic acid and methyl ricinoleate (biodiesel from castor oil), using immobilized commercial lipases (Novozym 435, Lipozyme RM-IM, and Lipozyme TL-IM) in a solvent-free medium was investigated. Acid value was used to monitor the reaction progress by determining the consumption of acid present in the medium. Novozym 435 showed the best performance. Water removal improved the conversion. Novozym 435 was more active at atmospheric pressure. Novozym 435 was reused four times with conversion reaching 15% after the fourth reaction at 80°C. Estolides produced under the reaction conditions used in this work presented good properties, such as, low temperature properties as pour point (−24°C), viscosity (23.9 cSt at 40°C and 5.2 cSt at 100°C), and viscosity index (153).

Hydrolysis of new phthalimide-derived esters catalyzed by immobilized lipase

The last step of the production of four phthalimide-derived acids, designed to act as antiasthma drugs, was performed by enzymatic hydrolysis of the respective methyl or ethyl esters. The esters 4-ethyl-[2-(1,3-dioxo-1,3-dihydro- 2-isoindoylyl)]-phenoxyacetic methyl ester (PHT-MET), 4-ethyl-[2- (1,3-dioxo-1,3-dihydro-2-isoindoylyl)]-phenoxyacetic ethyl ester, 4-(1, 3-dioxo-1,3-dihydro-2-isoindoylyl)-phenoxyacetic ethyl ester, and 2-(1,3-dioxo-1, 3-dihydro-2-isoindoylyl)-phenoxyacetic ethyl ester were hydrolyzed by immobilized lipase. The enzymatic reaction could be used only to produce the desired 4-substituted compounds. The best result that was found to hydrolysis of PHT-MET, and, therefore, that ester was selected for optimization experiments in a three-phase system. Reactions were performed with solid biocatalyst (Lipozyme RM IM), organic solvent phase (ethyl acetate), and aqueous phase (saturated Na2CO3 solution). To optimize the reaction conditions, an experimental design optimization procedure was used. The variables studied were the amount of enzyme, the temperature, and the volume of the aqueous solution. Time course experiments were then performed for different initial enzyme concentrations (0.5, 0.9, and 1.4 UH/mL of solvent). The optimized reaction conditions found were 20 mg of Lipozyme (0.9 UH/mL solvent) and 5.0 mL of Na2CO3(sat) at 40 degrees C for 6 h.