Cleide Mara Faria Soares

Characterization and Utilization of Candida rugosa Lipase Immobilized on Controlled Pore Silica

Candida rugosa lipase was immobilized by covalent binding on controlled pore silica (CPS) using glutaraldehyde as cross-linking agent under aqueous and nonaqueous conditions. The immobilized C. rugosa was more active when the coupling procedure was performed in the presence of a nonpolar solvent, hexane. Similar optima pH (7.5-8.0) was found for both free and immobilized lipase. The optimum temperature for the immobilized lipase was about 10 degrees C higher than that for the free lipase. The thermal stability of the CPS lipase was also greater than the original lipase preparation. Studies on the operational stability of CPS lipase revealed good potential for recycling under aqueous (olive-oil hydrolysis) and nonaqueous (butyl butyrate synthesis) conditions.

Production and purification of an extracellular lipolytic enzyme using ionic liquid-based aqueous two-phase systems

The ability of ionic liquid-based aqueous two-phase systems (ATPS) to purify lipase produced by fermentation is here evaluated and compared against conventional PEG-based ATPS systems. Four ionic liquids, chosen after screening of a larger number of ionic liquids are evaluated, with the maximum purification and higher recovery being obtained for the systems based on [C8mim]Cl. It is shown that IL-based ATPS have a performance superior to PEG-based ATPS for the purification of this enzyme.

Purification of lipase produced by a new source of Bacillus in submerged fermentation using an aqueous two-phase system

This work discusses the application of an aqueous two-phase system for the purification of lipases produced by Bacillus sp. ITP-001 using polyethylene glycol (PEG) and potassium phosphate. In the first step, the protein content was precipitated with ammonium sulphate (80% saturation). The enzyme remained in the aqueous solution and was dialyzed against ultra-pure water for 18 h and used to prepare an aqueous two-phase system (PEG/potassium phosphate). The use of different molecular weights of PEG to purify the lipase was investigated; the best purification factor (PF) was obtained using PEG 20,000g/mol, however PEG 8000 was used in the next tests due to lower viscosity. The influence of PEG and potassium phosphate concentrations on the enzyme purification was then studied: the highest FP was obtained with 20% of PEG and 18% of potassium phosphate. NaCl was added to increase the hydrophobicity between the phases, and also increased the purification factor. The pH value and temperature affected the enzyme partitioning, with the best purifying conditions achieved at pH 6.0 and 4°C. The molecular mass of the purified enzyme was determined to be approximately 54 kDa by SDS-PAGE. According to the results the best combination for purifying the enzyme is PEG 8000g/mol and potassium phosphate (20/18%) with 6% of NaCl at pH 6.0 and 4°C (201.53 fold). The partitioning process of lipase is governed by the entropy contribution.

Protic ionic liquid as additive on lipase immobilization using silica sol-gel.

Ionic liquids (ILs) have evolved as a new type of non-aqueous solvents for biocatalysis, mainly due to their unique and tunable physical properties. A number of recent review papers have described a variety of enzymatic reactions conducted in IL solutions, on the other hand, to improve the enzymes activity and stability in ILs; major methods being explored include the enzyme immobilization (on solid support, sol-gel, etc.), protic ionic liquids used as an additive process. The immobilization of the lipase from Burkholderia cepacia by the sol-gel technique using protic ionic liquids (PIL) as additives to protect against inactivation of the lipase due to release of alcohol and shrinkage of the gel during the sol-gel process was investigated in this study. The influence of various factors such as the length of the alkyl chain of protic ionic liquids (monoethanolamine-based) and a concentration range between 0.5 and 3.0% (w/v) were evaluated. The resulting hydrophobic matrices and immobilized lipases were characterised with regard to specific surface area, adsorption-desorption isotherms, pore volume (V(p)) and size (d(p)) according to nitrogen adsorption and scanning electron microscopy (SEM), physico-chemical properties (thermogravimetric - TG, differential scanning calorimetry - DSC and Fourier transform infrared spectroscopy - FTIR) and the potential for ethyl ester and emulsifier production. The total activity yields (Y(a)) for matrices of immobilized lipase employing protic ionic liquids as additives always resulted in higher values compared with the sample absent the protic ionic liquids, which represents 35-fold increase in recovery of enzymatic activity using the more hydrophobic protic ionic liquids. Compared with arrays of the immobilized biocatalyst without additive, in general, the immobilized biocatalyst in the presence of protic ionic liquids showed increased values of surface area (143-245 m(2) g(-1)) and pore size (19-38 Å). Immobilization with protic ionic liquids also favoured reduced mass loss according to TG curves (always less than 42.9%) when compared to the immobilized matrix without protic ionic liquids (45.1%), except for the sample containing 3.0% protic ionic liquids (46.5%), verified by thermogravimetric analysis. Ionic liquids containing a more hydrophobic alkyl group in the cationic moiety were beneficial for recovery of the activity of the immobilized lipase. The physico-chemical characterization confirmed the presence of the enzyme and its immobilized derivatives obtained in this study by identifying the presence of amino groups, and profiling enthalpy changes of mass loss.

Lipase purification using ionic liquids as adjuvants in aqueous two-phase systems

Aqueous two-phase systems (ATPS) are efficient, environmentally friendly, and “biocompatible” separation processes, which allow the recovery of enzymes. The most common systems are based on polymers and salts, and recently, to overcome the low polarity difference between the phases of the polymeric systems, ATPS based on ionic liquids (ILs) were proposed and have been successfully applied in this field. This work discusses the use of imidazolium-based ILs not as phase forming compounds but as adjuvants (5 wt%) in ATPS of polyethylene glycol systems (1500, 4000, 6000 and 8000 g mol−1) with potassium phosphate buffer at pH 7, in the extraction and purification of a lipase produced by submerged fermentation by Bacillus sp. ITP-001. An initial optimization study was carried out with the commercial lipase B from Candida antarctica (CaLB) allowing us to further purify the commercial CaLB (purification factor = 5.2). Using the optimized conditions, a purification factor of 245 for the lipase from Bacillus sp. ITP-001 was achieved with 1-hexyl-3-methyl imidazolium chloride. The high purification factor is a consequence of the favorable interactions between the IL and the contaminant proteins that migrate for the PEG-rich phase, where the IL also concentrates preferentially, while the enzyme remains in the salt-rich phase.

Increased significance of food wastes: Selective recovery of added-value compounds

A single-step selective separation of two food additives was investigated using alcohol-salt aqueous two-phase systems (ATPS). The selective partitioning of two of the most used additives from a processed food waste material, vanillin and l-ascorbic acid, was successfully accomplished. The results obtained prove that alcohol-salt ATPS can be easily applied as cheaper processes for the selective recovery of valuable chemical products from food wastes and other sources. As a first approach, the phase diagrams of ATPS composed of different alcohol+inorganic salt+water were determined at 298 (± 1)K and atmospheric pressure. The influence of methanol, ethanol, 1-propanol, and 2-propanol and K(3)PO(4), K(2)HPO(4) or KH(2)PO(4)/K(2)HPO(4) in the design of the phase diagrams was addressed. After the evaluation of the phase diagrams behaviour, the influence of the phase forming constituents was assessed towards the partition coefficients and recovery percentages of vanillin and l-ascorbic acid among the coexisting phases. Both model systems and real processed food waste materials were employed. Using these ATPS as partitioning systems it is possible to recover and separate vanillin, which migrates for the alcohol-rich phase, from l-ascorbic acid, which preferentially partitions for the salt-rich phase.

Covalent Coupling Method for Lipase Immobilization on Controlled Pore Silica in the Presence of Nonenzymatic Proteins

Candida rugosa lipase was covalently immobilized on silanized controlled pore silica previously activated with glutaraldehyde in the presence of nonenzymatic proteins. This strategy is suggested to protect the enzyme from aggregation effects or denaturation that occurs as a result of the presence of silane precursors used in the formation of the silica matrix. The immobilization yield was evaluated as a function of the lipase loading and the additive type (albumin and lecithin) using statistical concepts. In agreement with the mathematical model, the maximum coupling yield (32.2%) can be achieved working at high lipase loading (450 units·g‐1 support) using albumin as an additive. In these conditions, the resulting immobilized lipase exhibits high hydrolytic (153.2 U·mg‐1) and esterification (337.6 mmol·g‐1·min) activities. The enhanced activity of the final lipase derivative is the sum of the benefits of the immobilization (that prevents enzyme aggregation) and the lipase coating by additives that increases the accessibility of active sites to the substrate.

Studies on immobilized lipase in hydrophobic sol-gel

The hydrolysis of tetraethoxysilane using the sol-gel process was used to produce silica matrices, and these were tested for the immobilization of lipase from Candida rugosa by three methods: physical adsorption, covalent binding, and gel entrapment in the presence and absence of polyethylene glycol (PEG-1450). The silica matrices and their derivatives were characterized regarding particle size distribution, specific surface area, pore size distribution (Brunauer, Emmett, and Teller [B.E.T.] method), yield of grafting (thermogravimetric analyzer [TGA]), and chemical composition (Fourier transform infrared). Immobilization yields based on recovered lipase activity varied from 3.0 to 32.0%, and the highest efficiency was attained when lipase was encapsulated in the presence of PEG.

Extraction and Recovery of Rutin from Acerola Waste using Alcohol-Salt-Based Aqueous Two-Phase Systems

Extraction of rutin from acerola waste was investigated using alcohol-salt-based aqueous two-phase systems (ATPS). Initially, the partitioning was studied using model systems with pure and commercial rutin. The impact of the ATPS constituents and composition, initial amount of rutin, temperature and addition of electrolytes was evaluated. Rutin can be recovered either in the alcohol-or-salt-rich phase depending on the salt used. To validate the optimization process, rutin extraction from acerola waste was carried out further. The results obtained with the real samples are in close agreement with the model systems and validate the optimization tests and support their applicability in bioresource-related processes.

Ionic liquid-based aqueous biphasic systems as a versatile tool for the recovery of antioxidant compounds

The comparative evaluation of distinct types of ionic liquid‐based aqueous biphasic systems (IL‐ABS) and more conventional polymer/salt‐based ABS to the extraction of two antioxidants, eugenol and propyl gallate, is focused. In a first approach, IL‐ABS composed of ILs and potassium citrate (C6H5K3O7/C6H8O7) buffer at pH 7 were applied to the extraction of two antioxidants, enabling the assessment of the impact of IL cation core on the extraction. The second approach uses ABS composed of polyethylene glycol (PEG) and potassium phosphate (K2HPO4/KH2PO4) buffer at pH 7 with imidazolium‐based ILs as adjuvants. Their application to the extraction of the compounds allowed the investigation of the impact of the presence/absence of IL, the PEG molecular weight, and the alkyl side chain length of the imidazolium cation on the partition. It is possible to maximize the extractive performance of both antioxidants up to 100% using both types of IL‐ABS. The IL enhances the performance of ABS technology. The data puts in evidence the pivotal role of the appropriate selection of the ABS components and design to develop a successful extractive process, from both environmental and performance points of view.