RESUMO
A new design of cross-linked enzyme aggregates (CLEAs) of Burkholderia cepacia lipase (BCL) based mainly on the use of lignocellulosic residue of palm fiber as an additive was proposed. Different parameters for the preparation of active CLEAs in the hydrolysis of olive oil, such as precipitation agents, crosslinking agent concentration, additives, and coating agents were investigated. The highest activity yield (121.1 ± 0.1%) and volumetric activity (1578.1 ± 2.5 U/mL) were achieved for CLEAs prepared using the combination of a coating step with Triton® X-100 and polyethyleneimine plus the use of palm fiber as an additive. The variations of the secondary structures of BCL-CLEAs were analyzed by second-derivative infrared spectra, mainly indicating a reduction of the α-helix structure, which was responsible for the lipase activation in the supramolecular structure of the CLEAs. Thus, these results provided evidence of an innovative design of BCL-CLEAs as a sustainable and biocompatible opportunity for biotechnology applications.
Assuntos
Proteínas de Bactérias/química , Burkholderia cepacia/enzimologia , Enzimas Imobilizadas/química , Lipase/química , Estabilidade Enzimática , CinéticaRESUMO
In order to recover biomolecules, a single downstream processing step is carried out. In this sense, an aqueous two-phase system (ATPS) containing polyethylene glycol (PEG) and potassium phosphate salts is used. Intending the purification of Burkholderia cepacia (Bc) lipase, the effects of the molecular masses of 1500 (PEG 1500), 4000 (PEG 4000), and 6000 (PEG 6000), pH (6, 7, and 8) and distinct tie line lengths are perfomed. Although this is reasonable reported in literature, a study covering an economical production aspect considering the Bc is scarce. This characterizes a novelty proposed in this investigation. Lipase is recovered in a polymer phase at lower pH value. PEG 1500/phosphate salt ATPS at pH 6 is considered a good method with ~ 98% of the extraction efficiency. Another contribution of this proposed investigation concerns to a biotechnological material synthesis, which is applied in several advanced and revolutionize engineering practices. Additionally, an economic analysis of the proposed method indicates a minimal sale price (~ US$410/L) inducing to a future and potential commercial application.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Burkholderia cepacia/enzimologia , Lipase/química , Lipase/isolamento & purificação , Proteínas de Bactérias/economia , Concentração de Íons de Hidrogênio , Lipase/economia , Polietilenoglicóis/químicaRESUMO
This study is focussed on a biocatalysing chemical synthesis in order to produce a green apple flavour (ethyl valerate) using an immobilised lipase from Burkholderia cepacia. A strategy to improve the lipase stability during the esterification is used. In order to increase the ethyl valerate efficiency, an alternative method using the buffer pH to dissolve the lipase into alginate is proposed. Parameters of the immobilised lipase such as pH, temperature, activation energy and stirring speed are evaluated. The optimal condition using the substrate concentration and the lipase loading is provided. After 5 recyclability cycles, the immobilised lipase reveals a decreasing â¼25% in the ethyl valerate yield. An economical ester synthetising associated with the esterification efficiency is evidenced. This induces that a potential industrial application can be considered. This due to the demand for ethyl valerate in the flavour industry is required.
Assuntos
Burkholderia cepacia/enzimologia , Enzimas Imobilizadas/metabolismo , Aromatizantes/metabolismo , Lipase/metabolismo , Valeratos/metabolismo , Alginatos/química , Biotecnologia , Enzimas Imobilizadas/química , Esterificação , Lipase/químicaRESUMO
In this work, the effect of several phosphonium-based ionic liquids (ILs) on the activity of lipase from Burkholderia cepacia (BCL) was evaluated by experimental assays and molecular docking. ILs comprising different cations ([P4444 ]+ , [P444(14) ]+ , [P666(14) ]+ ) and anions (Cl- , Br- , [Deca]- , [Phosp]- , [NTf2 ]- ) were investigated to appraise the individual roles of IL ions on the BCL activity. From the activity assays, it was found that an increase in the cation alkyl chain length leads to a decrease on the BCL enzymatic activity. ILs with the anions [Phosp]- and [NTf2 ]- increase the BCL activity, while the remaining [P666(14) ]-based ILs with the Cl- , Br- , and [Deca]- anions display a negative effect on the BCL activity. The highest activity of BCL was identified with the IL [P666(14) ][NTf2 ] (increase in the enzymatic activity of BCL by 61% at 0.055 mol·L-1 ). According to the interactions determined by molecular docking, IL cations preferentially interact with the Leu17 residue (amino acid present in the BCL oxyanion hole). The anion [Deca]- has a higher binding affinity compared to Cl- and Br- , and mainly interacts by hydrogen-bonding with Ser87, an amino acid residue which constitutes the catalytic triad of BCL. The anions [Phosp]- and [NTf2 ]- have high binding energies (-6.2 and -5.6 kcal·mol-1 , respectively) with BCL, and preferentially interact with the side chain amino acids of the enzyme and not with residues of the active site. Furthermore, FTIR analysis of the protein secondary structure show that ILs that lead to a decrease on the α-helix content result in a higher BCL activity, which may be derived from an easier access of the substrate to the BCL active site.