In this communication, lipase A from
(CALA) was immobilized by covalent bonding on magnetic nanoparticles coated with chitosan and activated with glutaraldehyde, labelled CALA-MNP, (immobilization ...parameters: 84.1% ± 1.0 for immobilization yield and 208.0 ± 3.0 U/g ± 1.1 for derivative activity). CALA-MNP biocatalyst was characterized by X-ray Powder Diffraction (XRPD), Fourier Transform Infrared (FTIR) spectroscopy, Thermogravimetry (TG) and Scanning Electron Microscope (SEM), proving the incorporation of magnetite and the immobilization of CALA in the chitosan matrix. Besides, the immobilized biocatalyst showed a half-life 8-11 times higher than that of the soluble enzyme at pH 5-9. CALA showed the highest activity at pH 7, while CALA-MNP presented the highest activity at pH 10. The immobilized enzyme was more active than the free enzyme at all studied pH values, except pH 7.
Penicillin G acylase (PGA) from
was immobilized on vinyl sulfone (VS) agarose. The immobilization of the enzyme failed at all pH values using 50 mM of buffer, while the progressive increase of ionic ...strength permitted its rapid immobilization under all studied pH values. This suggests that the moderate hydrophobicity of VS groups is enough to transform the VS-agarose in a heterofunctional support, that is, a support bearing hydrophobic features (able to adsorb the proteins) and chemical reactivity (able to give covalent bonds). Once PGA was immobilized on this support, the PGA immobilization on VS-agarose was optimized with the purpose of obtaining a stable and active biocatalyst, optimizing the immobilization, incubation and blocking steps characteristics of this immobilization protocol. Optimal conditions were immobilization in 1 M of sodium sulfate at pH 7.0, incubation at pH 10.0 for 3 h in the presence of glycerol and phenyl acetic acid, and final blocking with glycine or ethanolamine. This produced biocatalysts with stabilities similar to that of the glyoxyl-PGA (the most stable biocatalyst of this enzyme described in literature), although presenting just over 55% of the initially offered enzyme activity versus the 80% that is recovered using the glyoxyl-PGA. This heterofuncionality of agarose VS beads opens new possibilities for enzyme immobilization on this support.
The synthesis of ethyl butyrate catalyzed by lipases A (CALA) or B (CALB) from
immobilized onto magnetic nanoparticles (MNP), CALA-MNP and CALB-MNP, respectively, is hereby reported. MNPs were ...prepared by co-precipitation, functionalized with 3-aminopropyltriethoxysilane, activated with glutaraldehyde, and then used as support to immobilize either CALA or CALB (immobilization yield: 100 ± 1.2% and 57.6 ± 3.8%; biocatalysts activities: 198.3 ± 2.7 U
/g and 52.9 ± 1.7 U
/g for CALA-MNP and CALB-MNP, respectively). X-ray diffraction and Raman spectroscopy analysis indicated the production of a magnetic nanomaterial with a diameter of 13.0 nm, whereas Fourier-transform infrared spectroscopy indicated functionalization, activation and enzyme immobilization. To determine the optimum conditions for the synthesis, a four-variable Central Composite Design (CCD) (biocatalyst content, molar ratio, temperature and time) was performed. Under optimized conditions (1:1, 45 °C and 6 h), it was possible to achieve 99.2 ± 0.3% of conversion for CALA-MNP (10 mg) and 97.5 ± 0.8% for CALB-MNP (12.5 mg), which retained approximately 80% of their activity after 10 consecutive cycles of esterification. Under ultrasonic irradiation, similar conversions were achieved but at 4 h of incubation, demonstrating the efficiency of ultrasound technology in the enzymatic synthesis of esters.
► C. vulgaris and C. gracilis displayed the best suitability toward oil production. ► Tetraselmis sp., Isochrysis sp. andT. chui were unsuitable for oil production. ► Dunaliella sp. and T. ...weissflogii were unsuitable for large scale oil production. ► The salinity of the medium showed high influence on production of biomass and oil. ► The species Chaetoceros gracilis was not affected by salinity.
Microalgae have the ability to grow rapidly, synthesize and accumulate large amounts (approximately 20–50% of dry weight) of lipids. A successful and economically viable algae based oil industry depends on the selection of appropriate algal strains. In this study ten species of microalgae were prospected to determine their suitability for oil production: Chaetoceros gracilis, Chaetoceros mulleri, Chlorella vulgaris, Dunaliella sp., Isochrysis sp., Nannochloropsis oculata, Tetraselmis sp., Tetraselmis chui, Tetraselmis tetrathele and Thalassiosira weissflogii. The study was carried out in 3L glass flasks subjected to constant aeration and controlled artificial illumination and temperature at two different salinities. After harvesting, the extraction of oil was carried out using the Bligh and Dyer method assisted by ultrasound. Results showed that C. gracilis presented the highest oil content and that C. vulgaris presented the highest oil production.
Lipase from
Rhizomucor miehei
(RML) was immobilized onto chitosan support in the presence of some surfactants added at low levels using two different strategies. In the first approach, the enzyme was ...immobilized in the presence of surfactants on chitosan supports previously functionalized with glutaraldehyde. In the second one, after prior enzyme adsorption on chitosan beads in the presence of surfactants, the complex chitosan beads-enzyme was then cross-linked with glutaraldehyde. The effects of surfactant concentrations on the activities of free and immobilized RML were evaluated. Hexadecyltrimethylammonium bromide (CTAB) promoted an inhibition of enzyme activity while the nonionic surfactant Triton X-100 caused a slight increase in the catalytic activity of the free enzyme and the derivatives produced in both methods of immobilization. The best derivatives were achieved when the lipase was firstly adsorbed on chitosan beads at 4 °C for 1 h, 220 rpm followed by cross-link the complex chitosan beads-enzyme with glutaraldehyde 0.6% v.v
−1
at pH 7. The derivatives obtained under these conditions showed high catalytic activity and excellent thermal stability at 60° and 37 °C. The best derivative was also evaluated in the synthesis of two flavor esters namely methyl and ethyl butyrate. At non-optimized conditions, the maximum conversion yield for methyl butyrate was 89%, and for ethyl butyrate, the esterification yield was 92%. The results for both esterifications were similar to those obtained when the commercial enzyme Lipozyme® and free enzyme were used in the same reaction conditions and higher than the one achieved in the absence of the selected surfactant.
Lipase B from
was immobilized on heterofunctional support octyl agarose activated with vinyl sulfone to prevent enzyme release under drastic conditions. Covalent attachment was established, but the ...blocking step using hexylamine, ethylenediamine or the amino acids glycine (Gly) and aspartic acid (Asp) altered the results. The activities were lower than those observed using the octyl biocatalyst, except when using ethylenediamine as blocking reagent and
-nitrophenol butyrate (
) as substrate. The enzyme stability increased using these new biocatalysts at pH 7 and 9 using all blocking agents (much more significantly at pH 9), while it decreased at pH 5 except when using Gly as blocking agent. The stress inactivation of the biocatalysts decreased the enzyme activity versus three different substrates (
NPB, S-methyl mandelate and triacetin) in a relatively similar fashion. The tryptophane (Trp) fluorescence spectra were different for the biocatalysts, suggesting different enzyme conformations. However, the fluorescence spectra changes during the inactivation were not too different except for the biocatalyst blocked with Asp, suggesting that, except for this biocatalyst, the inactivation pathways may not be so different.
The lipase from
(PFL) has been immobilized on octyl-agarose beads under 16 different conditions (varying pH, ionic strength, buffer, adding some additives) at two different loadings, 1 and 60 mg of ...enzyme/g of support with the objective of check if this can alter the biocatalyst features. The activity of the biocatalysts versus
-nitrophenyl butyrate and triacetin and their thermal stability were studied. The different immobilization conditions produced biocatalysts with very different features. Considering the extreme cases, using 1 mg/g preparations, PFL stability changed more than fourfolds, while their activities versus
NPB or triacetin varied a 50-60%. Curiously, PFL specific activity versus triacetin was higher using highly enzyme loaded biocatalysts than using lowly loaded biocatalysts (even by a twofold factor). Moreover, stability of the highly loaded preparations was higher than that of the lowly loaded preparations, in many instances even when using 5°C higher temperatures (e.g., immobilized in the presence of calcium, the highly loaded biocatalysts maintained after 24 h at 75°c a 85% of the initial activity, while the lowly loaded preparation maintained only 27% at 70°C). Using the highly loaded preparations, activity of the different biocatalysts versus
NPB varied almost 1.7-folds and versus triacetin 1.9-folds. In this instance, the changes in stability caused by the immobilization conditions were much more significant, some preparations were almost fully inactivated under conditions where the most stable one maintained more than 80% of the initial activity. Results suggested that immobilization conditions greatly affected the properties of the immobilized PFL, partially by individual molecule different conformation (observed using lowly loaded preparations) but much more relevantly using highly loaded preparations, very likely by altering some enzyme-enzyme intermolecular interactions. There is not an optimal biocatalyst considering all parameters. That way, preparation of biocatalysts using this support may be a powerful tool to tune enzyme features, if carefully controlled.
► Bligh and Dyer method assisted by ultrasound resulted in the highest extraction of oil. ► Addition of powder silica did not improve the extraction of oil. ► Diffusion mechanism was more important ...than cell rupture during extraction. ► Polar solvent systems improve the extraction of microalgae oil.
Microalgae have the ability to grow rapidly, synthesize and accumulate large amounts (approximately 20–50% of dry weight) of lipids. A successful and economically viable algae based oil industry will depend on the selection of appropriate microalgal strains and the selection of the most suitable lipid extraction method. In this paper, five extraction methods were evaluated regarding the extraction of lipids from Chlorella vulgaris: Bligh and Dyer, Chen, Folch, Hara and Radin, and Soxhlet. Furthermore, the addition of silica powder was studied to evaluate the introduction of more shear stress to the system as to increase the disruption of cell walls. Among the studied methods, the Bligh and Dyer method assisted by ultrasound resulted in the highest extraction of oil from C. vulgaris (52.5% w/w). Addition of powder silica did not improve the extraction of oil.
This study reports an alternative strategy for the expression of a recombinant
l
-AI from
Enterococcus faecium
DBFIQ E36 by auto-induction using glucose and glycerol as carbon sources and residual ...whey lactose as inducer agent. Commercial lactose and isopropyl
β
-
d
-1-thiogalactopyranoside (IPTG) were also evaluated as inducers for comparison of enzyme expression levels. The enzymatic extracts were purified by affinity chromatography, characterized, and applied in the bioconversion of
d
-galactose into
d
-tagatose.
l
-AI presented a catalytic activity of 1.67 ± 0.14, 1.52 ± 0.01, and 0.7 ± 0.04 U/mL, when expressed using commercial lactose, lactose from whey, and IPTG, respectively. Higher activities could be obtained by changing the protocol of enzyme extraction and, for instance, the enzymatic extract produced with whey presented a catalytic activity of 3.8 U/mL. The specific activity of the enzyme extracts produced using lactose (commercial or residual whey) after enzyme purification was also higher when compared to the enzyme expressed with IPTG. Best results were achieved when enzyme expression was conducted using 4 g/L of residual whey lactose for 11 h. These results proved the efficacy of an alternative and economic protocol for the effective expression of a recombinant
l
-AI aiming its high-scale production.
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