Production, immobilization and synthesis of pharmacological derivatives of lipase B from Candida antarctica in Pichia pastoris

Lipase B from Candida antarctica (CALB) is widely used because of its excellent enantioselectivity. Producing this recombinant lipase in Pichia pastoris has advantages since it can be cultured in simple media and can reach high cell densities. This capability is especially important when using a constitutive promoter for lipase production, as here. The PPGK promoter is similar to the well-known PGAP promoter and also circumvents the need for inducing production with methanol, which is a hazard when used on a large scale and would increase the downstream production costs, which could be prohibitive for pharmaceutical products. This study tested two main fermentation strategies: continuous and fed-batch. In both cultures, different specific growth rates occurred (0.05, 0.10, 0.15 and 0.18h–1), and process parameters (qP, qS, YX/S, YP/X, YP/S) were evaluated in order to properly compare them. The highest specific production rate achieved with a continuous culture was 57.71 U/gX.h with µ=0.15 h–1 and 16 U/gX.h with µ=0.14 h–1 for a fed-batch culture. Productivity decreased dramatically near the µmax (0.18 h–1) for P. pastoris (57.6% lower). The best strategy for production was calculated over a three-month period. In both cases, the enzyme is secreted to the supernatant and purification is needed to ensure that only LIPB participates in further reactions. The immobilization process is ideal because purification and concentration is achieved in only one step, reusability is made possible, and in certain cases, stability and efficiency are boosted. Hydrophobic core-shell polymeric supports synthesized by a combined suspension and emulsion polymerization process have shown good potential for lipase immobilization procedures and were used in this study, compared to traditional supports such as Accurel, in order to determine their efficiency. After the enzyme was immobilized, the reactions included the resolution of (±)-1,3,5-O-benzyl-myo-inositol (DL-1) via acylation using vinyl acetate in hexane, and resolution of (±)-1,2-O- isopropylidene-3,6-di-O-benzyl-myo-inositol (DL-2) via acylation using vinyl acetate (solvent-free system). The support used directly affected the reaction, but trends were observed. In general, the recombinant lipase produced (LIPB) had higher resolutions than the commercial lipase (CALB, Novozym 435). In the resolution of DL-1 and DL-2 via transesterification (using different media), LIPB immobilized in Accurel or PS-co-DVB/PS-co-DVB showed more activity per enzyme molecule than CALB immobilized in similar supports, while when immobilized in PMMA-co-DVB/ PMMA-co-DVB the activities of the two enzymes were similar. The recombinant LIPB immobilized on PS-co-DVB proved to be the most efficient in the enantioselective resolution of both racemic derivatives, DL-1 and DL-2. The productivity for DL-2 resolution was 50% higher than the commercial Novozym 435, and the new derivative was operationally more stable than Novozym 435. The products obtained had a high level of purity (ee of 99% for both derivatives). Both products of the enantio-selective reaction, L-2 and L-5, obtained from the racemic derivatives (DL-1 and DL-2, respectively), are intermediates from different pharmacological pathways involved in the synthesis of building blocks for drugs that inhibit the etiological agent of Chagas disease, Trypanosoma cruzi

Immobilization of lipases on hydrophobic supports involves the open form of the enzyme

The lipases from Thermomyces lanuginosus and Pseudomonas cepacia have been immobilized on octyl and cyanogen bromide (CNBr) agarose beads. The immobilization on octyl-agarose is slowed with increasing ionic strength, The lipases from Thermomyces lanuginosus and Pseudomonas cepacia have been immobilized on octyl and cyanogen bromide (CNBr) agarose beads. The immobilization on octyl-agarose is slowed with increasing ionic strength, while the immobilization on CNBr is not significantly affected by the ionic strength. The inhibition of the immobilized preparations with diethyl p-nitrophenylphosphate (D-pNPP) was analyzed. The inhibition was more rapid using octyl-lipase preparations than using covalent preparations, and the covalent preparations were much more sensitive to the reaction medium. The addition of detergent increased the inhibition rate of the covalent preparation while an increase on the ionic strength produced a slowdown of the inhibition rate by D-pNPP for both lipases. The effect of the medium on the activity versus fully soluble substrate (methyl mandelate) was in the same direction. The octyl preparations presented a slight decrease in activity when comparing the results using different concentrations of sodium phosphate buffer (between 0.025 and 1 M), while the CNBr preparations suffered drastic drops in its activity at high ionic strength.The results confirm that the lipases immobilized on octyl agarose presented their open form stabilized while the covalent preparation maintains a closing/opening equilibrium that may be modulated by altering the medium.We gratefully recognize the support from the MINECO of Spanish Government, by the grant CTQ2013-41507-R, CNPq (PVE 301139/2014-8) and FAPERJ. The predoctoral fellowships for Ms. Rueda (Colciencias, Colombian Government) and Mr. dos Santos (CNPq, Brazil) are also recognized.Peer Reviewe

Accurel MP 1000 as a support for the immobilization of lipase from Burkholderia cepacia: Application to the kinetic resolution of myo-inositol derivatives

Lipase from Burkholderia cepacia (PS) has been immobilized on Accurel MP 1000, and their performance has been compared to that of the most widely used immobilized commercial preparation of this enzyme. The maximum loading was 18 mg protein/g support, and its thermal and solvent stability was much higher than that of the commercial. PS preparations were used in hydrolysis of triacetin and methyl mandelate, in the esterification of oleic acid and ethanol and in the kinetic resolution of 1,3,4-tri-O-benzyl-myo-inositol (DL-1) using vinylacetate as activated acyl donor. For all reactions studied, PS on Accurel was more active than PS-IM. The conversion in the kinetic resolution of racemic DL-1 was optimized using response surface methodology. Optimal conditions were determined to be 2.0 mg/mL of substrate, temperature of 40 °C, 2.0 mL of vinyl acetate and without water addition. Under these conditions, maximum loaded Accurel-PS preparation permitted to improve the activity in this kinetic resolution compared to the PS commercial preparation by a 55-fold factor, and compared to Novozym 435 (the most active described in literature for this reaction) by a 23-fold factor. The conversion attained was 49.9% ± 0.3 of conversion and ee of 99% after 24 h. The reusability studies showed maintenance of conversion and ee during eight cycles.We gratefully recognize the support from the MINECO of Spanish Government, by the grant CTQ2013-41507-R, and Brazilian FAPERJ, CNPq and CAPES for funding and/or fellowships, including a PVE project for R. Fernandez-Lafuente.Peer Reviewe

Phytoremediation of polycyclic aromatic hydrocarbons (PAH) by cv. Crioula: A Brazilian alfalfa cultivar

<p>This work aimed to evaluate the phytoremediation capacity of the alfalfa cultivar Crioula in soils contaminated with polycyclic aromatic hydrocarbons (PAHs), primary pollutants with mutagenic and carcinogenic potential. Alfalfa was grown from seed for 40 days on soil amended with anthracene, pyrene, and phenanthrene. Soil and plant tissue was collected for biometric assay, dry mass analysis, and PAH analysis by liquid chromatography. Increased total PAH concentration was associated with decreases in plant biomass, height, and internode length. The Crioula cultivar had a satisfactory phytoremediation effect, reducing total PAH concentration (300 ppm) in the experimental soil by 85% in 20 days, and by more than 95% in 40 days. The PAH showed a tendency to be removed in the temporal order: phenanthrene before pyrene before anthracene, and the removal ratio was influenced by the initial soil concentration of each PAH.</p> <p>Extracts containing PAH were obtained from soils cultivated with Medicago sativa L. (cv. Crioula) in the presence and absence of 150, 300, and 450 ppm total of PAH (phenanthrene, anthracene, and pyrene). After ultrasonication steps and purification of PAH by silica adsorption chromatography, samples containing PAH were quantified by high-performance liquid chromatography in reverse phase. At the end of this study, it was determined that the cv. Crioula showed a satisfactory phytoremediation potential. It presented the reduction of the total of PAH at around 85% in 20 days. </p

Preparation of core-shell polymer supports to immobilize lipase B from Candida antarctica: Effect of the support nature on catalytic properties

Core–shell supports have been prepared and utilized to immobilize lipase B from Candida antarctica. The hydrophobic nature of the supports permitted to immobilize the enzyme via interfacial activation at low ionic strength. Different supports were prepared having different hydrophobicity and crosslinking degree, and compared to the commercially available. Accurel MP 1000 (hydrophobic macroporous polymer of propylene) is a commercial support described as advantageous in different circumstances and it was used as comparative control in the process of immobilization. The immobilized lipase preparations were evaluated in the hydrolysis of p-nitro-phenyl laurate and the esterification of oleic acid with ethanol. On the kinetic resolution of (±)-1,2-O-isopropylidene-3,6-di-O-benzyl-myo-inositol, vinyl acetate was used as activated acyl donor. Results were very diverse, as the lipase properties may be easily tuned via immobilization, and some of the supports permitted to obtain activities even a two fold factor higher than the same amount of lipase immobilized in Accurel MP 1000. Moreover, in many instances, the loading of the support with enzyme produced reduced total activity in some reactions while not in other. This was explained by changes in the physical properties of the support surface that may alter the entry of substrates. Supports PS-co-DVB/PS-co-DVB 25% and PMMA-co-DVB/PMMA-co-DVB 25% presented very good features to immobilize CALB.This work was supported by grants from Petrobras, CNPq and CTQ2009-07568 from Spanish Ministerio de Ciencia e Innovación.Peer Reviewe

Preparation of core-shell polymer supports to immobilize lipase B from Candida antarctica: Effect of the support nature on catalytic properties

Core–shell supports have been prepared and utilized to immobilize lipase B from Candida antarctica. The hydrophobic nature of the supports permitted to immobilize the enzyme via interfacial activation at low ionic strength. Different supports were prepared having different hydrophobicity and crosslinking degree, and compared to the commercially available. Accurel MP 1000 (hydrophobic macroporous polymer of propylene) is a commercial support described as advantageous in different circumstances and it was used as comparative control in the process of immobilization. The immobilized lipase preparations were evaluated in the hydrolysis of p-nitro-phenyl laurate and the esterification of oleic acid with ethanol. On the kinetic resolution of (±)-1,2-O-isopropylidene-3,6-di-O-benzyl-myo-inositol, vinyl acetate was used as activated acyl donor. Results were very diverse, as the lipase properties may be easily tuned via immobilization, and some of the supports permitted to obtain activities even a two fold factor higher than the same amount of lipase immobilized in Accurel MP 1000. Moreover, in many instances, the loading of the support with enzyme produced reduced total activity in some reactions while not in other. This was explained by changes in the physical properties of the support surface that may alter the entry of substrates. Supports PS-co-DVB/PS-co-DVB 25% and PMMA-co-DVB/PMMA-co-DVB 25% presented very good features to immobilize CALB.This work was supported by grants from Petrobras, CNPq and CTQ2009-07568 from Spanish Ministerio de Ciencia e Innovación.Peer Reviewe

Revisiting the Heterogeneous IFN-γ Response of Bacille of Calmette-Guérin (BCG)-Revaccinated Healthy Volunteers in a Randomized Controlled Trial: Effect of the Body Mass Index and of the <i>IFNG</i>+874 A/T Polymorphism - Fig 1

<p><b>Genotype distribution of IFN-γ production (A, C, D) or T2/T0 IFN-γ ratio (B) in whole blood cultures stimulated with mycobacterial antigen.</b> (A, B) BCG-revaccinated subjects; A depicts IFN-γ production at baseline. (C) LTBI volunteers. (D) TB volunteers.</p