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

Potential for Waste Reduction in Activated Sludge Systems: Evaluation of the Initial Conditions of a Rapid Test with Rhamnolipid Biosurfactant

Adding chemicals that alter the microbial metabolism and reduce cell growth, without affecting organic matter removal, is an alternative technology to reduce sludge production. This study was intended to determine the best initial conditions to conduct a rapid test (4 h), which evaluates the potential of chemicals to reduce microbial growth and chemical oxygen demand removal. A commercial biosurfactant was used as model product and a central composite design (face centered) was performed using substrate/inoculum ratio and rhamnolipid/inoculum ratio as independent variables, and cellular yield coefficient, substrate consumption rate, and specific oxygen uptake rate for exogenous respiration as response variables. Lower values of substrate/inoculum ratio permitted larger reductions of cellular yield coefficient with lower rhamnolipid concentrations. The best condition was 1.06 g chemical oxygen demand/g total suspended solids and 25 mg rhamnolipid/L or 25 mg rhamnolipid/g total suspended solids, which achieved a reduction of 50-75% in cellular yield coefficient

Novel CaLB-like Lipase Found Using ProspectBIO, a Software for Genome-Based Bioprospection

Enzymes have been highly demanded in diverse applications such as in the food, pharmaceutical, and industrial fuel sectors. Thus, in silico bioprospecting emerges as an efficient strategy for discovering new enzyme candidates. A new program called ProspectBIO was developed for this purpose as it can find non-annotated sequences by searching for homologs of a model enzyme directly in genomes. Here we describe the ProspectBIO software methodology and the experimental validation by prospecting for novel lipases by sequence homology to Candida antarctica lipase B (CaLB) and conserved motifs. As expected, we observed that the new bioprospecting software could find more sequences (1672) than a conventional similarity-based search in a protein database (733). Additionally, the absence of patent protection was introduced as a criterion resulting in the final selection of a putative lipase-encoding gene from Ustilago hordei (UhL). Expression of UhL in Pichia pastoris resulted in the production of an enzyme with activity towards a tributyrin substrate. The recombinant enzyme activity levels were 4-fold improved when lowering the temperature and increasing methanol concentrations during the induction phase in shake-flask cultures. Protein sequence alignment and structural modeling showed that the recombinant enzyme has high similarity and capability of adjustment to the structure of CaLB. However, amino acid substitutions identified in the active pocket entrance may be responsible for the differences in the substrate specificities of the two enzymes. Thus, the ProspectBIO software allowed the finding of a new promising lipase for biotechnological application without the need for laborious and expensive conventional bioprospecting experimental steps

Measurement of the cosmic ray spectrum above 4×10184{\times}10^{18} eV using inclined events detected with the Pierre Auger Observatory

A measurement of the cosmic-ray spectrum for energies exceeding $4{\times}10^{18}$ eV is presented, which is based on the analysis of showers with zenith angles greater than $60^{\circ}$ detected with the Pierre Auger Observatory between 1 January 2004 and 31 December 2013. The measured spectrum confirms a flux suppression at the highest energies. Above $5.3{\times}10^{18}$ eV, the "ankle", the flux can be described by a power law $E^{-\gamma}$ with index $\gamma=2.70 \pm 0.02 \,\text{(stat)} \pm 0.1\,\text{(sys)}$ followed by a smooth suppression region. For the energy ($E_\text{s}$) at which the spectral flux has fallen to one-half of its extrapolated value in the absence of suppression, we find $E_\text{s}=(5.12\pm0.25\,\text{(stat)}^{+1.0}_{-1.2}\,\text{(sys)}){\times}10^{19}$ eV.Comment: Replaced with published version. Added journal reference and DO

Energy Estimation of Cosmic Rays with the Engineering Radio Array of the Pierre Auger Observatory

The Auger Engineering Radio Array (AERA) is part of the Pierre Auger Observatory and is used to detect the radio emission of cosmic-ray air showers. These observations are compared to the data of the surface detector stations of the Observatory, which provide well-calibrated information on the cosmic-ray energies and arrival directions. The response of the radio stations in the 30 to 80 MHz regime has been thoroughly calibrated to enable the reconstruction of the incoming electric field. For the latter, the energy deposit per area is determined from the radio pulses at each observer position and is interpolated using a two-dimensional function that takes into account signal asymmetries due to interference between the geomagnetic and charge-excess emission components. The spatial integral over the signal distribution gives a direct measurement of the energy transferred from the primary cosmic ray into radio emission in the AERA frequency range. We measure 15.8 MeV of radiation energy for a 1 EeV air shower arriving perpendicularly to the geomagnetic field. This radiation energy -- corrected for geometrical effects -- is used as a cosmic-ray energy estimator. Performing an absolute energy calibration against the surface-detector information, we observe that this radio-energy estimator scales quadratically with the cosmic-ray energy as expected for coherent emission. We find an energy resolution of the radio reconstruction of 22% for the data set and 17% for a high-quality subset containing only events with at least five radio stations with signal.Comment: Replaced with published version. Added journal reference and DO

Measurement of the Radiation Energy in the Radio Signal of Extensive Air Showers as a Universal Estimator of Cosmic-Ray Energy

We measure the energy emitted by extensive air showers in the form of radio emission in the frequency range from 30 to 80 MHz. Exploiting the accurate energy scale of the Pierre Auger Observatory, we obtain a radiation energy of 15.8 \pm 0.7 (stat) \pm 6.7 (sys) MeV for cosmic rays with an energy of 1 EeV arriving perpendicularly to a geomagnetic field of 0.24 G, scaling quadratically with the cosmic-ray energy. A comparison with predictions from state-of-the-art first-principle calculations shows agreement with our measurement. The radiation energy provides direct access to the calorimetric energy in the electromagnetic cascade of extensive air showers. Comparison with our result thus allows the direct calibration of any cosmic-ray radio detector against the well-established energy scale of the Pierre Auger Observatory.Comment: Replaced with published version. Added journal reference and DOI. Supplemental material in the ancillary file