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L-arginine deiminase (ADI, EC 3.5.3.6) hydrolyzes arginine to ammonia and citrulline which is a natural supplement in health care. ADI was purified from Penicillium chrysogenum using 85% ammonium sulfate, DEAE-cellulose and Sephadex G200. ADI was purified 17.2-fold and 4.6% yield with a specific activity of 50 Umg- 1 protein. The molecular weight was 49 kDa. ADI expressed maximum activity at 40oC and an optimum pH of 6.0. ADI thermostability was investigated and the values of both t0.5 and D were determined. Kd increased by temperature and the Z value was 38oC. ATP, ADP and AMP activated ADI up to 0.6 mM. Cysteine and dithiothreitol activated ADI up to 60 µmol whereas the activation by thioglycolate and reduced glutathione (GSH) prolonged to 80 µmol. EDTA, α,α-dipyridyl, and o-phenanthroline inactivated ADI indicating that ADI is a metalloenzyme. N-ethylmaleimide (NEM), N-bromosuccinimide (NBS), butanedione (BD), dansyl chloride (DC), diethylpyrocarbonate (DEPC) and N-acetyl-imidazole (NAI) inhibited ADI activity indicating the necessity of sulfhydryl, tryptophanyl, arginyl, lysyl, histidyl and tyrosyl groups, respectively for ADI catalysis. The obtained results show that ADI from P. chrysogenum could be a potential candidate for industrial and biotechnological applications.

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Microorganisms produce secondary metabolites to survive under stressful conditions. The effect of drought and heat stress on fungi isolated from Arabian desert soil during the hot (ca 40°C) and cool (ca 10°C) seasons was studied using the genome mining approach. The presence of three stress-related genes (calmodulin, polyketide synthase and beta tubulin) was analyzed molecularly using specific primers. The presence of the genes in desert fungi was compared to their antimicrobial (ten bacterial or fungal pathogens) and anticancer (liver, cervical and breast) properties and the production of thermostable enzymes (phytase and xylanase). The genes appeared to be present in the fungal sequence obtained during the summer, while none of the genes were present during winter. Appreciable differences were observed in enzyme activities, with summer activities high and winter low. The antagonistic activities of A. niger were relatively stable and varying, while those of P. chrysogenum were consistently higher in summer than in winter. The presence of the three genes seemed to correlate with the highly antagonistic activities of P. chrysogenum, while A. niger had relatively active winter isolates without any of the genes. The hot season in deserts yields fungal isolates with biological activities useful in biotechnological solutions.

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Real-time measurements and adjustments of critical process parameters are essential for the precise control of fermentation processes and thus for increasing both quality and yield of the desired product. However, the measurement of some crucial process parameters such as biomass, product, and product precursor concentrations usually requires time-consuming offline laboratory analysis. In this work, we demonstrate the in-line monitoring of biomass, penicillin (PEN), and phenoxyacetic acid (POX) in a Penicilliumchrysogenum fed-batch fermentation process using low-cost microspectrometer technology operating in the near-infrared (NIR). In particular, NIR reflection spectra were taken directly through the glass wall of the bioreactor, which eliminates the need for an expensive NIR immersion probe. Furthermore, the risk of contaminations in the reactor is significantly reduced, as no direct contact with the investigated medium is required. NIR spectra were acquired using two sensor modules covering the spectral ranges 1350-1650 nm and 1550-1950 nm. Based on offline reference analytics, partial least squares (PLS) regression models were established for biomass, PEN, and POX either using data from both sensors separately or jointly. The established PLS models were tested on an independent validation fed-batch experiment. Root mean squared errors of prediction (RMSEP) were 1.61 g/L, 1.66 g/L, and 0.67 g/L for biomass, PEN, and POX, respectively, which can be considered an acceptable accuracy comparable with previously published results using standard process spectrometers with immersion probes. Altogether, the presented results underpin the potential of low-cost microspectrometer technology in real-time bioprocess monitoring applications. Graphical abstract.

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Fourier transform near-infrared (FT-NIR) spectroscopy combined with multivariate analysis has been applied in bioprocesses for a couple of decades. Nevertheless the papers published in this field are case-specific and do not focus on providing the community generic workflows to conduct experiments, especially as a standard Design of Experiment (DoE) for a multi-analyte process might require overwhelming amount of measurements. In this paper, a workflow for feasibility studies and inline implementation of FT-NIR spectrometer in multi-analyte fermentation processes is presented. The workflow is applied to Penicillium crysogenum fermentation, where the similarities in chemical structures and growth trends between the key analytes together with the aeration and growing fungi make the task challenging: first, the pure analytes are measured off-line with FT-NIR and clustered using principal component analysis. To study the separability of the gained clusters, a DoE approach by spiking is applied. The multivariate modelling of the separable analytes is conducted using the off-line and inline data followed by a comparison of the properties of the different models. Finally, the model output constraints are set by means of outlier diagnostics. As a result, biomass, penicillin (PEN), phenoxyacetic acid (POX), ammonia and biomass were shown to be separable with root mean square error of predictions of 2.62 g/l, 0.34 g/l, 0.51 g/l and 18.3 mM, respectively. Graphical abstract Flowchart illustrating the workflow for feasibility studies and implementation of models for inline monitoring of Ammonia, Biomass, Phenoxyacetic acid and Penicillin.

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The aim of present work was to effectively remediate grease waste by Penicillium chrysogenum. For efficient degradation, grease waste was pre-treated using various lipases, among them lipolase was the best. The pretreated grease was used as a substrate by P. chrysogenum resulting into the production of fatty acids. Process was optimized by response surface methodology (RSM) using four variables viz; FeCl2 (mM), spore concentration (spores/ml), time period (days) and amount of grease (g). The optimized conditions viz; FeCl2 1.25mM, culture amount 5×1011spores/ml and time period 16days led to the production of 6.6mg/g fatty acid from 10.0g of pre-treated grease mixed with 5.0g wheat bran in 10.0ml czapek-dox medium under solid state fermentation. The fermented media was extracted with hexane and subjected to GCMS analysis, which showed the presence of higher amount of palmitic acid. It was purified by crystallization method and 2.8g of palmitic acid was recovered from 1.0kg grease waste in tray fermentation.

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The scale-up of fermentation processes frequently leads to a reduced productivity compared to small-scale screening experiments. Large-scale mixing limitations that lead to gradients in substrate and oxygen availability could influence the microorganism performance. Here, the impact of substrate gradients on a penicillin G producing Penicillium chrysogenum cultivation was analyzed using an intermittent glucose feeding regime. The intermittent feeding led to fluctuations in the extracellular glucose concentration between 400 µM down to 6.5 µM at the end of the cycle. The intracellular metabolite concentrations responded strongly and showed up to 100-fold changes. The intracellular flux changes were estimated on the basis of dynamic (13) C mass isotopomer measurements during three cycles of feast and famine using a novel hybrid modeling approach. The flux estimations indicated a high turnover of internal and external storage metabolites in P. chrysogenum under feast/famine conditions. The synthesis and degradation of storage requires cellular energy (ATP and UTP) in competition with other cellular functions including product formation. Especially, 38% of the incoming glucose was recycled once in storage metabolism. This result indicated that storage turnover is increased under dynamic cultivation conditions and contributes to the observed decrease in productivity compared to reference steady-state conditions.

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This paper presents the quantification of Penicillin V and phenoxyacetic acid, a precursor, inline during Pencillium chrysogenum fermentations by FTIR spectroscopy and partial least squares (PLS) regression and multivariate curve resolution - alternating least squares (MCR-ALS). First, the applicability of an attenuated total reflection FTIR fiber optic probe was assessed offline by measuring standards of the analytes of interest and investigating matrix effects of the fermentation broth. Then measurements were performed inline during four fed-batch fermentations with online HPLC for the determination of Penicillin V and phenoxyacetic acid as reference analysis. PLS and MCR-ALS models were built using these data and validated by comparison of single analyte spectra with the selectivity ratio of the PLS models and the extracted spectral traces of the MCR-ALS models, respectively. The achieved root mean square errors of cross-validation for the PLS regressions were 0.22 g L(-1) for Penicillin V and 0.32 g L(-1) for phenoxyacetic acid and the root mean square errors of prediction for MCR-ALS were 0.23 g L(-1) for Penicillin V and 0.15 g L(-1) for phenoxyacetic acid. A general work-flow for building and assessing chemometric regression models for the quantification of multiple analytes in bioprocesses by FTIR spectroscopy is given.

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Species classified in Penicillium sect. Chrysogena are primary soil-borne and the most well-known members are P. chrysogenum and P. nalgiovense. Penicillium chrysogenum has received much attention because of its role in the production on penicillin and as a contaminant of indoor environments and various food and feedstuffs. Another biotechnologically important species is P. nalgiovense, which is used as a fungal starter culture for the production of fermented meat products. Previous taxonomic studies often had conflicting species circumscriptions. Here, we present a multigene analysis, combined with phenotypic characters and extrolite data, demonstrating that sect. Chrysogena consists of 18 species. Six of these are newly described here (P. allii-sativi, P. desertorum, P. goetzii, P. halotolerans, P. tardochrysogenum, P. vanluykii) and P. lanoscoeruleum was found to be an older name for P. aethiopicum. Each species produces a unique extrolite profile. The species share phenotypic characters, such as good growth on CYA supplemented with 5 % NaCl, ter- or quarterverticillate branched conidiophores and short, ampulliform phialides (< 9 µm). Conidial colours, production of ascomata and ascospores, shape and ornamentation of conidia and growth rates on other agar media are valuable for species identification. Eight species (P. allii-sativi, P. chrysogenum, P. dipodomyis, P. flavigenum, P. nalgiovense, P. rubens, P. tardochrysogenum and P. vanluykii) produce penicillin in culture.