Síntesis enzimàtica d'un aromatitzant

Investigadors del Departament d'Enginyeria Química de la UAB han dut a terme amb èxit la síntesis enzimàtica d'un aromatitzant. Aquesta síntesis permet obtindre compostos aromatitzants "naturals" sense que suposin un cost tan elevat com els compostos extrets directament del producte natural. Els científics, que han sintetitzat enzims que s'utilitzen com a biocatalitzadors per obtenir aromatitzants, també han determinat diferents paràmetres, com la temperatura o la concentració d'enzim, que permetran establir les condicions òptimes per a la producció industrial d'aquests aromatitzant.Investigadores del Departamento de Ingeniería Química de la UAB han llevado a cabo con éxito la síntesis enzimática de un aromatizante. Esta síntesis permite obtener compuestos aromatizantes "naturales" sin que supongan un coste tan elevado como los compuestos extraídos directamente del producto natural. Los científicos, que han sintetizado enzimas que se utilizan como biocatalizadores para obtener aromatizantes, también han determinado los diferentes parámetros, como la temperatura o la concentración de enzima, que permitirán establecer las condiciones óptimas para la producción industrial de estos aromatizantes.Researchers at the Department of Chemical Engineering at UAB have carried out successfully a flavor enzymatic synthesis. This synthesis allows to obtain a "natural" flavor compound without the high cost of the natural product directly extracted compound. Scientists have synthesized enzymes that are used to produce flavors and also have stated the parameters, such as temperature or the concentration of enzyme, that will provide the optimal conditions for the industrial production of these flavorings

Recent advances in lipases and their applications in the food and nutraceutical industry

This research was funded by the Spanish Ministry of Science and Innovation (Project PID2019-104666GB-100) (AEI/10.13039/501100011033).Lipases are efficient enzymes with promising applications in the nutraceutical and food industry, as they can offer high yields, pure products under achievable reaction conditions, and are an environmentally friendly option. This review addresses the production of high-value-added compounds such as fatty acid esters, with the potential to be used as flavoring agents or antioxidant and antimicrobial agents, as well as structured lipids that offer specific functional properties that do not exist in nature, with important applications in different food products, and pharmaceuticals. In addition, the most recent successful cases of reactions with lipases to produce modified compounds for food and nutraceuticals are reported

Constitutive expression in komagataella phaffii of mature rhizopus oryzae lipase jointly with its truncated prosequence improves production and the biocatalyst operational stability

Altres ajuts: Eusko Jaurlaritza/PRE_2017_1_0110Rhizopus oryzae lipase (ROL) containing 28 C-terminal amino acids of the prosequence fused to the N-terminal mature sequence in ROL (proROL) was successfully expressed in the methylotrophic yeast Komagataella phaffii (Pichia pastoris) under the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (PGAP). Although the sequence encoding the mature lipase (rROL) was also transformed, no clones were obtained after three transformation cycles, which highlights the importance of the truncated prosequence to obtain viable transformed clones. Batch cultures of the K. phaffii strain constitutively expressing proROL scarcely influenced growth rate and exhibited a final activity and volumetric productivity more than six times higher than those obtained with proROL from K. phaffii under the methanol-inducible alcohol oxidase 1 promoter (PAOX1). The previous differences were less marked in fed-batch cultures. N-terminal analysis con-firmed the presence of the 28 amino acids in proROL. In addition, immobilized proROL exhibited increased tolerance of organic solvents and an operational stability 0.25 and 3 times higher than that of immobilized rROL in biodiesel and ethyl butyrate production, respectively. Therefore, the truncated prosequence enables constitutive proROL production, boosts bioprocess performance and provides a more stable biocatalyst in two reactions in which lipases are mostly used at industrial level, esterification (ethyl butyrate) and transesterification (biodiesel)

Second- and third-generation biodiesel production with immobilised recombinant Rhizopus oryzae lipase : Influence of the support, substrate acidity and bioprocess scale-up

Altres ajuts: Beca del Gobierno Vasco para la formación de investigadores predoctorales PRE_2017_1_0110Altres ajuts: Acord transformatiu CRUE-CSICRhizopus oryzae lipase immobilised onto differently functionalised polymethacrylate (Purolite®) and magnetite superparamagnetic supports was assessed as a catalyst for biodiesel production with pomace oil. The presence of surface hydrocarbon chains increased the operational stability of the biocatalysts supported on Purolite® and superparamagnetic particles up to 9 and 2 times, respectively. By contrast, the presence of functional groups had no effect on the initial transesterification rate, which was twice higher with the lipase immobilised onto Purolite®. Also, functionalising Purolite® with epoxide and octadecyl groups led to the highest biodiesel and volumetric productivity. This biocatalyst with other substrates including makauba, jatropha, waste cooking oil, and microbial oil, led to similar initial reaction rates. However, simply raising substrate acidity from 0.5 to 2% increased the operational stability of the biocatalysts 15 times. A synergistic effect between acyl-acceptor concentration and substrate acidity was observed. The transesterification reaction was successfully scaled up to 50 mL

De la cuina al laboratori : Aplicacions de les microones en química analítica

Mesurar el contingut de nitrogen d'una mostra té importants aplicacions en el tractament d'aigües residuals, o en la indústria alimentaria, per posar uns exemples. Sovint s'utilitza l'anomenat mètode de Kjeldahl, que requereix l'aplicació de calor. Científics de la UAB han investigat l'ús de microones per generar aquesta calor.Medir el contenido de nitrógeno de una muestra tiene importantesaplicaciones en el tratamiento de aguas residuales, o en la industriaalimentaria, por poner unos ejemplos. A menudo se utiliza el llamadométodo de Kjeldahl, que requiere la aplicación de calor. Científicos de laUAB han investigado el uso de microondas para generarlo

A step forward to improve recombinant protein production in Pichia pastoris : from specific growth rate effect on protein secretion to carbon-starving conditions as advanced strategy

The recombinant protein production platform based on the GAP promoter and Pichia pastoris as a host has become a very promising system from an industrial point of view. The need for highly productive bioprocesses gives grounds for the optimization of fermentation strategies maximizing yields and/or productivities, which are often associated with cell growth. Coherent with previous studies, a positive effect of high specific growth rate (μ) on the productivity was observed in carbon-limited chemostat cultivations secreting an antibody fragment. Notably, no significant impact of this factor could be observed in the balance intra- and extracellular of the product. Accordingly, fed-batch cultures operating at a constant high μ were conducted. Furthermore, short carbon-starving periods were introduced along the exponential substrate feeding phase. Strikingly, it was observed an important increase of specific production rate (qP) during such short carbon-starving periods in relation to the exponential substrate feeding intervals. Therefore, the application of carbon-starving periods as an innovative operational strategy was proposed, resulting into increments up to 50% of both yields and total production. The implementation of the proposed substrate feeding profiles should be complementary to cell engineering strategies to improve the relation qP vs μ, thereby enhancing the overall bioprocess efficiency

Co-composting of sewage sludge : fats mixtures and characteristics of the lipases involved

Co-composting of sewage sludge and animal fat mixtures was studied in order to determine the possibility of using this technology to recycle fat-enriched wastes. A maximum fat content of 30% in fat:sludge mixtures is recommended to achieve the international sanitation requirements on compost quality and to avoid an excessive thermophilic composting time. Under these conditions a fat content reduction of 85% was achieved. Biological activity was highly dependent on the moisture content as shown by the respiratory quotient values. Moisture content is a critical control factor because of the hydrophobic nature of fats and should be maintained above 40% in the composting of fats. Biological indices of the compost obtained after 69 days of process (maturity grade: IV; respiration index: 1.1 mg O₂ g OM⁻¹ h⁻¹) indicated a high stability and maturity degree of the material. Lipases responsible for fat hydrolysis were monitored during the composting process and a sample from the thermophilic period was characterized in terms of stability in front of pH and temperature. Optimal conditions for lipase stability were found at 38.3 °C and pH 7.97, however, the maximum lipolytic activity was observed at thermophilic temperatures. Lipases from the thermophilic period were purified by anion exchange chromatography and visualised by SDS-PAGE. Two major bands were observed at molecular weights of 29 and 62 kDa. These bands could not be identified precisely by N-terminal sequence analysis

Rational development of bioprocess engineering strategies for recombinant protein production in Pichia pastoris (Komagataella phaffii) using the methanol-free GAP promoter. Where do we stand?

The increasing demand for recombinant proteins for a wide range of applications, from biopharmaceutical protein complexes to industrial enzymes, is leading to important growth in this market. Among the different efficient host organism alternatives commonly used for protein production, the yeast Pichia pastoris (Komagataella phaffii) is currently considered to be one of the most effective and versatile expression platforms. The promising features of this cell factory are giving rise to interesting studies covering the different aspects that contribute to improving the bioprocess efficiency, from strain engineering to bioprocess engineering. The numerous drawbacks of using methanol in industrial processes are driving interest towards methanol-free alternatives, among which the GAP promoter-based systems stand out. The aim of this work is to present the most promising innovative developments in operational strategies based on rational approaches through bioprocess engineering tools. This rational design should be based on physiological characterization of the producing strains under bioprocess conditions and its interrelation with specific rates. This review focuses on understanding the key factors that can enhance recombinant protein production in Pichia pastoris; they are the basis for a further discussion on future industrial applications with the aim of developing scalable alternative strategies that maximize yields and productivity

Effect of acyl-acceptor stepwise addition strategy using alperujo oil as a substrate in enzymatic biodiesel synthesis

Background: using renewable feedstock sources for biodiesel production seems to be a promising strategy and even more so when enzymatic catalysis with lipases are used. However, it is well known that these enzymes could be inactivated by reaction conditions such as temperature or alcohol concentration. In this work, the effect of temperature and initial water activity (aw) value on immobilised recombinant Rhizopus oryzae lipase (rROL) were studied. Methanolysis and ethanolysis reactions using alperujo oil with three different stepwise addition strategies were employed. - Results: recombinant 1,3-positional selective rROL covalently immobilised on polymethacrylate amino-epoxy activated support showed maximum initial reaction rate at low aw value (0.093). It was found that 30 °C was the optimal temperature in terms of biocatalyst stability during transesterification reactions. Adding alcohol at once, ethanol was clearly a better acyl-acceptor in terms of stability than methanol. Productivity was found to be 2-fold higher when five pulses of ethanol were used instead of methanol. - Conclusions: alperujo oil has great potential as a low cost feedstock for biodiesel production through enzymatic catalysis using a nearly semi-continuous alcohol addition strategy

Continuous Cultivation as a Tool Toward the Rational Bioprocess Development With Pichia Pastoris Cell Factory

The methylotrophic yeast Pichia pastoris (Komagataella phaffii) is currently considered one of the most promising hosts for recombinant protein production (RPP) and metabolites due to the availability of several tools to efficiently regulate the recombinant expression, its ability to perform eukaryotic post-translational modifications and to secrete the product in the extracellular media. The challenge of improving the bioprocess efficiency can be faced from two main approaches: the strain engineering, which includes enhancements in the recombinant expression regulation as well as overcoming potential cell capacity bottlenecks; and the bioprocess engineering, focused on the development of rational-based efficient operational strategies. Understanding the effect of strain and operational improvements in bioprocess efficiency requires to attain a robust knowledge about the metabolic and physiological changes triggered into the cells. For this purpose, a number of studies have revealed chemostat cultures to provide a robust tool for accurate, reliable, and reproducible bioprocess characterization. It should involve the determination of key specific rates, productivities, and yields for different C and N sources, as well as optimizing media formulation and operating conditions. Furthermore, studies along the different levels of systems biology are usually performed also in chemostat cultures. Transcriptomic, proteomic and metabolic flux analysis, using different techniques like differential target gene expression, protein description and 13 C-based metabolic flux analysis, are widely described as valued examples in the literature. In this scenario, the main advantage of a continuous operation relies on the quality of the homogeneous samples obtained under steady-state conditions, where both the metabolic and physiological status of the cells remain unaltered in an all-encompassing picture of the cell environment. This contribution aims to provide the state of the art of the different approaches that allow the design of rational strain and bioprocess engineering improvements in Pichia pastoris toward optimizing bioprocesses based on the results obtained in chemostat cultures. Interestingly, continuous cultivation is also currently emerging as an alternative operational mode in industrial biotechnology for implementing continuous process operations