A modified Pseudomonas aeruginosa strain capable of overexpressing the estA gene, an encoding gene for a membrane-bound esterase, was constructed and its rhamnolipid (RML) production was studied. ...Fermentations using wild-type (WT) and modified P. aeruginosa strains were conducted until exhaustion of glycerol in Medium Salt Production, using two different C/N ratios. At a C/N of 83.2, the modified strain produced up to 3.9 times more RMLs than the WT, yielding a maximum concentration of 14.62 g/L RML when measured by HPLC and 22 g/L by the orcinol assay. Cell-free supernatant from the modified strain reduced surface tension to 29.4 mN/m and had a CMC of 240 mg/L and CMD of 56.05. This is the first report on the construction of an estA-based recombinant strain for RML production.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Two strategies were investigated to perform the controlled degradation of two grades of poly(butylene succinate) (PBS): the chemical and enzymatic degradation routes. PBS chemical degradation assays ...were carried out in alkaline and acidic media at the temperature range between 40 and 80 °C. Additionally, the effects of distinct enzymes (cutinase, lipase, cellulase, protease, and amylase) that exhibit distinct hydrolysis activities were investigated on the enzymatic degradation of PBS. Two grades of PBS were compared: PBS microparticles manufactured through suspension polycondensation (PBS1) and; commercial PBS (PBS2) manufactured in bulk processes. Polymer degradation was evaluated through high-performance liquid chromatography, gel permeation chromatography, weight loss analyses, and determination of the pH of the supernatant. It was observed that PBS1 was subject to high rates of chemical degradation at higher temperatures, especially when degradation was conducted in alkaline media. On the other hand, the analyzed operation conditions did not affect the degradability of PBS2 significantly, which fluctuated around 5% of weight loss after 4 weeks. The degradability of both polymer matrices was much more intense when enzymes were used. For PBS1, the enzyme degradation effect was significant (weight loss of 57%, 32%, 25% after 4 weeks of experimentation, using cutinase, lipase, and amylase, respectively); for PBS2, after 1 month of experimentation, the weight loss using cutinase and lipase approached respectively 100% and 81%. It can be concluded that the application of enzymes can be beneficial for the controlled degradation of PBS at mild process conditions, allowing the development of environmental-friendly strategies for the controlled degradation of this material, and that both chemical and enzymatic degradations are sensitive to the characteristics of the analyzed samples, which must be carefully considered during the process development stage.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The side streams derived from the palm oil production process, namely palm kernel cake, palm pressed fibre, palm kernel shells and empty fruit bunches, were evaluated as sources of phenolic ...compounds. Among these streams, kernel cake had the highest total phenolic content (in mg of gallic acid equivalents (GAE) per g of dry sample) with a value of 5.19, whereas the empty fruit bunches had the lowest value (1.79). The extraction time and liquid-to-solid ratio were investigated to optimize the phenolic extraction. Kernel cake exhibited the highest total phenolic content (5.35 mg/g) with a liquid-to-solid ratio of 40:1 during 20 min of extraction. The main phenolic compounds of the extracts deriving from all byproduct streams were also identified and quantified with HPLC-DAD. Pyrogallol, 4-hydroxybenzoic acid, gallic acid and ferulic acid were the main compounds found in kernel cake extracts. Empty fruit bunch and pressed fibre extracts were also rich in 4-hydroxybenzoic acid, while pyrogallol was the predominant compound in kernel shell extracts. All extracts showed antioxidant activity as it was indicated from the results of DPPH analysis and subsequently tested in sunflower oil aiming to prolong its shelf life. The addition of 0.8% kernel cake extract increased the induction time of sunflower oil more than 50%. According to the results obtained in this study, kernel cake extracts could be considered as a value-added co-product with a potential application as antioxidants in the food industry.
ABSTRACT
A recombinant lipase B from Candida antarctica (LipB) in Pichia pastoris was synthesized through submerged fermentation using crude glycerin as substrate. The immobilization of this enzyme ...on the core–shell polymeric supports is an effective alternative for its application. The supports with distinct levels of hydrophobicity were produced through combined suspension and emulsion polymerization in pilot scale. Particles with distinct compositions were synthesized (PMMA/PMMA; PMMA‐co‐DVB/PMMA‐co‐DVB; and PS‐co‐DVB/PS‐co‐DVB) and employed on the immobilization of the produced lipase (LipB) and the commercial enzyme (CalB). The morphological properties (specific area, average pore diameter, specific volume of pores, and hydrophobicity level) and the influence of the polymerization conditions on the morphology of the supports were studied. The thermal stability of such biocatalysts was also investigated in the presence of calcium cation (Ca+2), maintained 100% of the activity after 3 h at 50°C when the PMMA‐co‐DVB/PMMA‐co‐DVB was employed. The synthesized enzyme and supports manufactured in pilot scale were employed successfully for production of esters using residual fatty acids as substrates, adding value to these raw materials and increasing the ranges of possible applications.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Flavonoids are one of the most important and diversified phenolic groups among products of natural origin. An important property of this metabolite class is the antioxidant action. This study ...evaluated the antioxidant and cytotoxic activities and oxidative stress of transesterification products of the flavonoid rutin, catalyzed by Novozym® 435. The presence of monoacetate and diacetate was confirmed by quantitative evaluation of the retention times (rutin, 15.68 min; rutin monoacetate, 18.14 min; and rutin diacetate, 18.57 min) and by the data from LC-MS and NMR 1H and 13C. The experiment showed excellent conversion values of 96% in total acetates (rutin monoacetate and diacetate). These results confirmed that rutin derivatives have antioxidant potential, as evaluated by the ORAC method (rutin standard: 0.53 ± 0.08 μM Trolox/g and rutin derivatives: 2.33 ± 1.08 μM Trolox/g) and also show low cytotoxicity in human and animal cells. Rutin derivatives reduced the production of reactive oxygen species in RAW macrophages as well. Many qualities attributed to rutin derivatives make them promising potential candidates for use as nutraceuticals, including their high amounts of antioxidants, biological potential and low toxicity, which contribute to the reduction of oxidative stress.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Biofilm formation on exposed surfaces is a serious issue for the food industry and medical health facilities. There are many proposed strategies to delay, reduce, or even eliminate biofilm formation ...on surfaces. The present study focuses on the applicability of fire ant venom alkaloids (aka 'solenopsins', from
) tested on polystyrene and stainless steel surfaces relative to the adhesion and biofilm-formation by the bacterium
. Conditioning with solenopsins demonstrates significant reduction of bacterial adhesion. Inhibition rates were 62.7% on polystyrene and 59.0% on stainless steel surfaces. In addition, solenopsins drastically reduced cell populations already growing on conditioned surfaces. Contrary to assumptions by previous authors, solenopsins tested negative for amphipathic properties, thus understanding the mechanisms behind the observed effects still relies on further investigation.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Yeast Surface Display (YSD) is a strategy to anchor proteins on the yeast cell wall which has been employed to increase enzyme stability thus decreasing production costs. Lipase B from Candida ...antarctica (LipB) is one of the most studied enzymes in the context of industrial biotechnology. This study aimed to assess the biochemical features of this important biocatalyst when immobilized on the cell surface of the methylotrophic yeast Pichia pastoris using the YSD approach. For that purpose, two anchors were tested. The first (Flo9) was identified after a prospection of the P. pastoris genome being related to the family of flocculins similar to Flo1 but significantly smaller. The second is the Protein with Internal Repeats (Pir1) from P. pastoris. An immunolocalization assay showed that both anchor proteins were able to display the reporter protein EGFP in the yeast outer cell wall. LipB was expressed in P. pastoris fused either to Flo9 (FLOLIPB) or Pir1 (PIRLIPB). Both constructions showed hydrolytic activity towards tributyrin (>100 U/mgdcw and >80 U/mgdcw, respectively), optimal hydrolytic activity around 45°C and pH 7.0, higher thermostability at 45°C and stability in organic solvents when compared to a free lipase.
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•Production of environmentally friendly polyol esters (biolubricants) through non-conventional catalysis.•The reaction was performed in a solvent-free system using a side stream from ...palm-oil refining (PFAD).•NMR analysis of the products showed that the PFAD-TMP and PFAD-DIP esters were composed mostly of triesters and diesters,•Properties of these esters were characterized, including DSC analysis, viscosity, pour point and oxidative stability.•The polyol esters showed promising lubricant properties within the acceptable range for many applications.
This contribution describes a sustainable, environmentally benign process for the production of biolubricants. The reaction was performed in a solvent-free system using a side stream from palm-oil refining (palm fatty-acid distillate, PFAD) and polyols as substrates for enzymatic catalysis. Biocatalysts consume less energy than chemical processes, and produce esters with good lubricant properties and environmentally friendly characteristics such as high biodegradability and low toxicity. The effects of different parameters (molar ratio, temperature and enzyme concentration) were evaluated during esterification of PFAD with neopentyl glycol (NPG) or trimethylolpropane (TMP) to produce polyol esters. The products obtained through esterification of PFAD with NPG and TMP in the stoichiometric molar ratio at 45 °C, using 4% (w/w) of Candida rugosa lipase, attained a modification of around 90% of hydroxyl groups (OH) at 10 and 8 h, respectively. These conditions were used to reach the maximum OH esterification for PFAD-TMP esters (94%) and PFAD-NPG esters (87%). NMR analysis of the final ester composition showed that the PFAD-TMP and PFAD-NPG esters were composed mostly of triesters and diesters, respectively. The properties of these esters were also characterized, including fusion and crystallization temperatures, viscosity, pour point and oxidative stability. The polyol esters showed promising lubricant properties, such as oxidative stability and viscosity within the acceptable range for many applications. This study demonstrated the potential of using PFAD for the production of environmentally benign polyol esters through non-conventional catalysis.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
The production of extracts containing a pool of enzymes for extensive biomass deconstruction can lead to significant advantages in biorefinery applications. In this work, a strain of Aspergillus ...awamori IOC-3914 was used for the simultaneous production of five groups of hydrolases by solid-state fermentation of babassu cake. Sequential experimental design strategies and multivariate optimization using the desirability function were first used to study temperature, moisture content, and granulometry. After that, further improvements in product yields were achieved by supplementation with other agro-industrial materials. At the end of the study, the production of enzymes was up to 3.3-fold increased, and brewer’s spent grains and babassu flour showed to be the best supplements. Maximum activities for endoamylases, exoamylases, cellulases (CMCases), xylanases, and proteases achieved were 197, 106, 20, 835, and 57 U g⁻¹, respectively. The strain was also able to produce β-glucosidases and debranching amylases (up to 35 and 43 U g⁻¹, respectively), indicating the potential of its enzyme pool for cellulose and starch degradation.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Summary
The use of the methylotrophic yeast Pichia pastoris (Komagataella phaffi) to produce heterologous proteins has been largely reported. However, investigations addressing the potential of this ...yeast to produce bulk chemicals are still scarce. In this study, we have studied the use of P. pastoris as a cell factory to produce the commodity chemical 3‐hydroxypropionic acid (3‐HP) from glycerol. 3‐HP is a chemical platform which can be converted into acrylic acid and to other alternatives to petroleum‐based products. To this end, the mcr gene from Chloroflexus aurantiacus was introduced into P. pastoris. This single modification allowed the production of 3‐HP from glycerol through the malonyl‐CoA pathway. Further enzyme and metabolic engineering modifications aimed at increasing cofactor and metabolic precursors availability allowed a 14‐fold increase in the production of 3‐HP compared to the initial strain. The best strain (PpHP6) was tested in a fed‐batch culture, achieving a final concentration of 3‐HP of 24.75 g l−1, a product yield of 0.13 g g−1 and a volumetric productivity of 0.54 g l−1 h−1, which, to our knowledge, is the highest volumetric productivity reported in yeast. These results benchmark P. pastoris as a promising platform to produce bulk chemicals for the revalorization of crude glycerol and, in particular, to produce 3‐HP.
Production of 3‐HP through the Malonyl‐CoA pathway was achieved in Pichia pastoris. Expressing the two subunits of the Malonyl‐CoA reductase separately led to a substantial increase in 3‐HP production. Further metabolic engineering to increase precursors availability increased the 3‐HP titre, achieving a final product concentration of 24.75 g l−1 in fed‐batch cultures, and the highest 3‐HP volumetric productivity value reported for yeast to date (0.54 g l−1 h−1).
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FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK
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