The Experts below are selected from a list of 258 Experts worldwide ranked by ideXlab platform
Maria A.m. Reis - One of the best experts on this subject based on the ideXlab platform.
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Low Temperature Dissolution of Yeast Chitin-Glucan Complex and Characterization of the Regenerated Polymer.
Bioengineering (Basel Switzerland), 2020Co-Authors: Diana Araújo, Elvira Fortunato, Maria A.m. Reis, Vítor D. Alves, Ana C. Marques, Filomena FreitasAbstract:Chitin-glucan complex (CGC) is a copolymer composed of chitin and glucan moieties extracted from the cell-walls of several yeasts and fungi. Despite its proven valuable properties, that include antibacterial, antioxidant and anticancer activity, the utilization of CGC in many applications is hindered by its insolubility in water and most solvents. In this study, NaOH/urea solvent systems were used for the first time for solubilization of CGC extracted from the yeast Komagataella pastoris. Different NaOH/urea ratios (6:8, 8:4 and 11:4 (w/w), respectively) were used to obtain aqueous solutions using a freeze/thaw procedure. There was an overall solubilization of 63–68%, with the highest solubilization rate obtained for the highest tested urea concentration (8 wt%). The regenerated polymer, obtained by dialysis of the alkali solutions followed by lyophilization, formed porous macrostructures characterized by a chemical composition similar to that of the starting co-polymer, although the acetylation degree decreased from 61.3% to 33.9–50.6%, indicating that chitin was converted into chitosan, yielding chitosan-glucan complex (ChGC). Consistent with this, there was a reduction of the crystallinity index and thermal degradation temperature. Given these results, this study reports a simple and green procedure to solubilize CGC and obtain aqueous ChGC solutions that can be processed as novel biomaterials.
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Co-production of chitin-glucan complex and xylitol by Komagataella pastoris using glucose and xylose mixtures as carbon source.
Carbohydrate polymers, 2017Co-Authors: Diana Araújo, Christian Grandfils, Filomena Freitas, Chantal Sevrin, Maria A.m. ReisAbstract:Abstract Komagataella pastoris was cultivated in glucose/xylose mixtures for production of chitin-glucan complex (CGC), a cell-wall polysaccharide. The culture preferred glucose as substrate for growth, resulting in high biomass yields (0.46–0.54 g/g). After glucose depletion, xylose was consumed but no cell growth was observed, indicating K. pastoris was unable to use it for growth. Interestingly, concomitant with xylose consumption, xylitol synthesis was noticed, reaching a maximum concentration of 7.64 g/L, with a yield on xylose of 0.52 g/g. Lower CGC production was reached as the xylose content was increased in the substrate mixtures, due to the lower biomass production. Moreover, cultivation in the presence of xylitol resulted in CGC enriched in chitin with higher molecular weight. These results suggest the possibility of using K. pastoris for the co-production of CGC and xylitol using glucose/xylose-rich substrates. It may also be a strategy to tailor CGC composition and average molecular weight.
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Implementation of a repeated fed-batch process for the production of chitin-glucan complex by Komagataella pastoris.
New biotechnology, 2016Co-Authors: Inês Farinha, Filomena Freitas, Maria A.m. ReisAbstract:The yeast Komagataella pastoris was cultivated under different fed-batch strategies for the production of chitin-glucan complex (CGC), a co-polymer of chitin and β-glucan. The tested fed-batch strategies included DO-stat mode, predefined feeding profile and repeated fed-batch operation. Although high cell dry mass and high CGC production were obtained under the tested DO-stat strategy in a 94h cultivation (159 and 29g/L, respectively), the overall biomass and CGC productivities were low (41 and 7.4g/Lday, respectively). Cultivation with a predefined profile significantly improved both biomass and CGC volumetric productivity (87 and 10.8g/Lday, respectively). Hence, this strategy was used to implement a repeated fed-batch process comprising 7 consecutive cycles. A daily production of 119-126g/L of biomass with a CGC content of 11-16wt% was obtained, thus proving this cultivation strategy is adequate to reach a high CGC productivity that ranged between 11 and 18g/Lday. The process was stable and reproducible in terms of CGC productivity and polymer composition, making it a promising strategy for further process development.
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chitin glucan complex production by Komagataella pastoris downstream optimization and product characterization
Carbohydrate Polymers, 2015Co-Authors: Inês Farinha, Paulo Duarte, Ana Pimentel, Evgeniya Plotnikova, Ferreira Chagas Bárbara, Luís Mafra, Christian Grandfils, Filomena Freitas, Elvira Fortunato, Maria A.m. ReisAbstract:Abstract Purified chitin–glucan complex (CGC pure ) was extracted from Komagataella pastoris biomass using a hot alkaline treatment, followed by neutralization and repeated washing with deionized water. The co-polymer thus obtained had a β-glucan:chitin molar ratio of 75:25 and low protein and inorganic salts contents (3.0 and 0.9 wt%, respectively). CGC pure had an average molecular weight of 4.9 × 10 5 Da with a polydispersity index of 1.7, and a crystallinity index of 50%. Solid-state NMR provided structural insight at the co-polymer. X-ray diffraction suggests that CGC pure has α-chitin in its structure. CGC pure presented an endothermic decomposition peak at 315 °C, assigned to the degradation of the saccharide structures. This study revealed that K. pastoris CGC has properties similar to other chitinous biopolymers and may represent an attractive alternative to crustacean chitin derived-products, being a reliable raw material for the development of new/improved pharmaceutical, cosmetic or food products.
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Chitin–glucan complex production by Komagataella pastoris: Downstream optimization and product characterization
Carbohydrate polymers, 2015Co-Authors: Inês Farinha, Paulo Duarte, Ana Pimentel, Evgeniya Plotnikova, Ferreira Chagas Bárbara, Luís Mafra, Christian Grandfils, Filomena Freitas, Elvira Fortunato, Maria A.m. ReisAbstract:Abstract Purified chitin–glucan complex (CGC pure ) was extracted from Komagataella pastoris biomass using a hot alkaline treatment, followed by neutralization and repeated washing with deionized water. The co-polymer thus obtained had a β-glucan:chitin molar ratio of 75:25 and low protein and inorganic salts contents (3.0 and 0.9 wt%, respectively). CGC pure had an average molecular weight of 4.9 × 10 5 Da with a polydispersity index of 1.7, and a crystallinity index of 50%. Solid-state NMR provided structural insight at the co-polymer. X-ray diffraction suggests that CGC pure has α-chitin in its structure. CGC pure presented an endothermic decomposition peak at 315 °C, assigned to the degradation of the saccharide structures. This study revealed that K. pastoris CGC has properties similar to other chitinous biopolymers and may represent an attractive alternative to crustacean chitin derived-products, being a reliable raw material for the development of new/improved pharmaceutical, cosmetic or food products.
Filomena Freitas - One of the best experts on this subject based on the ideXlab platform.
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Chitin-glucan complex - Based biopolymeric structures using biocompatible ionic liquids.
Carbohydrate polymers, 2020Co-Authors: Inês C. Ferreira, Filomena Freitas, Diana Araújo, Pierre Voisin, Vítor D. Alves, Andreia A. Rosatella, Carlos A. M. Afonso, Luísa A. NevesAbstract:Abstract This work explores the novelty of dissolving chitin-glucan complex (CGC), from two fungal strains, Komagataella pastoris (CGCP) and Aspergillus niger (CGCKZ) (KiOnutrime-CG™), using biocompatible ionic liquids (ILs). Three cholinium-based ILs were tested, choline acetate, choline propionate and choline hexanoate. Although all tested ILs resulted in the dissolution of the co-polymer at a concentration of 5 % (w/w), distinct polymeric structures, films or gels, were obtained from CGCP and CGCKZ, respectively. CGCP films were dense, flexible and elastic, with high swelling capacity (> 200 %). The IL anion alkyl chain length influenced the polymeric structures’ properties, namely, the CGCP films elongation at break and swelling degree. CGCKZ resulted in weak gels. For both polymeric structures, exposure to the ILs under the dissolution conditions caused significant changes in the co-polymers’ chemical structure, namely, reduction of their glucan moiety and reduction of the degree of acetylation, thus yielding chitosan-glucan complexes (ChGC) enriched in glucosamine (53.4 ± 0.3–60.8 ± 0.3 %).
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Low Temperature Dissolution of Yeast Chitin-Glucan Complex and Characterization of the Regenerated Polymer.
Bioengineering (Basel Switzerland), 2020Co-Authors: Diana Araújo, Elvira Fortunato, Maria A.m. Reis, Vítor D. Alves, Ana C. Marques, Filomena FreitasAbstract:Chitin-glucan complex (CGC) is a copolymer composed of chitin and glucan moieties extracted from the cell-walls of several yeasts and fungi. Despite its proven valuable properties, that include antibacterial, antioxidant and anticancer activity, the utilization of CGC in many applications is hindered by its insolubility in water and most solvents. In this study, NaOH/urea solvent systems were used for the first time for solubilization of CGC extracted from the yeast Komagataella pastoris. Different NaOH/urea ratios (6:8, 8:4 and 11:4 (w/w), respectively) were used to obtain aqueous solutions using a freeze/thaw procedure. There was an overall solubilization of 63–68%, with the highest solubilization rate obtained for the highest tested urea concentration (8 wt%). The regenerated polymer, obtained by dialysis of the alkali solutions followed by lyophilization, formed porous macrostructures characterized by a chemical composition similar to that of the starting co-polymer, although the acetylation degree decreased from 61.3% to 33.9–50.6%, indicating that chitin was converted into chitosan, yielding chitosan-glucan complex (ChGC). Consistent with this, there was a reduction of the crystallinity index and thermal degradation temperature. Given these results, this study reports a simple and green procedure to solubilize CGC and obtain aqueous ChGC solutions that can be processed as novel biomaterials.
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Optimization of medium composition for production of chitin-glucan complex and mannose-containing polysaccharides by the yeast Komagataella pastoris.
Journal of biotechnology, 2019Co-Authors: Inês Farinha, Diana Araújo, Filomena FreitasAbstract:Abstract Komagataella pastoris was recently proposed as a source of valuable polysaccharides, namely, the co-polymer chitin-glucan complex (CGC) and mannose-containing polymers (mannans), that are extracted from its cell-wall. In this study, a novel cultivation medium, Medium K, was developed envisaging the simultaneous production of both types of cell-wall polysaccharides. The use of Medium K for the cultivation of K. pastoris resulted in high contents of CGC (19 wt%) and mannans (21 wt%) in the biomass, corresponding to significantly higher products’ volumetric productivities (17.5 and 19.2 g/L day, respectively) compared to previous studies. The produced CGC had a chitin:β-glucan molar ratio of 12:88, similarly to previously reported values for K. pastoris CGC (11:89–19:81), while the mannans were mainly composed of mannose units, with a protein content of 10 wt%. These results demonstrated that the developed optimized medium was suitable for cultivation of K. pastoris and the production of both CGC and mannans. It comprised fewer components with considerably reduced salts content, thus representing a significant simplification of the bioprocess with no precipitation problems, without impacting on the polymers’ composition.
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Co-production of chitin-glucan complex and xylitol by Komagataella pastoris using glucose and xylose mixtures as carbon source.
Carbohydrate polymers, 2017Co-Authors: Diana Araújo, Christian Grandfils, Filomena Freitas, Chantal Sevrin, Maria A.m. ReisAbstract:Abstract Komagataella pastoris was cultivated in glucose/xylose mixtures for production of chitin-glucan complex (CGC), a cell-wall polysaccharide. The culture preferred glucose as substrate for growth, resulting in high biomass yields (0.46–0.54 g/g). After glucose depletion, xylose was consumed but no cell growth was observed, indicating K. pastoris was unable to use it for growth. Interestingly, concomitant with xylose consumption, xylitol synthesis was noticed, reaching a maximum concentration of 7.64 g/L, with a yield on xylose of 0.52 g/g. Lower CGC production was reached as the xylose content was increased in the substrate mixtures, due to the lower biomass production. Moreover, cultivation in the presence of xylitol resulted in CGC enriched in chitin with higher molecular weight. These results suggest the possibility of using K. pastoris for the co-production of CGC and xylitol using glucose/xylose-rich substrates. It may also be a strategy to tailor CGC composition and average molecular weight.
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Implementation of a repeated fed-batch process for the production of chitin-glucan complex by Komagataella pastoris.
New biotechnology, 2016Co-Authors: Inês Farinha, Filomena Freitas, Maria A.m. ReisAbstract:The yeast Komagataella pastoris was cultivated under different fed-batch strategies for the production of chitin-glucan complex (CGC), a co-polymer of chitin and β-glucan. The tested fed-batch strategies included DO-stat mode, predefined feeding profile and repeated fed-batch operation. Although high cell dry mass and high CGC production were obtained under the tested DO-stat strategy in a 94h cultivation (159 and 29g/L, respectively), the overall biomass and CGC productivities were low (41 and 7.4g/Lday, respectively). Cultivation with a predefined profile significantly improved both biomass and CGC volumetric productivity (87 and 10.8g/Lday, respectively). Hence, this strategy was used to implement a repeated fed-batch process comprising 7 consecutive cycles. A daily production of 119-126g/L of biomass with a CGC content of 11-16wt% was obtained, thus proving this cultivation strategy is adequate to reach a high CGC productivity that ranged between 11 and 18g/Lday. The process was stable and reproducible in terms of CGC productivity and polymer composition, making it a promising strategy for further process development.
Inês Farinha - One of the best experts on this subject based on the ideXlab platform.
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Optimization of medium composition for production of chitin-glucan complex and mannose-containing polysaccharides by the yeast Komagataella pastoris.
Journal of biotechnology, 2019Co-Authors: Inês Farinha, Diana Araújo, Filomena FreitasAbstract:Abstract Komagataella pastoris was recently proposed as a source of valuable polysaccharides, namely, the co-polymer chitin-glucan complex (CGC) and mannose-containing polymers (mannans), that are extracted from its cell-wall. In this study, a novel cultivation medium, Medium K, was developed envisaging the simultaneous production of both types of cell-wall polysaccharides. The use of Medium K for the cultivation of K. pastoris resulted in high contents of CGC (19 wt%) and mannans (21 wt%) in the biomass, corresponding to significantly higher products’ volumetric productivities (17.5 and 19.2 g/L day, respectively) compared to previous studies. The produced CGC had a chitin:β-glucan molar ratio of 12:88, similarly to previously reported values for K. pastoris CGC (11:89–19:81), while the mannans were mainly composed of mannose units, with a protein content of 10 wt%. These results demonstrated that the developed optimized medium was suitable for cultivation of K. pastoris and the production of both CGC and mannans. It comprised fewer components with considerably reduced salts content, thus representing a significant simplification of the bioprocess with no precipitation problems, without impacting on the polymers’ composition.
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Implementation of a repeated fed-batch process for the production of chitin-glucan complex by Komagataella pastoris.
New biotechnology, 2016Co-Authors: Inês Farinha, Filomena Freitas, Maria A.m. ReisAbstract:The yeast Komagataella pastoris was cultivated under different fed-batch strategies for the production of chitin-glucan complex (CGC), a co-polymer of chitin and β-glucan. The tested fed-batch strategies included DO-stat mode, predefined feeding profile and repeated fed-batch operation. Although high cell dry mass and high CGC production were obtained under the tested DO-stat strategy in a 94h cultivation (159 and 29g/L, respectively), the overall biomass and CGC productivities were low (41 and 7.4g/Lday, respectively). Cultivation with a predefined profile significantly improved both biomass and CGC volumetric productivity (87 and 10.8g/Lday, respectively). Hence, this strategy was used to implement a repeated fed-batch process comprising 7 consecutive cycles. A daily production of 119-126g/L of biomass with a CGC content of 11-16wt% was obtained, thus proving this cultivation strategy is adequate to reach a high CGC productivity that ranged between 11 and 18g/Lday. The process was stable and reproducible in terms of CGC productivity and polymer composition, making it a promising strategy for further process development.
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chitin glucan complex production by Komagataella pastoris downstream optimization and product characterization
Carbohydrate Polymers, 2015Co-Authors: Inês Farinha, Paulo Duarte, Ana Pimentel, Evgeniya Plotnikova, Ferreira Chagas Bárbara, Luís Mafra, Christian Grandfils, Filomena Freitas, Elvira Fortunato, Maria A.m. ReisAbstract:Abstract Purified chitin–glucan complex (CGC pure ) was extracted from Komagataella pastoris biomass using a hot alkaline treatment, followed by neutralization and repeated washing with deionized water. The co-polymer thus obtained had a β-glucan:chitin molar ratio of 75:25 and low protein and inorganic salts contents (3.0 and 0.9 wt%, respectively). CGC pure had an average molecular weight of 4.9 × 10 5 Da with a polydispersity index of 1.7, and a crystallinity index of 50%. Solid-state NMR provided structural insight at the co-polymer. X-ray diffraction suggests that CGC pure has α-chitin in its structure. CGC pure presented an endothermic decomposition peak at 315 °C, assigned to the degradation of the saccharide structures. This study revealed that K. pastoris CGC has properties similar to other chitinous biopolymers and may represent an attractive alternative to crustacean chitin derived-products, being a reliable raw material for the development of new/improved pharmaceutical, cosmetic or food products.
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Chitin–glucan complex production by Komagataella pastoris: Downstream optimization and product characterization
Carbohydrate polymers, 2015Co-Authors: Inês Farinha, Paulo Duarte, Ana Pimentel, Evgeniya Plotnikova, Ferreira Chagas Bárbara, Luís Mafra, Christian Grandfils, Filomena Freitas, Elvira Fortunato, Maria A.m. ReisAbstract:Abstract Purified chitin–glucan complex (CGC pure ) was extracted from Komagataella pastoris biomass using a hot alkaline treatment, followed by neutralization and repeated washing with deionized water. The co-polymer thus obtained had a β-glucan:chitin molar ratio of 75:25 and low protein and inorganic salts contents (3.0 and 0.9 wt%, respectively). CGC pure had an average molecular weight of 4.9 × 10 5 Da with a polydispersity index of 1.7, and a crystallinity index of 50%. Solid-state NMR provided structural insight at the co-polymer. X-ray diffraction suggests that CGC pure has α-chitin in its structure. CGC pure presented an endothermic decomposition peak at 315 °C, assigned to the degradation of the saccharide structures. This study revealed that K. pastoris CGC has properties similar to other chitinous biopolymers and may represent an attractive alternative to crustacean chitin derived-products, being a reliable raw material for the development of new/improved pharmaceutical, cosmetic or food products.
Ursula Rinas - One of the best experts on this subject based on the ideXlab platform.
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Fate of the UPR marker protein Kar2/Bip and autophagic processes in fed-batch cultures of secretory insulin precursor producing Pichia pastoris
Microbial Cell Factories, 2018Co-Authors: Gustavo Roth, Ana Letícia Vanz, Heinrich Lünsdorf, Manfred Nimtz, Ursula RinasAbstract:Background Secretory recombinant protein production with Pichia ( syn. Komagataella ) pastoris is commonly associated with the induction of an unfolded protein response (UPR) usually apparent through increased intracellular levels of endoplasmic reticulum (ER) resident chaperones such as Kar2/Bip. During methanol-induced secretory production of an insulin precursor (IP) under industrially relevant fed-batch conditions the initially high level of intracellular Kar2/Bip after batch growth on glycerol unexpectedly declined in the following methanol fed-batch phase misleadingly suggesting that IP production had a low impact on UPR activation. Results Analysis of the protein production independent level of Kar2/Bip revealed that high Kar2/Bip levels were reached in the exponential growth phase of glycerol batch cultures followed by a strong decline of Kar2/Bip during entry into stationary phase. Ultra-structural cell morphology studies revealed autophagic processes (e.g. ER phagy) at the end of the glycerol batch phase most likely responsible for the degradation of ER resident chaperones such as Kar2/Bip. The pre-induction level of Kar2/Bip did not affect the IP secretion efficiency in the subsequent methanol-induced IP production phase. During growth on methanol intracellular Kar2/Bip levels declined in IP producing and non-producing host cells. However, extracellular accumulation of Kar2/Bip was observed in IP-producing cultures but not in non-producing controls. Most importantly, the majority of the extracellular Kar2/Bip accumulated in the culture supernatant of IP producing cells as truncated protein (approx. 35 kDa). Conclusions Rapid growth leads to higher basal levels of the major UPR marker protein Kar2/Bip independent of recombinant protein production. Entry into stationary phase or slower growth on poorer substrate, e.g. methanol, leads to a lower basal Kar2/Bip level. Methanol-induced secretory IP production elicits a strong UPR activation which counteracts the reduced UPR during slow growth on methanol. The major ER chaperone Kar2/Bip is found together with recombinant IP in the culture medium where full-length Kar2/Bip accumulates in addition to large amounts of truncated Kar2/Bip. Thus, for judging UPR activating properties of the produced protein it is important to additionally analyze the medium not only for intact Kar2/Bip but also for truncated versions of this UPR reporter protein.
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Fate of the UPR marker protein Kar2/Bip and autophagic processes in fed-batch cultures of secretory insulin precursor producing Pichia pastoris.
Microbial cell factories, 2018Co-Authors: Gustavo Roth, Ana Letícia Vanz, Heinrich Lünsdorf, Manfred Nimtz, Ursula RinasAbstract:Secretory recombinant protein production with Pichia (syn. Komagataella) pastoris is commonly associated with the induction of an unfolded protein response (UPR) usually apparent through increased intracellular levels of endoplasmic reticulum (ER) resident chaperones such as Kar2/Bip. During methanol-induced secretory production of an insulin precursor (IP) under industrially relevant fed-batch conditions the initially high level of intracellular Kar2/Bip after batch growth on glycerol unexpectedly declined in the following methanol fed-batch phase misleadingly suggesting that IP production had a low impact on UPR activation. Analysis of the protein production independent level of Kar2/Bip revealed that high Kar2/Bip levels were reached in the exponential growth phase of glycerol batch cultures followed by a strong decline of Kar2/Bip during entry into stationary phase. Ultra-structural cell morphology studies revealed autophagic processes (e.g. ER phagy) at the end of the glycerol batch phase most likely responsible for the degradation of ER resident chaperones such as Kar2/Bip. The pre-induction level of Kar2/Bip did not affect the IP secretion efficiency in the subsequent methanol-induced IP production phase. During growth on methanol intracellular Kar2/Bip levels declined in IP producing and non-producing host cells. However, extracellular accumulation of Kar2/Bip was observed in IP-producing cultures but not in non-producing controls. Most importantly, the majority of the extracellular Kar2/Bip accumulated in the culture supernatant of IP producing cells as truncated protein (approx. 35 kDa). Rapid growth leads to higher basal levels of the major UPR marker protein Kar2/Bip independent of recombinant protein production. Entry into stationary phase or slower growth on poorer substrate, e.g. methanol, leads to a lower basal Kar2/Bip level. Methanol-induced secretory IP production elicits a strong UPR activation which counteracts the reduced UPR during slow growth on methanol. The major ER chaperone Kar2/Bip is found together with recombinant IP in the culture medium where full-length Kar2/Bip accumulates in addition to large amounts of truncated Kar2/Bip. Thus, for judging UPR activating properties of the produced protein it is important to additionally analyze the medium not only for intact Kar2/Bip but also for truncated versions of this UPR reporter protein.
Diana Araújo - One of the best experts on this subject based on the ideXlab platform.
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Chitin-glucan complex - Based biopolymeric structures using biocompatible ionic liquids.
Carbohydrate polymers, 2020Co-Authors: Inês C. Ferreira, Filomena Freitas, Diana Araújo, Pierre Voisin, Vítor D. Alves, Andreia A. Rosatella, Carlos A. M. Afonso, Luísa A. NevesAbstract:Abstract This work explores the novelty of dissolving chitin-glucan complex (CGC), from two fungal strains, Komagataella pastoris (CGCP) and Aspergillus niger (CGCKZ) (KiOnutrime-CG™), using biocompatible ionic liquids (ILs). Three cholinium-based ILs were tested, choline acetate, choline propionate and choline hexanoate. Although all tested ILs resulted in the dissolution of the co-polymer at a concentration of 5 % (w/w), distinct polymeric structures, films or gels, were obtained from CGCP and CGCKZ, respectively. CGCP films were dense, flexible and elastic, with high swelling capacity (> 200 %). The IL anion alkyl chain length influenced the polymeric structures’ properties, namely, the CGCP films elongation at break and swelling degree. CGCKZ resulted in weak gels. For both polymeric structures, exposure to the ILs under the dissolution conditions caused significant changes in the co-polymers’ chemical structure, namely, reduction of their glucan moiety and reduction of the degree of acetylation, thus yielding chitosan-glucan complexes (ChGC) enriched in glucosamine (53.4 ± 0.3–60.8 ± 0.3 %).
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Low Temperature Dissolution of Yeast Chitin-Glucan Complex and Characterization of the Regenerated Polymer.
Bioengineering (Basel Switzerland), 2020Co-Authors: Diana Araújo, Elvira Fortunato, Maria A.m. Reis, Vítor D. Alves, Ana C. Marques, Filomena FreitasAbstract:Chitin-glucan complex (CGC) is a copolymer composed of chitin and glucan moieties extracted from the cell-walls of several yeasts and fungi. Despite its proven valuable properties, that include antibacterial, antioxidant and anticancer activity, the utilization of CGC in many applications is hindered by its insolubility in water and most solvents. In this study, NaOH/urea solvent systems were used for the first time for solubilization of CGC extracted from the yeast Komagataella pastoris. Different NaOH/urea ratios (6:8, 8:4 and 11:4 (w/w), respectively) were used to obtain aqueous solutions using a freeze/thaw procedure. There was an overall solubilization of 63–68%, with the highest solubilization rate obtained for the highest tested urea concentration (8 wt%). The regenerated polymer, obtained by dialysis of the alkali solutions followed by lyophilization, formed porous macrostructures characterized by a chemical composition similar to that of the starting co-polymer, although the acetylation degree decreased from 61.3% to 33.9–50.6%, indicating that chitin was converted into chitosan, yielding chitosan-glucan complex (ChGC). Consistent with this, there was a reduction of the crystallinity index and thermal degradation temperature. Given these results, this study reports a simple and green procedure to solubilize CGC and obtain aqueous ChGC solutions that can be processed as novel biomaterials.
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Optimization of medium composition for production of chitin-glucan complex and mannose-containing polysaccharides by the yeast Komagataella pastoris.
Journal of biotechnology, 2019Co-Authors: Inês Farinha, Diana Araújo, Filomena FreitasAbstract:Abstract Komagataella pastoris was recently proposed as a source of valuable polysaccharides, namely, the co-polymer chitin-glucan complex (CGC) and mannose-containing polymers (mannans), that are extracted from its cell-wall. In this study, a novel cultivation medium, Medium K, was developed envisaging the simultaneous production of both types of cell-wall polysaccharides. The use of Medium K for the cultivation of K. pastoris resulted in high contents of CGC (19 wt%) and mannans (21 wt%) in the biomass, corresponding to significantly higher products’ volumetric productivities (17.5 and 19.2 g/L day, respectively) compared to previous studies. The produced CGC had a chitin:β-glucan molar ratio of 12:88, similarly to previously reported values for K. pastoris CGC (11:89–19:81), while the mannans were mainly composed of mannose units, with a protein content of 10 wt%. These results demonstrated that the developed optimized medium was suitable for cultivation of K. pastoris and the production of both CGC and mannans. It comprised fewer components with considerably reduced salts content, thus representing a significant simplification of the bioprocess with no precipitation problems, without impacting on the polymers’ composition.
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Co-production of chitin-glucan complex and xylitol by Komagataella pastoris using glucose and xylose mixtures as carbon source.
Carbohydrate polymers, 2017Co-Authors: Diana Araújo, Christian Grandfils, Filomena Freitas, Chantal Sevrin, Maria A.m. ReisAbstract:Abstract Komagataella pastoris was cultivated in glucose/xylose mixtures for production of chitin-glucan complex (CGC), a cell-wall polysaccharide. The culture preferred glucose as substrate for growth, resulting in high biomass yields (0.46–0.54 g/g). After glucose depletion, xylose was consumed but no cell growth was observed, indicating K. pastoris was unable to use it for growth. Interestingly, concomitant with xylose consumption, xylitol synthesis was noticed, reaching a maximum concentration of 7.64 g/L, with a yield on xylose of 0.52 g/g. Lower CGC production was reached as the xylose content was increased in the substrate mixtures, due to the lower biomass production. Moreover, cultivation in the presence of xylitol resulted in CGC enriched in chitin with higher molecular weight. These results suggest the possibility of using K. pastoris for the co-production of CGC and xylitol using glucose/xylose-rich substrates. It may also be a strategy to tailor CGC composition and average molecular weight.
