Odabir plijesni Aspergillus fumigatus za proizvodnju prebiotičkih ksilooligosaharida na podlozi od šećerne trske

Sugarcane bagasse is an important lignocellulosic material studied for the production of xylooligosaccharides (XOS). Some XOS are considered soluble dietary fibre, with low caloric value and prebiotic effect, but they are expensive and not easily available. In a screening of 138 fungi, only nine were shortlisted, and just Aspergillus fumigatus M51 (35.6 U/mL) and A. fumigatus U2370 (28.5 U/mL) were selected as the most significant producers of xylanases. These fungi had low β-xylosidase activity, which is desirable for the production of XOS. The xylanases from Trichoderma reesei CCT 2768, A. fumigatus M51 and A. fumigatus U2370 gave a significantly higher XOS yield, 11.9, 14.7 and 7.9 % respectively, in a 3-hour reaction with hemicellulose from sugarcane bagasse. These enzymes are relatively thermostable at 40–50 °C and can be used in a wide range of pH values. Furthermore, these xylanases produced more prebiotic XOS (xylobiose and xylotriose) when compared with a commercial xylanase. The xylanases from A. fumigatus M51 reached a high level of XOS production (37.6 %) in 48–72 h using hemicellulose extracted from sugarcane bagasse. This yield represents 68.8 kg of prebiotic XOS per metric tonne of cane bagasse. In addition, in a biorefinery, after hemicellulose extraction for XOS production, the residual cellulose could be used for the production of second-generation ethanol.Šećerna trska je važan lignocelulozni materijal koji se koristi za ispitivanje proizvodnje ksilooligosaharida. Neki se ksilooligosaharidi upotrebljavaju u prehrani kao topljiva vlakna niske kalorijske vrijednosti s prebiotičkim učinkom, ali su skupi i teško dostupni. Od 138 plijesni u uži je izbor ušlo samo njih devet, od kojih su odabrana samo dva soja što su proizvela najviše ksilanaza, i to Aspergillus fumigatus M51 (35,6 U/mL) i A. fumigatus U2370 (28,5 u/mL). Aktivnost β-ksilozidaze u tim sojevima bila je vrlo slaba, što je pogodovalo nastanku ksilooligosaharida. Djelovanjem ksilanaza su nakon tri sata iz šećerne trske razgradnjom hemiceluloze dobivene veće količine ksilooligosaharida, i to 11,9 % s pomoću plijesni Trichoderma reesei CCT 2768; 14,7 % s pomoću A. fumigatus M51 i 7,9 % s pomoću A. fumigatus U2370. Ti su enzimi relativno termostabilni na temperaturama od 40 do 50 °C, a mogu se koristiti pri različitim pH-vrijednostima. Osim toga, u usporedbi s komercijalnom ksilanazom, ove su ksilanaze proizvele više prebiotičkih ksilooligosaharida (ksilobioze i ksilotrioze). Razgradnjom hemiceluloze izolirane iz šećerne trske pomoću ksilanaze iz plijesni A. fumigatus M51 dobivena je znatna količina (37,6 %) ksilooligosaharida nakon 48-72 sata hidrolize, što odgovara prinosu od 68,8 kg prebiotičkih ksilooligosaharida po toni šećerne trske. Osim toga, nakon izdvajanja hemiceluloze za proizvodnju ksilooligosaharida, preostali dio celuloze može se upotrijebiti u rafineriji za proizvodnju druge generacije biogoriva

Diversity of amylolytic potential in filamentous fungi: purification and characterization of a glucoamylase from Aspergillus brasiliensis

O Brasil apresenta cerca de 10 a 17,6% da biodiversidade mundial e apenas uma fração dela é conhecida. Os fungos filamentosos são bons produtores de enzimas e despertam um grande interesse biotecnológico. O amido é o principal carboidrato de reserva das plantas. Dentre as enzimas amilolíticas estão as glucoamilases, que catalisam a hidrólise das ligações -1,4 e -1,6 das extremidades da cadeia do amido liberando glucose. Neste trabalho foram isolados 25 fungos filamentosos de amostras de materiais em decomposição da Mata Atlântica. Dos micro-organismos com alta atividade amilolítica foram selecionados e identificados Aspergillus brasiliensis e Rhizopus oryzae. Foi realizada a otimização do cultivo e caracterização das amilases do extrato bruto de ambos os fungos. Após a obtenção destes dados foi selecionado A. brasiliensis, pois, sua amilase é mais termoestável e ainda não reportada na literatura. Após purificação a enzima foi identificada como glucoamilase, a qual é monomérica com 69 kDa e contém aproximadamente 21% de carboidratos. Apresenta um domínio de ligação ao amido na porção terminal e estrutura secundária rica em -hélice. Sua atividade ótima ocorre em pH 4,5 a 60°C, seu pI é de 3,21, pode ser ativada com a adição de Mn2+, e é inibida por glucose em concentrações maiores que 0,1 M. A glucoamilase apresenta excelente estabilidade ao pH e boa estabilidade a temperatura (a 50°C mantém 67% de atividade após 7 horas; a 55°C a meia vida é de 147 minutos). Com amido de batata a enzima apresentou as seguintes constantes cinéticas (km 2,21 mg/mL; Vmáx 155 U/mg; kcat 179 s-1; kcat/km 81,06). A glucoamilase foi imobilizada em DEAE-PEG com ativação de 12 vezes e possibilidade de reuso de 10 vezes com perda de apenas 31% de atividade. O derivado demostrou maior facilidade para hidrolisar a amilopectina do que à amilose. Também foi realizada uma análise de neighbor joining, que agrupou a glucoamilase de A. brasiliensis próxima às glucoamilases de espécies de Aspergillus, que são consideradas as mais derivadas.Brazil holds about 10-17.6% of the world\'s biodiversity and just a percentage of it is known. Filamentous fungi are enzyme producers that have great biotechnological application. Starch is the main reserve carbohydrate in plants. Among the amylolytic enzymes there are the glucoamylases, that catalyze the hydrolysis of -1,4 and -1,6 linkages of the end of starch chains, and releases glucose. In this research 25 filamentous fungi from Atlantic forest decaying material samples were isolated. Among microorganisms with high amylolytic activity Aspergillus brasiliensis and Rhizoupus oryzae were selected and identified. The cultivation parameters were optimized and the enzymes of crude extract were characterized. Considering the previous data Aspergillus brasiliensis was selected because its amylases are more thermostable and it has not been described in the literature yet. After purification the enzyme was identified as a glucoamylase, which is monomeric with 69 kDa and about 21% of carbohydrates in its composition. The enzyme has a starch binding domain in the terminal position and its secondary structure is rich in -helix. The optimum pH for glucoamylase activity is 4.5, the temperature is 60ºC and its pI is 3.21. The enzyme can be activated by the addition of Mn+2, and inhibited in concentrations above 0,1M glucose. The glucoamylase has an excellent pH stability and a good temperature stability (at 50ºC 67% of the activity was retained after 7 hours; at 55°C its half-life was 147 minutes). The best kinetic values were obtained with potato starch (km 2.21 mg/mL; Vmax 155 U/mg; kcat 179 s-1; kcat/km 81,06). The glucoamylase was immobilized on DEAE-PEG, with an activation of 12 times and enzyme reuse 10 times with just 31% loss of its activity. The immobilized enzyme has a greater activity on amylopectin than amylose. A neighbor joining analysis with glucoamylases from filamentous fungi species was made and Aspergillus brasiliensis glucoamylase was grouped close to the glucoamylases of Aspergillus species, which are considered the most derivative

Cellulolytic system enzymes cloning and heterologous expression aiming the lignocellulosic biomass degradation

O Brasil possui um grande potencial para a produção de biocombustíveis a partir de biomassa lignocelulósica. Neste contexto para o desenvolvimento de um bioprocesso limpo e sustentável faz-se necessária a utilização de grandes quantidades de enzimas, sobretudo as celulases. As celulases são responsáveis pela quebra da cadeia de celulose em unidades de glucose. Para baratear e ampliar a produção destas enzimas uma das estratégias que podem ser adotadas é a expressão heteróloga. Neste trabalho foram expressas em Escherichia coli uma endoglucanase de Trichoderma reesei (TR), uma endoglucanase de Eubacterium cellulosolvens com a estrutura completa (MI+MII) e parcial (MII) e uma ?-glucosidase de Anoxybacillus thermarum (BG). A enzima TR, que foi expressa com a cepa de E. coli Origami2, destacou-se por ter pH ótimo baixo, de 3,5 e temperatura ótima de 65°C, ter uma excelente estabilidade entre os pH 2,5 e 7, e ser ativada em 88% por Mn2+. As endoglucanases MI+MII e MII se destacaram pela produção fácil e rápida, gerando grande quantidade de enzimas com baixo custo, possuem pH ótimo de 4,5 e temperatura de 45°C para MI+MII e 50°C para MII. Ambas em extrato bruto são estáveis nos pH de 4 a 7 e temperaturas de 40°C e 50°C por até 24 horas. A BG destacouse, além da produção rápida e pouco dispendiosa, pela excelente tolerância ao produto, sendo a mesma ativada em concentrações de até 0,4 M de glucose. Seu pH ótimo foi de 7 e a temperatura de 65°C, o km de 0,35 mM de ?NPG e Vmáx cerca de 7000 U/mg, variando pouco conforme as diferentes metodologias aplicadas. A BG foi estável entre os pH 5,5 a 8 e na temperatura de 50°C por até 48 horas. As enzimas TR e BG foram aplicadas na degradação de bagaço-de-cana e capim-elefante, in natura ou tratados por autohidrólise, juntamente com um extrato bruto de enzimas provenientes do cultivo de Aspergillus brasiliensis. Os melhores rendimentos foram obtidos na hidrólise com o capim-elefante in natura, resultando na produção até 30 µmol/mL de açúcares redutores.Brazil has great potential for the biofuels production from lignocellulosic biomass. In this context, the development of a clean and sustainable bioprocess requires the use of large quantities of enzymes, especially cellulases. Cellulases are responsible for the breakdown of cellulose chain into glucose units. To increase the production of enzymes and turn the process cheaper one of the strategies that can be adopted is the heterologous expression. In this work, an endoglucanase from Trichoderma reesei (TR), another endoglucanase from Eubacterium cellulosolvens, with complete (MI+MII) and partial (MII) structure, and a ?-glucosidase from Anoxybacillus thermarum (BG) were expressed in Escherichia coli. The enzyme TR, which was expressed with E. coli Origami2, stood out to have a low optimal pH of 3.5 and an optimal temperature of 65°C, to have an excellent pH stability, among 2.5 and 7, and to be activated in 88% by Mn2+. The MI+MII and MII endoglucanases were characterized by easy and fast production, generating a large quantity of enzymes with low cost, having an optimum pH of 4.5, and an optimal temperature of 40°C for MI+ MII, and 50°C for MII. The crude extract of both were stable at pH 4 to 7, and 40ºC and 50°C for up to 24 hours. The BG had a fast and inexpensive production, and an excellent tolerance to the product. BG was activated up to 0.4 M of glucose. Its optimum pH was 7 and the temperature was 65°C. The km was calculated as 0.35 mM ?NPG and Vmax about 7000 U/mg, varying little according to the methodology applied. BG was stable among pH 5.5 to 8 and at 50 ° C for up to 48 hours. The TR and BG were added to a crude extract of enzymes from Aspergillus brasiliensis, and applied in the degradation of sugarcane bagasse and elephantgrass, both in natura or treated by autohydrolysis. The best hydrolysis yields were obtained with elephantgrass in natura, resulting in the production up to 30 µmol/mL of reducing sugars

Trametes versicolor laccase production using agricultural wastes: a comparative study in Erlenmeyer flasks, bioreactor and tray

Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00449-019-02245-z) contains supplementary material, which is available to authorized users.Laccases are very interesting biocatalysts of recognized importance for several industrial applications. Its production by Trametes versicolor, a white-rot fungus, was induced by a combination of cotton gin wastes (1\%), a lignocellulosic waste, and vinasse (15\%), an industrial by-product from sugarcane industry. The use of these agro-industrial wastes are interesting, since it helps in reducing the enzyme production costs, due to their low cost and wide availability, as well as the environmental contamination issues, due to their improper disposal. Thus, laccase production was studied in submerged fermentation of T. versicolor using these agro-industrial wastes (cotton gin waste and vinasse) as carbon source and an additional nitrogen source (0.1\\% peptone). Three different bioreactors were evaluated for laccase production, such as BioFlo 310 bioreactor, aluminium tray and Erlenmeyer flasks to achieve high levels of laccase production. The highest specific production of laccase was found in BioFlo 310 bioreactor with 12 days of fermentation (55.24 U/mg prot.), which has been shown to be closely related to the oxygen supply to the microorganism through aeration of the fermentation medium. This study brings new insights into green biotechnology regarding vinasse utilization, which is frequently discharged in soils, rivers, and lakes causing adverse effects on agricultural soils and biota, as well as the cotton gin waste recovery.The authors are grateful to Mariana Cereia and Maurício de Oliveira for their technical assistance. This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, processes 2010/52322-3; 2014/50884-5; 2018/07522-6), and V. E. P. was the recipient of a FAPESP fellowship (Process 2015/23200-0).info:eu-repo/semantics/publishedVersio

Screening of Xylanolytic Aspergillus fumigatus for Prebiotic Xylooligosaccharide Production Using Bagasse

Sugarcane bagasse is an important lignocellulosic material studied for the production of xylooligosaccharides (XOS). Some XOS are considered soluble dietary fibre, with low caloric value and prebiotic effect, but they are expensive and not easily available. In a screening of 138 fungi, only nine were shortlisted, and just Aspergillus fumigatus M51 (35.6 U/mL) and A. fumigatus U2370 (28.5 U/mL) were selected as the most significant producers of xylanases. These fungi had low β-xylosidase activity, which is desirable for the production of XOS. The xylanases from Trichoderma reesei CCT 2768, A. fumigatus M51 and A. fumigatus U2370 gave a significantly higher XOS yield, 11.9, 14.7 and 7.9 % respectively, in a 3-hour reaction with hemicellulose from sugarcane bagasse. These enzymes are relatively thermostable at 40–50 °C and can be used in a wide range of pH values. Furthermore, these xylanases produced more prebiotic XOS (xylobiose and xylotriose) when compared with a commercial xylanase. The xylanases from A. fumigatus M51 reached a high level of XOS production (37.6 %) in 48–72 h using hemicellulose extracted from sugarcane bagasse. This yield represents 68.8 kg of prebiotic XOS per metric tonne of cane bagasse. In addition, in a biorefinery, after hemicellulose extraction for XOS production, the residual cellulose could be used for the production of second-generation ethanol

Enzymatic activity enhanced by high hydrostatic pressure: Trichoderma reesei endoglucanase II catalytic module

Trabajo presentado al the 60th Anniversary Meeting of the British Biophysical Society goes virtual, celebrado del 14 al 17 de septiembre de 2020.Peer reviewe

Heterologous production and biochemical characterization of a new highly glucose tolerant GH1 β-glucosidase from Anoxybacillus thermarum

The enzymatic lignocellulosic biomass conversion into value-added products requires the use of enzyme-rich cocktails, including β-glucosidases that hydrolyze cellobiose and cellooligosaccharides to glucose. During hydrolysis occurs accumulation of monomers causing inhibition of some enzymes; thus, glucose/xylose tolerant β-glucosidases could overcome this drawback. The search of new tolerant enzymes showing additional properties,such as high activity, wide-pH range, and thermal stability is very relevant to improve the bioprocess. We describe a novel β-glucosidase GH1 from the thermophilic Anoxybacillus thermarum (BgAt), which stood out by the robustness combination of great glucose/xylose tolerance, thermal stability, and high Vmax. The recombinant his-tagged-BgAt was overexpressed in Escherichia coli, was purified in one step, showed a high glucose/xylose tolerance, and activity stimulation (presence of 0.4M glucose/1.0M xylose). The optimal activity was at 65 °C - pH 7.0. BgAt presented an extraordinary temperature stability (48 h – 50 °C), and pH stability (5.5–8.0). The novel enzyme showed outstanding Vmax values compared to other β-glucosidases. Using p-nitrophenyl-β-D-glucopyranoside as substrate the values were Vmax (7614 U/mg), and KM (0.360 mM). These values suffer a displacement in Vmax to 14,026 U/mg (glucose), 14,886 U/mg (xylose), and KM 0.877mM (glucose), and 1.410mM (xylose).This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, process no 2018/07522-6) and a scholarship to Tassio B. Oliveira (grant 2017/09000-4). MLTMP is a Research Fellow of Conselho de Desenvolvimento Científico e Tecnológico (CNPq, process 301963/2017-7). The project also received grants from National Institute of Science and Technology of Bioethanol,INCT, CNPq 465319/2014-9/FAPESP nº 2014/50884-5). PZA was a fellow from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) funding code 001 and Programa de Doutorado Sanduiche no Exterior, PDSE no 88881.135684/2016-01.Peer reviewe

Bioprospection and characterization of the amylolytic activity by filamentous fungi from Brazilian Atlantic Forest

<div><p>Abstract Filamentous fungi are widely diverse and ubiquitous organisms. Such biodiversity is barely known, making room for a great potential still to be discovered, especially in tropical environments - which are favorable to growth and species variety. Filamentous fungi are extensively applied to the production of industrial enzymes, such as the amylases. This class of enzymes acts in the hydrolysis of starch to glucose or maltooligosaccharides. In this work twenty-five filamentous fungi were isolated from samples of decomposing material collected in the Brazilian Atlantic Forest. The two best amylase producers were identified as Aspergillus brasiliensis and Rhizopus oryzae. Both are mesophilic, they grow well in organic nitrogen-rich media produce great amounts of glucoamylases. The enzymes of A. brasiliensis and R. oryzae are different, possibly because of their phylogenetical distance. The best amylase production of A. brasiliensis occurred during 120 hours with initial pH of 7.5; it had a better activity in the pH range of 3.5-5.0 and at 60-75°C. Both fungal glucoamylase had wide pH stability (3-8) and were activated by Mn2+. R. oryzae best production occurred in 96 hours and at pH 6.5. Its amylases had a greater activity in the pH range of 4.0-5.5 and temperature at 50-65ºC. The most significant difference between the enzymes produced by both fungi is the resistance to thermal denaturation: A. brasiliensis glucoamylase had a T50 of 60 minutes at 70ºC. The R. oryzae glucoamylase only had a residual activity when incubated at 50°C with a 12 min T50.</p></div

Production of omegas-6 and 9 from the hydrolysis of açaí and buriti oils by lipase immobilized on a hydrophobic support

This article belongs to the Special Issue Enzyme Immobilization and Its Applications.This paper describes a bioprocess to obtain omegas-6 and 9 from the hydrolysis of Açaí (Euterpe oleracea Martius) and Buriti (Mauritia flexuosa) oils by lipases immobilized on octyl-sepharose. For this, oils and butters were initially selected as the carbon source which resulted in higher production of lipases in Beauveria bassiana and Fusarium oxysporum cultures. The carbon source that provided secretion of lipase by B. bassiana was Açaí oil, and for F. oxysporum, Bacuri butter. Lipases obtained under these conditions were immobilized on octyl-sepharose, and both, the derivatives and the crude extracts were biochemically characterized. It was observed that the immobilization promoted an increase of stability in B. bassiana and F. oxysporum lipase activities at the given temperatures and pH. In addition, the immobilization promoted hyperactivation of B. bassiana and F. oxysporum lipase activities being 23.5 and 11.0 higher than free enzyme, respectively. The hydrolysis of Açaí and Buriti oils by the derivatives was done in a biphasic (organic/aqueous) system, and the products were quantified in RP-HPLC. The results showed the potential of these immobilized lipases to obtain omegas-6 and 9 from Brazilian natural oils. This work may improve the enzymatic methodologies for obtaining foods and drugs enriched with fatty acids.This research was supported by “Development of immobilization strategies and the use of enzymes as biocatalysts for industrial applications: Biodiesel production and functional ingredients”. Funded by FAPESP, 2013/50892-5; and “Obtainment of ethyl and methyl esters from filamentous fungi lipases and genetic engineering techniques, using residual oils, jatropha and macaúba pulp: biotechnological viability to obtain Biodiesel”. Funded by CNPq 406838/2013-5. This study was also financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brazil (CAPES)–Finance Code 001. This work was supported by grants from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Mobility Program Santander-University of São Paulo. My thanks to Professor Glória Fernandez Lorente and Paz García of the Department of Biotechnology and Food Microbiology, Food Sciences Institute, CIAL-CSIC, Campus UAM, Cantoblanco, Spain and Alejandro Herrera Orrego of the Department of Biocatalysis, Institute of Catalysis and Petroleoquímica - CSIC, Campus UAM, Cantoblanco, Spain. We thank Mauricio de Oliveira and Mariana Cereia for the technical assistance. Acknowledgments for CBMSO colleagues, The Proteomic Analysis: “Protein Identification and Quantitation by LC-MS/MS” was carried out at the “CBMSO PROTEIN CHEMISTRY FACILITY”, which belongs to ProteoRed, PRB2-ISCIII, supported by grant PT13/0001”.Peer reviewe