Imobilização e caracterização de uma nova lipase obtida por prospecção metagenômica

Resumo: Um nova lipase (LipC6G9) obtida por prospecção metagenômica foi imobilizada e seu potencial de aplicação em biocatálise foi avaliado. Três diferentes suportes com propriedades distintas em relação à forma de interação com a enzima (adsorção física e ligação covalente) foram comparados. Foram investigados os principais parâmetros do processo de imobilização: eficiência de imobilização (E), retenção de atividade (R) e atividade de esterificação da enzima imobilizada nos diferentes suportes. As eficiências de imobilização foram 100% em apenas 6 h para Immobead, 87% em 48 h para Accurel e 13% em 48 h para Celite. As atividades de hidrólise contra trioleína em n-heptano para LipC6G9 imobilizada em Accurel, Immobead e Celite foram de 160 U g-1, 26 U g-1 e 22 U g-1, respectivamente. Entre os três preparados enzimáticos avaliados, apenas a lipase imobilizada em Accurel apresentou atividade de esterificação, com um valor de 16 U g-1 na síntese de oleato de etila em n-heptano, com 90% de conversão em 8 h, a 40 °C. O suporte Accurel foi selecionado para a imobilização de LipC6G9 e sua posterior caracterização em meio orgânico. A enzima imobilizada manteve 100% de sua atividade residual após incubação a 30 °C e mais de 70% quando incubada a 40 °C e 50 °C, por 8 h em n-heptano. LipC6G9 imobilizada também se mostrou estável em diferentes solventes orgânicos como n-heptano (log P 4,0), n-hexano (log P 3,5), etanol (log P -0,23) e acetona (log P -0,31), mantendo mais de 80% de sua atividade residual após 8 h de incubação a 30 °C. A atividade de síntese de ésteres etílicos foi investigada com ácidos graxos de diferentes comprimentos de cadeia. Melhores conversões (90% em 3 h) foram obtidas para ácidos graxos saturados de cadeia média e longa (C8, C14 e C16), com máxima atividade (29 U g-1) obtida para ácido palmítico (C16). Em relação à regiosseletividade, LipC6G9 foi caracterizada como uma lipase 1,3 específica. A enantiosseletividade de LipC6G9 foi avaliada por meio de reações de transesterificação do álcool (R,S)-1-fenil-1-etanol com acetato de vinila e da hidrólise do éster análogo, (R,S)-acetato de 1-feniletila. Foram obtidos excelentes valores de excesso enantiomérico (ee >95%) e enantiosseletividade (E > 200) para o isômero (R) de ambos os substratos avaliados. Os resultados reportados neste trabalho são promissores e fundamentam estudos futuros para o desenvolvimento de aplicações LipC6G9 imobilizada em Accurel em biocatálise

New Tailor-made Alkyl-aldehyde Bifunctional Supports For Lipase Immobilization

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Immobilized and stabilized lipases are important biocatalytic tools. In this paper, different tailor-made bifunctional supports were prepared for the immobilization of a new metagenomic lipase (LipC12). The new supports contained hydrophobic groups (different alkyl groups) to promote interfacial adsorption of the lipase and aldehyde groups to react covalently with the amino groups of side chains of the adsorbed lipase. The best catalyst was 3.5-fold more active and 5000-fold more stable than the soluble enzyme. It was successfully used in the regioselective deacetylation of peracetylated d-glucal. The PEGylated immobilized lipase showed high regioselectivity, producing high yields of the C-3 monodeacetylated product at pH 5.0 and 4 degrees C.612Ramon Areces FoundationCNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico), a Brazilian government agency for the advancement of science [201757/2015-0, 201688/2015-8]Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Cloning, purification, and biochemical characterization of an esterase from Aspergillus nidulans

A large accumulation of agro-industrial waste from different segments is generated daily and is often not properly managed. There are now other fronts in research to give a destination to these residues; these studies are generally aimed at obtaining new and better enzymes and the formulation of enzymatic cocktails that contain (for example, cellulases and hemicellulases) responsible for the degradation of lignocellulosic material. The plant cell wall is mainly composed of cellulose, hemicellulose, and lignin, forming a complex structure. Xylan is one of the main constituents of hemicellulose. To degrade this structure, enzymatic hydrolysis must occur synergistically with xylanolytic enzymes, such as endo-beta-1,4-xylanases, -xylosidases, and acetyl xylan esterase (AXE). In the current work, we reported the purification and biochemical characterization of an acetyl xylan esterase (AxeCE3) from Aspergillus nidulans. The axeCE3 gene was cloned into the pEXPYR vector and transformed into A. nidulans A773 for protein expression. The enzyme AxeCE3 was purified and characterized for its biochemical properties. AxeCE3 showed activity over a wide range of pH (3.0-9.0) and temperature (30-70 °C), with maximum activity at 55 °C, pH 7.0. Regarding the stability at temperature, AxeCE3 showed values above 90% of residual activity after 24 h of incubation at 45 and 50 °C. In relation to stability at pH, AxeCE3 maintained more than 90% of its residual activity after being incubated at 25 °C for 24 h between the pH range 3.0 to 9.0. It was also verified the effect of possible inhibitors (ethylenediamine tetraacetic acid (EDTA), Furfural, and 5- Hydroxymethylfurfural (5-HMF)) on the enzyme activity. AxeCE3 maintained 88% of relative activity at 5 mM EDTA, 43% and 82% at 50 mM furfural and 5-HMF, respectively. The results showed that AxeCE3 has interesting properties to use in the development in the formulation of enzymatic cocktails for the hydrolysis of lignocellulosic residues.The work was supported by the following: FAPESP (São Paulo Research Foundation, grants: 2014/50884 and 2018/07522-6) and National Institute of Science and Technology of Bioethanol, INCT, CNPq (grant: 465319/2014-9) and process 301963/2017-7. Research scholarships were granted to RCA and DA by FAPESP (Grant No: 2020/00081-4 and No: 2020/15510-8), to GSA by CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Finance Code 001).info:eu-repo/semantics/publishedVersio

Challenges of biomass utilization for bioenergy in a climate change scenario

The climate changes expected for the next decades will expose plants to increasing occurrences of combined abiotic stresses, including drought, higher temperatures, and elevated CO2 atmospheric concentrations. These abiotic stresses have significant consequences on photosynthesis and other plants’ physiological processes and can lead to tolerance mechanisms that impact metabolism dynamics and limit plant productivity. Furthermore, due to the high carbohydrate content on the cell wall, plants represent a an essential source of lignocellulosic biomass for biofuels production. Thus, it is necessary to estimate their potential as feedstock for renewable energy production in future climate conditions since the synthesis of cell wall components seems to be affected by abiotic stresses. This review provides a brief overview of plant responses and the tolerance mechanisms applied in climate change scenarios that could impact its use as lignocellulosic biomass for bioenergy purposes. Important steps of biofuel production, which might influence the effects of climate change, besides biomass pretreatments and enzymatic biochemical conversions, are also discussed. We believe that this study may improve our understanding of the plant biological adaptations to combined abiotic stress and assist in the decision-making for selecting key agronomic crops that can be efficiently adapted to climate changes and applied in bioenergy production.info:eu-repo/semantics/publishedVersio

Development of new eco-friendly supports for immobilization of enzymes based on cellulose residues

The pulp and paper industry generates a high volume of solid wastes that are usually burned to obtain energy, directed to landfills, or incinerated. Among the wastes generated in this process is the paper sludge, a residue rich in cellulose with low lignin content making it a useful raw material to produce high-value products such as cheap immobilization supports. In the current work, paper sludge was activated using different functional groups (amino, epoxy, and aldehyde). The xylanase GH10 from Malbranchea pulchella was used as a model enzyme for the immobilization assays. The enzyme was efficiently immobilized through reversible immobilization on aminated support monoaminoethyl-N-ethyl (MANAE) and polyethyleneimine (PEI), achieving yields of more than 90 %. Furthermore, the yield and activity of the biocatalyst immobilized with paper sludge using groups glyoxyl and epoxy (irreversible immobilization) were higher than the enzyme immobilized on agarose supports. The biocatalyst immobilized on paper sludge-epoxy presented the best results, reaching 12.54 U.g-1 of support. Therefore, the use of paper sludge, such as backbone of different immobilization supports, was an efficient method and promising approach for the immobilization of enzymes such as xylanase. More studies are necessary to optimize the displayed potential for future applications as tests in other enzymes of different characteristics and their behavior with bifunctional reagents as the glutaraldehyde. Furthermore, the valorization of these residues in a biorefinery context holds great socio-economical relevance for Portugal and Brazil. Thus, our perspectives are the development of a hybrid biocatalyst using magnetics nanoparticles and paper sludge.info:eu-repo/semantics/publishedVersio

Immobilization and stabilization of the endo-1,4-beta-xylanase of Malbranchea pulchella for production of the xylooligosaccharides

Over the last few years, the lignocellulosic biorefinery concept has been extended beyond the application of biofuel production. Innovative and efficient technologies for lignin, cellulose, and hemicellulose fractionation allow the implementation of integrated processes for the co-production of bioenergy and higher value-added bioproducts. Among the different approaches, the use of Endo-1,4--xylanases (EC 3.2.1.8) in the hydrolysis of rich-xylan feedstocks has increased in the integrated process to produce fermentable and xylooligosaccharides (XOS). Nowadays, XOS has been preferentially used as prebiotic components in the development of new functional foods for presenting additional biological benefits such as antioxidant, inflammatory, and immunomodulatory activities. In the current work, we immobilized an endo-1,4-beta-xylanase of Malbranchea pulchella (Mpxyn10) and evaluated its potential in the production of XOS from xylan from various sources. Mpxyn10 was immobilized on agarose-activated supports (Glyoxyl-, MANAE-, GLUT- and PEI-agarose) and commercial Purolite support. Values >90% of immobilization yield were obtained on aminoactivated supports (Purolite, MANAE, and PEI-agarose) after 120 min, and the highest values of activity recovery were obtained for MANAE-MpXyn10 (137%) and Purolite-MpXyn10 (142%) derivatives. MANAE- and Purolite-MpXyn10 derivatives maintained more than 90% of their activity after 24 h of incubation at 70 °C, while the residual activity of free MpXyn10 was only 11%. MpXyn10 derivatives were also active and stable over a wide range of pH (4.0-6.0) and in the presence of furfural and HMF compounds. MpXyn10 derivatives were tested to produce XOS from xylan from various sources. Maximum values of XOS (xylobiose and xylotriose) were found for xylan beechwood at 8.1 mg mL-1, birchwood at 8.6 mg mL-1, and wheat arabinoxylan at 8.9 mg mL-1 after 3 h of reaction, at 50 °C, using Purolite-MpXyn10. This derivative was reused in various reaction cycles, maintaining more than 80% of yield XOS after 6 cycles of reaction. The results obtained in this work provide a basis for the development of applications of immobilized MpXyn10 to XOS production and other high value-added product in the lignocellulosic biorefinery field.The work was supported by the following: FAPESP (São Paulo Research Foundation, grants: 2014/50884 and 2018/07522-6; Process 2020/00081-4) and National Institute of Science and Technology of Bioethanol, INCT, CNPq (grant: 465319/2014-9) and Process 301963/2017-7.info:eu-repo/semantics/publishedVersio

Imobilização e caracterização de uma nova lipase obtida por prospecção metagenômica

Resumo: Um nova lipase (LipC6G9) obtida por prospecção metagenômica foi imobilizada e seu potencial de aplicação em biocatálise foi avaliado. Três diferentes suportes com propriedades distintas em relação à forma de interação com a enzima (adsorção física e ligação covalente) foram comparados. Foram investigados os principais parâmetros do processo de imobilização: eficiência de imobilização (E), retenção de atividade (R) e atividade de esterificação da enzima imobilizada nos diferentes suportes. As eficiências de imobilização foram 100% em apenas 6 h para Immobead, 87% em 48 h para Accurel e 13% em 48 h para Celite. As atividades de hidrólise contra trioleína em n-heptano para LipC6G9 imobilizada em Accurel, Immobead e Celite foram de 160 U g-1, 26 U g-1 e 22 U g-1, respectivamente. Entre os três preparados enzimáticos avaliados, apenas a lipase imobilizada em Accurel apresentou atividade de esterificação, com um valor de 16 U g-1 na síntese de oleato de etila em n-heptano, com 90% de conversão em 8 h, a 40 °C. O suporte Accurel foi selecionado para a imobilização de LipC6G9 e sua posterior caracterização em meio orgânico. A enzima imobilizada manteve 100% de sua atividade residual após incubação a 30 °C e mais de 70% quando incubada a 40 °C e 50 °C, por 8 h em n-heptano. LipC6G9 imobilizada também se mostrou estável em diferentes solventes orgânicos como n-heptano (log P 4,0), n-hexano (log P 3,5), etanol (log P -0,23) e acetona (log P -0,31), mantendo mais de 80% de sua atividade residual após 8 h de incubação a 30 °C. A atividade de síntese de ésteres etílicos foi investigada com ácidos graxos de diferentes comprimentos de cadeia. Melhores conversões (90% em 3 h) foram obtidas para ácidos graxos saturados de cadeia média e longa (C8, C14 e C16), com máxima atividade (29 U g-1) obtida para ácido palmítico (C16). Em relação à regiosseletividade, LipC6G9 foi caracterizada como uma lipase 1,3 específica. A enantiosseletividade de LipC6G9 foi avaliada por meio de reações de transesterificação do álcool (R,S)-1-fenil-1-etanol com acetato de vinila e da hidrólise do éster análogo, (R,S)-acetato de 1-feniletila. Foram obtidos excelentes valores de excesso enantiomérico (ee >95%) e enantiosseletividade (E > 200) para o isômero (R) de ambos os substratos avaliados. Os resultados reportados neste trabalho são promissores e fundamentam estudos futuros para o desenvolvimento de aplicações LipC6G9 imobilizada em Accurel em biocatálise

Produção e caracterização de novas lipases imobilizadas para utilização em biocatálise

Orientador : Profª. Drª. Nadia KriegerTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Ciências : Bioquímica. Defesa: Curitiba, 29/03/2017Inclui referências ao final de cada capítuloResumo: Este trabalho teve como objetivo geral a produção, imobilização e caracterização das lipases LipC12 e LipBC visando futuras aplicações em biocatálise. Para obtenção da lipase LipBC, os genes lipA e lipB que codificam para lipase (LipBC) e foldase (LifBC), respectivamente, foram identificados no genoma da bactéria Burkholderia contaminans LTEB11, e clonados em vetores de expressão. As proteínas LipBC e LifBC foram expressas, purificadas e ensaios de caracterização foram realizados. LipBC e LifBC apresentam massas moleculares correspondentes a 33 kDa e 37 kDa, respectivamente, e permanecem complexadas após a purificação. LipBC livre apresenta alta atividade específica para substratos de cadeia curta (tributirina, 1400 U mg-1) e longa (óleo de oliva, 845 U mg-1), além de atividade e estabilidade em uma ampla faixa de pH (6,5 - 10) e temperatura (25 - 45 °C). LipBC foi imobilizada no suporte Sepabeads e apresentou atividade de esterificação (4 U g-1) quando aplicada na síntese de oleato de etila em n-hexano, com 90% de conversão em 6 h. Este trabalho também teve por objetivo a síntese de novos suportes para imobilização de lipases, assim como o desenvolvimento de novos protocolos de imobilização. Novos suportes bifuncionais foram preparados a partir de agarose e a lipase LipC12 foi utilizada como modelo de estudo. Os novos suportes apresentam grupos hidrofóbicos (diferentes grupos alquila) para promover a adsorção da lipase e grupos aldeídos para promover ligações covalentes com a enzima adsorvida. A melhor preparação imobilizada, C12-aldeído-LipC12, apresentou uma hiperativação acima de 300% e um aumento significativo na estabilidade (5000 vezes) a 80 °C em comparação com a enzima livre. C12-aldeído-LipC12 foi aplicada com sucesso na hidrólise regiosseletiva do substrato peracetilado D-glucal, apresentando 69% de conversão do produto C-3 mono-desacetilado após 96 h. As diferentes estratégias de imobilização propostas nesse trabalho podem ser aplicadas em materiais como celulose, sílica ou resinas acrílicas, o que permite a obtenção de novos biocatalisadores com alta atividade e estabilidade para a aplicação em diferentes sistemas reacionais em biocatálise. Palavras-chave: Imobilização, lipases, hiperativação, agarose, novos suportes, regiosseletividade, biocatálise.Abstract: This work aimed the production, immobilization and characterization of the lipases LipC12 and LipBC for future applications in biocatalysis. To obtain the LipBC lipase, lipA and lipB genes encoding lipase (LipBC) and foldase (LifBC), respectively, were identified in the genome of the bacterium Burkholderia contaminans LTEB11. The genes were amplified and cloned into expression vectors. LipBC and LifBC proteins were expressed, purified and characterization assays were performed. LipBC and LifBC have molecular weights corresponding to 33 kDa and 37 kDa, respectively, and remained complexed after purification. LipBC has high specific activity against short (tributyrin, 1400 U mg-1) and long chain substrates (olive oil, 845 U mg-1), as well as activity and stability over a wide range of pH (6.5 - 10) and temperature (25 - 45 °C). LipBC was immobilized on Sepabeads and 90% of efficiency of immobilization was obtained in 1 h. The immobilized preparation catalyzed the synthesis of ethyl-oleate with an activity of 4 U g-1, with a yield of 90% obtained in 6 h. This work also aimed the synthesis of novel supports for lipase immobilization, as well as the development of new immobilization protocols. New bifunctional supports were prepared and LipC12 lipase was used as the study model. The new supports contained hydrophobic groups (different alkyl groups) to promote interfacial adsorption of the lipase and aldehyde groups to react covalently with the amino groups of side chains of the adsorbed lipase. The best immobilized preparation, C12-aldehyde-LipC12 was 3.5-fold more active (hyperactivation) and 5000-fold more stable (at 80 °C ) than the soluble enzyme. C12-aldehyde-LipC12 was successfully applied in the regioselective hydrolysis of peracetylated D-glucal, producing high yields (69%) of the C-3 monodeacetylated product after 96 h of reaction. The different strategies of immobilization proposed in this work can be applied in materials such as cellulose, silica or acrylic resins, which allows the production of new biocatalysts with high activity and stability for the application in biocatalysis. Key-words: Immobilization, lipases, hyperactivation, agarose, new supports, glutaraldehyde, regioselectivity, biocatalysis

Recent Trends in Biomaterials for Immobilization of Lipases for Application in Non-Conventional Media

© 2020 by the authors.The utilization of biomaterials as novel carrier materials for lipase immobilization has been investigated by many research groups over recent years. Biomaterials such as agarose, starch, chitin, chitosan, cellulose, and their derivatives have been extensively studied since they are non-toxic materials, can be obtained from a wide range of sources and are easy to modify, due to the high variety of functional groups on their surfaces. However, although many lipases have been immobilized on biomaterials and have shown potential for application in biocatalysis, special features are required when the biocatalyst is used in non-conventional media, for example, in organic solvents, which are required for most reactions in organic synthesis. In this article, we discuss the use of biomaterials for lipase immobilization, highlighting recent developments in the synthesis and functionalization of biomaterials using different methods. Examples of effective strategies designed to result in improved activity and stability and drawbacks of the different immobilization protocols are discussed. Furthermore, the versatility of different biocatalysts for the production of compounds of interest in organic synthesis is also described.This research was funded by Spanish Government projects (AGL2017-84614-C2-1-R), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico-CBAB/CABBIO-Brazilian-Argentine Center for Biotechnology, grant No:441015/2016-6) and FAPESP (São Paulo Research Foundation, grant No: 2018/07522-6) for financial support. Research scholarships were granted to R.C.A. by FAPESP (Grant No: 2020/00081-4), to J.M.A. and N.K. (grant No 303287/2019-5 by CNPq, and to L.A.S. and R.C.A. by CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), a Brazilian government agency for the development of personnel in higher education. This study was financed in part by CAPES-Finance Code 001.Peer reviewe