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Kinetic resolution of racemates with lipases is the preferred method for producing bioactive compounds. One strategy for obtaining highly enantioselective lipases that are stable in the organic media that are often used in these reactions is to improve existing lipases by protein engineering. In this work, we engineered the lipase LipC12, which has good stability in organic media, but only moderate enantioselectivity. Molecular docking with LipC12 identified V261 as a key position influencing, first, enantioselectivity in the transesterification of (RS)-1-phenylethanol and, second, activity in the hydrolysis of p-nitrophenyl octanoate. Variants were then obtained by site-directed mutagenesis, expressed in Escherichia coli, and their performance in these reactions was evaluated. Enzymes immobilized on Immobead 150 were used in the transesterification while free enzyme was used in the hydrolysis reaction. It was not possible to increase the hydrolytic activity and enantioselectivity simultaneously: some variants had increased enantioselectivity but lower hydrolytic activity, and others had increased hydrolytic activity but lower enantioselectivity. The best result for enantioselectivity was obtained for LipC12V261Q, with an increase of the E-value (for (R)-1-phenylethanol) from 46 to 110, however, its hydrolytic activity decreased 4-fold in comparison to LipC12wt. The highest hydrolytic activity was obtained for LipC12V261F, with a value almost 6-fold higher than that of LipC12wt. This variant also had an inverted enantiopreference (i.e. for (S)-1-phenylethanol), but with a very low E-value of only 4.

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/publishedVersion

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/publishedVersion

Orientador : Profª. Drª. Nadia Krieger Tese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Ciências : Bioquímica. Defesa: Curitiba, 29/03/2017 Inclui referências ao final de cada capítulo Resumo: 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.

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.

Orientadora: Profa. Dra. Nadia Krieger Coorientador: Dr. Robson Carlos Alnoch Dissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Ciências - Bioquímica. Defesa : Curitiba, 26/08/2021 Inclui referências: p. 66-72 Resumo: Esta dissertação relata análises in silico e a expressão e caracterização de lipase e foldase produzidas por Burkholderia contaminans LTEB11. O trabalho consistiu, na parte in silico, das análises de sequência, modelagem por homologia e análise filogenética; na parte laboratorial, consistiu de ensaios de clonagem, expressão e caracterização de lipase denominada LipE e sua foldase correlata, LifE. As análises de sequência mostraram que os genes lipE e lifE estão localizados dentro de um operon denominado lipEF e a construção do modelo de homologia mostrou que LipE possui a topologia a/p-hidrolase característica de lipases, além de presença de uma "lid " formada pela a-hélice a5. Análises de estrutura secundária demonstraram que LipE apresenta características típicas de lipases, como o pentapeptídeo conservado, a tríade catalítica, no caso de LipE formada pelos resíduos His284, Ser86 e Asp262, além de um sítio de ligação ao Ca2+ e dois resíduos de cisteína, indicando a existência de uma ponte dissulfeto. As análises filogenéticas classificaram LipE na subfamília I.2 de lipases bacterianas, que apresentam a necessidade de uma foldase específica para o correto enovelamento. Ensaios de cross-refolding com foldases relacionadas mostraram que apenas outra foldase produzida por B. contaminans LTEB11, LifBC, foi capaz de ativar LipE. LipE e LifE foram coexpressas usando os vetores pET-28a e pT7-7, respectivamente. A clonagem foi satisfatória, com clones apresentando atividade em placas de Petri contendo tributirina. Entretanto, depois da expressão, o extrato contendo o complexo LipE-LifE apresentou baixa atividade de hidrólise contra ésteres de p-nitrofenila (0,01 a 0,04 U mg-1), e nenhuma atividade contra triglicerídeos em meio líquido, indicando a necessidade de otimização da expressão de LipE para possibilitar sua correta caracterização. Abstract: This dissertation reports in silico analyses and the expression and characterization of lipase and foldase produced by Burkholderia contaminans LTEB11. The work consisted, in the in silico part, of sequence analysis, homology modeling and phylogenetic analysis; in the laboratory part, it consisted of cloning, expression and characterization assays of a lipase called LipE and its correlated foldase, LifE. Sequence analysis showed that lipE and lifE genes are located within an operon called lipEF and the construction of the homology model showed that LipEhas the a/p-hydrolase fold characteristic of lipases, in addition to the presence of a formed "lid"by the a-helix a5. Secondary structure analyzes showed that LipE has characteristics typical of lipases, such as the conserved pentapeptide, the catalytic triad, in the case of LipE formed byresidues His284, Ser86 and Asp262, in addition to a Ca2+ binding site and two cysteine residues ,indicating the existence of a disulfide bridge. Phylogenetic analyzes classified LipE in the I.2 subfamily of bacterial lipases, which need the presence of a specific foldase for correct folding. Cross-refolding assays with related foldases showed that only another foldase produced by B. contaminans LTEB11, LifBC, was able to activate LipE. LipE and LifE were co-expressed using vectors pET-28a and pT7-7, respectively. Cloning was satisfactory, with clones showingactivity in Petri dishes containing tributyrin. However, after expression, the extract containing the LipE-LifE complex showed low hydrolysis activity against p-nitrophenyl esters (0.01 to0.04 U mg-1), and no activity against triglycerides in liquid medium, indicating the need for optimization of LipE expression to enable its correct characterization.