Melhoramento por evolução dirigida da termoestabilidade da xilanase xynA de Orpinomyces sp. PC-2

• Ano de defesa: 2009
• Autor(a) principal: Trevizano, Larissa Mattos
• Orientador(a): Guimarães, Valéria Monteze
• Banca de defesa: Fietto, Luciano Gomes , Passos, Flávia Maria Lopes
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Viçosa
• Programa de Pós-Graduação: Mestrado em Bioquímica Agrícola
• Departamento: Bioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animal
• País: BR
• Palavras-chave em Português: Evolução dirigida; Xilanase; Orpinomyces sp.
• Palavras-chave em Inglês: Directed evolution; Xylanase; Orpinomyces sp.
• Área do conhecimento CNPq: CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA
• Link de acesso: http://locus.ufv.br/handle/123456789/2408

Xylan is the most common hemicellulosic polysaccharide in cell walls of land plants. This polysaccharide is composed of a main-chain polymer made up exclusively of β-1,4 xylose residues. This backbone possesses branches containing arabinofuranosyl, acetyl, and glucuronosyl residues. Enzymatic hydrolysis of xylans is brought about by a variety of enzyme activities that are grouped under the generic term hemicellulases. Endoxylanases (EC 3.2.1.8) hydrolyze internal β-1,4 bonds in the main-chain and generate soluble xylooligosaccharides. A variety of microorganisms, including bacteria, yeast and filamentous fungi, have been reported to produce xylanase, in which the most potent producers are fungi. Researchers are especially interested in fungal xylanases because they are secreted extracellularly and their activity is much higher than the xylanases from yeasts and bacteria. Among the biotechnological applications of xylanases, it can be outstanding the bioconversion of lignocellulosic materials into fermentative products, improvement of digestibility of animal feedstock, clarification of juices, facilitating the release of lignin from the pulp and reducing the amount of chlorine required for bleaching in pulp in the paper industry. Over the last few decades, there has been a growing interest in lignocellulose bioconversion as a renewable energy source and xylanases can be effectively used with cellulases to hydrolyze the lignocellulosic biomass and bioethanol production. Most of the fungal xylanases show optimal activities at neutral or acidic pH and at temperatures below 45 °C. Xylanase (XynA), originally isolated from the anaerobic fungus Orpinomyces PC-2, has been tested for several industrial applications, in pilot scale. This enzyme presented extremely high activity with the xylan substrate of animal ration, cellulose pulp and other biomass types. Studies demonstrated that the use of the native enzyme has been more efficient in optimal conditions as pH 5.0-5.5 and temperature of 50-55 °C. However, the industrial process is usually accomplished at higher temperature and alkaline pH, what justifies the interest for xylanases that are active in these conditions. Directed evolution has emerged as a successful alternative in the genetic engineering of enzymes and it has been used to improve the functional properties of those molecules. In this study, error-prone PCR was used to improve the thermostability of endo-β-1,4-xylanase from Orpinomyces PC-2. The constructed library of xylanase (xynA) mutants was submitted to several screening cycles. The transformants were first exposed to 60 ºC during one hour and then the thermostable mutants were selected with the azo-xylan-agarose 0.2% pH 6.5 as substrate. Six mutants selected were sequenced and these amino acid sequences were analyzed to identify the mutations. Two mutants displayed higher thermal stability than the xylanase without mutations (wild-type). Whereas the wild-type lost 60% of its activity after 10 min at 60 ºC, mutants M4 and M6 showed enhanced thermostability and retained approximately 50% of its activities after treatment at 60 ºC for 60 min. In addition, M4 retained about of 40% of its initial activity after incubation at 75 ºC for 60 min. In order to evaluate the mutation effects in the enzyme properties, these ones were characterized for the optimum temperature, optimum pH and substrate specificity. The mutants and the wild-type showed an optimal temperature and pH for xylanase activity at 60 ºC and pH range of 5.0-7.0. The enzymes showed activity against the two tested substrates. An initial structural modeling study was accomplished with the wild-type and two mutants, which presented higher thermostability, seeking the best understanding of the relationship between structure and function of those xylanases.