Expressão heteróloga do gene Hxyn2 do fungo Humicola grisea var. thermoidea em Pichia pastoris: Produção e purificação da enzima HXYN2r e aplicação em testes de panificação
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2008-05-30
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Universidade Federal de Goiás
Resumo
Endoxylanases are the main group of enzymes involved in the hydrolysis of xylan. This enzymes have application in industrial proposes, like as drink, food, feed, clothes industries and for bleaching cellulose paper pulps. In bread making the xylanases have been used to improve processing and product quality of loaf, leading soft dough and loaf with larger volume as well as an improved crumb structure. The xylanolitic enzymes produced by filamentous fungi constitute an enzymatic pool with distinct activities which make their use in industrial process more difficult, the obtainment of recombinant microorganisms constitutes a strategy to achieve suitable enzymes to industrial process. The thermophilic fungus Humicola grisea var. thermoidea has proved to be a good source of xylanolitic enzymes. The gene Hxyn2 from H. grisea codes a xylanase with 23 kDa that belongs to G/11 family of glycosil-hydrolases. Its cDNA was cloned in the vector pHILD2 and expressed in yeast Pichia pastoris under the control of the promoter AOX1. The transformants were chosen trough genetic stability and capacity to produce and secrete the enzyme HXYN2r into culture medium. The purified HXYN2r showed a high xylanolitic activity with an optimum temperature of 60 ºC and an optimum pH value of 6.5. The aim of this project was the production, purification and application of HXYN2r enzyme in bread making tests. The production in the optimized medium obtained 100 mg of active xylanase per liter of medium BMMY-U. This quantify of enzyme presented 40% of the total proteins in culture supernatants. The proteins of culture supernatants was concentrated by liofylization and fractionated by into a chromatographic column of gel filtration Sephacryl S-100. The purified xylanase presented specific activity of 2250 U/mg and the purification profit was 6.4%. The 23 kDa protein was confirmed by the activity on Zymogram assay. The pure xylanase was added at the rate of 45 and 90 U/Kg of wheat flour in the bread making tests. In this rates it was not observed any effect of xylanase in specific volume either in crumb structure. The HXYN2r enzyme was partially purified by a heat treatment (45 min at 60 °C) and was concentrated by liofylization and a yield of 17.9% was obtained. The partially purified HXYN2r was added at the rates of 500, 1500, 3000, 4500 and 6000 U/kg of wheat flour. The added xylanase improved the specific volume and the crumb structure, but any effect was observed in the moisture content of the loaf. The best result was the rise of 16.0 % in loaf specific volume with the dose of 3000 U/kg of wheat flour. This effect was similar to the obtained with a commercial xylanase added to the dough in equal dose.
The results presented suggest that the xylanase from H. grisea can be used in bread making to improve specific volume.
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BASTOS, Fernando Medeiros. Heterologous expression of the gene Hxyn2 fungus Humicola grisea var. thermoidea in Pichia pastoris: production and purification of the enzyme HXYN2r testing and application in bakery. 2008. 99 f. Dissertação (Mestrado em Ciências Biolóicas) - Universidade Federal de Goiás, Goiânia, 2008.