Produção e caracterização da Beta-frutofuranosidase de linhagem mutante de Aspergillus niger e sua aplicação na produção de frutooligossacarideos

AUTOR(ES)
DATA DE PUBLICAÇÃO

1998

RESUMO

Fructooligosaccharides (or Neosugar) are sucrose oligomers, which are composed of glucose attached via a β-(2-1) linkage to 2, 3 or 4 fructose units. The resulting structures are designated as l-kestose (GF2), nystose (GF3) and lf-fructofuranosylnystose (GF4), respectively. It is known that fructooligosaccharides exist naturally in many kinds of plants. They can also be prepared from sucrose through the transfructosylating action of enzyme β-fructofuranosidase (E.C. 3.2.1.26) obtained from microorganisms. Fructooligosaccharides have become of major interest due to their growth stimulation of beneficial bifidobacteria in the digestive tract, decrease of total cholesterol and lipid in the serum, relief of constipation, and general improvement of human health. Neosugar is a mixture of the fructooligosaccharides, l-kestose (GF2), nystose (GF3) and 1f-fructofuranosylnystose (GF4), and is commercially produced from sucrose using the enzyme β-frutofuranosidase enzyme obtained from A. niger ATCC 20611. The objective of this work was to induce a mutant of Aspergillus niger, using M-Methyl N -Nitro N-Nitrosoguanidine (M.N.N.G.) and ultraviolet ligth (UV) producing higher β-fructofuranosidase activity for the production of fructooligosaccharides than the parent strain and to study the characteristics of the enzyme. A mutant strain(nº 12) of A. niger, obtained by treatment with M.N.N.G.-UV, was selected, producing 100% more β-fructofuranosidase than the parent strain. The mutant produced less conidia on PDA and was morphologically different: brownish white colonies. The extracellular β-fructofuranosidase of the mutant strain was purified about 15.8-fold (40.2% yield) with respect to the crude enzyme preparation, showing a specific activity of 4.26 units/mg protein. It was found that the optimum pH and temperature for activity were 5.5-6.0 and 50-55°C, respectively. The enzyme was stable from pH 5.0 to 6.0 and at temperatures below 55°C. The Km and Vmáx values of the β-fructofuranosidase for sucrose were 0.47 M and 1.02 µmol/ml/min., respectively. The activity of the enzyme was inhibited by 1 mM by N-bromosuccinimide, β-mercaptoethanol, HgCl2 and I2, and by 10 mM AgNO3 after 30 minutes at 55°C. The extracellular β-fructofuranosidase parent strain was purified about 15.4-fold (38.6 % yield) with respect to the crude enzyme preparation showing a specific activity of 1.66 units/mg protein. It was found that the optimum pH and temperature for activity were 5.0-6.0 and 50-55°C, respectively. The enzyme was stable from pH 5.0 to 6.0 and at temperatures below 55°e. The enzyme was stable from pH 5.0 to 6.0 and at temperatures below 55 °e. The Km and Vmax values of the β -fructofuranosidase for sucrose were 0.37 M and 1.12 µmol/ml/min.., respectively. The activity of the enzyme was inhibited by 1 mM by N-bromosuccinimide, β-mercaptoethanol, HgCl2 and I2, and by 10 mM AgNO3 after 30 minutes at 55°C. Maximum conversion yeld from sacarose to fructo-oligosaccharides (54.41%) was obtained when added β-frutofuranosidase in 30% sucrose, at 55°C and pH 5,5, for 6 hrs incubation. The production efficienceis of GF2/GF3/GF4 depending on sucrose concentration and the reaction time.

ASSUNTO(S)

mutação (biologia) aspergillus niger

ACESSO AO ARTIGO

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