Full Length Research Paper
Abstract
The β-D-fructofuranosidases are enzymes with biotechnological potential that can be used in different industrial sectors as food and beverage. In this context, microorganisms are important producers of these biomolecules, especially filamentous fungi. The production of extracellular β-D-fructofuranosidase fromAspergillus parasiticus using sugarcane bagasse as a carbon source under submerged fermentation was optimized by factorial design and high levels of enzyme were obtained in 24 h-old cultures at 30°C using 1.5% sugarcane bagasse under agitation. The extracellular β-D-fructofuranosidase was purified 119-fold using diethyl aminoethyl (DEAE)-cellulose and Sephacryl S-200 chromatographic columns withrecovery of 16%. The native molecular mass was estimated as 136 kDa with two subunits of 63 kDa determined by 7% sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE) and 64% carbohydrate content. The purified enzyme showed optimal temperature of activity from 38-56°C and optimum pH from 4.5 to 6.2 determined by experimental design (CCRD), with half-life of 25 min at 50°C. It was stable from pH 5.0-10.0. The extracellular enzyme activity was stimulated by Ba2+ and Mg2+, and it was not affected by urea, silver and ethylenediaminetetraacetic acid(EDTA). The K0.5 and Vmax values were 10 mM and 1565 U/mg of protein, and 19 mM and 1965 U/mg of protein for sucrose and raffinose, respectively.
Key words: Invertase, fructofuranosidase, Aspergillus parasiticus, sugarcane bagasse, factorial design.
Abbreviation
Abbreviations: FOS, Fructooligosaccharides; PB, Plackett and Burman; CCRD,central composite rotatable design; DEAE, diethyl aminoethyl; DNS, 3’,5’-dinitrosalisilic acid; BSA, bouvine serum albumin; SDS-PAGE, sodium dodecyl sulfate poly-acrylamide gel electrophoresis; EDTA, ethylenediaminetetraacetic acid.
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