Alfredo Eduardo Maiorano

Microbial production of fructosyltransferases for synthesis of pre-biotics.

Fructooligosaccharides (FOS) are prebiotic substances found in several vegetable or natural foods. The main commercial production of FOS comes from enzymatic transformation of sucrose by the microbial enzyme fructosyltransferase. The development of more efficient enzymes, with high activity and stability, is required and this has attracted the interest of biotechnologists and microbiologists with production by several microorganisms being studied. This article reviews and discusses FOS chemical structure, enzyme characteristics, the nomenclature, producer microorganisms and enzyme production both in solid state fermentation and submerged cultivation.

Screening of β-fructofuranosidase-producing microorganisms and effect of pH and temperature on enzymatic rate

Seventeen different strains of filamentous fungi were grown in batch cultures to compare their abilities for the production of β-fructofuranosidase. Three of them, Aspergillus oryzae IPT-301, Aspergillus niger ATCC 20611 and strain IPT-615, showed high production with total fructosyltransferase activity higher than 12,500 units l−1. In addition, the β-fructofuranosidases of those strains have a high fructosyltransferase activity-to-hydrolytic activity ratio. The temperature and pH effects on the sucrose-β-fructofuranosidase reaction rate were studied using a 22 factorial experimental design. The comparative analysis of the tested variable coefficients shows that the variable pH contributes mostly to the changes in the fructosyltransferase and hydrolytic rates and in the Vt/Vh ratio. At 40 and 50°C, there were no significant differences between the fructosyltransferase and hydrolytic velocities of these enzymes.

Media optimization for β-Fructofuranosidase production by Aspergillus oryzae

β-Fructofuranosidase production by Aspergillus oryzae IPT301 was maximized in shake flasks. Response Surface Methodology (RSM) involving Small Central Composite Design was adopted to evaluate the fructosyltransferase (FTase) activity by changing three medium component concentrations: sucrose, urea and yeast extract. The optimal set of conditions for maximum fructosyltransferase production was as follows: sucrose 320.5 g/L, urea 7.13 g/L and yeast extract 2.11 g/L. In this optimal condition, the following improvements were achieved: an increase of 48.8% in cell growth, 112% and 62% in micelial and free FTase activities, respectively, 62.8% in the ratio of fructosyltransferase/hydrolytic activities for enzyme linked to mycelium and 67.5% for free enzyme.

Mutagenesis of Aspergillus oryzae IPT-301 to improve the production of β-fructofuranosidase

Aspergillus oryzae IPT-301, previously reported as a β-fructofuranosidase producing microorganism, was successfully mutated using UV irradiation at 253.7 nm followed by the screening of survivors resistant to certain stress conditions. Strains were first subjected to the β-fructofuranosidase activity assay using a portion from the colony grown in Petri dish as the enzyme source. Seven mutants with βfructofuranosidase activity values relative to the parent culture between 140 - 190% were selected from survivors grown at temperature of 40oC or 0.018% (w/v) sodium dodecyl sulfate concentration. They were cultivated on a rotary shaker to characterize mycelium and extracellular fructosyltransferase activities. Three mutants named IPT-745, IPT-746 and IPT-748 showed the highest amount of mycelium activity whose values increased 1.5 - 1.8 fold, compared with the parent strain. It was found that more than 55% of total enzyme activity (mycelium- plus extracellular- activity) from these strains was detected in the mycelium fraction. Only one mutant, IPT-747, exceeded the amount of extracellular enzyme exhibited by the parent strain (1.5 times). This mutant also showed the highest value of total fructosyltransferase activity.