Microbial Fructosyltransferase: Production by Submerged Fermentation and Evaluation of pH and Temperature Effects on Transfructosylation and Hydrolytic Enzymatic Activities
Abstract
Fructosyltransferases (FTase, E.C. 2.4.1.9) are enzymes that catalyze transfructosylation reactions obtaining, as final product, fructose oligomers. In terms of industrial production, the use of microbial enzymes is interesting, especially those produced by Aspergillus sp. The aim of the work was to study the production of FTase by submerged fermentation and evaluation of pH and temperature effects on fructosyltransferase activity. The enzyme was produced by Aspergillus oryzae IPT 301 in a 10-liter bioreactor with growth medium containing sucrose as main carbon source. The assay was performed at 800 rpm, 30 °C, 0.75 vvm and pH 4.5. Transfructosylation (At) and hydrolytic (Ah) activities were determined in the temperature range from 35 to 65 °C and pH range from 3.5 to 6.0. It was observed that mycelial is increases with temperature, holding the maximum value at 50 – 65 ºC, while the optimum pH value were 5.0. The optimum temperature for extracellular At ranged from 55 to 65 °C and the optimum pH ranged from 5.0 to 6.0. Furthermore, the optimum temperature for mycelial Ah was 65 °C and optimum pH 4.5 - 5.0. For extracellular Ah, the optimum temperature and optimum pH were 55 to 65 °C and 3.5 to 6.0, respectively.
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Introduction
Fructooligosaccharides (FOS) are oligosaccharides of fructose containing a single glucose moiety [1,2]. They are mainly composed of 1-kestose (GF2), nystose (GF3), and 1-β-fructofuranosyl nystose (GF4), in which fructosyl units (F) are bound at the β (2→1) position of the sucrose molecule (GF) [2,3,4,5]. FOS with low polymeric degree has better therapeutic properties than those with a high polymeric degree [6]. These fructose oligomers are classified as prebiotics and have numerous beneficial properties for human health [1,2,7]. They are non-cariogenic sweeteners of low caloric value, safe for diabetes, as they are not hydrolyzed by the gastro-intestinal enzymes, selectively promoting the growth of Bifidobacterium in the colon [8,9,10] thus helping to eliminate microorganisms which are pathogenic to human health and preventing colonic carcinogenesis [11,12], and increase the adsorption of calcium and magnesium [13]. In addition, FOS favor the reduction of plasma levels of cholesterol, triglycerides and phospholipids [14,15,16]. They are about 0.4 and 0.6 times as sweet as sucrose and have been widely utilized in food and pharmaceutical industries as a functional sweetener [2,6,17].
Although FOS can be produced by the action of enzymes present in some plants, the commercially available FOS are produced mainly by enzymatic synthesis from sucrose by microbial enzymes called β-fructofuranosidases (FFases, E.C.3.2.1.26) and fructosyltransferases (FTases, E.C.2.4.1.9) [18,5] which have been found in several fungal strains, such as Penicillium sp. [19,20,21,22], Aureobasidium sp. [23,24,25], Fusarium sp. [26,27,28,29] and mainly Aspergillus sp. [3,6,9,17,18,30,31,32,33,34,35,36]. FOS-producing enzymes from microorganisms are excreted either outside the cell as extracellular enzymes or retained within the cell as intracellular enzymes [9,35,37]. FFase enzymes catalyse hydrolytic and transfructosylating reactions, but their ability for transfer is only with high sucrose amounts. On the other hand, FTases possess transfructosylating activity, acting on the β(2→1) link of sucrose and transfer a fructose molecule to an acceptor such as another sucrose molecule, leading to the generation of FOS with a different chain length and release of glucose in the reaction as a by-product [22,36,38]. FTases shows a little affinity towards water as an acceptor, which means that the hydrolases activity of enzyme is relatively low [38].
Conclusion
FOS production using Aspergillus oryzae IPT 301 by submerged fermentation results in a 45.8% yield after 24 h of fermentation, and maximum extracellular FTase activity of 36 U.mL -1 at 40 h. The temperature used in fermentation (30 °C) is very different from the one 55 °C resulting in the maximum transfructosylation activity and (At/Ah) ratio. The optimum temperature and pH for transfructosylation activity, ideal for FOS production, are different for the extracellular and mycelial enzymes. Using a proper pH value and temperature, it is possible to maximize the transfructosylation activity and minimize the hydrolytic activity for FOS production using the FTase from the microorganism studied.