Enzymes fructosyltransferase

P. S. J. Cheetam, A. J. Hacking, and M. Vlitos, Synthesis of novel disaccharides by a newly isolated fructosyltransferase from Bacillus subtilis, Enzyme Microb. Technol., 11 (1989) 212-219. 

This enzyme [EC 2.4.1.9], also referred to as sucrose 1-fructosyltransferase, catalyzes the reaction of sucrose with [(2,l)-/3-D-fructosyl] to produce D-glucose and [(2,l)-/3-D-fructosyl]( +1). Thus, this enzyme participates in the conversion of sucrose into inulin and glucose. Other fructosyl-containing sugars can serve as substrates as well instead of sucrose. 

Inulin biosynthesis in Jerusalem artichoke occurs via the combined action of two enzymes l-sucrose sucrose fructosyltransferase (1-SST) and l-fructan fructan fructosyltransferase (1-FFT). 1-SST catalyzes the synthesis of inulins of a low degree of polymerization, while 1-FFT catalyzes the synthesis of ffuctans of a degree of polymerization up to 50 (Sevenier et al., 2002a). High... 

Koops, A.J. and Jonker, H.H., Purification and characterization of the enzymes of fructan biosynthesis in tubers of Helianthus tuberosus Columbia. 1. Fructan fructan 1-fructosyltransferase, J. Exp. Bot., 45, 1623-1631, 1994. 

Nagaraj, V.J., Altenbach, D., Galati, V., Luscher, M., Meyer, A.D., Boiler, T., and Wiemken, A., Distinct regulation of sucrose sucrose-l-fructosyltransferase (1-SST) and sucrose fructan-6-fructosyltrans-ferase (6-SFT), the key enzymes of fructan synthesis in barley leaves 1-SST as the pacemaker, New Phytol., 161, 735-748, 2004. 

Keywords Biocatalysis, Enzyme engineering, Fructosyltransferases, Glucosyl-transferases, Immobilization, Oligosaccharides, Reaction engineering... 

Pectinex Ultra SP-L is a commercial enzyme preparation from Aspergillus acu-leatus that is used in the food industry for reducing viscosity in fruit juice processing. It contains different pectinolytic and cellulolytic enzymes [29]. In addition, the existence of fructosyltransferase activity in Pectinex Ultra SP-L has been reported by several authors [30-32]. In recent years, we have investigated the purification, characterization, and application of the fructosyltransferase from A. aadeatus contained in this commercial preparation [33]. 

The fructosyltransferase from A. aculeatus displayed activity without the addition of any metal ions. However, some effects were observed in its susceptibUity to mono- and divalent cations [33]. For ejample, Mn, K, and Co caused a 1.4—1.9-fold increase in the activity, whereas low concentrations of Hg and Zn produced 35-60% inhibition. It was also found that the enzyme was slightly activated by several non-ionic and anionic surfactants such as sodium dodecylsulphate (1.5-fold at lOmM), sodium deoxycholate (1.4-fold at ImM), and Triton X-100 (1.4-fold at 5% w/v). Moreover, it was resistant to low concentrations (1-lOmM) of reducing agents such as dithiothreitol and P-mercaptoethanol. 

Both fructooligosaccharide synthesis and sucrose hydrolysis are catalyzed by most of the fructosyltransferases and 3-fmctofuranosidases (invertases) in the presence of sucrose. The transferase hydrolase raho, which determines the maximum yield of fructooligosaccharide, depends basically on two parameters the concentra-hon of sucrose and the intrinsic enzyme properties, that is its ability to bind the nucleophile (to which a fructose is transferred) and to exclude H2O from the acceptor binding site [11]. 

Figure 11.2 Kinetic plots (initial rate versus sucrose concentration) of fructosyltransferase from A aculeatus. Transfer activity ( ) and hydrolytic activity (o). Reactions were carried out in 0.2 M sodium acetate buffer (pH 5.5) at 60°C. Kinetic parameters were calculated estimating a molecular mass of 135 kDa for the active enzyme. Adapted from Ref [33].

The fructosyltransferase mined from the commercial preparation Pectinex Ultra SP-L is quite stable towards pH, temperature, and the presence of chemicals. The A aculeatus fructosyltransferase showed a high transferase hydrolase raho that provides it with great potential for oligosaccharide synthesis. The enzyme can be easily immobilized on epoxy-activated supports for better performance. Further studies are required to draw mechanishc conclusions on the nature of the kinetics observed with this fructosyltransferase. 

Only the fructosyltransferase (FTF, EC 2. 4. 1. 9) is required in bacteria for the synthesis of bacterial inulin. The enzyme shuffles fructosyl units from one sucrose molecule (acting as druior) to another sucrose molecule, 1-kestose, and higher polymeric p-(2 1) linked fructan molecules, respectively (acting as acceptor). This enzyme partly leads to p-(2—>6) linkages, which results in branches within the inulin molecule [130, 133]. 

Levan has a backbone of p-(2 6) linked o-fructose and occurs as high molecular weight polysaccharide in microorganisms. It is accessible from sucrose by use of the enzyme levansucrase (sucrose 6-fructosyltransferase (FTF, EC 2.4.1.10)). Either culture broth of bacterial strains like Bacillus or Zymomonas or the cell-free supernatant can be used for the enzymatic reaction with sucrose. The molecular weight and the viscosity of levan depend on the strain used and the reaction conditions [131, 134]. Even though levan has interesting properties, it has never gained extensive industrial use up to now [134]. 

Plant synthates, primarily intended for the developing seed, are the main nutrition source to the fungus (Mower and Hancock, 1975) which obviously is exploited at about 5 dpi depending on the ergot species. To use this natural sink, several enzymes are secreted such as the cell-wall bound inducible fructosyltransferase (invertase) (Bassett et ah, 1972 Taber, 1985 Tudzynski et ai, 1995) and the fungal foot is developed structurally for attaching and absorbing (Luttrell, 1980). While intense exudation of honeydew is reported to occur without penetration... 

Yun JW, Song SK. The production of high-content fructo-ohgosaccharides from sucrose by the mixed-enzyme system of fructosyltransferase and glucose-oxidase. Biotechnol Lett 1993 15(6) 573—6. 

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