Cloning and Expression of Genes Encoding Fructosyltransferases from Higher Plants in Food Technology

Recently, we have investigated enzymic production of non-digestible oligosaccharides which have several functional activities as “tertiary functional ingredients” of foods. Fructo-oligosaccharides synthesized from sucrose by Eurotium repense fructosyltransferase had the effect of no elevation of blood glucose or insulin concentrations in rats. Fructosylxyloside formed from sucrose and xylose with fructosyltransferase action of Scopulariopsis brevicaulis cells suppressed serum glucose and insulin responses and / or promoted the absorption of calcium and magnesium ions in rats administered with sucrose. On the other hand, we studied purification and characterization of several fructosyltransferases; sucrose: sucrose 1-fructosyltransferase (1-SST), fructan: fructan 1-fructosyltransferase (1-FFT) and a new enzyme, fructan: fructan 6G-fructosyltransferase (6G-FFT) from asparagus roots. Previously, we reported that the asparagus 1-FFT synthesized new functional oligosaccharides elongated with one or two additional fructose units by fructosyltransfer from 1-kestose to 4G-β-D-galactosylsucrose and the saccharides selectively stimulated growth for Bifidobacteria. In this study, we have tried the isolation and expression of cDNAs encoding 6G-FFT, 1-FFT and 1-SST from asparagus for industrial use. The cDNAs encoding 6G-FFT, 1-FFT and 1-SST were isolated from a cDNA library of asparagus leaves or roots. The isolated cDNAs were named AoFT 1, AoFT 2 and AoFT 3, respectively. The deduced amino acid sequences of these cDNAs showed a high homology with those of plant fructosyltransferases. Expression of these cDNAs was done using Pichia pastoris. The recombinant protein from Pichia transformed with AoFT 1 produced 1F,6G-di-β-D-fructofuranosylsucrose, neokestose and sucrose from 1-kestose, while the transformant with an empty vector produced no saccharides. These results show that 6G-FFT is expressed in P. pastoris. In the same way, recombinant protein from Pichia transformed with AoFT 2 produced nystose from 1-kestose and AoFT 3 recombinant protein produced 1-kestose from sucrose. These results also show that 1-FFT and 1-SST are expressed in P. pastoris. Three recombinant proteins had enzymatic properties similar to those of 6G-FFT, 1-FFT and 1-SST from asparagus roots, respectively.

[1]  K. Imada,et al.  Purification and properties ofβ-fructofuranosidase fromAureobasidium sp. ATCC 20524 , 1991, Journal of Industrial Microbiology.

[2]  上野敬司,et al.  アスパラガス由来sucrose:sucrose 1 fructosyltransferase遺伝子のクローニング , 2004 .

[3]  E. Fukushi,et al.  NMR analysis of mono‐ and difructosyllactosucrose synthesized by 1F‐fructosyltransferase purified from roots of asparagus (Asparagus officinalis L.) , 2002 .

[4]  N. Shiomi,et al.  Two Novel Oligosaccharides Formed by 1F-Fructosyltransferase Purified from Roots of Asparagus (Asparagus officinalis L.) , 2002, Bioscience, biotechnology, and biochemistry.

[5]  N. Shiomi,et al.  Fructo‐oligosaccharide content and fructosyltransferase activity during growth of onion bulbs , 1997 .

[6]  Hiroyuki Takeda,et al.  Production of Fructosylxylosides by Scopulariopsis brevicaulis sp. , 1995 .

[7]  H. Sasaki,et al.  Production of 1-kestose by Scopulariopsis brevicaulis , 1994 .

[8]  M. Kikuchi,et al.  Purification and Some Properties of β-Fructofuranosidase from Bifidobacterium adolescentis G1 , 1993 .

[9]  N. Shiomi.  Structure of fructopolysaccharide (asparagosin) from roots of asparagus (Asparagus officinalis L.) , 1993 .

[10]  T. Housley,et al.  Purification and Characterization of Wheat beta(2-->1) Fructan:Fructan Fructosyl Transferase Activity. , 1992, Plant physiology.

[11]  K. Fujita,et al.  Transfructosylation Catalyzed by β-Fructofuranosidase I from Arthrobacter sp. K-1 , 1990 .

[12]  秀一 小野寺,et al.  Two Oligosaccharides Formed by Onion Sucrose : sucrose 1^F-β-D-fructosyltransferase from Onion Seeds , 1989 .

[13]  N. Shiomi.  Properties of Fructosyltransferases Involved in the Synthesis of Fructan in Liliaceous Plants , 1989 .

[14]  M. Hirayama,et al.  A fructooligosaccharide-producing enzyme from Aspergillus niger ATCC 20611. , 1988 .

[15]  T. Yamano,et al.  Identification of Neokestose Produced from Sucrose by an Enzyme of Penicillium oxalicum , 1986 .

[16]  N. Shiomi,et al.  Purification and properties of sucrose:sucrose 1F-β-d-fructosyltransferase in onion seeds , 1985 .

[17]  N. Shiomi.  Purification and characterisation of 6G-fructosyltransferase from the roots of asparagus (Asparagus officinalis L.) , 1981 .

[18]  N. Shiomi.  Two novel hexasaccharides from the roots of Asparagus officinalis , 1981 .

[19]  N. Shiomi,et al.  Purification and Characterization of Sucrose: sucrose 1-fructosyltransferase from the Roots of Asparagus (Asparagus officinalis L.) , 1980 .

[20]  J. Edelman,et al.  THE MECHANISIM OF FRUCTOSAN METABOLISM IN HIGHER PLANTS AS EXEMPLIFIED IN HELIANTHUS TUBEROSUS , 1968 .