&bgr;-fructofuranosidase and gene thereof

Details &bgr;-fructofuranosidase and gene thereof &bgr;-fructofuranosidase and gene thereof

2000-03-09

2003-05-20

Saidha, Tekchand (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C435S183000, C435S193000, C530S350000, C536S063000

Reexamination Certificate

active

06566111

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a &bgr;-fructofuranosidase having a fructose transferase activity, which is useful for the industrial production of fructooligosaccharides, and its gene and use.
2. Description of the Related Art
The molecular structure of a fructooligosaccharide is the same as that of sucrose, except that the fructose half of a fructooligosaccharide is coupled with another one to three fructose molecules at positions C1 and C2 via a &bgr;-bond. Fructooligosaccharides are indigestible sugars known for their physiological advantages, such as the facilitation of Bifidobacterial growth in the intestines, metabolic stimulation for cholesterols and other lipids, and little cariosity.
Fructooligosaccharides are found in plants, such as asparagus, onion, Jerusalem-artichoke and honey. They are also synthesized from sucrose by the newly industrialized mass production technique using fructosyltransfer reaction which is catalyzed by a &bgr;-fructofuranosidase derived from a microorganism.
The molecular structure of 1-kestose and nystose, which make up component of industrially produced fructooligosaccharide mixtures of today, are the same as that of sucrose except that their fructose half is coupled with one and two molecules of fructose, respectively. It has been found recently that their high-purity crystals exhibit new desirable characteristics both in physical properties and food processing purpose while maintaining the general physiological advantages of fructooligosaccharides (Japanese Patent Application No. 222923/1995, Japanese Laid-Open Publication No. 31160/1994). In this sense, they are fructooligosaccharide preparations having new features.
In consideration of the above, some of the inventors have already proposed an industrial process for producing crystal 1-ketose from sucrose (Japanese Patent Application No. 64682/1996, Japanese Patent Application No. 77534/1996, and Japanese Patent Application No. 77539/1996). According to this process, a &bgr;-fructofuranosidase harboring fructosyltransferase activity is first allowed to act on sucrose to produce 1-kestose; the resultant 1-kestose is fractionated to a purity of 80% or higher by chromatographic separation; then, using this fraction as a crystallizing sample, crystal 1-kestose is obtained at a purity of 95% or higher. The &bgr;-fructofuranosidase harboring fructosyltransferase activity used in this process should be able to produce 1-kestose from sucrose at a high yield while minimizing the byproduct nystose, which inhibits the reactions in the above steps of chromatographic separation and crystallization. In the enzyme derived from
Aspergillus niger
, which is currently used for the industrial production of fructooligosaccharides mixtures, the 1-kestose yield from sucrose is approximately 44%, while 7% is turned to nystose (Japanese Patent Application No.64682/1996). These figures suggest that the enzyme has room for improvement in view of the industrial production of crystal 1-ketose.
As a next step, some of the inventors have successfully screened new enzymes having more favorable characteristics from
Penicillium rogueforti
and
Scopulariopsis brevicaulis
. These enzymes were able to turn 47% and 55% of sucrose into 1-kestose, respectively, and 7% and 4% to nystose (Japanese Patent Application No. 77534/1996, and Japanese Patent Application No. 77539/1996). These enzymes are inferior in productivity and stability to the enzyme derived from
Aspergillus niger
, and have room for improvement in view of the industrial production of crystal 1-ketose.
Thus, some of the inventors had paid attention to the procedure of genetic engineering as a process for improving the productivity of the enzyme, isolated the gene encoding &bgr;-fructofuranosidase from
Penicillium roqueforti
and
Scopulariopsis brevicaulis
, respectively, and conducted the structure analysis (PCT/JP97/00757). As a result, the translation regions encoding 565 amino acids and 574 amino acids as a mature protein were respectively deduced in the &bgr;-fructofuranosidase genes from
Penicillium roqueforti
and
Scopulariopsis brevicaulis
and their expression products were shown to have &bgr;-fructofuranosidase activity, like the &bgr;-fructofuranosidase gene from
Aspergillus niger
(L.M. Boddy et al., Curr. Genet., 24, 60-66 (1993)).
SUMMARY OF THE INVENTION
The inventors have now found that the addition of 38 and 39amino acids to the C-terminal of the &bgr;-fructofuranosidase genes from
Penicillium roqueforti
and
Scopulariopsis brevicaulis
, which were previously found by some of the inventors, improves its activity.
Thus, an object of the present invention is to provide a novel &bgr;-fructofuranosidase and its gene.
The novel &bgr;-fructofuranosidase according to the present invention is a polypeptide comprising the amino acid sequence of SEQ ID No. 1 or 3 or a homologue thereof.
Furthermore, the gene according to the present invention is a DNA encoding the above polypeptide.
The amino acid sequence of SEQ ID No. 1 or 3 according to the present invention is constructed by adding 38 and 39 amino acids to the C-terminals of the &bgr;-fructofuranosidase genes from
Penicillium roqueforti
and
Scopulariopsis brevicaulis
, which were previously found by some of the inventors as described above. It has been found that an intron actually exists at the region of the &bgr;-fructofuranosidase gene, which was presumed to encode the C-terminal amino acids by some of the present inventors and that the &bgr;-fructofuranosidase genes further encode 38 and 39 amino acids of the C-terminal. Surprisingly, the &bgr;-fructofuranosidase activity was remarkably improved by adding these amino acids to the C-terminal, as compared with the protein to which these sequences are not added.


REFERENCES:
patent: 6337201 (2002-11-01), Yanai et al.
patent: 97/34004 (1997-09-01), None
Hatakeyama et al. [J. Ferment. Bioeng., 1996, 81(6) : 518-23, Abstract only].*
L. M. Boddy et al., “Purification and Characterization of an Aspergillus nigar invertase and its DNA sequence”, Curr. Genet., vol. 24, pp. 60-66, 1993.

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