Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1998-09-10
2002-01-08
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S183000, C435S193000, C530S350000
Reexamination Certificate
active
06337201
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a &bgr;-fructofuranosidase gene, a process for isolating the gene, and a system for producing a &bgr;-fructofuranosidase. More particularly, the present invention relates to a novel &bgr;-fructofuranosidase, a DNA encoding it, and a process for isolating a DNA encoding &bgr;-fructofuranosidase; a novel mold fungus having no &bgr;-fructofuranosidase and a process for producing a recombinant &bgr;-fructofuranosidase using the mold fungus as a host; and a &bgr;-fructofuranosidase variant which selectively and efficiently produces a specific fructooligosaccharide such as 1-kestose from sucrose.
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 B 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. However, as &bgr;-fructofuranosidase preparations which are currently used for the industrial production of fructooligosaccharides is a cell-bound &bgr;-fructofuranosidase derived from
Asperglllus niger
. They contain a relatively large proportion of proteins as impurities. Therefore, a need still exists for a high-purity &bgr;-fructofuranosidase preparation with little unwanted proteins and a high titer. Further, an extracellular &bgr;-fructofuranosidase is desired in an attempt to improve efficiently by using it in a fixed form, as an extracellularly available enzyme is more suitable for fixation.
Genes encoding &bgr;-fructofuranosidase have been isolated from bacteria (Fouet, A., Gene, 45, 221-225 (1986), Martin, I. et al., Mol. Gen. Genet., 208, 177-184 (1987), Steininctz, M. et al., Mol. Gen. Genet., 191, 138-144 (1983), Scholle, R. et al., Gene, 80,49-56 (1989), Aslanidis, C. et al., J. Bacteriol., 171, 6753-6763 (1989), Sato, Y. and Kuramitsu, H. K., Infect. Immun., 56, 1956-1960 (1989), Gunasekaran, P. et al., J. Bacteriol., 172, 6727-6735 (1990)); yeast (Taussing, R, and M. Carlson, Nucleic Acids Res., 11, 1943-1954 (1983), Laloux, O. et al., FEBS Lett., 289, 64-68 (1991); mold (Boddy, L. M. et al., Curr, Genet., 24, 60-66 (1993); and plants (Arai, M. et al., Plant Cell Physiol., 33, 245-252 (1992), Unger, C. et al. Plant Physiol., 104, 1351-1357 (1994), Elliott, K. et al., Plant Mol. Biol., 21, 515-524 (1993), Sturm, A. and Chrispeels, M. J., Plant Cell, 2, 1107-1119 (1990)). However, to the best knowledge of the inventors, no gene has been found which encodes a &bgr;-fructofuranosidase having transferase activity and is usable for the industrial production of fructooligosaccharides.
If a &bgr;-fructofuranosidase gene usable for the industrial production of fructooligosaccharides is obtained, other functionally similar genes may be isolated, making use of their homology to the former. To the best knowledge of the inventors, no case has been reported on the screening of a new &bgr;-fructofuranosidase gene using this technique. A process for isolating a &bgr;-fructofuranosidase gene by this approach may also be applied to the screening of &bgr;-fructofuranosidase enzyme to achieve significantly less effort and time than in conventional processes: first, using a &bgr;-fructofuranosidase gene as a probe, a similar &bgr;-fructofuranosidase gene is isolated, making use of its homology to the former; then, the isolated gene is introduced and expressed in a host which does not metabolize sucrose, such as
Trichoderma viride
, or a mutant yeast which lacks sucrose metabolizing capability (Oda, Y. and Ouchi, K., Appl. Environ. Microbiol., 1989, 55, 1742-1747); a homogeneous preparation of &bgr;-fructofuranosidase is thus obtained as a genetic product with significantly less effort and time of screening. Furthermore, if the resultant &bgr;-fructofuranosidase exhibits desirable characteristics, its encoding gene may be introduced in a safe and highly productive strain to enable the production of the desired &bgr;-fructofuranosidase.
In addition, for producing such desirable &bgr;-fructofuranosidase, designing a system for production, particularly a host which does not metabolize sucrose, is an important consideration. Using a host which intrinsically has &bgr;-fructofuranosidase activity would result in a mixture of the endogenous &bgr;-fructofuranosidase of the host and the &bgr;-fructofuranosidase derived from the introduced gene. In this case, to take advantage of the &bgr;-fructofuranosidase derived from the introduced gene, it must be isolated from the endogenous &bgr;-fructofuranosidase of the host before application. On the contrary, using a host which lacks &bgr;-fructofuranosidase activity would eliminate the need for enzyme isolation. In other words, the resultant unpurified enzyme would show the desirable characteristics of the &bgr;-fructofuranosidase derived from the introduced gene. Known examples of microorganisms which do not have &bgr;-fructofuranosidase activity include the Trichoderma strains and yeast mutants lacking sucrose metabolizing capability (Oda, Y. Ibid.) as described above. However, considering that the resultant &bgr;-fructofuranosidase will be applied in food industry, a better candidate for a host would be a strain having no &bgr;-fructofuranosidase selected from Aspergillus mold fungi which have been time-tested for safety through application to foods and industrial production of enzymes.
Furthermore, if a &bgr;-fructofuranosidase gene usable for the industrial production of fructooligosaccharides is obtained, it may enable the development of a mutant with improved characteristics. For example, &bgr;-fructofuranosidase which produces 1-kestose selectively and efficiently would provide the following advantage:
The molecular structures of 1-kestose and nystose, which make up part 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 Patent 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 proposed an industrial process for producing crystal 1-kestose 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 fructooligosaccharide mixtures, the 1-kestose yield from sucrose is approximately 44%, whi
Baba Yuko
Hirayama Masao
Nakamura Hirofumi
Nakane Akitaka
Watabe Akemi
Hutson Richard
Meiji Seika Kaisha Ltd.
Prouty Rebecca E.
Wenderoth , Lind & Ponack, L.L.P.
LandOfFree
&bgr;-fructofuranosidase and its gene, method of isolating... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with &bgr;-fructofuranosidase and its gene, method of isolating..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and &bgr;-fructofuranosidase and its gene, method of isolating... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2822994