&bgr;-fructofuranosidase gene

Details &bgr;-fructofuranosidase gene &bgr;-fructofuranosidase gene

2000-02-14

2001-09-04

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

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

Hydrolase

C435S069100, C435S183000, C435S252300, C435S320100

Reexamination Certificate

active

06284510

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a &bgr;-fructofuranosidase gene. &bgr;-Fructofuranosidase (EC3.2.1.26.) is an enzyme that hydrolyses sucrose and other &bgr;-fructofuranosides to release fructose residue.
This enzyme has an action of transferring a &bgr;-fructofuranosyl group from a substrate to water to hydrolyze the substrate. In addition, it has an action of transferring a &bgr;-fructofuranosyl group to acceptors having a hydroxyl group such as other saccharides, alcohols, and phenol. Utilizing this action, the enzyme is used in the synthesis of transfructosylated oligosaccharides such as lactosucrose.
BACKGROUND OF THE INVENTION
Transfructosylated oligosaccharides such as lactosucrose have an activity of propagating bifidobacterium and currently attract attention in the fields of foods and drugs as a new sweetener that is a substitute for sucrose.
Transfructofuranosylated oligosaccharide, one of transfructosylated oligosaccharides, is usually produced by allowing &bgr;-fructofuranosidase to act on a raw material such as sucrose, starch sugar, lactose, etc.
Microorganisms, such as Arthrobacter, produce &bgr;-fructofuranosidase. However, said microorganisms are each low in &bgr;-fructofuranosidase productivity. Therefore, there is a problem in that microorganisms must be cultivated in large amounts to produce transfructofuranosylated oligosaccharides on a large scale.
Incidentally, current progress in genetic engineering techniques has made it possible to obtain a large amount of an enzyme relatively easily even if the amino acid sequence of the enzyme has not been elucidated yet. This is achieved by isolating the gene coding for the enzyme, determining the base sequence of the enzyme, producing a recombinant DNA containing the gene coding for the enzyme, incorporating the recombinant DNA into microorganism or animal or plant cells, and cultivating the obtained transformants.
Accordingly, it has been a desire for ascertaining the gene coding for &bgr;-fructofuranosidase and determining its base sequence.
SUMMARY OF THE INVENTION
An object of the present invention is to identify the structure of the gene coding for a polypeptide having a &bgr;-fructofuranosidase activity, thereby providing a mass production system for a transfructofuranosylated oligosaccharide using the enzyme.
Elucidation of the gene coding for a polypeptide having a &bgr;-fructofuranosidase activity is considered to give a possibility for obtaining the enzyme in a large amount. Also, it is considered to be useful for the development of a variant enzyme whose heat resistance and transfer ratio are increased by means of genetic engineering techniques.
The inventors of the present invention have made research on the synthesis of transfructosylated oligosaccharides for several years continuously. During the research, the inventors have made it clear that
Arthrobacter
sp. K-1 strain produces &bgr;-fructofuranosidase having substrate specificity that is different from that of other &bgr;-fructofuranosidase and that exhibits high transfer efficiency in the presence of an acceptor.
That is,
Arthrobacter
sp. K-1 strain can produce &bgr;-fructofuranosidase outside the cell without addition of sucrose to the culture medium and it does not synthesize high molecular fructan. Therefore, it is easy for handling.
Therefore, the present inventors have paid their attention onto the structural gene coding for the &bgr;-fructofuranosidase, and have made intensive study. As a result, the inventors have analyzed the structure of the &bgr;-fructofuranosidase gene.
The first aspect of the present invention provides a &bgr;-fructofuranosidase gene coding for a protein having an amino acid sequence described in Seq. I.D. No. 2 in the Sequence Listing.
DETAILED DESCRIPTION OF THE INVENTION
First, an outline on the approach to the present invention will be described.
&bgr;-fructofuranosidase which was obtained from the culture of a bacterium belonging to the genus
Arthrobacter
and having a productivity of &bgr;-fructofuranosidase was highly purified and the amino acid sequence of its N-terminal was determined.
Further, the &bgr;-fructofuranosidase was enzymatically decomposed to prepare peptide fragments, and their amino acid sequences were determined.
Then, a primer was prepared based on the base sequence confirmed from the amino acid sequence. Using this primer, there was practiced a polymerase chain reaction (PCR) method using the primer and chromosomal DNA extracted from a bacterial strain belonging to the genus
Arthrobacter
as a template to obtain a clear band of 221 bp.
After TA cloning of the obtained PCR product, the base sequence thereof was analyzed using a DNA sequencer. The obtained base sequence was translated to amino acids and homology search was performed to find that it has a high homology with enzymes acting on sucrose. Therefore, the DNA was considered to be a portion of the &bgr;-fructofuranosidase gene.
Hence, cloning of the &bgr;-fructofuranosidase gene was practiced using the PCR product as a probe.
First, the chromosomal DNA extracted from a bacterium belonging to the genus
Arthrobacter
was digested with restriction enzymes and then subjected to Southern blot hybridization. As a result, the presence of the target &bgr;-fructofuranosidase gene which in an about 9 Kbp DNA fragment was confirmed. After cutting out the DNA fragment from the gel and purifying it, the DNA was in vitro packaged in a cosmid vector and a partial DNA library was constructed.
Out of the library was screened
Escherichia coli
having a &bgr;-fructofuranosidase gene by colony hybridization. From positive clone was extracted a gene consisting of 1917 bases in total. That is, this gene is the &bgr;-fructofuranosidase gene of the present invention.
Hereafter, the present invention will be described in detail.
As described above, the &bgr;-fructofuranosidase gene of the present invention is derived from a microorganism of
Arthrobacter
having a &bgr;-fructofuranosidase producing ability. Such a microorganism of
Arthrobacter
having a &bgr;-fructofuranosidase producing ability includes
Arthrobacter
sp. K-1 strain and variant strains thereof. Any mutants of the present bacterium obtained naturally or by an artificial means are all embraced by the present invention so far as they have the above-described ability.
Arthrobacter
sp. K-1 strain was isolated from the soil of Osaka prefecture and deposited at National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry at 1-3 Higashi 1-chome, Tsukuba-shi, Ibaraki-ken 305, JAPAN. The Accession Number is FERM BP-3192. The bacteriological properties of the said bacterium are described in Japanese Patent No. 2781412.
The &bgr;-fructofuranosidase can be obtained from the above-mentioned microbial strain. Specifically, after cultivating the above strain in a nutrient medium, microbial cells are removed by centrifugation or the like to recover the culture medium. Subsequently, using purification means such as salting out or various types of column chromatography, a highly purified &bgr;-fructofuranosidase can be obtained.
Next, the amino acid sequence of N-terminal of the purified &bgr;-fructofuranosidase is determined using a Gas Phase Protein Sequencer “476A” manufactured by Applied Biosystems. The determined amino acid sequence consists of 9 amino acids as shown in Seq. I.D. No. 3 in the Sequence Listing. Further, the &bgr;-fructofuranosidase is enzymatically decomposed to prepare peptide fragments and their amino acid sequences are determined (Seq. I.D. Nos. 4 to 6 in the Sequence Listing).
Based on the information on the decoded amino acid sequences, primers areprepared (Seq. I.D. Nos. 7and 8 inthe Sequence Listing). PCR is performed using the primers and the chromosomal DNA extracted from the strain of
Arthrobacter
as a template.
As a result, a DNA amplified product of 221 bp is obtained. This DNA is purified and TA cloned. Thereafter, its base sequence is decoded using a DNA seque

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