Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2001-07-20
2004-11-09
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S203000, C435S320100, C435S252300, C435S254110, C536S023200, C536S023700, C536S023740
Reexamination Certificate
active
06815191
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a novel endo-&bgr;-N-acetylglucosaminidase gene, and specifically relates to a gene where said gene is derived from the genus
Mucor.
Further, the present invention relates to a recombinant plasmid which comprises said gene, an organism transformed with said plasmid, and a method of producing a novel endo-&bgr;-N-acetylglucosaminidase using said transformant.
BACKGROUND ART
Glycoproteins are widely found in animal tissue, plant tissue, and the cell membrane and cell wall etc. of eukaryotic microorganisms.
In recent years, it has become increasingly clear that the sugar chains of glycoproteins play an important role in mechanisms such as cell differentiation, carcinogenesis and intercellular recognition. In order to clarify these mechanisms, research into the correlation between sugar chain structure and its function is proceeding. As a means of achieving this objective, when cleaving a sugar chain from a glycoprotein or when identifying the structure of a sugar chain, various glycosidases are employed. Among these, endo-&bgr;-N-acetylglucosaminidase acts on the asparagine-linked sugar chain (N-linked sugar chain, N-sugar chain) and has the action of cleaving the diacetyl-chitobiose portion that exists within the sugar chain thereby liberating the sugar chain.
Since endo-&bgr;-N-acetylglucosaminidase can liberate the sugar portion of a glycoprotein from the protein portion, it is thought to be important in the analysis of the function and structure of sugar chains in glycoproteins.
Asparagine-linked sugar chains may be classified by their structure as high mannose type (mannane type sugar chain), hybrid type, or complex type.
Known endo-&bgr;-N-acetylglucosaminidases include Endo H (A. L. Tarentino and F. Maley,
J. Biol. Chem
., 249, 811 (1974)), Endo F (K. Takegawa, et al.,
Eur. j. Biochem
., 202, 175 (1991)), and EndoA (K. Takegawa, et al.,
Appl. Environ. Microbiol
., 55, 3107 (1989)). However, these enzymes only act upon sugars with specific structures, and do not act upon glycoproteins except in the presence of a denaturing agent.
Endo-&bgr;-N-acetylglucosaminidase derived from
Mucor hiemalis
is capable of cleaving tri-antennary complex type sugar chains in respect of not only high mannose type (mannane type sugar chain) and hybrid type, but also complex type chains. Further, with the asialylated type, cleavage ability extends to tetra-antennary heteroglycan chains. Further, it is known that it is possible to free sugar chains from glycoproteins without subjecting the protein to denaturization treatment. (S. Kadowaki, et al.,
Agric. Biol. Chem
., 54, 97 (1990)). Therefore, endo-&bgr;-N-acetylglucosaminidase derived from
Mucor hiemalis
can be said to be useful in the research of the functional and physiological role of sugar chains and proteins of glycoproteins.
On the other hand, conversion of mannane type sugar chains derived from yeast into sugar chains in a form compatible with humans, is extremely significant industrially. As methods for this conversion, in vivo conversion through improvement of the yeast sugar chain biosynthetic system by genetic manipulation, and in vitro conversion using the trans-glycosylation reaction can be considered. For the purpose of sugar conversion, endo-&bgr;-N-acetylglucosaminidase is required to have as properties, 1) the substrate specificity, i.e an ability to cleave both mannane and complex types; and, 2) the ability to perform the trans-glycosylation reaction, which is the reverse reaction of the decomposition reaction. Therefore, endo-&bgr;-N-acetylglucosaminidase derived from
Mucor hiemalis
can be said to be an appropriate enzyme for the practice of said conversion.
The present inventors, have proposed a sugar chain conversion technique using endo-&bgr;-N-acetylglucosaminidase derived from
Mucor hiemalis
which can alter yeast sugar chains to human-compatible form. (Japanese Patent Application Laid-Open No: Hei 7-59587)
To perform sugar chain conversions such as the above, an authentic enzyme preparation of high purity is required in great amounts. In this case, improvement of enzyme productivity by conventional breeding methods using mold cells has been considered. However, since conventional breeding methods are limited predominantly to the method of selecting such an enzyme from mutant strains obtained using ultraviolet light or mutagens, the isolation of stable mutants is difficult. Further, conventional breeding methods are often accompanied by unfavorable transformations. Further, since molds generally produce protease enzymes, they are unfavorable for production of an enzyme for the purpose of sugar conversion. Since, in order to overcome these problems it is necessary to proceed through a number of purification steps, the work is complicated and the yield is low. For example, culturing a microorganism belonging to the genus
Mucor
which is a type of filamentous mold, even if purification of the supernatant of this culture is performed, contamination with protease cannot be prevented and preparation in large amounts is difficult because of low enzyme productivity of the microorganism, and thus this method was of little practical value.
Given the above, it is desired for the purpose of mass producing endo-&bgr;-N-acetylglucosaminidase, that the gene for said enzyme be obtained and produced through the use of genetic engineering. Further, if the gene can be obtained, it can be expected that an enzyme with improved heat resistance and pH resistance and increased reaction rate can be obtained using protein engineering techniques. However, there have been no reported attempts at gene cloning to date.
DISCLOSURE OF INVENTION
It is an object of the present invention to provide an endo-&bgr;-N-acetylglucosaminidase, an endo-&bgr;-N-acetylglucosaminidase gene, a recombinant vector which comprises said gene, an organism transformed with said vector, and a method of producing endo-&bgr;-N-acetylglucosaminidase.
The present inventors, as a result of their intensive research directed to solving the above problem, based on partial amino acid sequence information of said endo-&bgr;-N-acetylglucosaminidase derived from
Mucor hiemalis,
have succeeded in obtaining the gene that encodes said enzyme from a cDNA library prepared from
Mucor hiemalis
which is a bacteria which produces said enzyme, have further succeeded in expressing this gene in yeast, and thereby completed the present invention.
Thus, the present invention provides the recombinant protein of (a) or (b) below:
(a) a protein comprising the amino acid sequence represented by SEQ ID NO: 3; and,
(b) a protein comprising an amino acid sequence derived from the amino acid sequence represented by SEQ ID NO: 3 by deletion, substitution, insertion, or addition of at least one amino acid and having the activity of endo-&bgr;-N-acetylglucosaminidase.
Further, the present invention provides a endo-&bgr;-N-acetylglucosaminidase gene which encodes the protein of (a) or (b) below, and a gene that hybridizes with said gene under stringent conditions, and which comprises DNA encoding a protein that has endo-&bgr;-N-acetylglucosaminidase activity.
(a) a protein comprising the amino acid sequence represented by SEQ ID NO: 3; and,
(b) a protein comprising an amino acid sequence derived from the amino acid sequence represented by SEQ ID NO: 3 by deletion, substitution, insertion, or addition of at least one amino acid and having the activity of endo-&bgr;-N-acetylglucosaminidase.
Further, the present invention provides a gene comprising the DNA of (c) or (d) below:
(c) a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 2; and,
(d) a DNA which hybridizes under stringent conditions with a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 2, and encodes a protein having the activity of endo-&bgr;-N-acetylglucosaminidase.
Said gene includes a gene derived from a microorganism belonging to the genus
Mucor.
Further, the present invention provides a recombinant vector which comprises said gene.
Further,
Iwamatsu Akihiko
Kobayashi Kazuo
Kumagai Hidehiko
Takeuchi Makoto
Takeuchi Yoriko
Foley & Lardner LLP
Kirin Beer Kabushiki Kaisha
Prouty Rebecca E.
Steadman David J
Takeuchi Yoriko
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