Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2001-08-08
2003-07-29
Slobodyansky, Elizabeth (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S252300, C435S252330, C435S320100, C536S023200
Reexamination Certificate
active
06599729
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to an &agr;-agarase and a method for producing the same. Specifically, the present invention relates to an &agr;-agarase, which is useful for producing agarooligosaccharides with low degrees of polymerization having various physiological activities from agarose, and a method for producing the &agr;-agarase as well as use of the enzyme. The present invention also relates to a polypeptide having an &agr;-agarase activity and a gene encoding said polypeptide. Specifically, the present invention relates to an amino acid sequence of an &agr;-agarase, which is useful for producing agarooligosaccharides with low degrees of polymerization having various physiological activities from agarose, and a nucleotide sequence encoding the amino acid sequence. Furthermore, the present invention relates to a method for producing a polypeptide having an &agr;-agarase activity by genetic engineering. In addition, the present invention relates to a method for producing an agarooligosaccharide using a polypeptide having an &agr;-agarase activity.
2. Description of Related Art
Agarose is the principal constituent of agar. Agarose is a polysaccharide that has a structure in which D-galactose and 3,6-anhydro-L-galactose are alternately linked together through &agr;-1,3 bonds and &bgr;-1,4 bonds. One must degrade agarose into smaller molecules in order to produce oligosacchaides from agar. For this purpose, methods in which agarose is chemically degraded and methods in which agarose is enzymatically digested are known. In a chemical degradation method, agarose can be hydrolyzed using an acid. In this case, &agr;-1,3 bonds are mainly cleaved. Two enzymes, &bgr;-agarase which cleaves &bgr;-1,4 bonds in agarose and &agr;-agarase which cleaves &agr;-1,3 bonds in agarose, are known to digest agarose.
Oligosaccharides obtained by cleaving agarose at &bgr;-1,4 bonds are called as neoagarooligosaccharides. Neoagarooligosaccharides have D-galactose at their reducing ends and their degrees of polymerization are expressed by even numbers. On the other hand, oligosaccharides obtained by cleaving agarose at &agr;-1,3 bonds are called as agarooligosaccharides. Agarooligosaccharides have 3,6-anhydro-L-galactose at their reducing ends and their degrees of polymerization are expressed by even numbers. Recently, it was shown that agarooligosaccharides which have 3,6-anhydro-L-galactose at their reducing ends have physiological activities such as an apoptosis-inducing activity, a carcinostatic activity, various antioxidant activities, an immunoregulatory activity, an antiallergic activity, an anti-inflammatory activity and an activity of inhibiting &agr;-glycosidase (WO99/24447, Japanese Patent Application No. 11-11646). Based on the physiological activities, pharmaceutical compositions and functional foods or drinks containing the agarooligosaccharides as their active ingredients can be provided.
It is difficult to control the size of produced oligosaccharides in a method in which agarose is chemically degraded. In particular, it is quite difficult to selectively produce smaller oligosaccharides with low degrees of polymerization (e.g., T. Tokunaga et al., Bioscience & Industry, 49:734 (1991)). If &bgr;-agarase is used, only neoagarooligosaccharides which do not have the above-mentioned physiological activities can be obtained because this enzyme cleaves only &bgr;-1,4 bonds.
It is expected that agarooligosaccharides having physiological activities are produced by using &agr;-agarase which has an activity of cleaving &agr;-1,3 bonds. Known &agr;-agarases include enzymes produced by a marine Gram-negative bacterial strain GJ1B (Carbohydrate Research, 66:207-212 (1978); this strain is indicated as
Alteromonas agarlyticus
strain GJ1B in European Journal of Biochemistry, 214:599-607 (1993)) and a bacterium of genus Vibrio (JP-A 7-322878; strain JT0107-L4) However, it is impossible to produce agarobiose which has notable physiological activities by using the &agr;-agarase derived from Alteromonas agarlyticus strain GJ1B because the enzyme cannot digest hexasaccharides or shorter oligosaccharides. Furthermore, the &agr;-agarase derived from a bacterium of genus Vibrio cannot be used for the production of agarooligosaccharides using agarose as a raw material because this enzyme exhibits its activity only on hexasaccharides and shorter oligosaccharides and does not act on agarose at all.
As described above, prior art has problems regarding the production of smaller agarooligosaccharides such as agarobiose and agarotetraose which have 3,6-anhydro-L-galactose at their reducing ends and have various physiological activities.
The main object of the present invention is to provide a polypeptide having an &agr;-agarase activity which can be used for efficient production of smaller agarooligosaccharides, an amino acid sequence of the polypeptide, a gene encoding the polypeptide, a method for producing the polypeptide and a method for producing the smaller agarooligosaccharides.
SUMMARY OF THE INVENTION
In view of the problems as described above, the present inventors have studied intensively and conducted search in order to obtain an enzyme that cleaves &agr;-1,3 bonds in agarose and generates agarooligosaccharides having notable physiological activities. As a result, the present inventors have successfully found two microbial strains that produce enzymes having properties suitable for this purpose. The enzymes produced by these microorganisms were isolated and their physical and chemical as well as enzymatic properties were elucidated. Furthermore, the present inventors have successfully isolated genes for the enzymes, and found a method for readily producing polypeptides having &agr;-agarase activities by means of genetic engineering using the genes, thereby completing the present invention.
The present invention is outlined as follows. The first aspect of the present invention relates to a novel &agr;-agarase having the following physical and chemical properties:
(1) action: hydrolyzing an &agr;-1,3 bond between 3,6-anhydro-L-galactose and D-galactose;
(2) substrate specificity: acting on agarose, agarohexaose and agarooligosaccharides longer than agarohexaose but not on agarotetraose;
(3) optimal temperature: exhibiting its enzymatic activity at a temperature of 55° C. or below; and
(4) heat stability: retaining 20% or more of its activity after treatment at 48° C. for 30 seconds.
Such &agr;-agarases are exemplified by an enzyme that contains an amino acid sequence consisting of 749 residues from amino acid number 177 to amino acid number 925 in the amino acid sequence of SEQ ID NO:14, or an amino acid sequence in which one or more amino acids are substituted, deleted, added and/or inserted in said amino acid sequence consisting of 749 residues, or an enzyme that contains an amino acid sequence consisting of 767 residues from amino acid number 184 to amino acid number 950 in the amino acid sequence of SEQ ID NO:15, or an amino acid sequence in which one or more amino acids are substituted, deleted, added and/or inserted in said amino acid sequence consisting of 767 residues.
The second aspect of the present invention relates to a gene encoding a polypeptide having an &agr;-agarase activity, which encodes the &agr;-agarase of the first aspect. Such genes are exemplified by a gene that contains a nucleotide sequence consisting of 2247 bases from base number 529 to base number 2775 in the nucleotide sequence of SEQ ID NO:12, or a nucleotide sequence in which one or more bases are substituted, deleted, added and/or inserted in said nucleotide sequence consisting of 2247 bases, or a gene that contains a nucleotide sequence consisting of 2301 bases from base number 550 to base number 2850 in the nucleotide sequence of SEQ ID NO:13, or a nucleotide sequence in which one or more bases are substituted, deleted, added and/or inserted in said nucleotide sequence consisting of 2301 bases.
The third aspect of the present invention relates to a gene that is hy
Kato Ikunoshin
Nomura Yoshiko
Sagawa Hiroaki
Sakai Takeshi
Tomono Jun
Browdy and Neimark , P.L.L.C.
Slobodyansky Elizabeth
Takara Shuzo Co. Ltd.
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