Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-04-29
2001-09-11
Lilling, Herbert J. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
Reexamination Certificate
active
06287819
ABSTRACT:
DESCRIPTION
1. Technical Field
The present invention relates to a process for producing uridine diphosphate-N-acetylglucosamine (UDPAG), an important substrate in the synthesis of oligosaccharides.
2. Background Art
Recent remarkable progress in sugar chain science has clarified some of sugar's physiological roles, making possible the development of pharmaceuticals and functional materials based on oligosaccharides possessing physiological activities. However, currently only limited types of oligosaccharides are commercially available, and those which are commercially available are extremely expensive. Moreover, these oligosaccharides may be produced only on a reagent level, and a mass production method therefor has not necessarily been fully established.
Conventionally, oligosaccharides have been produced by extraction from natural substances, chemical synthesis, enzymatic synthesis, or a combination of these methods. Among these processes, enzymatic synthesis has been considered best suited for mass production, for the following reasons: (1) enzymatic synthesis does not require intricate procedures, such as protection and removal of protection, which are required for chemical synthesis, and thus can provide oligosaccharides of interest with ease; and (2) substrate specificity of an enzyme enables synthesis of oligosaccharides having highly structural specificity. In addition, recent advances in recombinant DNA technology have made possible economical mass production of various types of enzymes, also contributing to establishing the superiority of enzymatic synthesis over other processes.
Two processes for the synthesis of oligosaccharides through enzymatic synthesis are available: a process making use of the reverse reaction of hydrolase, and a process making use of glycosyltransferase. The former has an advantage in that it may employ inexpensive monosaccharides as the substrate, but, because it employs the reverse reaction to the hydrolysis reaction, it is not necessarily the beat process in terms of synthesis yield and application to oligosaccharides of complicated structure.
In contrast, the latter process makes use of glycosyltransferase and has an advantage over the former in terms of the synthesis yield and application to the synthesis of oligosaccharides of complicated structure. Moreover, due to recent progress in recombinant DNA techniques, mass-production of various types of glycosyltransferase has also contributed to realization of this process.
However, sugar nucleotides, which are sugar donors and used in a synthesis making use of glycosyltransferase are, with few exceptions, still expensive, and are provided only in small amounts on reagent levels. For example, although there has been reported a process for preparing uridine diphosphate-N-acetylglucosamine (UDPAG)—which is a donor of N-acetylglucosamine contained in the core portion of any of a variety of physiologically active sugar chains—through a method which makes use of an osmolarity-resistant yeast (Japanese Patent Application Laid-Open (kokai) No. 8-23993), studies are required before realization of industrial production thereof.
The present inventors have conducted extensive studies on the biosynthesis route of UDPAG, and considered that the synthesis rate is determined by the acetylation reaction of glucosamine-6 phosphate to N-acetylglucosamine-6-phosphate, which occurs in a series of reactions starting from glucosamine to glucosamine-6 phosphate then to N-acetylglucosamine-6 phosphate and finally to N-acetylglucosamine-1 phosphate. If this is the case, use of N-acetylglucosamine-6 phosphate as a substrate may improve the yield in synthesis of UDPAG. However, at present, a large quantity of this substance is not available at a low price.
Consequently, it was presumed that if N-acetylglucosamine, which is currently available in large quantities at a low cost, is used as a substrate, the aforementioned rate-determining step may be eliminated, and N-acetylglucosamine may serve as a better substrate than glucosamine. Thus, the present inventors examined the UDPAG production process proposed by Tochikura at al. (Japanese Patent Publication (kokoku) No. 49-8278, Tochikura Process), in which UDPAG is produced with yeast cells from uridylic acid (UMP) and N-acetylglucosamine serving as substrates. However, their studies ended up with confirmation of the finding by Tochikura et al. that use of N-acetylglucosamine as a substrate resulted in lower, or even nil or very little, yield of UDPAG as compared with the case when glucosamine is used as a substrate.
Accordingly, the present invention is directed to a process for producing UDPAG at high yield, even in the case when N-acetylglucosamrine is used as a substrate.
DISCLOSURE OF THE INVENTION
The inventors of the present invention have carried out careful studies to achieve the aforementioned objectives, and found that (1) yeast cells have no or very little enzymatic activity for phosphorylation of N-acetylglucosamine, so that N-acetylglucosamine may not be a candidate substrate; however, if N-acetylglucosamine kinase, which is a phosphokinase for N-acetylglucosamine, is added to the reaction, UDPAG may be synthesized efficiently; (2) furthermore, addition of N-acetylglucosamine phosphate mutase and/or uridine diphosphate-N-acetylglucosamine pyrophosphorylase improves the yield of UDPAG, as compared with the case which makes use of N-acetylglucosamine kinase alone; and (3) UDPAC is synthesized efficiently from uridine triphosphate (UTP) by addition of N-acetylglucosamine kinase, N-acetylglucosamine phosphate mutase, and uridine diphosphate-N-acetylglucosamine pyrophosphorylase; thus completing the invention.
Accordingly, the present invention provides a process of producing UDPAG from UMP and N-acetylglucosamine by use of microorganism cells, which process is characterized by the addition of N-acetylglucosamine kinase in the process.
Further, the present invention provides a process of producing UDPAG from UTP and N-acetylglucosamine by use of enzyme, which process is characterized by combined use of N-acetylglucosamine kinase, N-acetylglucosamine phosphate mutase, and uridine diphosphate-N-acetylglucosamine pyrophosphorylase as enzymes.
REFERENCES:
patent: 4569909 (1986-02-01), Seno et al.
patent: 4604349 (1986-08-01), Seno et al.
patent: 5674715 (1997-10-01), Tomita et al.
patent: 49-8278 (1974-02-01), None
patent: 8-23993 (1996-01-01), None
patent: 10-28594 (1998-02-01), None
Tatsurokuro Tochikura et al.; Agricultural and Biological Chemistry, vol. 35, pp. 163-176, 1971 XP000912295.
Hamamoto Tomoki
Ishige Kazuya
Midorikawa Yuichiro
Noguchi Toshitada
Okuyama Kiyoshi
Lilling Herbert J.
Wenderoth , Lind & Ponack, L.L.P.
Yamasa Corporation
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