Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing heterocyclic carbon compound having only o – n – s,...
Reexamination Certificate
1997-04-24
2002-03-26
Marx, Irene (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing heterocyclic carbon compound having only o, n, s,...
C435S117000, C435S252330
Reexamination Certificate
active
06361978
ABSTRACT:
This invention relates to a fermentative process for the production of biotin from desthiobiotin.
BACKGROUND OF THE INVENTION
Biotin is one of the essential vitamins for nutrition of animals, both human and non -human, plants, and microorganisms, and very important as a medicine or food additive.
There are many studies on fermentative production of biotin. Escherichia strains are known as microorganisms which can be used for the above process [see Japanese Patent Publication (Kokai) No. 149091/1986, WO 87/01391 and Japanese Patent Publication (Kokai) No. 155081/1987 ]. In addition to the above-mentioned strains, Bacillus strains [Japanese Patent Publication (Kokai) No. 180174/1991), Serratia strains [Japanese Patent Publication (Kokai) No. 27980/1990] and Brevibacterium strains [Japanese Patent Publication (Kokai) No. 240489/1991] are also known. But these processes have not yet been suitable for industrial use because of the low efficiency of carbon recovery from the nutrients into biotin and, in some cases, the accumulation of the direct intermediate, desthiobiotin. It is therefore desirable to improve the efficiency of the conversion of desthiobiotin to biotin. A conversion reaction of desthiobiotin to biotin using the resting cell system of
Escherichia coli
(Antimicrob. Agents Chemother. 21, 5, 1982) and one using cell-free extract of
Escherichia coli
[J. Biol. Chem., 270, 19158 (1995); Biosci. Biotechnol. Biochem., 56, 1780 (1992); Eur. J. Biochem., 224, 173 (1994); Arch. Biochem. Biophys., 326, 48 (1996)] are known. According to these publications, it has been clarified that protein factors such as ferredoxin-NADP reductase and flavodoxin together with biotin synthase are involved in the biotin formation from desthiobiotin. Nevertheless, only limited effect has been observed for biotin production from desthiobiotin under these conditions. It was simply speculated that another unknown protein should be involved in this reaction to more efficiently convert desthiobiotin to biotin.
Furthermore. a conversion reaction by using the purified biotin synthase of
Bacillus sphaietricus
with photoreduced deazaflavin as an artificial electron donor instead of using physiological electron transfer system of ferredoxin-NADP reductase and flavodoxin has recently been reported [Biochem. Biophys.
Res. Commun
., 217, 1231 (1995)]. But the reported reaction efficiency is not high enough for the reaction to be usable in the industrial production of biotin.
An object of the present invention is to find a more efficient process of producing biotin from desthiobiotin, and to this end there have been elucidated various protein factors. It has been found that nifU and nifS gene products (hereinafter referred to as NIFU and NIFS), which are suggested to be involved in the mobilization of the iron and sulfide necessary for nitrogenase metallocluster core formation [J. Bacteriology, 175, 6737 (1993)], are significantly effective for the production of higher amount of biotin from desthiobiotin. The present invention is based upon these findings.
Accordingly, the present invention provides a process for the production of biotin from desthiobiotin which comprises contacting desthiobiotin with an enzyme reaction system containing bioB gene product (which encodes biotin synthase; hereafter referred to as BIOB) and also NIFU and/or NIFS, and isolating the resulting biotin from the reaction mixture, especially such a process wherein BIOB is derived from
Escherichia coli
and NIFU and/or NIFS are derived from
Klebsiella pneumoniae
, or a process as described before wherein the enzyme reaction mixture further contains S-adenosylmethionine, L-cysteine and an electron supplying system, e.g. wherein the electron supplying system comprises NADPH, ferredoxin-NADP reductase and flavodoxin or wherein the electron supplying system comprises deazariboflavin or a functional equivalent component thereof selected from deazaflavin (5-deazaflavin) [J. Biol. Chem., 268, 2296 (1993)] and 8-hydroxy-5-deazaflavin [J. Bacteriology, 172, 6061 (1990)].
It is furthermore an object of the present invention to provide a process as described above wherein the reaction is effected at a pH of from about 6.0 to about 8.5, preferably from about 7.0 to about 8.0, and in a temperature range of from about 20 to about 45 C, preferably from about 25 to about 40 C.
Furthermore, the present invention also provides a fermentative process for the production of biotin from desthiobiotin which comprises cultivating a microorganism, which has been transformed by the DNA sequences encoding BIOB and NIFU and/or NIFS itself or comprised by a single or independent from each other by several plasmids in the presence of desthiobiotin and in an aqueous nutrient medium, and isolating the resulting biotin from the culture medium, especially such a process wherein the microorganism is selected from the genus Escherichia and specifically a process wherein the cultivation is effected for from about 1 to about 5 days, preferably from about 1 to about 3 days, at a pH of from about 5 to about 9, preferably from about 6 to about 8, and in a temperature range of from about 10 to about 45 C, preferably from about 25 to about 40 C.
SUMMARY OF THE INVENTION
The present invention provides a process for making biotin which comprises contacting desthiobiotin with an enzyme reaction mixture comprising a bioB gene product and an additional gene product selected from nifU gene product, nifS gene product, and a combination thereof to form biotin and then isolating the biotin from the reaction mixture. One preferred reaction mixture contains the bioB gene product and the nifU gene product and another preferred reaction mixture contains the bioB gene product and the nifS gene product. The most preferred reaction mixture contains the bioB gene product, the nifU gene product, and the nifS gene product. The reaction mixture can further contain S-adenosylmethionine, L-cysteine, and an electron supplying system selected from NADPH, ferredoxin-NADP reductase, flavodoxin and deazariboflavin or its functional equivalent component selected from deazaflavin and 8-hydroxy-5-deazaflavin.
It is preferred that the reaction mixture contains the bioB gene product, the nifU gene product, and the nifS gene product. The bioB gene product preferably is from
Escherichia coli
and the nifU and nifS gene products are preferably from
Klebsiella pneumoniae.
Preferably, the process occurs at a temperature of from about 25° C. to about 45° C., more preferably from about 25° C. to about 40° C., and a pH of from about 6.0 to about 8.5, more preferably from about 7.0 to about 8.0.
The present invention also provides a process for making biotin by fermentation comprising the steps of cultivating, in an aqueous nutrient medium, a microorganism transformed with a plasmid containing the DNA encoding bioB gene product and additional DNA selected from DNA encoding nifU gene product, DNA encoding nifS gene product and both the DNA encoding nifu gene product and the DNA encoding nifS gene product with desthiobiotin, producing and accumulating biotin in the aqueous medium, and isolating the biotin from the aqueous medium. The plasmid containing the DNA encoding bioB gene product preferably additionally contains the DNA encoding nifU gene product and the DNA encoding nifS gene product.
Preferably, the cultivation occurs at a time of from about 1 to about 5 days, preferably from about 1 to about 3 days, at a pH of from about 5 to about 9, preferably from about 6 to about 8, and at a temperature of from about 10° C. to about 45° C., preferably from about 25° C. to about 40° C.
Additionally, the present invention provides a process for making biotin by fermentation comprising the steps of cultivating, in an aqueous nutrient medium, a microorganism transformed with a plasmid containing the DNA encoding bioB gene product, the DNA encoding nifU gene product, and the DNA encoding nifS gene product with destiobiotin, producing an
Asakura Akira
Hoshino Tatsuo
Kiyasu Tatsuya
Nagahashi Yoshie
Bryan Cave LLP
Marx Irene
Roche Vitamins Inc.
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