Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing a carotene nucleus
Reexamination Certificate
1998-11-23
2001-04-10
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing a carotene nucleus
C435S069100, C435S189000, C435S252300, C435S252330, C435S320100, C536S023200
Reexamination Certificate
active
06214575
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a &bgr;-catotene hydroxylase, a DNA coding for the &bgr;-catotene hydroxylase, a recombinant vector comprising the DNA, a transformant transformed with the vector, a method for preparing the &bgr;-catotene hydroxylase and a method for preparing &bgr;-cryptoxanthin.
2. Description of the Prior Art
In carotenoids synthesized by animals, plants and microorganisms, there are a group of compounds with a hydroxyl group(s) generically called xanthophyll. These compounds are generated from carotenoids (starting substances) by the catalytic action of hydroxylase. For example, one hydroxyl group is introduced into &bgr;-carotene to yield &bgr;-cryptoxanthin, into which another hydroxyl group is introduced to yield zeaxanthin via the biosynthetic pathway shown below (see arrow (1) in FIG.
1
):
&bgr;-carotene →&bgr;-cryptoxanthin →zeaxanthin
This &bgr;-cryptoxanthin is obtained by introducing a hydroxyl group into one of the two ionone rings present in &bgr;-carotene. When another hydroxyl group is introduced into a position symmetric to the former position, zeaxanthin is produced (FIG.
1
).
In a large number of plants and microorganisms, metabolism proceeds from &bgr;-carotene to zeaxanthin, producing little &bgr;-cryptoxanthin, the intermediate into which only one hydroxyl group is introduced.
This reaction is controlled by a hydroxylase gene called Crt Z. In this enzyme reaction, it is considered that two hydroxyl groups are introduced almost simultaneously. For example, under the control of a hydroxylase gene cloned from a bacterium belonging to the genus Erwinia, zeaxanthin is produced which is obtainable by introducing two hydroxyl groups into &bgr;-carotene.
In
Citrus unshiu
(
Satsuma mandarine
) which is a major citrus fruit in Japan, &bgr;-cryptoxanthin obtainable by introducing one hydroxyl group into &bgr;-carotene is considered to be one of the most important carotenoids. In particular, &bgr;-cryptoxanthin occupies 60-70% of the total carotenoid content in the edible part of this fruit.
Considering this high &bgr;-cryptoxanthin content of
Citrus unshiu
, it is hard to think that the &bgr;-cryptoxanthin in
Citrus unshiu
is produced by a gene involved in the above-mentioned metabolic pathway. Also, it is still unknown whether &bgr;-cryptoxanthin is produced by those genes which have been already cloned.
OBJECTS AND SUMMARY OF THE INVENTION
It is an object of the present invention to provide a &bgr;-carotene hydroxylase and a gene coding for the enzyme.
As a result of intensive and extensive researches toward the solution of the above problem, the present inventors have succeeded in isolating from a citrus-derived cDNA library a DNA coding for a &bgr;-carotene hydroxylase. Thus, the present invention has been achieved.
The present invention relates to the following recombinant protein (a) or (b):
(a) a protein consisting of the amino acid sequence as shown in SEQ ID NO: 2;
(b) a protein which consists of the amino acid sequence as shown in SEQ ID NO: 2 having deletion, substitution or addition of one or several amino acids and which has &bgr;-carotene hydroxylase activity.
The present invention further relates to a DNA coding for the following protein (a) or (b):
(a) a protein consisting of the amino acid sequence as shown in SEQ ID NO: 2;
(b) a protein which consists of the amino acid sequence as shown in SEQ ID NO: 2 having deletion, substitution or addition of one or several amino acids and which has &bgr;-carotene hydroxylase activity.
Further, the present invention relates to a DNA coding for a &bgr;-carotene hydroxylase, comprising the nucleotide sequence as shown in SEQ ID NO: 1.
Further, the present invention relates to a recombinant vector comprising the above DNA.
Further, the present invention relates to a transformant which is transformed with the above vector.
Further, the present invention relates to a method for preparing a &bgr;-carotene hydroxylase comprising culturing the above transformant in a medium and recovering the &bgr;-carotene hydroxylase from the resultant culture.
Further, the present invention relates to a method for preparing &bgr;-cryptoxanthin comprising culturing the above transformant in a medium and recovering &bgr;-cryptoxanthine from the resultant culture.
REFERENCES:
patent: 0 747 483 A2 (1996-11-01), None
patent: WO 91/13078 (1991-09-01), None
patent: WO 97/36998 (1997-10-01), None
A. Ruther et al., “Production of zeaxanthin inEscherichia colitransformed with different carotenogenic plasmids” Appl. Microbiol. Biotechnol. (1997) 48:162-167.
Sun et al., “Cloning and Functional Analysis of the -Carotene Hydroxylase ofArabidopsis thaliana” The Journal of Biological Chemistry, vol. 271, No. 40, Issue of Oct. 4, pp. 24349-24352 (1996).
Hundle et al., “In vitro expression and activity of lycopene cyclase and -carotene hydroxylase fromErwina herbicola” FEBS 11977, vol. 315, No. 3, 329-334.
Ikoma Yoshinori
Komatsu Akira
Omura Mitsuo
Yano Masamitsu
Achutamurthy Ponnathapu
Director General of National Institute of Fruit Tree Science, Mi
Fish & Richardson P.C.
Moore William W.
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