Mutant strain of penicillium citrinum and use thereof for...

Details Mutant strain of penicillium citrinum and use thereof for... Mutant strain of penicillium citrinum and use thereof for...

1999-06-17

2001-11-27

Marx, Irene (Department: 1651)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Fungi

C435S125000, C435S441000

Reexamination Certificate

active

06323021

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a novel microorganism and uses thereof in preparation of antihypercholesterolemic agents. More specifically, it relates to a mutant strain of
Penicillium citrinum
ATCC 38065, and its use in preparation of compactin.
2. Description of the Related Arts
High blood cholesterol levels are recognized as being one of the main causes of cardiopathy, e.g. cardiac infarction, arteriosclerosis or hyperlipaemia. Cholesterol biosynthesis is greatly reduced by inhibiting the activity of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, which is a rate controlling enzyme. As a result, considerable research has been undertaken with a view to discover physiologically acceptable substances which are capable of inhibiting cholesterol biosynthesis and thus are capable of reducing blood cholesterol levels.
Many antihypercholesterolemic agents are presently used to cure cardiopathy disease and one of the best-known antihypercholesterolemic agents is pravastatin, which is prepared from modifying compactin, the only precursor of pravastatin. Thus, a process that can prepare compactin at a high concentration level is urgently required.
Compactin was first purified from the nutrient medium of
Penicillium brevicocompactum
and found to be an antifugal metabolite, as disclosed by Brown et al in 1975. Later, Endo et al in Japan Sankyo Company found that compactin exists in the fermentation broth of
Penicillium citrinum.
They also discovered compactin has blood cholesterol reducing ability.
U.S. Pat. No. 3,983,140 (Sep. 28, 1976) and U.S. Pat. No. 4,049,495 (Sep. 20, 1977) patented by A. Endo et al disclosed fermentative preparation of compactin by cultivation of a microorganism belonging to
Penicillium citrinum.
U.S. Pat. No. 5,691,173 (Nov. 25, 1997) patented by Scott Primrose et al disclosed the preparation of compactin by fermentation using a microorganism belonging to
Penicillium adametzoides.
In prior patents, compactin is prepared at an extremely low concentration level. Therefore, a novel microorganism or a new preparation is required to produce compactin at a high concentration level.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a novel microorganism capable of producing compactin using fermentation.
An another object of the present invention is to provide a process of producing compactin using the novel microorganism.
According to one aspect of the present invention, a mutant strain
Penicillium citrinum
ATCC 38065, which is capable of secreting compactin, is deposited at the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, Va. 20110-2209, and is given an accession number of PTA-3429, before the issuance of the patent, under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the purpose of Patent Procedure. A viable sample of this mutant strain is currently deposited at the Food Industry Research Development Institute (FIRDI), R.O.C., under accession No. CCRC 930024.
According to another aspect of the invention, a process of preparing compactin using
Penicillium citrinum
CCRC 930024 which comprises cultivating
Penicillium citrinum
CCRC 930024 in a fermentation medium and recovering compactin from the fermentation broth is provided.
Preferably, before cultivation in the fermentation medium,
Penicillium citrinum
CCRC 930024 is cultivated in a spore medium to germinate spores and the spores are cultivated in a seed medium.
Other objects, features, and advantages of the present invention will become apparent from the following detailed description.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is based on the observation that compactin is produced by a novel microorganism very different from that employed by prior patents. The novel microorganism is a mutant strain of
Penicillium citrinum
ATCC 38065. This mutant strain is capable of secreting compactin. A viable sample of this mutant strain is currently deposited at the Food Industry Research Development Institute (FIRDI), R.O.C., under accession No. CCRC 930024. A viable sample of this mutant strain (CCRC 930024) is deposited at the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, Va. 20110-2209, and is given an accession number of PTA-3429, before the issuance of the patent, under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the purpose of Patent Procedure.
Penicillium citrinum
CCRC 930024 can be obtained by applying a conventional mutation-inducing technique to the parent strain,
Penicillium citrinum
ATCC 38065. For example, irradiation of the microorganisms with gamma rays or ultraviolet light, or treatment with a mutagen, such as N-methyl-N′-nitro-N-nitrosoguanidine, ethylmethane sulfonate and hydroxylamine are suitable methods of inducing mutation. Then mutant strains which are filipine-resistant can be selected and the desired mutant,
Penicillium citrinum
CCRC 930024, is obtained as a strain capable of producing compactin at the highest concentration level among all mutant strains.
In this embodiment, all microorganisms are cultivated by the same process for producing compactin at the highest concentration level, the process comprising the steps of: (a) germinating spores in a spore medium; (b) cultivating the spores in a seed medium; and (c) cultivating the seed culture in the fermentation medium. But for simplifying the production process, microorganisms can be directly cultivated in the fermentation medium to produce compactin.
The spore medium can be malt-extract-agar (MEA) medium or potato-dextrose-agar (PDA) medium. The MEA can be composed of malt extract, peptone, and agar. Preferably, MEA contains 2% of malt extract, 0.1% peptone, and 2% of agar. The PDA medium can contain potato extract, D(+)glucose, agar and glycerol. Preferably, the PDA medium contains 20% of potato extract, 2% of D(+)glucose, 2% of agar and 5-10% of glycerol.
Researchers found that the spore-producing ability was increased in proportion to the content of glycerol in the spore medium. Therefore, the spore-producing activity of the
Penicillium citrinum
CCRC 930024 can be increased, depending on the amount of glycerol in the spore medium.
The seed medium can contain glucose, glycerol, corn steep liquor and yeast power. Preferably, the seed medium contains 2% of glucose, 1% of glycerol, 2% of corn steep liquor, and 0.5% of yeast powder and has a pH about 4-9.
The fermentation medium can contain glucose, glycerol, pharmamedia®, soybean meal, corn steep liquor, KH
2
PO
4
, K
2
HPO
4
, FeSO
4
and CaCO
3
. Preferably, the fermentation medium contains 7% of glucose, 3% of glycerol, 2% of pharmamedia®, 1% of soybean meal, 1% of corn steep liquor, 0.15% of KH
2
PO
4
, 0.05% of K
2
HPO
4
, 0.003% of FeSO
4
and 0.2% of CaCO
3
and has a pH about 4-7.
MEA medium
PDA medium
Malt extract
2%
Potato extract
20%
Peptone
0.1%
D(+) glucose
20%
Agar
2%
Agar
2%
Glycerol
1%
Seed medium
Fermentation medium
Glucose
2%
Glucose
7%
Glycerol
1%
Glycerol
3%
Corn steep liquor
2%
Pharmamedia ®
Yeast powder
0.5%
2%
Soybean meal
Corn steep liquor
1%
KH
2
PO
4
0.15%
K
2
HPO
4
0.05%
FeSO
4
0.003%
CaCO
3
0.2%
Compactin titer in the fermentation broth is determined by HPLC under mobile conditions of 20% of methanol and 80% of water throughout a Merck RP-18 (254×4 mm) column. The Detection wavelength was 237nm. Compactin is identified by the retention time in comparison with a standard obtained from Sigma Company.
Without intending to limit it in any manner, the present invention will be further illustrated by the following examples.


REFERENCES:
patent: 3983140 (1976-09-01), Endo
patent: 4231938 (1980-11-01), Monaghan et al.
patent: 5362638 (1994-11-01), Dahiya
patent: 5409820 (1995-04-01), Gerson et al.

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