Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Reexamination Certificate
1998-07-07
2001-02-06
Witz, Jean C. (Department: 1651)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
C424S094400, C435S252500, C435S264000
Reexamination Certificate
active
06184014
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a bacterial polyphenol oxidase, to a process of making the enzyme, to a bacterial strain that produces the enzyme, and to processes using the enzyme. More specifically, the present invention provides a novel enzyme source of polyphenol oxidase for use in the oxidation of colored substances and oxidation of polyphenol-containing substances and also for use in cleaning.
BACKGROUND ART
Polyphenol oxidase and laccase are known as enzymes that oxidize polyphenols. Various studies have been made on the use of polyphenol-oxidizing enzymes. For example, WO 94-29510 reports the delignination in the field of paper and pulp; and WO 91-05839, EP 91619032, DE 4008894 and JP-A 64-60693 report the use of the enzymes for bleaching in laundry washing.
However, the only known microbial sources for polyphenol oxidase and laccabe are mold fungi such as Basidiomycetes and Deuteromycetes., and it is generally difficult to cultivate large amounts of such fungi and, in addition, the rate of growth of the cells during cultivation is not high. Improving the enzyme yield by mutation or by use of genetic engineering technology is more difficult with these fungi than with bacteria, since the fungi have complicated life cycles and have complicated gene structures comprising introns, etc. For these reasons, it is difficult to stably and inexpensively obtain large amounts of polyphenol oxidases from such fungi, and bacteria-derived polyphenol oxidases are desired in order to apply them to practical use.
It is the object of the present invention to provide a polyphenol oxidase to be produced by bacteria, the bacteria that produce the enzyme and the use of the enzyme, thus providing a novel enzyme source of polyphenol oxidase for use in the oxidation of colored substances and oxidation of polyphenol-containing substances and also for use in cleaning.
STATEMENT OF THE INVENTION
We, the present inventors have assiduously searched various bacteria for extracellular products that catalyze the oxidation of polyphenol substances. Though being extremely difficult, our search for such products has at last resulted in the finding of the fact that bacteria belonging to the genus Bacillus can extracellularly produce the intended enzyme. On the basis of this finding, we have completed the present invention.
Accordingly, the present invention provides the following:
A polyphenol oxidase which is derived from a bacterium.
A method for oxidizing a phenolic compound, an alkoxy group-containing aromatic compound, a halogenated phenolic compound or an aromatic amine compound, which comprises treating said compound with said polyphenol oxidase in the presence of oxygen.
A method for bleaching a colored substance, which comprises treating the colored substance with said polyphenol oxidase in the presence of oxygen.
A method for inhibiting the transfer of a textile dye from a dyed fabric to another fabric when said fabrics are washed together in a wash liquor, which method comprises treating the wash liquor with said polyphenol oxidase in the presence of oxygen.
A method for bleaching colored waste water, which comprises treating the colored waste water with said polyphenol oxidase in the presence of oxygen.
A method for inactivating a microorganism or virus, which comprises treating the microorganism or virus with said polyphenol oxidase in the presence of oxygen.
A method for bleaching of lignin-containing material, which comprises treating the lignin-containing material with said polyphenol oxidase in the presence of oxygen.
A detergent composition comprising said polyphenol oxidase.
A method for producing a polyphenol oxidase, which comprises cultivation of a polyphenol oxidase-producing bacterium of the genus Bacillus in a suitable nutrient medium, followed by recovery of the polyphenol oxidase.
Bacillus licheniformis
strain SD3003 (FERM P-15383) which is productive of polyphenol oxidase.
Now, the present invention is described in detail hereinunder.
DETAILED DESCRIPTION OF THE INVENTION
Enzyme-producing Bacteria
According to the invention, polyphenol oxidase is derived from a bacterial strain, preferably a strain of the genus Bacillus. Any and every strain of the genus Bacillus having the ability to produce polyphenol oxidase can be used herein to obtain the polyphenol oxidase of the invention, and there is not any other specific; limitation on the bacteria to be used herein. The enzyme-producing bacteria employable herein include, for example, those of
Bacillus alcalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus firmus, Bacillus licheniformis, Bacillus natto, Bacillus pumilus, Bacillus sphaericus, Bacillus subtilis,
etc. Some preferred species are
B. licheniformis, B. natto
and
B. sphaericus,
particularly
B. licheniformis.
Some preferred strains are Bacillus sp. NCIB 10314,
B. licheniformis
NCIB 8059, NCIB 8061, ATCC 6634, ATCC 9945a, ATCC 11945 and SD3003,
B. natto
SN AKU 0205 and
B. sphaericus
IFO 3341.
The strains NCIB 10314, NCIB 8059 and NCIB 8061 are freely available from National Collections of Industrial and Marine Bacteria Limited (NCIMB, previously NCIB), 23 St. Machar Drive, Aberdeen AB2 1RY, Scotland, United Kingdom.
The strains
B. licheniformis
ATCC 6634, ATCC 9945a and ATCC 11945 are freely available from American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852, United States of America.
The strain
B. natto
SN AKU 0205 is freely available from the culture collection of the Agricultural Faculty of Kyoto University, Kyoto, Japan.
The strain
B. sphaericus
IFO 3341 is freely available from the Institute for Fermentation (IFO), 17-85 Juso-honmachi 2-chome, Yodogawa-ku, Osaka 532, Japan.
The strain
B. licheniformis
SD3003 was deposited as FERM P-15383 on Dec. 28, 1995 at the National Institute of Bioscience and Human-Technology, Ministry of International Trade and Industry, 1-3 Higashi 1-chome, Tsukuba-shi, Ibaraki-ken 305, Japan. The deposit was subsequently transferred to an international deposit under the Budapest Treaty on Jan. 28, 1997 under the deposit number FERM BP-5801. The deposit was made by Showa Denko K. K., Japan, and is being assigned to Novo Nordisk A/S. The following results were found from morphological observations and physiological tests with this representative strain according to the invention:
Property
Results
Morphology
rod shaped
Gram-staining Ability
+
Spores
+
Shape
Oval
Position
Central to Semi-peripheral
Sporangia
No Evagination
Motility
+
Behavior Toward Oxygen
Anaerobic to Aeration
Catalase
+
Growth in Anaerobic
+
Condition
V-P Reaction
+
pH in V-P Broth
5.2
Production of Acid
+
from Glucose
Production of Gas
−
from Glucose
Liquefaction of Gelatin
+
Decomposition of Starch
+
Utilization of Citrate
+
Utilization of Propionate
+
Egg Yolk Reaction
−
Reduction of Nitrate
+
Growth at pH 6.8 (in
+
nutrient broth)
Growth at pH 5.7
+
Growth in the Presence
+
of 5 NaCl
Growth in the Presence
+
of 7 NaCl
Growth at 10° C.
−
Growth at 30° C.
+
Growth at 55° C.
+
Growth at 65° C.
−
GC content (mol %) of
46
Intracellular DNA(*)
(*)Measured through HPLC.
From the above-mentioned results and with reference to “Bergey's Manual of Systematic Bacteriology”, Vol. 2, 1986, Williams & Wilkins, and “The Genus Bacillus”, 1973, U.S. Department of Agriculture, this strain is classified as Bacillus licheniformis SD3003.
Preparation of Enzyme
The polyphenol oxidase of the present invention can be obtained by cultivating the cells of strains belonging to the genus Bacillus such as those mentioned hereinabove, or those of mutants thereof. In addition, it can also be obtained by cultivating transformants from such strains to be prepared through genetic engineering. For example, host cells as transformed with an expression vector to be prepared by inserting a DNA that codes for the polyphenol oxidase of the invention, along with suitable promoter, operator and terminator DNAs that func
Echigo Takashi
Ohno Ritsuko
Green, Esq. Reza
Novo Nordisk A S
Witz Jean C.
Zelson, Esq. Steve T.
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