Claims
- 1. A method for the preparation of alkali-tolerant enzymes comprising:
- culturing bacteria in a culture medium;
- separating the bacteria from the culture medium; and
- recovering said alkali-tolerant enzymes from the culture medium, wherein said bacteria are a pure bacterial culture consisting of aerobic, Gram-negative, rod-shaped, obligate alkaliphilic bacteria having the following characteristics:
- a) form cream-colored, circular colonies;
- b) grow optimally between pH 9 and pH 10;
- c) give a positive response to the following tests:
- 1) Leucine arylamidase
- 2) Valine arylamidase
- 3) Phosphohydrolase
- 4) Polymixin;
- d) give a negative response to the following tests:
- 1) N-acetylglucosamine
- 2) Maltose
- 3) Propionate
- 4) Caprate
- 5) Valerate
- 6) Citrate
- 7) Histidine
- 8) Glycogen
- 4) 4-hydroxybenzoate
- 10) .beta.-galactosidase.
- 2. A method for the preparation of alkali-tolerant enzymes comprising:
- culturing bacteria in a culture medium;
- separating the bacteria from the culture medium; and
- recovering said alkali-tolerant enzymes from the culture medium, wherein said bacteria are a pure bacterial culture consisting of aerobic, Gram-negative, rod-shaped, obligate alkaliphilic bacteria having the following characteristics:
- a) form small, cream-colored colonies;
- b) grow optimally between pH 7.8 and pH 11.2;
- c) give a positive response to the following tests:
- 1) Starch
- 2) Acetate
- 3) Propionate
- 4) Valerate
- 5) Proline
- 6) Lipase
- 7) Oxidase (response within 10 seconds);
- d) give a negative response to the following tests:
- 1 ) N-acetylglucosamine
- 2) Saccharose
- 3) Histidine
- 4) 2-ketogluconate
- 5) 4-hydroxybenzoate
- 6) .alpha.-glucosidase
- 7) .beta.-glucosidase
- 8) Fusidic Acid.
- 3. A method for the preparation of alkali-tolerant enzymes comprising:
- culturing bacteria in a culture medium;
- separating the bacteria from the culture medium; and
- recovering said alkali-tolerant enzymes from the culture medium, wherein said bacteria are a pure bacterial culture consisting of aerobic, Gram-negative, rod-shaped, obligate alkaliphilic bacteria having the following characteristics:
- a) form cream-colored, opaque colonies;
- b) grow optimally between pH 8.5 and pH 10.7;
- c) contain ubiquinone 6 as a major respiratory quinone;
- d) give a positive response to the following tests:
- 1) Acetate
- 2) Lactate
- 3) Propionate
- 4) Valerate
- 5) Citrate
- 6) 3 -hydroxybenzoate
- 7) Proline
- 8) Leucine arylamidase;
- e) give a negative response to the following tests:
- 2) Phosphohydrolase
- 2) .alpha.-galactosidase
- 3) Fusidic Acid
- 4) Tetracycline
- 5) Vancomycin
- 6) Bacitracin.
- 4. A method for the preparation of alkali-tolerant enzymes comprising:
- culturing bacteria in a culture medium; separating the bacteria from the culture medium; and
- recovering said alkali-tolerant enzymes from the culture medium, wherein said bacteria are a pure bacterial culture consisting of aerobic, Gram-negative, rod-shaped, obligate alkaliphilic bacteria having the following characteristics:
- a) form beige to brown-colored, opaque colonies;
- b) grow optimally between pH 7.5 and pH 10.9;
- c) contain ubiquinone 9 as a major respiratory quinone;
- d) give a positive response to the following tests:
- 1) Lactate
- 2) Alanine
- 3) 3-hydroxybutyrate
- 4) Valine arylamidase
- 5) Polymixin;
- e) give a negative response to the following tests:
- 1) Histidine
- 2) Ampicillin
- 3) Naladixic acid
- 4) Triimethoprim
- 5) Penicillin G
- 6) Methicillin.
- 5. A method for the preparation of alkali-tolerant enzymes comprising:
- culturing bacteria in a culture medium;
- separating the bacteria from the culture medium; and
- recovering said alkali-tolerant enzymes from the culture medium, wherein said bacteria are a pure bacterial culture consisting of aerobic, Gram-negative, rod-shaped, obligate alkaliphilic bacteria having the following characteristics:
- a) form bright yellow-colored colonies;
- b) grow optimally between pH 8 and pH 10.5;
- c) give a positive response to the following tests:
- 1) Phosphohydrolase
- 2) .alpha.-galactosidase
- 3) .beta.-galactosidase
- 4) Ampicillin
- 5) Fusidic Acid
- 6) Methicillin
- 7) Tetracycline
- 8) Vancomycin
- 9) Bacitracin;
- d) give a negative response to the following tests:
- 1) N-acetylglucosamine
- 2) Lactate
- 3) L-alanine
- 4) Mannitol
- 5) Propionate
- 6) Caprate
- 7) Valerate
- 8) Histidine
- 9) 3 -hydroxybenzoate
- 10) 3-hydroxybutyrate
- 11 ) 4-hydroxybenzoate
- 12) Polymixin.
- 6. A method for the preparation of alkali-tolerant enzymes comprising:
- culturing bacteria in a culture medium;
- separating the bacteria from the culture medium; and
- recovering said alkali-tolerant enzymes from the culture medium, wherein said bacteria are a pure bacterial culture consisting of aerobic, Gram-negative, rod-shaped, obligate alkaliphilic bacteria having the following characteristics:
- a) form cream to beige-colored, irregular, flat colonies;
- b) grow optimally between pH 8.2 and pH 10.9;
- c) give a positive response to the following tests:
- 1) Starch
- 2) N-acetylglucosamine
- 3) Saccharose
- 4) Maltose
- 5) Acetate
- 6) Alanine
- 7) Citrate
- 8) Glycogen
- 9) 3 -hydroxybutyrate
- 10) Penicillin G
- 11) Fusidic Acid
- 12) Methicillin
- 13 ) Tetracycline
- 14) Bacitracin;
- d) give a negative response to the following tests:
- 1) Pyruvate
- 2) 4-hydroxybenzoate
- 3) Leucine arylamidase
- 4) Valine arylamidase
- 5) .alpha.-galactosidase
- 6) Polymixin.
- 7. A method for the preparation of alkali-tolerant enzymes comprising:
- culturing bacteria in a culture medium;
- separating the bacteria from the culture medium; and
- recovering said alkali-tolerant enzymes from the culture medium, wherein said bacteria are a pure bacterial culture consisting of aerobic, Gram-negative, motile, rod-shaped, obligate alkaliphilic bacteria having the following characteristics:
- a) cells frequently in pairs;
- b) grow optimally between pH 9 and pH 10;
- c) on alkaline agar, form smooth, translucent, beige colored colonies, 1-2 mm in diameter which are circular, convex with an entire margin;
- d) in alkaline broth growth at 37.degree. C. is flocculent with a ring or surface pellicle and formation of a sediment;
- e) grow optimally at 20.degree. C. to 30.degree. C;
- f) do not grow at 15.degree. C. or 40.degree. C.;
- g) KOH test is positive;
- h) aminopeptidase test is weak positive;
- i) oxidase test is weak positive;
- j) catalase test is positive;
- k) obligate halophile;
- l) grow optimally at 4% NaCl;
- m) do not grow at 0% or 8% NaCl;
- n) hydrolysis of gelatin test is slow positive;
- o) hydrolysis of starch is positive;
- p) does not grow on simple sugars;
- q) does not grow on organic acids;
- r) grow on yeast extract and peptones.
- 8. A method for the preparation of alkali-tolerant enzymes comprising:
- culturing bacteria in a culture medium;
- separating the bacteria from the culture medium; and
- recovering said alkali-tolerant enzymes from the culture medium, wherein said bacteria are a pure bacterial culture consisting of aerobic, Gram-negative, motile, rod-shaped, obligate alkaliphilic bacteria having the following characteristics:
- a) grow optimally between pH 8.2 and pH 10.9;
- b) on alkaline agar, form smooth, opaque, beige or brown colored colonies, 2-4 mm in diameter which are circular in form, convex in elevation, with an entire margin;
- c) in alkaline broth, growth at 37.degree. C. is heavy and flocculent with a sediment and surface pellicle;
- d) grow optimally between 20.degree. C. and 37.degree. C.;
- e) do not grow at 80.degree. C. or at 40.degree. C. or above;
- f) KOH test is positive;
- g) aminopeptidase test is positive;
- h) oxidase test is very weakly positive;
- i) catalase test is positive;
- j) grow at a NaCl concentration of between 0% and 15%;
- k) do not grow at 20% NaCl;
- l) hydrolysis of gelatin test is negative;
- m) hydrolysis of starch is negative;
- n) grow on yeast extract;
- o) grow on organic acids selected from the group consisting of succinate, pyruvate, citrate, malonate, acetate and lactate;
- p) grow on fatty acids selected from the group consisting of propionate, valerate and suberate;
- q) grow on amino acids selected from the group consisting of proline, serine, histidine and lysine.
- 9. A method for the preparation of alkali-tolerant enzymes comprising:
- culturing bacteria in a culture medium;
- separating the bacteria from the culture medium; and
- recovering said alkali-tolerant enzymes from the culture medium, wherein said bacteria are a pure bacterial culture consisting of aerobic, Gram-negative, rod-shaped, obligate alkaliphilic bacteria having the following characteristics:
- a) grow optimally between pH 9 and pH 10.5;
- b) on alkaline agar, form smooth, opaque, brown colored colonies, 3-4 mm in diameter which are fairly irregular in form, generally flat to slightly umbonate in elevation with a lobate margin;
- c) in alkaline broth, growth at 37.degree. C. is moderate to heavy, becoming flocculent with a sediment and surface pellicle;
- d) grow optimally between 20.degree. C. and 40.degree. C.;
- e) do not grow at 45.degree. C.;
- f) KOH test is positive;
- g) aminopeptidase test is positive;
- h) oxidase test is negative;
- i) catalase test is positive;
- j) grow at a NaCl concentration 0% to 12%;
- k) do not grow at 20% NaCl;
- l) hydrolysis of gelatin test is positive;
- m) hydrolysis of starch is weakly positive;
- n) does not grow on simple sugars;
- o) grow on yeast extract;
- p) grow on organic acids selected from the group consisting of pyruvate, citrate, acetate and lactate;
- q) grow on fatty acids selected from the group consisting of propionate, caprate and valerate;
- r) grow on amino acids selected from the group consisting of proline, alanine and lysine.
- 10. A method for the preparation of alkali-tolerant enzymes comprising:
- culturing bacteria in a culture medium;
- separating the bacteria from the culture medium; and
- recovering said alkali-tolerant enzymes from the culture medium, wherein said bacteria are a pure bacterial culture consisting of aerobic, Gram-negative, rod-shaped, obligate alkaliphilic bacteria having the following characteristics:
- a) does not grow below pH 7.5;
- b) on alkaline agar, form smooth, cream colored colonies, initially translucent but becoming opaque;
- c) on alkaline agar, the colonies develop from circular, entire and become irregular, lobate in form, with a convex elevation;
- d) in alkaline broth, growth at 37.degree. C. is slow, slight, flocculent with a sediment but no surface pellicle;
- e) grow optimally between 10.degree. C. and 40.degree. C.;
- f) do not grow at 8.degree. C. or 45.degree. C.;
- g) KOH test is positive;
- h) aminopeptidase test is negative;
- i) oxidase test is positive;
- j) catalase test is positive;
- k) grow at an NaCl concentration of between 0% to 15%;
- l) do not grow at 20% NaCl;
- m) hydrolysis of gelatin test is positive;
- n) hydrolysis of starch is weakly positive;
- o) grow on yeast extract and peptones;
- p) grow on sugars;
- q) grow on organic acids;
- r) grow on amino acids.
- 11. A method for the preparation of alkali-tolerant enzymes comprising:
- culturing bacteria in a culture medium;
- separating the bacteria from the culture medium; and
- recovering said alkali-tolerant enzymes from the culture medium, wherein said bacteria are a pure bacterial culture consisting of aerobic, Gram-negative, small, rod-shaped, obligate alkaliphilic bacteria having the following characteristics:
- a) cells frequently form short chains;
- b) does not grow below pH 8;
- c) on alkaline agar, form smooth, circular, convex colonies with an entire margin, about 1 mm in diameter which are initially transparent, cream beige in color, the colonies become opaque and brown in color with age;
- d) in alkaline broth, growth 37.degree. C. is initially evenly turbid with a sediment but no surface pellicle becoming flocculent with formation of a pellicle;
- e) grow optimally between 30.degree. C. and 37.degree. C.;
- f) do not grow at 40.degree. C.;
- g) KOH test is positive;
- h) aminopeptidase test is positive;
- i) oxidase test is positive;
- j) catalase test is positive;
- k) obligate halophile;
- l) grow at 4% NaCl;
- m) do not grow at 0% or 8% NaCl;
- n) hydrolysis of gelatin test is slow positive;
- o) hydrolysis of starch is negative;
- p) grow on yeast extract and peptones;
- q) grow on sugars;
- r) grow on organic acids;
- s) grow on fatty acids;
- t) grow on amino acids.
- 12. The method of claim 1, wherein said culture medium is alkaline.
- 13. The method of claim 2, wherein said culture medium is alkaline.
- 14. The method of claim 3, wherein said culture medium is alkaline.
- 15. The method of claim 4, wherein said culture medium is alkaline.
- 16. The method of claim 5, wherein said culture medium is alkaline.
- 17. The method of claim 6, wherein said culture medium is alkaline.
- 18. The method of claim 7, wherein said culture medium is alkaline.
- 19. The method of claim 8, wherein said culture medium is alkaline.
- 20. The method of claim 9, wherein said culture medium is alkaline.
- 21. The method of claim 10, wherein said culture medium is alkaline.
- 22. The method of claim 11, wherein said culture medium is alkaline.
Parent Case Info
This application is a divisional of U.S. Ser. No. 08/122,745, filed 16 Sep. 1993 now U.S. Pat. No. 5,545,062, which is a continuation-in-part of U.S. Ser. No. 08/046,878, filed Apr. 8, 1993, now U.S. Pat. No. 5,571,716, which is a continuation-in-part of U.S. Ser. No. 07/903,786, filed Jun. 24, 1992 now U.S. Pat. No. 5,540,165, which is a continuation-in-part of U.S. Ser. No. 07/719,307, filed Jun. 24, 1991, now abandoned, which is a continuation-in-part of U.S. Ser. No. 07/562,863, filed Aug. 6, 1990 now abandoned all of which are incorporated herein reference.
Non-Patent Literature Citations (9)
| Entry |
| Grant, W.D. et al., FEMS Microbiology Reviews (1990) 75:255-270. |
| Grant, W.D. and Tindall, B.J., Microbes in Extreme Environments, eds., R.A. Herbert and G.A. Codd, London: Academic Press (1986) 22-54. |
| Tindall, B.J., Halophilic Bacteria, eds. Rodriguez-Valera, Boca Raton, FL: CRC Press, Inc. (1988) 31-70. |
| Horikoshi, K. et al., Alkalophilic Microorganisms, Berlin, Heidelberg, NY: Springer-Verlag (1982). |
| Shiba, H., Superbugs (Horikoshi and Grant (eds.)), Tokyo: Japan Scientific Societies Press and Berline, Heidelberg, NY: Springer-Verlag (1991) 191-211. |
| Nakatsugawa, N., Superbugs (Horikoshi and Grant (eds.)), Tokyo: Japan Scientific Societies Press and Berline, Heidelberg, NY: Springer-Verlag (1991) 212-220. |
| Wang, D. et al., "Natronobacterium from Soda Lakes of China," Recent Advances in Microbial Ecology, Proceedings of the 5th International Symposium on Microbial Ecology, T. Hattori et al. (eds.), Tokyo: Japan Scientific Societies Press, (1989) 68-72. |
| Morth, S. and Tindall, B.J., Sys. Appl. Microbiol. (1985) 6:247-250. |
| Upasani, V. and Desai, S., Arch. Microbiol (1990) 154:589-593. |
Divisions (1)
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Number |
Date |
Country |
| Parent |
122745 |
Sep 1993 |
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Continuation in Parts (4)
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Number |
Date |
Country |
| Parent |
46878 |
Apr 1993 |
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| Parent |
903786 |
Jun 1992 |
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| Parent |
719307 |
Jun 1991 |
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| Parent |
562863 |
Aug 1990 |
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Patent Grant
5719047
