Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2000-10-30
2002-08-13
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S209000, C435S210000, C435S200000, C510S392000
Reexamination Certificate
active
06432689
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The present invention relates to novel alkaliphilic and thermophilic microorganisms and to novel enzymes obtained therefrom.
BACKGROUND OF THE INVENTION
Alkaliphiles are a heterogeneous group of microorganisms spread over many taxonomic groups which exhibit optimum growth in an alkaline pH environment (Jones, B. E. et al, (1994) Alkaliphiles: diversity and identification, in “
Microbial Diversity and Identification
” (F. G. Priest et al, Eds.) Plenum Press, New York and London, pages 195-230), generally in excess of pH 8. Obligate alkaliphiles generally have a pH optimum for growth between pH 9 and pH 10, and are incapable of growth at neutral pH. Alkalitolerant (or facultatively alkaliphilic) microorganisms are less exacting and although they are capable of growth at alkaline pH values, their optima lie in the neutral to acid pH range.
Thermophiles are also a very heterogeneous collection of microorganisms defined as having an optimum growth temperature in excess of 50° C. For moderate thermophiles the maximum growth temperature usually lies below 70° C. An organism with a growth minimum above 40° C., an optimum above 65° C., and a growth maximum above 70° C. is defined as an extreme thermophile (Cowan, D. A. (1992) Biochemistry and molecular biology of extremely thermophilic archeaobacteria, in “
Molecular Biology and Biotechnology of Extremophiles
” (R. A. Herbert and R. J. Sharp, Eds.), Blackie & sons Ltd., Glasgow and London, pages 1-43).
The combined phenotype, alkaliphily and thermophily appears to have only rare occurrence. Only two such microorganisms, both isolated from sewage digestion plants, have been well described and both were assigned to the genus Clostridium of the Gram-positive bacteria. One of the organisms,
Clostridium paradoxum
, is obligately alkaliphilic growing between pH 7.3 and pH 11.0, with an optimum around pH 10. It can however, only be classified as a moderate thermophile since it has an optimum growth temperature of 55° C. and a maximum at 63° C. (Youhong Li et al (1992) Int. J. Syst. Bacteriol. 43, 450-460). A second organism,
Clostridium thermoalcaliphilum
is a facultative alkaliphile or alkalitolerant organism growing between pH 7 and pH 11, with an optimum between pH 9.5 and pH 10. With an optimum growth temperature of 50° C. and maximum at 57° C. this bacterium can only be classified as a very moderate thermophile or as thermotolerant (Youhong Li et al (1994) Int. J. Syst. Bacteriol. 44, 111-118).
Among the known types of thermophilic bacteria several species belong to the order Thermotogales. This distinct group of mainly extreme thermophilic bacteria has been shown by sequencing of the ribosomal RNA genes to be phylogenetically distant from all other bacteria, and to represent one of the deepest branches and most slowly evolving lineages within the Domain Bacteria. Bacteria of the Thermotogales are characteristically, Gram-negative, rod-shaped, anaerobic, fermentative bacteria with an outer sheath-like envelope (“toga”); their growth is inhibited by molecular hydrogen (Huber, R. and Stetter, K. O. (1992) The order Thermotogales, in “
The Prokaryotes
” (A. Balows et al, Eds.), Springer-Verlag, New York, pages 3809-3815).
At present, the Thermotogales are represented by five genera. The genera Thermotoga, Thermosipho and Fervidobacterium comprise the known extreme thermophilic species, while the more distantly related (on the basis of 16S rRNA analysis) genera Geotoga and Petrotoga represent the more mesophilic species. None of the known species is noticeably alkaliphilic in nature. Most of the extant species of extreme thermophilic Thermotogales have been isolated from active geothermal aquatic environments such as shallow and deep-sea marine hydrothermal systems or from low-salinity continental solfatara springs. More recently less thermophilic strains, particularly those of the genera Geotoga and Petrotoga have been isolated from deep sub-surface oil fields (Huber, R. and Stetter, K. O. (1992) ibid; Davey, M. E. et al, (1993) Syst. Appl. Microbiol. 16, 191-200; Ravot, G. et al, (1995) Int. J. Syst. Bacteriol. 45, 308-314).
Although the different members of the Thermotogales may be partially differentiated on the basis of phenotypic characteristics such as temperature, pH and NaCl ranges permitting growth (Table 1, Ravot, G. et al (1995) Int. J. Syst. Bacteriol. 45, 308-314), their classification is largely based on a comparison of similarity between nucleotide sequences on the 16S rRNA genes and DNA-DNA hybridisation studies. Stackebrandt and Goebel (Int. J. Syst. Bacteriol. 44, 846-849, 1994) suggest that strains of microorganisms having more than 97% 16S rRNA sequence identity may be considered members of the same species, provided that other criteria are also met. It has been shown that the 16S rRNA sequences of
Fervidobacterium islandicum
and
Fervidobacterium nodosum
are 95.3% similar which is typical of different species within the same genus (Huber, R. et al, (1990) Arch. Microbiol. 154, 105-111), but that these differ by 10-15% with strains of Thermotoga and Thermosipho. Within the Thermotogales sequence differences of up to about 8% have generally qualified for placing the strains in the same genus. 16S rRNA sequence differences of greater than about 10%, together with differences in phenotype have frequently been used as compelling arguments for placing different isolates of Thermotogales in separate genera (Huber, R. et al, (1989) Syst. Appl. Microbiol. 12, 32-37; Davey, M. E. et al, (1993) Syst. Appl. Microbiol. 16, 191-200; Ravot, G. et al, (1995) Int. J. Syst. Bacteriol. 45, 308-314).
TABLE 1
Some characteristics that differentiate members of the Thermotogales
NaCl
TEMPERATURE
CONCENTRATION
G + C
° C.
pH
(%)
CONTENT
GENUS
SPECIES
RANGE
OPTIMUM
RANGE
OPTIMUM
RANGE
OPTIMUM
(mol %)
REFERENCE
Thermotoga
maritima
55-90
80
5.5-9
6.5
0.25-3.75
2.7
46
1
neapolitana
55-90
80
5.5-9
7
41
2
thermarum
55-84
70
5.5-9
7
0.2-0.55
0.35
40
3
elfii
50-72
66
5.5-8.7
7.5
0-2.8
1.2
39.6
4
sp. FjSS3
55-90
80
4.8-8.2
7
45.8
5
Thermosipho
africanus
35-77
75
6-8
7.2
0.11-3.6
29
6
Fervidobacterium
nodosum
41-79
70
6-8
7
0.1
33.7
7
islandicum
50-80
65
6-8
7.2
0.2
41
8
pennavorens
70
6.5
40
9
Petrotoga
miotherma
35-65
55
5.5-9
6.5
0.5-10
2
39.8
10
Geotoga
petraea
30-55
50
5.5-9
6.5
0.5-10
3
29.5
10
subterranea
30-60
45
5.5-9
6.5
0.5-10
4
29.9
10
Thermopallium
natronophilum
52-78
70
7.2>10.5
9.2
0-5
1
36.3
11
1 Huber, R. et al (1986) Arch. Microbiol. 144, 324-333.
2 Jannasch, H. et al (1988) Arch. Microbiol. 150, 103-104.
3 Windburger, E. et al (1989) Arch. Microbiol. 151, 506-512.
4 Ravot, G. et al (1995) Int. J. Syst. Bacteriol. 45, 308-314.
5 Huser, B. A. et al (1986) FEMS Microbiol. Letts. 37, 121-127; Janssen, P. H. and Morgan, H. W. (1992) FEMS Microbiol. Letts. 96, 213-218.
6 Huber, R. et al (1989) Syst. Appl. Microbiol. 12, 32-37.
7 Patel, B. K. et al (1985) Arch. Microbiol. 141, 63-69.
8 Huber, R. et al (1990) Arch. Microbiol. 154, 105-111.
9 WO 93/18134.
10 Davey, M. E. et al (1993) Syst. Appl. Microbiol. 16, 191-200.
11 The microorganisms of the present invention.
SUMMARY OF THE INVENTION
The present invention provides novel thermophilic alkaliphilic bacteria of the novel genus Thermopallium, more specifically of the novel species
Thermopallium natronophilum
, and novel polypeptides obtainable from these bacteria. In a more specific aspect, the invention provides novel alkaline pullulanase and amylase preparations from these novel bacteria.
In a third aspect, the invention provides a composition comprising a novel polypeptide according to the invention.
In a fourth aspect, the invention provides an isolated DNA fragment encoding a polypeptide according to the invention, recombinant DNA comprising such DNA fragment, host cells transformed with such recombinant DNA and a culture of such host cells.
In another aspect, the invention provides a method
Danson Michael J.
Herweijer Margareta A.
Hough David W.
Jones Brian E.
Thompson Carl R.
Genencor International Inc.
Genencor International Inc.
Prouty Rebecca E.
Steadman David J
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