DNA fragment containing gene for alkaline pullulanase

Details DNA fragment containing gene for alkaline pullulanase DNA fragment containing gene for alkaline pullulanase

1994-09-28

2003-01-21

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C435S183000, C435S252300, C435S320100, C435S069100

Reexamination Certificate

active

06509183

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a DNA fragment containing a gene for alkaline pullulanase derived from an alkalophilic strain of the genus Bacillus to a recombinant vector with said DNA fragment inserted therein., and further to a transformant microorganism transformed with said vector.
The present invention also relates to alkaline pullulanase for use as an effective additive in detergents, which is obtained by a recombinant technique, and to a method of preparing the pullulanase.
2. Description of the Related Art
Pullulanase is an enzyme which cuts specifically the alpha-1,6-glucoside bond present in the molecule of pullulan to generate maltotriose as the major product. This enzyme was first found by Bender and Wallenfels [Biochem. Z., 334, 79, (1961)] in culture of
Aerobacter aerogenes
. Since then, various microorganisms have been reported to produce alkaline pullulanases, which include Bacillus sp. [J. Jpn. Soc. Starch Sci., 30, 200, (1983)],
Bacillus acidopullulyticus
[Agric. Biol. Chem., 52, 2293, (1984)],
Bacillus stearothermophilus
[Eur. J. Appl. Microbiol. Biotechnol., 17, 24, (1983)],
StrePtococcus mitis
[Biochem. J., 10, 33, (1968)],
Lactobacillus
[Starch Science, 28, 72, (1987)],
Clostridium thermohydrosulfuricum
[Appl. Environ. Microb., 49, 5, (1985); Biochem.J., 246, 193, (1987)], Thermus sp. [J. Jpn. Soc. Starch Sci., 34, 1, (1987)], and
Clostridium thermosulfurogenes
[Appl. Microb. Biotechnol., 33, 511 (1990)].
Presently, pullulanase is known to hydrolyze the alpha-1,6-glucoside linkages not only of pullulan, but also of starch, glycogen, amylopectin, and branched oligosaccharides, and is called a “debranching enzyme”. This enzyme is also known to produce glucose, maltose and maltooligosaccharides (such as maltotriose, maltotetraose, maltopentaose and maltohexaose), from starch with high yield when used in combination with endo-type amylase and with exo-type amylase. In recent years, therefore, pullulanase is highlighted in starch-manufacturing industry.
Based on the characteristics of the pullulanase, the inventors of the present invention incorporated pullulanase and alpha-amylase into detergents. They discovered that the detergent power of the compositions was markedly enhanced especially against starch soil, and filed a patent application (Japanese Patent Application Laid-open (Kokai) No. 132193/1990).
Enzymatic components of detergents must exhibit their maximal activities in an alkaline pH range, or must be resistant to alkaline washwater. However, almost all pullulanases found hitherto in the natural world are so-called neutral or acid pullulanase, and exhibit their maximal and steady enzymatic activities in the neutral or acidic pH range. There have been found very few alkali-resistant pullulanases which are resistant to alkaline environment. In this specification, the term “alkaline pullulanase” is used for pullulanase having its optimum pH in the alkaline range, and the term “alkali-resistant pullulanase” is used for pullulanase having its optimum pH in the neutral to acidic range while having a sufficient and steady activity in the alkaline pH range when compared with the optimum activity. The term “neutral” indicates a pH range of 6 to 8. The term “alkaline” indicates a pH range higher than the neutral range.
Only two publications have reported a method of producing alkaline or alkali-resistant pullulanases: Horikoshi et al. cultured an alkalophilic strain of the genus Bacillus (Bacillus sp. 202-1) and produced alkaline pullulanase (Japanese Patent Publication (Kokoku) No. 277786/1978); and the inventors of the present invention produced alkaline pullulanase from Bacillus sp. KSM-AP 1876 (Japanese Patent Application Laid-open (Kokai) No. 87176/1991).
The gene for alkaline pullulanase from an alkalophilic Bacillus has not yet been obtained, and therefore, mass-production of the enzyme by genetic engineering and improvement of the enzyme by protein engineering have been difficult to carry out.
SUMMARY OF THE INVENTION
Accordingly, an object of the present invention is to provide a DNA fragment containing gene for alkaline pullulanase derived from an alkalophilic bacterial strain which belongs to the genus Bacillus, to a recombinant vector with said DNA fragment inserted therein, and further to transformant microorganism transformed with said recombinant vectors.
Another object of the present invention is to provide alkaline pullulanase by a genetic approach, and a method of mass-producing alkaline pullulanase by a genetic approach.
Generally speaking, in order to enhance the titer of an enzyme, culture conditions of productive microorganisms are suitably selected, and breeding based on mutation is followed. Breeding by recombinant techniques, which has progressed markedly in recent years, has received attention as it may enable a direct manipulation of the gene. In such breeding by recombinant techniques, it is essential that the alkaline pullulanase gene be isolated. The inventors of the present invention, in an attempt to carry out breeding by recombinant techniques, first prepared a recombinant bacterial strain which belongs to the genus Escherichia and which produces alkaline pullulanase, and isolated therefrom a DNA fragment containing the alkaline pullulanase gene. The obtained DNA fragment was analyzed comparing the gene with other pullulanase genes which have been isolated from various bacterial strains which belong to the genus Bacillus, and as a result, it was revealed that the obtained DNA fragment was a new fragment having a unique restriction enzyme map. The inventors continued their research and succeeded in cloning of the alkaline pullulanase gene by means of gene recombination using chromosome of an alkalophilic bacterial strain which belongs to the genus Bacillus as a starting material, and opened up a way to the mass-production of a new alkaline pullulanase by the use of this gene. The present invention was accomplished based on these findings.
Accordingly, in one aspect of the present invention, there is provided a DNA fragment coding for an alkaline pullulanase which comprises about 6.3 kilo base pairs and has a restriction map shown in
FIG. 2
, a recombinant vector which contains the DNA fragment, and a recombinant microorganism carrying the recombinant vector.
In another aspect of the present invention, there is provided a method of producing an alkaline pullulanase, which comprises culturing a transformant microorganism transformed with a recombinant vector which contains the above-described DNA fragment, and harvesting alkaline pullulanase from the culture.
The above and other objects, features and advantages of the present invention will become apparent from the following description.


REFERENCES:
patent: 5147796 (1992-09-01), Ara et al.
Igarashi et al,Biosci. Biotech. Biochem., 56 (3): 514-516, 1992.
Ara, et al.,Biosci. Biotech. Biochem, 56 (1): 62-65, 1992.
Sambrook, et al., “Molecular Cloning: A Laboratory Manual”, Cold Spring Harbor Press, p. 11.3-11.19, 1989.

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