Process for obtaining glucose dehydrogenase and micro-organism therefor

The present invention provides a process for obtaining glucose dehydrogenase by culturing a micro-organism in an appropriate nutrient medium and obtaining the enzyme from the cell mass or from the culture medium, wherein the micro-organism cultured is Bacillus cereus DSM 1644. The nutrient medium used contains a carbon source which consists predominantly or solely of glycerol.

This invention relates to a process for obtaining glucose dehydrogenase and 
to a new micro-organism especially suited for this purpose. 
Glucose dehydrogenase (Gluc-DH) (E.C.1.1.1.47) catalyses the following 
reaction: 
##STR1## 
Since NADH.sub.2 can easily be determined, this reaction can be used for 
the analytical determination of glucose. 
A disadvantage of this process is that the enzyme has hitherto been 
difficult to obtain and was, therefore, very expensive. This is due, on 
the one hand, to disturbing impurities which are difficult to remove 
(resistant spores, NADH oxidases) and, on the other hand, to too low 
yields. It is admittedly known (see "Abstracts of the 5th International 
Fermentation Symposium," Berlin, 1976, page 260) to suppress spore 
formation by culturing Bacillus megaterium M 1286, the yield thereby being 
said to be about 2 U/ml. However, our own experiments gave a yield of at 
most 0.25 U/ml. and very high contamination with NADH oxidases. 
It is also known to use a strain of Bacillus cereus which, however, only 
produces about 0.1U glucose-dehydrogenase/ml. of culture and displays very 
considerable spore formation. 
It is an object of the present invention not only to improve the known 
process for obtaining glucose dehydrogenase, especially with regard to 
enzyme yield, but also with regard to sporulation (spore formation up to 
the formation of maximum glucose dehydrogenase activity is undesired), as 
well as elimination of interfering activities, especially of NADH 
oxidases. 
Thus, according to the present invention, there is provided a process for 
obtaining glucose dehydrogenase by culturing a micro-organism in an 
appropriate nutrient medium and obtaining the enzyme from the cell mass or 
from the nutrient medium, wherein the micro-organism cultured is Bacillus 
cereus DSM 1644. 
The micro-organism Bacillus cereus DSM 1644 used in the process according 
to the present invention is new and is obtained by a mutation process 
involving a suitable parent strain. Bacillus cereus DSM 1730 is the 
preferable parent strain but Bacillus cereus DSM 345 and Bacillus cereus 
ATCC 10987 also proved to be useful. 
For mutation, the parent strain is incubated in the presence of methyl or 
ethyl methanesulphonate for 10 to 90 minutes at 28.degree. C. Mutagenesis 
is then stopped by washing in phosphate buffered saline and single clones 
of mutagen treated cells are isolated in surface culture. With these, 
overnight cultures are prepared which, after incubation for 12 hours, are 
examined microscopically to obtain asynchronous cultures in which 
spore-free, endospore-containing cells, as well as the first free spores, 
are visible. These are simultaneously selected and used for a fresh batch. 
After 5 to 7 growth cycles a pure culture of Bacillus cereus DSM 1644 was 
obtained, which is also the subject of the present invention. 
The micro-organism according to the present invention is a lad-mutant (late 
abnormal development), the spore development of which proceeds normally up 
to developmental stage III but which is then inhibited for up to about 20 
hours, whereafter it resumes development until the appearance of 
heat-resistant spores. 
According to a taxonomic description the microorganism consists of 
individual cells with a tendency to chain formation. The cell size is 
1.times.4 to 6.mu. (growth-phase dependent). The spores have an oval shape 
and are terminally arranged. Gram positive. Growth at 25.degree. to 
40.degree. C. in aerobic or anaerobic condition. .beta.-Haemolysis on 
blood agar. Further positive reactions include: glucose, maltose, salicin, 
nitrate, Voges-Proskauer, citrate and catalase. Negative tests are indole, 
oxidase, lysine decarboxylase, lactose, arabinose, saccharose, mannose and 
xylose. The definition of the growth phases corresponds to that given in 
Biochem. J., 109, 819/1968, and in Adv. Genet., 18, 69/1976. 
For the culturing, there can be used the known nutrient media which are 
suitable for the genus Bacillus. A nutrient medium is preferred in which 
the carbon source consists of glycerol instead of glucose. An especially 
preferred nutrient medium contains, per liter: 
5 to 50 g. glycerol, 
1 to 10 g. yeast extract, 
1 to 50 g. peptone, 
1 to 20 g. ammonium sulphate, 
0.2 to 5 g. dipotassium phosphate, 
0.1 to 5 g. magnesium sulphate, 
1 to 10 g. sodium chloride, as well as trace elements (manganese, zinc, 
copper, calcium and iron). 
The pH is adjusted to a value of from 7 to 8, preferably by the addition of 
an aqueous solution of sodium hydroxide. 
To achieve maximum glucose dehydrogenase activity, aerobic growth is 
carried out for 12 to 20 hours at 30.degree. C., After centrifugation the 
supernatant is discarded while the enzyme is obtained from the biomass. 
Normally, the glucose dehydrogenase is found intracellularly and the 
harvested cells are, therefore, disrupted by methods conventionally used 
to prepare a cell-free extract. Appropriate methods for this purpose 
include ultrasonic treatment, lysozyme digestion, glass bead disruption 
(Vibrogen Mill) and the like. The maximum enzyme yield is 2000 U/liter of 
culture medium. Further purification can be carried out by known methods, 
for example, as described in J. Bacteriol., 132, 282-293/1977. 
An special advantage of the process according to the present invention is 
that, as a result of retarded sporulation, which, during growth until 
harvest does not lead to the formation of heat-resistant spores, 
contamination problems are avoided. This substantially simplifies the 
culturing and obtaining of the enzyme. 
In contrast thereto, using parent strain Bacillus cereus DSM 1730, massive 
sporulation occurs already after 15 hours and the biomass obtained 
therefrom always contains a considerable amount of heat-resistant spores. 
This shows that, according to the present invention, the enzyme yield can 
be increased and, at the same time, the process is substantially 
simplified. An additional advantage is that undesired enzyme activities, 
especially NADH oxidase activity, which is particularly undesirable when 
the enzyme is used for determining glucose, can be separated off very 
easily, for example by a heating step.

The following Example is given for the purpose of illustrating the present 
invention: 
EXAMPLE 
A nutrient medium of the following composition: 
2.8 g. yeast extract, 
4 g. ammonium sulphate, 
1.0 g. dipotassium phosphate, 
0.8 g. magnesium sulphate, 
4.8 g. sodium chloride, 
5 g. glycerol (87%). 
1.6 g. peptone, as well as 
trace amounts of manganese sulphate, zinc sulphate, copper sulphate, 
calcium chloride and ferric chloride (pH 7.4), 
was sterilized and then inoculated with Bacillus cereus DSM 1644. After 
incubating for 8 hours at 30.degree. C., the medium of the main culture, 
having the same composition as above, was inoculated with 1% of 
pre-culture and aerated. The main culture was cultured for 19 hours at 
30.degree. C., with aeration and shaking. Determination of glucose 
dehydrogenase at this time gave an activity of 1800 U/liter. The cell mass 
was then centrifugated and frozen. After thawing cells were disrupted by 
ultrasonic treatment and a clear solution was obtained by centrifugation. 
The activity determination was carried out in the following manner: 
Enzyme assay: 
2.1 ml. 0.05 M tris-manganese buffer+12 mg./liter manganese sulphate 
hydrate (pH 8.5), 
0.3 ml. 1 M glucose solution 
0.4 ml. NAD solution (10 mg./ml.) 
The reaction was started by the addition of sample (crude extract). 
Temperature 25.degree. C.; the increase of extinction was measured at 365 
nm. 
It will be understood that the specification and examples are illustrative 
but not limitative of the present invention and that other embodiments 
within the spirit and scope of the invention will suggest themselves to 
those skilled in the art.