The present invention relates to alginase produced by bacteria belonging to the genus and a process for production thereof and bacteria as well as a method for decomposing alginic acid.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to alginase produced by bacteria belonging to 
the genus Enterobacter and a process for production thereof and bacteria 
used, as well as a method for decomposing alginic acid. 
The alginase of the present invention can be produced in remarkable 
quantities by Enterobacter cloacae M-1 strain. Therefore, alginic acid can 
be partially decomposed or fully decomposed using this enzyme to modify 
its physical properties, whereby new products of alginic acid can be 
prepared or such is helpful for studies on the structure of alginic acid; 
etc. Thus, the present invention is extremely useful. 
2. Prior art and Problem 
It is heretofore known that alginase can be produced by bacteria belonging 
to the genus Flavobacterium, bacteria belonging to the genus Pseudomonas 
(Japanese Patent Laid-Open No. 59-143597), bacteria belonging to the genus 
Vibrio (Nippon Suisan Gakkaishi, 55(4), 709-713 (1989)), etc. 
However, a demand for alginase is recently increasing in processing, 
treatment, etc. of alginic acid and an improved method for the production 
of alginase in a large scale has been desired. 
SUMMARY OF THE INVENTION 
As a result of extensive investigations with an attempt to survey a 
bacterial strain capable of producing alginase in a marked quantity, the 
present inventors have come to isolate one bacterial strain capable of 
producing alginase in a marked quantity from the soil waste of alginic 
acid extraction residue. 
An object of the present invention is to provide an alginase in an 
industrial scale. Another object of the present invention is to provide a 
novel strain capable of producing an alginase. 
A further object of the present invention is to provide a method for 
producing an alginase using the novel strain. 
A still further object of the present invention is to provide a method for 
decomposing alginic acid using the alginase. 
That is, the present invention is directed to: 
an alginase produced by an alginase-producing bacterium belonging to the 
genus Enterobacter and capable of splitting off and decomposing alginic 
acid lyase-wise; 
a method for producing an alginase which comprises culturing an 
alginase-producing bacterium belonging to the genus Enterobacter and 
collecting the alginase from the culture; 
alginase-producing bacterium M-1 strain belonging to the genus 
Enterobacter; and, 
a method for decomposing alginic acid which comprises adding to and acting 
on a solution containing sodium alginate the culture of an 
alginase-producing bacterium belonging to the genus Enterobacter, treated 
products thereof, crude enzyme or purified enzyme.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The novel strain isolated from the soil waste of alginic acid extraction 
residue has been named M-1 strain and taxonomical studies have been made. 
It has thus been recognized that the strain belongs to Enterobacter 
cloacae. The M-1 strain has been deposited under Accession No. FERM 
BP-2577 in the Fermentation Research Institute of the Agency of Industrial 
Science and Technology of Japan. 
Bacteriological properties of Enterobacter cloacae M-1 strain are as 
follows. 
______________________________________ 
Morphology rod 
Gram staining - 
Spore - 
Mobility + 
Oxidase - 
Catalase + 
OF F 
Production of gas from glucose 
+ 
Production of indole - 
Methyl red - 
V-P + 
Utilization of citrate 
+ 
Production of hydrogen sulfide 
- 
Decomposition of urea 
- 
Deamination of phenylalanine 
- 
Decarboxylation of lysine 
- 
Arginine dihydrolase + 
Decarboxylation of ornithine 
+ 
Liquefaction of gelatin 
- 
Reduction of nitric acid 
+ 
ONPG + 
Production of yellow pigment 
- 
Production of acid: 
Adonitol - 
Arabinose + 
Inositol - 
Sucrose + 
Dulcitol - 
Sorbitol + 
Mannitol + 
Melibiose + 
Lactose + 
Ramnose + 
______________________________________ 
Enterobacter cloacae M-1 strain proliferates extremely rapidly. With the 
proliferation of the strain, the activity of alginase increases and 
reaches the maxim 12 hours after the initiation of the incubation. 
As a culture medium of the present bacteria, there is used a medium 
containing a suitable quantity of sodium alginate and also containing 
nitrogen sources and inorganic acid salts. Examples of the nitrogen 
sources are peptone, yeast extract, ammonium sulfate, ammonium nitrate, 
etc. As the inorganic acid salts, potassium monophosphate, potassium 
diphosphate, magnesium sulfate and the like can be suitably used. 
The culture temperature is between 30.degree. and 40.degree. C. Culture is 
carried out by aerial spinner culture until the quantity of the enzyme 
produced reaches the maximum in about 12 hours. 
The alginase is produced outside the cells. Thus, the resulting culture 
solution is centrifuged at about 10,000 G and the supernatant is obtained 
as an enzyme solution. 
The enzyme can be purified from the obtained supernatant by means of 
fractionation with acetone, fractionation with ammonium sulfate, ion 
exchange chromatography using DEAE-Sephadex A-50, CM-Sephadex C-50, etc., 
gel filtration using Bio-Gel P-100, adsorption chromatography using 
Hydroxyapatite; etc. 
Fractionation with acetone was performed by slowly adding 1 of crude enzyme 
solution to 2 of cold acetone. However, alginic acid remained in the crude 
enzyme solution precipitated at the same time so that a recovery rate was 
as considerably low as below 10%. 
Fractionation with ammonium sulfate was performed using ammonium sulfate to 
saturation degrees of 20, 40, 60, 80 and 90%. As the result, the best 
recovery rate was obtained by precipitation fractionation with 90% 
saturated ammonium sulfate. The recovery rate was approximately 70%. 
However, also in this case, about 25% of the activity remained in the 
supernatant after fractionation with ammonium sulfate. 
Ion exchange chromatography was carried out by equilibrating the column 
with 0.02 M potassium monophosphate buffer (pH 7.8) and supplying the 
crude enzyme solution dialyzed to the buffer. Elution was effected by NaC1 
gradient with the same buffer. As the result, the enzyme was eluted as the 
non-adsorbed fraction by DEAE-Sephadex A-50. Its recovery rate was 65% and 
a magnification of purification was 15 times. In the case of CM-Sephadex 
C-50, the enzyme was eluted as the adsorbed fraction. Its recovery rate 
was 25% and a magnification of purification was 35 times. Since the enzyme 
was requiring for metal ions, 1 mM calcium was added to the buffer, which 
was subjected to chromatography using CM-Sephadex C-50 in s similar 
manner. As the result, the recovery rate and magnification of purification 
increased to 45% and 90 times, respectively. Gel filtration using Bio-Gel 
P-100 was carried out by equilibrating the column with 0.02 M potassium 
monophosphate buffer (pH 7.8) and then supplying the crude enzyme 
solution. As the result, its recovery rate was 42% and a magnification of 
purification was 34 times. 
Adsorption chromatography was carried out by equilibrating the column with 
0.02 M potassium monophosphate buffer (pH 7.8) and then supplying the 
crude enzyme solution dialyzed to the buffer. Elution was effected by 0.2 
M potassium monophosphate buffer (pH 7.8). As the result, its recovery 
rate was 80% and a magnification of purification was 3.3 times. 
In the present invention, the culture supernatant of the enzyme solution, 
concentrates, crude enzyme in the acetone fractionation, etc., purified 
enzyme by various means for purification, etc. can all be the alginase of 
the present invention. They can be used depending upon use. 
Next, physicochemical properties of the alginase of the present invention 
determined by using the supernatant of the alginase solution obtained in 
Example 1 are shown below. 
1. Activity 
It acts on alginic acid and splits off and decomposes alginic acid 
lyase-wise. 
2. Substrate Specificity 
It splits off and decomposes alginic acid lyase-wise and produces 
unsaturated uronic acid or oligosaccharide having an unsaturated uronic 
acid residue. 
1% sodium alginate (M/G=0.93) solution (pH 7.8) was mixed with the crude 
enzyme solution, which had been inactivated by heating at 100.degree. C. 
for 5 minutes, in a ratio of 1:1. The mixture was made a blank. The 
reaction solution was subjected to sampling of 200 .mu.l each every 
definite time. Subsequently, each sample was measured by TBA reaction. 
That is, 0.25 ml of 0.125 N H.sub.2 SO.sub.4 solution containing 0.025 N 
HIO.sub.4 was added to 200 .mu.l of the sample, which was settled for 20 
minutes to allow the sample to oxidize with periodic acid. Then, 0.5 N HC1 
solution containing 2% sodium arsenite was added to the mixture. By 
allowing to settle for 2 minutes, the reaction was terminated. Then, 2 ml 
of 0.3% thiobarbituric acid solution was added thereto and the mixture was 
heated on a hot bath of 100.degree. C. for 10 minutes to perform 
condensation. The reaction solution colored red was allowed to cool and 
absorbancy was measured at 548 nm. In case that the absorbancy at 548 nm 
exceeded 1.0, the enzyme reaction solution was appropriately diluted with 
water so that absorbancy by the TBA reaction was adjusted to 1.0 or less, 
which was provided for measurement. 
The results are shown in FIG. 1. 
As is evident from FIG. 1, the formation of unsaturated uronic acid or 
oligosaccharide having an unsaturated uronic acid with the present enzyme 
is almost completed in 6 hours. 
Furthermore, the red compound formation system from alginic acid is as 
shown below. 
##STR1## 
3. Acting pH and Stable pH 
It is shown in FIG. 2 that the optimum pH of the enzyme reaction is 7.8, 
and stable pH is shown in FIG. 2. In FIG. 2,B, the residual activity of 
the enzyme after treating at various pH values at 30.degree. C. for 3 
hours was stable at pH of about 8 but the stability decreased to less than 
50% at pH below 6.5 and pH above 9. 
4. Acting Temperature and Temperature Stability 
As shown in FIG. 2,C, the optimum temperature of the enzyme reaction is 
35.degree. C. The residual activity of the enzyme after treating at 
various temperatures at pH of 7.8 for 3 hours was stable up to 30.degree. 
C. but as the temperature increased, the residual activity decreased and 
was completely lost at 60.degree. C. (FIG. 2,D). 
5. Influence of Metal Salts 
Influence of metal salts on the enzyme activity is shown in FIG. 3. The 
activity of the enzyme was inhibited more strongly by the addition of 1 mM 
EDTA. However, the activity was markedly activated by the addition of 2 mM 
cadmium or calcium, etc. 
6. Reduction in Viscosity of Sodium Alginate by the Enzyme Action 
To 5 ml of 1% sodium alginate solution (pH 7.8) was added 5 ml of the 
supernatant enzyme solution. Change in viscosity with passage of time was 
measured at 35.degree. C. with Ostwald viscometer. 
The results are shown in FIG. 4. Five minutes after, the specific viscosity 
(.alpha.) of the reaction solution was 0.83 and 60 minutes after, the 
specific viscosity reached 0.97. Finally, the viscosity was reduced to 
almost the same level of pure water. 
In FIG. 4: 
##EQU1## 
7. Decomposition Products of Sodium Alginate 
To 5 ml of 1% sodium alginate solution (pH 7.8) was added 5 ml of the 
enzyme solution. The mixture was enzymatically reacted at 35.degree. C. 
The reaction solution was collected with the passage of time and the 
change of the products was traced by TLC (developing solvent was 
n-butanol: acetic acid: water=5:2:3). The results are shown in FIG. 5. 
8. Thiobarbituric Acid Reaction of the Decomposition Products of Sodium 
Alginate 
To 5 ml of 1% sodium alginate solution (pH 7.8) was added 5 ml of the 
enzyme solution. The mixture was enzymatically reacted at 35.degree. C. 
The reaction solution was collected with the passage of time and the red 
compound formed by the thiobarbituric acid reaction was measured at 548 
nm. The results are shown in FIG. 1. As the reaction proceeded, the 
absorbancy at 548 nm rapidly increased. It is thus recognized that the 
present enzyme is lyase which splits off and decomposes alginic acid. 
Next, the present invention is described in more detail by referring to the 
examples below. 
EXAMPLE 1 
Composition of medium B: 1.0% sodium alginate, 0.6% peptone, 0.3% yeast 
extract, 0.5% KH.sub.2 PO.sub.4 and 0.05% MgSO.sub.4.7H.sub.2 O. 
Enterobacter cloacae M-1 strain, FERM BP-2577, was inoculated on medium B 
having the above composition and shake cultured at 35.degree. C. for 24 
hours to give a seed culture solution. 
In a culture tank of 10 l was charged 6.4 l of medium B having the above 
composition. After sterilizing at 120.degree. C. for 20 minutes in an 
autoclave, the system was cooled to 35.degree. C. 
To the medium was inoculated 600 ml of the seed culture solution described 
above followed by culturing at 35.degree. C. and 350 to 450 rpm in an 
aerial amount of 1.5 l/min. 
The production of the enzyme with passage of time is shown in FIG. 6. 
The enzyme production reached the maximum 12 hours after the culture 
started. After completion of the culture, centrifugation was performed at 
10,000 G for 20 minutes to remove the cells. The resulting supernatant was 
made the alginase solution. 
The enzyme activity of the obtained alginase solution was 
20.times.10.sup.-3 u/ml. 
The activity of the alginase solution was determined by the following 
method. In Trishydrochloride buffer containing 1 mM CaC1.sub.2 was 
dissolved sodium alginate in 1% concentration and, 0.5 ml of the enzyme 
solution was added to 0.5 ml of the resulting solution. The mixture was 
reacted at 35.degree. C. for 30 minutes and the activity was determined by 
the thiobarbituric acid method. The enzyme activity was made one unit when 
the enzyme formed the product corresponding to 1 .mu.mol of uronic acid 
for one minute under the same conditions. 
EXAMPLE 2 
To 5 ml of 1% aqueous solution (pH 7.8) of sodium alginate having M/G ratio 
of D-mannuronic acid (M) and L-gluronic acid (G) of 0.93 was added 5 ml of 
the supernatant of the alginase solution obtained in Example 1 followed by 
enzyme reaction at 35.degree. C. 
The reaction solution was collected with passage of time and the red 
compound formed by the thiobarbituric acid method was measured at 548 nm. 
The results obtained are shown in FIG. 1. 
While the invention has been described in detail and with reference to 
specific embodiments thereof, it is apparent to one skilled in the art 
that various changes and modifications can be made therein without 
departing from the spirit and the scope of the present invention.