JPH0455670B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a new thermophilic bacterium of the genus Bacillus that produces cyclodextrin synthase in a state bound to the bacterial body. [Prior Art] Bacillus macellans, circulans, megaterium, obensis, and stearothermophilus have been known as bacteria producing cyclodextrin synthase (cyclodextrin glucanotransferase). The bacteria produce the enzyme outside the bacterial body, which is disadvantageous for recovery and immobilization of the enzyme. Furthermore, if a high temperature of 65° C. or higher can be used during bacterial culture, bacterial contamination can be prevented, which is advantageous. In this respect, stearothermophilus can be used because it grows at 50 to 65°C, but no bacterial strain is known that produces the enzyme in a state bound to bacterial cells. [Means for Solving the Problems] Therefore, the present inventors proposed that the growth temperature be 65°C or higher,
We searched hot spring soil for bacteria that produce cell-bound cyclodextrin synthase, and found more than a dozen strains that were suitable for this purpose. Among these, three strains with strong enzymatic activity were selected and their mycological properties were investigated.The results are described below. These three strains are bacilli, form spores, have positive Gram staining, and are aerobic, so they belong to the genus Bacillus. Other properties are as shown in the table.

[Table] Bergey's Manual
Manual of Determinative Bacteriology 8th
edition) and according to Cowan and Steel's classification (Manual for the ldentifcation of Medical
Bacteria, 2nd edition, Cambridge Univ.
Press, 1974), and are closely related to stearothermophilus in terms of growth temperature; however, strains KF5-1 and KF9-10 differ in growth pH, salt tolerance, and nitrate reducing ability; -10 differs from stearothermophilus in its resistance to sodium azide and salt. Furthermore, these strains differ from Megatherium, Macerans, and Circulans not only in growth temperature but also in the utilization of citric acid, hydrolysis of casein, hydrolysis of gelatin, and production of acid from arabinose. Furthermore, although S. obensis, Megaterium, and Circulans produce β-cyclodextrin synthase, the strain of the present invention mainly produces an enzyme that synthesizes α-cyclodextrin, and is therefore different from the above-mentioned known bacteria in this respect as well. Based on these results, the present inventors recognized the three strains as new bacteria belonging to Bacillus stearothermophilus var., and identified them as KF5-1, KF9-10,
It was named KF53-10. These strains have been deposited with the National Institute of Microbial Technology, Agency of Industrial Science and Technology, and their accession numbers are FERM P-8716, FERM P-8718, and FERM P-8718, respectively.
8717. This strain was added to corn steep liquor, soluble starch,
Mixed medium of ammonium sulfate and calcium carbonate or potato,
It grows well when cultured in a mixed medium of oatmeal, calcium chloride, and salt, and when cultured at 65-75°C for 12-72 hours, cyclodextrin synthase is produced in a state bound to the bacterial cells. Culture temperature 65℃
The enzyme is still produced even if the amount is below, but more extracellular enzymes are produced, for example, KF9
-10 produces 10 THU (Tilden-Hudson units) of activity outside the bacterial cells when cultured at 50°C. This tendency is also observed in Macellas and Stearothermophilus, but the bacterial cell binding enzyme activity is less than 1/10 compared to the bacteria of the present invention, and the activity of extracellular enzymes is also significantly lower than that of the bacteria of the present invention. low. The optimal medium may be used by combining various inorganic, organic, carbon sources, nitrogen sources, salts, vitamins, amino acids, etc., but economically, corn stew liquor, blackstrap molasses, bran, starch, ammonium sulfate, etc. are recommended. is advantageous. 65 ~ using a medium containing corn steep liquor
When culturing at 75°C, there is no need to use a sealed jar fermenter; the bacteria can grow by blowing air into the heated container, and since the bacteria tend to settle, it is easy to collect the bacteria. Therefore, the bacteria of the present invention can be grown continuously while adding a medium and can be taken out continuously, and the bacteria of the present invention are also advantageous for continuous culture. After washing the cultured bacterial cells with water, they are added to a starch suspension and subjected to a shaking reaction at 65°C or higher, whereby the starch granules are gradually dissolved and at the same time subjected to enzyme action. Continuous reaction can be carried out by removing the supernatant of the reaction solution and adding starch, but the enzyme will be released from the bacterial cells little by little, and if live bacterial cells are used in batches, the reaction will be repeated 3 to 4 times (24 hours/once). ), the amount of bound enzyme is halved. Therefore, we reinforced the bond with glutaraltehyde and it was able to withstand more than 10 uses. Other methods for preparing immobilized enzymes can also be used to reinforce enzyme bonds, such as using sodium alginate, polyacrylamide, nylon, cellulose nitrate, and polyethyleneimine, or by mixing two or more of these or using them alone. It can be used in Furthermore, this immobilized bacterial enzyme can be used in bioreactors, such as cell culture reactors, column reactors, model reactors, and the like. [Effects of the Invention] As described above, the thermophilic bacteria in which the cyclodextrin synthase of the present invention is bound to the cells are very advantageous for the production of cyclodextrin, and the production of cyclodextrin can be made continuous.
Its production costs are significantly reduced. Next, the present invention will be explained in more detail with reference to examples, but the present invention is not limited thereto. Example 1 Corn steep liquor 1.0%, soluble starch 1.0%
A culture medium containing 0.5% ammonium sulfate and 0.5% calcium carbonate in tap water was autoclaved at 120°C for 15 minutes, and then dispensed in 100ml portions into 500ml Erlenmeyer flasks in the laboratory.
One platinum loop of bacterial cells KF5-1 (FERM
P-8716) was taken and inoculated, then covered with aluminum foil and cultured at 65°C for 2 days. After the culture was completed, the mixture was left at room temperature for 1 hour, the supernatant was removed, and the precipitate was collected in a glass filter and washed with tap water. The yield of bacterial cells is 2 to 3 g/100 ml of wet culture solution, and the activity is 100 to 300 THU per g of wet bacterial cells. This bacterial enzyme is
It does not become inactivated even if left overnight at 65-70℃, and is stable at pH 5.0 or higher. 1g of this moist bacteria contains 20g of corn starch
When added to 100ml of starch suspension and shaken for 24 hours, most of the starch granules undergo a liquefaction reaction, with α-cyclodextrin at 20-30% and β-cyclodextrin at 10%.
The result is a cyclodextrin starch syrup containing ~15% and 5% γ-cyclodextrin. This reaction solution was removed by centrifugation, 100 ml of starch suspension was newly added, and the reaction was carried out in the same manner. The liquefaction and reaction proceeded satisfactorily up to three times. Example 2 The same procedure as in Example 1 was carried out except that KF9-10 (FERM P-8718) was used as the bacterial cells to obtain 1 to 2 g of wet bacterial cells/100 ml of culture solution. The activity is 100-150 THU/g of wet bacterial cells. This bacterial enzyme is 65
It does not become inactivated even if left as a single solution at ~75℃, and is stable at pH 5.0 or higher. 1.5 g of this wet bacterial cell was added to 100 ml of a starch suspension containing 20 g of corn starch and shaken at 70° C. for 24 hours to obtain the same results as in Example 1. Example 3 The same procedure as in Example 1 was carried out except that KF53-10 (FERM P-8717) was used as the bacterial cells to obtain 1 to 2 g of wet bacterial cells/100 ml of culture solution. The activity is 200-300 THU per gram of wet bacterial cells.
This bacterial enzyme does not become inactivated even if left overnight at 65-70℃, and is stable at pH 6.0 or higher. 1.0 g of this wet bacterial cell was added to 100 ml of a starch suspension containing 20 g of corn starch, and the mixture was shaken at 70° C. for 24 hours to obtain the same results as in Example 1.

Claims (1)

[Scope of Claims] 1. A thermophilic bacterium that belongs to Bacillus stearothermophilus var. and produces cyclodextrin synthase bound to its bacterial cells at a temperature of 65°C or higher. 2 Bacillus KF5-1 (FERM P-1) is a thermophilic bacterium that belongs to Bacillus stearothermophilus var. and produces cyclodextrin synthase.
8716), Bacillus KF9-10 (FERMP-8718), and Bacillus KF53-10 (FERM P-8717). 3. The bacterium according to claim 1 or 2, which is a bacterium that has a growth pH lower limit of 5 or 6, and/or a salt tolerance upper limit of 4 to 6%, and/or grows in a 0.02% sodium azide medium. .