JPH0315384A - Novel cyclodextrinase and its preparation - Google Patents

Abstract

PURPOSE:To enable to prepare a cyclodextrinase having physicochemical properties such as the cleavage of cyclodextrin to produce oligo saccharides, by culturing a microorganism belonging to the genus Bacillus train and collecting the miroorganism from the cultured product. CONSTITUTION:Bacillus sphaericus E-244 (FERM 2458) separated from soil is subjected to a shaking culture of to an air-bubbling stirring culture in a medium containing a suitable nitrogen source, carbon source, vitamins, minerals, cyclodextrin, etc., at a pH of 6-8 at a temperature of 20-40 deg.C. The bacterial cells are collected from the culture by a centrifugal method, etc., and the bacterial cells are crushed by an ultrasonic treatment, etc., followed by removing the residues of the cells to provide a crude raw solution of the enzyme. The crude enzyme is treated by an ion exchange chromatography, etc., to provide the pure product of the enzyme having physicochemical properties such as the production of oligo saccharides originated from the glucose polymerization degree of the cyclodextrin. Malto oligosaccharide can thereby be efficiently prepared industrially.

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a novel cyclodextrinase and a method for producing the same. [Prior Art] Conventionally, cyclodextrinase [cyclomaltodextrinase (cyclomsltod@xtr+na
ss) Also known as EC 3.2.1.54. ]
There are very few reports on the product produced by Bacillus macsrans [Biochemistry].
), Volume 7, No. 121-124 members, 196B]
, Bacillus coagulans
The only known products are those produced by G. sgulans [Gric, Biol, Chew., Vol. 47, No. 1441-1447, 19831].

[The problem that the invention tries to solve! I】

An object of the present invention is to provide a novel cyclodextrinase and a method for producing the same. [Means for Solving the Problem] As a result of screening soil for microorganisms that produce cyclodextrinase, the present inventors found that a strain belonging to the genus Bacillus is a novel strain that is completely different from known cyclodextrinases. They discovered that cyclodextrinase can be produced and completed the present invention. That is, the present invention provides a novel cyclodextrinase having the following physical and chemical properties. ■Action: Cleaves cyclodextrin to generate oligosaccharides derived from the degree of glucose polymerization of cyclodextrin. ■Substrate specificity: The water lysis rate or affinity for cyclodextrin is
It is higher than that of linear oligosaccharides with the same degree of glucose polymerization as polysaccharides or cyclodextrins. ■Optimal pH and stable pH range: When β-cyclodextrin is used as a substrate, the optimal pH is around 8.0, and the stable p}l range is 5.0. 5-
9. It is 5. ■ Range of suitable temperature for action: The suitable temperature for action is around 40°C. ■ Conditions for deactivation due to temperature, etc.: Almost all deactivation is achieved by treatment at 50°C or higher for 15 minutes. ■Inhibition and activation: Inhibited by more than 90% by Hg", Cu", 2n", Ni'+, F1+, etc., and activated by In-30% by C1+ and Mal+. ■Molecular weight: In gel filtration method 144,000 and SDS PAGE
72,000 according to the method, and the present invention involves culturing a microorganism belonging to the genus Bacillus and having a novel cyclodextrinase-producing ability in a medium, producing and accumulating a novel cyclodextrinase, and producing and accumulating the novel cyclodextrinase from the culture. This is a novel method for producing cyclodextrinase, which is characterized by the step of collecting cyclodextrinase. First, the physicochemical properties of the present enzyme according to the present invention will be described. (1) Action: Acts on cyclodextrin to produce maltooligosaccharides derived from the degree of glucose polymerization of the cyclodextrin. (2) Substrate specificity: The substrate specificity is summarized in Table 1. Further, reaction rate parameters for cyclodextrins and maltooligosaccharides are shown in Table 2. Table 1 Table 2 (3) Optimal pH and stable pH range: The optimal pH is as shown in Figure 1, and is around pH 8.0 when 1% 6-cyclodextrin is used as a substrate. . Stable pH! The i range is as shown in Figure 2, and each pH
The residual activity was determined by measuring the residual activity after treatment at 25° C. for 24 hours. As is clear from Figure 2,
Stable pH range is 5. 5-9. It is 5. ? 4) Titer measurement method: 100 mM phosphate III buffer (p
H 7.5) After mixing 500 yen and reacting at a temperature of 40°C for an appropriate period of time, the reaction was stopped by boiling for 10 minutes, and the resulting mixture was produced using a high performance liquid chromatography (hereinafter abbreviated as HPLC) method. Maltohebutaose was quantified. In addition, when the amount of enzyme was small, the reducing power was determined by the Somogyi-Nelson method using glucose as the standard. The enzyme unit of this enzyme was defined as the amount of enzyme that produces 1 μO mole of maltopentaose per minute. (5) Range of suitable temperature for action As shown in FIG. 3, this enzyme has a suitable temperature for action around 40°C. (6) Conditions for temperature-induced inactivation As shown in Figure 4, the activity of this enzyme was stable up to 45°C when treated in I00mM phosphate buffer (pH 7.5) for 15 minutes. It was deactivated at temperatures above 50°C. (7) Inhibition and activation: The results of the study on the effect of metal ions on the activity of this enzyme were
Shown in the table. As is clear from Table 3, this enzyme is a divalent metal ion}+9 2 +. cυ2+In'
", old 2" and Fe"+ etc. inhibited the sale by almost 90% or more,
Ca'' and Mg''ltJ: About In-3096 was activated. Table 3 (8) Purification method: As described in Examples. (9) Molecular weight: It is 72,000 in the SOS PAGE method and 144,000 in the gel filtration method, so this enzyme is a dimer consisting of subunits with a molecular weight of 72,000. Table 4 shows the differences in physical and chemical properties between this enzyme and conventionally known cyclodextrinases. As detailed above, this enzyme is a completely new enzyme in that its properties are different from conventionally known cyclodextrinases, and in particular it acts best on cyclodextrins. By using this enzyme, it is possible to efficiently industrially produce maltooligosaccharides from cyclodextrin with a chain length derived from the degree of glucose polymerization of the cyclodextrin. Next, the method for producing this enzyme will be described. The microorganism used in the present invention may be any microorganism as long as it belongs to the genus Bacillus and produces the present enzyme, such as Bacillus sphaericus (Bacillus sphaericus).
lus sphaericus) E-244 strain. Bacillus sphaericus ε-244 strain is a wild strain obtained from soil. The mycological properties of this strain are shown below. ◎Mycological properties of Bacillus sphaericus E-244 strain (a) Morphology ■Cell shape and size 0.6-0.8 X 1.6
- It is a 4.0 micron bacillus. ■ Presence or absence of cell pleomorphism ■ Presence or absence of motility. Perifelgate ■ Presence of spores Sporangia size Miku O-shaped Oval position Subterminal ■ Durham staining Positive ■ Acid-fast Negative (b) Growth status in each medium ■ Juicy agar plate culture Forms colorless, diffusive colonies. The settlement is smooth with smooth edges. No pigment production is observed. ■Juice agar slant culture Fungal moss is smooth with smooth edges. No pigment production is observed. ■ Broth liquid culture There is growth throughout the medium. Bulge 0.8-0.9 X 1.1-1.2 Not observed. It has periflagellates, and transport is observed, but no precipitation is observed. ■Meat juice gelatin puncture culture Growth occurs only in the upper part of the medium, and no liquefaction is observed. ■Litmus milk No coagulation is observed, and no acid or alkali production is observed. Physiological properties Reduction of nitrate Denitrification MR test VP test Production of indole Production of hydrogen sulfide Hydrolysis of starch Utilization of citric acid Utilization of inorganic nitrogen sources Production of pigments Urease Oxidase Catalase Range of growth Temperature No reduction. none. Does not produce negative results. Not generated. Do not disassemble. Not used. Not used. Not generated. Negative Positive Positive +3- 38℃ pH [Phase] Attitude towards oxygen [Phase] O-F test not accepted) ■Attitude towards sugars L-7 Rabinose, 0-xyO-su, 0-glucose,
No acid production or gas production from romannose, O-fructose, O-galactose, maltose, sucrose, lactose, trehalose, O-sorbitol, O-mannite, inosit, glycerin, and starch was observed. (d) Deamination reaction of phenylalanine The positive Bacillus sphaericus E-244 strain was identified as a bacterium belonging to the genus Bacillus since it was a Durham-positive bacillus that formed spores. Furthermore, since the production of acid and gas from sugar was not observed, the pH of the VP broth was 7.0 or higher, and the de-7-mino reaction of phenylalanine was observed, Purge Aids Manual of Systematics Bacteriology Volume 2, 1984, 6
Based on the production of −10.5 aerobic negative <U, it was identified as a bacterium belonging to the species Sphaericus of the genus Bacillus. In addition, Bacillus sphaericus E-244 was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, as part of the Microbiological Research Institute No. 2458 (F
ERM BP-2458). Cultivation of the bacterial strain in the method of the present invention is in principle the same as the method used for aerobic cultivation of general microorganisms, but usually a shaking culture method using a liquid medium or an aerated agitation culture method is used. . The medium used includes a suitable nitrogen source, carbon source, vitamins, minerals, etc., and cyclodextrin, which is an inducing substrate for this enzyme. pH
may be in any pH range in which this bacterium grows, but is usually preferably in the range of 6-8. The culture conditions are, for example, usually 20-40'C, 16 hours to 4 days of shaking culture or aerated agitation culture. Using the culture obtained as described above, a purified product of the present enzyme is obtained, for example, by the enzyme collection process shown below. To obtain the enzyme from the above culture, the bacteria are collected by centrifugation or membrane concentration, and then the cells are crushed by ultrasonication or surfactant treatment, and the bacterial residue is removed by centrifugation to obtain the crude enzyme. Get the liquid. A purified product of the enzyme is obtained by subjecting the crude enzyme solution to an appropriate combination of column chromatography such as ion exchange chromatography, hydrophobic chromatography, and gel filtration. [Example] Next, the method of the present invention will be explained in more detail with reference to Examples. Example 1% 6-cyclodextrin, 1% pebtone, 0.5%
Liquid medium consisting of NaCI and 0.1% yeast extract (tap water used, pH 7.0) +00 Tllll
was placed in a 500 ml Sakaguchi flask and sterilized at 120°C for 20 minutes. One platinum loop of Bacillus sphaericus strain E-244 (FERM BP-2458) was inoculated into this from a preserved slant, and cultured with shaking at 30°C for 1 day. 50 ml of the main culture solution was inoculated into a 3-containing mini jar containing a 2°C medium prepared with the same medium composition and sterilization conditions as above, and incubated at 30°C, 1 vvm, 350 r.
．． p. m. Aerated agitation culture was carried out for 2 days under the following conditions, and after the culture was completed, the culture solution was drained at 8000 r.p.m. p. m. The bacterial cells were separated by centrifugation for 20 minutes, and 2% Triton x 1
IOmM phosphate buffer containing 00 (pH 7.0
) The bacterial cells were suspended in 500 ral2 and stirred at 25°C for 1 day. The suspension was heated to 1200 O r. p. m. So 2
After removing bacterial cell residue by centrifugation for 0 minutes,
The supernatant was dialyzed against 1 OmM phosphate buffer (pH 7.0) for 16 hours. The dialysate was heated at 12000 r.p. p. m
．． The mixture was centrifuged for 20 minutes to remove insoluble matter, and the supernatant was used as a crude enzyme solution (1). Next, about 5011III2 of this crude enzyme solution (1) (total activity 200 units, specific activity 0.1, p}17.0) was dissolved in lO
A DEAE cef7os loading force ram (Φ34 x 17
0 mm) to adsorb this enzyme, and then
．． Elution was performed with a gradient gradient of 5M NaCI. The elution pattern of this DEAE Cef7 loin chromatography is shown in FIG. Collect the active fractions and make crude enzyme solution (2) 105ITLi! (total activity 1
45 units, specific activity 0.5B, yield 72.5%). Next, this crude enzyme solution (2) was equilibrated with IM sodium sulfate-containing 100 mM phosphate buffer (pH 7.0) using an ether spw packing force ram (φ21.5 x 150
After adsorbing this enzyme, IM-OM
Elution was performed with a sodium sulfate gradient. This elution pattern is shown in FIG. Collect active fractions and make crude enzyme solution (3) 50 rats! (Total activity: 72 units, specific activity: 2.93, yield: 36%). Next, this crude enzyme solution (3) was ultraconcentrated to 1.5 ml using a collodion bag, and then 0.2 rILi
! was subjected to gel filtration using TSK gel G3000SW, and 100mM phosphoric acid containing 0.2M NaCl! 1
It was eluted with '# solution (pH 7.0). This elution pattern is shown in FIG. Collect the active fractions and make 1.4 nti of purified enzyme solution! (
Total activity 2.2 units, protein amount 0.24 mg, specific activity 9.1
7, yield 1%). This enzyme was found to be unique by SDS PAGE. (Figure 8)

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the optimum pH of this enzyme, FIG. 2 is a diagram showing the stability pH of this enzyme, and FIG. 3 is a diagram showing the operating temperature of this enzyme, Figure 4 is a diagram showing the conditions for inactivation of this enzyme by temperature, and Figure 5 is a diagram showing the conditions for deactivation of this enzyme by temperature.
FIG. 6 is a diagram showing the results of column chromatography of this enzyme using Cef7rose, and FIG.
●This is a diagram showing the results of HPLC using Iether 5PW.
FIG. 8 is a diagram showing the results of }IPLc with W, and FIG. 8 is a diagram showing the results of SOS PAGE of the present enzyme. Engraving of the drawings (no changes to the contents) YO 醇 迒 filtration Masue's review of the procedures stsi Day 0 00 Fi LI+J11 0 Mitomi Metaima 11 Day Nippon Yume Morofuka y diagram procedural amendment (method) Winter Ichiichi Takumen. (1--wipe 1 makku? Furin-1--●λ\sπ, reeler B 114111●ku7redami) Order! i human 1νr'-7
-Se71+-Recommendation 1 gamer C" 1 ←Tria 5 冫a) Hishi 1゛da- October 23, 1999

Claims (2)

[Claims] (1) A novel cyclodextrinase having the following physical and chemical properties. [1] Action Cyclodextrin is cleaved to produce oligosaccharides derived from the degree of glucose polymerization of cyclodextrin. [2] The water lysis rate or affinity for substrate specific cyclodextrin is
It is higher than that of linear oligosaccharides with the same degree of glucose polymerization as polysaccharides or cyclodextrins. [3] Optimal pH and stable pH range: When β-cyclodextrin is used as a substrate, the optimal pH is around 8.0, and the stable pH range is 5.5-9.5.
It is. [4] Range of suitable temperature for action: The suitable temperature for action is around 40°C. [5] Conditions for deactivation due to temperature, etc.: Almost all deactivation occurs by treatment at 50° C. or higher for 15 minutes. [6] Inhibition and activation: Hg^2^+, Cu^2^+, Zn^2^+, Ni^2
It is inhibited by more than 90% by ^+ and Fe^2^+, etc., and Ca^
It is activated by 10-30% by 2^+ and Mg^2^+. [7] Molecular weight: 144,000 by gel filtration method, SDS PAGE
According to the law, it is 72,000. (2) A novel method characterized in that a microorganism belonging to the genus Bacillus and having a novel cyclodextrinase-producing ability is cultured in a medium to produce and accumulate a novel cyclodextrinase, and the enzyme is collected from the culture. A method for producing cyclodextrinase.