JP2785323B2 - β-glucosidase and method for producing the same - Google Patents

Description

The present invention relates to a novel β-glucosidase and a method for producing the same. β-glucosidase is an enzyme that hydrolyzes cellooligosaccharide, which is an intermediate hydrolyzate of cellulose that can be an inexpensive glucose source, to produce glucose.In the present invention, β-glucosidase has properties extremely suitable for this use. Is produced using a microorganism belonging to the thermophilic bacillus genus.

[Prior Art] β-Glucosidase is one of enzymes that saccharify biomass (cellulose resources) by an enzymatic method together with other cellulases. Of the series of cellulolytic enzymes, β-glucosidase is an important enzyme responsible for the final step of degrading cellooligosaccharides, which are intermediate products of cellulolysis, to glucose.

By the way, the cellulosic substance has a strong crystal structure and forms a complex with lignin, hemicellulose and the like. Therefore, pretreatment such as alkali treatment is performed since cellulase is unlikely to act as it is (Japanese Patent Application Laid-Open No.
-98093). The alkaline pretreated product is washed with a large amount of water in order to adjust it to the action pH range of the cellulase, but this washing water is subjected to wastewater treatment, and it is desirable to reduce the washing water as much as possible.

Therefore, the use of cellulase containing β-glucosidase which is active even in alkaline conditions for saccharification of cellulose can reduce the amount of wastewater, which is extremely industrially beneficial.

Alkaline and active cellulases have been developed as follows. Alkaline cellulase A (JP-B 50-285)
No. 15), alkaline cellulase 301A (JP-A-58-224686)
), Cellulase produced by Streptomyces sp. (JP-A-61-19483), alkaline cellulase K (JP-A-63-1)
09776). These include CMCase, A, which degrades long-chain cellulose into cellooligosaccharides among cellulase activities.
Vicelase activity is high but β-glucosidase activity is very low. Therefore, in order to completely degrade cellulose to glucose, an alkaline and active β-glucosidase is additionally required.

[Problems to be Solved by the Invention] Alkaline and active β-glucosidase is β-glucosidase (Agric. Biol. Chem., 45 , 571-577, (1981)) produced by Mucor miehei YH-10. )
There is. This β-glucosidase is heat-resistant and has an activity even in alkaline, but the enzyme source is a filamentous fungus,
Since a solid culture method is used, special equipment is required, and it is not easy to scale up the production method. In addition, there is a disadvantage that it takes a long time of 4 to 5 days to culture the microorganism.

Other β-glucosidases include thermus species ( Ther
mus sp.) produced β-glucosidase (Appl. Microbio
l. Biotechnol., 29 , 55-60, (1988)) has heat resistance,
The time required for culturing is short, but the activity range in alkaline is insufficient.

Therefore, there is a demand for β-glucosidase which is 1) alkaline and active, 2) easy to culture and scale up microorganisms, and 3) can be produced in a short time.

[Means for Solving the Problems] The present inventors have intensively studied to solve the above-mentioned problems, separated microorganisms from many soils, had alkalinity and activity, and were easy to culture and scale up. A β-glucosidase-producing strain that can be produced in a short time was searched. As a result, a microorganism belonging to the thermophilic Bacillus genus, Bacillus stearothermophilus ( Bacillus starotherm)
ophilus ) TB-636 (hereinafter referred to as TB-636) produces the desired β-glucosidase, and the present invention has been completed based on such findings.

That is, the present invention provides a β-glucosidase which has an action of hydrolyzing cellooligosaccharides to produce glucose, acts in an alkaline region, and is easy to culture and scale up microorganisms, and can be produced in a short time. Yes,
A method for producing β-glucosidase, comprising culturing a microorganism belonging to the genus Thermophilic Bacillus and capable of producing β-glucosidase in a nutrient medium, producing β-glucosidase in the culture, and collecting the β-glucosidase. Is provided.

The β-glucosidase-producing bacterium used in the present invention may be any thermophilic microorganism which belongs to the genus Thermophilic Bacillus and can produce the above-mentioned β-glucosidase. Therefore, TB
In addition to the -636 bacteria, natural or artificial mutants thereof and various organisms into which the genes of these strains have been transferred can also be
-It can be used in the present invention as long as it has glucosidase producing ability.

Next, the bacteriological properties of TB-636 are shown. The mycological properties were examined using the methods and medium compositions described in "Separation and Identification of Microorganisms" (edited by Takeharu Hasegawa, University of Tokyo Press) and "Microbial Identification Method" (Japan Society of Sanitation Technology). Was.

[Morphological findings] (cultured at 55 ° C for 18 hours) 1. Shape and size of cells: 0.25 to 0.3 µm x 0.6 x 1.0 µm
2. Polymorphism: None 3. Motility: None 4. Spore: A cylindrical endospore is formed at the center or tip of the cell. The spore swells.

5. Gram staining: Positive 6. Acid-fast: None 7. Capsule: None 8. Metastaining granules: None [Growth state] (cultivation at 55 ° C, 18 hours) 1. Gravy agar plate culture Shape: Circumference Perimeter: All edges or Slightly wavy Ridge: Flat Gloss: Yes Surface: Smooth Color: translucent 2. Gravy agar slant medium Growth: Poor Shape: Slightly spread 3. Gravy liquid medium Surface growth: None Turbidity: Transparent Sediment: Trace color, Decolorization: None 4. Broth gelatin stab culture (addition of 30% gelatin, timely culture at 55 ℃, then cool to judge the solidification state) Does not grow.

5. Gravy agar puncture culture Form: surface growth only Surface growth: poor 6. Litmus milk No litmus discoloration, no pH change, no milk coagulation, liquefaction [Physiological properties] (55 ℃, 1-2 days culture) 1. Whether nitrate is reduced: No 2. MR test: negative 3. VP test: negative 4. Indole formation: no 5. Hydrogen sulfide formation: no 6. Starch hydrolysis: yes 7. Use of citric acid: no 8. Use of ammonium salt: None 9. Production of pigment: None 10: Oxidase activity: Available 11. Catalase activity: Available 12. Growth pH: 5.5 to 8.5, Optimum pH range: 5.5 to 7.0 13. Growth temperature: 33 -55 ° C, optimal growth temperature range: 42-55 ° C 14. Growth on anaerobic medium: none 15. Growth on Sabouraud dextrose agar: none 16. Medium containing sodium azide 0.02% at 55 ° C Growth potential in the presence of: 17. Viability in the presence of 0.001% lysozyme (45 ° C 18.) Deamination of phenylalanine: none 19. Sodium chloride tolerance: grows at 2.5%, but does not grow at 4.5%.

20. Vitamin requirement: yes 21. Tyrosine degradability: no [Utilization of carbon source] Fructose, D-glucose, inositol, D-xylose, arabinose, sucrose, D-galactose, D-mannitol And proliferate to produce acids.

From the properties of the microbiological fluids of the microorganisms described above, Bergey's manual of systematic bacteriology (19)
When the TB-636 strain was searched according to the classification method of 84), the TB-636 strain almost coincided with Bacillus stearothermophilus, but was different from known strains in that it had no motility and did not degrade gelatin. Furthermore, none of the standard strains Bacillus stearothermophilus IAM11001, 11002, 11003, 11004, 12043 (stocks of the Institute of Applied Microorganisms, University of Tokyo) produced β-glucosidase by the culture method described below.

As apparent from the above, TB-636 is distinguished from known strains, and it was concluded that it is appropriate to set this as a new strain.

TB-636 has been deposited with the Institute of Microbial Industry and Technology of the National Institute of Advanced Industrial Science and Technology as Microbial Bacteria No. 10523 (FERM P-10523).

The microorganism having the ability to produce β-glucosidase of the present invention is cultured in a nutrient medium, the β-glucosidase is produced in the culture, and the β-glucosidase is collected by collecting the β-glucosidase.
Glucosidase is obtained.

The nutrient medium used in the present invention may be any of natural and synthetic media as long as it contains a carbon source, a nitrogen source, an inorganic substance and, if necessary, a trace nutrient required by the used strain.

As the carbon source, sugars usually used for culturing microorganisms, for example, starch, maltose, sucrose, glucose and the like can be used.
That is, the present enzyme can be more effectively induced and produced by using cellooligosaccharides such as cellobiose, lamina ribose, laminarin, and salicin as the main carbon sources.

As a component other than the carbon source, a medium containing organic nitrogen compounds such as peptone, meat extract, yeast extract, malt extract, soybean protein hydrolyzate, cottonseed meal, and trace nutrients such as vitamins as appropriate may be used.

As the culture method, either solid culture or liquid culture is possible, but aeration and agitation culture is most suitable industrially.

The cultivation can be performed in the range of 33 to 55 ° C.,
A range of 55 ° C. is preferred. The pH is preferably neutral to weakly acidic.

Enzyme production varies depending on conditions, but usually 4
The culture is stopped when the production of β-glucosidase is confirmed, preferably when the production reaches a maximum.

Collection of β-glucosidase from the culture may be carried out by appropriately combining known methods. For example, after completion of the culture, the cells are separated from the culture by centrifugation or the like.
Then, when obtaining extracellular β-glucosidase, the previously centrifuged supernatant may be used as it is, and when acquiring β-glucosidase in the intracellular cells, an appropriate centrifuged cell may be used from the previously centrifuged cells. A β-glucosidase may be extracted by a means, and this extract may be further treated by centrifugation or the like to use a clarified liquid from which insoluble components have been removed.

The resulting enzyme solution is purified by salting out, dialysis, and various types of chromatography (ion exchange method, gel filtration method, hydrophobic chromatography method, etc.).
-Glucosidase can be obtained.

Next, properties of the β-glucosidase of the present invention will be described. In addition, the enzyme obtained in Example 1 described later was used as a standard.

β-glucosidase activity was determined by reacting 2 ml of a 0.5 M glycine-NaOH buffer solution (pH 8.0) containing 5 mM p-nitrophenyl-β-D-glucoside and the enzyme at 37 ° C. for 15 minutes.
The reaction was stopped by adding 0.2 M Na ₂ CO ₃ ml, and the resulting p-
The amount of nitrophenol was determined by the absorbance at 400 nm.
Under these conditions, the amount of enzyme that produces 1 μmol of p-nitrophenol per minute was defined as 1 unit.

(1) Action and substrate specificity The reactivity of this enzyme preparation with various substrates was examined. The measurement was performed by reacting a reaction solution having a total volume of 2 ml containing the substrate at various concentrations shown in the table, 0.32 m unit of the enzyme preparation, and 0.5 M glycine-NaOH buffer (pH 8.0) for 1 hour at 37 ° C. The resulting glucose was measured by colorimetry using glucose oxidase. The numbers in the table are p-
The relative activity of each substrate was expressed as the activity when nitrophenyl-β-glucoside was used as the substrate.

As is clear from the table, this enzyme works well on cello-oligosaccharides with β-1,4 linkage and lamina ribose with β-1,3 linkage, but not on gentiobiose with β-1,6 linkage. .

(2) Optimum pH The enzyme preparation of the present invention was added to various pH buffers shown in Fig. 1.
The reaction was carried out by dissolving to a concentration of 0.16 m unit / ml. 5 for substrate
Using mM p-nitrophenyl-β-D-glucoside, the relative activity was determined by measuring the amount of p-nitrophenol produced after reaction at 37 ° C. for 15 minutes by measuring the absorbance at 400 nm. The buffer used was 0.1 M acetate buffer at pH 4-6.5, and glycine-NaOH buffer at pH 7-10.

The activity at the pH showing the maximum activity of this enzyme is 100, and each pH
Are shown in FIG. As is apparent from the figure, the enzyme preparation of the present invention has a wide range of action pH range and showed almost 100% activity at pH 5-8.

(3) Stable pH The enzyme preparations of the present invention were dissolved in various pH buffers shown in FIG. 2 and kept at 37 ° C. for 1 hour. Next, an enzyme solution was added to give a 5 mM p-nitrophenyl-β-D-glucoside, 0.5 M glycine-NaOH buffer (pH 8.0) to prepare a reaction solution, and the enzyme activity was measured. FIG. 2 shows the residual activity in each pH treatment when the activity at the start of the test (that is, no treatment) was set to 100.

 The enzyme completely maintained its activity between pH 5 and 10.

(4) Optimum temperature 0.32 m unit of the enzyme preparation of the present invention was added to 2 ml of 0.5 M glycine-NaOH buffer (pH 8.0) containing 5 mM p-nitrophenyl-β-D-glucoside, and FIG. The reaction was performed in the temperature range shown. The activity at the temperature showing the maximum activity is defined as 100, and the relative activity at each temperature is shown in FIG.

 The optimum temperature of this enzyme was found to be around 55 ° C.

(5) Heat stability 0.32m unit of the enzyme preparation was added to 0.5M glycine-NaOH buffer (pH
8.0) After dissolving in 2 ml and keeping at the temperature range shown in FIG. 4 for 1 hour or 24 hours, the residual activity was measured at 37 ° C.

The relative activity at each temperature is shown in FIG. 4 with the untreated enzyme activity as 100.

This enzyme does not deactivate at all when kept at 37 ° C for 24 hours.
Even when kept at 50 ° C. for 100 hours, 100% activity was maintained.

(6) Influence of metal 1 mM Fe ³⁺ , Fe ²⁺ , Pb ²⁺ has a slight inhibitory effect, and 1 mM
Completely inhibited by Hg ²⁺ .

(7) Molecular weight The molecular weight of this enzyme determined by gel filtration is about 74,000.

Although the β-glucosidase of the present invention has an activity even on the alkaline side, it has an optimum pH for alkalinity produced from Mucor (Agric. Biol. Chem., 45 , 571-577, (1981)) by solid culture. Β-glucosidase having the molecular weight of about 220,000 is different from the enzyme of the present invention.

In addition, the thermophilic genus Thermos (Appl. Microbiol. Bio
technol., 29 , 55-60, (1988)) has an optimum action temperature of 80-85 ° C. and a residual activity at pH 8.0 of about 60%. Different from β-glucosidase.

[Examples] Next, the present invention will be described in more detail with reference to examples.

Example 1 Cellobiose 0.5%, Peptone 0.5%, Yeast extract 0.5
%, KH ₂ PO ₄ 0.3%, K ₂ HPO ₄ 0.1%, MgSO ₄ 0.02% and 3 l of medium consisting of tap water are put into a 5 l jar fermenter,
After sterilization by a conventional method, the pH was adjusted to 6.0, and Bacillus stearothermophilus TB-636 (FERM P-10523) was inoculated as an inoculum, and incubated at 50 ° C, 500 rpm, and aeration rate of 1 / l. Aeration and agitation culture was performed for a period of time.

After completion of the cultivation, β-glucosidase activity was measured for the supernatant liquid from which the cells were removed by centrifugation.
t / ml.

Ammonium sulfate was gradually added to the supernatant to achieve 60% saturation, salting out was performed, and a small amount of precipitate was obtained by centrifugation.
This precipitate is washed with 20 mM Tris-HCl (pH 7.0) containing 1.5 M ammonium sulfate.
It was redissolved in a buffer and then applied to a Phenyl Toyopearl column which had been equilibrated with the same buffer. Subsequently, gradient elution was performed using the same buffer and a buffer not containing ammonium sulfate, and a β-glucosidase active fraction was collected. This active fraction was dialyzed according to a conventional method, and further 10 mM Trial
DEAE-Toyopearl equilibrated with s-HCl buffer (pH 8.0)
, And gradient elution is performed using the same buffer and the same buffer containing 1 M NaCl.
-The glucosidase active fraction was collected. And 0.2M NaC
l equilibrated with 10 mM Tris-HCl buffer (pH 8.0)
The previously active fraction was added to a column of opearl HW-55 superfine, and gel filtration chromatography was performed. The active fractions were collected to obtain a purified preparation. This purified sample was confirmed as a single substance by electrophoresis, and as a result of measurement by the enzyme activity measurement method described above, a result of 3.6 unit / mg (protein) was obtained. The amount of protein was determined by the method of Lowry et al. (J. Biol. Chem., 193 ,
265 (1951)). The yield of the purified enzyme was 13% assuming that the total enzyme activity in the culture supernatant was 100.

[Effects of the Invention] The β-glucosidase of the present invention has an optimum pH of pH 5 to 8, so it works well even on the alkaline side, and has properties such as excellent stability because it is derived from a thermophilic microorganism. . Further, since the source of the present enzyme is an aerobic thermophilic bacterium, deep submerged culture, which is one of aeration and stirring cultures, can be adopted, and the production method can be easily scaled up and can be produced efficiently in a short time.

Therefore, the β-glucosidase of the present invention is useful for various industrial uses and the like, and can be effectively used, for example, for enzymatic saccharification of a cellulose material that has been subjected to alkali pretreatment.

[Brief description of the drawings]

FIG. 1 shows the optimum pH of the β-glucosidase of the present invention.
The figure shows the stable pH range of the β-glucosidase, FIG. 3 shows the optimal temperature of the β-glucosidase, and FIG. 4 shows the β-glucosidase.
3 shows a stable temperature range of glucosidase.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int. Cl. ⁶ , DB name) C12N 9/42 BIOSIS (DIALOG) WPI / L (QUESTEL)

Claims (3)

(57) [Claims] 1. A β-glucosidase having the following physicochemical properties. 1) Action Hydrolyze cellooligosaccharides to produce glucose. 2) Working pH and optimum pH The working pH is 3.5-10 and the optimum pH is 5-8. 3) Stable pH The stable pH when left at 37 ° C for 1 hour is 5-10. 4) Working temperature range and optimum temperature It works in a wide temperature range of 20 to 80 ° C, and has an optimum temperature around 55 ° C. 5) Thermal stability No inactivation when kept at 37 ° C for 24 hours, and no inactivation even when kept at 50 ° C for 1 hour. 6) Molecular weight (gel filtration method) About 74,000 2. A microorganism capable of producing β-glucosidase having the enzymatic property according to claim 1 which belongs to the genus thermophilic Bacillus and cultured in a nutrient medium, and the β-glucosidase is produced in the culture. A method for producing β-glucosidase, which comprises collecting the same. 3. The method according to claim 2, wherein the microorganism belonging to the thermophilic genus Bacillus and capable of producing β-glucosidase is Bacillus stearothermophilus TB-636 (FERM P-10523).