JPH05227957A - Purification of cellulase and production of cellobiose - Google Patents

Abstract

PURPOSE:To obtain pure cellulase giving cellobiose in a high yield by applying a specific treatment to crude cellulase to readily remove beta-glucosidase from the cellulase without employing an effective separative extraction agent. CONSTITUTION:Cellulose derivatives, a cellulose ester and/or a cellulose ether ester (preferably hydroxypropylmethylcellulose phthalate, etc.,) and cellulase are dissolved in an aqueous medium, preferably an aqueous medium having a pH of 5-7, and the solution is then held for a constant time, preferably at 20-30 deg.C for 1-3hr. The pH of the solution is changed preferably into a pH of 3-4 to form the cellulose derivative in the solution into a solid fraction. Finally, the solid fraction adsorbing the purified enzyme component is separated from the solution containing the beta-glucosidase.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for purifying cellulase and a method for producing cellobiose. Specifically, the present invention relates to a method for removing β-glucosidase from cellulase using a cellulose derivative and a method for producing cellobiose using the enzyme purified by this method. Cellobiose is a disaccharide in which D-glucoses are bound to each other by β, 1-4, and naturally exists in pine needles, corn stalks and the like. Cellobiose is an oligosaccharide with a sweet taste, but it is not decomposed in the human body. Therefore, if it is possible to economically produce cellobiose, it is considered to be effective as a health food, a diet food, or a sweetener for diabetic patients who are said to have a modern disease.

[0002]

2. Description of the Related Art Conventionally, cellobiose has been produced by oxidatively decomposing cellulose with dilute hydrochloric acid or the like to saccharify it and then purifying it by a separation method such as gel chromatography. The saccharification rate by this oxidative decomposition method is relatively fast, but it requires high pressure and acid resistance equipment.
There are drawbacks such as a decrease in cellobiose yield due to secondary decomposition.

On the other hand, according to the method using an enzyme,
Although the saccharification rate is relatively slow, it can be carried out under mild conditions, which is advantageous in terms of equipment and has the advantage that the enzyme that serves as a catalyst can be recovered and reused. However, the commercially available cellulase preparations are complex enzyme systems and are roughly classified into 3
Endo-β-1,4-glucanase, which randomly cleaves different types of enzyme components, namely, cellulose molecules with low crystallinity to give cellulose with a lower degree of polymerization, and exo that sequentially decomposes cellulose molecules from their non-reducing ends into cellobiose units. It is known that -β-1,4-glucanase and β-glucosidase that further hydrolyze cellobiose into glucose are contained (Z. Physi.
ol. Chem. , 78 , 266 (1912), J. Am. B
actiol. , 59 , 485 (1950), J. Am.
Am. Chem. Soc. , 33 , 181 (197)
9). In particular, β-glucosidase decomposes cellobiose in a high yield, so that only a small amount of cellobiose is produced.

Therefore, when cellobiose is produced by the enzymatic method, β-glucosidase is removed from the components of cellulase, or only β-glucosidase is selectively inhibited and inactivated, and then, it is allowed to act on cellulose. Is considered to be effective. However, a method for inhibiting and inactivating only β-glucosidase has not been reported so far.

Regarding the method of separating β-glucosidase, a method of separating it by chromatography using an ion exchange resin or the like is reported (J. Appl. Bi.
ochem. , 6 , 336 (1984), Eur. J.
Biochem. , 146 , 301 (1985), Miyadai Agricultural Bulletin 36 , 271 (1989), Agric. Bio
l. Chem. , 54 , 301 (1990)). However, a separating and extracting agent such as an ion-exchange resin is expensive and complicated in operation, so that it is not practical and a method for economically removing β-glucosidase is not known.

[0006] Further, endo-β-1,4-glucanase and exo-β-1,4-glucanase are adsorbed on cellulose due to their specific adsorptivity for the substrate of the enzyme, but β-glucosidase is difficult to adsorb. Is widely known (Biotechnol. Bioen.
g. , 26 . , 488 (1984), Biotechn.
ol. Bioeng. , 24 , 2383 (1983),
Biotechnol. Bioeng. , 28 , 172
7 (1986), Appl. Microbiol. Bi
otechnol. , 25 , 256 (1986), Bi
otechnol. Bioeng. , 30 , 251 (1
987)) and JP-A-63-226294 reported a method for producing cellobiose utilizing this property. However, in this case, as the β-glucosidase separating agent, since the insoluble cellulose that directly serves as the substrate is used, the separation carrier itself is hydrolyzed, and if repeated reuse as an immobilized enzyme is considered, it is stable. There is a problem of lack of sex. Further, regarding the adsorptivity of various enzymes to cellulose derivatives such as cellulose ester or cellulose ether ester, JP-A-48-286.
No. 86, but nothing is mentioned about selective adsorption of cellulase components.

[0007]

In view of the present state of the art as described above, a purification method for removing β-glucosidase from the components of cellulase by utilizing the specific adsorptivity of the enzyme to the cellulose derivative, and the purification thereof. There is room for development of a method for producing cellobiose using an enzyme.

Therefore, the present invention is a cellulose ester or a cellulose ether ester which is soluble in the optimum pH range of cellulase enzyme action, but is insoluble outside the optimum pH range and in the pH range in which the enzyme is not deactivated. Selective β using the specific adsorption of enzyme to
-The purpose of the present invention is to find a method for purifying cellulase which removes glucosidase, and to efficiently produce cellobiose using the purified enzyme component obtained by this method.

[0009]

Means for Solving the Problems The present inventor has confirmed that an alkali-soluble cellulose derivative having a carboxyl group as a free radical and a cellulase preparation can be used as a cellulose derivative within the optimum pH range of the enzyme. Add to an aqueous medium of pH condition to dissolve, mix well, change pH to acid side within the range where enzyme is not inactivated, insolubilize the cellulose derivative, and then use supernatant by centrifugation or filtration. It was found that β-glucosidase can be selectively removed by removing the liquid. In addition, the insolubilized product on which the purified enzyme component is adsorbed and the cellulose are treated with the optimal p of the enzyme.
It has been found that cellobiose can be efficiently produced by reacting in an aqueous medium in a pH condition where the insoluble matter is solubilized within the H range.

That is, in the present invention, either or both of cellulose ester or cellulose ether ester and cellulase are dissolved in an aqueous medium to form a solution, the solution is kept warm for a certain period of time, and then the pH of the solution is changed,
Producing an insolubilized solid fraction, after which by separating the solid fraction and the solution, a method for purifying cellulase, characterized in that β-glucosidase contained in cellulase is selectively removed, and The solid fraction obtained by this method and cellulose are added to an aqueous medium to form a suspension, and the suspension is kept warm for a certain period of time. Then, cellobiose is produced and accumulated in the suspension to obtain cellobiose. Provided is a method for producing cellobiose, which comprises collecting the cellobiose.

The cellulose derivative or cellulose ether ester which is a cellulose derivative used in the method of the present invention has a free carboxylic acid content of 0.05-0.5 mol, preferably 0.3-, per anhydrous glucose unit.
A pH-responsive soluble insoluble reversible polymer that contains 0.4 mol and reversibly loses water solubility under pH conditions of pH 4.5 or less, that is, hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, cellulose acetate phthalate, etc. It can be illustrated.

The cellulose used in the method of the present invention is obtained from the woody parts of hardwoods and conifers,
Other examples include those obtained from straw, which is an agricultural waste, sugarcane, which is a monosaccharide-based raw material, cotton, which is a hydrocarbon plant, and giant kelp, which is an aqueous plant.

The enzyme used in the method of the present invention, that is, cellulase, is obtained from filamentous fungi such as Trichoderma spp., Asbergillus spp., Penicillium spp., Pericularia spp., Acremonium spp. And Humicola spp.
Examples thereof include those obtained from aerobic bacteria such as Bacillus and Cellulomonas, anaerobic bacteria such as Clostridium and Luminococcus, and actinomycetes such as Streptomyces.

As a method for purifying cellulase in the present invention, 1 to 5% by weight of a cellulose derivative in an aqueous medium and 0.5 to 2.5% by weight of a cellulase in an aqueous medium are used. Of pH 5-9, preferably p
Dissolved in an aqueous medium of H5-7, at 5-60 ° C, preferably 20-30 ° C for 0.1-10 hours, preferably 1-
After incubating for 3 hours, the pH in the solution is changed to pH 2 to 4, preferably pH 3 to 4, and the insolubilized solid fraction is separated by a means such as centrifugation or filtration to collect the solid fraction. By performing the above operation, 90% by weight or more of β-glucosidase in the cellulase component can be removed. That is, the purified enzyme component of cellulase from which β-glucosidase has been removed is bound to the insolubilized cellulose derivative as a solid carrier and is carried in the solid fraction.

As the method for producing cellobiose in the present invention, the solid fraction obtained by the above cellulase purification method is 0.1 to 2% by weight with respect to the aqueous medium, and the cellulose powder is 1 to 20 with respect to the aqueous medium. Wt%, suspended in an aqueous medium of pH 5 to 7, preferably pH 5 to 6 so that the ratio of the solid fraction to cellulose is 0.05 to 0.2, and after incubating at 20 to 60 ° C. for a certain period of time, The solid fraction and the solution are separated by centrifugation, filtration or the like, and the solution fraction is collected to obtain a cellobiose solution. The cellobiose content in the total sugar produced in this solution is 70% by weight or more. The heat retention time is preferably 1 hour or more, but there is no particular upper limit. Further, as a method for recovering the enzyme when producing cellobiose by repeatedly reusing the enzyme, the solution fraction obtained by the above cellobiose production method is changed to pH 2 to 4, preferably pH 3 to 4, The cellulose derivative is insolubilized, and the solid fraction is collected by centrifugation, filtration or the like. By using this recovery method, continuous production of cellobiose becomes possible.

The aqueous medium used in the method of the present invention includes:
Examples include water, salt solutions such as NaCl and KCl, and buffer solutions containing phosphoric acid, acetic acid, citric acid and the like. The concentration of solutes such as salts or acids in the aqueous medium is 0.0
It is preferably 1M to 1M.

[0017]

[Operation] According to the present invention, by using an alkali-soluble cellulose ester or cellulose ether ester having a free carboxylic acid, the activity ratio contained in the components of cellulase is 90% or more. The β-glucosidase can be easily removed without using an expensive separating and extracting agent such as an ion-exchange resin by a slight pH change that does not inactivate the enzyme activity. Therefore, the complicated refining process is simplified, and the time required for the refining process is less than half that of the conventional one. In addition, cellobiose can be produced in a high yield of 70% by weight or more by hydrolyzing cellulose using a purified cellulase component from which β-glucosidase has been removed. The purified enzyme component in the solid fraction can be extracted from the solid fraction and used by a means known to those skilled in the art.

When the purified cellulase component is used as the immobilized enzyme in a state of being adsorbed on the cellulose derivative, the cellulose derivative serving as the immobilization carrier is stable without being decomposed by the cellulase, and the cellulose derivative is soluble in water. The enzyme reaction is carried out in this state. For this reason, the diffusion of the substrate into the carrier, which is a drawback of the immobilization method in which an enzyme is entrapped in an insoluble carrier (polyacrylamide, polystyrene, ion exchange resin), which has been widely used in recent years, is rate-determining and the apparent activity is remarkably increased. The problem of decrease (Acta. Chem. Scand., 25 , 1129).
(1971), Biochem. J. , 119 , 447
(1970), Biochem. Biophys. Re
s. Commun. , 44 , 426 (1971)). Furthermore, since the enzyme can be reused repeatedly, a sufficient economic effect can be obtained compared to the conventional acid decomposition method, and cellobiose can be used for industrial purposes as a sweetener for health foods, diet foods, or diabetics. Leads to expansion.

[0019]

EXAMPLES Example 1 Hydroxypropyl methylcellulose acetate succinate (A coat AS-LF, manufactured by Shin-Etsu Chemical Co., Ltd.) 2
and 10 g of cellulase preparation of Trichoderma origin (Meiselase CEP-6130, manufactured by Meiji Seika Co., Ltd.)
Dissolve in 0 ml of 0.1 M acetate buffer pH 6.0,
After keeping the temperature at 0 ° C. for 2 hours, the pH of the solution was adjusted to pH 3.5 to make the cellulose derivative insoluble. afterwards,
The solid fraction and the solution were centrifuged to collect the solid fraction. The residual activity ratio of β-glucosidase contained in the solid fraction was 7.2%, and 90% or more of β-glucosidase could be removed. Then, 1 g of the solid fraction and 10 g of commercially available cellulose powder were added to 100 ml of the same buffer p.
It was suspended in H5.0 and kept at 30 ° C. for 10 hours. As a comparative example, 0.5 g of a cellulase preparation that does not purify cellulase using a cellulose derivative and 10 g of cellulose powder are suspended in 100 ml of the same buffer pH 5.0,
The temperature was kept at 30 ° C. for 10 hours, and the cellobiose content in each obtained saccharified solution was quantified.

The results are shown in Table 1.

[Table 1]

In this example and the following examples, high performance liquid chromatography was used to quantify cellobiose. That is, Shim-Pack CLC
-NH ₂ eluted with (6.0 mm × 15cm) column, it was quantitated using a differential refractometer. In addition, β-glucosidase activity can be measured by p-nitrophenyl-β-
P-Nitrophenol produced using glucopyranoside as a substrate was converted into O. D. This was done by measuring the absorbance at 420. The total reducing sugar produced is quantified by the Somogene Nelson method (J. Biol. Chem., 195 , 19).
(1952)). D. The absorbance at 660 was measured.

Example 2 Cellulase was purified under the same conditions as in Example 1. afterwards,
The pH of each saccharified solution obtained by performing the saccharification reaction
Was adjusted to pH 3.5, the cellulose derivative was insolubilized, and the solid fraction and the solution were centrifuged to collect the solid fraction. Then, the saccharification reaction was repeated again under the same conditions as in Example 1 to quantify the cellobiose content.

The results are shown in Table 2.

[Table 2]

Example 3 Hydroxypropyl methylcellulose phthalate (HP
2 g of MC HP-55, manufactured by Shin-Etsu Chemical Co., Ltd.) was used to purify 1 g of macerase under the same conditions as in Example 1. The β-glucosidase residual activity ratio in the purified enzyme was 7.0%. Then, the saccharification reaction was performed under the same conditions as in Example 1, the purified enzyme was further recovered under the same conditions as in Example 2, and the saccharification reaction was repeated to quantify the cellobiose content.

The results are shown in Table 3.

[Table 3]

[0026]

INDUSTRIAL APPLICABILITY According to the present invention, β-glucosidase contained in a cellulase component can be easily removed by slightly changing the pH of the solution without using an expensive separating and extracting agent. .. Also, by hydrolyzing cellulose with the enzyme component from which β-glucosidase has been removed as a catalyst, cellobiose can be produced in a high yield, and when it is used as an immobilized enzyme, the enzyme can be repeatedly reused. Compared with the oxidative decomposition method, which is the manufacturing method of (3), is more advantageous in terms of cost and operation, and leads to the expansion of use in industrial applications.

Cellulose, which is a raw material, occupies most of the biomass (wood, manure, agricultural waste, seaweed, etc.) produced throughout the earth, and the present invention is also effective in terms of effective utilization thereof. It is effective.

Claims (7)

[Claims] 1. Either or both of cellulose ester or cellulose ether ester, which is a derivative of cellulose, and cellulase are dissolved in an aqueous medium to form a solution, the solution is kept warm for a certain period of time, and then the pH of the solution is changed. , Producing an insolubilized solid fraction, after which
A method for purifying cellulase, which comprises selectively removing β-glycosidase contained in cellulase by separating the solid fraction from the solution. 2. Cellulose ester or cellulose ether ester which is a derivative of cellulose contains 0.05 to 0.5 mol of free carboxylic acid per anhydroglucose unit and loses water solubility reversibly under pH condition of pH 4.5 or less. The method according to claim 1, which is a pH-responsive soluble insoluble reversible polymer. 3. The cellulose ester or cellulose ether ester which is a derivative of cellulose is added in an amount of 1 to 5% by weight based on the aqueous medium, and the amount of cellulase added is 0.5 to 2.5% by weight based on the aqueous medium. %, And the ratio of cellulase to the cellulose derivative is 0.3 to
The method according to claim 1 or 2, which is 0.7. 4. The pH for keeping a solution of the cellulose derivative and cellulase in an aqueous medium at pH 5 to 9, the temperature at 5 to 60 ° C., and the keeping time for 0.1 to 10 hours. The method according to any one of 1 to 3. 5. The solid fraction obtained by the method according to any one of claims 1 to 4 and cellulose are added to an aqueous medium to form a suspension, and the suspension is kept warm for a certain period of time. And then producing and accumulating cellobiose in the suspension and collecting cellobiose, the method for producing cellobiose. 6. The cellulose is added in an amount of 1 to 20% by weight based on the aqueous medium, and the solid fraction is added in an amount of 0.1 to 2% by weight based on the aqueous medium. The method according to claim 5, wherein the ratio is 0.05 to 0.2. 7. The pH for keeping the suspension warm is pH 5 to 5.
7. The method according to claim 5 or 6, wherein the temperature is 20 to 60 ° C.