JPS59213396A - Saccharification of cellulose - Google Patents

Abstract

PURPOSE:To enable the effective utilization of enzyme, by removing a sugar- containing liquid from the reaction mixture obtained by the saccharification reaction, and further carrying out the saccharification of cellulose using the residual solid obtained by the above process as a substitute for enzyme. CONSTITUTION:An aqueous solution (adjusted to about 3-8pH with an acidic buffer solution) containing insoluble cellulose (e.g. agricultural wastes such as wood chips, pulp, rice straw, etc.) is saccharified with an enzyme (e.g. cellulase prepared from the cells of microorganisms belonging to Aspergillus genus). After the completion of the decomposition and saccharification treatment, the sugar-containing liquid is removed from the reaction mixture, and the resultant insoluble component (containing adhered enzyme) is added with an aqueous buffer solution containing insoluble cellulose to effect the saccharification and decomposition of the cellulose without addition of additional enzyme.

Description

[Detailed Description of the Invention] The present invention provides a method for further saccharifying a cellulose-containing substance by using an enzyme attached to a solid substance remaining in the reaction mixture when enzymatically saccharifying a cellulose-containing substance. It is related to.

In recent years, from the viewpoint of depletion of petroleum resources and environmental problems, research on the saccharification of cellulosic substances has been actively conducted. The enzymatic agglomeration method, which does not further decompose the superproduced sugar, is attracting attention as an effective method.

In this enzymatic saccharification method, it is generally said that the cost of producing cellulases such as cellulases and hemicellulases used as enzymes accounts for about 50% of the total cost, so it is difficult to recover and reuse these expensive enzymes. This has become an extremely important issue.

However, although many proposals and implementations have been made regarding methods for recovering cellulases remaining in the solution obtained by centrifugation or other methods of centrifugation of the decomposed and saccharified solution of cellulosic substances, There is no known method for reusing cellulases remaining in the solid matter other than the method for recovering cellulases previously proposed by the present inventors. In addition, in conventional enzymatic saccharification methods for cellulosic substances, except for the method previously proposed by the inventors, the recovery rate of cellulases is only about 35-45%.

By the way, when the inventors measured the amount of cellulases remaining in the solid matter separated from the solution after decomposition and saccharification of cellulosic substances using a circular dichroism measuring device, they found that The amount of remaining cellulases is greater than the amount of cellulases remaining in the solution separated from the solution after completion of decomposition and saccharification, and the activity of cellulases remaining in this solid substance is It was found that, like the remaining cellulases, it was almost similar to the original ioo.

Therefore, the inventors conducted intensive research to develop an economical method for saccharifying cellulose by making effective use of the enzymes contained in this solid material. It has been discovered that the solid matter remaining after separating and removing the sugar-containing liquid from the liquid can be used in place of the IT fermentation to carry out an enzymatic reaction, and based on this knowledge, the invention has been made.

That is, the non-invention is that when saccharifying a raw material consisting of a water bath solution containing insoluble cellulose and an acidic buffer using an enzyme, the sugar-containing liquid is separated and removed from the reaction mixture after the completion of the separation treatment. The present invention provides a method for saccharifying cellulose, which is characterized in that raw materials are added to the remaining insoluble portion and decomposition and saccharification is carried out without newly supplying enzymes.

Insoluble cellulose used in the uninvented method includes:
Examples include all types of wood such as wood chips, sawdust, bark, and waste wood obtained from coniferous trees, broad-leaved trees, southern and northern woods, and rice straw and sugarcane, which require expense to dispose of as agricultural waste. Examples include waste materials such as corn, and paper materials such as newspaper and cardboard. However, water-insoluble polysaccharides and oligosaccharides that undergo the same enzymatic action as cellulose can also be used.

Enzymes, ie, cellulases, used in the uninvented method include, for example, cellulases produced from cells of the genus Aspergillus, or enzymes containing cellulases and hemicellulases. In addition, in the non-invention, these enzymes may be used singly or two or more of them may be used at the same time, and furthermore, a bacterial cell extract containing these enzymes or a bacterial cell extract containing these enzymes may be used as the enzyme source. A bacterial culture solution can also be used.

In the uninvented method, the decomposed saccharification-completed liquid is separated into a solution and a solid by means such as p-filtration or centrifugation, and then
The solid substance may be brought into contact with insoluble cellulose in an aqueous solution or a mild pH buffer solution, or, if desired, in order to simplify the operation, the residual liquid from which only sugars have been separated from the solution after decomposition and saccharification may be made into a solid substance. It is also possible to bring the enzyme into contact with newly supplied insoluble cellulose without separating the substance and the solution, and recycle the enzyme remaining in both the solution and the solid substance. In the former method of separating the solution and the solid, the enzyme contained in the solution is recovered by a commonly used method such as ultrapolar filtration. The recovery rate at this time is 90 to 100 times the amount remaining in the solution. In addition, the enzyme in the solid material transfers to the insoluble cellulose in the newly added raw material due to its affinity with the insoluble cellulose, and its activity is recycled by 65 to 100 percent.

The acidic buffer in the uninvented method is used to increase the stability of the enzyme during reuse, and the preferred pH range is 3.0 to 8.0. This buffer is preferably an acetate buffer.

In the uninvented method, cellulases remaining in the solid matter in the decomposed and saccharified solution, which has never been used, are mixed with insoluble cellulose in an aqueous solution and an acidic buffer at a temperature of room temperature to 55°C. 65 to 100 times the activity of cellulases remaining in the solid can be reused by an extremely simple operation.

Cellulases remaining in the solution of insoluble cellulose decomposition and saccharification are reduced to 90 to 10% of the remaining amount by a conventional method.
Although 0.00% can be recovered, this capacity is only 35 to 45% of the cellulases originally used. On the other hand, in the uninvented method, 55
The remaining cellulases in the solid matter equivalent to ~65% can be reused, with an activity of 65% to 100%. By applying the method, the cost can be significantly reduced.

Next, the invention will be explained in more detail with reference to Examples.

Example 1 100 ml of an acetate buffer solution with pH 4.5 was mixed with 4 g of ground red pine wood powder, and then commercially available Trichoderma
Cellulase originating from Viride (Cellulase Onozuka RIO
) After shaking 200 m1i+ at 7IO and 40°C for 24 hours, the reaction-completed liquid in which 40 parts of wood flour had been decomposed was separated into a bath liquid and 2.4 g of solid matter by centrifugation.

2.4 g of this solid was added to pH 4.5 acetic acid buffer with 55 ml of
1 (2.5 g of red pine wood flour was freshly added to the cutting edge and the reaction was carried out again at 40°C. The activity of cellulase in this reaction was determined by the amount of glucose produced per 1 mg of cellulase). 15.5 μmol, compared to 16.0 gmol in the original reaction, could be reused.

Example 2 A 100 mA water bath nutrient medium containing an appropriate amount of cellulose powder as a carbon source was placed in a soocc flask and sterilized.
were inoculated and cultured under aerobic conditions at a temperature of 30°C for 6 days. The pH of the culture solution was always adjusted to 5.4.

5. Finely ground beech wood powder with a particle size of 20-50μ in another 300CC flask. ! 9, and add the above culture solution 50-
(enzyme content: 12C1i) was added and decomposed at 40° C. for 48 hours to decompose the 56 fractions of wood flour, and the reaction-completed liquid was separated into a bath liquid and solid matter by centrifugation. Add 35 - of water to this solid substance, and then add 4 g of beech wood flour and add 4 g of
Decomposition was carried out at 0°C for 48 hours. As a result, 54 particles of wood powder were decomposed.

Example 3 Dry newspaper and use 2g as a square piece with 3 sides on 50
-, 100 my of cellulosin AO was added thereto and decomposition was carried out at 40°C for 24 hours, and the reaction-completed liquid was centrifuged +/C to separate IgM and 1.4 g of solid matter. 1.4 g of this solid was added to pH 5.0 acetate buffer 30
ml, 1.2 g of red pine wood powder was added, and the reaction was carried out at 40"C.The amount of glucose produced per hour was 7.5 μmol, compared to the original 11.3 μmol. Compared to the above, it was possible to utilize the 67th activity.

Example 4 1. 100 g of powder obtained by drying and pulverizing corn stems
m1! 100 mg of cellulase originating from Trichoderma viride (Cellulase Onodzuka RIO) and 100 mg cellulase originating from Aspergillus niger (Cellulozin AC) were added to the suspension at 35°C for 24 hours. After time resolution, the reaction solution was separated by centrifugation. 32g of separated solid was added to 50ml of 1
The suspension was suspended in 1H5,0 acetic acid and a protective buffer solution, and 20I of Japanese red pine wood flour was added thereto to carry out a reaction at 40°C. Cellulase] Tng/Glucose production amount per hour is 175μ
mole, and the activity recovery rate was 97% based on the initial value of 18.0 g mole.

Claims (1)

[Claims] 1. When saccharifying a raw material consisting of an aqueous suspension containing insoluble cellulose and an acidic buffer using an enzyme, the remaining sugar-containing liquid is separated and removed from the reaction mixture after completion of the decomposition and saccharification treatment A method for saccharification of cellulose, which is characterized by adding raw materials to the insoluble portion of cellulose and allowing decomposition and saccharification to occur without the need to newly supply enzymes. 2 Claim 1 in which the insoluble cellulose is wood
The method described in section. 6. The method according to claim 1, wherein the insoluble cellulose is pulp. 4. The method according to claim 1, wherein the insoluble cellulose is agricultural waste.