JPH0683674B2 - Cellobiose manufacturing method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention is directed to a case where a cellulase-containing enzyme agent is allowed to act on insoluble cellulose and a cellulose-containing substance for saccharification, and after a short reaction, the The present invention relates to a method for producing cellobiose by washing solids and then reacting only solid fractions.

In recent years, the obesity population has been increasing due to the improvement of eating habits, which causes various diseases, and the number of those suffering from the diseases is also increasing. For this reason, people's dietary habits are changing, and the demand for diet foods is rapidly increasing in accordance with health consciousness, and the development of technology for stable supply of the ingredients is in progress. Cellobiose produced in the present invention is a disaccharide composed only of glucose, is a natural product existing in corn stalk, and has no problem in stability as a food, and is normally digested in the body. It has a function as a diet food because it is hard to be eaten. Therefore, cellobiose can be used as a food material. On the other hand, the raw material cellulose is the most mass-produced biomass on earth,
Most of vegetable foods such as vegetables, fruits, and grains are contained. On the other hand, unused cellulose as agricultural waste and forest waste also occupies a considerable part. Therefore, the present invention is extremely important industrially in terms of effective utilization of these biomasses.

<Prior Art> Cellobiose is industrially produced by chemically decomposing cellulose. On the other hand, cellobiose production by enzymatic decomposition of cellulose is also generally known, but mainly glucose is produced by the decomposition of cellulose by a commercially available cellulase preparation, for example, a cellulase produced by Trichoderma or Aspergillus, and all of the cellobiose is produced. The ratio to the produced sugar is about 15% or less.

<Problems to be Solved by the Invention> Cellulase is a complex enzyme system, and at least three kinds of enzymes, namely C ₁ , C _X and β-glucosidase, are involved in the hydrolysis of cellulose to glucose by cellulase. ing. Of these enzymes, C ₁ and C _X break down cellulose into cellobiose. Another enzyme, β-glucosidase, decomposes cellobiose and converts it into glucose. Thus inhibiting β-glucosidase,
It should be possible to produce only cellobiose from cellulose by deactivating or removing it. However, a method for selectively inhibiting and inactivating only β-glucosidase has not been known so far. A method using an ion exchange resin is known as a method for separating β-glucosidase, but the operation is complicated. That is, there is no known method for inexpensively and industrially removing β-glucosidase. It is also known that C ₁ and C _X are adsorbed on cellulose, but β-glucosidase is difficult to be adsorbed, but a method for producing cellobiose utilizing this property is not known.

<Means for Solving Problems> The inventors of the present invention have conducted extensive studies to solve such problems, and as a result, have found that the difference in the cellulose adsorption force between C ₁ , C _X and β-glucosidase of the cellulase complex enzyme system. It was found that a cellulosic acid as a main component and a cellulose decomposition product can be obtained by utilizing the above, and the present invention has been completed. That is, the present invention, after incubating the insoluble cellulose or cellulose-containing substance and cellulase in an aqueous medium, the solid fraction is separated,
A method for producing cellobiose, comprising adding an aqueous solution to the solid fraction, keeping it warm for a certain period of time to allow cellobiose to be produced and accumulated in the aqueous solution, and collecting the cellobiose.

As the insoluble cellulose and the insoluble content cellulose used in the method of the present invention, for example, chips obtained from coniferous hardwood, sawdust, all wood materials such as waste wood, and agricultural by-products such as rice straw, fir, sugarcane, corn, etc. are discarded. And pulps and powders thereof, which have been delignified.

Examples of the enzyme used in the method of the present invention, that is, cellulase include, for example, Sporotrichum, Trichoderma
Cellulase produced by bacteria such as Aspergillus spp. And Irpex spp. Further, in the present invention, these enzymes may have different origins individually or may be used in combination of two or more kinds.
As the enzyme source, a cellulase preparation which is generally commercially available, a culture solution of the above-mentioned bacterium, or a persolution thereof can be directly used.

As the β-glucosidase removal method in the method of the present invention, cellulase and an excess amount of insoluble cellulose are adjusted to pH 2 to 9,
After incubating at 0 to 60 ° C, preferably 30 to 50 ° C for 1 to 4 hours in an aqueous medium having a pH of 3 to 7, the solid fraction and the solution fraction are separated by means such as a hyper-centrifugation method. . Since a small amount of β-glucosidase remains in this solid fraction, in order to remove it as much as possible, this solid fraction was adjusted to pH 2 to 1
Wash with an aqueous solution of 0, preferably pH 3-7.
By this operation, 95% or more of β-gl in the cellulase preparation was
Can remove ucosidase.

As a method for producing cellobiose in the method of the present invention, the solid fraction after washing is suspended in an aqueous solution having a pH of 2 to 10, preferably a pH of 3 to 7 and heated at 20 to 60 ° C. without newly adding cellulase. To do. After a certain period of time, the solid fraction and the solution are separated again by a hyper-centrifugation method or the like, and the solid fraction is suspended again in an aqueous solution and kept warm. This operation is repeated and the resulting solution fractions are collected to obtain a cellobiose forming solution. The content of cellobiose in the total saccharides produced in this solution is 80% or more. In this operation, the concentration of insoluble cellulose decreases as the saccharification proceeds, so that the unadsorbed portion of cellulase increases and the cellulase gradually migrates to the saccharified solution. The enzyme in this solution can be recovered by newly adding cellulose after the reaction and before separating the solid fraction and the solution. In addition, cellobiose can be continuously produced by using a membrane-attached reaction tank.

The aqueous medium and the aqueous solution used in the present invention include water and NaC.
Examples thereof include salt solutions of l, KCl, (NH ₄ ) ₂ SO ₄ , Na ₂ CO ₃ , and the like, and buffer solutions containing phosphoric acid, acetic acid, citric acid, glycine, and the like. The concentration of these is 0 to 0.5M, preferably 0 to 0.1M.

As a method of recovering cellobiose in the method of the present invention,
As the simplest operation, the cellobiose-producing solution can be separated by cooling or freezing to 0 ° C. or lower to insolubilize the cellobiose. When a large amount of impurities are present, it can be desalted with an ion exchange resin or the like, the polymer substance is removed by an ultrafiltration method, and after concentration, an organic solvent such as acetone is added to insolubilize cellobiose to separate it.

The detection of saccharides in the present invention uses thin layer chromatography, that is, a saccharide solution is spotted on a silica gel thin layer as a solvent (n-butanol: acetic acid: ether: water = 9: 6: 3:
After development in 1), the color was developed using a naphthresorcin-phosphate reagent. High-performance liquid chromatography was used to quantify cellobiose. That is, it was performed using a Showdex Ionpack S-801 (φ8 mm × 50 cm) column, eluting with water at 40 ° C., and using a differential refractometer. The β-glucosidase activity was measured by measuring the absorbance at 400 nm of p-nitrophenol produced using p-nitrophenyl-β-glucoside as a substrate.

This will be described in detail in the following examples.

Example A liquid medium having the following composition was prepared, dispensed into a 500 ml Sakaguchi flask in an amount of 75 ml, and sterilized by heating at 120 ° C. for 10 minutes.

Sporotrichum cellulofilm ATCC 20494 in this medium
And shake culture at 48 ° C for 72 hours, separate the culture broth and use an ultrapermeabilizer (MW = 6000 cut) for about 20
It was concentrated twice and lyophilized to obtain a crude enzyme powder of cellulase.

Next, 0.5 g of this cellulase powder and 5 g of cellulose powder (Whatman CC41) were suspended in 100 ml of 0.1 M acetate buffer pH 5.0.
After incubating at 50 ° C for 2 hours, the solid fraction and the solution were separated by centrifugation, the solid fraction was washed 3 times with 50 ml of the same buffer, and then suspended again in 100 ml of the same buffer at 50 ° C for 16 hours. Kept warm. As a control experiment, cellulase and cellulose were directly incubated for 16 hours without separating the solid fraction, and cellobiose and glucose in the obtained saccharified solutions were quantified. The results are shown in the table below.

Example 2 20 g of cellulose powder (Whatman CC41) and 2 g of the above cellulase were suspended in 400 ml of 0.1 M acetate buffer pH 5.0.
Incubate for 2 hours in the same manner as above and wash the solid fraction.
I kept it warm for an hour. 2 more steps of this 16-hour incubation
Repeated three times (total three times), a total volume of 1160 ml of saccharified solution was obtained. This saccharified liquid (6.2 g of cellobiose and 0.75 g of glucose)
Amber Light IRA-410 and Amber Light I
Desalted with RA 120B, the polymer components were removed by ultrafiltration (molecular weight cut to 5000) and concentrated to 40 ml. Acetone (30 ml) was added thereto, and the mixture was allowed to stand at 4 to 7 ° C. and the precipitated crystals were collected by filtration and vacuum dried to obtain 4.1 g of white powder. No glucose was detected in this preparation.

Example 3 0.5 g of a commercially available cellulase (Meisserase P-1) derived from Trichoderma konnigii was added to cellulose powder (Whatman CC).
41) 5 g was allowed to act under the same conditions as in Example 1. The results are shown in the table below.

Example 4 Saccharification was carried out under the same conditions as in Example 1 using various commercially available cellulose powders and celluloses of Sporotrichum cellulofilm ATCC 20494. The results are shown in the table below.

<Effects of the Invention> As described in detail above, the method of the present invention makes it possible to selectively and efficiently produce cellobiose over insoluble cellulose. This suggests the possibility that cellobiose can be used as a new food material, and is extremely important industrially.

Claims (1)

[Claims] 1. Insoluble cellulose or a substance containing insoluble cellulose and cellulase in an aqueous medium at 30 to 60 ° C.
After incubating for a period of time, the solid fraction in the aqueous medium is separated before completion of the degradative saccharification reaction, an aqueous solution is added to the solid fraction without adding a new substrate, and the mixture is incubated for a certain period of time and cellobiose is added to the aqueous solution. A method for producing cellobiose, which comprises collecting and producing cellobiose.