JPH02222694A - Enzymatic hydrolysis of cellulose-containing material - Google Patents

Abstract

PURPOSE:To obtain glucose or oligosaccharides thereof in a high yield by acting a cellulase on a cellulose-containing material in a specified method and hydrolyzing the subject material at a high speed without requirement of a complicated pretreatment such as a blasting process. CONSTITUTION:A cellulose-containing material is initially dispersed into a saturated hydrocarbon (preferably hexane) and a surfactant (preferably dodecyl sulfate, etc.) is added thereto at the same time. To the resultant dispersion of the cellulose-containing material in the saturated hydrocarbon, an aqueous cellulase (preferably Aspergillus niger-derived cellulase) solution is then added for emulsification, thus hydrolyzing the above-mentioned cellulose-containing material. In addition, 22-55 deg.C is recommended as the reaction temperature in the hydrolysis.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention involves dispersing a cellulose-containing material in a saturated hydrocarbon liquid containing a surfactant, and adding water and a cellulose hydrolase to the liquid. The present invention relates to an enzymatic hydrolysis method comprising emulsifying a cellulose-containing material and an enzyme as a core.

[Prior Art] Many attempts have been made to hydrolyze cellulose-containing materials into glucose or its oligosaccharides using cellulose hydrolase. However, cellulose-containing materials are sparsely soluble substrates, and in order to hydrolyze them with cellulose hydrolases, complex pretreatments such as blasting or steaming are required, and the rate of hydrolysis is slow, resulting in the production of reducing sugars. The yield is low, and a practical hydrolysis method using enzymes has not yet been proposed.

[Problems to be Solved by the Invention] The present invention provides cellulose that does not require complicated pretreatment such as blasting treatment, has a high hydrolysis rate, and can obtain glucose or its oligosaccharides in high yield from a cellulose-containing material. A method for enzymatic hydrolysis of contained materials is provided.

Furthermore, the present invention provides a method for hydrolyzing a cellulose-containing material using an enzyme, which contains reducing sugar at a high concentration and which does not cause contamination by bacteria even at a high concentration.

CT! Means for solving JAR] The present invention involves adding a cellulose hydrolase dissolved in water to a saturated hydrocarbon solution containing a surfactant in which a cellulose-containing material is dispersed, and then emulsifying the solution. Provided is a method for enzymatic hydrolysis of cellulose-containing materials comprising:

Cellulose-containing materials used in the present invention include various valves derived from various plants such as wood, herbs, algae, etc., such as ground wood pulp, sulfite valves, sulfate valves, and paper such as newspaper, medium-weight paper, Examples include various materials such as high-quality paper and P paper. These materials have appropriate dimensions,
For example, it is preferable to use it by pulverizing it to about 20 to 40 meshes or even finer sizes, since this can improve the hydrolysis rate and reducing sugar yield.

The surfactants used in the present invention include anionic surfactants, cationic surfactants, nonionic surfactants,
Any amphoteric surfactant can be applied. Some examples of preferred surfactants are as follows.

Amine salts such as alkyl amine salts, amide bonded amine salts, ester bonded amine salts, alkylammonium salts, amide bonded ammonium salts, ester bonded ammonium salts,
Examples include cationic surfactants such as quaternary ammonium salts such as ether-bonded ammonium salts, pyridinium salts such as alkylpyridinium salts, amide-bonded pyridinium salts, ether-bonded pyridinium salts, and ester-bonded pyridinium salts; Specifically, n-decyltrimethylammonium bromide, dodecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, n-hexadecyltrimethylammonium bromide, tetradecyldimethylbenzylammonium chloride, cetyldimethylethylammonium bromide, bis(2-ethylhexyl) Examples include cationic surfactants such as dium sulfosuccinate. Examples of anionic surfactants include Vi oxidized oil, alkyl sulfates, alkyl sulfonates, alkylnaphthalene sulfonates, and more specifically dodecyl sulfate, 1-tridecane sulfate, tetradecane sulfate, 1-
Examples include sodium salts such as pentadecane sulfate, n-hexadecyl sulfate, and 1-octadecane sulfate. Nonionic surfactants include ethylene oxide type such as alkyl allyl ether type, alkyl ether type, and alkyl amine type, polyhydric alcohol fatty acid ester type such as fatty acid glycerin ester type, anhydrone rubitol fatty acid ester type, and polyethylene. Examples include imine type, fatty acid alkylolamide type, etc. 1 For example, sorbitan monobalmitate 1 Ampholytic surfactants include carboxyl group, sulfonic acid group, sulfate ester group in the anionic part, and amine group in the cationic part. Examples include those having the following.

The surfactant prevents enzyme deactivation and promotes emulsification of the enzyme and cellulose-containing material as a core. The amount of surfactant added is 0.
Amounts of 1 to 20 weight Ji/volume %, preferably 1 to 15 weight/volume 1%, more preferably 1 to 8 weight/volume % are preferably used.

The HLB of the surfactant is preferably selected appropriately depending on the type of surfactant. For example, for sorbitan monobalmitate, which is a nonionic surfactant, the optimal HLB is around 7.

The saturated hydrocarbon used in the present invention has 5 to 20 carbon atoms.
, more preferably 5 to 13, and various examples include hexane, heptane, octane, isooctane, dodecane, and tridecane. Since saturated hydrocarbons with a large number of carbon atoms tend to have lower reducing sugar yields than hexane and heptane, hexane can be exemplified as the most preferred saturated hydrocarbon. However, the method of the present invention is significantly affected by the treatment temperature, and is superior to enzymatic hydrolysis methods in aqueous systems at temperatures between 10°C and 58°C, preferably between 20°C and 55°C, and most preferably between 25°C and 55°C. One of its characteristics is that it exhibits reducing sugar yield. Therefore, when performing enzymatic hydrolysis at relatively high temperatures, it is preferable to use saturated hydrocarbons with a relatively large number of carbon atoms, such as dodecane and tridecane, taking into consideration the boiling point of saturated hydrocarbons. The reducing sugar yield in the permeate water system has relatively little difference depending on temperature, and the reducing sugar yield is significantly inferior to that of the method of the present invention.

The enzyme used in the present invention is cellulose hydrolase (hereinafter referred to as cellulase),
ger, Pencillus + funiculos
Cellulases derived from various producing countries can be used, such as us+ and Trichodermaviride. In particular, cellulase derived from Aspergillus niger is preferably used because it is inexpensive, easily available, and has a high hydrolytic ability.

The cellulase is dissolved in water and then added to the saturated hydrocarbon solution containing the cellulose-containing material.

The water content in the saturated hydrocarbon liquid is 16% by volume or less,
Preferably it is 10% by volume or less, most preferably 6% by volume or less. As the water content increases, the reducing sugar yield decreases,
When the amount exceeds 16% by volume, no significant increase in reducing sugar yield is observed compared to the aqueous reducing sugar yield.

A buffer solution may be used as the water for dissolving the cellulase. Examples of the buffer solution include a weakly basic solution such as tris(hydroxymethyl)aminomethane to which a strong acid is added, succinic acid, potassium citrate, and boron. Examples include various buffer solutions such as acid, a weakly acidic solution of phosphoric acid to which a strong alkaline dilute solution is added, an acetic acid-sodium acetate system, and a mixture of the above-mentioned buffers. The optimal pt-i determined by the above buffer solution in combination with various surfactants and water content.
By adjusting the amount, the yield of reducing sugar can be increased. The ionic strength of the buffer solution is preferably around 1M, but may be a very low value, and is not limited to this value.

Reducing sugar yield, optimal pH, optimal water content, and residual enzyme activity in water (containing buffer) system, hexane-water (containing M'S solution) system, and hexane-water (containing buffer)-surfactant system An example is as follows. The data below is based on a processing time of 200 hours.

Hana Wataru Nino Basket length submersion system -10% Hexane system 27% Surfactant system 5pan10 32%^OT
38% Tetramethylammonium chloride 30% Amhitol 86B 32% Tabi' 1 and Dan 4.0 5.0 5.0 5.0 6.8 6.8 Water-based hexane surfactant system 5pan-40 ＾OT Remaining Enzyme Shino Less than 10% on I-KX 88.4 3% 97.6 3% 100 , sorbitan monopalmitate sodium salt (I-ILB6.7), manufactured by Tokyo Kasei Kogyo Co., Ltd., nonionic surfactant, ^OT, trade name, bis(2-ethylhexyl) sodium sulfosuccinate, Wako Pure Chemical Industries, Ltd. cationic surfactant, Anjotoll 86B; trade name, CH.

C, H2□+ N’ CH2C0O-SCH.

(In the formula, n=14 is about 3% by weight, n-16 is about 17% by weight
, n = 18 is approximately 80% by weight), manufactured by Kao Corporation, amphoteric surfactant, measurement of residual enzyme activity: by spectrophotometer using varanitrophenyl glucoside, an analog substrate. The change in wavelength at 420 nm was measured.

[Example] The amount of reducing sugar produced in this example is So+wogy
Nelson method or high performance liquid chromatography (
Column: SI+odex Sugar St”101
0, column temperature = 70°C).

Example 1 Acetic acid-sodium acetate at ionic strength IM, pH 5.0!
! Cellulase derived from Aspgillus niger (manufactured by Sigma) was added at a rate of 0.13 g to 1 ml of the buffer solution, and dissolved with stirring to prepare an enzyme aqueous solution.

Bexa 7285 in an Erlenmeyer flask with a capacity of 500ml
Avicel (crystalline cellulose, manufactured by Sigma) Ig in m1
After adding and dispersing 17.6 g of surfactant AOT (trade name), 15 ml of the above enzyme aqueous solution was added,
The mixture was stirred at 600 rpm for a predetermined time. Stirring treatment for each predetermined time Stop stirring and remove 0.1 m of sample solution from the aqueous system.
1 was collected and subjected to analysis.

Fig. 1 shows the reducing sugar yield after each treatment time.

Comparative Example 1 15 ml of the same enzyme aqueous solution used in Example 1 was added to 285 ml of the W solution and dispersed in the same manner as in Example 1. A sample was taken and submitted for analysis.

Fig. 1 shows the reducing sugar yield after each treatment time.

Comparative Example 2 15 ml of the same enzyme aqueous solution used in Example 1 and 15 ml of the same buffer solution used in Example 1 were mixed with 27 ml of hexane.
In the same manner as in Example 1 except that 0ml was added and dispersed,
Samples were taken after each predetermined period of stirring and subjected to analysis.

Fig. 1 shows the reducing sugar yield after each treatment time.

The standardized reducing sugar concentration in FIG. 1 was calculated as follows in order to compare the concentrations of Avicel and cellulase in water at the same level.

Example 1 in which a surfactant was added had a water content of 5% by volume,
These measured values are shown as they are in the figure.

Since Comparative Example 1, which consists of only a buffer solution, has a water content of 100% by volume, the measured value is expressed as a value multiplied by 20 in order to keep the concentration of Avicel etc. at the same level.

Hexane-M? Comparative Example 2 consisting of II liquid system has a water capacity of 1
Since it was 0%, the measured value was doubled and displayed.

The yield of reducing sugars in hexane-surfactant-buffer is superior to that in hexane-buffer and M buffer systems. The concentration of reducing sugars is also extremely high according to the method of the present invention.

Example 2 In Example 1, surfactant AOT was replaced with surfactant 5p.
The reducing sugar yield was examined in the same manner as in Example 1 except that an40 was used.

Fig. 2 shows the reducing sugar yield after each treatment time.

The standardized concentrations in FIG. 2 were calculated as follows. Measured values of hexane-Mm liquid system (moisture 10% by volume) (Comparative Example 1
Measured value of
0%) (measured value of Comparative Example 2) was expressed as 10@.

According to the method of the present invention, both the reducing sugar yield and the reducing N concentration are lower than those of the hexane-buffer system and M? Superior to R liquid system.

Example 3 The reducing sugar yield was investigated in the same manner as in Example 1 except that the surfactant AOT was changed to tetramethylammonium chloride.

Fig. 3 shows the reducing sugar yield after each treatment time.

Examples 4-6 Hexane 15.8 in a Erlenmeyer flask with a capacity of 20 m1
ml, crush P paper and pass through a 20 mesh sieve,
After adding and dispersing 0.05 g of the V paper powder remaining on the 0 mesh sieve and the surfactant AOT, 0.8 ml of the same enzyme aqueous solution used in Example 1 was added, and the mixture was heated at 20°C and 60°C.
The mixture was stirred at 0 rpm for 166 hours. After the treatment, it was subjected to analysis to examine the yield of reducing sugar.

Tissue paper (Example 5) and newspaper (Example 6) were treated in the same manner as above, and the reducing sugar yield and standardized reducing sugar concentration were also examined.

The results are shown in Tables 1 and 2.

Comparative Examples 3-5 The same as that used in Example 1 [R liquid 15.8 ml was placed in an Erlenmeyer flask with a capacity of 20 ml, P paper (Comparative Example 3) crushed in the same manner as in Examples 4-6, and tissue paper were added. (Comparative Example 4) and newspaper (Comparative Example 5) were added and dispersed, and then 0.8 g of the same enzyme aqueous solution used in Example 1 was added.
ml and stirred at 20° C. and 600 rpm for 166 hours. After treatment, it was subjected to analysis to examine the yield of reducing sugar and the standardized concentration of reducing sugar.

The results are shown in Tables 1 and 2.

Comparative Examples 6-8 Hexane 15.0 in an Erlenmeyer flask with a capacity of 20 m1
ml of R paper crushed in the same manner as Examples 4 to 6 (Comparative Example 6
), tissue paper (Comparative Example 7) and newspaper (Comparative Example 8)
) were added and dispersed, then 0.8 ml of the same enzyme aqueous solution used in Example 1 and 0.8 ml of the same buffer solution used in Example 1 were added, and the mixture was heated at 20°C and 600 ml.
The mixture was stirred for 166 hours under the condition of rpm. After the treatment, it was subjected to analysis to examine the reducing sugar yield and standardized reducing sugar concentration.

The results are shown in Tables 1 and 2.

The standardized reducing sugar concentration is based on the reducing sugar concentration in hexane (water content 10% by volume), the reducing sugar concentration in the buffer solution (water content 100% by volume) is increased by 10 times, and the surfactant system (
The reducing sugar concentration in water (5% by volume) was multiplied by 1/2 and displayed, respectively.

Table 1 (Standardized reducing sugar concentration mg/di) Table 2 (Reducing sugar yield, weight %) ) - Riedi I Enotan" Gokerei Examples 4-6 Comparative Examples 3-5 N8 24.5 2.6 19.1 39.8 7.4 28.4 28.8 3.9 18.0 [Effects of the Invention] According to the present invention, a method for hydrolyzing a cellulose-containing material that does not require pretreatment such as blasting treatment or steaming treatment is provided.

Furthermore, according to the present invention, hexane-uj liquid system and Mf
A method for hydrolyzing a cellulose-containing material is provided that provides a reducing sugar yield that is extremely superior to that in an R liquid system.

According to the present invention, there is provided a method for enzymatic hydrolysis of a cellulose-containing material, which allows reducing sugars to be obtained from the cellulose-containing material at a high reduction rate and at a high concentration.

According to the present invention, enzymatic hydrolysis of a cellulose-containing material allows the cellulose-containing material to be saccharified without being contaminated by bacteria by enzymatically hydrolyzing the cellulose-containing material in a buffer system emulsified in a saturated hydrocarbon solution. A decomposition method is provided.

According to the present invention, a method for enzymatic hydrolysis of cellulose-containing materials is provided, which allows reducing sugars to be obtained in high yield over a wide temperature range.

[Brief explanation of the drawing]

FIG. 1 shows the yield and standardized concentration of reducing sugars obtained in Example 1 and Comparative Examples 1 and 2. Figure 2 shows the yield and standardized concentration of reducing sugar obtained in Example 2 and Comparative Examples 1 and 2. Figure 3 shows the yield and standardized concentration of reducing sugar obtained in Example 3. show. 1: Yield and standardized concentration of reducing sugar obtained in Example 1 2: Yield and standardized concentration of reducing sugar obtained in Comparative Example 1 3: Yield and standardized concentration of reducing sugar obtained in Comparative Example 2 Concentration 4: Yield and standardized concentration of reducing sugar obtained in Example 2

Claims (1)

[Claims] A method for enzymatic hydrolysis of a cellulose-containing material, which comprises adding a cellulose hydrolase dissolved in water to a saturated hydrocarbon solution containing a surfactant in which the cellulose-containing material is dispersed to form an emulsion.