JPS63226294A - Production of cellobiose - Google Patents

Abstract

PURPOSE:To obtain cellobiose useful as a raw material for food, by saccharifying an insoluble cellulose and a cellulose-containing substance with a cellulase- containing enzyme agent, wherein a solid component is washed after reacting the above components for a short time and reacting only the solid fraction thereafter. CONSTITUTION:Cellulase produced by microorganisms of Sporotrichum genus, Trichoderma genus, Aspergillus genus, Irpex genus, etc., and excess amount of insoluble cellulose are heated in an aqueous medium of pH 2-9, preferably 3-7 at 0-60 deg.C, preferably 30-50 deg.C for 0.1-24hr, preferably 1-4hr and the solid fraction is separated from the solution fraction e.g. by centrifugal separation. After removing residual beta-glucosidase from the solid fraction e.g. by washing, the fraction is suspended in a water-soluble solution of pH 2-10, preferably 3-7 and heated at 20-60 deg.C without adding cellulase. After a prescribed period, the solid fraction is separated from the solution, suspended again in a water-soluble solution and heated. The operations are repeated to obtain cellobiose.

Description

Detailed Description of the Invention [Objective of the Invention] [Industrial Field of Application] The present invention provides a method for saccharifying insoluble cellulose and cellulose-containing substances by acting on them with a cellulase-containing fermentation agent. - It relates to a method for producing cellobiose by washing the carrier and solid matter and then reacting only the solid fraction.

In recent years, obesity has been on the rise due to improvements in dietary habits, and this has led to various diseases, and the number of people suffering from them has also increased. For this reason, people's eating habits are changing, and as people become more health-conscious, the demand for diet foods is rapidly increasing, and the development of technologies to stably supply these ingredients is progressing. Cellobiose produced in the present invention is a disaccharide composed only of glucose, and is a natural product that is also present in the stalks of corn, so there is no problem with its safety as a food, and it is not normally found in the body. It functions as a diet food because it is difficult to digest. Therefore, cellobiose can be used as a food material.

On the other hand, cellulose, the raw material, is the most abundant biomass produced on earth and is widely used in plant foods such as beans, vegetables, fruits, and grains. On the other hand, unused cellulose also accounts for a large portion of agricultural waste and forest waste. Therefore, the present invention is of great industrial importance also in terms of the effective use of biomass from wild boars.

<Prior Art> Cellobiose is industrially produced by chemical decomposition of cellulose. On the other hand, cellobiose production through enzymatic decomposition of cellulose is also generally known; however, when cellulose is decomposed using commercially available cellulase preparations, such as cellulases produced by Trichoderma sp. and Aspergillus sp., glucose is mainly produced, and cellobiose is The proportion of the total sugar produced is about 15% or less.

<Problems to be solved by the invention> Cellulase is a complex enzyme system, and at least three types of enzymes, namely C1°CX and β-glucosidase, are involved in the hydrolysis of cellulose to glucose by cellulase. . Among cholera enzymes, C
4 and Cx degrade cellulose to cellobiose.

Another enzyme, β-glucosidase, breaks down cellobiose and converts it to glucose. Therefore, it should be possible to generate only cellobiose from cellulose by inhibiting, inactivating, or removing β-glucosidase. However, no method for selectively inhibiting and deactivating only β-glucosidase is known so far. β−
Regarding the separation method of glucosidase, a method using an ion exchange resin is known, but the operation is complicated. That is, there is no known method for industrially removing β-glucosidase at low cost. It is also known that C4 and CI are adsorbed to cellulose, but β-glucosidase is difficult to adsorb, but there is no known method for producing cellobiose utilizing this property.

Means for Solving the Problems> As a result of intensive studies to solve these problems, the present inventors have developed a method that utilizes the difference in cellulose adsorption power between CI+CX and β-glucosidase of the cellulase complex enzyme system. They discovered that a cellulose decomposition product containing cellobiose as a main component can be obtained and completed the present invention. That is, in the present invention, after incubating insoluble cellulose or a cellulose-containing substance and cellulase in an aqueous medium, separating a solid fraction, adding an aqueous solution to the solid fraction, and keeping it warm for a certain period of time, cellobiose is added to the aqueous solution. This method of producing cellobiose is characterized by producing and accumulating cellobiose and collecting it.

Insoluble cellulose and insoluble-containing cellulose used in the method of the present invention include, for example, all wood materials such as chips obtained from coniferous broad-leaved trees, sawdust, and waste wood, as well as agricultural byproducts such as rice straw, fir, sugarcane, and horse maize. and other wastes, as well as their delignified derivatives and their powders.

Next, the enzymes used in the method of the present invention, that is, cellulases, include, for example, Sporotrichum sp., Trichoderma sp.
Examples include cellulases produced by bacteria such as Aspergillus M, (RuR. It's okay.

As the enzyme source, commercially available cellulase preparations, culture solutions of the above-mentioned bacteria, and their filtrate can also be used directly.

The β-glucosidase removal method in the method of the present invention includes cellulase, an excess amount of insoluble cellulose, gold...2-9,
Preferably in an aqueous medium of pH 3-7 at 0-60°C, preferably 30-50°C for 0.1-24 hours, preferably 1
After incubating for ~4 hours, the solid fraction and solution fraction are separated by means such as p-filtration and centrifugation. This solid fraction contains β-
Since some glucosidase remains, this solid fraction is washed with an aqueous solution of -2 to 10, preferably -3 to 7, in order to remove as much glucosidase as possible. By this operation, more than 95% of β-gluco in the cellulase preparation
aidase can be removed.

As a method for producing cellobiose in the method of the present invention, the solid fraction after washing is adjusted to pH 2 to 10, preferably -3 to
7 in an aqueous solution and heated at 20 to 60°C without adding any additional cellulase. After a certain period of time, separate the solid fraction from the solution again by filtration or centrifugation,
The solid fraction is resuspended in an aqueous solution and kept warm. This operation is repeated and the resulting solution fractions are collected to obtain a cellobiose production solution. The content of cellobiose in all the sugars produced in this solution is 80q6 or more. In this operation, as the concentration of insoluble cellulose decreases as saccharification progresses, the unadsorbed portion of cellulase increases, and cellulase gradually moves toward the saccharification solution. The enzyme in this solution can be recovered by newly adding cellulose before separating the solid fraction from the solution after the reaction. Furthermore, if a detachable reaction tank is used, cellobiose can be continuously produced.

The aqueous medium and aqueous solution used in the present invention include water, N
aC1, KCl*(NH4)2804. Na2C
Examples include salt solutions such as O3, buffers containing phosphoric acid, acetic acid, citric acid, glycine, and the like. Moreover, the concentration of these is θ~0.5M, preferably 0~0.1M.

The method for recovering cellobiose in the method of the present invention is as follows:
As the simplest operation, cellobiose can be separated by cooling or freezing the molebiose-generating solution to 0° C. or below to insolubilize cellobiose. If many impurities are present, it can be desalted using an ion exchange resin or the like, remove polymeric substances by ultrafiltration, and after concentration, add an organic solvent such as acetone to insolubilize and separate cellobiose.

In the present invention, sugars were detected using thin layer chromatography, in which a sugar solution was spotted on a thin layer of silica glue using a solvent (n-butanol:acetic acid:ether:water=9:6:
3:1), followed by color development using a naphresorcinol-phosphate reagent. Furthermore, high performance liquid chromatography was used to quantify cellobiose. That is, Showdex Ionpaek S-801
(φ8m×50cm) column, elution was carried out using water at 40° C., and the analysis was performed using a differential refractometer. Also β
-111 determination of glucosidase activity - Using nitrophenyl-β-glucocytes as a substrate, p-nitrophenol produced was determined by measuring the absorbance at 400 nm.

This will be explained in detail in Examples below.

Example A liquid medium with the following composition was prepared, dispensed into 500 d capacity Sakaro flasks, and heated at 120° C. for 10 minutes to sterilize.

Medium composition (pi(6,2) cellulose powder 5011/1 peptone
12 ttKH2PO46# (NH4, SO24,5N Mg504-7H200,91 CaCt2 ・H2O0,91 Urea 0.37 Tween80 2 #ZnSO
4”7) I20 15 WQ/lFeS
O4・7H205g MnSO4・4H203g CoCt2 6 #This medium was inoculated with Sporotrichum cellobiose ATCC20494 and cultured with shaking at 48℃ for 72 hours. The cellulase was concentrated approximately 20 times using the same enzyme and lyophilized to obtain a crude cellulase enzyme powder.

Next, add 0.4 ml of this cellulase powder and cellulose powder (
What My CC41) 511 and 100WLIO0
, suspended in 1M acetate buffer -5,0 and kept warm at 50°C for 2 hours, separated the solid fraction and solution by centrifugation, washed the solid fraction three times with the same buffer 50d, and then resuspended it. 100Wi
The suspension was suspended in 1 of the same buffer and kept at 50°C for 16 hours. As a control experiment, cellulase and cellulose were directly incubated for 16 hours without separating the solid fraction, and cellobiose and glucose in each of the resulting saccharified solutions were quantified. The results are shown in the table below.

2 hour heat retention/washing 0.75 0.11 Control experiment 0.01 0.45 Example 2 Cellulose powder (Whatman CC41) 20 # and the above cellulase 211 were mixed at 400 itI! The suspension was suspended in 0.1 M acetate buffer pH 5.0 and kept warm for 2 hours in the same manner as in Example 1. The isomorphic fraction was washed and kept warm for 16 hours. .

This 16-hour incubation operation was repeated two more times (3 times in total) to obtain a saccharified solution with a total volume of 116 (ILA'). ) Amberlite IRA-
410 and Amberlite IRA 120B, and the polymer components were removed by ultrafiltration (molecular weight 5000 cut) and concentrated to 40m. This is a seven ton 3
0- was added and allowed to stand at 4 to 7°C. 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 Commercially available cellulase originating from Trichoderma koningii (Meicelase P-1) 0.5. li"i cellulose powder (Whatman CC41) 11 was treated under the same conditions as in Example 1. The results are shown in the table below.

Cellobiose Glucose 2-hour heat retention and washing 0.53 0.12 Control experiment 0.06 0.42 Example 4 Example 1 using various commercially available cellulose powders and cellulase of Sporotrichum cellulofilm ATCC 20494
Saccharification was performed under the same conditions. The results are shown in the table below.

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0.11 <Effects of the Invention> As detailed above, the method of the present invention makes it possible to efficiently and selectively produce cellobiose rather than cellulose-soluble cellulose. This suggests the possibility of using cellobiose as a new food material, which is extremely important industrially.

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Claims (1)

[Claims] Insoluble cellulose or an insoluble cellulose-containing substance and cellulase are kept warm in an aqueous medium, then the solid fraction in the aqueous medium is separated, an aqueous solution is added to the solid fraction, and kept warm for a certain period of time to produce cellobiose in the aqueous solution. Let it accumulate,
A method for producing cellobiose, which comprises collecting cellobiose.