JP2866157B2 - Method for producing cellooligosaccharide - Google Patents

Description

The present invention relates to a method for producing cellooligosaccharides such as cellobiose, and more particularly, to an enzymatic method for producing cellooligosaccharides. Cellooligosaccharides include, for example, glucose such as cellobiose, cellotriose, cellotetraose, cellopentaose, cellohexaose, and β-1,4.
It is a relatively low-polymerized oligosaccharide that is bound and is used as a substrate or inhibitor of cellulase, a starting material for synthetic products, and other general reagents. Furthermore, in recent years, its functionality has been attracting attention, and its use as a food additive is also expected.

[Prior Art] In order to produce cellooligosaccharides enzymatically, a method of reacting cellulase with cellulose and a method of reacting cellulose with a highly specific special cellulase that produces cellooligosaccharide at a high yield are known. It was known (Japanese Unexamined Patent Publication No.
256394).

[Problems to be Solved by the Invention] However, the enzymatic method using a commercially available cellulase has a disadvantage that the produced cellooligosaccharide is further dispersed and decomposed into glucose, and the yield of the target cellooligosaccharide is extremely low. there were. In addition, a method of acting a special cellulase having a high specificity requires culturing the producing bacteria to obtain the cellulase, requires equipment such as an ultrafiltration reactor, and requires a long reaction time. Has the disadvantage of

[Means for Solving the Problems] The present invention solves the above-mentioned problems, and industrially produces cellooligosaccharides in high yields using generally available cellulase preparations which are generally commercially available. The aim is to provide a method. That is, the present inventors have conducted intensive studies to solve the above-mentioned disadvantages, and as a result, have found a method for efficiently producing and accumulating cellooligosaccharides by a batch-type enzymatic reaction using an easily available cellulase preparation which is generally commercially available. The present invention has been achieved. That is, the present invention relates to a method for producing cellooligosaccharides from cellulosic substances by the action of cellulase, wherein the reaction is carried out in the presence of gluconolactone and / or gluconic acid. . Hereinafter, the present invention will be described in detail.

The cellulase used in the present invention need not be particularly limited, and any commercially available cellulase preparation can be used. The cellulosic material as the main raw material is not particularly limited, but a delignified and powdered material is more preferable. Also, short fibers produced as a by-product in the pulp manufacturing process, non-renewable cellulose generated in the used paper recycling process, and the like can be used. The reaction is carried out in an aqueous suspension containing gluconolactone and / or gluconic acid, an enzyme and a main raw material. At this time, the concentration of gluconolactone and / or gluconic acid in the reaction solution is 0.1 to 5 W / W%, preferably 0.5 to 5 W / W%.
It is in the range of 2W / W%. The enzyme concentration in the reaction solution is 0.01 to 5 W / W
%, Preferably 0.1 to 1 W / W%, and the concentration of the main raw material in the reaction solution is in the range of 0.1 to 15 W / W%, preferably 1 to 10 W / W%, and these are used in appropriate combination. The pH of the reaction solution
Is in the range of 3-6, preferably 4-5, and the reaction temperature is
The reaction is carried out at 30 to 60 ° C, preferably at 45 to 55 ° C. The reaction time is preferably terminated when the production of cellooligosaccharide is maximized, but is generally in the range of 1 to 10 hours. After completion of the enzymatic reaction, the unreacted residue is removed by centrifugation, diatomaceous earth filtration or the like to obtain a clear liquid. Next, deionization treatment with an ion exchange resin is performed, concentration is performed under reduced pressure, and cooling is performed to obtain cellooligosaccharide crystals. Further, if recrystallization is repeated, a trace amount of coexisting glucose can be completely removed.
In addition, a method of adding an alcohol such as ethanol and cooling in order to improve the crystal yield can also be applied.

[Action] The mechanism of cellooligosaccharide formation in the cellulase reaction according to the present invention is not clear, but the following was presumed. Cellulase is generally known as an enzyme that decomposes a cellulose component into glucose and cellooligosaccharide, and is said to contain C1 enzyme activity, CX enzyme activity, β-glucosidase activity and the like. In the process of cellulose degradation, it is said that C1 enzyme activity and CX enzyme activity act first to produce cellooligosaccharides, and β-glucosidase activity acts on these to decompose to glucose, which is the minimum unit of cellulose. It is considered that when gluconolactone and / or gluconic acid was added during the cellulase reaction, the β-glucosidase activity was inhibited thereby, and cellooligosaccharides were accumulated.

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples.

The method for separating and quantifying cellooligosaccharides in the examples was performed by the following high performance liquid chromatography (hereinafter abbreviated as HPLC).

(1) Measuring instrument HPLC pump: Waters MODEL 510 Differential refractive index detector: Showa Denko KK SHODEX RISE-6
1 Chromatographic data processing device: Hitachi 833A type (2) Measurement conditions Column: 4.6 mm ID × 25 cm Filler: TSKge1Amide-80 (5 μm) manufactured by Tosoh Corporation Column temperature: 80 ° C. Elution solvent: acetonitrile / water = 6 / 4 Flow rate: 1 ml / min Injection volume: 10 μl Example 1 2 g of cellulase (trade name: Meisserase, manufactured by Meiji Seika Co., Ltd.), 20 g of cellulose powder (trade name: KS powder 100 mesh, manufactured by Kojin Co., Ltd.), And 10 g of gluconolactone were added to 1 L of 0.1 M acetate buffer pH 5, and the temperature was adjusted to 50 ° C.
, And the enzyme reaction was carried out with stirring. After the reaction for 4 hours, a supernatant was obtained by centrifugation. The supernatant was further clarified by diatomaceous earth filtration using Radiolite # 500 to obtain 960 ml of a clear solution. As a result of HPLC analysis, the sugar composition of the clarified solution was glucose 1.04 mg / ml, cellobiose 6.29 mg / ml, and cellotriose 0.22 mg / ml. Next, the clarified solution was continuously passed through a column of Diaion SKIB (H type, manufactured by Mitsubishi Kasei) having a resin amount of 50 ml and a column of Diaion WA30 (OH type, manufactured by Mitsubishi Kasei) having a resin amount of 100 ml (SV = 5). ). The eluate obtained was concentrated under reduced pressure (40 ° C) by an evaporator to about 12
ml (Bx about 60 degrees). To this was added 4 ml of ethanol, and the mixture was allowed to stand at 4 ° C. After standing for one solution, 4.5 g of white crystals were obtained. Next, this was dissolved in about 9 ml of hot water and cooled in the same manner as above to obtain about 3.1 g of a recrystallized product. The result of HPLC analysis of this recrystallized product was as shown in FIG. That is, cellobiose was 96.6% and cellotriose was 3.4% as compared with the standard substance. In addition, the obtained crystals were completely decomposed into glucose by almond β-glucosidase (manufactured by Sigma), which means that they were cellooligosaccharides.

Example 2 A reaction was carried out under exactly the same conditions as in Example 1 except that cellulase Onozuka (produced by Yakult Co., Ltd.) was used as cellulase and gluconic acid was used instead of gluconolactone. 955m clear liquid by the method of
got l. The sugar composition of this clarified solution was determined by HPLC analysis to be glucose 0.82 mg / ml, cellobiose 5 mg / ml, cellotriose
It was 0.17 mg / ml. Thereafter, the same treatment as in Example 1 was performed to obtain 2 g of a recrystallized product. The saccharide composition of this product was almost the same as in Example 1 as a result of HPLC analysis.

Comparative Example 1 The reaction was carried out under exactly the same conditions as in Example 1 except that gluconolactone was not added. Next, 965 ml of a clear solution was obtained from the reaction solution in the same manner as in Example 1. As a result of HPLC analysis, the sugar composition of the clarified solution was 5.71 mg / ml for glucose and 0.96 mg / ml for cellobiose. Thus, the yield of the target cellobiose was low, and most of the cellobiose had been converted to glucose.

[Effects of the invention] As is clear from Comparative Example 1, in the cellulase reaction using a commercially available cellulase preparation as it is, the production of cellooligosaccharides is extremely low, which is unsuitable as a method for producing cellooligosaccharides. Cellooligosaccharides can also be produced by easy commercial cellulase preparations. Therefore, no special cellulase is required. In addition, equipment such as an ultrafiltration reactor is not particularly required, and the reaction time is short. Thus, it is extremely effective as an industrial production method of cellooligosaccharide.

[Brief description of the drawings]

FIG. 1 is an HPLC chromatogram showing the sugar composition of the final product obtained in Example 1.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int. Cl. ⁶ , DB name) C12P 19/14 CA (STN)

Claims (1)

(57) [Claims] 1. A method for producing cellooligosaccharides from cellulosic substances by the action of cellulase, wherein the reaction is carried out in the presence of gluconolactone and / or gluconic acid.