JPS637781A - Recovering method for cellulase - Google Patents

Abstract

PURPOSE:To efficiently recover cellulose, by treating the cellulose remaining in a liquor after completing hydrolysis and saccharification of a cellulosic substance with the cellulase, particularly in a solid material with ultrasonic waves. CONSTITUTION:A liquor after completing hydrolysis and saccharification of a cellulosic substance with cellulose is separated into a solution and a solid material by centrifugation, etc., and the cellulase contained in the solution is recovered by a conventional method, e.g. ultrafiltration, etc. On the other hand, the solid material is then dipped in an aqueous solution of a water-soluble polysachharide, oligosaccharide and a derivative thereof with a monosaccharide or a pH buffer solution or alcohol solution thereof and ultrasonic waves are applied thereto at room temperature for >=1min, normally about 5min to separated the solid material.

Description

DETAILED DESCRIPTION OF THE INVENTION A. Industrial Field of Application The present invention relates to a method for recovering cellulases, and more specifically, the present invention relates to a method for recovering cellulases, and more specifically, the present invention relates to a method for recovering cellulases, and more specifically, the present invention relates to a method for recovering cellulases. The present invention relates to a method for recovering cellulases with a high yield through extremely simple operations.

B. Conventional technology In recent years, research on the saccharification of cellulosic substances has been actively conducted from the viewpoints of energy issues, environmental pollution, and the effective use of unused resources. Moreover, enzymatic saccharification is attracting attention as an effective method because it can be carried out under mild conditions and the produced sugars are not further decomposed.

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 half of the total cost, so it is important to collect and reuse these expensive enzymes. has become an extremely important issue.

However, although various methods have been proposed in the past for recovering cellulases remaining in the separated solution by centrifuging the solution after decomposition and saccharification of cellulosic substances, There are still few reports on methods for recovering cellulases, and the recovery rate is at most 30% or less. Among these, the present inventor's earlier invention (Patent No. 1299068) (hereinafter referred to as the "prior invention") is particularly well known, and it is a method for decomposing and saccharifying a cellulosic substance by cellulases. To recover the cellulases, first, the solution after decomposition and saccharification is separated into a solution and a solid substance, and then the cellulases are recovered from the solution according to a conventional method, and the cellulases remaining in the solid substance are dissolved in water. It is characterized by being recovered by treating with an aqueous solution or PI (buffer solution) of polysaccharides, oligosaccharides, or alcohols. It is excellent in that it is extremely simple and easy to bring the solid matter into contact with the solution by gently stirring or shaking at a temperature of %, and it is desirable to further improve the recovery rate.

C6 Problems to be Solved by the Invention Therefore, the purpose of the present invention is to recover almost completely the enzyme remaining in the solid material, thereby further shortening and simplifying the operation and processing time.

Means for Solving the D0 Problem In order to achieve the above objective, the present inventors have made intensive studies from various angles and have found that by using ultrasonic waves, the bond between the enzyme, which is a glycoprotein, and unreacted solid matter. breaking of hydrogen bonds,
It has also been found that chemical binding and physical adsorption of enzymes to unreacted solids can be reduced by destroying the pores of unreacted solids. Furthermore, by using this treatment together with the inventors' prior invention, they found that the above chemical bonding and physical adsorption can be extremely easily reduced, and based on these findings, they have completed the present invention.

That is, in the method of the present invention, in order to recover the remaining cellulases from the solution after completion of decomposition and saccharification, the solution after completion of decomposition and saccharification is first separated into a solution and solid matter by means such as centrifugation, and then the solution is separated into solid matter. The cellulases contained in the cellulase are recovered by a commonly used method such as ultrafiltration. The recovery rate at this time was 90% of the amount remaining in the solution.
~100%. The cellulases remaining in the separated solid are impregnated with an aqueous solution or pH buffer solution of water-soluble polysaccharides, oligosaccharides, or alcohols, and ultrasonic waves are applied.

The temperature is from 5°C to 60°C, but it is usually performed at room temperature. The time required for the ultrasonic treatment varies somewhat depending on the state of the unreacted solids, but 1 minute or more is required, and usually about 5 minutes is sufficient.

The cellulosic material used in the method of the present invention is 1
Examples include all types of wood such as wood chips, sawnage shoulders, bark, and waste wood obtained from conifers, hardwoods, southern and northern woods, as well as 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.

Examples of cellulases used in the method of the present invention include cellulases produced from Aspergillus bacteria and Trichoderma bacteria, or enzymes containing cellulases and hemicellulases. In addition, in the present invention, these enzymes may be used singly or two or more of them may be used at the same time, and furthermore, bacterial cell extracts or bacterial cell extracts containing these enzymes may be used as enzyme sources. A culture solution can also be used.

Examples of water-soluble polysaccharides used to recover cellulases include glucomannan, oligosaccharides such as cellobiose, and alcohols such as monohydric alcohols such as ethanol and polyhydric alcohols such as ethylene glycol. can be mentioned. Also, glucose and sugar derivatives such as p-nitrophenyl-β-D-glucoside may be used. The concentration of the aqueous solution or pH buffer solution of water-soluble polysaccharides and oligosaccharides is 0.05 to 5.
A range of weighted background % is suitable. In addition, monosaccharide derivatives are 0.
Suitable conditions start from 0.02% by weight.

A pH buffer solution is used to increase the stability of the cellulases after recovery, and the preferred pH is in the range of 3.0 to 8.0. As this pH buffer solution, an acetic acid buffer solution is usually preferably used.

The recovered solution containing cellulases obtained using an aqueous solution of these saccharides or a pH buffer solution can be used as it is as a source for polysaccharides and oligosaccharides, and can also be used to decompose cellulosic substances. Can be reused as a source of saccharifying enzymes. The concentration of the aqueous alcohol solution or pH buffer solution is preferably in the range of 1 to 30% by weight.

E0 Effects of the Invention In the method of the present invention, the recovery rate of the enzyme adsorbed on unreacted solids is 90 to 95%, and therefore, in addition to recovery from the solution, it is possible to recover the enzyme used at a high rate. can. Moreover, since the operation is carried out at room temperature, no energy is required for heating, and the operation time can be significantly shortened. can save significantly.

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

Example-1 2 g of ball-milled red pine wood flour was added to 50 mQ of pH 4,5 acetate buffer solution, and commercially available cellulosin AP originating from Aspergillus niger and Trichoderma.
200 mg of a 1:1 mixture of cellulase Onodzuka R-10 derived from viride was added and shaken at 40° C. for 48 hours, and the reaction-completed liquid was filtered to separate the solution and 780 mg of solid matter.

61% of the wood flour was decomposed. 60% of the originally added enzyme remained in the solution. Place the solid in a 50mQ stoppered flask and add 1% carboxymethyl cellulose to pH 4.5.
A 25 mA acetate buffer solution was added, sonication was performed at room temperature for 5 minutes, and a supernatant was obtained by centrifugation. The enzyme contained in the supernatant was 36% of the enzyme originally used. 1 g of ball milled cellulose was added to the supernatant, and the mixture was shaken at 40°C. Glucose produced after 48 hours was quantified by the Nelson-5omogyi method and found to be 940 mg.

Example-2 A 500 cc flask was filled with 100 mm of an aqueous nutrient medium containing an appropriate amount of cellulose powder as a carbon source and sterilized. Trichoderma viride QM414 was inoculated into the flask, and the medium was incubated at a temperature of 30°C under aerobic conditions. The cells were cultured for 6 days. The pH of the culture solution was always adjusted to 5.4.

Put 2 g of ball milled beech wood flour into another room Q flask, and add 50 m m of the above culture solution to this.
(Enzyme content: 120 mg) was added and decomposed at 40° C. for 48 hours to decompose 56% of the wood flour. After one reaction was completed, the liquid was separated into a solution and a solid by centrifugation. 50 mg of enzyme remained in the solution, and 42 mg of enzyme was recovered by ultrafiltration. - The solid material was impregnated with 20 ml (l) of 0.8% carboxymethyl cellulose aqueous solution,
Ultrasound was applied for 0 minutes at room temperature. The filtrate from which the solid matter was filtered contained 65 mg of enzyme. Add a phosphate buffer solution of pH 4.8 to the filtrate to bring the total volume to 25 mQ, add 1 g of hydrogen peroxide-alkali cooking to make paper made from bagasse into strips (1 cm x 1 cm), and heat at 40'C.
It was shaken with After 8 hours, the paper was completely dissolved and Nel
The amount of glucose produced by the son-5omogyi method was 1200 mg.

Example 3 Dry newspaper and weigh 2 g of 50 square pieces with sides of 3 m.
Cellular Zee Onozuka R-1
After adding 0.100 mg of the solution and decomposing it at 40°C for 24 hours, the solution after one reaction was centrifuged to separate the solution and solid matter.
It was separated into g. 35 mg of cellulase remained in the solution.

Add 40 mM of a 15% by weight ethylene glycol aqueous solution to 1.4 g of the solid obtained above.

Ultrasound was applied for 10 minutes at room temperature. The ethylene glycol aqueous solution contained 62 mg of enzyme.

Example-4 2 g of ball milled yellow lauan wood flour
Pour into a m Erlenmeyer flask.

A 45mM PH5.0 acetate buffer solution was added and the mixture was heated at 45°C. Add 6% 5mM of cellulosin AP and meicelase of Trichoderma viride origin in a 1:l mixture;
After shaking for 48 hours, the mixture was filtered. In the filtrate,
1160 mg was detected in terms of glucose. In addition, the β-glucosidase activity of the filtrate was 80% of the initially added amount.
% was detected. 0.03% P for 40 mg of unreacted solids
-Nitrophenyl-β-D-glucoside was added to PH4,5 acetate buffer solution and subjected to ultrasonic treatment for 5 minutes, and after centrifugation, the supernatant was immersed at 40°C for 10 minutes to measure the eluted β-glucosidase activity. . 94% of the amount adsorbed on the solid material was eluted.

Patent applicant Kozo Iizuka, Director of the Agency of Industrial Science and Technology Designated agent: Ryozo Hayami, Director of the Osaka Institute of Industrial Science and Technology, Agency of Industrial Science and Technology

Claims (4)

[Claims] (1) Decomposition of cellulosic substances by cellulases In recovering the cellulases from the saccharification-completed liquid, first separate the decomposition-saccharification-completed liquid into a solution and solid matter, and then extract the cellulases from the solution according to a conventional method. At the same time, the cellulase remaining in the solid material is impregnated with an aqueous solution of a derivative of water-soluble polysaccharides, oligosaccharides, and monosaccharides, the above pH buffer solution, or an alcohol solution, and treated with ultrasound. 1. A method for recovering cellulases, the method comprising recovering cellulases. (2) Water-soluble polysaccharide or oligosaccharide aqueous solution or p
2. A method according to claim 1, wherein the concentration of the H buffer solution is 0.5 to 5% by weight. (3) The method according to claim 1, wherein the concentration of the aqueous alcohol solution or pH buffer solution is 1 to 30% by weight. (4) The method according to claim 1, wherein the pH of the pH buffer solution is 3.0 to 8.0.