JPS61170388A - Recovery of cellulase - Google Patents

Abstract

PURPOSE:To recover the titled enzyme in high efficiency, by decomposing a cellulosic material with a cellulase, acidifying the liquid obtained by the decomposition and saccharification, adding and dissolving chitosan to the liquid, and adjusting the mixture to alkaline state to effect the deposition and separation of chitosan adsorbing the cellulase. CONSTITUTION:The recovery of cellulase from the liquid obtained by the decomposition and saccharification of a cellulosic material with a cellulase can be carried out by (1) acidifying the liquid after the completion of the decomposition and saccharification process, (2) dissolving chitosan or partially deacetylated chitin in the liquid, (3) adjusting the mixture to alkaline state to effect the precipitation of chitosan or partially deacetylated chitin adsorbing the cellulase, and (4) separating the cellulase-carrying chitosan, etc., together with unreacted solid component.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial 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 remaining cellulases are recovered from a solution after decomposition and saccharification of cellulosic substances using cellulases such as cellulases and hemicellulases. The present invention relates to a method for efficiently recovering cellulases through extremely simple treatment.

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. Enzymatic saccharification is attracting attention as an effective method because it can be carried out under suitable conditions and the produced sugars are not further degraded.

In this enzymatic saccharification method, it is generally said that the cost of producing the cellulases used as enzymes, such as cellulases and hemicellulases, accounts for about half of the total cost, so it is extremely difficult to recover and reuse these expensive enzymes. This has become an important issue.

Therefore, as methods for recovering cellulases from the saccharification-finished liquid, methods using ultrafiltration and methods for recovering cellulases by precipitation using an organic solvent or the like have been proposed.

However, the former method requires a large amount of equipment and operation costs because it is carried out under pressure, and the latter method has problems with the cost and recovery of solvents. There are disadvantages such as a high rate of enzyme deactivation.

Problems to be Solved by the Invention The purpose of the present invention is to overcome the drawbacks of the conventional methods for recovering cellulases, and to remove remaining cellulases from the solution after decomposition and saccharification of cellulosic substances using cellulases. This provides an efficient and low-cost recovery method using extremely simple processing.

Means for Solving the Problems As a result of extensive research, the present inventors found that chitosan and partially deacetylated chitin, which are derived from chitin that is abundant in nature, are easily dissolved in acidic aqueous solutions, but in neutral or Focusing on the fact that they do not dissolve in alkaline aqueous solutions and have excellent affinity for proteins, they discovered that the above objectives could be achieved by using them as adsorbents for cellulases, and based on this knowledge, The present invention has now been completed.

That is, 1. In the present invention, when recovering cellulases from a solution that has completed decomposition and saccharification of cellulosic substances using cellulases, 1. The solution that has undergone decomposition and saccharification is adjusted to be acidic, and chitosan or partially deacetyl chitin is added thereto. A method for recovering cellulases, which comprises dissolving the cellulases, then adjusting the mixture to alkaline to precipitate chitosa/or partially deacetylated chitin that has adsorbed the cellulases, and separating this together with unreacted solids; After previously separating unreacted solids from the decomposed and saccharified solution, the solution is acidified, chitosan or partially deacetylated chitin is added and dissolved, and the mixture is then made alkaline to adsorb the cellulases. The present invention provides a method for recovering cellulases, which comprises precipitating and separating chitosan or partially deacetylated chitin.

The cellulosic substances used in the method of the present invention include:
Examples include all types of wood such as wood chips, sawdust, bark, and waste wood obtained from coniferous trees, broad-leaved trees, southern and northern woods, and 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 cells of the genus Aspergillus and bacteria of the genus Trichoderma, or enzymes containing cellulases and hemicellulases. Also, regarding the present invention V.

These enzymes may have different origins and may be used alone, or two or more of them may be used simultaneously.

Furthermore, bacterial cell extracts and bacterial culture fluids containing these enzymes can also be used as enzyme sources.

A feature of the method of the present invention is the use of chitosan or partially deacetylated chitosan as an adsorbent for cellulases. Chitosan is a substance that is deacetylated by repeatedly heating chitin, which is a component of the outer skin of crustaceans such as shrimp and crabs, with an aqueous alkaline solution with a concentration of 40 to 50% by weight at a temperature of 60°C or higher. and D
-It is a type of polysaccharide consisting of β-1,4 bonds with glucosamine as its basic unit. Partially deacetylated chitin is partially deacetylated chitin,
Obtained as an intermediate process in the production of chitosan. Such chitosan and partially deacetylated chitin have excellent affinity for proteins and are particularly effective as adsorbents for cellulases. Further, these have the property of being soluble in acidic aqueous solutions, but not in neutral or alkaline aqueous solutions. The method of the present invention utilizes such properties of chitosan and partially deacetylated chitin.

This is a method for recovering cellulases.

In the method of the present invention, in order to recover residual cellulases from a solution that has completed decomposition and saccharification of cellulosic substances using cellulases, the decomposition and saccharification of a cellulosic substance is first adjusted to acidity in a state that contains unreacted solids. Then, chitosan or partially deacetyl chitin is added and dissolved, or the decomposed and saccharified solution is separated into a solution and unreacted solids by means such as filtration or centrifugation, and then the solution is adjusted to be acidic. Then, chitosan or partially deacetylated chitin is added and dissolved. At this time, the pH of the decomposed saccharification-completed liquid or filtrate is usually 2.3 to 2.3 by adding acid to the extent that chitosan and partially deacetyl chitin can be dissolved and that their deterioration and enzyme deactivation will not occur. In general, the decomposition and saccharification of cellulosic substances by cellulases is adjusted to a range of 6.8.
Since the process is carried out in an acidic environment for the stability of the enzyme, there is no need to add any acid. When adjusting the pH, there are no particular restrictions on the acid used, but acetic acid and hydrochloric acid are preferred.

In addition, if a pH buffer solution in which chitosan or partially deacetylated chitin is difficult to dissolve is used for the decomposition and saccharification of cellulosic materials, acetic acid or hydrochloric acid? It is desirable to add. On the other hand, it is advantageous that the amount of chitosan or partially deacetylated chitin used is at least twice the amount of remaining cellulases.

Next, the decomposed and saccharified solution in which chitosan or partially deacetylated chitin was dissolved in this way or the filtrate was adjusted to pH 7.
, and adjust the alkalinity to about 0 to 10 to precipitate chitosan or partially deacetylated chitin that has adsorbed cellulases. There are no particular restrictions on the alkali used for this pH adjustment.

Preferred are sodium hydroxide and potassium hydroxide. Next, the precipitated chitosan or partially deacetylated chitin is separated by means such as filtration or centrifugation. At this time, if the decomposed saccharification-completed solution is used as it is, chitosan or partially deacetylated chitin that has adsorbed cellulases is separated together with unreacted solids. Further, the treatment temperature is generally operated in a temperature range of 1 to 60°C throughout the entire process.

By such a method of recovering cellulases, 90 to 95% of the cellulases remaining in the decomposed and saccharified solution or the filtrate can be
% is recovered. In addition, in the method of the present invention, after the decomposed saccharification-completed liquid is separated into a solution and an unreacted solid, if necessary, cellulases remaining in the solid are eluted according to a conventional method. It is also possible to add a dilute solution to the separation solution and recover cellulases from this mixed solution as described above.

Chitosan or partially deacetylated chitin adsorbing cellulases thus separated.

Alternatively, a mixture of this and unreacted solids is added to an aqueous solution or buffer solution containing a new substrate at pH 2.3 to 6.8, and if necessary, fresh cellulases are added to carry out the decomposition and saccharification reaction. The cellulases remaining in this decomposed and saccharified solution can be recovered by the same procedure as described above, without adding new chitosan or partially deacetylated chitin.

Thus, in the method of the present invention, chitosan or partially deacetylated chitin can be used repeatedly.

Effects of the Invention According to the method of the present invention, by using chitosan or partially deacetyl chitin as an adsorbent for cellulases, the cellulases can be extracted from the solution after the decomposition and saccharification of cellulosic substances using cellulases in an extremely simple process. can be recovered efficiently and at low cost, and by applying this method, the cost of saccharification of cellulosic substances can be significantly reduced.

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

Example 1 42 grams of crushed red pine wood flour was added to 100 grams of an acetate buffer solution with a pH of 4.5, and 300 grams of commercially available cellulase originating from Trichoderma viride (Cellulase Onozuka R-10) was added.
After adding ■ and shaking at 40℃ for 24 hours,
The reaction solution, in which % has been decomposed, is centrifuged to separate unreacted solids and solution. Analysis of this solution revealed that 60% of cellulase remained.

Next, after dissolving chitosan 400 Wli in the separated solution, the pH is adjusted to 9.0 with IN aqueous sodium hydroxide solution at room temperature. After the precipitate no longer formed, the precipitate was filtered off and the resulting precipitate was added to a 60-mL acetate buffer solution of pH 4.5 to dissolve it. When cellulase is quantified, it is 1
659 ml of cellulase was dissolved and 92 pieces of remaining cellulase were recovered. When 2.4 f of red pine wood flour was added to this solution and the mixture was shaken at 40'C for 24 hours, a decomposition rate of 41'l was obtained.

This decomposition and saccharification completed solution was adjusted to pH 9.5 again at room temperature, and when the formation of precipitate was completed, the precipitate was filtered off along with the unreacted residue, and the acetate buffer solution at pH 4.5 was again dissolved in 1i60
2.41 of fresh Japanese red pine wood flour was added and reacted at 40°C for 24 hours to obtain a decomposition rate of 38%.

Example 2 Sugarcane squeezed residue (bagasse) was delignified by alkali treatment, and about 20 mesh (pulverized powder 2
2 was added to 5° of a citrate buffer solution of pH 4.0, 200 μg of Aspergillus niger origin nocellulase (cellulosin AP) was added, and the mixture was shaken at 48°C for 24 hours. As a result, a decomposition rate of 50% was obtained. . When analyzed by circular dichroism measurement, it was found that cellulosin AP with a coefficient of 70 remained in the solution. Next, 380 ~ partially deacetyl chitin and 0.1-
of glacial acetic acid was added to this solution to dissolve it, the pH was adjusted to 8.0 with potassium hydroxide solution, and the resulting precipitate was filtered off. Quantitative analysis of the cellulase content in this filtrate again revealed that it was only a trace, and most of the cellulase was removed along with partially deacetylated chitin.

Example 3 A 100-volume water-soluble nutrient medium containing an appropriate amount of cellulose powder as a carbon source was placed in a 500-volume flask, sterilized, and then inoculated with Trichoderma viride Q, M414.
The cells were cultured for 6 days under aerobic conditions at a temperature of 0°C. p of culture solution
H was always adjusted to 5.4.

In another 100-volume flask, crushed beech wood flour (32 kg) was placed, and 50 kg of the above-mentioned culture solution (enzyme content: 120 kg) was added thereto and decomposed at 45°C for 24 hours to obtain a decomposition rate of 40°. The initially added cellulase 58 remained in the reaction solution. Next, the mixture was separated into a solid and a solution by centrifugation, and 1.8 ml of this solid was treated with 100 ml of water and 40 ml of an acetate buffer solution having a pH of 4.6.

40 μm of cellulase remaining in the solid matter was eluted.

A liquid i1140-, in which cellulase was eluted from the unreacted solids, was added to the supernatant of the centrifugation, and chitosan 3 was added to this.
After dissolving 00■, p! with sodium hydroxide aqueous solution.
(Adjust to 9.0. When the precipitate no longer forms, the precipitate is filtered out. This precipitate is again put into an acetate buffer solution of pH 4.0 and dissolved. After that, lauan material powder 1. 21 was added and decomposition was carried out at 45°C for 24 hours to obtain a decomposition rate of 42.

Example 4 Cellulosin AP75q and Cellulase Onozuka R-1075 were placed in a 100-capacity Erlenmeyer flask, 50 g of a phosphate buffer solution of pH 4.5 was added, and 2 tons of a sample obtained by drying and pulverizing newspaper was added. When the mixture was shaken at 45°C for 24 hours, a decomposition rate of 52% was obtained. Add 350 μl of partially deacetyl chitin to this solution and 1 m of 1NHC1O! After adding and dissolving, the pH was adjusted to 9.0 with 1N NaOH, and the resulting precipitate and unreacted solid were filtered out.
In addition to the acetate buffer solution of 4' and 8, the newspaper of 22 was added and mixed again at 45°C for 24 hours.
A decomposition rate of 9% was obtained.

Patent applicant: Tatsu Todoroki, 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 (1)

[Claims] 1. When recovering cellulases from a solution that has completed decomposition and saccharification of cellulosic substances using cellulases, the solution that has completed decomposition and saccharification is adjusted to be acidic, and chitosan or partially deacetylated chitin is added thereto. A method for recovering cellulases, which comprises: dissolving the cellulases in water, then adjusting the mixture to alkaline to precipitate chitosan or partially deacetylated chitin that has adsorbed the cellulases, and separating this together with unreacted solids. 2. When recovering cellulases from the solution after decomposition and saccharification of cellulosic substances using cellulases, unreacted solids are separated from the solution after completion of decomposition and saccharification, this solution is adjusted to acidity, and chitosan is added to it. Alternatively, a method for producing cellulases characterized by adding and dissolving partially deacetyl chitin, then adjusting the mixture to alkaline to precipitate chitosan or partially deacetyl chitin that has adsorbed the cellulases, and then separating this. Collection method.