JPS61162180A - Recovery of cellulase - Google Patents

Abstract

PURPOSE:To recover cellulase from the completely decomposed and saccharified liquid of a cellulosic substance, with simple operation, in high efficiency, by adding chitosan or a partially deacetylated chitin and a fresh substrate to the liquid. CONSTITUTION:A cellulosic substance is decomposed and saccharified with a cellulase containing at least one kind of cellulase originated from the cell of microorganism belonging to Aspergillus genus, and the cellulase is recovered from the completely decomposed and saccharified liquid by adding chitosan or partially deacetylated chitin and a fresh substrate to the liquid. Not only the cellulase originated from Aspergillus but also other cellulase can be recovered extremely efficiently by this process.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a method for recovering cellulases, and more specifically, to the completion of the decomposition and saccharification of cellulosic substances by cellulases containing at least one type of cellulase derived from Aspergillus microorganisms. The present invention relates to a method for selectively and efficiently recovering residual Aspergillus cellulase-derived cellulases from a liquid using extremely simple operations, and a method for efficiently recovering all cellulases.

Conventional technology In recent years, due to energy problems, environmental pollution, food problems, etc.
Research on the saccharification of cellulosic substances is being actively conducted in order to determine the effective use of unused resources. Enzymatic saccharification is attracting attention as an effective method because the produced sugars are not further degraded.

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.
Therefore, recovering and reusing this expensive enzyme has become an extremely important issue.

Therefore, conventionally, various methods have been proposed for recovering cellulases from the saccharification-completed liquid, including a method using ultrafiltration and a method of recovering cellulases by precipitation using an organic solvent or the like. However, in these methods,
Handling a large amount of saccharification-finished liquid requires a lot of processing time and equipment costs, and it also has drawbacks such as a high rate of enzyme deactivation during processing. Therefore, as a relatively simple method,
In recent years, a method of soaking a new cellulosic material in the saccharification-finished liquid to adsorb and recover cellulases has been attracting attention in recent years.

On the other hand, a method is known in which cellulase derived from Trichoderma and Aspergillus bacteria is used in combination to increase the decomposition rate of cellulosic substances and shorten the decomposition time. however.

In this case, the cellulase originating from Aspergillus spp.
Since the affinity for cellulose substances is extremely small compared to that derived from cells of the genus Trichoderma, even if one attempts to recover cellulase by adsorbing it to a new cellulose substance, the recovery rate of cellulases derived from Aspergillus will be extremely low. do not have.

Problems to be Solved by the Invention In view of the above-mentioned circumstances, the object of the present invention is to provide an extremely simple solution for decomposing and saccharifying a cellulosic substance using a cellulase containing at least one type of cellulase originating from the cells of the genus Aspergillus. By operation.

The object of the present invention is to provide a method for recovering cellulases originating from remaining cells of the genus Aspergillus at a high yield, and a method for recovering cellulases in general at a high yield.

As a result of extensive research by the present inventors, we found that chitosan and partially deacetylated chitin have a great affinity for cellulases originating from Aspergillus cells! However, it was discovered that the above object could be achieved by using these, and the present invention was completed based on this knowledge.

That is, one aspect of the present invention is to collect cellulases from a decomposed and saccharified solution of a cellulosic substance using cellulases containing at least one type of cellulase derived from cells of the genus Aspergillus, and to add chitosan or A method for recovering cellulases, which comprises adding partially deacetyl chitin, selectively adsorbing cellulases originating from Aspergillus cells, and then separating the cellulases, and adding chitosan or a partially deacetyl chitin to the decomposed saccharification solution. The present invention provides a method for recovering cellulases, which comprises adding deacetyl chitin and a new substrate to which the cellulases are adsorbed and then separating them.

The cellulosic substance used in the method of the present invention is 2
Examples include all types of wood such as wood chips, sawdust, bark, and waste wood obtained from conifers, hardwoods, southern and northern woods, as well as agricultural waste, sugarcane, corn, etc. Examples include papers such as

Furthermore, the cellulase enzymes used in the method of the present invention include at least one type of cellulase originating from the cells of the genus Aspergillus, such as cellulases alone originating from the cells of the genus Aspergillus; and cellulase derived from Trichoderma bacteria. These enzymes may also include hemicellulases. 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 that chitosan or partially deacetyl chitin is used as an adsorbent for adsorbing cellulase originating from Aspergillus cells. Chitosan is shrimp,
Chitin, which is a component of the outer skin of crustaceans such as crabs, is mixed with an alkaline aqueous solution with a concentration range of 40 to 50% by weight, for example.
6L of deacetylated material in warm water above 0°C.
It is a type of polysaccharide consisting of β-1,4 bonds with D-glucosamine as the basic unit. Moreover, partially deacetylated chitin is the chitin partially deacetylated.

Obtained as an intermediate process in the production of chitosan. Such chitosan or partially deacetylated chitin has excellent affinity for cellulases originating from Aspergillus cells.

In the method of the present invention, a cellulosic substance is degraded and saccharified by cellulases containing at least one type of cellulase derived from cells of the genus Aspergillus.

The treatment may be carried out by adding only chitosan or partially deacetylated chitin, or they may be added together with a new substrate. At this time, the decomposition and saccharification completed liquid may be used as it is containing solids, or if necessary, a solution after separating solids may be used. Further, the amount of chitosan or partially deacetylated chitin used is preferably equal to or more than the amount of cellulase originating from Aspergillus cells used for decomposition and saccharification of cellulosic substances, and the treatment temperature is 1 to 1. It is desirable that the treatment pH be at 60° C. and in the range of 4.0 to 10.0. If the pH is less than 4.0, chitosan or partially deacetylated chitin will dissolve, and l010
Exceeding this is not preferable since cellulases may be denatured. Generally, a treatment time of about 30 minutes to 3 hours is sufficient.

When a finished liquid obtained by decomposing and saccharifying cellulosic substances using a combination of cellulase originating from Aspergillus spp. cells and cellulase originating from Trichoderma spp. cells is treated by adding only chitosan or partially deacetyl chitin, Aspergillus spp. Only body-derived cellulases have a 50 to 7
On the other hand, when Chitosa/or partially deacetylated chitin is added and treated with a new substrate, 9 of the cellulase originating from Trichoderma cells remaining in the finished solution is
0 to 100 ots of cellulase and 60 to 5 ots of cellulase originating from Aspergillus cells are adsorbed.

In addition, when the finished liquid obtained by decomposing and saccharifying cellulosic substances using only cellulase originating from Aspergillus bacteria is treated by adding only chitosan or partially deacetyl chitin, the cellulase adsorbs 50 to 704% of the remaining amount. On the other hand, when chitosan or partially deacetylated chitin is added and treated with a new substrate, the cellulase is adsorbed by 60-80% of the remaining amount.

After adsorption treatment in this manner, the solid matter is removed by means such as centrifugation or filtration, and if a new substrate is used for the adsorption treatment, the solid matter is left as is in an aqueous solution or in a solution with a pH of 4.5 to 5.0. Suspend in an acetic acid or citric acid buffer solution, add new cellulases if necessary, and perform a decomposition and saccharification reaction according to a conventional method. In addition, when a new substrate is not used for the adsorption treatment, the filtered solid matter, a new substrate, and if necessary, new cellulases are added to the buffer solution, and the decomposition and saccharification reaction is performed.

Effects of the Invention According to the method of the present invention, chitosan or partially deacetylated chitin, which has a high affinity for cellulases originating from Aspergillus cells, is used, and a cellulosic substance is decomposed and saccharified using enzymes. The cellulase originating from Aspergillus cells can be selectively recovered by a simple operation, and by using chitosan or partially deacetylated chitin in combination with a new substrate.

Not only cellulases originating from cells of the genus Aspergillus but also other cellulases can be very effectively recovered from the solution after completion of decomposition and saccharification.

The method of the present invention is a method for very effectively recovering expensive cellulases through simple operations, 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 Pulverized red pine wood powder in 100% of acetate buffer at pH 4.5. In addition, commercially available Trichoderma pi 11th generation Cellular-W (+ Ruler ν, Sennozusada R-10)
150mg Toaspergillus niger origin nocellulase (cellulosin AP) 150 1llt - 24 at 40°C
After shaking for a period of time, the reaction finished liquid in which the 60 part of the wood flour was decomposed was centrifuged to separate the solid matter, and beech wood flour 41 and chitosan 300119 were added to the solution at room temperature and mixed. 1
After a period of time, the solid obtained was suspended in an acetic acid buffer solution of pH 4.5 and reacted again. A decomposition rate of 40% was obtained after 24 hours. When only beech wood flour was added without using chitosan, the decomposition rate in the re-reaction was 23%.

Example 2 Sample 2 was prepared by drying newspaper and pulverizing it into approximately 20 mesh pieces. was suspended in 50-g water, and cellulosin AP10011 was added to this.
1i and decomposition was carried out at 45°C for 24 hours.

The decomposition rate of newspaper was 35%. Analysis revealed that 70% of sicelulosin AP remained in the decomposition solution. Akamatsu 2 in the reaction solution? Add 500 μl of partially deacetyl chitin and shake at 40°C for 1 hour to remove cellulosin A in the solution.
Analysis of the remaining amount of P showed that it had decreased to 21%, indicating that 70% of the remaining amount was adsorbed.

Furthermore, when only partially deacetylated chitin was added without using Japanese red pine, 60% of the remaining amount of cellulosin AP was adsorbed.

Example 3 A 500 cc flask was charged with 100-ml water-soluble nutrient medium containing an appropriate amount of cellulose powder as a carbon source, and then sterilized. The cells were cultured under atmospheric conditions for 6 days. The pH of the culture solution was always adjusted to 5.4.

Delignified corn stalk powder 22 was placed in another 100 cc flask, and the culture solution 50
- (enzyme content 1zomy) and cellulosin AP 100
Wq was added and decomposition was carried out at 40°C for 24 hours to obtain a decomposition rate of 60. Just 2 more? Add Notori sorghum powder and chitosan 250111i and heat at 30°C for 2 hours from time to time.
Mix it up and then separate the solids □ and p
Suspend in H5,0 citrate buffer solution 50-50°C and incubate again at 40°C.
I reacted again. The decomposition rate after 24 hours was 37%.

Example 4 75 μm each of Cellulase Onozuka R-10 and Cellulosin AP and 2 tons of Japanese red pine wood flour were added to a 100-volume flask, and a 50-mL pH 4.5 acetic acid buffer solution was added.
After 4 hours of decomposition, the solution and residue were separated by centrifugation. Next, a 5% ethanol aqueous solution of 25- was added to this residue, and the mixture was stirred at 40°C for 5 hours to elute the amount of enzyme 50 adsorbed to the residue. In addition to this, add Red Pine Wood Flour 32 and Chitosan 600 TTli for 30 minutes at room temperature and shake.

The solid matter was then separated by centrifugation, suspended in a 50-pH acetate buffer solution, and resaccharified at 45° C. for 24 hours, and the decomposition rate of Japanese red pine flour was compared with that without using chitosan. When chitosan was used, the decomposition rate was 1.5 times higher than when chitosan was not used.

Example 5 Nakajiyun 5+1 1 L Hyakushokara IF I Now
7-ri・so+raku1σ) 4＼Rutsukiariik was performed, and when 55 pieces of wood flour were decomposed, the reaction solution was centrifuged to remove solid matter. Add 300μ of chitosan to this solution,
After shaking at 0°C for 1 hour, chitosan was filtered off. In this chitosan, only cellulosin AP was adsorbed to approximately 60% of the remaining cellulosin AP in the solution from which the solid matter had been separated. Director Ryozo Hayami

Claims (1)

[Scope of Claims] 1. In recovering cellulases from a solution that has completed decomposition and saccharification of cellulosic substances using cellulases containing at least one type of cellulases derived from cells of the genus Aspergillus,
A method for recovering cellulases, which comprises adding chitosan or partially deacetylated chitin to the decomposed saccharification solution, selectively adsorbing cellulases originating from cells of the genus Aspergillus, and then separating the cellulases. 2. In recovering cellulases from the solution after completion of decomposition and saccharification of cellulosic substances using cellulases containing at least one type of cellulases derived from cells of the genus Aspergillus,
A method for recovering cellulases, which comprises adding chitosan or partially deacetylated chitin and a new substrate to the decomposed saccharification-completed solution, and collecting the cellulases by adsorbing them onto these.