JPH01309683A - Separation of chitin-decomposition enzyme and chitosan-decomposition enzyme - Google Patents

Abstract

PURPOSE:To separate microbial cells from a decomposition enzyme and a decomposition product with a simple operation in high yield at a low cost by successively passing a mixture of the above components through plural ultrafiltration membranes having different fractionation molecular weights. CONSTITUTION:A mixed microbial system containing microorganisms capable of producing chitin decomposition enzyme and microorganisms capable of producing chitosan decomposition enzyme is incorporated with crust of crab, shrimp,. etc., as a substrate and cultured. The obtained cultivation liquid is centrifuged to separate the suspending materials and the liquid is charged into a stock liquid tank 1. The liquid is passed through the 1st ultrafiltration membrane apparatus 3 provided with an ultrafiltration membrane 2 having a fractionation molecular weight of 200,000-500,000 and separated into a concentrated liquid 5 of the microbial cells and a permeated liquid 4 containing the enzymes and decomposition products. The permeated liquid is supplied to the 2nd ultrafiltration apparatus 8 having a fractionation molecular weight of 50,000-100,000 and the permeated liquid of the apparatus is supplied to the 3rd ultrafiltration apparatus 13 having fractionation molecular weight of 10,000-30,000 to obtain a permeated liquid 14 containing the decomposition product of chitosan.

Description

[Detailed Description of the Invention] <Industrial Application Field> The present invention relates to an enzyme that decomposes chitin contained in the shells of crustaceans such as crabs and shrimp, krill, etc., and an enzyme obtained by deacetylating the chitin. This invention relates to a method for efficiently separating enzymes that degrade chitosan from the culture solution of microorganisms that produce them.

<Prior art> Chitin and chitosan are themselves useful as films, fibers, medical materials, cosmetic materials, carriers for various enzymes, flocculants, etc., and have recently attracted particular attention. N-acetylglycosamine monomers and polymers, and decomposed products such as glucosamine monomers and polymers obtained by enzymatically decomposing chitosan, are also extremely useful, and can be used, for example, in anticancer drugs and natural preservatives. How to use it is being researched in many fields, and some have already been put into practical use.

The chitin-degrading enzymes and chitosan-degrading enzymes used to degrade chitin and chitosan as described above are generally microorganisms that produce these enzymes, such as actinobacteria such as Streptomyces, bacteria such as Pseudomonas and Vibrio. It is obtained by culturing filamentous fungi such as , Aspergillus, etc. using the shells of crabs, shrimps, etc. as substrates. These enzymes are used not only as enzyme preparations for producing chitin decomposition products and chitosan decomposition products as mentioned above, but also as enzyme pesticides that strongly inhibit the growth and growth of Fusarium fungi, which are plant pathogens, It has been confirmed that it is useful as a cell wall-dissolving enzyme that decomposes chitin or chitosan in cell walls, and also as a medical enzyme agent, and is expected to be used widely and in great demand.

However, although much research has been conducted on microorganisms that produce such useful chitin-degrading enzymes and chitosan-degrading enzymes and methods for culturing these microorganisms, research on methods for separating or extracting the enzymes from microbial culture fluids has been limited. There are very few cases, and no separation or extraction method has been found that would enable mass production of these enzymes on an industrial scale.

For example, conventional extraction methods known for boat fishing include:
There is a method of extracting colloidal chitin by adding colloidal chitin to a culture solution and adsorbing a chitin-degrading enzyme to the colloidal chitin, and a method of extracting colloidal chitosan by adsorbing a chitosan-degrading enzyme to colloidal chitosan in the same way. .

<Problems to be Solved by the Invention> However, such an extraction method requires extremely complicated operations, and the yield of chitin-degrading enzyme is about 30%, and the yield of chitosan-degrading enzyme is about 20%. Therefore, although it is suitable as an extraction method on a laboratory scale, it is unsuitable for mass production on an industrial scale, and the production cost becomes significantly high when producing with this extraction method. It had a point.

Due to the above-mentioned circumstances, applied research on chitin and chitosan decomposition products or research on the enzymes themselves have been hindered in the past. , it was desired to establish an extraction method.

The present invention was made against the above-mentioned background, and it is possible to separate chitin-degrading enzymes and chitosan-degrading enzymes from a culture solution containing these enzymes with an extremely simple operation and in a higher yield than conventional methods. The purpose of this invention is to provide a separation method that can be carried out on an industrial scale.

Means for Solving Problems〉 As a result of extensive research into methods for separating chitin-degrading enzymes and chitosan-degrading enzymes from the culture fluid, the present inventors have found that the culture fluid is separated into multiple chitin-degrading enzymes and chitosan-degrading enzymes with different molecular weight fractions. It was discovered that the enzyme can be efficiently separated by an extremely simple operation of sequential treatment with an ultrafiltration membrane, and the present invention was completed based on this finding.

That is, in the present invention, a culture solution containing a chitin-degrading enzyme and a chitosan-degrading enzyme is first treated with an ultrafiltration membrane having a molecular weight cutoff of 200,000 to 500,000 to produce the enzymes that contain almost no of the enzymes. The resulting concentrated solution of microbial cells is separated into a permeate containing the enzymes and decomposed products of chitin and chitosan, and then the permeate is treated with an ultrafiltration membrane with a molecular weight cutoff of 50,000 to 100,000. Separate the tacondensate of the chitin-degrading enzyme and the permeate containing the chitosan-degrading enzyme and the decomposed products, and further treat the permeate with an ultrafiltration membrane with a molecular weight cutoff of 10,000 to 30,000 to remove the chitosan-degrading enzyme. This method is characterized in that it is separated into a concentrated liquid and a permeated liquid containing the decomposed products.

Effect〉 The present invention will be explained in detail below using the drawings.

For example, when separating chitin-degrading enzymes and chitosan-degrading enzymes from a culture solution in which microorganisms that produce chitin-degrading enzymes and microorganisms that produce chitosan-degrading enzymes are cultured using the shells of crabs, shrimps, etc. as substrates, the culture solution The culture solution is pretreated by centrifugation or other means to remove relatively coarse suspended matter such as shells and high-molecular proteins in the culture solution, and the culture solution with these removed is processed through the flow shown in the diagram. Process accordingly.

That is, a culture solution from which crab, shrimp shells, high molecular weight proteins, etc. have been removed is temporarily stored in a stock solution tank 1, and then the culture solution is passed through an ultrafiltration membrane 2 with a molecular weight cutoff in the range of 200,000 to 500,000. is fed to the first ultrafiltration membrane device 3 equipped with an ultrafiltration membrane device 3 for treatment. In this process, only extremely large molecules such as microbial cells, chitin, and chitosan in the culture solution are removed using an ultrafiltration membrane 2.
A permeated liquid 4 that does not contain these substances is obtained, and a non-permeated liquid 5 in which the blocked substances are concentrated is circulated to the stock solution tank 1. The obtained permeate 4 contains chitin-degrading enzymes, chitosan-degrading enzymes, and decomposed products of chitin and chitosan that have passed through the ultrafiltration membrane 2, so they are led to the subsequent intermediate tank 6 and temporarily stored. Then, it is subjected to the ultrafiltration membrane treatment in the next step.

Through this circulation process while taking out the permeated liquid 4, the microbial cells, etc. are concentrated in the stock solution tank 1, and the solution in the tank 1 contains almost no chitin-degrading enzyme and chitosan-degrading enzyme. Since it no longer exists, the process is stopped at that point.

The concentrated solution finally remaining in the stock solution tank 1 is used again, for example, for culturing microorganisms as described above.

In addition, in the so-called batch processing as described above, as the processing continues, the concentration of the solution in the stock solution tank 1 increases, and the amount of liquid permeated through the ultrafiltration membrane device 3 decreases, or microbial cells etc. In some cases, impurities with large particle sizes may easily leak out to the permeate side, but in order to eliminate this problem, temporarily add water to the stock solution tank 1.
Alternatively, the treatment may be performed while diluting the solution in the stock solution tank 1 by adding it continuously. If such an operation is performed, the concentrated liquid or permeated liquid finally obtained will be diluted. However, on the other hand, the enzymes in the stock solution tank 1 can be effectively "washed out" to the permeate side, and the amount of enzymes ultimately remaining in the stock solution tank 1 can be significantly reduced. Of course, this operation can also be applied to the subsequent ultrafiltration membrane treatment.

Here, the reason why the molecular weight cutoff of the ultrafiltration membrane 2 used in the first ultrafiltration membrane device 3 is set in the range of 200,000 to 500,000 is that the ultrafiltration membrane with a molecular weight cutoff exceeding 500,000 If used, microbial cells, etc. will leak into the permeate side, reducing the purity of the chitin-degrading enzyme or chitosan-degrading enzyme that is finally obtained, so it is not preferable. This is because if a membrane is used, part of the chitin-degrading enzyme and chitosan-degrading enzyme will be inhibited by the membrane and transferred to the concentrated liquid side, resulting in a decrease in the yield of these enzymes, which is undesirable.

Next, the permeated liquid 4 stored in the intermediate tank 6 is
It is fed to a second ultrafiltration membrane device 8 equipped with an ultrafiltration membrane 7 in the range of 10,000 to 100,000 for treatment.

In this treatment, chitin degrading enzymes such as chitinase and chitobiase with a molecular weight of about 110,000, which are contained in the permeate 4 of the first ultrafiltration membrane device 3, are blocked on the membrane surface of the ultrafiltration membrane 7. A permeated liquid 9 that does not contain the chitin-degrading enzyme is obtained, and a non-permeated liquid 10 in which the chitin-degrading enzyme is concentrated is circulated to the intermediate tank 6. Obtained permeate 9
It contains the chitosan-degrading enzyme that passed through the ultrafiltration membrane 7, and the decomposed products of chitin and chitosan.
This is led to the intermediate tank 11 in the latter stage, where it is temporarily stored and subjected to the ultrafiltration membrane treatment in the next step.

Through this circulation process, it is possible to obtain a concentrated solution containing chitinolytic enzymes in high concentration and purity in the intermediate tank 6. Therefore, the process is stopped at an appropriate time and the concentrated solution is transferred to the tank 6.
Take it outside. The concentrated solution can be used as it is as a chitinolytic enzyme solution, or it can be used directly as a chitinolytic enzyme solution. It is also possible to obtain a powdered enzyme that is further purified from the ffi condensate by freeze-drying or other means.

Here, the reason why the molecular weight cutoff of the ultrafiltration membrane 2 used in the second ultrafiltration membrane device 8 is set in the range of 50,000 to 100,000 is that an ultrafiltration membrane with a molecular weight cutoff exceeding 100,000 is used. If used, a part of the chitin-degrading enzyme will leak into the permeate side, reducing the purity of the chitosan-degrading enzyme obtained in the next step, so it is not preferable. This is because if a membrane is used, a part of the chitosan degrading enzyme is blocked by the membrane and transferred to the concentrated solution, which reduces the purity of the obtained chitin degrading enzyme solution, which is not preferable.

Furthermore, the permeated liquid 9 stored in the intermediate tank 11 has a molecular weight cut-off of 1.
The permeated liquid 9 of the second ultrafiltration membrane device 8 is fed to the third ultrafiltration membrane device 13 equipped with an ultrafiltration membrane 12 in the range of 10,000 to 30,000 and treated. The chitosan-degrading enzyme (chitosanase with a molecular weight of about 40,000) contained therein can be blocked on the membrane surface of the ultrafiltration membrane 12, and a permeated liquid 14 that does not contain the chitosan-degrading enzyme can be obtained. The concentrated non-permeate liquid 15 is transferred to the intermediate tank 11.
circulates.

Through this circulation process, it is possible to obtain a solution in tank m containing chitosan-degrading enzyme at a high concentration and with high purity in the intermediate tank 11. Therefore, the process is stopped at an appropriate time and the concentrated liquid in the intermediate tank 11 is removed. Take out as a chitosan degrading enzyme solution. As in the case of the chitinolytic enzyme solution described above, this solution also contains
This can be used as it is, or a powdered enzyme can be further purified from the concentrated solution by freeze-drying or the like. Note that the third ultrafiltration membrane device 13
The permeate liquid 14 contains very useful substances such as monomers and polymers of N-acetylglucosamine, which is a decomposition product of chitin, and monomers and polymers of acetylglucosamine, which is a decomposition product of chitosan. The permeate 14 can also be recovered and used.

Here, the reason why the molecular weight cutoff of the ultrafiltration membrane 12 used in the third ultrafiltration membrane device 13 is set in the range of 10,000 to 30,000 is that the ultrafiltration membrane with a molecular weight cutoff exceeding 30,000 However, if an ultrafiltration membrane with a cutoff molecular weight of less than 10,000 is used, the chitosan-degrading enzyme will leak into the permeate side, reducing the yield of the enzyme, and if an ultrafiltration membrane with a molecular weight cutoff of less than 10,000 is used, the membrane will cause This is because some of the decomposed products are blocked and transferred to the concentrated solution, which undesirably reduces the purity of the obtained chitosan-degrading enzyme solution.

In addition, the above operation is preferably performed at a low temperature of about 5 to 10° C. in order to minimize the deactivation of each enzyme during the operation.

In addition, in the above explanation, each ultrafiltration membrane treatment was explained as a complete batch process to facilitate understanding, but for example, while processing is performed by the first ultrafiltration membrane device, the second ultrafiltration membrane treatment It goes without saying that the membrane device may be used to treat the permeate from the first ultrafiltration membrane device.

The ultrafiltration membranes applicable to the present invention may be of any type as long as they have a molecular weight cut-off within the range described above, and their shape, material, etc. are not limited.

<Effects> According to the present invention, a chitin-degrading enzyme and a chitosan-degrading enzyme can be obtained by an extremely simple operation of sequentially treating a culture solution containing a chitin-degrading enzyme and a chitosan-degrading enzyme using a plurality of ultrafiltration membranes having different molecular weight cutoffs. It is possible to separate each of the chitosan-degrading enzymes independently, and it is also possible to obtain the chitosan degrading enzymes in a higher yield than in the conventional method. Therefore, the method of the present invention enables the mass production of these enzymes on an industrial scale, making it possible to produce these enzymes at a much lower cost than conventional methods. Furthermore, as mass production becomes possible, it will become easier to obtain these enzymes, and it is therefore expected that applied research on decomposed products of chitin and chitosan and research on the enzymes themselves will become more active. The contribution it makes to development is extremely large.

Furthermore, in the present invention, after separating the enzymes, N
- It is possible to obtain a solution containing a large amount of useful substances such as glucosamine and other impurities, and the solution has the advantage that it can also be effectively utilized.

<Example> The present invention will be explained in detail below.

A mixed microbial system of microorganisms that produce chitin-degrading enzymes and microorganisms that produce chitosan-degrading enzymes (a mixed system of one strain of Flavobacterium, one strain of Pseudomonas, and two unidentified strains) was decalcified using phosphate. The cells were cultured in a medium containing crab shell powder as the sole substrate at 30° C. for 10 days using a conventional method to obtain a culture solution containing chitin-degrading enzyme and chitosan-degrading enzyme. The obtained culture solution was treated with a centrifuge to remove relatively coarse suspended matter such as crab shells and high-molecular proteins, and this was sequentially processed according to the flow shown in the drawing. The ultrafiltration membrane devices used in the first to third ultrafiltration membrane devices were all manufactured by Romicon, and each had a large number of hollow fiber ultrafiltration membranes with an inner diameter of 1.1 mm and an outer diameter of 169 mm. It is packed in a 2-inch cartridge,
The membrane area is 0.46 rJ in both cases.

In addition, the ultrafiltration membrane used had a molecular weight cutoff of 500,000 (membrane material polysulfone) that of the first ultrafiltration membrane device.
, the second ultrafiltration membrane device has a molecular weight cutoff of 150,000 (the membrane material is a hydrophilic polymer), and the third ultrafiltration membrane device has a molecular weight cutoff of 10,000 (the membrane material is a hydrophilic polymer). polymer).

The processing conditions were a liquid temperature of 10°C, an operating pressure of each ultrafiltration membrane device, an inlet pressure of 1.75 kg/cd, and an outlet pressure of 1.75 kg/cd.
0kg/all, and the circulation flow rate of non-permeate is 900R/
I went as H. Note that the flow rate of the permeate at this time is
That of the ultrafiltration membrane device was about 18.417H1, that of the second ultrafiltration membrane device was about 13.8ffi/H1, that of the third ultrafiltration membrane device was about 32.21/H .

Under the above conditions, culture solution 4 (1) was first treated with the first ultrafiltration membrane device to obtain a 5I1 concentrate and a 35/3 permeate, and then the 35β j3 filtrate was was treated with the second ultrafiltration membrane device to obtain a concentrated liquid 5β in which the chitin-degrading enzyme was concentrated and a permeated liquid 301.The ?R condensate liquid obtained by the treatment with the second ultrafiltration membrane device The activity of the chitinolytic enzyme was measured and the yield of the enzyme was found to be approximately 40%.
Met.

Further, the 30 g of the permeate was treated with a third ultrafiltration membrane device to concentrate the chitosan-degrading enzyme, resulting in a concentrated liquid 51 and a permeated liquid 2.
51 was obtained. The chitosan degrading enzyme activity in the obtained concentrate was measured, and the yield of the enzyme was determined to be approximately 35%.

[Brief explanation of the drawing]

The drawing is an explanatory diagram of a flow showing an example of an embodiment of the present invention. 1... Stock solution tank 2... Ultrafiltration membrane 3...
First ultrafiltration membrane device 4... Permeated liquid 5... Non-permeated liquid 6... Intermediate tank 7... Ultrafiltration membrane 8... Second ultrafiltration membrane device 9...・Permeated liquid 10...Non-permeated liquid 11...
Intermediate tank 12... Ultrafiltration membrane 13... Third ultrafiltration membrane device

Claims (1)

[Claims] A culture solution containing chitin-degrading enzyme and chitosan-degrading enzyme,
First, a concentrated solution of microbial cells producing the enzymes, which is treated with an ultrafiltration membrane with a molecular weight cutoff of 200,000 to 500,000 and hardly contains the enzymes, and a decomposed product of the enzymes, chitin, and chitosan. The permeate is then treated with an ultrafiltration membrane with a molecular weight cutoff of 50,000 to 100,000 to obtain a concentrated solution of chitin-degrading enzyme and a permeate containing chitosan-degrading enzyme and the decomposed product. The permeated liquid is further treated with an ultrafiltration membrane having a molecular weight cutoff of 10,000 to 30,000 to separate it into a concentrated liquid of chitosan degrading enzyme and a permeated liquid containing the decomposed product. A method for separating chitin-degrading enzyme and chitosan-degrading enzyme.