JPH0160232B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for treating a crude pectinolytic enzyme solution. The present invention provides a method for treating a crude pectinolytic enzyme solution, which is characterized by ultrafiltering the crude pectinolytic enzyme solution in the presence of a nonionic or cationic surfactant. Pectin-degrading enzymes are widely distributed in microorganisms and plants, and are used in the food industry, cell fusion, etc., such as in the clarification of fruit juice and in the production of protoplasts. Ultrafiltration has conventionally been used to treat crude pectin-degrading enzyme solutions, but it is not successful because the pectin-degrading enzyme is adsorbed to the surface of the ultrafiltration membrane, resulting in a large loss of the enzyme. In the present invention, by adding a surfactant to the enzyme treatment solution or using a membrane treated with a surfactant, there is almost no loss of the pectin-degrading enzyme to be treated due to adsorption to the membrane, and pectin can be efficiently treated. This is based on the knowledge that degrading enzymes can process the process. It is an object of the present invention to provide an improved method for treating crude pectinolytic enzyme solutions by ultrafiltration. The method of the present invention is characterized by adding a nonionic or cationic surfactant to the crude pectin enzyme solution to be treated, or treating the ultrafiltration membrane with the above surfactant in advance. It is a feature. The present invention will be explained in detail below. The crude pectinolytic enzyme solution referred to in this specification is
For example, an enzyme solution is prepared by removing bacterial cells from a culture solution obtained by culturing microorganisms that produce pectin-degrading enzymes (e.g., Erwinia, Clostridium, Pseudomonas, Xanthomonas, Aeromonas, Bacillus, Asparagillus) in a pectin-containing medium. Of course, extracts from plants can also be used. For example, Erwinia
The main pectin-degrading enzyme produced by Carotovora is pectate lyase, but it also contains other enzymes such as pectin lyase. As the nonionic surfactant used in the present invention,
Polyoxyethylene alkyl ether (polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene stearyl ether, polyoxyethylene oleyl ether, etc.), polyoxyethylene alkyl phenyl ether (polyoxyethylene octylphenyl ether, polyoxy ethylene nonyphenyl ether, etc.), sorbitan fatty acid esters (sorbitan monolaurate, sorbitan monopalmitate,
sorbitan monostearate, sorbitan monooleate, etc.), polyoxyethylene sorbitan fatty acid esters (polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monooleate, etc.), polyethylene glycol fatty acid esters ( polyethylene glycol monolaurate, polyethylene glycol monostearate, etc.), fatty acid monoglycerides (glyceryl monostearate, glyceryl monooleate, etc.)
can be given. Examples of the cationic surfactant include alkylamine salts and quaternary ammonium salt type compounds, and more specifically, stearylamine acetate, coconut amine acetate,
Stearylamine hydrochloride, coconut amine hydrochloride, stearylamine oleate, lauryltrimethylammonium chloride, stearyltrimethylammonium chloride, distearyldimethylammonium chloride, lauryl betaine, stearyl betaine, lauryl dimethylamine oxide, benzalkonium chloride etc. The concentration of the surfactant used is 0.01 to 5% (weight %, same hereinafter). Next, treatment of the ultrafiltration membrane with a surfactant is
For example, do the following: The ultrafiltration membrane is treated with a chemical such as an aqueous solution of caustic soda alone, a solution prepared by adding sodium chloride to it, chlorine, sodium hypochlorite, an aqueous solution of chlorine dioxide, etc. to remove contaminants attached or adsorbed to the membrane surface. Next, an aqueous solution containing a nonionic or cationic surfactant is passed through the cleaned ultrafiltration membrane, or the membrane is immersed in the aqueous solution to modify the surface of the membrane. As the ultrafiltration membrane, membranes such as polysulfone and polyacrylonitrile, which are commonly used as ultrafiltration membranes, are used. Examples are shown below. Example 1 10ml of crude pectin-degrading enzyme solution obtained in Reference Example
Solution 10 to which 0.1 to 0.5% of Cortamine 24P [cationic surfactant (lauryl trimethyl ammonium chloride) manufactured by Kao Soap Co., Ltd.] was added.
ml of ultrafiltration membrane SEP1013 (polysulfon, manufactured by Asahi Kasei Corporation) equivalent to 50 cm ² of ultrafiltration membrane (inner diameter 0.8 mm, length equivalent to 2 m) is immersed. Next, the solution was shaken back and forth at 250 rpm for 24 hours at 7°C to sufficiently contact the membrane fibers, and then the amount of pectic acid lyase in the solution was measured. The recovery rate is defined as the ratio between the amount of pectate lyase remaining in the solution and the amount of pectate lyase present in the crude pectin-degrading enzyme solution. The results are shown in Table 1.

[Table] As is clear from Table 1, the recovery rate of pectate lyase increases in a nearly linear relationship with the concentration of cortamine 24P. Example 2 To 10 ml of a 0.1 to 0.7% aqueous solution of Cortamine 24P,
Soak an area equivalent to 50 cm ² of ultrafiltration membrane SEP1013 for 24 hours. The membrane was pulled up and shaken back and forth for 30 minutes with 10 ml of 2% sodium hypochlorite, then washed with water to firmly bind the surfactant to the membrane, and then washed 10 times with 10 ml of distilled water. do. Then,
The membrane was mixed with the crude pectin-degrading enzyme solution obtained in the reference example.
Pour into a test tube containing 10ml. After shaking at 7°C for 24 hours to bring the membrane fibers into sufficient contact with the enzyme, the amount of pectic acid lyase in the solution is measured. The recovery rate is defined as the ratio of the amount of pectate lyase remaining in the solution to the amount of pectate lyase present in the crude pectin-degrading enzyme solution. An untreated membrane of Cortamine 24P was used as a blank sample. The results are shown in Table 2.

[Table] As is clear from Table 2, the recovery rate of enzyme was significantly increased in the case of the treated membrane compared to the untreated membrane of Cortamine 24P. Example 3 Ultrafiltration membrane SEP1013 (equivalent to 50 cm ² of ultrafiltration membrane) was poured into 10 ml of solutions of Sanisol B-50 (benzalkonium chloride, manufactured by Kao Soap Co., Ltd.) with varying concentrations.
Shake back and forth for 24 hours at room temperature. This was taken out and added to 10ml of the crude enzyme solution obtained in the reference example, and heated to 7℃ for 24 hours.
After reciprocating shaking for an hour, determine the recovery rate of the enzyme.
The results are shown in Table 3.

[Table] The recovery rate is calculated based on the value obtained by maintaining the enzyme solution without adding an ultrafiltration membrane under the same conditions as 100%. Example 4 Ultrafiltration membrane SEP1013 (extra area equivalent to 50 cm ² ) was poured into 10 ml of solutions of Sanisol B-50 with different concentrations, shaken back and forth at room temperature for 24 hours, then taken out and diluted 50 times. Pour into 10ml of sodium hypochlorite and shake back and forth for 30 minutes. This was taken out and washed 10 times with 10 ml of distilled water, then placed in 10 ml of the crude enzyme solution obtained in the reference example, and after reciprocating shaking at 7°C for 24 hours, the recovery rate of the enzyme was examined. The results are shown in Table 4.

[Table] Comparing Examples 3 and 4, Sanizol B-
It is better to wash with sodium hypochlorite after treating with 50%
Enzyme adsorption is also suppressed when the concentration of the processing surfactant is low. Therefore, by washing with a sodium hypochlorite solution (50 times diluted) after treatment with Sanizol B-50, the surfactant can be firmly attached to the membrane surface. Examples 5 and 6 Instead of Cortamine 24P as a surfactant, Osban [cationic reverse soap (benzalkonium chloride 10% solution), manufactured by Daigo Nutrient Chemical Co., Ltd.] and Emulgen [nonionic surfactant (polyoxy The results shown in Table 5 were obtained in the same manner as in Example 1, except that ethyl lauryl ether (manufactured by Kao Soap Co., Ltd.) was used.

[Table] As shown in Table 5, the enzyme recovery rate was significantly improved by the addition of a surfactant. Example 7 Ultrafiltration membrane SEP1013 (excess area equivalent to ^50cm2 )
Pour into 10 ml of 0.4% Sanizol B-50 solution and shake reciprocally (250 rpm) at room temperature for 24 hours. Pull up the fiber and wash it with water. 10ml per serving
The enzyme titer was determined by using distilled water with different washing times, adding each to 10 ml of the crude enzyme solution obtained in the reference example, and shaking it back and forth at 7°C for 24 hours. The reduction was measured and the peeling status of the surfactant was investigated depending on the number of water washings. After 10 washes, the recovery rate decreased to 28.1%. Separately, after immersing in Sanizol B-50 under the same conditions and washing once with water, it was poured into 10 ml of commercially available sodium hypochlorite diluted 50 times (2%) and allowed to stand for 30 minutes at room temperature. Shake back and forth for a minute. After pulling up the membrane and washing it 10 times with 10ml of distilled water,
When the crude enzyme was poured into an enzyme solution in the same manner as above and the recovery rate of the crude enzyme was examined, there was almost no peeling of the surfactant due to water washing. The recovery rate at this time is
It was 83.0%. For these reasons, surfactant (Sanisol B
-50) treatment of the ultrafiltration membrane alone can prevent enzyme adsorption to the membrane surface, but cleaning with sodium hypochlorite further strengthens the bond between the surfactant and the membrane surface. . Example 8 Sanisol B-50 0.5% solution 1 was added to ultrafiltration membrane SEP1013 (over area 0.2 m ² , separated molecular weight 3000) for 24 hours.
After treatment by circulating at a flow rate of 280 ml/min for 30 minutes, 500 ml of 2% sodium hypochlorite was circulated for 30 minutes. Then, it is washed twice with distilled water. After using 500 ml for the first and second times and draining it, the crude enzyme solution 3 obtained in the reference example was subjected to ultra-superconcentration. After 456 minutes of circulation at a temperature of 2° C. and a flow rate of 230 ml/min, the recovery rate of pectate lyase was 98% at a concentration rate of 10 times. The results are shown in Table 6. On the other hand, when concentration was performed using Sanizol B-50 untreated membrane, the recovery rate after concentration under similar conditions was 81
It was %.

[Table] Reference example: Erwinia carotovora GIR1044 (Feikoken Bacteria No. 7576) was grown in a medium (composition: pectin 5.0g/,
L-glutamine monosodium 5.0g/,
(NH ₄ ) ₂ SO ₄ 3.0g/, KH ₂ PO ₄ 2.4g/,
Na ₂ HPO ₄ 0.8g/, MgSO ₄・7H ₂ O 0.2g/,
Incubate under aeration for 24 hours at 28 °C (PH6.8−7.0). Centrifuge the culture solution (12000 x g, 10 minutes),
Collect the supernatant and use it as a crude pectin-degrading enzyme solution.

Claims (1)

[Scope of Claims] 1. A method for treating a crude pectin-degrading enzyme solution, which comprises the step of ultrafiltering the crude pectin-degrading enzyme solution in the presence of a nonionic or cationic surfactant. 2. The method according to claim 1, wherein a surfactant is added to the crude pectinolytic enzyme solution and then ultrafiltered. 3. The method according to claim 1, which uses an ultrafiltration membrane treated with a surfactant.