JP2005052066A - Method for producing acylated sericin - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for acylating sericin under mild conditions. <P>SOLUTION: Paying attention to the fact that a lipase has been applied to synthesizing a sugar fatty acid ester as one of food emulsifiers, the fact is applied to acylating a cocoon-derived sericin. The method for producing the acylated sericin comprises reacting sericin isolated from cocoon with an organic acid in the presence of a lipase in a non-aqueous solvent. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for acylating sericin, and more particularly to a method for producing acylated sericin by acylating sericin, which is a residue obtained by removing silk from silkworms, under mild conditions in the presence of lipase.

Sericin is a water-soluble natural high molecular weight protein that constitutes the silk thread spouted from the silk gland of Bombyx mori. Approximately 70% of the total weight of the silk thread is made up of a protein called fibroin, which becomes silk thread, and the remaining 20-30% is sericin. The structure surrounds fibroin (Non-Patent Document 1). These two proteins account for about 97% of the silk thread, the remaining components are about 1.2-1.6% carbohydrates, 1% lipids, 1% pigments, 0.7% minerals, these proportions are the varieties of silkworms It will vary slightly. As for the amino acid composition of sericin, serine is very much contained at about 33%, and when combined with threonine and tyrosine, about 46% is a hydroxy amino acid.
Since silky sericin is unnecessary to produce its unique luster and texture as a silk product, most of it has been removed and discarded without being effectively used. The production of firewood is about 1 million tons worldwide, which is equivalent to 400,000 tons of dry firewood. About 50,000 tons of sericin is produced in this raw silk processing step (Non-patent Document 2). Discarding this is not only a loss of protein resources, but also raises the BOD (Biochemical Oxygen Demand) of the wastewater, which necessitates considerable costs for wastewater treatment. Therefore, there is an urgent need to develop a technology that effectively uses sericin as an unused protein resource.

As a method of acylating sericin and using it for cosmetics and drugs, it is known to hydrolyze silkworm residue of silkworm with acid and acylate the obtained peptide or amino acid with fatty acid or the like (Patent Document 1). ). However, in order to carry out hydrolysis, there are problems such as the need for a strong acid-resistant facility, and a method for acylating sericin under milder conditions has been demanded.
On the other hand, lipases have been applied to the synthesis of sugar fatty acid esters in food applications (Patent Documents 2, 3 etc.), but examples of applying lipases to acylation of peptides and amino acids such as sericin of the present invention are known. It wasn't.

Supervised by Kazuo Mama “Science of Silk” Culture Publishing Bureau (1990) Biotecnology Advances., 20, 91-100 (2002) JP 06-256274 JP 62-289190 JP 08-245680

  An object of the present invention is to provide a method for producing an acylated sericin, which can effectively utilize sericin, which is a silk residue of straw, and can acylate sericin under mild conditions.

The present inventors have noted that lipase has been applied to the synthesis of sugar fatty acid esters, which are one of food emulsifiers (Patent Documents 2, 3, etc.), and applied to acylation of sericin derived from koji. By thinking and studying the conditions, the present invention has been completed.
That is, the present invention is a process for producing an acylated sericin comprising reacting sericin separated from soot with an organic acid in a non-aqueous solvent in the presence of lipase.
Furthermore, the present invention is a surfactant, emulsifier or humidity control agent mainly composed of acylated sericin obtained by this production method.

The sericin used in the present invention is obtained by removing silk from cocoons. The method for separating sericin from the koji is not particularly limited, but can be performed, for example, as follows.
The cocoon or string is immersed in electrolytic alkaline water (pH 11.5), extracted by heating at 95 ° C. for 7 hours, cooled and dried. As the heat extraction time becomes longer, the molecular weight decreases and coloring occurs, so about 7 hours is appropriate. Drying can be performed by various methods such as freeze drying, spray drying, and reduced pressure drying, but the former two are desirable because the dried product is easy to handle in the form of cotton or powder. In the case of drying under reduced pressure, the dried product becomes a film.

The lipase used in the present invention is preferably a lipase derived from Candida antractica or Candida cylindrea. Especially as lipase, Candida
Novozym 435 (Novozymes Japan) derived from antractica and lipase OF (named sugar industry) derived from Candida cylindrea or their equivalents are preferred.
In addition, it is preferable to use a lipase immobilized so that the enzyme can be easily separated and recovered.

Using this lipase, hydroxyl groups such as serine of sericin are acylated with an organic acid. In the esterification reaction, removal of water is essential to suppress the hydrolysis reaction, and a non-aqueous solvent is used as the solvent. The non-water means 5% or less of moisture, preferably 1% or less, and most preferably means drying using a dehydrating agent such as molecular sieve. Examples of such a solvent include hexane, heptane, octane, isooctane, chloroform, ethyl acetate, acetone, benzene, petroleum ether and the like. However, acetone is not preferable because it contains a small amount of aldehyde and a side reaction product is produced and browns.
It is desirable to dehydrate the solvent during the reaction in order to dehydrate the solvent that dissolves the organic acid, particularly fatty acid, which is one of the substrates, and to remove the water produced by acylation. For example, the solvent is distilled or a dehydrating agent is added.
The amount of organic acid, particularly fatty acid, in the reaction solution is preferably about 1 to 5 times the equivalent of the hydroxyl group of sericin, and the amount of lipase is preferably about 1 to 10 times the weight of sericin. Since sericin is insoluble in organic solvents, for example, when the immobilized lipase and sericin come into contact, the acylation reaction is considered to proceed only when the sericin comes into contact with the hydroxyl group on the sericin surface. Is preferably increased at such a concentration that can always be maintained. Therefore, it is preferable to stir the reaction solution during the reaction.

The reaction temperature is preferably 35 to 75 ° C, more preferably 50 to 65 ° C.
The organic acid is an organic compound having a carboxyl group, and the kind thereof is not particularly limited, but is preferably a fatty acid. Examples of fatty acids include caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, caproleic acid, Linderic acid, myristoleic acid, palmitoleic acid, oleic acid, cadreic acid Acid, erucic acid, decadienoic acid, linoleic acid, hiragoic acid, linolenic acid, eicosatrienoic acid, docosatrienoic acid, hexadecatetraenoic acid, stearidonic acid, arachidonic acid, docosatetraenoic acid, eicosapentaenoic acid, succinic acid, sabinin Examples include acids, iprolic acid, yarapinolic acid, ricinoleic acid, and ferronic acid.
There is no particular limitation on the degree of esterification. In the examples described later, the reaction product (crude acylated sericin) is purified by salting out, but it is considered that it is practically effective without purification.
The acylated sericin obtained according to the present invention has excellent emulsifying properties, as shown in the examples described later, and therefore has an emulsifier for foods and cosmetics, moisture evaporation suppression, and high sorption to wool. And can be used as a hair conditioner, a surfactant, and the like.

The following examples illustrate the invention, but are not intended to limit the invention.

(1) Separation of sericin from rice cake The white birch layer (dry rice cake) is immersed in electrolytic alkaline water (pH 11.5), heated and extracted at 95 ° C for 7 hours, filtered with 10% ethanol added, and sericin solution (sericin MY -30N, Shinko Silk, Wakayama). After dialysis of this sample solution against distilled water, pH was adjusted using 0.1M sodium hydroxide.
The mixture was adjusted to 7.0 and incubated for 1 hour with stirring in a 60 ° C. water bath. Next, the supernatant collected by centrifugation (room temperature, 12,000 rpm, 30 minutes) is collected into a membrane filter (pore
size 0.45 μm), and the filtrate was freeze-dried to obtain sericin.

The obtained sericin was subjected to SDS-polyacrylamide electrophoresis (SDS-PAGE) and size exclusion chromatography. Sericin is composed of various molecules, and the state of sericin obtained varies depending on the conditions under which hot water is extracted from the silk thread. This sericin preparation also had a molecular weight that was not uniform and polydispersed, and had a molecular weight of about 10,000 to 120,000 Da and a main molecular weight of 18,700 Da.
The chemical characteristics of sericin are summarized in Table 1. The isoelectric point of sericin was 3.8, and fatty acids were present at a rate of 0.1% (w / w). The amino acid composition of this sericin preparation has been reported (Biosci.
Biotecnol. Biochem., 62 (1), 145-147 (1998)). Serine was 34.3%, and the total amount of hydroxyamino acids including serine was 45.9%. The total amount of polar neutral, acidic and basic amino acids was 43.8%, and the total amount of nonpolar amino acids was 11.2%. Since there are more acidic residues than basic amino acid residues, the isoelectric point is considered to be acidic.

(2) Acylation of sericin using lipase 1 g of sericin was weighed into a conical flask with a screw cap and dried by heating at 110 ° C. for 1 hour. After that, add 50 ml of hexane that has been dehydrated with molecular sieves (10 mg / ml) in advance, make sericin fine powder (24,000 rpm, about 1 minute) with Polytron PCU-2 (manufactured by KINEMATICA), then add about 2 g of molecular sieves. And deaerated. Oleic acid here
3.5 ml was added, and finally 10 g of Novozym 435 (Novozymes Japan) was added and purged with nitrogen and sealed. The Erlenmeyer flask was incubated at 60 ° C. for 96 hours while shaking at 60 times / min. After completion of the incubation, the reaction mixture was passed through a stainless steel mesh in hexane to remove the molecular sieve, and then the membrane filter (pore
The residue was collected by suction filtration at a size of 0.45 μm. This was immediately transferred to a beaker, mixed with chloroform / hexane (5: 1 (v / v)) and allowed to stand at room temperature for 1 hour, and then most of the enzyme floating on the liquid surface was removed together with the supernatant. In addition, a glass centrifuge tube (id
24 × 100 mm), and the mixture was added and centrifuged (room temperature, 3,000 rpm, 5 minutes) to remove the enzyme together with the supernatant. This operation was repeated until the enzyme particles were completely removed, and the precipitate was washed three times with hexane and then dried under reduced pressure. This is dissolved in distilled water, dialyzed against distilled water, and pH adjusted with 0.1M sodium hydroxide.
After adjusting to 7.0, suction filtration was performed with a membrane filter (pore size 0.45 μm). The yield of the crude acylated sericin preparation obtained by freeze-drying this filtrate was 84% (844 mg).
The crude acylated sericin was immediately put in a vacuum desiccator together with an oxygen absorber (A-2500HS, ISO Inc.), and then stored in a cold room until salting out.
In addition, it incubated similarly, without adding Novozym 435, and the sericin prepared by the same method was used as control sericin.

(3) Purification of crude acylated sericin Since the crude acylated sericin sample obtained was presumed to contain a large amount of unreacted sericin, it was purified by salting out.
Dissolve the crude acylated sericin in 0.2M acetic acid buffer (pH 4.5) to a concentration of 1%, gradually add ammonium sulfate with stirring to a final concentration of 35% and stir for 1 hour, then overnight. It was left in a low greenhouse. This is centrifuged (12,000
(rpm, 30 min, 4 ° C), the supernatant is left as it is, the precipitate is resuspended in distilled water, dialyzed, adjusted to pH 7.0 with 0.1 M sodium hydroxide, and the supernatant fraction is left as it is as a membrane filter (0.45). The precipitate fraction was dissolved by heating at 60 ° C., filtered in the same manner, and freeze-dried. The freeze-dried preparation of the salted out precipitate fraction was converted to acylated sericin, which was stored in a vacuum desiccator together with an oxygen absorbent until use. The yield of purified acylated sericin obtained by lyophilization was 8.4% (84 mg).

なお表中、Sericin-OAは、オレイン酸によりアシル化されたセリシンをいう。 Size exclusion chromatography was performed to determine the molecular weight of the resulting acylated sericin. Each sample was measured in triplicate, and the average value was defined as the main molecular weight. As shown in Table 2, the main molecular weight of acylated sericin was 22,000 Da.

In the table, Sericin-OA refers to sericin acylated with oleic acid.

The chemical analysis results of acylated sericin are shown in Tables 3 and 4.

表３の脂肪酸含量を見ると、アシル化セリシンはコントロールセリシン及びセリシンよりも脂肪酸含量が約10倍に増加している。また表４に示すように、アシル化セリシンのオレイン酸含量はコントロールセリシン及びセリシンより約19倍高かった。
これらの結果は、Novozym 435によるヘキサン中の固相エステル合成反応によりセリシンがアシル化されたことを示している。

Looking at the fatty acid content in Table 3, the acylated sericin has a fatty acid content approximately 10 times higher than the control sericin and sericin. Moreover, as shown in Table 4, the oleic acid content of acylated sericin was about 19 times higher than that of control sericin and sericin.
These results indicate that sericin was acylated by a solid phase ester synthesis reaction in hexane by Novozym 435.

Hereinafter, the surface active properties of the acylated sericin obtained in Example 1 were examined.
Test Example 1 Emulsification test Acylated sericin was dissolved in 0.1M phosphate-0.05M citrate buffer (McIlvaine buffer, pH 7.0) to a concentration of 0.1%. Take 2 ml of this solution as an aqueous phase and 0.5 ml of corn oil as an oil phase in a test tube.
An oil-in-water (o / w) emulsion was prepared by homogenizing at 24,000 rpm for 1 minute at 25 ° C. using Polytron PCU-2 (manufactured by KINEMATICA) equipped with 7).
After preparing the o / w emulsion, take 50 μl after 0, 10, 30, 60 and 120 minutes from the bottom of the aqueous phase while maintaining at 30 ° C using a thermostatic layer, and dilute 50 times with 0.1% SDS solution. After stirring, 500
The absorbance at nm was measured in triplicate to determine turbidity.
For comparison, sericin (the sericin before acylation), the control sericin and bovine serum albumin (BSA) (Wako Pure Chemical Industries) were used.
The emulsifying activity index (EAI) is determined by the method of Pearce and Kinsella (J. Agric. Food
Chem., 26, 716-723 (1978)).
EAI = 2 T / φC
T = 2.3 A / l (A = A ₅₀₀ , l = 10 ⁻² m (optical path length))
T: Turbidity, A: Absorbance at 500 nm taking into account dilution factor (A ₅₀₀ × 50)
φ = Oil phase ratio (= 0.2), C = Standard concentration (0.1% = 10 ³ g / m ³ )
The test results are shown in FIG. The acylation significantly increased the EAI value, indicating that the resulting acylated sericin has excellent emulsifying ability.

Test Example 2 Adsorption to oil droplets interface Emulsion was prepared in the same manner as in Test Example 1. Immediately after emulsification and 60 minutes after emulsification, 1.5 ml of the emulsion was taken from the bottom of the test tube and centrifuged at 1,000 rpm at 25 ° C for 5 minutes. Separation was carried out to separate an oil phase and an aqueous phase. Aliquot 1 ml of this water phase and again 20,000
Centrifugation was performed at rpm and 25 ° C. for 30 minutes, and another 0.7 ml was taken from this aqueous phase, diluted twice with the same buffer, and then centrifuged in the same manner. This operation was repeated twice, and the protein content of the obtained aqueous phase was determined by the microburette method (J.
Am. Oil Chem. Soc., 76, 1291-1295 (1999)), and the protein adsorption rate in the aqueous phase was calculated.
The results are shown in FIG. Immediately after emulsification (0 min), the added acylated sericin showed the highest adsorption rate of about 30%, followed by BSA (about 12%) and control sericin (about 9%). This result shows good adsorption of acylated sericin on the oil droplet interface.
From this result, it is considered that acylated sericin exhibits excellent emulsification activity and emulsion stability by adsorbing and orienting more molecules at the oil droplet interface.

Test Example 3 Moisturizing Test Acylated sericin and control sericin were each dissolved in distilled water, adjusted to pH 7.0 with 0.1M NaOH, stirred at 60 ° C. for 10 minutes, and then 18,000
Centrifuge for 10 minutes at rpm. The supernatant was filtered through a membrane filter (pore size 0.45 μm), then subjected to the microburette method, and the concentration was measured and adjusted to 2.0% (w / v). After collecting about 30 mg of this sample solution in a platinum open-type container, the differential thermothermal gravimetric simultaneous measurement device (TG / DTA
6200, Seiko Instruments Inc., Chiba), and the weight change of moisture content and the temperature difference from the reference were measured. The measurement conditions were from 30 ° C. to 180 ° C., a heating rate of 5 ° C./min, and 30 mg of sapphire plate (Al ₂ O ₃ ) was used as a reference without gas flow, and measurements were made in triplicate.
The evaporation rate at the peak top temperature of the differential thermogravimetric curve (DTG curve), which is a time differential curve of the TG curve useful for the analysis of the volatile sorbent desorption reaction, etc. was taken as the maximum evaporation rate (Vmax). If the sample has high hydrophilicity and moisture retention, TVmax shifts to a high temperature side in order to suppress moisture evaporation, and Vmax decreases because the amount of evaporation per unit time is also suppressed.
The results are shown in FIG. It can be seen that acylated sericin effectively suppresses evaporation of moisture.

It is a figure which shows the result of the emulsification test of the acylated sericin obtained in Example 1. It is a figure which shows the result of the adsorptivity test to the oil droplet interface of the acylated sericin obtained in Example 1. It is a figure which shows the result of the moisture retention test of the acylated sericin obtained in Example 1.

Claims (5)

A process for producing an acylated sericin comprising reacting sericin isolated from straw with an organic acid in a non-aqueous solvent in the presence of lipase. The process according to claim 1, wherein the lipase is a lipase derived from Candida antractica or Candida cylindrea. The process according to claim 1 or 2, wherein the organic acid is a fatty acid. The process according to any one of claims 1 to 3, wherein the reaction temperature is 35 to 75 ° C. A surfactant, emulsifier, or humidity control agent comprising as a main component acylated sericin obtained by the production method according to any one of claims 1 to 4.

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