KR102211892B1 - Silkpeptide production method using high pressure and enzyme treatment - Google Patents

Abstract

The present invention provides a method for producing silk peptides by a high pressure enzyme method, characterized in that the silk fibroin is hydrolyzed under a high pressure of 50 to 100 MPa using a protease, and according to the present invention, a protein for silk fibroin Single proteolytic enzymes or complex enzymes with weak decomposition power can also be used, thereby producing low-molecular peptides/proteins with a molecular weight of 4-10 kD, peptides with 1 to 2.5 kD, and silk amino acids from silk fibroin. It can provide an effect that can increase the decomposition efficiency by 10 to 40%.

Description

Production method of silk peptide by high pressure enzyme method {SILKPEPTIDE PRODUCTION METHOD USING HIGH PRESSURE AND ENZYME TREATMENT}

The present invention relates to a method for producing silk peptides, and more particularly, by a simple process using a single or complex enzyme at high pressure, a single proteolytic enzyme or a complex enzyme having weak proteolytic power for silk fibroin can be used, This allows the production of low molecular weight peptides/proteins with a molecular weight of 4 to 10 kD, peptides of 1 to 2.5 kD, and silk amino acids from silk fibroin, and provides the effect of increasing protein hydrolysis efficiency by 10 to 40%. It relates to a method for producing silk peptides by an enzyme method.

Silk (Cocoon) is composed of fibroin (75%) and sericin (25%), and is used as a food material and functional cosmetic material by manufacturing silk peptide or silk amino acid using fibroin (silk protein) from which sericin has been removed through a refining process. In addition, many studies are being conducted on silk proteins themselves as biomaterials with biocompatibility.

Silk protein (fibroin) is a protein that is insoluble and maintains a certain strength due to β-sheet form such as cellulose, and all silk peptides and amino acids are being used by acid-treated hydrolysis.

However, the acid-treated hydrolysis method produces silk peptides and silk amino acids through complex steps of neutralization, decolorization, deodorization, desalting, concentration, and drying for 7 days or more after decomposing silk proteins at high temperature and strong acid. It has problems such as protein denaturation by chemicals and high-temperature treatment, irregular molecular weight, and low production yield.

Accordingly, there is a need to develop a technology capable of producing eco-friendly, high-efficiency, and high-quality products from silk proteins with low molecular weight silk peptides and silk amino acids.

The present invention was conceived to solve the problems of the prior art as described above, and its object is a single proteolytic enzyme or complex with weak proteolytic power to silk fibroin by a simple process using a single or complex enzyme at high pressure. Enzymes can also be used, thereby producing low molecular weight peptides/proteins with a molecular weight of 4-10 kD, peptides of 1 to 2.5 kD, and silk amino acids from silk fibroin, and increasing the protein hydrolysis efficiency by 10 to 40%. It is to provide a method for producing silk peptides by a high-pressure enzyme method that provides a possible effect.

The technical problem of the present invention as described above is achieved by the following means.

(1) A method for producing silk peptides by a high-pressure enzyme method, characterized in that silk fibroin is hydrolyzed at a high pressure of 50 to 100 MPa using a protease.

(2) In the above (1), the protease is at least one selected from the group of alcalase, alpalase, food pro, protamex, flavozyme, pepsin, bromelain, and cholupurin. Silk peptide production method by the high-pressure enzyme method.

(3) The method for producing a silk peptide according to the above (1), wherein the proteolytic enzyme contains flavozyme as a complex enzyme.

(4) The method for producing a silk peptide according to the above (1), wherein the proteolytic enzyme contains any one selected from pepsin, bromelain, and cholupurine, which has a low degrading activity of silk fibroin at normal pressure.

(5) The method for producing a silk peptide according to (3) above, wherein the proteolytic enzyme contains any one selected from pepsin, bromelain, and cholupurine, which has a low degrading activity of silk fibroin at normal pressure.

(6) The method for producing silk peptides using acid-treated hydrolysis according to the above (1), wherein at least one selected from citric acid, gluconic acid, and tartaric acid is further included in the reaction.

(7) The method for producing silk peptides using acid-treated hydrolysis according to (1) above, wherein citric acid, gluconic acid, and tartaric acid are included in the reaction.

According to the configuration of the present invention as described above, by a simple process using a single or complex enzyme at high pressure, it is possible to use a single protease or a complex enzyme having a weak proteolytic power to silk fibroin, whereby the molecular weight from silk fibroin It can produce low molecular weight peptides/proteins of 4 to 10 kD, peptides of 1 to 2.5 kD, and silk amino acids, and provides the effect of increasing protein hydrolysis efficiency by 10 to 40%.

1 is a molecular weight measurement result of silk fibroin protein from SEC (molecular exclusion chromatography) using a solubilized silk fibroin solution.
2 is an SDS-PAGE of solubilized silk fibroin.
3 is a result of enzymatic hydrolysis of silk fibroin using a single enzyme.
Figure 4 is a molecular weight measurement result of silk fibroin hydrolyzate from SEC (molecular exclusion chromatography) using a single enzyme (alcalase, alpalase, food pro, protamex, flavozyme) at atmospheric pressure.
5 is a molecular weight measurement result of silk fibroin hydrolyzate from SEC (molecular exclusion chromatography) using a single enzyme (bromelain, pepsin, cholupurin, promod 278, flavozyme) at atmospheric pressure.
6 is a molecular weight measurement result of a silk fibroin solution solubilized from SEC (molecular exclusion chromatography) at high pressure compared to atmospheric pressure.
7 is a molecular weight measurement result of silk fibroin hydrolyzate using a single enzyme (flavozyme) at high pressure.
Figure 8 is the molecular weight measurement results of silk fibroin hydrolyzate using a single enzyme (Food Pro) at high pressure.
9 is a molecular weight measurement result of silk fibroin hydrolyzate using a single enzyme (alfalase) at high pressure.
Figure 10 is a molecular weight measurement result of silk fibroin hydrolyzate using a single enzyme (Protamex) at high pressure.
11 is a molecular weight measurement result of silk fibroin hydrolyzate using a single enzyme (cholupurin) at high pressure.
12 is a molecular weight measurement result of silk fibroin hydrolyzate using a single enzyme (bromelain) at high pressure.
13 is a result of molecular weight measurement of silk fibroin hydrolyzate using a single enzyme (pepsin) at high pressure.
14 is a measurement result of hydrolysis of silk fibroin using a complex enzyme at high pressure compared to atmospheric pressure.
15 is a molecular weight measurement result of silk fibroin hydrolyzate using a complex enzyme (food pro + flavozyme) at atmospheric pressure/high pressure.
Figure 16 is the molecular weight measurement results of silk fibroin hydrolyzate using a complex enzyme (cholupurin + flavozyme) at atmospheric pressure / high pressure.
17 is a result of molecular weight measurement of silk fibroin hydrolyzate using a single enzyme (Food Pro) at high pressure.
18 is a molecular weight measurement result of silk fibroin hydrolyzate using a single enzyme (flavozyme) at high pressure.
19 is a molecular weight measurement result of silk fibroin hydrolyzate using a complex enzyme (food pro + flavozyme) at high pressure.

The present invention provides a method for producing silk peptides and silk amino acids from silk fibroin by a simple process using protease and ultra-high pressure.

In the present invention, by selecting a solvent composition capable of solubilizing poorly soluble silk fibroin, the β-sheet form of silk fibroin is converted to an amorphous state as much as possible to maximize hydrolysis of protease. To this end, in the present invention, a low molecular weight silk peptide and a silk amino acid are prepared by maximizing the decomposition efficiency of the protease by relaxing the protein structure in the form of a β-sheet due to the treatment of ultra-high pressure, preferably 50 to 100 MPa.

In the present invention, the proteolytic enzyme does not require special limitation, and not only enzymes having high proteolytic efficiency for silk fibroin, but also low enzymes (eg, pepsin, bromelain, cholupurin, etc.) can be used. to be. For example, alcalase, alpalase, foodpro, protamex, flavozyme, pepsin, bromelain, cholupurin, or other enzymes alone or two or more complex enzymes may be used.

In the present invention, the protease preferably includes flavozyme as a complex enzyme.

In the present invention, the proteolytic enzyme may be added in an amount of 0.1 to 10% by weight, and if it is added in an amount of less than 0.1% by weight, there is a concern that the decomposition efficiency will be low. It is not enough and the above range is preferable.

In the present invention, preferably, at least one selected from citric acid, gluconic acid, and tartaric acid is included in order to accelerate the hydrolysis reaction. More preferably, citric acid, gluconic acid, and tartaric acid are used in a weight ratio of 0.5:1:0.5, and these are used in the range of 0.1 to 0.2% by weight, either individually or in a mixed state.

Hereinafter, the present invention will be described in more detail with the following experimental examples. However, this is to help easier understanding of the present invention, but is not intended to limit the present invention through these.

[Experiment method]

1. Analysis method for silk fibroin and hydrolyzed products

(1) Protein analysis of silk fibroin and hydrolysates

-Lowry method (J. Biol. Chem. 1951. 193:265-275) was used to quantitatively confirm protein degradation by enzymatic reaction.

-Add 1 mL of a mixture of 2% Na ₂ CO ₃ (0.1M NaOH), 1% CuSO₄, and 2% Potassium Sodium tartrate tetrahydrate in a ratio of 98:1:1 to 0.2 mL of the reaction solution that has undergone enzymatic reaction. After allowing to stand for 15 minutes, 0.1 mL of a 1:1 mixture of Folin-Ciocalteu's Reagent (Sigma-Aldrich, USA) and distilled water was added, and absorbance was measured at 595 nm after 30 minutes at room temperature. The standard material for measuring the protein concentration was BSA (Bovine serum albumin) Protein Standard (Sigma-Aldrich, USA).

(2) Analysis of free amino groups of silk fibroin and hydrolysates

-The free amino group, which is a decomposition product by enzymatic reaction, was quantified using the Ninhydrin method (J. Biol. Chem. 1915. 20:217-230).

-0.1 mL of Ninhydrin Reagent (Sigma-Aldrich USA) was mixed with 0.2 mL of the enzyme reaction solution, heated for 10 minutes, cooled to room temperature, and mixed with 0.5 mL of 95% ethanol, and the absorbance was measured at 570 nm. Glycine (Samchun, Korea) dissolved in 0.05% Acetic acid was used as a standard material for quantification of the free amino group.

(3) Analysis of amino acid composition of silk fibroin and enzyme degradation products

-The method of food code was applied and analyzed by Korea Health Functional Food Research Institute.

-Analysis of free amino acid composition

ㅇ HPLC system (1200 Series, Agilent Technologies)

ㅇ Column: ZORBOX Eclipse XDB C18 column (Agilent Technologies, 4.6 mm ㅧ 150 mm, 5 μm) at 40℃

ㅇ Fluorescence Detector (Excitation 340nm / Emission 435 nm)

ㅇ Mobile: A (40 mM Phosphate buffer, pH 7.8), B (ACN/MeOH/DW, 45/45/10, v/v)

ㅇ Gradient: An initial 1.90 min gradient from 100%→50% A (1.9 min), 50%→14.0 min from 50% A, 18.0 min from 0% A, and a final 22.0 min at 100% A at 2 mL/ min

-Analysis of constituent amino acid composition

ㅇ Sample pretreatment (0.2 g of sample was added with 40 mL of 6N HCl, filled and sealed with N ₂ gas, hydrolyzed at 110°C for 24 hours, and then vacuum concentrator (EYELA N-1100, Tokyo Rikakikai Co., Ltd., Tokyo, Japan), dilute 50 mL with 0.2M sodium citrate buffer and filter with a 0.45 μm syringe filter

ㅇ Automatic amino acid analyzer (Amino acid analyzer L-8900, Hitachi Co., Ltd., Tokyo, Japan)

(4) Molecular weight analysis of silk fibroin and hydrolysates using size exclusion chromatography (SEC)

-HPLC system (Atlus™, PerkinElmer)

-Column: 5Diol-300-II size exclusion column (COSMOSIL, 7.5 mm×300 mm, 300Å Pore size) at 30℃

-Photoarray Detector (A 280 nm)

-Mobile: 0.25M Citrate-Phosphate buffer (pH 6.0) at 1 mL/min

-Molecular weight standards: β-Amylase (200 kD), Alchohol dehydrogenase (150 kD), Albumin (66 kD), Carbonic anhydrase (29 kD), Cytochrome (12.4 kD)

(5) Molecular weight analysis of silk fibroin and hydrolyzate using SDS-PAGE

-4~20% Gradient Mini-PROTEAN ^ㄾ TGX™ Precast protein gel (BIO-RAD)

-Precision Plus Protein Standards (MW: 10 kD, 15 kD, 20 kD, 25 kD, 37 kD, 50 kD, 75 kD, 100 kD, 150 kD, 250 kD)

-Stain: Silver Staining Plus Kit (BIO-RAD)

(6) Molecular weight analysis of silk fibroin and hydrolyzate using MALDI-TOF/TOF

-Analysis using Pohang Technopark's MALDI-TOF/TOF to confirm the molecular weight of low molecular weight peptides/proteins and amino acids

-MALDI-TOF system (Autoflex speed MALDI-TOF/TOF, Bruker)

-Sample pretreatment (Mixed with sinapinic acid in 80% ACN: 0.1% TFA)

-Autoflex speed MALDI-TOF Pro linear mode

-Laser (355 nm, 2 kHz, 200 shots)

-Molecular weight scope: 800 ~ 60,000 Da

2. Enzymatic hydrolysis and high pressure treatment

(1) Selection of proteolytic enzymes for food and conditions of enzyme hydrolysis

-10 types were selected according to reaction pH (akaline, neutral, acidic protease) and decomposition method (Endo/Exo).

-The pH for the enzyme reaction was provided using 0.25M Citrate-phosphate buffer.

-The reaction temperature was controlled using a water-bath.

-The enzyme activity of the proteolytic enzyme was determined and applied at a ratio of 12.5 U-enzyme/g-silkfibroin.

(2) 50~100 MPa high pressure treatment method

-High pressure treatment of 50~100 MPa was performed using HHP machine (TFS-2L, Toyo-Koatsu Innoway Co. Ltd., Hiroshima, Japan).

-The temperature at high pressure was applied by selecting 50℃.

[Experimental Example]

[Experimental Example 1] Silk Fibroin Fiber and Solubilized Silk Fibroin Solution

1. Solubilization of purified silk fibroin

-Since degummed silk fibroin is insoluble, a solution containing CaCl ₂ and a heat treatment method were used as a prerequisite for food consumption, and the excess salt was removed using a dialysis method.

2. Amino Acid Composition of Silk Fibroin Fiber and Solubilized Silk Fibroin Solution (Table 1)

-The amino acid composition of silk fibroin fiber and solubilized silk fibroin solution was found to be similar.

-Free amino acids were not detected in the silk fibroin solution, and all amino acids were found to exist in the form of proteins.

ND: Not detected

3. Molecular weight of silk fibroin

-Molecular weight of silk fibroin protein from SEC using solubilized silk fibroin solution is widely distributed, retention time spans 7 to 11 minutes, and molecular weight is approximately 10 kD to 500 kD, making it difficult to determine the molecular weight. .(Figure 1)

-The molecular weight of silk fibroin solubilized in SDS-PAGE using 4-20% Gradient Gel showed a full range of molecular weight from high molecular weight to low molecular weight, so the molecular weight could not be specified (Fig. 2).

-As a result of SEC and SDS-PAGE, most of the solubilized silk fibroin were found to be more than 20 kD in a state in which most of the proteins do not exist in a single form but are physicochemically entangled with each other.

4. Characteristics of proteolytic enzymes for food

(1) Classification of proteolytic enzymes (Table 2)

-Bacteria, fungi, plants, animals and mixed enzymes by origin were secured.

-Alkaline, neutral and acidic enzymes were obtained according to the reaction pH.

-The endo-type and endo/exo mixed enzyme were secured according to the reaction.

(2) Enzymatic activity of proteolytic enzymes (Table 2)

-Enzyme activity was determined using casein as a substrate under optimal reaction conditions (temperature and pH).

NA: Specific enzyme activities were not available because the substrate was precipitated at pH 4.0.

[Experimental Example 2] Enzymatic hydrolysis of silk fibroin by single enzyme treatment

1. Enzymatic hydrolysis of silk fibroin using a single enzyme (Table 3) (Fig. 3)

-As a result of reacting for 5 hours at pH 7.0 and 50℃ at normal pressure and high pressure (50/75/100 MPa), most of the enzyme hydrolysis tended to increase due to the high pressure treatment.

-In the case of flavozyme in which endo/exo-type is mixed among 10 kinds of proteolytic enzymes, the free amino group of at least 30% (100 MPa) to 44% (75 MPa) increased by high pressure treatment, resulting in the increase of exo-type. It was more effective than protease.

-Alkaline proteolytic enzymes FoodPro (75 MPa), Alcalase (50 MPa), and Protamex (75 MPa), which have high decomposition efficiency for silk fibroin at normal pressure without high pressure treatment, up to 9% depending on the conditions of high pressure treatment , 14%, 12% of free amino groups were increased, and the effect of high pressure was the lowest.

-In the case of bromelain (100 MPa) and cholupurin (75 MPa), which had low decomposition efficiency for silk fibroin at normal pressure without high pressure treatment, the formation of free amino groups of up to 42% increased depending on the high pressure treatment conditions The effect was the best.

-The production of free amino groups by enzymatic hydrolysis tended to increase mostly by high pressure (50~100 MPa) treatment compared to normal pressure.

2. Amino Acid Composition of Enzymatic Hydrolyzate of Silk Fibroin Using Single Enzyme (FoodPro/Flavourzyme)

-As a result of reacting for 5 hours at pH 7.0 and 50℃ at normal pressure and high pressure (50/75/100 MPa), food pro and flavozyme, which are efficient in enzymatic hydrolysis by high pressure treatment, amino acids of hydrolyzed products using a single enzyme The composition was confirmed.

-As a result of hydrolysis using a single enzyme and Food Pro at normal pressure, the free amino acids present in the hydrolyzate are 0.38 mg/g, mostly in the form of low molecular weight peptides/proteins. Only 2.4% of the amino acids were broken down into the free amino acid form. (Table 4)

-As a result of hydrolysis using a single enzyme and Food Pro at high pressure (100 MPa), free amino acids were 0.45 mg/g, and only 2.4% of all amino acids were degraded into free amino acids. (Table 4)

-As a result of hydrolysis using a single enzyme and flavozyme at atmospheric pressure, the free amino acid present in the hydrolyzate was 2.90 mg/g, which was found to be higher than that of the endo-type Food Pro. As the enzyme reaction proceeded, the precipitate This was produced, and the specific gravity of free amino acids was meaningless. (Table 5)

-As a result of hydrolysis using a single enzyme and flavozyme at high pressure (100 MPa), the free amino acid was 4.0 mg/g, and the free amino acid was increased by 38% by high pressure treatment. (Table 5)

-Hydrolysis of silk fibroin by high pressure treatment was more effective in the endo/exo blended flavozyme compared to the endo-type Food Pro.

3. Molecular weight of silk fibroin hydrolyzate using a single enzyme at normal pressure

-The peak of silk fibroin broadened in hydrolysates by endo-type proteolytic enzymes (alcalase, alpalase, foodpro, protamex, flavozyme) in which enzymatic hydrolysis of silk fibroin is efficiently performed Disappeared, and the main peak in the 11th minute and minor peaks in the 11.5~13th minute were created. (Fig. 4)

-In the enzyme hydrolyzate by flavozyme, the main peak is confirmed at 11.1 minute, but this is thought to have changed the retention time due to the presence of the protein corresponding to the broad peak at 10.3 minute, and the free amino acid by exo-type is It was predicted to appear in Squad 13.

-As a result of hydrolysis using a single enzyme at atmospheric pressure, the broad peak of silk fibroin disappeared, low molecular weight protein/peptide at 11 minutes, low molecular weight peptides at 12 to 13 minutes, and free amino acids after 13 minutes. It was determined to correspond to the peak.

-The peak of broad silk fibroin disappeared in the hydrolyzate of endo-type proteolytic enzymes (bromelain, pepsin, cholupurin, promod 278, flavozyme) with relatively poor enzymatic hydrolysis to silk fibroin. However, with a considerable amount of broad peaks at 10.3 minute remaining, broad minor peaks between 11.5 and 13 minutes were generated along with the main peak at 10.8 minute. (Fig. 5)

-As a result of hydrolysis using a single enzyme at normal pressure, it is judged that normal enzymatic hydrolysis is performed only when the β-sheet, which is complexly related to silk fibroin, is decomposed. When enzymatic hydrolysis was efficient, the peak of silk fibroin, which had been widely distributed, was completely dissolved and a peak of a certain molecular weight was confirmed.

4. Molecular weight of silk fibroin hydrolyzate using single enzyme at high pressure

-As a result of treating silk fibroin at 50°C for 5 hours at 50, 75, and 100 MPa at normal pressure, it was confirmed that the peak in the 11th minute disappeared and the retention was finely shortened and the molecular weight increased. (Fig. 6)

-In the case of enzymatic hydrolysis of silk fibroin using flavozyme with active Exo-type activity alone, it was confirmed that the peaks of the 10.3 and 12.3 minutes that existed in the atmospheric pressure enzyme hydrolysis disappear from the high-pressure enzyme hydrolysis, and the 13th minute It was confirmed that the peak corresponding to the free amino acid of was increased as the applied pressure increased. (Fig. 7)

-In the case of silk fibroin hydrolysis using FoodPro as a single proteolytic enzyme, the peak at 12 minutes decreased sharply in high pressure enzyme hydrolysis, and the peak at 12.5 minutes increased as the applied pressure increased. (Fig. 8)

-In the case of enzymatic hydrolysis of silk fibroin using alpalase (FIG. 9), Protamex (FIG. 10), and alpalase alone as a single proteolytic enzyme, a similar tendency to that of Food Pro was confirmed.

-In the case of enzymatic hydrolysis of silk fibroin using cholupurin alone, the peak at 10.3 minute, which was present at atmospheric pressure enzymatic hydrolysis, was maintained at high pressure enzymatic hydrolysis, while the peak at 11.5 minute confirmed at normal pressure was subjected to high pressure treatment. It disappeared by and new peaks were generated at 12.6 and 13.5 minutes. (Fig. 11)

-In the case of enzymatic hydrolysis of silk fibroin using bromelain alone, the peak at 10.3 min, which was present in atmospheric pressure enzymatic hydrolysis, gradually decreases as the applied pressure increases in high pressure enzymatic hydrolysis, and high pressure treatment As a result, the peak at 11.5 to 12.5 min disappeared, but the peak at 12.9 min increased. (Fig. 12)

-In the case of enzymatic hydrolysis of silk fibroin using pepsin alone, the peak at 10.3 minute, which was present in atmospheric pressure enzymatic hydrolysis, gradually decreases as the applied pressure increases in high pressure enzyme hydrolysis. The peak in the 11.5-12.5 minute period disappeared. (Fig. 13)

5. Whether silk fibroin is self-degraded by protease during enzymatic hydrolysis

-In the given enzymatic reaction conditions, the self-degradation of 5 kinds of proteolytic enzymes that efficiently hydrolyze silk proteins was confirmed according to the presence of silk proteins.

-Five kinds of proteolytic enzymes with high silk protein decomposition efficiency degrade silk protein under the given reaction conditions, whereas self-decomposition that degrades the enzyme itself was found to be negligible. (Table 6)

[Experimental Example 3] Enzymatic hydrolysis of silk fibroin by complex enzyme treatment

1. Reaction process using complex enzymes to improve the hydrolysis efficiency of silk fibroin at normal pressure

-For the hydrolysis efficiency of silk fibroin at normal pressure, Step 1: Food Pro (pH 8, 60°C) → Step 2: Plavozyme (pH 7, 50°C) promoted the second-stage enzymatic reaction. Free amino groups increased step by step.

-Hydrolysis of silk fibroin using a complex enzyme is performed at normal pressure, pH 7.0, 50℃ using a 1:1 mixture with other enzymes based on flavozyme in order to utilize the endo-type and exo-type protease activity. Enzymatic hydrolysis was performed for 5 hours. (Table 7)

-6,400 mg/L of free amino groups were produced in all of the hydrolysis of the complex enzymes food pro, alpalase, protamex and flavozyme, which are alkaline proteolytic enzymes with high decomposition efficiency for silk fibroin at normal pressure. Good results were also confirmed in complex enzyme hydrolysis.

-Only the complex enzymes of alpalase and flavozyme, which are neutral proteolytic enzymes for silk fibroin, were hydrolyzed at a level similar to that of alkaline protease.

-The complex enzyme of bromelain and flavozyme, which are neutral proteolytic enzymes for silk fibroin at normal pressure, shows a low decomposition efficiency at 66% of the result of hydrolysis using alkaline protease, but compared to the proteolytic efficiency of individual enzymes. By using the complex enzyme, the protein degradation efficiency was increased.

2. Reaction process using complex enzyme at high pressure to improve the hydrolysis efficiency of silk fibroin

-Hydrolysis of silk fibroin using a complex enzyme at high pressure compared to normal pressure all increased (Table 7) (Fig. 14)

-In the case of alcalase, alfalase, foodpro, and protamex, which have excellent hydrolysis results of silk fibroin by a complex enzyme used in combination with flavozyme at normal pressure, an increase of up to 12~16% by high pressure treatment Free amino groups were created.

-In the case of pepsin, bromelain, and cholupurine whose hydrolysis result of silk fibroin by a complex enzyme mixed with flavozyme at normal pressure was low, free amino groups increased by 31-40% by high pressure treatment were produced. .

-It was interpreted that the high-pressure treatment of 50~100 MPa for enzymatic hydrolysis of silk fibroin loosened the molecular structure constituting the β-sheet structure so that the proteolytic enzyme could be accessed.

3. Amino acid composition of enzyme hydrolyzate of silk fibroin using complex enzyme (Food Pro/Flavozyme)

-At high pressure (100 MPa) compared to normal pressure, the free amino acid present in the decomposition product of silk fibroin using a complex enzyme increased by 13% from 4.30 mg/g to 4.84 mg/g. (Table 8)

-The proportion of free amino acids present in the hydrolyzed product at high pressure compared to normal pressure was 25%, and it was confirmed that 75% of the peptide/protein forms of lower molecular weight than the free amino acids were present by enzymatic hydrolysis.

4. Molecular weight of silk fibroin hydrolyzate using complex enzyme at normal/high pressure

-The molecular weight change of the enzyme hydrolyzate due to high-pressure enzymatic hydrolysis using a complex enzyme mixed with flavozyme was confirmed using SEC.

-As a result of enzymatic hydrolysis of silk fibroin by FoodPro + flavozyme complex enzyme, the broad peak of 12.3 min, which was present in normal pressure/complex enzyme hydrolysis, decreases with increasing applied pressure in high pressure/complex enzyme hydrolysis, The peak at 13 minutes increased with the high pressure treatment. (Fig. 15)

-Enzymatic hydrolysis of silk fibroin by alpalase+flavozyme, protamex+flavozyme, alcalase+flavozyme, and complex enzymes showed similar tendency to foodpro+flavozyme.

-As a result of enzymatic hydrolysis of silk fibroin by cholupurin + flavozyme (Fig. 16), bromelain + flavozyme, pepsin + flavozyme, and complex enzymes, the 10.3 component present in normal pressure/complex enzyme hydrolysis and It was found that the peak at 12.3 min decreased in high pressure/enzymatic hydrolysis, and the peak at 13 min increased according to the high pressure treatment.

[Experimental Example 4] Determination of molecular weight of enzyme hydrolyzate using MALDI-TOF/TOF

1. Molecular weight of silk fibroin hydrolyzate using a single enzyme at high pressure

-As a result of analyzing the molecular weight of silk fibroin hydrolyzate at high pressure (100 Mpa)/single enzyme (Food Pro) using MALDI??TOF/TOF, the molecular weight of the hydrolyzate of silk fibroin by Food Pro is 3.8 to 10 kD It was composed of a low molecular weight peptide/protein corresponding to and a large amount of peptides corresponding to 0.85 to 2.5 kD. (Fig. 17)

-From the molecular weight and distribution of the low molecular weight peptide (0.85 ~ 2.5 kD), it was determined that peptides constituting various amino acids were produced due to endo-type enzyme decomposition.

-As a result of determining the molecular weight of silk fibroin high pressure (100 Mpa)/single enzyme (flavozyme) hydrolyzate using MALDI??TOF/TOF, it was found to be similar to the results obtained by FoodPro. (Fig. 18)

-The content of low molecular weight peptides/proteins (3.8-10 kD) and peptides (0.85-2.5 kD) in silk fibroin hydrolyzate using a single flavozyme enzyme was 80% and 40, respectively, compared to the hydrolyzate produced by Food Pro. % Level.

-Due to the exo-type of flavozyme enzyme, low molecular weight peptides were already decomposed into amino acids of 0.8 kD or less, and it was determined that the amount of low molecular weight peptides/proteins was relatively low.

2. Molecular weight of silk fibroin hydrolyzate using complex enzyme (Food Pro/Flavozyme) at high pressure

-As a result of analyzing the molecular weight of silk fibroin at high pressure (100 Mpa)/complex enzyme hydrolyzate using MALDI-TOF/TOF, the molecular weight of silk fibroin hydrolyzate is low molecular weight peptide/protein corresponding to 3.8 to 10 kD. Peptides corresponding to 0.85~2.5 kD showed a similar tendency to the molecular weight of the hydrolyzate by a single enzyme. (Fig. 19)

-The content of low molecular weight peptides/proteins (3.8~10 kD) and peptides (0.85~2.5 kD) in silk fibroin hydrolysates by complex enzymes is 50% and 40%, respectively, compared to hydrolysates produced by Food Pro. appear.

-The content of low molecular weight peptide/protein (3.8~10 kD) and peptide (0.85~2.5 kD) in silk fibroin hydrolyzate by complex enzyme is 60% and 80%, respectively, compared to hydrolyzate by flavozyme. Showed.

-As silk fibroin is hydrolyzed by complex enzymes, the amount of low molecular weight peptides/proteins corresponding to 3.8 to 10 kD is reduced, and low molecular weight peptides corresponding to 0.85 to 2.5 kD due to the exo-type activity of flavozyme are the lowest level. Decreased to.

[Experimental Example 5]

When the enzymatic reaction was carried out in the same manner as in Experimental Example 3 3, except that citric acid, gluconic acid, and tartaric acid were added in a weight ratio of 0.5:1:0.5 to 0.1% by weight, the free amino acid It was confirmed that the content increased to 4.976 mg/g [Glycine:1.41, Alanine: 1.20, Serine: 0.532, Tyrosine: 0.574, Valine: 0.324, Aspartic acid: 0.038, Glutamic acid: 0.108, Phenylalanine: 0.176, Threonine: 0.122. , Arginine: 0.131, Isoleucine: 0.138, Tryptophan: 0.053, Leucine: 0.067, Lysine: 0.037, Histidine: 0.031, Methionine: 0.035, Proline: 0.033, Cystine: 0.000)]

[Experiment result]

1. Silk peptide manufacturing method using an enzyme hydrolysis method that replaces acid-treated hydrolysis

(1) Substitution of silk amino acid production method through acid-treated hydrolysis

-Traditional acid-treatment hydrolysis uses 3M HCl for a long time (1~2 days) at 100℃ or higher. After hydrolysis of silk fibroin, neutralization → decolorization → desalting → concentration → drying to produce silk amino acids. As a result, more than 70% of the hydrolyzed product is decomposed into free amino acids by acid-treated hydrolysis, and the content of free amino acids in the final dried product is 90%.

(2) Silk peptide/amino acid manufacturing method through enzymatic hydrolysis

-After hydrolyzing the solubilized silk fibroin solution at 50°C, pH 7.0 (neutral) for 5 hours using various proteolytic enzymes, it is concentrated without additional work → After drying, a low molecular weight peptide/protein having a molecular weight of 4-10 kD and Produces peptides of 1 to 2.5 kD and silk amino acids.

-Using the enzyme hydrolysis method using a single or complex enzyme mixed with a flavozyme of proteolytic enzymes such as alcalase, alpalase, food pro, and protamex, silk amino acids present in the enzyme hydrolyzate The content was adjusted to 2 to 30%, and a technology for producing a hydrolyzate containing 70 to 98% of a low molecular weight silk peptide/protein having various functions was secured.

-Compared to the acid-treated hydrolysis method, it is possible to secure competitiveness by shortening the manufacturing process and minimizing process steps.

2. Silk peptide manufacturing method using high pressure/enzymatic hydrolysis method

(1) Silk fibroin is hydrolyzed at a high pressure of 50 to 100 MPa using a single or a complex enzyme mixed with flavozyme of proteolytic enzymes such as cholupurine, bromelain, and pepsin, which have weak proteolytic power to silk fibroin. By decomposition, the technology to produce low molecular weight peptides/proteins with a molecular weight of 4 to 10 kD, peptides with 1 to 2.5 kD, and silk amino acids was secured.

(2) A technology to increase protein hydrolysis efficiency by 10-40% by using single or complex enzymes of silk fibroin at high pressure (50-75 MPa) was secured.

As described above, the present invention has been described with reference to preferred embodiments of the present invention, but those skilled in the art can variously modify and change the present invention within the scope not departing from the spirit and scope of the present invention described in the following claims. You will understand that you can do it.

Claims (7)

Silk fibroin at a high pressure of 50 to 100 MPa using a complex enzyme consisting of at least one selected from the group of alcalase, alpalase, food pro, protamex, pepsin, bromelain, and cholupurin and flavozyme The production method of silk peptide by the high-pressure enzyme method, characterized in that hydrolyzed. delete delete delete delete The method of claim 1, wherein the reaction further comprises at least one selected from citric acid, gluconic acid, and tartaric acid. The method of claim 1, wherein the reaction comprises citric acid, gluconic acid, and tartaric acid.

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