TW581811B - Process for preparing lysozyme - Google Patents

Abstract

The present invention provides a process for preparing lysozyme characterized by mixing the egg white or its diluted solution with diatomaceous earth, kaolin, zeolite or the mixtures thereof, and followed by eluting the adsorbed lysozyme with a salt solution. The diatomaceous earth, kaolin and zeolite can specifically adsorb the lysozyme in egg white, and the adsorbed lysozyme can be easily eluted with a salt solution.

Description

581811 Description of the invention: The present invention provides a simple and effective method for preparing lysozyme. Lysozyme has a wide range of uses in food and medicine. It is generally used for, preservation and processing. Common products include aquatic products such as oysters, shrimps, fresh products such as beans, vegetables, fish, fruits, sushi, boiled noodles, fish balls, shell balls, and fish plates. In addition, the addition of lysozyme has also been widely used in the production of cheese, lysozyme has the role of rennet, and makes the protein in cow's milk unstable and produces rennet effect. The purified lysobacteria obtained through repeated crystallization, purification, desalting, and drying. 酉 Mother II: made into the form of ingots, granules, tongues, etc. and used. For example, lysozyme can be used as a therapeutic agent for wounds of chronic paranasal inflammation, and as a preservative for medical supplies such as eye drops. ,

The egg white contains about 3.5% soluble gg | (dry weight basis), which is the main raw material for the preparation of soluble gluten. The method of producing lysozyme in Muli industry is mainly by direct crystallization [Alderton, and Fev (Dld H L

Direct crystallization of lys〇zyme fr〇m egg white and some crystalline salts of lysozyme_ βι〇1_

Chem. 1 04: 1 (1 946)] 'This method is to add sodium chloride to the chicken protein solution, adjust the pH to 9.5, add a small amount of seeds, and then crystallize at 4 ° C. The lysozyme recovery rate is about 60 to 80%. However, the recovery rate of this method may be greatly affected by the amount of raw materials. Zhang et al. [Zhang Zhentian et al. Study on Purification of Chicken Protein Lysozyme by Ultrafiltration. The Chinese Agricultural Chemical Society, 24 (0:86 (1 986), Taipei] uses 15564 chicken egg white solution in the same formula; and each .., ^, the results of the test are shown in male and male. ： The recovery rate of the first crystallization is 43. 0%, the purification times | & π ΙΛ- ^ number is 4.9 times; the recovery rate during the two crystallizations is reduced to 12 · 4%, and the purification is performed. & Men Congxuan ★, the number is 7.9 times; the recovery rate during the three crystallizations is 11.8%, the purification factor is AQ, and the denier is 4. a 1σ sigh is 9.1 times. This result shows a larger: == It may significantly reduce its recovery rate, and the purification factor is not very reasonable. / The biggest disadvantage is: a large amount of protein solution and ιb after the lysozyme is separated; the second is the emulsified sodium, so it cannot be used effectively. To solve the problem of the amount of salt, many studies have attempted to dissolve the salt by the ultra-thin method (see above), which shows that the yield of a single ultrafiltration combined with 47 is 47%, and the purification factor is 3.0; the research by Chiang et al. hiang, B'H · 'et Egg White lysozyme 7lflCatl0n by ultl-afntrat10n and affxmty c romatography. J. Food Sci. 58 (2): 303 (1993)] pointed out that the molecule 篁 3 was removed by a knife. The recovery rate of lysozyme after 〇000 ultrafiltration membrane treatment is 96%, but the purification factor is only 6 times. It shows that the purification effect is irrelevant. In addition, the biggest disadvantage of the method is that the filter membrane is easily clogged. Hinder mass production.-In addition, there are also studies on the purification of lysozyme using adsorbents,

Alderton # [Alderton, etal. Isolation of lysozyme fr⑽ egg white. J. Bl0l · Chem · 1 57:43 (1 945)] found that t soil (bentoni te) has the property of adsorbing lysozyme, but it is not easy to wash out. 5% acetic acid in pyridine solution is not suitable for industrial production. In order to solve the above-mentioned problems, the inventors have conducted in-depth research. First, they found that diatomite, kaolin, and zeolite have excellent selective adsorption of lysozyme, and the adsorbed lysozyme can be easily washed out with an ionic solution. In addition, the protein solution after adsorption treatment has the same processing characteristics as the original protein solution, without any contamination by additives, and can fully maintain its usability. The present invention has been completed based on the above findings. Explanation of Formula 1 The first figure shows the effect of the amount of adsorbent on the recovery of lysozyme. Figure 2 shows the SDS-PAGE spectra of the protein stock solution and the products treated with the three adsorbents. M shows labeled protein; i shows protein stock solution; 2 shows protein stock solution treated with diatomaceous earth; 3 shows protein stock solution treated with kaolin; 4 shows protein stock solution treated with zeolite. Figure 3 shows the SDS-PAGE spectra of lysozyme standard (1), protein stock solution (2), kaolin-treated product (3), and product recrystallized (4) or anion exchange resin. Analytical method 1 · Determination of relative amount of protein After proper dilution of the protein solution, the absorbance at 280 nm (A280) was measured with an 11-1100 Spectrophotometer manufactured by Hitachi, and the relative value of A28G was used to calculate the protein adsorption percentage. 2. Measurement of protein concentration The Lowry method was used for measurement. Solution A: Na2C03 1 0g and NaHC03 2g are dissolved into 500mL. Solution B: 1.6g of CuS04.5H20 and 2.3g of sodium citrate were dissolved into an aqueous solution. Liquid C ... Take 50 mL of liquid A and 1 mL of liquid B and mix before use.

Fol in-Ciocal teu ’s S: Dilute it by adding 2 volumes of Luohe water before use. Take 0.2 niL of the appropriately diluted sample solution, add 1 mL of solution C, mix with 15564 7 and then react at room temperature for 30 minutes, then add Folln, s solution 0 · 1 ml L 'to react at room temperature 6 At 0 minutes, the absorbance at 650 nm was measured and compared with the standard curve to determine the protein content in the sample. The standard curve was prepared using bovine serum albumin at 50 to 400 / zg / niL as the standard solution. 3 · Determination of lysozyme activity

Micrococcus lysodeikticus cells were suspended in 0.067M phosphate buffer solution (20mg / 100niL, ρΗ6.3 · 3), and 2.97mL of the cell liquid was taken out and placed in Shiyang '. Add 3 # L enzyme solution, mix well, The change in absorbance at 450nm was measured at room temperature, recorded every 30 seconds, and held for 20 minutes. * One unit of lysozyme activity refers to the amount of enzyme that can hydrolyze bacteria to reduce the absorbance per minute under the above conditions.

0.001 OD / minx 0.03mL 4. SDS-PAGE electrophoresis analysis The colloid concentration of 15% is used for separating colloids, and the colloid concentration of coke colloid is 4%. Take ΙΟ "! sample solution (concentration of about 4mg / mL), add 1/10 sample buffer (Note 丨) and 4v 丨 trace dye solution (Note2), and then heat at 100 ° C for 5 minutes After that, it was cooled to room temperature and analyzed by electrophoresis. The protein bands in the colloid were stained with CoomassieBrilliantBlueR and decolorized with acetic acid-methanol-water (10: 20: 7) to have a transparent background ', then dried and stored. Note 1: SDS-PAGE sample buffer solution (2χ)

TrisCTrizma Base, 125mMx2) 3. Ogm 8 15564 581811 EDTA-2Na (2mMx2) 14.8mg SDS (2% x2) 4.0gm —2-mercaptoethanol_i 〇_ 〇mi dissolved in 80ml water, the pH was adjusted to 6.8 , Add water to 100ml and dilute it twice before use. Note 2: Trace dye solution: Bromophenol blue is about 1 mg (we don't need to be very accurate for weighing). Dissolve in 5m 1 of water, add 5m 1 of glycerin and mix well. 5 · Functional determination of waste protein after adsorption (A) Determination of protein foaming power and stability of vesicles Take 50ΐ: liter of protein solution after adsorption is placed in a milliliter volumetric cylinder, sealed with stone soil paper, and then shaken by hand The method oscillates vertically back and forth for 1 minute (2 times / second), and the obtained foam volume (mL) is the foam expansion (foam expansion). Measure the residual foam volume after standing for 30 minutes. The ratio of π residual volume to the original volume indicates its foam stability. 0 (B) Determination of protein emulsification stability. Take 25 ml of the adsorbed protein solution and place it in a 50 mL measuring cylinder. Add 25 ml of soybean oil, homogenize at 12,000 rpm for 1 minute, immediately measure the water layer height (Mo), then leave it at room temperature for 30 minutes, then measure the water layer height (Mt), and then calculate it by the following formula Its emulsification stability. 50-Mt Emulsification stability (%) = '^ X 100% 50-Mo Examples and test examples 9 15564 The following 11 examples and test examples make the content of the present invention further-specific. Brother, but the present invention is not limited to every embodiment. Anyone who uses the spirit of the present invention should fall within the scope of the present invention. Dilute the chicken protein solution with a base solution, dilute it 5 times with water, stir and mix with the gauze pass rate and the lysozyme specific activity of the adsorbed protein and the theoretical purification multiple. The results are shown in Table 1.

Sorbent Unabsorbed protein Percentage (%) Unadsorbed lysozyme Percentage (%) Protein Adsorption rate (%) Lysozyme activity Adsorption rate (%): In addition to the sticky mass f, it can be centrifuged if necessary. Take protein dilute OOmL, add 5g of different adsorbents (bentonite, diatomaceous earth, and =, and "Fu Shi, all products of Sl§me company), and treat 301 side occasionally at room temperature. Shake to mix. The supernatant was separated by centrifugation (5, n). Measure the lysozyme activity and absorbance at 28nm of the supernatant and the original protein solution. According to the measured value, the adsorption rate of different proteins is deduced; the bacterial enzyme activity is adsorbed and purified. The original solution is bentonite siliceous soil kaolin zeolite 100 100 47.18 96.73 94.02 95.82 0 0 0 49 52.82 3.27 5.98 4.18

100 100 100 51 1.89 26.88 16.72 12.20 15564 10 581811 From the results in Table 1, it can be seen that ο, but although the adsorption to lysozyme is reported to be strong, the performance is not good, and the adsorption of tadpoles is also strong, so choose high To U9 times. : Yes: The specific activity of the lysozyme that breaks the adsorbed protein is mostly lysozyme, and ‘::, double kaolin and fossil adsorbed protein times, times and 12.2. Times :::: r: Xuan Yu Fu has excellent selectivity, also g τ ★: a kind of adsorbent for lysozyme. And in the preparation of lysozyme. ', P The two adsorbents mentioned above can be effectively used. Example 2 Take 100mL each and dilute 5 仵 to remove the protein solution borrowed, using diatomaceous earth, kaolin, and Buddhist stones according to Example g ...丨 丨 Order, main, and six methods are used for adsorption: Lysozyme activity 'is used to calculate the percentage of lysobacteria adsorbed. In addition, each adsorbent of Shen Dian Centrifuge was centrifuged: 1M NaCl was used to elute lysozyme (room temperature, 7 d, e, a palpitate (to / dish, shake at 75 rpm for 30 minutes). Centrifugal wash = 1 to determine its dissolution_ Activity. Then calculate the recovery rate of lysozyme after treatment with each adsorbent and the elution rate of self-adsorbent. The results are shown in Table 2 _Table 2 Preliminary ^ purified lysozyme sorbent screening test sorbent lysozyme adsorption Rate (%) Bentonite diatomaceous earth zeolite 100 100 100 50.88 Recovery rate of lysozyme (%) 82.23 90.70 49.05 Extraction rate of lysozyme (%) 0 82.23 90.70 96.40 15564 11 The results in Table 2 above show that I soil ^ Wei, 1 ,, f> glutenase has a very strong adsorption force,…, and washed out with 1M NaCl. Shi Xi algae soil, Mo Ling soil and Na Shi Temple used in the present invention for a mountain, wave industry μ. The lysozyme adsorbed by the adsorbent is very easy to wash out. The elution rate is 82.23%, 90.70 // and 96.40% in order. Example 3 Take l. 〇mL diluted 5 times the protein solution, eight # _, add 1 to 5 g of silica bath or kaolin, or 2 to 12 洮 r, gf stone, and then according to Example 1 Adsorption treatment was performed in the same way. After the supernatant was separated by centrifugation, each adsorbent was used to extract the adsorbed lysozyme (75_,… 25 c) with 1M NaC1 of m company. The eluate was collected by centrifugation, and its lysozyme activity was measured. The results of the “recovery rate” are shown in FIG. I. The results of the “i” graph show that for each protein solution diluted 5 times, the diatomite and kaolin can recover almost 1GG% of lysozyme by using 4 g (That is, the ratio of protein stock solution to adsorbent :,) 'Fu Shi requires 8 g (that is, the ratio of protein stock solution to adsorbent j is 10.1) to achieve the highest lysozyme recovery, approximately 50% Example 4 Take 100 mL of a 5-fold diluted protein solution, add 4 g of diatomite or Nanling'or zeolite, and then perform adsorption and elution treatment in the same manner as in Example 3, but The eluent used was changed to different concentrations of MaCl / Valley. Then, the recovery of lysozyme in the eluent was measured, and the results are not as shown in Table J. The results in Table 3 show that the elution using 1M NaCl can be recovered. Most adsorbed lysozyme, using diatomite, kaolin and zeolite / Tongue of sequential recoveries were 86〇1%, 90.12% and 51.69% 1215564581811 Table 3 Road Temple gasification satisfied - Effect of an e-wash effect of lytic

NaCl concentration (M) 0 0.25 2 7 0.50 4 0.75 43 1.00 86 f Example 5 Recovery rate of lysozyme (%)

0 0 Kaolin 29.18 58.10 78.96 90.12 34.27 42.13 41.57 51.69 Take 2OmL of chicken egg homogeneous solution diluted 5 times + a, and add 8 Shi Xi algae soil, 8 g kaolin clay or 16 g Buddha stone, put them in the water bath and shake them up Shake the reaction (75rpmx30min, 25 ° C, ^ C) 'Centrifuge (1,000gx 5min), water sink, centrifuge, and then wash the egg protein repeatedly with 15Gml 1MNaa three times to determine the total protein concentration and Lysozyme activity was analyzed by electrophoresis. 1. The results are shown in Table 4. Using diatomite, kaolin and sea stone as adsorbents, the enzyme recovery rates were 82%, 98%, and 54%, respectively; the purification multiples were 17.05 times, M, respectively. "10 and-乜. The three adsorptive sentences have excellent purification effect. Table 4 Isolation of lysozyme from a five-fold diluted egg solution by comparing three adsorbents respectively 15564 13 Specific activity of the test sample (U / mg) Purification factor lysozyme recovery rate (%) > Double dilution of the white liquor 516 1 diatomite kaolin clay; fusi ^-8798 10273 1 0757 17.05 19.91 20.85 82 98 54 Purified using the above three sorbents The SDS-pAGE〆 analysis profile of lysozyme is shown in Figure 2. Figure i shows the protein stock solution 厶 圜. The color band is mainly composed of the band near UKd, and is near l4.4Kd = lysozyme Enzymes hardly show a visible color band, indicating that the amount of enzyme in the protein stock solution is relatively small. Samples 2, 3, and 4 are SDS-PAGE of the refined products treated with diatomaceous earth and zeolite, respectively. The figure shows that the two stilbene shows that the lysozyme band near l4.4Kd is the main component, while the original The color band near 43 Kd of the component becomes a trace component. This result shows that the above three adsorbents have excellent selective adsorption of lysozyme. FExample 6 Using kaolin as an adsorbent, the same method as in Example 5 was used Preparation of lysozyme. Then proceed to anion exchange resin treatment or lysozyme crystallization treatment according to the following methods. (1) Embed 1 g of anion exchange resin (Amberlyst A-207, 0H-type, (Organo Co., Ltd.) 'washed with distilled water to a pH of about 8 to 9, and added 10 ml of the enzyme solution that has been purified, purified and dialyzed', and processed at room temperature for 15 hours at 14 pm 14. Take the supernatant and measure the total protein mass and The lysozyme activity was analyzed by electrophoresis. (2) Crystallization of lysozyme: Take the enzyme solution purified and adsorbed and bound, and #, Jie, Qu W /, and two ultrafiltration) to reduce the enzyme solution 6 times, adjust the P value to 9 5 Add J final crystalline lysozyme, place 4 times, and wait for the results ... 'Centrifuge to collect the lysozyme crystals, and determine the protein content and glutenase activity. Where: The protein of the treated sample Amount and lysozyme activity ... After ° 10: Its specific activity, purification factor and recovery of lysozyme. The results are shown in Table 5. This result shows that the above two treatments can further purify the lysozyme, and the purification factor can be further improved. Increase to "to 28 times. The purified lysozyme was subjected to SDS weaving analysis. As shown in the figure, the results showed that the above-mentioned locustases treated with anion exchange resin or crystallized all showed a single color band, and were commercially available with purified lysozyme a Division production deduction) same. This result indicates that the two products purified above were lysozyme. & Specific activity 辺 / mg) 722 Purification multiple --------_ Recovery rate --_ 1 3 527 18.74 88.04 20375 28.22 80.98 1 9724 27.32 68.27 Stock solution Southern Territories adsorption Refined Kaolin adsorption Refined +

Amberlyst A- 27 (〇Η ·) treatment kaolin adsorption purification + crystallization treatment 15564 15 581811 Test example The processing functionality of the three proteins after the treatment with the three adsorbents used in the present invention and the method described in the following is described, that is, Foaming power, foam, foam stability and emulsification stability, and compared with the original protein solution. The results are shown in Table 6. The results in Table 6 show that the above-mentioned protein solutions after adsorption treatment still have fairly good processing functionality. Furthermore, the adsorbents used in the present invention are all food processing mixtures, and the protein solution treated by such adsorbents can still be used safely. Because it has no added salt or any other additives, it can still be widely used as a human. 、 、、, and counterfeit ingredients Table ·… 6 Comparison of functional properties before and after adsorption '' ~ --- --- Functional egg protein types

17.5 64 7 1-43

Protein solution liquid stone diatomaceous earth processor Kaolin clay processor 72〉 Effective effect of the stone processor ^ The three adsorbents used by the present invention, oblique J, main compound / combined mother, have good adsorption ability, and can be easily and effectively added Use ·, choose% clothing to prepare lysozyme. Use chicken gluten two raw materials' using the above three adsorbents to improve the specific activity to 17 仃 lysozyme to increase the specific activity to 17 俾, and K Yier ^, can be "above" and the recovery of lysozyme It has a high rate of 15564 16 581811, which is about 80% or more (when using diatomite), 90% or more (when using kaolin and 50% or more (when using zeolite). Further crystallization treatment and sub-exchange resin treatment can be prepared. The purified lysozyme can be obtained in a band of SDS-PAGE analysis map (in the case of soil) or in a shade of 17 15564

Claims (1)

5mii with bulletin m * m w · PI II < 1 丨 · _ Patent application No. 86 1 1 808 1. Amended version of patent application scope (October 21, 1992) 1. A method for preparing lysozyme, characterized by using diatomite, kaolin, zeolite or mixture thereof It adsorbs the lysozyme in the protein and then elutes it with a neutral salt solution. 2. The method for preparing a lysozyme as described in item 1 of the scope of the patent application, wherein the solution of the salt is a solution of sodium chloride. 3. The method for preparing lysozyme as described in item 1 or 2 of the scope of patent application, wherein it is further purified by crystallization. 4. The method for preparing a lysozyme as described in item 1 or 2 of the scope of patent application, wherein the method is further purified by an anion exchange resin. 15564