Extraction process of papain

Abstract

The invention relates to the technical field of enzyme extraction, in particular to an extraction process of papain. The invention discloses an extraction process of papain. The extraction process comprises the following steps: (1) taking supernate from immature papaya; (2) stirring and mixing the supernatant and the aqueous two-phase liquid, and standing for phase splitting to obtain a lower phase; (3) collecting a lower phase, and removing impurities through ultrafiltration to obtain a concentrated solution; and freeze-drying the concentrated solution to obtain enzyme powder. According to the extraction process disclosed by the invention, an organic solvent is replaced by a two-aqueous-phase liquid, mild phase splitting is realized through the synergistic effect of a surfactant and salt, and the damage to an enzyme structure is reduced; and further purifying and maintaining the enzyme activity by combining ultrafiltration and freeze drying.

Description

Technical Field

[0001] This invention relates to the technical field of enzyme extraction, and in particular to an extraction process for papain. Background Technology

[0002] Papaya, also known as caraway or longevity fruit, belongs to the Caricaceae family. It is rich in sugars, proteins, fats, various vitamins, and organic acids, making it highly nutritious. Papain is a sulfhydryl (-SH) endopeptidase widely found in the roots, stems, leaves, and fruits of papaya, with the highest concentration in the unripe latex. It possesses both protease and esterase activities, exhibiting broad specificity and a strong hydrolytic ability on animal and plant proteins, polypeptides, esters, and amides. In medicine and healthcare, papain can double the extraction yield of cod liver oil while simultaneously increasing the content of vitamins A and C.

[0003] Traditional papain extraction processes include organic solvent precipitation. This method lowers the dielectric constant of the solution by adding an organic solvent, increasing the intermolecular forces between protein particles and causing them to aggregate and precipitate. The solvent used in organic solvent precipitation must be water-miscible and not react with the enzyme; acetone and ethanol are commonly used. Organic solvent precipitation does not require special removal of the precipitant after precipitation; it is simply removed by natural evaporation. However, a disadvantage is that organic solvents can disrupt the hydrogen bonds of proteins, easily causing denaturation and inactivation.

[0004] Therefore, this application provides a papain extraction process. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, this invention provides a papain extraction process that solves the problem of enzyme inactivation in traditional methods and achieves enzyme activity retention through gentle phase separation and low-temperature drying.

[0006] The technical solution adopted by this invention to solve its technical problem is:

[0007] An extraction process for papain includes the following steps:

[0008] (1) Take the supernatant from an unripe papaya;

[0009] (2) Stir and mix the supernatant and the aqueous two-phase liquid, let them stand to separate the phases, and obtain the lower phase;

[0010] (3) Collect the lower phase, remove impurities by ultrafiltration to obtain a concentrated solution; the concentrated solution is then freeze-dried to obtain enzyme powder.

[0011] In some specific embodiments, in step (1), the preparation process of the supernatant from the unripe papaya is as follows: take fresh unripe green papaya, peel and chop it, add ice water and crush it; filter, collect the supernatant, and obtain the supernatant from the unripe papaya.

[0012] In some specific embodiments, in step (2), the preparation process of the aqueous two-phase liquid is as follows: preparing sodium dodecyl sulfate solution with a concentration of 0.10-0.13 mol / L, hexadecyltrimethylammonium bromide solution with a concentration of 0.10-0.13 mol / L, and potassium bromide solution with a concentration of 0.05-0.06 mol / L, respectively;

[0013] Mix 0.10-0.13 mol / L sodium dodecyl sulfate solution and 0.10-0.13 mol / L hexadecyltrimethylammonium bromide solution at a volume ratio of 1:1, then add an equal volume of 0.05-0.06 mol / L potassium bromide solution and mix. After mixing, let stand at 30-35℃ for 2-3 hours to obtain an aqueous two-phase liquid.

[0014] In some specific embodiments, in step (2), the volume ratio of the supernatant to the aqueous two-phase liquid is 1:2.5.

[0015] In some specific embodiments, in step (2), the pH of the aqueous two-phase liquid is adjusted to 7.8-8.0 using Tris-HCl buffer, and then the supernatant and the aqueous two-phase liquid are stirred and mixed.

[0016] In some specific embodiments, in step (2), the mixture is stirred at 20-35°C at a speed of 200-1000 r / min for 30-150 min.

[0017] In some specific embodiments, in step (3), the process of removing impurities by ultrafiltration is as follows: the lower phase is filtered using an ultrafiltration membrane to obtain a concentrated solution.

[0018] In some specific embodiments, the ultrafiltration membrane has a molecular weight cutoff of 10-30 kDa.

[0019] In some specific embodiments, in step (3), the concentrate is pre-frozen at -80°C to -70°C for 2 hours and then vacuum dried to obtain enzyme powder.

[0020] In some specific embodiments, in step (3), the activity of the enzyme powder is 115,950-131,576 units / mg.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The extraction process described in this application uses an aqueous two-phase liquid to replace organic solvents, and achieves gentle phase separation through the synergistic effect of surfactants and salts, reducing damage to the enzyme structure; then, it is further purified by ultrafiltration and freeze drying to maintain enzyme activity. Attached Figure Description

[0023] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0024] Figure 1 This is the standard curve diagram described in this application. Detailed Implementation

[0025] To facilitate understanding by those skilled in the art, the present invention will be further described below with reference to embodiments. The content mentioned in the embodiments is not intended to limit the present invention.

[0026] As used herein, “and / or” includes all combinations of any one or more of the associated listed items. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention. As used herein, the singular forms “a,” “an,” “an,” and “the” are also intended to include the plural forms unless the context clearly indicates otherwise. Further understanding is needed; when used in this specification, “comprising” designates the stated features, integers, steps, operations, elements, and / or components, but does not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or combinations thereof.

[0027] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Further understanding is that terms, such as those defined in common dictionaries, are interpreted in accordance with their meaning in the context of the relevant field and are not idealized or overly formal, unless expressly defined herein.

[0028] The exemplary invention described herein may suitably omit any one or more limiting elements, which are not specifically disclosed herein. Therefore, terms such as “comprising,” “including,” “containing,” etc., should be interpreted broadly and non-limitingly. Furthermore, the terminology used herein is for descriptive purposes without limitation, and it is unintentional to use terms that do not include any equivalent characteristics, but only to describe a portion of their characteristics; however, various modifications are possible within the scope of the invention according to the claims. Therefore, while the invention has been specifically disclosed through preferred embodiments and optional features, variations of the invention embodied by the modifications disclosed herein may be noted by those skilled in the art, and such modifications and variations are considered to be within the scope of the invention.

[0029] Tris-HCl buffer is a buffer system prepared from tris(hydroxymethyl)aminomethane (Tris) and hydrochloric acid (HCl) with a pH range of 7.0-9.0. In this application, it is used to maintain the pH of the aqueous two-phase system at 7.8-8.0 to ensure the activity and structural stability of papain. Tris in the Tris-HCl buffer can bind metal ions, reducing enzyme oxidative inactivation.

[0030] An extraction process for papain includes the following steps:

[0031] (1) Take the supernatant from an unripe papaya;

[0032] (2) Stir and mix the supernatant and the aqueous two-phase liquid, let them stand to separate the phases, and obtain the lower phase;

[0033] (3) Collect the lower phase, remove impurities by ultrafiltration to obtain a concentrated solution; the concentrated solution is then freeze-dried to obtain enzyme powder.

[0034] Specifically, in step (1), the preparation process of the supernatant from the unripe papaya is as follows: take fresh unripe green papaya, peel and chop it, add ice water and crush it; filter, collect the supernatant, and obtain the supernatant from the unripe papaya.

[0035] In this application, chopped papaya is crushed using ice water at 2-4°C, which can destroy cell structure and inhibit enzyme activity loss. After filtration, solid impurities are removed to obtain supernatant from unripe papaya.

[0036] A two-phase liquid is formed by using surfactants (SDS, CTAB) and salt (KBr). Papain is selectively partitioned to the lower phase through hydrophobic and electrostatic interactions. Adjusting the pH to 7.8-8.0 can maintain enzyme activity and optimize phase partitioning efficiency.

[0037] Specifically, in step (2), the preparation process of the aqueous two-phase liquid is as follows: prepare sodium dodecyl sulfate solution with a concentration of 0.10-0.13 mol / L, hexadecyltrimethylammonium bromide solution with a concentration of 0.10-0.13 mol / L, and potassium bromide solution with a concentration of 0.05-0.06 mol / L, respectively;

[0038] Mix 0.10-0.13 mol / L sodium dodecyl sulfate solution and 0.10-0.13 mol / L hexadecyltrimethylammonium bromide solution at a volume ratio of 1:1, then add an equal volume of 0.05-0.06 mol / L potassium bromide solution and mix. After mixing, let stand at 30-35℃ for 2-3 hours to obtain an aqueous two-phase liquid.

[0039] Specifically, in step (2), the volume ratio of the supernatant to the aqueous two-phase liquid is 1:2.5.

[0040] Specifically, in step (2), the pH of the aqueous two-phase liquid is adjusted to 7.8-8.0 using Tris-HCl buffer, and then the supernatant and the aqueous two-phase liquid are stirred and mixed.

[0041] Specifically, in step (2), the mixture is stirred at 20-35°C at a speed of 200-1000 r / min for 30-150 min.

[0042] Furthermore, in step (2), the aqueous two-phase liquid is formed by mixing sodium dodecyl sulfate (SDS), hexadecyltrimethylammonium bromide (CTAB), and potassium bromide (KBr) solutions. The aqueous two-phase liquid consists of two immiscible aqueous phases, and the selective partitioning and separation of biomolecules are achieved by utilizing the synergistic effect of surfactants and salts.

[0043] In an aqueous two-phase liquid, papain selectively partitions into the lower phase due to its specific water solubility and charge properties. By adjusting the pH value and stirring conditions, the partitioning efficiency of papain can be optimized. The upper phase mainly contains unbound impurities and a small amount of enzyme. Phase separation can effectively remove these impurities and improve the purity of papain.

[0044] More specifically, in aqueous two-phase liquids, sodium dodecyl sulfate (SDS) acts as an anionic surfactant, participating in the formation of the aqueous two-phase system; SDS can reduce the surface tension between the aqueous phases, promoting the formation of the aqueous two-phase system. The hydrophobic portion of SDS interacts with the hydrophobic region of papain, promoting the partitioning of the enzyme into the lower phase.

[0045] Hexadecyltrimethylammonium bromide (CTAB), as a cationic surfactant, participates in the formation of aqueous two-phase systems. CTAB can reduce the surface tension between the aqueous phases, promoting the formation of aqueous two-phase systems. The cationic portion of CTAB interacts with the negatively charged region of papain, promoting the partitioning of the enzyme into the lower phase.

[0046] Potassium bromide (KBr) modulates the ionic strength of the aqueous two-phase system, affecting the partitioning behavior of papain.

[0047] Tris-HCl buffer is used to adjust the pH of the aqueous two-phase system. It maintains the pH of the aqueous two-phase system at 7.8-8.0, ensuring the activity and structural stability of papain. Simultaneously, the Tris in the buffer can bind metal ions, reducing oxidative inactivation of the enzyme.

[0048] More specifically, pH (7.8-8.0) maintains the activity and structural stability of papain. Within the pH range of 7.8-8.0, papain exhibits the highest partitioning efficiency, effectively partitioning into the lower phase. Excessively high pH values ​​may lead to enzyme denaturation and inactivation, while excessively low pH values ​​may affect enzyme activity.

[0049] Use a stirring speed of 200-1000 rpm to ensure thorough mixing of the supernatant and the aqueous two-phase liquid. An appropriate stirring speed ensures thorough mixing of the supernatant and the aqueous two-phase liquid, improving the partitioning efficiency of papain. Excessive stirring speed may cause liquid to splash or generate too much foam, affecting the phase separation effect.

[0050] Stirring time (30-150 min) ensures that papain is fully distributed into the lower phase. Appropriate stirring time ensures sufficient distribution of papain into the lower phase, improving extraction efficiency. Excessive stirring time may lead to enzyme inactivation or remixing of impurities.

[0051] Temperature (20-35℃) is crucial for maintaining the stability of the aqueous two-phase system. Within this temperature range, the stability of the aqueous two-phase system is optimal, and the partitioning efficiency of papain is highest. Excessively high temperatures may lead to enzyme denaturation and inactivation, while excessively low temperatures may affect the formation and partitioning efficiency of the aqueous two-phase system.

[0052] Specifically, in step (3), the process of removing impurities by ultrafiltration is as follows: the lower phase is filtered using an ultrafiltration membrane to obtain a concentrated solution. The molecular weight cutoff of the ultrafiltration membrane is 10-30 kDa.

[0053] Specifically, in step (3), the concentrate is pre-frozen at -80°C to -70°C for 2 hours and then vacuum dried to obtain enzyme powder.

[0054] In step (3), the activity of the enzyme powder is 115,950-131,576 units / mg.

[0055] Furthermore, in step (3), papain is further purified by ultrafiltration and freeze-drying to obtain highly active enzyme powder; Ultrafiltration: using the retention effect of the ultrafiltration membrane, impurities in the concentrate are removed to obtain a relatively pure papain concentrate. By freezing the concentrate at low temperature and then sublimating and drying it under vacuum conditions, highly active papain enzyme powder is obtained.

[0056] Ultrafiltration membranes are used to remove impurities from the concentrate. With a molecular weight cutoff of 10-30 kDa, ultrafiltration membranes can effectively retain papain while removing smaller impurity molecules. Therefore, ultrafiltration can significantly improve the purity of papain, ensuring high activity of the enzyme powder obtained through subsequent freeze-drying.

[0057] The concentrate, used as a raw material for freeze-drying, contains a high concentration of papain, which can be freeze-dried to obtain highly active enzyme powder. Pre-freezing at -80℃ to -70℃ for 2 hours can effectively protect the activity of papain and prevent enzyme inactivation during the drying process.

[0058] Drying at a vacuum of 0.08-0.09 MPa and a temperature of -50°C can quickly remove moisture from the concentrate, yielding dried enzyme powder.

[0059] The extraction process described in this application will be further illustrated by the following examples and comparative examples;

[0060] Example 1

[0061] An extraction process for papain includes the following steps:

[0062] (1) Take fresh, unripe green papaya, peel and chop it to obtain raw material to be processed; add 5 times the mass of ice water at 2-4℃ (containing about 40% ice) to the raw material to be processed, crush it; filter it to obtain filtrate, collect the supernatant of the filtrate to obtain supernatant from unripe papaya.

[0063] (2) Adjust the pH of the aqueous two-phase liquid to 7.8-8.0 using Tris-HCl buffer, and then mix the supernatant obtained in step (1) with the aqueous two-phase liquid with pH controlled at 7.8-8.0. The volume ratio of the supernatant to the aqueous two-phase liquid with pH controlled at 7.8-8.0 is 1:2.5.

[0064] Then, the mixture was stirred at 800 r / min for 100 min at 30℃; after mixing, it was allowed to stand at room temperature for 2 h to separate the phases, resulting in the lower phase and the upper phase.

[0065] The preparation process of the aqueous two-phase liquid is as follows: prepare sodium dodecyl sulfate solution with a concentration of 0.11 mol / L, hexadecyltrimethylammonium bromide solution with a concentration of 0.11 mol / L, and potassium bromide solution with a concentration of 0.055 mol / L, respectively;

[0066] A 0.11 mol / L sodium dodecyl sulfate solution and a 0.11 mol / L hexadecyltrimethylammonium bromide solution were mixed at a volume ratio of 1:1. Then, an equal volume of 0.055 mol / L potassium bromide solution was added and mixed. After mixing, the mixture was allowed to stand at 32 °C for 2.5 h to obtain an aqueous two-phase liquid.

[0067] (3) Collect the lower phase and filter it using an ultrafiltration membrane with a molecular weight cutoff of 20 kDa to obtain a concentrated solution. Pre-freeze the concentrated solution at -75°C for 2 hours, and then dry it at a vacuum of 0.08-0.09 MPa and a temperature of -50°C to obtain enzyme powder.

[0068] Example 2

[0069] An extraction process for papain includes the following steps:

[0070] (1) Take fresh, unripe green papaya, peel and chop it to obtain raw material to be processed; add 5 times the mass of ice water at 2-4℃ (containing about 40% ice) to the raw material to be processed, crush it; filter it to obtain filtrate, collect the supernatant of the filtrate to obtain supernatant from unripe papaya.

[0071] (2) Adjust the pH of the aqueous two-phase liquid to 7.8-8.0 using Tris-HCl buffer, and then mix the supernatant obtained in step (1) with the aqueous two-phase liquid with pH controlled at 7.8-8.0. The volume ratio of the supernatant to the aqueous two-phase liquid with pH controlled at 7.8-8.0 is 1:2.5.

[0072] Then, the mixture was stirred at 1000 r / min for 150 min at 35 °C; after mixing, it was allowed to stand at room temperature for 2 h to separate the phases, resulting in the lower phase and the upper phase.

[0073] The preparation process of the aqueous two-phase liquid is as follows: prepare sodium dodecyl sulfate solution with a concentration of 0.13 mol / L, hexadecyltrimethylammonium bromide solution with a concentration of 0.13 mol / L, and potassium bromide solution with a concentration of 0.06 mol / L, respectively;

[0074] A 0.13 mol / L sodium dodecyl sulfate solution and a 0.13 mol / L hexadecyltrimethylammonium bromide solution were mixed at a volume ratio of 1:1. Then, an equal volume of 0.06 mol / L potassium bromide solution was added and mixed. After mixing, the mixture was allowed to stand at 35 °C for 3 h to obtain an aqueous two-phase liquid.

[0075] (3) Collect the lower phase and filter it using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to obtain a concentrated solution. Pre-freeze the concentrated solution at -70°C for 2 hours, and then dry it at a vacuum of 0.08-0.09 MPa and a temperature of -50°C to obtain enzyme powder.

[0076] Example 3

[0077] An extraction process for papain includes the following steps:

[0078] (1) Take fresh, unripe green papaya, peel and chop it to obtain raw material to be processed; add ice water (containing 40% ice) with a mass ratio of 5 to the raw material to be processed, crush it; filter it to obtain filtrate, collect the supernatant of the filtrate to obtain supernatant from unripe papaya.

[0079] (2) Adjust the pH of the aqueous two-phase liquid to 7.8-8.0 using Tris-HCl buffer, and then mix the supernatant obtained in step (1) with the aqueous two-phase liquid with pH controlled at 7.8-8.0. The volume ratio of the supernatant to the aqueous two-phase liquid with pH controlled at 7.8-8.0 is 1:2.5.

[0080] Then, the mixture was stirred at 200 r / min for 30 min at 20℃; after mixing, it was allowed to stand at room temperature for 2 h to separate the phases, resulting in the lower phase and the upper phase.

[0081] The preparation process of the aqueous two-phase liquid is as follows: prepare sodium dodecyl sulfate solution with a concentration of 0.10 mol / L, hexadecyltrimethylammonium bromide solution with a concentration of 0.10 mol / L, and potassium bromide solution with a concentration of 0.05 mol / L, respectively;

[0082] A 0.10 mol / L sodium dodecyl sulfate solution and a 0.10 mol / L hexadecyltrimethylammonium bromide solution were mixed at a volume ratio of 1:1. Then, an equal volume of 0.05 mol / L potassium bromide solution was added and mixed. After mixing, the mixture was allowed to stand at 30 °C for 2 h to obtain an aqueous two-phase liquid.

[0083] (3) Collect the lower phase and filter it using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to obtain a concentrated solution. Pre-freeze the concentrated solution at -80°C for 2 hours, and then dry it at a vacuum of 0.08-0.09 MPa and a temperature of -50°C to obtain enzyme powder.

[0084] Example 4

[0085] An extraction process for papain includes the following steps:

[0086] (1) Take fresh, unripe green papaya, peel and chop it to obtain raw material to be processed; add ice water (containing 40% ice) with a mass ratio of 5 to the raw material to be processed, crush it; filter it to obtain filtrate, collect the supernatant of the filtrate to obtain supernatant from unripe papaya.

[0087] (2) Adjust the pH of the aqueous two-phase liquid to 7.8-8.0 using Tris-HCl buffer, and then mix the supernatant obtained in step (1) with the aqueous two-phase liquid with pH controlled at 7.8-8.0. The volume ratio of the supernatant to the aqueous two-phase liquid with pH controlled at 7.8-8.0 is 1:2.5.

[0088] Then, the mixture was stirred at 1000 r / min for 30 min at 20℃; after mixing, it was allowed to stand at room temperature for 2 h to separate the phases, resulting in the lower phase and the upper phase.

[0089] The preparation process of the aqueous two-phase liquid is as follows: prepare sodium dodecyl sulfate solution with a concentration of 0.10 mol / L, hexadecyltrimethylammonium bromide solution with a concentration of 0.13 mol / L, and potassium bromide solution with a concentration of 0.05 mol / L, respectively;

[0090] A 0.10 mol / L sodium dodecyl sulfate solution and a 0.13 mol / L hexadecyltrimethylammonium bromide solution were mixed at a volume ratio of 1:1. Then, an equal volume of 0.05 mol / L potassium bromide solution was added and mixed. After mixing, the mixture was allowed to stand at 30 °C for 3 h to obtain an aqueous two-phase liquid.

[0091] (3) Collect the lower phase and filter it using an ultrafiltration membrane with a molecular weight cutoff of 30 kDa to obtain a concentrated solution. Pre-freeze the concentrated solution at -80°C for 2 hours, and then dry it at a vacuum of 0.08-0.09 MPa and a temperature of -50°C to obtain enzyme powder.

[0092] Example 5

[0093] An extraction process for papain includes the following steps:

[0094] (1) Take fresh, unripe green papaya, peel and chop it to obtain raw material to be processed; add ice water (containing 40% ice) with a mass ratio of 5 to the raw material to be processed, crush it; filter it to obtain filtrate, collect the supernatant of the filtrate to obtain supernatant from unripe papaya.

[0095] (2) Adjust the pH of the aqueous two-phase liquid to 7.8-8.0 using Tris-HCl buffer, and then mix the supernatant obtained in step (1) with the aqueous two-phase liquid with pH controlled at 7.8-8.0. The volume ratio of the supernatant to the aqueous two-phase liquid with pH controlled at 7.8-8.0 is 1:2.5.

[0096] Then, the mixture was stirred at 200 r / min for 40 min at 35 °C; after mixing, it was allowed to stand at room temperature for 2 h to separate the phases, resulting in the lower phase and the upper phase.

[0097] The preparation process of the aqueous two-phase liquid is as follows: prepare sodium dodecyl sulfate solution with a concentration of 0.13 mol / L, hexadecyltrimethylammonium bromide solution with a concentration of 0.10 mol / L, and potassium bromide solution with a concentration of 0.06 mol / L, respectively;

[0098] A 0.13 mol / L sodium dodecyl sulfate solution and a 0.10 mol / L hexadecyltrimethylammonium bromide solution were mixed at a volume ratio of 1:1. Then, an equal volume of 0.06 mol / L potassium bromide solution was added and mixed. After mixing, the mixture was allowed to stand at 35 °C for 2 h to obtain an aqueous two-phase liquid.

[0099] (3) Collect the lower phase and filter it using an ultrafiltration membrane with a molecular weight cutoff of 15 kDa to obtain a concentrated solution. Pre-freeze the concentrated solution at -75°C for 2 hours, and then dry it at a vacuum of 0.08-0.09 MPa and a temperature of -50°C to obtain enzyme powder.

[0100] Comparative Example 1

[0101] Papain was extracted using a traditional organic solvent precipitation method (such as acetone).

[0102] Steps: Take fresh, unripe green papaya, peel and chop it, add ice water and mash it, then filter and collect the supernatant. Add acetone to the supernatant in a volume equal to 3 times the volume of the supernatant, gently stir and mix at room temperature for 30 minutes, let it stand to allow the protein to precipitate, centrifuge to separate the protein, collect the precipitate and obtain the filtrate.

[0103] Comparative Example 2

[0104] The extraction process of Example 1 was adopted, but the molecular weight cutoff of the ultrafiltration membrane in step (3) was 35 kDa. That is, after collecting the lower phase, the lower phase was filtered using an ultrafiltration membrane with a molecular weight cutoff of 35 kDa to obtain a concentrated solution, which was then freeze-dried.

[0105] Comparative Example 3

[0106] The process of Example 1 is used, but in step (2), the pH of the aqueous two-phase liquid is not adjusted to 7.8-8.0 using Tris-HCl buffer, and the prepared aqueous two-phase liquid and supernatant are directly stirred and mixed.

[0107] 1. Enzyme activity

[0108] The enzyme activity of each comparative example and embodiment was determined using spectrophotometry. The enzyme activity was evaluated by measuring the amount of tyrosine produced in the enzymatic hydrolysis reaction, as detailed below:

[0109] ①. Prepare reagents and materials

[0110] Tyrosine standard solution: Weigh tyrosine, dissolve it in 0.2N HCl, and bring the volume to 100ml. Then dilute it 5 times with water to obtain a tyrosine solution of 200μg / ml.

[0111] Casein solution: Weigh 2g of casein, adjust the pH to 9.0 with 0.2mol / L Na2HPO4, add water to 100ml, and filter to remove particles.

[0112] Enzyme solution: Accurately weigh 1.0g of the sample to be tested, add it to 500ml of buffer solution, and let it stand at room temperature for 1 hour while stirring. Filter the enzyme extract, take 1ml of the filtrate and dilute it with buffer solution to 100ml, which is the enzyme solution diluted 500 times.

[0113] ②. Construction of Standard Curve

[0114] Add different amounts of tyrosine standard solution (1.0 ml, 0.8 ml, 0.6 ml, 0.4 ml, 0.2 ml) to test tubes, and bring the volume to 1.0 ml with distilled water. Then add 0.4 ml of Folin-Ciocalteu reagent, and after color development, incubate at 20°C for 30 minutes. Read the optical density (OD value) at 680 nm using a 721 spectrophotometer. Plot a standard curve with optical density as the ordinate and tyrosine concentration as the abscissa, as shown below. Figure 1 As shown.

[0115] ③. Protease activity assay

[0116] Take three stoppered test tubes and add 5 ml of casein solution to each. Incubate in a 40°C water bath for 10 minutes. Add different amounts of enzyme solution (0.5 ml, 1.0 ml, and 1.5 ml) respectively, shake well, and react in a 40°C water bath for 1 hour. Immediately add 5 ml of 10% trichloroacetic acid solution, shake vigorously to mix well, and let stand for 30–40 minutes to precipitate the protein. Filter, take 1 ml of the filtrate, add 5 ml of 0.4 mol / L Na₂CO₃ and 1 ml of Folin-Ciocalteu reagent, develop color, and incubate in a 40°C water bath for 20 minutes. Read the optical density at a wavelength of 680 nm on a 721 spectrophotometer.

[0117] ④. Calculate enzyme activity

[0118] Find the corresponding tyrosine content (in grams) from the standard curve using the optical density value obtained in the measurement, and calculate the enzyme activity according to the following formula:

[0119] Papain activity unit = tyrosine micrograms / OD value.

[0120] 2. Enzyme extraction rate

[0121] The formula for calculating enzyme extraction rate is: Enzyme extraction rate (%) = (Total enzyme activity in enzyme powder / Total enzyme activity in supernatant) × 100%

[0122] ① Determine the total enzyme activity in the supernatant:

[0123] Take the supernatant from step (1), dilute it by a certain factor (e.g., 10 times), and use spectrophotometry to determine the activity of papain; calculate the total enzyme activity (U) in the supernatant.

[0124] ② Determine the total enzyme activity in the enzyme powder:

[0125] For example, a certain amount of enzyme powder obtained in step (3) is accurately weighed, dissolved in buffer solution, diluted by a certain factor, and the activity of papain is determined by spectrophotometry to calculate the total enzyme activity (U) in the enzyme powder.

[0126] ③ Calculate the extraction rate using the formula above.

[0127] The calculated enzyme activity and extraction rate are summarized in Table 1.

[0128] Table 1

[0129]

[0130]

[0131] As shown in Table 1, the enzyme activity of the examples all reached between 115,950 and 131,576 units / mg, with an extraction rate of over 92%, and a maximum of 98%.

[0132] Compared with the examples, the performance of Comparative Examples 1-3 was significantly worse:

[0133] Low enzyme activity: The enzyme activity of Comparative Example 1 was only 38,950 units / mg, far lower than the lowest value of 115,950 units / mg in the examples. The enzyme activities of Comparative Examples 2 and 3 were also low, at 65,760 units / mg and 58,950 units / mg, respectively.

[0134] Low extraction rate: The extraction rate of Comparative Example 1 was only 30%, while the extraction rates of Comparative Example 2 and Comparative Example 3 were 50% and 45%, respectively, both significantly lower than the lowest extraction rate of 92% in the examples.

[0135] In Comparative Example 1, the traditional organic solvent precipitation method uses acetone as the organic solvent for precipitation. Although this method can effectively precipitate proteins, acetone disrupts the hydrogen bonds of proteins, leading to partial enzyme denaturation and inactivation, thereby reducing the enzyme activity and extraction rate. Both enzyme activity and extraction rate are significantly lower than in the example.

[0136] In Comparative Example 2, the ultrafiltration membrane had an excessively high molecular weight cutoff. In step (3), the ultrafiltration membrane had a molecular weight cutoff of 35 kDa, which was larger than the 10-30 kDa range used in the examples. This larger molecular weight cutoff resulted in some papain being retained on the membrane and failing to enter the concentrate, thus reducing the enzyme concentration and activity in the concentrate. Although the enzyme activity in Comparative Example 2 was improved, it was still far lower than in the examples, and the extraction rate was also low.

[0137] In Comparative Example 3, the pH of the aqueous two-phase liquid was not adjusted. In step (2), the pH of the aqueous two-phase liquid was not adjusted to 7.8-8.0 using Tris-HCl buffer. Instead, the prepared aqueous two-phase liquid and the supernatant were directly stirred and mixed. A pH value outside the optimal range can affect the stability and partitioning efficiency of papain, and may lead to enzyme inactivation or reduced activity.

[0138] The enzyme activity and extraction rate of Comparative Example 3 were significantly lower than those of the Example, indicating that pH value has an important influence on enzyme activity and stability.

[0139] It is evident that using aqueous two-phase liquids instead of organic solvents: Compared to traditional organic solvent precipitation methods such as acetone, aqueous two-phase systems achieve gentle phase separation through the action of surfactants (such as sodium dodecyl sulfate SDS and hexadecyltrimethylammonium bromide CTAB) and salts (such as potassium bromide KBr), reducing damage to enzyme structure and thus maintaining higher enzyme activity.

[0140] Optimize pH and stirring conditions: Adjust the pH of the aqueous two-phase liquid to 7.8-8.0 using Tris-HCl buffer to ensure the activity and structural stability of papain. Simultaneously, mixing is performed under appropriate temperature (20-35℃), stirring speed (200-1000 r / min), and time (30-150 min) conditions to ensure thorough mixing of the supernatant and the aqueous two-phase liquid, thereby improving enzyme partitioning efficiency.

[0141] Application of ultrafiltration and freeze-drying technologies: Impurities are removed using ultrafiltration membranes with a molecular weight cutoff of 10-30 kDa, and highly active enzyme powder is obtained through low-temperature pre-freezing and vacuum drying. This step not only removes small molecule impurities but also effectively protects the enzyme's activity.

[0142] The above embodiments are preferred implementations of the present invention. In addition, the present invention can be implemented in other ways. Any obvious substitutions without departing from the concept of the present invention are within the protection scope of the present invention.

Claims

1. A papain extraction process, characterized in that, Includes the following steps: (1) Take the supernatant from an unripe papaya; (2) Stir and mix the supernatant and the aqueous two-phase liquid, let them stand to separate the phases, and obtain the lower phase; (3) Collect the lower phase, remove impurities by ultrafiltration to obtain a concentrated solution; the concentrated solution is then freeze-dried to obtain enzyme powder.

2. The extraction process according to claim 1, characterized in that, In step (1), the preparation process of the supernatant from the unripe papaya is as follows: take fresh unripe green papaya, peel and chop it, add ice water and crush it; filter, collect the supernatant, and obtain the supernatant from the unripe papaya.

3. The extraction process according to claim 1, characterized in that, In step (2), the preparation process of the aqueous two-phase liquid is as follows: prepare sodium dodecyl sulfate solution with a concentration of 0.10-0.13 mol / L, hexadecyltrimethylammonium bromide solution with a concentration of 0.10-0.13 mol / L, and potassium bromide solution with a concentration of 0.05-0.06 mol / L, respectively; Mix 0.10-0.13 mol / L sodium dodecyl sulfate solution and 0.10-0.13 mol / L hexadecyltrimethylammonium bromide solution at a volume ratio of 1:1, then add an equal volume of 0.05-0.06 mol / L potassium bromide solution and mix. After mixing, let stand at 30-35℃ for 2-3 hours to obtain an aqueous two-phase liquid.

4. The extraction process according to claim 1, characterized in that, In step (2), the volume ratio of the supernatant to the aqueous two-phase liquid is 1:2.

5.

5. The extraction process according to claim 1, characterized in that, In step (2), the pH of the aqueous two-phase liquid is adjusted to 7.8-8.0 using Tris-HCl buffer, and then the supernatant and the aqueous two-phase liquid are stirred and mixed.

6. The extraction process according to claim 1, characterized in that, In step (2), the mixture is stirred at 20-35℃ at a speed of 200-1000r / min for 30-150min.

7. The extraction process according to claim 1, characterized in that, In step (3), the process of removing impurities by ultrafiltration is as follows: the lower phase is filtered using an ultrafiltration membrane to obtain a concentrated solution.

8. The extraction process according to claim 7, characterized in that, The molecular weight cutoff of ultrafiltration membranes is 10-30 kDa.

9. The extraction process according to claim 1, characterized in that, In step (3), the concentrate is pre-frozen at -80°C to -70°C for 2 hours and then vacuum dried to obtain enzyme powder.

10. The extraction process according to claim 1, characterized in that, In step (3), the activity of the enzyme powder is 115,950-131,576 units / mg.

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