Method for purifying sweet protein thaumatin I
By combining high-speed centrifugation, D201 resin treatment, activated carbon removal, three-stage plate and frame filtration, and membrane encapsulation, the problem of removing impurities and pigments from Thaumatin I protein in Trichoderma reesei fermentation broth was solved, and high-purity Thaumatin I protein was purified.
Patent Information
- Application Number
- CN202510778592.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2024-10-25
- Filing Date
- 2025-06-11
- Publication Date
- 2026-01-20
AI Technical Summary
Existing technologies are insufficient to efficiently remove impurities and pigments from Thaumatin I protein in Trichoderma reesei fermentation broth, thus affecting its purification effect.
Thaumatin I protein was purified stepwise using a combination of high-speed centrifugation, D201 resin treatment, activated carbon removal, three-stage plate and frame filtration, 30kDa and 10kDa membrane encapsulation, and freeze-drying.
High-purity Thaumatin I protein was obtained by removing pigments, inorganic salts and impurities from the fermentation broth, thus improving purification efficiency.
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Figure CN121362236A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the field of biotechnology, and particularly relates to a purification method of sweet protein Thaumatin I BACKGROUND
[0002] Trichoderma reesei is a thermophilic saprophytic fungus widely distributed in the soil of tropical and subtropical regions, and is one of the main strains for industrial production of cellulase and hemicellulase. The cellulase and hemicellulase of Trichoderma reesei have high efficiency and wide application potential, and play an important role in many industries such as food, feed, textile, agriculture and bioenergy. These enzymes can convert plant biomass into fermentable sugars, and then produce biofuels and bio-chemical products, showing its important position in modern bioeconomy. In addition, the safety and convenience of genetic manipulation of Trichoderma reesei make it a valuable microbial resource in the field of biotechnology. At the same time, Trichoderma reesei is a food safety level industrial production strain certified by the US FDA, and the proteins produced by it can be used in food, brewing and feed protein industries.
[0003] Sweeteners are a kind of widely used food additives, which can be divided into two categories: synthetic sweeteners and natural sweeteners. With the development of organic chemical technology, synthetic sweeteners currently occupy a dominant position in the sweetener market. However, more and more studies have shown that some mainstream synthetic sweeteners pose safety risks, so many developed countries have banned the use of these sweeteners. Therefore, it is of great significance to develop natural non-sugar sweeteners such as sweet proteins.
[0004] Sweet proteins are a class of proteins found in plants that have sweet taste and are composed entirely of amino acids. The first sweet protein discovered is miraculin protein, which has the function of converting sour taste into sweet taste. Subsequently, a variety of sweet proteins have been discovered in other plants. Compared with traditional sweeteners, sweet proteins have the advantages of high safety, high sweetness (usually thousands of times sweeter than sucrose), long duration of sweet taste, etc., and have broad market application value.
[0005] Thaumatin, also known as African bamboo sweetener, etc., is a very high sweet protein found in West African tropical plants (Thaumatococcus daniellii), which is 3000 times as sweet as sucrose. Related studies have shown that the thaumatin family contains two main proteins, thaumatin I and thaumatin II, and three trace proteins, thaumatin a, thaumatin b and thaumatin c. The molecular weight of thaumatin I and thaumatin II proteins is 22.2 kDa, and the amino acid sequences of the two are composed of 207 amino acids, but there are differences in individual amino acids. Because the protein structure of thaumatin contains 16 cysteines to form 8 disulfide bonds, it has good stability: not only can it exist under acidic pH conditions, but also has good heat resistance. In addition, thaumatin protein is not easily soluble in organic solvents, but is easily soluble in water.
[0006] As a super sweet protein, thaumatin has similar taste characteristics to carbohydrate sweeteners, and its safety has been proven and approved by the European Union as a food additive. In 2014, China has officially approved the use of plant-extracted thaumatin as a food additive. Thaumatin protein is often used as a sweetener and flavor enhancer in the food industry, and as a component of special low-calorie diet in pharmaceuticals. The traditional way to obtain thaumatin protein is to extract it from plants, which has the disadvantages of high cost and low yield, greatly limiting its development and utilization. The rapid development of synthetic biology technology provides a very useful tool for the development and production of thaumatin protein. The use of engineered bacteria to produce thaumatin protein opens up a fast and effective way for the commercial production of thaumatin.
[0007] Due to the strong protein secretion ability of the fungus Trichoderma reesei, the engineered Trichoderma reesei secreting thaumatin Thaumatin I protein will also secrete many heteroproteins and pigment molecules while secreting thaumatin Thaumatin I protein. Therefore, it is very important to develop a method for purifying thaumatin Thaumatin I protein from which heteroproteins and pigments are removed. SUMMARY
[0008] Therefore, the present application is proposed. In order to achieve the above-mentioned purpose, the present application provides a method for purifying sweet protein thaumatin from Trichoderma reesei fermentation broth, which can effectively remove pigments, inorganic salts and heteroproteins in the fermentation broth, and obtain high-purity thaumatin Thaumatin I protein. The method comprises the following steps:
[0009] (1) The solid-liquid separation is realized by high-speed centrifugation, and then the sweet protein thaumatin Thaumatin I fermentation broth supernatant from which the bacterial bodies are removed is obtained from the Trichoderma reesei fermentation broth;
[0010] (2) The pH of the supernatant of the fermentation broth obtained in step (1) is adjusted to 5-7, and the supernatant is treated with D201 resin to remove part of the impurities and most of the pigments, and then activated carbon is used to remove the residual pigments in the fermentation broth;
[0011] (3) The residual activated carbon in the fermentation broth is removed by high-speed centrifugation and three-stage plate and frame filtration to obtain a crude Thaumatin I protein solution;
[0012] (4) The crude Thaumatin I protein solution is subjected to interception and concentration using 30 kDa and 10 kDa membrane packages to obtain a Thaumatin I protein concentrate;
[0013] (5) The Thaumatin I protein concentrate is freeze-dried to obtain Thaumatin I protein dry powder.
[0014] Further, the concentration of activated carbon used to remove the residual pigments in the fermentation broth in step (2) is 0.5-1.5%.
[0015] Further, in step (3), the residual activated carbon in the fermentation broth is removed by three-stage plate and frame filtration. The first filter membrane is a medium-speed qualitative filter paper, the second filter membrane is a 1 μm filter membrane, and the third filter membrane is a 0.22 μm filter membrane.
[0016] Further, in step (4), the 30 kDa and above impurities in the crude Thaumatin I protein solution are intercepted using a 30 kDa membrane package to obtain a permeate containing Thaumatin I protein.
[0017] Further, when intercepting impurities using a 30 kDa membrane package, the operating pressure is controlled at 0.2-0.3 MPa, and the operating temperature is room temperature.
[0018] Further, after the 30 kDa membrane package is used up, it is cleaned with 0.3 M NaOH solution.
[0019] Further, in step (4), the permeate containing Thaumatin I protein is concentrated using a 10 kDa membrane package to obtain a Thaumatin I protein concentrate.
[0020] Further, when concentrating using a 10 kDa membrane package, the operating pressure is controlled at 0.2-0.3 MPa, and the operating temperature is room temperature.
[0021] Further, after the 10 kDa membrane package is used up, it is cleaned with 0.3 M NaOH solution. BRIEF DESCRIPTION OF DRAWINGS
[0022] Figure 1 SDS-PAGE verification chart for 30 kDa membrane package retention effect;
[0023] Figure 2 SDS-PAGE verification chart for 10 kDa membrane package retention effect;
[0024] Figure 3 Freeze-dried sample chart. DETAILED DESCRIPTION
[0025] The present application is further described in detail by the following specific examples. The present application can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and fully convey the scope of the application to those skilled in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.
[0026] The experimental methods in the following examples are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified.
[0027] Example 1
[0028] The removal of pigment residues in the fermentation broth is carried out according to the following steps:
[0029] (1) Solid-liquid separation is achieved by high-speed centrifugation, and then Thaumatin I protein fermentation broth supernatant is obtained from the Trichoderma reesei fermentation broth, and the fermentation broth supernatant containing Thaumatin I protein is collected;
[0030] (2) The pH of the collected fermentation broth supernatant is adjusted to 5-7, and D201 resin is used for treatment to remove part of the impurities and most of the pigments, and then activated carbon is used to remove the residual pigments in the fermentation broth;
[0031] (3) High-speed centrifugation and three-stage plate and frame filtration are used to remove the residual activated carbon in the fermentation broth to obtain Thaumatin I protein crude extract; wherein the first filter membrane is a medium-speed qualitative filter paper, the second filter membrane is a 1 μm filter membrane, and the third filter membrane is a 0.22 μm filter membrane;
[0032] Example 2
[0033] The removal of impurities in the fermentation broth and the obtaining of freeze-dried powder are carried out according to the following steps:
[0034] (1) using 30kDa membrane package to protein crude extract of 30kDa above impurities for intercept. Operating pressure control at 0.2-0.3MPa, operating temperature is room temperature, get containing Thaumatin I protein permeate. 30kDa membrane package using after, with 0.3M NaOH solution is cleaned. By Figure 1 It can be seen that the 30kDa membrane package permeate protein is mainly Thaumatin I protein, no obvious detection of 25kDa above impurities, indicating that 25kDa above impurities are successfully intercepted by 30kDa membrane package.
[0035] (2) using 10kDa membrane package to contain Thaumatin I protein permeate is concentrated. Operating pressure control at 0.2-0.3MPa, operating temperature is room temperature, get Thaumatin I protein concentrate. 10kDa membrane package using after, with 0.3M NaOH solution is cleaned. By Figure 2 It can be seen that the intercept liquid contains a large number of Thaumatin I protein, and the purity is very high, indicating that Thaumatin I protein is successfully intercepted by 10kDa membrane package.
[0036] (3) Thaumatin I protein concentrate is freeze-dried, get Thaumatin I protein dry powder, Figure 3 The display is the Thaumatin I protein sample after freeze-drying.
[0037] The above only for the embodiment of the present application, not therefore limit the patent range of the present application, any equivalent structure or equivalent process transformation, or directly or indirectly used in other related technical fields, are also included in the patent protection scope of the present application.
Claims
1. A method for purifying a sweet protein thaumatin I from Thaumatin I, characterized by, The method comprises the following steps: (1) solid-liquid separation is realized by high-speed centrifugation, and then the Thaumatin I protein fermentation supernatant is obtained from the Trichoderma reesei fermentation liquor by removing the bacteria; (2) the pH of the fermentation supernatant obtained in step (1) is adjusted to 5-7, and D201 resin is used for treatment to remove part of the impurities and most of the pigments, and then activated carbon is used to remove the residual pigments in the fermentation liquor; (3) high-speed centrifugation and three-stage plate and frame filtration are used to remove the residual activated carbon in the fermentation liquor, and the Thaumatin I protein crude extract is obtained; (4) the Thaumatin I protein crude extract is subjected to cutting and concentration using 30kDa and 10kDa membrane packages, and the Thaumatin I protein concentrated solution is obtained; (5) the Thaumatin I protein concentrated solution is freeze-dried to obtain Thaumatin I protein dry powder.
2. The method of claim 1, wherein, The concentration of activated carbon used for removing the residual pigments in the fermentation liquor in step (2) is 0.5-1.5%.
3. The method of claim 1, wherein, In step (3), three-stage plate and frame filtration is used to remove the residual activated carbon in the fermentation liquor. The first-stage filter membrane is a medium-speed qualitative filter paper, the second-stage filter membrane is a 1μm filter membrane, and the third-stage filter membrane is a 0.22μm filter membrane.
4. The method of claim 1, wherein, In step (4), the 30kDa and above impurities in the Thaumatin I protein crude extract are cut using a 30kDa membrane package, and the permeate containing Thaumatin I protein is obtained.
5. The method according to any one of claims 1 to 5, characterized in that, When the 30kDa membrane package is used to cut the impurities, the operating pressure is controlled at 0.2-0.3MPa, and the operating temperature is room temperature.
6. The method according to any one of claims 1 to 6, characterized in that, After the 30kDa membrane package is used, it is cleaned with 0.3M NaOH solution.
7. The method of claim 1, wherein, In step (4), the permeate containing Thaumatin I protein is concentrated using a 10kDa membrane package, and the Thaumatin I protein concentrated solution is obtained.
8. The method according to any one of claims 1 to 8, characterized in that, When the 10kDa membrane package is used for concentration, the operating pressure is controlled at 0.2-0.3MPa, and the operating temperature is room temperature.
9. The method according to any one of claims 1 to 9, characterized in that, After the 10kDa membrane package is used, it is cleaned with 0.3M NaOH solution.