Hydrophobin HFBII purification method

Abstract

The invention relates to the technical field of biology, in particular to a hydrophobin HFBII purification method. The invention provides a method for purifying hydrophobin from trichoderma reesei fermentation liquor, which can effectively remove pigments, inorganic salts and impure proteins to obtain high-purity hydrophobin. In specific implementation, solid-liquid separation is realized through multiple times of high-speed centrifugation, and a Trichoderma reesei fermentation broth supernatant containing hydrophobin is collected. Adjusting the pH value, removing residual pigments in the fermentation liquor by using D201 resin and activated carbon, and removing the residual activated carbon through high-speed centrifugation and three-stage filtration to obtain a crude extract; and then, concentrating the crude extract by using a 3kDa membrane, adjusting the pH value, carrying out heating treatment, collecting the supernatant, and carrying out freeze drying to finally obtain the hydrophobin freeze-dried powder. The method is simple to operate, convenient and fast, and has the application prospect of large-scale production of hydrophobin.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a purification of hydrophobin HFB II. The protein can be widely applied to food, biological medicine and detection fields. BACKGROUND

[0002] Trichoderma reesei is a kind of filamentous fungi, belonging to the genus Trichoderma of Moniliales, and is an important industrial microorganism. It is widely used because of its high cellulase production capacity. At the same time, Trichoderma reesei is also an FDA certified food safety level industrial production strain. Trichoderma reesei can produce a variety of enzymes, including cellulase, hemicellulase, protease, amylase, etc. These enzymes have important applications in the industries of biofuels, food, textiles, feed and agriculture. In industrial production, the genetic modification of Trichoderma reesei has also been widely concerned. Through the genetic modification of the strain, the efficiency and yield of enzyme production can be improved. For example, the cbh1 gene of Trichoderma reesei encodes a major cellulase, cellobiohydrolase 1, which accounts for 50% of the total amount of extracellular secretory proteins of Trichoderma reesei. Therefore, the cbh1 promoter is a very strong promoter, which is often used to construct vectors to guide the secretory expression of recombinant proteins. Trichoderma reesei not only has the ability to synthesize and secrete proteins, but also has eukaryotic secretion mechanism, which may have similar protein modification properties as mammalian systems, such as high mannose type and N-glycosylation. These characteristics make Trichoderma reesei an effective way to produce homologous or heterologous secretory proteins. Trichoderma reesei is a filamentous fungus with important industrial value, and has broad application prospects in the field of biotechnology.

[0003] Hydrophobins are a class of surface-active proteins with relatively small molecular weight (7-10 kDa), but strong function. This class of proteins mainly exists in filamentous fungi, is rich in cysteine, and is closely related to the growth and development of fungal hyphae. In addition, this class of proteins not only has good heat resistance, but also can self-assemble to form amphiphilic membranes. Based on the morphological and functional characteristics of the self-assembled membrane and the uniformity of the amino acid sequence, fungal hydrophobins can be divided into two types: type I and type II. In the field of food, hydrophobins can be used as emulsifiers or foam stabilizers; in the field of biological medicine, hydrophobins have the functions of prolonging the half-life of drugs and enhancing the solubility of drugs due to the self-assembly characteristics; in the field of detection, hydrophobins are mainly used in the preparation of biosensors, which have the advantages of fast response rate, good stability and simple operation.

[0004] Studies have shown that Trichoderma reesei can produce two types of II hydrophobic proteins: HFB I and HFB II. When glucose is used as a carbon source, HFB I can be expressed in the form of mycelium binding; when lactose is used as a carbon source, HFB II can be expressed in a free manner and secreted into the culture medium, which is very conducive to the separation and purification of HFB II, and the secretion level of HFB II can be greatly improved through genetic engineering of Trichoderma reesei.

[0005] Due to the strong protein secretion ability of Trichoderma reesei itself, the Trichoderma reesei engineering bacteria secreting and expressing hydrophobic proteins will also secrete a large number of impurities and pigment molecules at the same time, and how to purify the hydrophobic proteins from the fermentation broth becomes a major problem. Therefore, it is very important to develop a protein purification method for removing impurities and pigments from the hydrophobic protein HFB II. SUMMARY

[0006] Therefore, the present application is proposed. To achieve the above-mentioned object, the present application provides a method for purifying hydrophobic protein HFB II from Trichoderma reesei fermentation broth, which can effectively remove pigments, inorganic salts and impurities in the fermentation broth, and obtain high-purity hydrophobic protein, and the method comprises the following steps

[0007] A hydrophobic protein HFB II purification method, characterized in that the method comprises the following steps:

[0008] (1) solid-liquid separation is realized by high-speed centrifugation, and then the hydrophobic protein fermentation broth supernatant free of bacteria is obtained from the Trichoderma reesei fermentation broth;

[0009] (2) the pH of the fermentation broth supernatant obtained in step (1) is adjusted to 5-7, D201 resin is used for treatment, part of the impurities and most of the pigments are removed, and then activated carbon is used to remove the residual pigments in the fermentation broth;

[0010] (3) high-speed centrifugation and three-stage plate frame are used to remove the residual activated carbon in the fermentation broth, and the crude hydrophobic protein solution is obtained;

[0011] (4) the crude hydrophobic protein solution is filtered and concentrated by using a 3kDa membrane bag, and the hydrophobic protein concentrate is obtained;

[0012] (5) the impurities in the hydrophobic protein concentrate are removed by adjusting the pH, water bath heating and high-speed centrifugation;

[0013] (6) the hydrophobic protein concentrate free of impurities is freeze-dried to obtain hydrophobic protein freeze-dried powder.

[0014] Further, the concentration of activated carbon used for removing the residual pigments in the fermentation broth in step (2) is 0.5-1.5%.

[0015] Further, the step (3) uses three-stage plate and frame filtration to remove the residual activated carbon in the fermentation broth. 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, when using a 3 kDa membrane package for concentration, the operating pressure is controlled at 0.2-0.3 MPa, and the operating temperature is room temperature.

[0017] Further, after the use of the 3 kDa membrane package, 0.3 M NaOH solution is used for cleaning.

[0018] Further, in the step (5), the pH of the hydrophobic protein concentrate is adjusted to below 7, and the temperature is controlled at 70-90°C for water bath heating. The heating time is 20-30 min. After high-speed centrifugation, the supernatant is collected to obtain the hydrophobic protein concentrate with removed impurities. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 SDS-PAGE verification diagram for 3KD membrane package retention effect

[0020] Figure 2 SDS-PAGE verification diagram for heating to remove impurities effect

[0021] Figure 3 Hydrophobic protein HFB II freeze-dried sample diagram DETAILED DESCRIPTION

[0022] The present application will be further described below in conjunction with specific embodiments. The present application can be realized in many different forms and is not limited to the embodiments described herein. Rather, the purpose of the embodiments is to make the disclosure of the present application more thorough and complete. 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 the present application belongs. The terms used in the specification of the present application herein are only for the purpose of describing specific embodiments and are not intended to limit the present application.

[0023] 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.

[0024] Example 1

[0025] The removal of pigment residues in the fermentation broth is carried out according to the following steps:

[0026] (1) Solid-liquid separation is achieved by high-speed centrifugation, and then the hydrophobic protein fermentation broth supernatant containing hydrophobic proteins is obtained from the Trichoderma reesei fermentation broth from which the bacterial bodies are removed;

[0027] (2) Adjust the pH of the collected fermentation broth supernatant to 5-7, use D201 resin for treatment, remove part of the impurities and most of the pigment, and then use activated carbon to remove the residual pigment in the fermentation broth;

[0028] (3) Remove the residual activated carbon in the fermentation broth by high-speed centrifugation and three-stage plate and frame filtration to obtain a crude hydrophobic protein 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;

[0029] Example 2

[0030] Remove the impurities in the fermentation broth and obtain a freeze-dried powder, according to the following steps:

[0031] (1) Use a 3 kDa membrane package to filter and concentrate the crude hydrophobic protein extract, and control the operating pressure at 0.2-0.3 MPa and the operating temperature at room temperature to obtain a concentrated solution containing hydrophobic proteins. After the 3 kDa membrane package is used up, it is cleaned with a 0.3 M NaOH solution. Figure 1 It can be seen that the concentrated solution of the 3 kDa membrane package contains a large amount of protein, and no protein is detected in the permeate, indicating that the 3 kDa membrane package successfully concentrates the protein in the crude extract.

[0032] (2) Remove the impurities in the hydrophobic protein concentrate by adjusting the pH, water bath heating and high-speed centrifugation. Among them, the pH of the hydrophobic protein concentrate is adjusted to below 7, and after water bath heating at 70-90℃ for 20-30 min, high-speed centrifugation is carried out, and the supernatant is collected to obtain a hydrophobic protein concentrate from which the impurities are removed. Figure 2 It can be seen that after heating treatment, the impurities are precipitated, and the hydrophobic protein stably exists in the supernatant.

[0033] (3) Freeze-dry the hydrophobic protein concentrate from which the impurities are removed to obtain a hydrophobic protein dry powder, Figure 3 The freeze-dried hydrophobic protein freeze-dried powder is shown.

[0034] The above is only an embodiment of the present application, and does not limit the patent scope of the present application, and any equivalent structure or equivalent process transformation using the content of the present application specification, or direct or indirect application in other related technical fields, are also included in the patent protection scope of the present application.

Claims

1. A method for purifying hydrophobin HFB II, characterized by, The method comprises the following steps: (1) solid-liquid separation is achieved by high-speed centrifugation, and then the hydrophobic protein fermentation supernatant is obtained from the Trichoderma reesei fermentation broth by removing the bacterial bodies; (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 broth; (3) high-speed centrifugation and three-stage plate frame filtration are used to remove the residual activated carbon in the fermentation broth to obtain the crude hydrophobic protein extract; (4) the crude hydrophobic protein extract is filtered and concentrated using a 3kDa membrane package to obtain a hydrophobic protein concentrate; (5) impurities in the hydrophobic protein concentrate are removed by adjusting the pH, water bath heating and high-speed centrifugation; (6) the hydrophobic protein concentrate after removing the impurities is freeze-dried to obtain a hydrophobic protein freeze-dried powder.

2. The method of claim 1, wherein, The concentration of activated carbon used in step (2) for removing residual pigments in the fermentation broth is 0.5-1.5%.

3. The method of claim 1, wherein, In step (3), three-stage plate frame filtration is used to remove the residual activated carbon in the fermentation broth. 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, When using a 3kDa membrane package for concentration, the operating pressure is controlled at 0.2-0.3MPa, and the operating temperature is room temperature.

5. The method according to claim 1 or 4, characterized in that, After the 3kDa membrane package is used, it is cleaned with 0.3M NaOH solution.

6. The method of claim 1, wherein, In step (5), the pH of the hydrophobic protein concentrate is adjusted to below 7, the temperature is controlled at 70-90℃ for water bath heating, the heating time is 20-30min, and after high-speed centrifugation, the supernatant is collected to obtain the hydrophobic protein concentrate after removing the impurities.

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