CN111793665B - Production method of biological surface active functional protein and culture medium used by same - Google Patents
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Abstract
The invention discloses a production method of biological surface active functional protein and a culture medium used by the same. The invention provides a fungus fermentation medium, which contains 0.1-1g/L corn steep liquor, 5-20g/L peptone and 20-40g/L lactose (or whey). The culture medium can be used for producing hydrophobin in trichoderma and can also be used for producing other proteins in trichoderma. Filamentous fungi have similar culture conditions, and thus the culture medium provided by the present invention is also suitable for expression of hydrophobins or other proteins in other fungi. The invention has simple fermentation process, only needs to inoculate the spore liquid into the fermentation culture medium, does not need to transfer, has cheap raw materials, greatly reduces the cost, and is suitable for industrial production and application.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a production method of a biosurfactant functional protein and a culture medium used by the biosurfactant functional protein.
Background
The culture medium is artificially prepared nutrient for the growth and development of microorganisms, plants and animal tissues. Growth has certain requirements on the proportion of nutrient substances in a culture medium and the pH value, for example, the carbon-nitrogen ratio has great influence on the growth of microorganisms, and yeasts and molds usually grow in the pH range of 4.5-6. Under different culture medium conditions, the cell products produced by the same microorganism are very different, and different culture media are generally selected for realizing different functions of the microorganism. Therefore, the selection of a highly efficient medium is critical to the microbial fermentation industry.
In the field of microbial fermentation engineering, a large class of fermentation media for microbial fermentation contains a certain amount of solids, and most of the solids are obtained from biological product raw materials, such as solid cellulose culture commonly used in trichoderma culture media. The culture medium with solid matters with larger particle sizes has larger influence on industrial production. High density fermentation of pure liquids is a pursued goal for industrial fermentation.
Biosurfactant functional proteins are small molecule secreted proteins with molecular structures very similar to the surfactant functional domain and therefore have properties similar to surfactants. Surfactants have properties of reducing surface tension, foaming, emulsifying, dispersing, wetting, solubilizing, penetrating, and antistatic properties by virtue of their unique amphiphilic structures, and are gaining importance in a variety of industrial and consumer product applications. At present, the world consumption of the surfactant is about 900 million tons, wherein the industrial consumption accounts for 55 percent, and the surfactant is widely applied to the industrial fields of coal dressing, textile, food, material preparation, paper making, pharmacy and the like.
Trichoderma filamentous fungi have a very strong ability to synthesize and secrete proteins. The hydrophobic protein derived from trichoderma is functional protein with biological surface activity, and has wide application prospect in the aspects of industry, medicine health and food. The coating can reduce pollutant adsorption after being coated on the surface of glass, and is more environment-friendly compared with chemical coatings. Can be used on the surface of medical equipment, and can reduce nonspecific protein binding and reduce the possibility of microbial infection. When the protein is used in a biosensor, the stability of the protein on the surface of the sensor can be increased, and the effective period of the sensor can be prolonged. The amphiphilic active protein can be used for wrapping some medicines which are difficult to dissolve in water and increasing the solubility of the medicines in the field of medicine. It can be used in daily chemical and cosmetic industries, and can be used as foaming agent and adhesion agent.
The characteristics of the biological surface active function of the hydrophobin determine that the hydrophobin has great application potential, researchers find the existence of the hydrophobin in various filamentous fungi successively, the hydrophobin derived from trichoderma reesei is proved to have extremely strong surface active function, but the low expression level of the natural biological surface active functional protein becomes the biggest bottleneck for hindering the application of the hydrophobin.
Disclosure of Invention
The invention aims to provide a method for producing a biosurfactant functional protein and a culture medium used by the same.
The invention firstly provides a fungus fermentation culture medium which is a fungus fermentation culture medium A or a fungus fermentation culture medium B.
The fungus fermentation medium A contains 0.1-1g/L corn steep liquor, 5-20g/L peptone and 20-40g/L lactose.
The fungus fermentation medium B contains 0.1-1g/L of corn steep liquor, 5-20g/L of peptone and 20-40g/L of whey.
The fungus fermentation medium A contains 1g/L of corn steep liquor, 5g/L of peptone and 40g/L of lactose.
The fungus fermentation medium B contains 1g/L of corn steep liquor, 5g/L of peptone and 40g/L of whey.
The fungus fermentation medium A contains 1g/L of corn steep liquor, 10g/L of peptone and 40g/L of lactose.
The fungus fermentation medium A contains 1g/L of corn steep liquor, 20g/L of peptone and 40g/L of lactose.
The fungus fermentation medium A contains 0.1g/L corn steep liquor, 5g/L peptone and 40g/L lactose.
The fungus fermentation medium A contains 0.5g/L corn steep liquor, 5g/L peptone and 40g/L lactose.
The fungus fermentation medium A contains 1g/L of corn steep liquor, 5g/L of peptone and 20g/L of lactose.
The fungus fermentation medium A contains 1g/L of corn steep liquor, 5g/L of peptone and 30g/L of lactose.
The fungus fermentation medium B contains 1g/L of corn steep liquor, 10g/L of peptone and 40g/L of whey.
The fungus fermentation medium B contains 1g/L of corn steep liquor, 20g/L of peptone and 40g/L of whey.
The fungus fermentation medium B contains 0.1g/L of corn steep liquor, 5g/L of peptone and 40g/L of whey.
The fungus fermentation medium B contains 0.5g/L of corn steep liquor, 5g/L of peptone and 40g/L of whey.
The fungus fermentation medium B contains 1g/L of corn steep liquor, 5g/L of peptone and 20g/L of whey.
The fungus fermentation medium B contains 1g/L of corn steep liquor, 5g/L of peptone and 30g/L of whey.
The fungus fermentation medium A consists of a solute and a solvent; the solute is corn steep liquor, peptone and lactose; the solvent is water.
The fungus fermentation medium B consists of a solute and a solvent; the solute is corn steep liquor, peptone and whey; the solvent is water.
The pH of any one of the above fungal fermentation media is 4.8-5.0.
The invention also protects the application of any one of the fungus fermentation culture media in inducing the expression of proteins in fungi.
The invention also provides a method for inducing protein expression in fungi, which comprises the following steps: inoculating the fungus into any one of the fungus fermentation culture media for fermentation culture, and inducing protein expression.
In the method, the fermentation culture condition can be specifically culture at 28 ℃ and 200 r/min.
The culture time may be specifically 5 to 7 days.
The invention also provides a method for inducing protein expression in fungi, which comprises the following steps:
(1) inoculating the fungus into a PDA solid culture medium for spore production culture to obtain a spore suspension;
(2) after the step (1) is finished, inoculating the spore suspension into any one of the fungus fermentation culture media for fermentation culture, and inducing protein expression.
In the step (1), the culture temperature may be specifically 28 ℃. The culture time may be 10 to 15 days.
In the step (2), the fermentation culture condition can be specifically culture at 28 ℃ and 200 r/min. The culture time may be specifically 5 to 7 days.
The invention also provides a kit for inducing protein expression in fungi, which comprises a PDA solid culture medium and any one of the fungus fermentation culture media.
Any of the fungi described above may be trichoderma. The trichoderma may be trichoderma reesei.
Any of the above proteins may be hydrophobins. The hydrophobin may be hydrophobin hfb II.
The expression of one protein has necessary expression elements on the DNA level, the corresponding elements can respond to some substance induction in the culture medium, the target gene in the expression elements is replaced by the coding gene of other proteins, the expression of other proteins in trichoderma can be successfully induced theoretically, for example, cellulase expression elements commonly used in trichoderma are sensitive to cellulose response in the culture medium, and other proteins such as heterologous endochitinase, glucoamylase, heterologous lipase and the like can be produced by the elements and the culture medium containing cellulose. The culture medium of the invention can therefore be used not only for the production of hydrophobins in Trichoderma but also for the production of other proteins (which may in particular be proteins expressed using hydrophobin elements). Filamentous fungi have similar culture conditions, and thus the culture medium provided by the present invention is equally applicable to the expression of hydrophobins or other proteins in other fungi (other proteins may in particular be proteins which are expressed using hydrophobin elements).
The culture medium provided by the invention can induce and express the trichoderma biological hydrophobin, has a simple purification method, and is suitable for industrial production and downstream application. The pigment can have great influence on the laboratory and industrial application downstream operation of the fermentation product, the color of the culture condition provided by the invention is obviously latent, and the subsequent purification process is reduced. The culture medium with solid substances with larger granularity has larger influence on industrial production, the traditional culture medium raw materials have solid substances which influence the high-density fermentation of pure liquid, and the raw materials used by the culture medium provided by the invention are all soluble in water, thereby facilitating industrial fermentation and purification. The invention has simple fermentation process, only needs to inoculate the spore liquid into the fermentation culture medium, does not need to transfer, has cheap raw materials, greatly reduces the cost, and is suitable for industrial production and application.
Drawings
FIG. 1 is a photograph showing the results of SDS-PAGE electrophoresis and fermentation in a groping experiment of peptone concentration.
FIG. 2 is a photograph showing the results of SDS-PAGE electrophoresis and fermentation in a groping experiment of corn steep liquor concentration.
FIG. 3 is a photograph showing the results of SDS-PAGE electrophoresis and fermentation in a blinding experiment of lactose concentration.
FIG. 4 shows the result of SDS-PAGE in the optimum concentration experiment.
FIG. 5 is a photograph showing the results of fermentation in the optimum concentration test.
FIG. 6 shows SDS-PAGE results of fermentation products of the medium of the present invention and the prior art medium.
FIG. 7 shows the result of SDS-PAGE electrophoresis of protein purification.
FIG. 8 shows the results of mass spectrometric identification of the target protein.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
The trichoderma reesei strains used in this patent were purchased from ATCC in the united states (strain number:
46480TM)。
PDA solid medium: 20g/L of glucose, 200g/L of potato and 20g/L of agar powder, and adding deionized water to a constant volume. Sterilizing with high pressure steam at 115 deg.C for 20min under natural pH.
Corn steep liquor: hebei kang Xin pharmaceuticals, Inc., analytically pure.
Peptone, OXOID, Inc., analytically pure.
Agar powder, BD, analytically pure.
Example 1 Induction of expression of Trichoderma reesei hydrophobin
Spore-producing culture of Trichoderma reesei strain
Inoculating the Trichoderma reesei strain on a PDA solid culture medium, culturing for 10-15 days in a constant-temperature incubator at 28 ℃ until conidia are generated, and eluting the conidia by deionized water to obtain a spore suspension.
Fermentation culture of Trichoderma reesei strain
1. Groping of peptone concentration in fermentation liquid medium
Respectively inoculating the spore suspension obtained in the step one into a fermentation liquid culture medium A, a fermentation liquid culture medium B and fermentation liquidIn body Medium C (final spore concentration of 10)5-106one/mL), culturing at 28 deg.C and 200r/min for 5-7 days, collecting culture system when hypha growth is observed to be dense and sporulation begins, filtering with 4 layers of sterilized gauze, collecting filtrate to obtain fermentation liquor containing hydrophobin, and identifying target protein by SDS-PAGE and mass spectrometry.
The fermentation liquid culture medium A consists of solute and solvent; the solute and the concentration thereof in the fermentation liquid culture medium A are as follows: 1g/L corn steep liquor, 5g/L peptone and 40g/L lactose; the solvent is deionized water; adjusting the pH value to 4.8-5.0.
The fermentation liquid culture medium B consists of solute and solvent; the solute and the concentration thereof in the fermentation liquid culture medium B are as follows: 1g/L corn steep liquor, 10g/L peptone and 40g/L lactose; the solvent is deionized water; adjusting the pH value to 4.8-5.0.
A fermentation liquid culture medium C and a solvent; solutes and their concentrations in fermentation broth C were: 1g/L corn steep liquor, 20g/L peptone and 40g/L lactose; the solvent is deionized water; adjusting the pH value to 4.8-5.0.
The mass spectrometric identification results are shown in FIG. 8. The results of SDS-PAGE are shown in FIG. 1. FIG. 1a is a SDS-PAGE result, and the arrow indicates the target protein. FIG. 1b is a photograph of the fermentation system at the end of fermentation. In FIG. 1, M is marker, 0.5% is the result of fermentation broth A, 1% is the result of fermentation broth B, and 2% is the result of fermentation broth C.
The above results indicate that the target protein is hydrophobin hfb II (size 7.2 KDa). The hydrophobin yield decreases gradually with increasing peptone content. The low-concentration peptone promotes spore germination, the high-concentration peptone inhibits induction expression of hydrophobic protein, and the subsequent experiment selects 5g/L as the optimal concentration of the peptone.
2. Groping of corn steep liquor concentration in fermentation liquid culture medium
Respectively inoculating the spore suspension obtained in the step one into a fermentation liquid culture medium D, a fermentation liquid culture medium E and a fermentation liquid culture medium F (the final concentration of spores is 10)5-106one/mL), cultured at 28 ℃ and 200r/min for 5-7 days, when the hyphae grow densely and begin to sporulateCollecting a culture system, filtering and collecting filtrate by using 4 layers of sterilized gauze to obtain fermentation liquor containing hydrophobin, and identifying the target protein by SDS-PAGE.
The fermentation liquid culture medium D consists of solute and solvent; the solute and the concentration thereof in the fermentation liquid medium D are as follows: 0.1g/L corn steep liquor, 5g/L peptone and 40g/L lactose; the solvent is deionized water; adjusting the pH value to 4.8-5.0.
The fermentation liquid culture medium E consists of solute and solvent; the solutes and their concentrations in fermentation broth E were: 0.5g/L corn steep liquor, 5g/L peptone and 40g/L lactose; the solvent is deionized water; adjusting the pH value to 4.8-5.0.
A fermentation liquid culture medium F and a solvent; solutes and their concentrations in fermentation broth F were: 1g/L corn steep liquor, 5g/L peptone and 40g/L lactose; the solvent is deionized water; adjusting the pH value to 4.8-5.0.
The results are shown in FIG. 2. FIG. 2a is a SDS-PAGE result, in which the arrow indicates the target protein. FIG. 2b is a photograph of the fermentation system at the end of fermentation. In FIG. 2, M is marker, 0.01% is the result of fermentation broth D, 0.05% is the result of fermentation broth E, and 0.1% is the result of fermentation broth F.
The results show that with the increase of the concentration of the corn steep liquor, the content of the carbon source is increased, the growth of cells is promoted, the expression capacity of the hydrophobin is gradually enhanced, and in subsequent experiments, 1g/L is selected as the optimal concentration of the corn steep liquor.
3. Investigation of lactose concentration in fermentation liquid medium
Respectively inoculating the spore suspension obtained in the step one into a fermentation liquid culture medium G, a fermentation liquid culture medium H and a fermentation liquid culture medium I (the final concentration of spores is 10)5-106one/mL), culturing at 28 deg.C and 200r/min for 5-7 days, collecting culture system when hypha growth is observed to be dense and sporulation begins, filtering and collecting filtrate with 4 layers of sterilized gauze to obtain fermentation liquor containing hydrophobin, and identifying target protein by SDS-PAGE.
The fermentation liquid culture medium G consists of solute and solvent; solutes and their concentrations in fermentation broth G were: 1g/L corn steep liquor, 5g/L peptone and 20g/L lactose; the solvent is deionized water; adjusting the pH value to 4.8-5.0.
The fermentation liquid culture medium H consists of solute and solvent; solutes and their concentrations in fermentation broth H were: 5g/L corn steep liquor, 5g/L peptone and 30g/L lactose; the solvent is deionized water; adjusting the pH value to 4.8-5.0.
A fermentation liquid culture medium I and a solvent; solutes and their concentrations in fermentation broth I were: 1g/L corn steep liquor, 5g/L peptone and 40g/L lactose; the solvent is deionized water; adjusting the pH value to 4.8-5.0.
The results are shown in FIG. 3. FIG. 3a is a SDS-PAGE result, in which the arrow indicates the target protein. FIG. 3b is a photograph of the fermentation system at the end of fermentation. In FIG. 3, M is marker, 2% is the result of fermentation broth G, 3% is the result of fermentation broth H, and 4% is the result of fermentation broth I.
The results show that the induction effect of lactose with different concentrations is seen from SDS-PAGE results, the hydrophobin concentration difference generated by each result is small, but through repeated experiments, the fermentation result of 4% lactose content is stable, the growth of hyphae is fast, a dominant flora is easily formed in production, and the culture medium is not easily polluted, so that 40g/L is selected as the optimal concentration of lactose in subsequent experiments.
Through the exploration, the concentration of the corn steep liquor in the determined optimal liquid fermentation medium is 1g/L, the concentration of the peptone is 5g/L, and the concentration of the lactose is 40 g/L.
4. Experiment of optimum concentration of fermentation liquid culture medium
Inoculating the spore suspension obtained in the step one into an optimal fermentation liquid culture medium (the final concentration of spores is 10)5-106one/mL), culturing at 28 deg.C and 200r/min for 5-7 days, collecting culture system when hypha growth is observed to be dense and sporulation begins, filtering and collecting filtrate with 4 layers of sterilized gauze to obtain fermentation liquor containing hydrophobin, and identifying target protein by SDS-PAGE.
The optimal fermentation liquid culture medium consists of solute and solvent; solutes and their concentrations in the optimal fermentation broth were: 1g/L corn steep liquor, 5g/L peptone and 40g/L lactose; the solvent is deionized water; adjusting the pH value to 4.8-5.0.
The results of SDS-PAGE are shown in FIG. 4. In FIG. 4, M is marker, 1 is the result of the optimal fermentation broth, and the arrow indicates the target protein. The results of photographing the fermentation culture system are shown in FIG. 5. FIG. 5a is a photograph of a flask for 6 days of fermentation; fig. 5b shows the filtrate after filtration with 4 layers of gauze.
The results show that the trichoderma hydrophobin is successfully expressed and has high expression quantity, and the target band is identified as the hydrophobin through mass spectrum. The color of the fermentation liquor is light, and the method is suitable for subsequent purification and other operations in a laboratory and industrial production application.
5. Comparison of the Effect of Using whey instead of lactose
Inoculating the spore suspension obtained in the first step into an optimal liquid fermentation medium (lactose is replaced by whey, and the concentration is constant) (the final concentration of the spores is 10)5-106one/mL), culturing at 28 deg.C and 200r/min for 5-7 days, collecting culture system when hypha growth is observed to be dense and sporulation begins, filtering and collecting filtrate with 4 layers of sterilized gauze to obtain fermentation liquor containing hydrophobin, and identifying target protein by SDS-PAGE. The results show that the concentration of hydrophobin does not change significantly with whey instead of lactose, and that whey can be used industrially instead of lactose.
Third, comparison with existing Medium
1. And (5) operating according to the step loaded in the step two 4.
2. Inoculating the spore suspension obtained in the first step into a culture medium recorded in the existing literature, culturing at 30 ℃ and 200r/min for 5 days, collecting a culture system, filtering and collecting filtrate by using 4 layers of sterilized gauze to obtain fermentation liquor containing hydrophobin, and identifying the target protein by SDS-PAGE.
The literature states that the culture medium consists of solutes and solvents; solutes and their concentrations in the media described in the literature are: 40g/L whey, 40g/L cereal, 15g/L KH2PO4And 5g/L (NH)4)2SO4(ii) a The solvent is deionized water; the pH was 5.5.
The results are shown in FIG. 6. In FIG. 6, M is marker, 1 is the result of the optimum fermentation broth, 2 is the result of the literature-described medium, and the arrow indicates the target protein.
The results show that the optimal fermentation liquid culture medium provided by the invention is beneficial to the production of hydrophobin, has higher yield and less impurity protein, and is beneficial to subsequent purification.
The highest trichoderma hydrophobin yield of 240mg/L is reported in the literature (Bailey M, Askolin S,
N,et al.Process technological effects of deletion and amplification of hydrophobins I and II in transformants ofTrichoderma reesei[J]appl Microbiol Biotechnol,2002,58(6): 721-. The invention is the original strain fermentation, the yield of the shake flask is estimated to be 0.2-0.4mg/mL by SDS-PAGE fermentation, and the advantages of the culture medium can be seen.
Example 2 verification of the purification Effect of hydrophobin Using different purification methods
1. And (3) heat treatment: and (3) heating the fermentation liquor prepared in the step two of the step (4) in the embodiment 1 in a water bath at 80 ℃ for 30 minutes, centrifuging at 11000r for 10 minutes, and taking the supernatant to obtain a protein solution with higher purity.
2. And (3) extraction: and (3) adding 40 mu L of Triton X-114 into 1ml of fermentation liquor prepared in the step two in the example 1, fully and uniformly mixing, standing for 30min at 35 ℃, discarding the supernatant, adding 400 mu L of isobutanol, fully and uniformly mixing, centrifuging, layering, discarding the supernatant, and collecting the residual solution to obtain the protein solution with higher purity.
3. And (3) identifying the target protein by SDS-PAGE of the protein solutions obtained in the step 1 and the step 2.
The results are shown in FIG. 7. In FIG. 7, the arrow indicates the target protein, wherein 1 is the fermentation broth obtained in step two, 4, of example 1; 2, the result of heating the fermentation liquor in 80 ℃ water bath for 30 minutes is shown, and the protein has extremely high thermal stability and can be well purified by using the thermal stability; 3 is the result of protein solution purified by common extraction method.
The results show that the culture medium and the culture method can obtain protein solution with higher concentration and purity, and can be used for subsequent experiments or industrial application.
Claims (7)
1. The fungus fermentation culture medium is a fungus fermentation culture medium A or a fungus fermentation culture medium B;
the fungus fermentation medium A contains 0.1-1g/L corn steep liquor, 5-20g/L peptone and 20-40g/L lactose;
the fungus fermentation medium B contains 0.1-1g/L of corn steep liquor, 5-20g/L of peptone and 20-40g/L of whey.
2. The fungal fermentation medium of claim 1, wherein:
the fungus fermentation medium A contains 1g/L of corn steep liquor, 5g/L of peptone and 40g/L of lactose;
the fungus fermentation medium B contains 1g/L of corn steep liquor, 5g/L of peptone and 40g/L of whey.
3. The fungal fermentation medium of claim 1 or 2, wherein:
the fungus fermentation medium A consists of a solute and a solvent; the solute is corn steep liquor, peptone and lactose; the solvent is water; the fungus fermentation medium B consists of a solute and a solvent; the solute is corn steep liquor, peptone and whey; the solvent is water.
4. Use of a fungal fermentation medium according to any one of claims 1 to 3 for inducing the expression of a protein in a fungus, said fungus being Trichoderma reesei and said protein being hydrophobin hfb II.
5. A method of inducing protein expression in fungi comprising the steps of: inoculating the fungus into a fungus fermentation culture medium of any one of claims 1 to 3 for fermentation culture, and inducing protein expression, wherein the fungus is trichoderma reesei, and the protein is hydrophobin hfb II.
6. A method of inducing protein expression in fungi comprising the steps of:
(1) inoculating the fungus into a PDA solid culture medium for spore production culture to obtain a spore suspension;
(2) after step (1) is completed, inoculating the spore suspension into the fungus fermentation culture medium of any one of claims 1 to 3 for fermentation culture, and inducing protein expression;
the fungus is trichoderma reesei, and the protein is hydrophobin hfb II.
7. A kit suitable for inducing protein expression in fungi comprising PDA solid medium and a fungal fermentation medium according to any of claims 1 to 3.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001014521A1 (en) * | 1999-08-20 | 2001-03-01 | Valtion Teknillinen Tutkimuskeskus | A method for decreasing the foam formation during cultivation of a microorganism |
| CN101538604A (en) * | 2009-04-09 | 2009-09-23 | 上海交通大学 | Online production method of rhamnolipid biosurfactant in cellulose hydrolyzation |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001014521A1 (en) * | 1999-08-20 | 2001-03-01 | Valtion Teknillinen Tutkimuskeskus | A method for decreasing the foam formation during cultivation of a microorganism |
| CN101538604A (en) * | 2009-04-09 | 2009-09-23 | 上海交通大学 | Online production method of rhamnolipid biosurfactant in cellulose hydrolyzation |
Non-Patent Citations (1)
| Title |
|---|
| Two crystal structures of Trichoderma reesei hydrophobin HFBI—The structure of a protein amphiphile with and without detergent interaction;JOHANNA HAKANPA¨;《Protein Science》;20060608;第83卷;第2129–2140页 * |
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