CN110760550A - Method for producing amino acid by hydrolyzing feather with keratinase - Google Patents
Method for producing amino acid by hydrolyzing feather with keratinase Download PDFInfo
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- CN110760550A CN110760550A CN201911199170.6A CN201911199170A CN110760550A CN 110760550 A CN110760550 A CN 110760550A CN 201911199170 A CN201911199170 A CN 201911199170A CN 110760550 A CN110760550 A CN 110760550A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
- C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
Abstract
The invention discloses a method for producing amino acid by hydrolyzing feathers with keratinase, belonging to the technical field of enzyme engineering. According to the method, the hydrolysis efficiency of producing amino acid by hydrolyzing feathers with keratinase is greatly improved by additionally adding sodium sulfite, and the keratinase and the sodium sulfite are simultaneously added into a reaction system containing feathers for reaction for 4 hours, so that the content of the amino acid in a reaction solution can reach 56.6g/L, and the conversion rate of the amino acid can reach 56.6%.
Description
Technical Field
The invention relates to a method for producing amino acid by hydrolyzing feathers with keratinase, belonging to the technical field of enzyme engineering.
Background
Keratin is present in the exoskeleton structure of many vertebrates, such as feathers, hair, scales, hooves, horns, and claws, and is a tough, insoluble protein. In addition, keratin is contained in the exoskeleton structure of various vertebrates in a high content of about 80%.
China has abundant keratin resources, particularly in the prior agriculture, large-scale poultry cultivation generates a large amount of keratin wastes which are mainly feathers of poultry, most of the keratin wastes are not fully utilized, and some of the keratin wastes even pollute the environment to cause public nuisance. If these keratin wastes are disposed of, they can be converted into good resources.
Keratinase is a specific protease capable of hydrolyzing keratin, which can hydrolyze keratin into a wide range of amino acids and polypeptides. Therefore, the keratin hydrolysis by the keratinase is a good method for realizing the keratin resource utilization.
However, wild keratinases have poor performance and very low hydrolysis efficiency of keratin by using these wild keratinases (see the references: W.H.Lo, J.R.Too, J.Y.Wu, Production of Keratino enzym by an indigo tissue strain Bacillus cereus Wu2, J bioscience Bioeng,114(2012)640-647.), and the requirements of industrial Production are far from being met.
Therefore, there is an urgent need to find a method for improving the hydrolysis efficiency of keratinase to hydrolyze keratin.
Disclosure of Invention
[ problem ] to
The invention aims to solve the technical problem of providing a method for producing amino acid by hydrolyzing feathers with keratinase, which has high hydrolysis efficiency.
[ solution ]
In order to solve the technical problems, the invention provides a method for producing amino acid, which comprises the steps of adding keratinase and sodium sulfite into a reaction system containing feather for reaction at the same time to obtain a reaction solution, and then extracting the reaction solution to obtain the amino acid.
In one embodiment of the present invention, the amount of the keratinase added to the reaction system is 4000 to 40000U/mL.
In one embodiment of the present invention, the keratinase is added to the reaction system in an amount of 40000U/mL.
In one embodiment of the invention, the addition amount of the sodium sulfite in the reaction system is 0.05-3 g/L.
In one embodiment of the present invention, the amount of sodium sulfite added to the reaction system is 1 g/L.
In one embodiment of the invention, the concentration of the feathers in the reaction system is 100-250 g/L.
In one embodiment of the present invention, the concentration of feathers in the reaction system is 100 g/L.
In one embodiment of the present invention, the temperature of the reaction is 40 to 70 ℃.
In one embodiment of the invention, the temperature of the reaction is 60 ℃.
In one embodiment of the invention, the reaction has a pH of 6 to 11.
In one embodiment of the invention, the pH of the reaction is 7.
In one embodiment of the present invention, the rotation speed of the reaction is 200 to 250 rpm.
In one embodiment of the invention, the reaction is carried out at a speed of 220 rpm.
In one embodiment of the invention, the keratinase is produced by inoculating recombinant bacillus subtilis to a fermentation medium for fermentation to obtain a fermentation broth, centrifuging the fermentation broth to obtain a fermentation supernatant, and extracting the fermentation supernatant to obtain the keratinase.
In one embodiment of the invention, the recombinant Bacillus subtilis uses a pP43NMK plasmid as an expression vector and uses Bacillus subtilis WB600 as an expression host to express a gene encoding keratinase.
In one embodiment of the invention, the amino acid sequence of the protease is shown in SEQ ID NO. 1.
In one embodiment of the invention, the nucleotide sequence of the gene encoding keratinase is shown in SEQ ID No. 2.
The invention also provides the application of the method in the aspect of producing amino acid.
[ advantageous effects ]
According to the method, the hydrolysis efficiency of producing amino acid by hydrolyzing feathers with keratinase is greatly improved by additionally adding sodium sulfite, and the keratinase and the sodium sulfite are simultaneously added into a reaction system containing feathers for reaction for 4 hours, so that the content of the amino acid in a reaction solution can reach 56.6g/L, and the conversion rate of the amino acid can reach 56.6%.
Drawings
FIGS. 1 to 3: degree of hydrolysis of feathers under different treatment regimes.
FIG. 4: scanning electron micrographs of feathers under different treatment modes; wherein (a) and (f) are untreated feathers, (b) and (g) are 4h feathers treated with 4000U/mL keratinase, (c) and (h) are 4h feathers treated with 1% sodium sulfite, (d) and (i) are 4h feathers treated with 40000U/mL keratinase, and (e) and (j) are 4h feathers treated with 4000U/mL keratinase and 1% sodium sulfite in a synergistic manner.
FIG. 5: influence of the amount of reducing agent added on the hydrolysis efficiency of feather production by keratinase hydrolysis.
FIG. 6: influence of pH on the hydrolysis efficiency of feather production of amino acids by keratinase hydrolysis.
FIG. 7: effect of temperature on hydrolysis efficiency of feather to produce amino acids using keratinase.
FIG. 8: influence of feather addition on hydrolysis efficiency of amino acid production by hydrolysis of feathers with keratinase.
Detailed Description
Escherichia coli JM109 to which the following examples refer was obtained from North Navy organisms, pP43NMK plasmid to which the following examples refer was obtained from Fenghui organisms, Bacillus subtilis WB600 to which the following examples refer was described in patent application publication No. CN102492645A, feathers to which the following examples refer were obtained from the vegetable market, and sodium sulfite, β -mercaptoethanol, and Dithiothreitol (DTT) to which the following examples refer were obtained from Taihe (Shanghai) chemical industry development Co., Ltd.
The media referred to in the following examples are:
LB liquid medium: yeast powder 5.0 g.L-1Tryptone 10.0 g.L-1、NaCl 10.0g·L-1Kanamycin 100 mg. L-1。
LB solid medium: yeast powder 5.0 g.L-1Tryptone 10.0 g.L-1、NaCl 10.0g·L-115g/L agar powder and 50 mg/L kanamycin-1。
Seed culture medium: yeast powder 5 g.L-1Peptone 10 g. L-1、NaCl 5g·L-1。
Fermentation medium: peptone 20 g.L-1Yeast powder 10 g.L-120 g.L of sucrose-1、KH2PO43 g·L-1、Na2HPO46g·L-1、MgSO40.3 g·L-1。
The detection methods referred to in the following examples are as follows:
the enzyme activity of the keratinase is detected by the following method:
taking 50 μ L of the fermentation supernatant diluted properly, adding 150 μ L of 50mM Gly/NaOH solution as buffer and 100 μ L of 2.5% water-soluble keratin (purchased from Taishiai (Shanghai) chemical industry development Co., Ltd., product code: K0043) as substrate, mixing, and reacting at 40 deg.C for 20 min; the reaction was stopped by adding 200. mu.L of 4% (w/v) trichloroacetic acid (TCA) and centrifuged at 8000r/min at room temperature for 3 min. The supernatant was taken to 200. mu.L, and 1mL of 4% (w/v) Na was added2CO3Mixing with 200 μ L of Folin phenol reagent, mixing, developing at 50 deg.C for 10min, and measuring clear solution light absorption value at 660nm with 0.5cm quartz cuvette; 3 experimental groups are paralleled, the blank control is that the reaction terminator TCA is added before the substrate is added, and the rest operations are the same as above;
definition of enzyme activity: the amount of enzyme required to convert a keratin substrate to produce 1. mu. mol tyrosine per minute under these conditions is one unit of enzyme activity (1U).
The detection method of the amino acid content and the amino acid conversion rate comprises the following steps:
adding sulfosalicylic acid into the reaction solution according to the volume ratio of 1:1 for precipitation, after precipitating for 4h, centrifuging the reaction solution at 12000g for 10min, and keeping the supernatant; diluting the supernatant with 0.02M hydrochloric acid to obtain a diluted solution containing 0.1mM amino acid; detecting the amino acid content in the diluted solution with an amino acid analyzer (Hitachi L-8900, Tokyo, Japan);
the calculation formula of the amino acid conversion rate is as follows:
conversion (%) × 100 (amino acid content/dry weight of feathers).
The keratinase enzymes referred to in the following examples were produced as follows:
chemically synthesizing a gene which has a nucleotide sequence shown as SEQ ID NO.2 and codes keratinase (the amino acid sequence of the protease is shown as SEQ ID NO. 1); connecting the obtained gene with a pP43NMK plasmid by using a homologous recombination kit (Clonexpress II One Step cloning kit), converting the connecting product into Escherichia coli JM109, coating the converted product on an LB solid culture medium, culturing for 8-10 h at 37 ℃, selecting 5 transformants on the LB solid culture medium, inoculating the transformants into an LB liquid culture medium for culturing, extracting the plasmid after culturing for 10h at 37 ℃, performing enzyme digestion verification and sequencing verification on the extracted plasmid, and obtaining a recombinant plasmid pP43NMK-ker after verification is correct; transforming Bacillus subtilis WB600 with the correctly verified recombinant plasmid pP43NMK-ker, coating the transformed product on an LB solid culture medium, culturing at 37 ℃ for 8h, selecting a transformant on the LB solid culture medium, inoculating the transformant to an LB liquid culture medium for culturing, culturing at 37 ℃ for 10h, extracting a plasmid, and performing sequence determination on the plasmid to obtain the Bacillus subtilis engineering bacteria containing the gene for encoding keratinase.
Coating the obtained bacillus subtilis engineering bacteria containing the gene for encoding the keratinase on an LB solid culture medium, and culturing at 37 ℃ for 8-10 h to obtain a single colony; selecting a single colony, inoculating the single colony into a seed culture medium, and culturing at 37 ℃ and 220rpm for 14h to obtain a seed solution; inoculating the seed solution into a fermentation medium according to the inoculation amount of 5% (v/v), and culturing at 37 ℃ and 220rpm for 28h to obtain a fermentation solution; the fermentation broth was centrifuged at 12000rpm for 20min at 4 ℃ to obtain a fermentation supernatant. The enzyme activity in the fermentation supernatant was: 40000U/mL.
Example 1: effect of different reducing Agents on the degree of hydrolysis of feather by keratinase hydrolysis
The method comprises the following specific steps:
the method comprises the steps of reacting a reaction system containing 2g/L feather and 40000U/mL keratinase for 4 hours at the temperature of 60 ℃, the pH value of 7 and the rotation speed of 220rpm to obtain a reaction liquid A, adding sodium sulfite, β -mercaptoethanol and Dithiothreitol (DTT) into the reaction system containing only 2g/L feather in an adding amount of 1g/L, reacting for 4 hours at the temperature of 60 ℃, the pH value of 7 and the rotation speed of 220rpm to obtain a reaction liquid B, adding sodium sulfite, β -mercaptoethanol and Dithiothreitol (DTT) into the reaction system containing 2g/L feather and 40000U/mL keratinase in an adding amount of 1g/L, and reacting for 4 hours at the temperature of 60 ℃, the pH value of 7 and the rotation speed of 220rpm to obtain a reaction liquid C.
The detection results are shown in figures 1 and 4 by naked eyes and scanning electron microscope observation.
The degree of hydrolysis of feathers in the reaction solution A, B, C before and after the reaction,
reacting a reaction system containing 10g/L feather and 40000U/mL keratinase for 12h under the conditions of 60 ℃, pH 7 and rotating speed of 220rpm to obtain a reaction solution D; sodium sulfite was added to a reaction system containing only 10g/L feathers and 40000U/mL keratinase in an amount of 1g/L, and the mixture was reacted at 60 ℃ and pH 7 at a rotation speed of 220rpm for 12 hours to obtain a reaction solution E.
The hydrolysis degree of the feather in the reaction solution D, E before and after the reaction was observed by naked eyes and a scanning electron microscope, and the detection results are shown in FIGS. 2 to 4.
As can be seen from FIGS. 1 to 4, sodium sulfite, β -mercaptoethanol, and Dithiothreitol (DTT) alone did not have the ability to hydrolyze feathers, keratinase alone partially hydrolyzed feathers, feathers in the reaction solution to which sodium sulfite was added were completely hydrolyzed, feathers in the reaction solution to which β -mercaptoethanol was added were hydrolyzed into fragments, which were slightly more hydrolyzed than when keratinase alone was used, and feathers in the reaction solution to which Dithiothreitol (DTT) was added were partially hydrolyzed, which were also slightly more hydrolyzed than when keratinase alone.
Example 2: influence of addition amount of reducing agent on hydrolysis efficiency of producing amino acid by hydrolyzing feather with keratinase
The method comprises the following specific steps:
sodium sulfite was added to a reaction system containing 3g/L feathers and 40000U/mL keratinase in amounts of 0.05, 0.1, 0.5, 1, 2, and 3g/L, respectively, and reacted at 60 ℃ and pH 7 at 220rpm for 4 hours to obtain a reaction solution.
The content of amino acids in each reaction solution was measured, and the results are shown in FIG. 5.
As can be seen from FIG. 5, the reaction solution to which 0.05g/L of sodium sulfite was added had an amino acid content of 0.95 g/L; the amino acid content in the reaction solution added with 0.1g/L of sodium sulfite alcohol is 1.07 g/L; the amino acid content in the reaction solution to which 0.5g/L of sodium sulfite alcohol was added was 1.39 g/L; the amino acid content in the reaction solution added with 1g/L of sodium sulfite alcohol is 1.76 g/L; the amino acid content in the reaction solution added with 2g/L of sodium sulfite alcohol is 1.417 g/L; the amino acid content in the reaction solution to which 3g/L of sodium sulfite alcohol was added was 1.35 g/L. It can be seen that the highest effect of improving the hydrolysis efficiency of producing amino acids by hydrolyzing keratin with keratinase is obtained when the amount of sodium sulfite added is 1 g/L.
Example 3: effect of pH on hydrolysis efficiency of feather production of amino acids Using keratinase
The method comprises the following specific steps:
in addition to example 2, the amount of sodium sulfite added was controlled to 1g/L, and the pH of the reaction was changed to 6, 7, 8, 9, 10, and 11, respectively, to obtain reaction solutions.
The results of the amino acid content measurement in each reaction solution are shown in FIG. 6.
As is clear from FIG. 6, the content of amino acids in the reaction mixture obtained by the reaction at pH 6 was 1.43 g/L; when the pH value is 7, the content of amino acid in the reaction liquid obtained by the reaction is 2.06 g/L; when the pH value is 8, the content of amino acid in the reaction liquid obtained by the reaction is 1.93 g/L; when the pH value is 9, the content of amino acid in the reaction liquid obtained by the reaction is 1.83 g/L; when the pH value is 10, the content of amino acid in the reaction liquid obtained by the reaction is 1.35 g/L; the content of amino acids in the reaction mixture obtained at pH 11 was 1.16 g/L. It can be seen that the hydrolysis efficiency of amino acids produced by hydrolyzing keratin with keratinase is highest at pH 7.
Example 4: effect of temperature on hydrolysis efficiency of producing amino acids by hydrolyzing feathers with keratinase
The method comprises the following specific steps:
in addition to example 2, the amount of sodium sulfite added was controlled to 1g/L, the pH of the reaction was controlled to 7, and the reaction temperatures were changed to 40, 45, 50, 55, 60, 65, and 70 ℃.
The results of the amino acid content measurement in each reaction solution are shown in FIG. 7.
As is clear from FIG. 7, the content of amino acids in the reaction mixture obtained by the reaction at 40 ℃ was 1.07 g/L; the content of amino acid in the reaction solution obtained by the reaction at the temperature of 45 ℃ is 1.23 g/L; the content of amino acid in the reaction solution obtained by the reaction at the temperature of 50 ℃ is 1.56 g/L; the content of amino acid in the reaction solution obtained by the reaction at the temperature of 55 ℃ is 1.68 g/L; the content of amino acid in the reaction solution obtained by the reaction at the temperature of 60 ℃ is 1.78 g/L; the content of amino acid in the reaction solution obtained by the reaction at the temperature of 65 ℃ is 1.55 g/L; the content of amino acids in the reaction mixture obtained by the reaction at 70 ℃ was 1.42 g/L. It can be seen that the hydrolysis efficiency for producing amino acids by hydrolyzing keratin with keratinase is the highest at a temperature of 60 ℃.
Example 5: influence of substrate addition on hydrolysis efficiency of feather production by hydrolysis with keratinase
The method comprises the following specific steps:
in addition to example 4, 250g/L, 200g/L, 125g/L, 100g/L feathers and 250g/L, 200g/L feather powder (obtained by pulverizing feathers) were replaced with 3g/L feathers, respectively, to obtain a reaction solution.
The content of amino acids in each reaction solution and the conversion rate of amino acids in the reaction solution were measured, and the results of the measurements are shown in FIG. 8.
As can be seen from FIG. 8, when the amount of the substrate added was 100g/L and the substrate was feathers, the amino acid content and the conversion rate in the reaction solution were the highest, and reached 56.60g/L and 56.60%, respectively. As can be seen, 100g/L should be selected as substrate addition.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Sequence listing
<110> university of south of the Yangtze river
<120> a method for producing amino acid by hydrolyzing feather with keratinase
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Claims (10)
1. A method for producing amino acid is characterized in that keratinase and sodium sulfite are added into a reaction system containing feather at the same time for reaction to obtain reaction liquid, and then the reaction liquid is extracted to obtain the amino acid.
2. The method for producing an amino acid according to claim 1, wherein the keratinase is added in an amount of 4000 to 40000U/mL in the reaction system.
3. The method for producing an amino acid according to claim 1 or 2, wherein the amount of sodium sulfite added to the reaction system is 0.05 to 3 g/L.
4. The method for producing an amino acid according to any one of claims 1 to 3, wherein the concentration of feathers in the reaction system is 100 to 250 g/L.
5. The process for producing an amino acid according to any one of claims 1 to 4, wherein the reaction temperature is 40 to 70 ℃.
6. The process for producing an amino acid according to any one of claims 1 to 5, wherein the reaction has a pH of 6 to 11.
7. The process for producing an amino acid according to any one of claims 1 to 6, wherein the reaction is carried out at a rotation speed of 200 to 250 rpm.
8. The method for producing amino acids according to any one of claims 1 to 7, wherein the keratinase is produced by inoculating recombinant Bacillus subtilis to a fermentation medium to perform fermentation to obtain a fermentation broth, centrifuging the fermentation broth to obtain a fermentation supernatant, and extracting the fermentation supernatant to obtain the keratinase.
9. The method for producing an amino acid according to claim 8, wherein the recombinant Bacillus subtilis expresses the gene encoding the keratinase with a pP43NMK plasmid as an expression vector and Bacillus subtilis WB600 as an expression host.
10. Use of a method according to any one of claims 1 to 9 for the production of amino acids.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112662653A (en) * | 2021-01-26 | 2021-04-16 | 江南大学 | Keratinase mutant with improved low-temperature enzymolysis performance and application thereof |
| CN112662653B (en) * | 2021-01-26 | 2022-03-04 | 江南大学 | Keratinase mutant with improved low-temperature enzymolysis performance and application thereof |
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