CN111763678B - Promoter for improving activity of heterologous expression enzyme of keratinase - Google Patents
Promoter for improving activity of heterologous expression enzyme of keratinase Download PDFInfo
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
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- 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)
Abstract
The invention discloses a promoter for improving the activity of heterologous expression enzyme of keratinase, belonging to the technical field of industrial biology. According to the invention, keratinase recombinant bacteria carrying 16 different promoter sequences are successfully constructed, wherein 7 keratinase expression levels can be improved, the highest enzyme activity level of the recombinant bacteria modified by the PaprE promoter reaches 2605U/mL, and the enzyme activity level is improved by 20 times compared with that of a control bacterium. The activity of the keratinase in fermentation liquor on a 5L fermentation tank is as high as 7176U/mL, which is the highest level of recombinant keratinase expression reported in the literature at present and can better serve the practical application. The invention provides an effective strategy for research on high-efficiency expression and enzyme production of keratinase. The traditional genetic engineering modification usually needs to construct a high-throughput screening method for screening a large amount of libraries, and has the advantages of large workload, long period and high cost.
Description
The application has the following application numbers: 201810933416.7, the filing date is: 8, month 16, 2018, with the application name: a promoter for heterologous expression of keratinase and divisional application of application thereof.
Technical Field
The invention relates to a promoter for improving the activity of heterologous expression enzyme of keratinase, belonging to the technical field of industrial biology.
Background
Keratin is an insoluble structural protein with high stability due to high cross-linking of disulfide bonds and hydrogen bonds. As a byproduct in the development process of animal husbandry, leather industry and textile industry, a large amount of hair is abandoned in the nature every year, and huge pollution is caused to the environment. At present, the hair is treated by mainly physical and chemical methods such as high temperature, high pressure, acid, alkali, oxidation and the like, but the methods can consume useful amino acids in keratin, and have low efficiency, high energy consumption and large pollution. Keratinase opens complex disulfide bonds in keratin by disulfide bond reduction, and then realizes degradation of keratin by proteolysis. The keratin degradation by adopting the keratinase is a green degradation process, and the environment-friendly biological treatment mode has wide prospect from the aspects of economy and environmental protection.
However, in summary, it has been found that the enzymatic activity and thermal stability of keratinase are generally poor. Although many documents report that the keratinase genes from different microorganisms successfully realize heterologous expression, the keratinase expression level of most genetic engineering bacteria is not broken through, so that the realization of the wide application of the keratinase and the satisfaction of the industrial large-scale use requirement become difficulties. In recent years, researchers gradually shift the research focus to the heterogenous high-efficiency expression of keratinase, and a genetic engineering modification means is a key method for improving the expression level of recombinant keratinase. Radha achieves efficient expression of keratinase in Bacillus megaterium by using inducible promoters PxylA and Pamyl, so that the expression level of recombinase is about 3 times higher than that of a control bacterium, and the enzyme activity reaches 168.6U/mL (Radha S, et al. Bioresource Technol,2008, 99. Wang et al integrated multiple copies of the keratinase gene into the Bacillus licheniformis genome to construct a recombinant strain that stably produces keratinase in high yields (Wang J, et al, biotechnol Bioeng,2004, 87. However, the high-efficiency expression of the keratinase realized by the transformation of a promoter is not reported in any literature at present, which has great significance for realizing the deep and wide application of the keratinase.
Disclosure of Invention
In order to solve the problems, the invention provides a promoter for high-efficiency heterologous expression of keratinase, which improves the expression quantity and enzyme activity of the keratinase.
The first purpose of the invention is to provide a promoter, the promoter is a promoter of a keratinase gene, and the nucleotide sequence of the promoter is shown as SEQ ID NO.2, SEQ ID NO.6, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.15 or SEQ ID NO. 16.
In one embodiment of the invention, the nucleotide sequence of the keratinase gene is a nucleotide sequence shown as SEQ ID NO.17, SEQ ID NO.18 or SEQ ID NO.19, or a nucleotide sequence having more than 95% homology with SEQ ID NO. 17-19.
The second object of the present invention is to provide an expression vector containing the above promoter.
In one embodiment of the invention, the vector is a vector suitable for expression by bacillus.
The third purpose of the invention is to provide a recombinant bacterium for expressing keratinase, wherein the recombinant bacterium contains the promoter.
In one embodiment of the present invention, the host of the recombinant bacterium includes, but is not limited to, bacillus subtilis, bacillus cereus, bacillus licheniformis, bacillus megaterium, bacillus fragilis, bacillus clausii, bacillus alcalophilus, or bacillus thuringiensis.
The fourth purpose of the invention is to provide a preparation method of the recombinant bacterium, which comprises the following steps:
(1) Taking a bacillus subtilis genome as a template, amplifying a promoter sequence, and carrying out mutation to obtain a promoter sequence with a nucleotide sequence shown as SEQ ID No.2, SEQ ID No.6, SEQ ID No.10, SEQ ID No.12, SEQ ID No.13, SEQ ID No.15 or SEQ ID No. 16;
(2) Recovering the promoter fragment in the step (1), and constructing the promoter fragment into a plasmid carrying a keratinase gene to obtain a recombinant plasmid;
(3) And (3) transforming the recombinant plasmid in the step (2) into host bacteria to obtain recombinant bacteria.
The fifth purpose of the invention is to provide a method for producing keratinase by the recombinant bacteria, wherein the recombinant bacteria are inoculated into a fermentation tank in an inoculation amount of 1-10%, and are fermented and produced under the conditions of fermentation temperature of 28-40 ℃, rotation speed of 100-1000 rpm, ventilation volume of 0.5-3 vvm and pH value of 5.5-8.5, so as to obtain the keratinase.
The sixth purpose of the invention is to provide the application of the promoter or the recombinant bacterium in animal husbandry, leather making industry or textile industry.
In one embodiment of the invention, the application is to construct a recombinant bacterium by using the promoter, and produce the keratinase degradation keratin by fermentation.
The invention has the beneficial effects that: the keratinase recombinant strain carrying 16 different promoter sequences is successfully constructed, wherein the enzyme activity level of the recombinant strain modified by the PaprE promoter is the highest and reaches 2605U/mL, and is improved by 20 times compared with a control strain. The fermentation research strategy of culture medium nutrient component optimization is adopted, the yield of the mutant transformed recombinant bacterium keratinase is improved, the activity of the keratinase in fermentation liquor on a 5L fermentation tank is as high as 7176U/mL, the activity is the highest level of the expression of the recombinant keratinase reported in the current literature, and the recombinant keratinase can be better served for practical application. The invention provides an effective strategy for the research of the high-efficiency expression and the enzyme production of the keratinase. The traditional genetic engineering modification usually needs to construct a high-throughput screening method for screening a large amount of libraries, and has the advantages of large workload, long period and high cost.
Drawings
FIG. 1 is a double restriction enzyme-digestion-verified electrophoresis of recombinant plasmid; m: DL 10000 DNA Marker; CT: pMA5-kerBv initial control plasmid; 1-16: transforming recombinant plasmids by using each promoter;
FIG. 2 shows the keratinase enzyme activity of the promoter-modified recombinant bacteria; CT: initial control strain containing pMA5-kerBv plasmid; 1-16: recombinant bacteria containing modified recombinant plasmids of each promoter;
FIG. 3 is SDS-PAGE analysis of recombinant bacteria enzyme production by promoter modification; m: protein molecular weight standards; CT1: plasmid b.subtilis WB600 containing pMA 5; CT2: an initial enzyme-producing strain containing the pMA5-kerBv plasmid; 1-16 recombinant bacteria containing various promoter modified recombinant plasmids;
FIG. 4 is a graph of the effect of different nutrient components on enzyme production by fermentation;
FIG. 5 shows the amplified enzyme production results in a 5L fermenter.
Detailed Description
The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.
The enzyme activity detection method of the keratinase comprises the following steps:
the enzyme activity of the keratinase is detected by taking soluble keratin which is sold in the market as a substrate.
Preparation of 1% keratin substrate solution: first, 0.1M Tris-HCl (pH 9.0) solution was prepared, then 5% soluble keratin stock solution was added, and deionized water was added to dilute the Tris-HCl solution to 0.05M and the keratin solution to 1%.
Enzyme reaction: taking 100 mu L of moderately diluted enzyme solution, adding 100 mu L of substrate solution, placing in a water bath at 50 ℃ for accurate reaction for 15min, and immediately adding 200 mu L of 5% (w/v) TCA to terminate the reaction after the reaction is finished. In the control group, 200. Mu.L of TCA was added, and 100. Mu.L of substrate solution was added after the completion of the enzyme reaction. After the above reaction, all samples were centrifuged at 12000rpm for 5min, then 200. Mu.L of the supernatant was aspirated into a new centrifuge tube, and 1mL of 0.4M Na was added 2 CO 3 Then 200 mul of forskolin phenol solution is added, and the mixture is placed in a water bath at 40 ℃ for color reaction for 20min, and then the light absorption value is detected at 680 nm.
Definition of enzyme activity: under the above reaction conditions, the difference in absorbance at 680nm of the substrate hydrolyzed by the enzyme solution was defined as one unit of enzyme activity U.
The enzyme activity calculation formula is as follows: u = (A-B). Times.100 XN
A: the absorbance value of the experimental group at 680nm is shown; b: represents the absorbance value of the control group at 680 nm; 100: the difference in absorbance is 1 multiplied by 100; n: the dilution factor is indicated.
Example 1: promoter molecule modification for improving enzyme activity and expression quantity of keratinase
The promoter nucleotide sequence is obtained by amplification from the bacillus genome and is subjected to mutation modification. In the research, all the promoters selected and modified are constitutive promoters or self-inducible promoters without adding an inducer, and are promoters of trnQ, sigX, yolA, wapA, gapB, cdd, veg, mpr, nprE, aprE, epr, bpr, nprB, pst, gsiB and srfA genes respectively, and the base sequences are shown as SEQ ID NO:1-SEQ ID NO:16, based on the promoter sequences of these genes, primer designs are shown in Table 1.
TABLE 1 promoter primer design
In the research of screening high-efficiency expression promoter of keratinase, a promoter screening system is constructed in a bacillus subtilis expression host, and the promoter can be inserted into a target gene coding sequence such as SEQ ID NO:17 in a recombinant expression plasmid for keratinase. The bacillus subtilis genome is taken as a template, a promoter sequence fragment is amplified by using a specific primer with an enzyme cutting site, and then the promoter fragment is recovered to construct a recombinant plasmid. Fig. 1 shows a promoter-modified recombinant plasmid double-restriction enzyme validation electrophoresis chart, and the recombinant plasmid is transformed into b.subtiliss wb600 after sequencing validation to construct a recombinant bacterium. As the selected promoters are all constitutive promoters or self-inducible promoters without adding an inducer, the constructed recombinant bacteria can be directly used for fermentation enzyme production research in a TB culture medium.
The recombinant strain can release active enzyme protein into a culture medium by using a signal peptide of the keratinase, so that the activity of the keratinase can be detected by directly taking a fermentation supernatant of the recombinant strain. And (3) fermenting and culturing each constructed recombinant bacterium in a TB culture medium for 30 hours, and taking fermentation supernatant to perform keratinase activity determination and SDS-PAGE analysis. The detection results of the enzyme activity of each recombinant strain are shown in figure 2, compared with a control strain, the expression level of keratinase of 7 recombinant strains is obviously improved, particularly, the keratinase activity in a fermentation supernatant of the recombinant strain modified by a PaprE promoter is 2605U/mL, which is 20 times of the extracellular keratinase activity (131U/mL) of the control strain. In addition, the expression level of keratinase of recombinant bacteria modified by PsigX and PsrfA promoters is also greatly improved, the keratinase activity in fermentation supernatant respectively reaches 2119U/mL and 1622U/mL which are respectively 16 times and 12 times of that of control bacteria, and the keratinase activity in fermentation supernatant of Pbpr, pnprB and PgsiB respectively reaches 749U/mL, 449U/mL and 567U/mL which are respectively 5.7 times, 3.4 times and 4.3 times of that of the control bacteria. And performing SDS-PAGE analysis on fermentation supernatant of the recombinant bacteria modified by each promoter. As a result, as shown in FIG. 3, a band of active protein produced by degradation of leader peptide was observed, and the difference in the shade of this band was consistent with the magnitude of the activity of keratinase in the fermentation supernatant of each recombinant bacterium, and the difference in the expression level of keratinase was confirmed. The promoter of the present invention is applied to the promoter as shown in SEQ ID NO:18-SEQ ID NO:19, the enzyme activity can be improved by more than 10% to different degrees.
Example 2: shake flask fermentation condition of recombinant bacteria modified by PaprE promoter
The method comprises the steps of selecting nutritional components such as glycerol, maltose, sucrose, beef extract and corn steep liquor, adding the nutritional components into a TB culture medium in an adding amount of 20g/L, inoculating a PaprE promoter to modify recombinant bacteria, and detecting enzyme production levels of the recombinant bacteria in different culture media. As can be seen from FIG. 4, the keratinase production was highest in TB medium when the fermentation time reached 30h, while the lower keratinase production in the group of experiments with simultaneous nutrient addition was probably due to the prolonged growth cycle of the recombinant strain. When fermented for 48h, all the groups supplemented with nutrients showed relatively higher keratinase yields compared to the control TB medium, in which the keratinase activity reached more than 4000U/mL in both the glycerol supplemented TBG medium and the maltose supplemented TBM medium.
Example 3: paprE promoter modified recombinant bacterium 5L fermentation tank fermentation condition
Further investigating the condition of fermenting and producing enzyme of recombinant bacteria modified by the PaprE promoter on a 5L tank. During the fermentation process, after inoculation with the inoculation amount of 5%, the pH value in the fermentation liquor is gradually reduced, the fermentation is carried out for about 18 hours, the pH value begins to be gradually increased, and the production of a large amount of recombinant keratinase is accompanied. The cell density in the fermentation liquid is increased faster in the early stage, and when the fermentation is carried out to 30h 600 A maximum of 32.2 was reached, which is about 2.2 times that of shake flask cultures. The keratinase activity in the fermentation broth reaches the highest value when the fermentation is carried out for 36h, is 7176U/mL (figure 5), and is about 1.6 times of the shake flask level. The yield of recombinant keratinase obtained in the 5L fermentor of this study is the highest level reported in the literature.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Sequence listing
<110> university of south of the Yangtze river
<120> a promoter for improving the activity of heterologous expression enzyme of keratinase
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caat 424
<210> 17
<211> 1149
<212> DNA
<213> (Artificial sequence)
<400> 17
gttcgtggca aaaaagtttg gatctctctt cttttcgctc ttgctcttat cttcacaatg 60
gctttcggct ctacaacatc tgctcaagct gctggcaaat ctaacggcga aaaaaaatac 120
atcgttggct tcaaacaaac aatgtctaca atgtctgctg ctaaaaaaaa agatgttatc 180
tctgaaaaag gcggcaaagt tgaaaaacaa ttcaaatacg ttgatgctgc ttctgctaca 240
cttaacgaaa aagctgttaa agaacttaaa aaagatcctt ctgttgctta cgttgaagaa 300
gatcatgttg ctcaagctta cgctcaatct gttccttacg gcgtttctca aatcaaagct 360
cctgctcttc attctcaagg cttcacaggc tctaacgtta aagttgctgt tatcgattct 420
ggcatcgatt cttctcatcc tgatcttaaa gttgctggcg gcgcttctat ggttccttct 480
gaaacaaacc ctttccaaga taacaactct catggcacac atgttgctgg cacagttgct 540
gctcttaaca actctgttgg cgttcttggc gttgctcctt ctgcttctct ttacgctgtt 600
aaagttcttg gcgctgatgg ctctggccaa tactcttgga tcatcaacgg catcgaatgg 660
gctatcgcta acaacatgga tgttatcaac atgtctcttg gcggcccttc tggctctgct 720
gctcttaaag ctgctgttga taaagctgtt gcttctggcg ttgttgttgt tgctgctgct 780
ggcaacgaag gcacatctgg cggctcttct acagttggct accctggcaa atacccttct 840
gttatcgctg ttggcgctgt taactcttct aaccaacgtg cttctttctc ttctgttggc 900
tctgaacttg atgttatggc tcctggcgtt tctatccaat ctacacttcc tggcaacaaa 960
tacggcgctt acaacggcac atctatggct tctcctcatg ttgctggcgc tgctgctctt 1020
atcctttcta aacatcctaa ctggacaaac acacaagttc gttcttctct tgaaaacaca 1080
acaacaaaac ttggcgatgc tttctactac ggcaaaggcc ttatcaacgt tcaagctgct 1140
gctcaataa 1149
<210> 18
<211> 1341
<212> DNA
<213> (Artificial sequence)
<400> 18
gaagaacgtg atatccctaa ccgtgataaa atcatctcta aaaaatgggt ttactaaaac 60
atcatccctt ctatcacaat caactctcaa aactctcttc tttctaaatc tacacttaac 120
ttccttaaag gcgaaggcaa agaataagaa gctaaaaact gcggctctgc ttgctgcctt 180
cgttaacgtt aatctcttcg ttggcgttct gctacatgcc ttcgtcgtct tcctgaaaaa 240
gctgttcaaa aacgtaacac actttctgat ctttctcgtc aataagttcc ttaagttcct 300
cctcgtaaac gtatgctttt ccttaaaaaa gctgaacgtt tcaaatctaa cctttctatg 360
cttacacgtc ctcaacaaca ttggatgaaa aaactttaaa aaaactaaaa aaaaatccgt 420
acacttcata tgtggaaaaa aatcatcctt catatgaaca tgcgtaacct tttccttatg 480
gctttcctta aacttaaacg tcgtcttttc acacttaaag ctacacaagc tcttacataa 540
aaataacttc tttctacagc tgaacttaca cttcttatcc ttacataaac atctgaagct 600
gaacaagctt ctttccttct taaacaaaca catacacgta cagctgttct tacagttcgt 660
atgtctcctg ttcgtcttcc tcttcttatc acacaatctg ttttctgggc ttaacgtcaa 720
gctcatcatt acatgcaata aaaatgcctt atccaacaag aagctgctaa catcgctggc 780
cttcttacag ctctttctgg ccctttccct acaatctgga tgctttctac ataagctctt 840
gctgatcttc ttgttcttca acgttaaaaa caataactta tcaaacgttt ccctgctgtt 900
tcttctcttc ttcctcaacc tgaaacaaaa gttcatcctg aagctcaagc tcaatctgct 960
acacttcaaa acatccttct tcttcttcaa taagttcgtt aaacagctgc tacaaaagaa 1020
cttcattctc ctgctcaagt tctttctctt atgtaatggc ttcttgcttg cccttctaaa 1080
gctcatttcc ttgaagctct tacagctctt atcacagaac gtccttggcg tcttcttaca 1140
cttcctgaac aacaacgtta attcttcctt tctacacgtc ttggccaaac acgtaaatct 1200
gttatcgttt aaaaagctct tcaacataca cttgaagctc tttctacaat ggaaaaaggc 1260
taatctacat acaaacaact tcataacaac atcaaaaaaa aacaagctcc tccttacctt 1320
cttttctact gccaacttgt t 1341
<210> 19
<211> 1152
<212> DNA
<213> (Artificial sequence)
<400> 19
atgtgcgtta aaaaaaaaaa cgttatgaca tctgttcttt gggctgttcc tcttcttttc 60
tctgctggct tcggcggctc tatggctaac gctgaaacag cttctaaatc tgaatctgaa 120
aaatcttaca tcgttggctt caaagcttct gctacaacaa actcttctaa aaaacaagct 180
gttacacaaa acggcggcaa acttgaaaaa caataccgtc ttatcaacgc tgctcaagtt 240
aaaatgtctg aacaagctgc taaaaaactt gaacatgatc cttctatcgc ttacgttgaa 300
gaagatcata aagctgaagc ttacgctcaa acagttcctt acggcatccc tcaaatcaaa 360
gctcctgctg ttcatgctca aggctacaaa ggcgctaacg ttaaagttgc tgttcttgat 420
acaggcatcc atgctgctca tcctgatctt aacgttgctg gcggcgcttc tttcgttcct 480
tctgaaccta acgctacaca agatttccaa tctcatggca cacatgttgc tggcacaatc 540
gctgctcttg ataacacaat cggcgttctt ggcgttgctc cttctgcttc tctttacgct 600
gttaaagttc ttgatcgtta cggcgatggc caatactctt ggatcatctc tggcatcgaa 660
tgggctgttg ctaacaacat ggatgttatc aacatgtctc ttggcggccc taacggctct 720
acagctctta aaaacgctgt tgatacagct aacaaccgtg gcgttgttgt tgttgctgct 780
gctggcaact ctggctctac aggctctaca tctacagttg gctaccctgc taaatacgat 840
tctacaatcg ctgttgctaa cgttaactct aacaacgttc gtaactcttc ttcttctgct 900
ggccctgaac ttgatgtttc tgctcctggc acatctatcc tttctacagt tccttcttct 960
ggctacacat cttacacagg cacatctatg gcttctcctc atgttgctgg cgctgctgct 1020
cttatccttt ctaaataccc taacctttct acatctcaag ttcgtcaacg tcttgaaaac 1080
acagctacac ctcttggcaa ctctttctac tacggcaaag gccttatcaa cgttcaagct 1140
gcttctaacg gc 1152
Claims (9)
1. A promoter is characterized in that the promoter is a promoter of a keratinase gene, and the nucleotide sequence of the promoter is shown as SEQ ID No. 12.
2. The promoter according to claim 1, wherein the nucleotide sequence of the keratinase gene is a nucleotide sequence shown as SEQ ID No.17, SEQ ID No.18 or SEQ ID No.19, or a nucleotide sequence having homology of 95% or more with SEQ ID No.17 to 19.
3. An expression vector comprising the promoter of claim 1 or 2.
4. The expression vector of claim 3, wherein the vector is a vector suitable for expression in Bacillus.
5. A recombinant bacterium which expresses keratinase and which comprises the promoter according to claim 1.
6. The recombinant bacterium of claim 5, wherein the host of the recombinant bacterium is Bacillus subtilis, bacillus cereus, bacillus licheniformis, bacillus megaterium, bacillus fragilis, bacillus clausii, bacillus alkalophilus, or Bacillus thuringiensis.
7. A method for producing a recombinant bacterium according to claim 5 or 6, comprising the steps of:
(1) Amplifying a promoter sequence by taking a bacillus subtilis genome as a template to obtain a promoter sequence with a nucleotide sequence shown as SEQ ID No. 12;
(2) Recovering the promoter fragment in the step (1), and constructing the promoter fragment into a plasmid carrying a keratinase gene to obtain a recombinant plasmid;
(3) And (3) transforming the recombinant plasmid in the step (2) into host bacteria to obtain recombinant bacteria.
8. The method for producing keratinase by using the recombinant strain as claimed in claim 5 or 6, wherein the recombinant strain is inoculated into a fermentation tank at an inoculation amount of 1-10%, and the fermentation temperature is 28-40% o C. Fermenting under the conditions that the rotating speed is 100 to 1000rpm, the ventilation volume is 0.5 to 3vvm and the pH value is 5.5 to 8.5 to obtain the keratinase.
9. Use of a promoter according to claim 1 or 2 to promote expression of keratinase.
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| CN113403315B (en) * | 2021-07-15 | 2023-06-30 | 江南大学 | Gene expression cassette for improving thallus growth and generating biological enzyme potential |
| CN114574469B (en) * | 2022-03-21 | 2023-06-02 | 江南大学 | Keratin mutant based on directed evolution transformation and application thereof |
| CN114958897B (en) * | 2022-06-14 | 2023-12-22 | 中农华威生物制药(湖北)有限公司 | Construction method of bacillus subtilis capable of efficiently expressing feed low-temperature keratinase |
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| CN104212830B (en) * | 2014-09-03 | 2016-09-07 | 江南大学 | A kind of bacillus subtilis Self-controlled expression system and construction method thereof and application |
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| CN111763677B (en) | 2022-10-04 |
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| CN111763678A (en) | 2020-10-13 |
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