CN114015676B - Construction method of cellulase adapting to traditional Chinese medicine feed additive - Google Patents
Construction method of cellulase adapting to traditional Chinese medicine feed additive Download PDFInfo
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- 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)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
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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
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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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
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/101—Plasmid DNA for bacteria
Abstract
The invention discloses a construction method of cellulase adapting to a traditional Chinese medicine feed additive, and relates to the technical field of genetic engineering. The nucleotide sequence of the cellulase is shown as SEQ ID NO. 3; the amino acid sequence is shown as SEQ ID NO. 4. The invention obtains the monomer cellulase with higher enzymatic activity under the gastrointestinal environment temperature. The constructed enzyme is novel, and can efficiently promote the dissolution of cellulose in traditional Chinese medicine at the temperature of the gastrointestinal environment, thereby promoting the rapid release of active ingredients.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a construction method of cellulase adapting to a traditional Chinese medicine feed additive.
Background
The traditional Chinese medicine fermented feed has great advantages in China. The traditional Chinese medicine is safe, nontoxic and multiple in targets, and has wide prospect as a feed additive.
The fermentation of the traditional Chinese medicine can improve the dissolution efficiency of the effective components of the traditional Chinese medicine, improve the efficacy and the taste, and the synergistic effect of the traditional Chinese medicine with the bacterial enzymes can better improve the immunity of animals and the absorption efficiency of nutrition.
Cellulase is often required when fermenting traditional Chinese medicines. The optimal enzyme activity of the existing cellulase is about 50 ℃. When the traditional Chinese medicine is fermented, the temperature is generally about 37 ℃, and the existing cellulose exonuclease is not suitable for the fermentation of the traditional Chinese medicine.
In the gastrointestinal tract of animals, the temperature of the digestive environment is typically around 37 ℃. Cellulases degrade fiber components in plants in vivo, requiring a high enzymatic activity at about 37 ℃.
Therefore, how to provide a cellulase which is adapted to a traditional Chinese medicine feed additive is a problem which needs to be solved by the person skilled in the art.
Disclosure of Invention
In view of the above, the invention provides a construction method of cellulase adapting to a traditional Chinese medicine feed additive.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a cellulase suitable for Chinese medicinal feed additive has a nucleotide sequence shown in SEQ ID NO. 3.
Preferably: the amino acid sequence is shown as SEQ ID NO. 4.
The invention also provides a construction method of the cellulase adapting to the traditional Chinese medicine feed additive, which comprises the following steps:
(1) Error-prone PCR amplification is carried out by taking a synthetic sequence with a nucleotide sequence shown as SEQ ID NO.1 as a template;
(2) Double-enzyme cutting is carried out on the amplified product by utilizing restriction enzymes BamHI and EcoRI, and a gene fragment is recovered;
(3) Double-digestion of the expression vector pRSFdure-1 with the same restriction enzymes as in step (2);
(4) Connecting the gene fragment recovered in the step (2) with the vector subjected to double digestion in the step (3) to obtain a recombinant expression vector pRSFdure-1-SQ, then, transferring the recombinant expression vector pRSFdure-1-SQ into E.coli DH5 alpha competent cells, extracting expression vector plasmids, identifying and numbering the transformed strains in sequence;
(5) The transformed strain is subjected to protein expression, purification and enzyme activity analysis to screen target cellulase.
Preferably: the error-prone PCR reaction system in step (1): error-prone PCR Mix 3.0. Mu.L, error-prone PCR dNTP 3.0. Mu.L, 5mM MnCl 23.0. Mu.L, 10. Mu.g/. Mu.L DNA template 1. Mu.L, 10. Mu.M PCR primer 2. Mu.L, taq DNA polymerase 1. Mu.L, sterilized double distilled water 17. Mu.L; the procedure is as follows: pre-denaturation at 95℃for 3min; denaturation at 94℃for 1min; annealing at 45 ℃ for 1min; extending at 72 ℃ for 4min; after 32 cycles, the extension was carried out at 72℃for 10min.
Preferably: and (3) enzyme digestion systems in the steps (2) and (3): 11. Mu.L of ultrapure water, 1. Mu.L of BamHI, 1. Mu.L of EcoRI, 3. Mu.L of Buffer and 14. Mu.L of the target gene.
The invention also provides application of the cellulase adapting to the traditional Chinese medicine feed additive in fermentation.
Compared with the prior art, the invention discloses a construction method of cellulase adapting to a traditional Chinese medicine feed additive, and the obtained technical effect is that the invention obtains the monomeric cellulase with higher enzymatic activity at the gastrointestinal ambient temperature. The constructed enzyme is novel, and can efficiently promote the dissolution of cellulose in traditional Chinese medicine at the temperature of the gastrointestinal environment, thereby promoting the rapid release of active ingredients.
Detailed Description
The following description will clearly and fully describe the technical solutions of the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The embodiment of the invention discloses a construction method of cellulase adapting to a traditional Chinese medicine feed additive.
The biological agents involved in the examples are all commercially available, for example:
e, purchasing the color DH5 a in a commercial way and preserving in a laboratory; restriction enzymes, takaRa; plasmid extraction kit, tiangen Biochemical technology (Beijing) Co., ltd; the ready-to-use error-prone PCR kit is available from Beijing Tianen technologies Co., ltd; BCA protein concentration assay kit, fozhou omnirange laboratories, inc; plasmid miniprep extraction kit, gel recovery kit, tiangen Biochemical technology (Beijing) limited; plasmid pRSFduet-1 was purchased from Coley Biotech Co.Ltd; the sequence SQ2-1 gene is synthesized completely, prime department biological organisms synthesizing by a company limited;
Primer F:GGATCCATGAAACGTAACCTGTT；
PrimerR: GAATTC TTAATGGTGGTGGTGGT the number of the individual pieces of the plastic, the Optimus of Opt synthesizing by a company limited;
culture medium and buffer used in protein expression and purification:
LB medium: 10g/L tryptone, 10g/L sodium chloride, 5g/L yeast extract powder:
washing buffer: 10mM imidazole, 50mM K 2 HPO 4 -KH 2 PO 4 300mM NaCl,10% glycerol, pH 8.0:
elution buffer: 200mM imidazole, 50mM K 2 HPO 4 -KH 2 PO 4 300mM NaCl,10% glycerol, pH 8.0:
preservation buffer: 50mM K 2 HPO 4 -KH 2 PO 4 300mM NaCl,10% glycerol, pH 8.0.
Example 1
Error-prone PCR
The synthesized Primer F and Primer R are used as primers, and the synthesized sequence SQ1-1 #GGATCCATGAAACGTAACCTGTTCCGCATCGTTTCTCGTGTTGTACTGATTGCTTTCATCGCATCCATCTCTCTGGTGGGTGCTATGAGCTATTTCCCGGTAGAAACCCAGGCGGCCCCGGATTGGTCTATTCCGTCCCTGTGCGAATCTTATAAAGATGACTTTATGATCGGTGTTGCGATCCCTGCCCGTTGTCTGAGCAACGATACCGACAAACGCATGGTTCTGAAACATTTCAACTCTATCACCGCCGAAAACGAAATGAAGCCAGAAAGCCTGCTGGCCGGTCAGACTAGCACCGGCCTGTCTTACCGCTTTTCCACTGCAGACGCTTTTGTGGATTTCGCGAGCACTAACAAAATTGGCATCCGCGGCCATACCCTGGTTTGGCACAACCAGACCCCGGATTGGTTCTTCAAAGATTCTAACGGTCAGCGTCTGTCCAAGGACGCACTGCTGGCTCGTCTGAAACAGTACATCTATGATGTTGTGGGCCGTTACAAGGGCAAAGTGTATGCGTGGGATGTAGTGAACGAGGCAATCGACGAAAACCAACCGGACAGCTACCGTCGCTCTACCTGGTACGAGATCTGTGGTCCGGAATACATTGAGAAAGCCTTCATCTGGGCCCACGAGGCTGACCCGAATGCGAAACTGTTTTACAACGATTATAATACCGAGATCAGCAAGAAACGTGACTTTATCTACAACATGGTTAAAAACCTGAAATCCAAGGGCATCCCTATTCACGGCATCGGTATGCAGTGTCATATTAATGTGAACTGGCCGTCCGTTAGCGAAATTGAAAACTCTATCAAACTGTTCTCCAGCATCCCGGGTATCGAAATTCATATCACTGAACTGGACATGTCTCTGTATAACTACGGCTCTAGCGAAAACTACTCCACCCCGCCGCAGGACCTGCTGCAGAAGCAAAGCCAGAAATACAAGGAAATTTTTACTATGCTGAAAAAATACAAAAACGTTGTTAAGAGCGTTACCTTCTGGGGTCTGAAAGACGACTACTCTTGGCTGCGCAGCTTCTACGGCAAAAACGACTGGCCGCTGCTGTTCTTCGAAGACTACTCTGCTAAACCGGCTTATTGGGCGGTCATCGAAGCTAGCGGCGTGACTACCTCCTCCCCGACCCCGACTCCGACCCCGACCGTAACCGTTACTCCGACCCCGACTCCGACTCCGACCCCAACTGTGACCGCGACTCCTACTCCGACCCCTACCCCGGTCTCCACTCCGGCAACTGGCGGCCAAATCAAAGTGCTGTACGCTAATAAGGAAACCAACTCCACCACGAATACCATCCGTCCGTGGCTGAAGGTTGTTAACAGCGGCTCCAGCAGCATCGACCTGTCTCGCGTTACTATCCGCTATTGGTATACCGTTGACGGTGAACGTGCCCAGTCCGCAGTCTCTGATTGGGCGCAGATCGGTGCTTCCAACGTCACTTTCAAATTTGTAAAACTGAGCTCTAGCGTGAGCGGTGCTGACTACTACCTGGAAATCGGTTTCAAGAGCGGTGCTGGTCAGCTGCAGCCGGGTAAAGACACTGGCGAGATCCAGATCCGTTTCAACAAATCTGACTGGTCTAACTATAACCAGGGCAACGACTGGTCTTGGCTGCAGTCCATGACCTCTTACGGTGAAAACGAGAAAGTTACTGCGTACATTGATGGTGTTCTGGTGTGGGGCCAGGAACCGTCTTGGGACATTAGCGAACTGTCTATTTCTGGTGAATACGTTCGCAGCCGTATCAAAGGTATCCCATACCAGCCGATCGAACGTACCCTGAAAATCTCCCAGGACCAGGTGGCATGTGCACCGATTGGCCAGCCGATCCTGCCGTCTGACTTCGAGGACGGTACCCGTCAGGGCTGGGATTGGGACGGCCCGTCCGGTGTTAAAGGTGCTCTGACCATTGAAGAAGCGAATGGCTCCAACGCTCTGAGCTGGGAAGTTGAGTACCCGGAAAAAAAACTGCAGGATGGCTGGGCATCTGCTCCTCGCCTGATCCTGCGTAATATTAACACCACGCGTGGTGATTGTAAATACCTGTGCTTCGACTTCTATCTGAAGCCGAAGCAGGCGACCAAAGGCGAACTGGCGATCTTCCTGGCGTTTGCTCCTCCGAGCCTGAACTACTGGGCTCAAGCTGAAGACTCCTTCAACATCGACCTGACTAATCTGTCTACTCTGAAGAAAACCCCGGATGATCTGTACTCTTTCAAAATTTCTTTCGATCTGGATAAGATCAAAGAAGGCAAAATCATCGGCCCTGACACCCACCTGCGTGACATCATCATCGTTGTAGCAGACGTGAACTCTGACTTCAAAGGTCGTATGTATCTGGACAATGTGCGTTTCACGAACATGCTGTTCGAAGATGTAACGCCGCAGACTACGGGTTACGAAGCGATCTCTAAACTGTACTCTAAGAAAATTGTTAACGGCATCAGCACCAACCTGTTCGGCCCGGAGAAAGCTGTTACTCGCGCAGAAGTGGCGGCTATGGCAGTCCGTCTGCTGGACCTGCAAGAAGAATCTTACAACGGTGAGTTTGTAGACGTATCTAAAAACTCCTGGTACGCGAACGAAGTATCTACTGCGTACAAAGCAGGTATCATTCTGGGCGACGGTAAGTACATCAAACCGGAGAAAGCCGTTACTCGCGAGGAAATGGCGGTCTTCGCGATGCGTATCTACCGTATCCTGACTGACGAAAAAGCAGAAGCGACCGAAGAAATTGCAATTTCTGACAAAAACAGCATTTCTTCCTGGGCGCGTCAAGATGTAAACGCTGCTATCTCTCTGGGCCTGATGGACGTCTTCACTGACGGTTCTTTCGGCCCGAAGGTGAAAGTGACTCGTGCCGAAGCGACTCAGATTATTTATAAGATCCTGGAACTGACTGGTAAAATGCACCACCACCACCACCATTAAGAATTCSEQ ID NO. 1) as a template (comprising restriction enzyme cleavage sites and terminator TAA), error-prone PCR was performed using the following reaction system:
error-prone PCR Mix 3.0. Mu.L, error-prone PCR dNTP 3.0. Mu.L, 5mM MnCl 2 3.0. Mu.L, 10. Mu.g/. Mu.L of DNA template 1. Mu.L, 10. Mu.M PCR primer 2. Mu.L, taq DNA polymerase 1. Mu.L, sterilized double distilled water 17. Mu.L.
Error-prone PCR procedure was as follows:
pre-denaturation at 95℃for 3min;
denaturation at 94℃for 1min;
annealing at 45 ℃ for 1min;
extending at 72 ℃ for 4min;
after 32 cycles, the extension was carried out at 72℃for 10min.
Enzyme digestion and transformation:
adding double distilled water, an endonuclease buffer solution, an enzyme cutting substrate and a restriction endonuclease into a PCR small tube respectively to carry out double enzyme cutting reaction, wherein the adding sequence is from more to less. The target gene and pRSFduet-1 are subjected to double digestion at 37 ℃, and the reaction system is as follows:
synthesizing a SQ1-1 sequence double enzyme digestion system:
11. Mu.L of ultrapure water, 1. Mu.L of BamHI, 1. Mu.L of EcoRI, 3. Mu.L of Buffer and 14. Mu.L of the target gene.
pRSFduet-1 double cleavage system:
pRSFduet-1 plasmid 43. Mu.L, bamHI 1. Mu.L, ecoRI 1. Mu.L, buffer 5. Mu.L.
After the double digestion reaction for 3 hours at 37 ℃ using BamHI and EcoRI restriction enzymes, loading Buffer was added to terminate the reaction, and the double digested product was purified and recovered according to the gel recovery kit instructions.
After cleavage with the same restriction enzyme, the double digested products have the same cohesive ends and can be ligated into a complete plasmid by DNA ligase. The synthetic SQ1-1 sequence and pRSFdure-1 double cleavage product containing the same cohesive end are placed in the same PCR small tube, the ligation reaction adopts a 10 mu L system, and the target gene and plasmid cleavage product are subjected to a 3:1, and 1. Mu. L T4 DNA ligase, and ligating overnight at 16 ℃. The ligated plasmid was designated pRSFduet-1-SQ.
The conversion step:
the ligation product was transformed into E.coli DH 5. Alpha. Competent cells as follows:
1) The temperature of the thermostatic water bath was first adjusted to 42 ℃.
2) A tube (100. Mu.l) of competent bacteria was removed from an ultra-low temperature freezer at-80℃and immediately thawed using a finger, and then inserted into ice for 10min ice bath.
3) Mu.l of ligation product pRSFduet-1-SQ was added and left on ice for 20min after gentle shaking.
4) After shaking gently, the mixture was inserted into a water bath at 42℃and subjected to heat shock for 90s, and then rapidly returned to ice and allowed to stand for 5min.
5) 900. Mu.L of LB medium without antibiotics was added to each of the above tubes, gently mixed, and then fixed on a spring holder of a shaker, and shaken at 37℃for 50min.
6) To solid LB plate dishes containing appropriate antibiotics, 300. Mu.l of the above-mentioned transformation mixture taken out of the ultra-clean bench was added dropwise, respectively, and the mixture was uniformly coated using a glass coating rod burned by an alcohol burner and cooled.
7) The coated culture dish is marked, firstly placed in a constant temperature incubator at 37 ℃ for 30-60 min, and then placed in the constant temperature incubator at 37 ℃ for overnight in a reverse way after the surface liquid permeates into the culture medium.
The single colony is identified by recombinant plasmid:
picking single colony grown on the LB solid medium, inoculating into 5mL of LB medium containing kanamycin, and culturing at 37deg.C for 200 r.min -1 Culturing overnight under the condition, taking part of bacterial liquid, preserving the strain with a glycerol tube, and numbering the strain as a transformed strain.
The transformed strain was inoculated in 6mLLB medium (containing 50mg/L kanamycin sulfate) and cultured overnight at 37℃at 220rpm for 14 hours;
according to the following steps of 1:100 transferring the seeds to 500mL LB culture medium, culturing at 37 ℃ and 150rpm until reaching OD 600 =0.5; induction of gene expression with 0.7mM IPTG; culturing at 18 ℃ for 24 hours at 150rpm overnight after induction;
4000g of the cells were collected by centrifugation at 4℃for 5min, resuspended in 30mL of preservation buffer, and then disrupted by an ultrasonic disrupter (working for 3s, at 7s intervals, for 10min, with an ultrasonic power of about 200W);
the crushed cell suspension is centrifuged for 30min at 12,000rpm and 4 ℃, and the obtained supernatant is crude protease solution.
Purification
The protein was purified using a Ni-NTA purification medium (south kyi gold sri biotechnology limited) from Shanghai:
in step 1, 6mL of Ni-NTA purification medium was added to a 50mL empty column, the medium allowed to settle freely, and the stock solution was drained.
Step 2, adding 4 times of column volume of washing buffer solution to balance the chromatography medium.
And 3, loading the crude protein enzyme solution into a column, wherein the flow rate is controlled to be 0.5mL/min.
Step 4, washing the column with washing buffer solution with a flow rate of 1mL/min, and removing the impurity protein by a flow rate of 10 times of the volume of the column.
And step 5, eluting with 5 times of column volume eluting buffer solution at the flow rate of 0.5-1 mL/min, and collecting the eluent.
Step 6, collecting target protein eluent, desalting and concentrating the purified protein by a Millipore ultrafiltration tube (15 mL of 100 kDa), and replacing the imidazole-containing elution buffer by a preservation buffer.
Enzyme Activity assay
The cellulase enzyme activities were measured in the same literature (Feng Yue, jiang Jianxin, julia Wei, tube red epitaxy, research on cellulase activity and synergistic effects of mixed cellulases; university of Beijing forestry, 2009, volume 31, journal 1) except that the measurement temperature was 37 ℃.
Purified protein concentration the BCA protein concentration assay kit was used.
Sequence screening
Determining to obtain initial cellulase with amino acid sequence SQ1 (MKRNLFRIVSRVVLIAFIASISLVGAMSYFPVETQAAPDWSIPSLCESYKDDFMIGVAIPARCLSNDTDKRMVLKHFNSITAENEMKPESLLAGQTSTGLSYRFSTADAFVDFASTNKIGIRGHTLVWHNQTPDWFFKDSNGQRLSKDALLARLKQYIYDVVGRYKGKVYAWDVVNEAIDENQPDSYRRSTWYEICGPEYIEKAFIWAHEADPNAKLFYNDYNTEISKKRDFIYNMVKNLKSKGIPIHGIGMQCHINVNWPSVSEIENSIKLFSSIPGIEIHITELDMSLYNYGSSENYSTPPQDLLQKQSQKYKEIFTMLKKYKNVVKSVTFWGLKDDYSWLRSFYGKNDWPLLFFEDYSAKPAYWAVIEASGVTTSSPTPTPTPTVTVTPTPTPTPTPTVTATPTPTPTPVSTPATGGQIKVLYANKETNSTTNTIRPWLKVVNSGSSSIDLSRVTIRYWYTVDGERAQSAVSDWAQIGASNVTFKFVKLSSSVSGADYYLEIGFKSGAGQLQPGKDTGEIQIRFNKSDWSNYNQGNDWSWLQSMTSYGENEKVTAYIDGVLVWGQEPSWDISELSISGEYVRSRIKGIPYQPIERTLKISQDQVACAPIGQPILPSDFEDGTRQGWDWDGPSGVKGALTIEEANGSNALSWEVEYPEKKLQDGWASAPRLILRNINTTRGDCKYLCFDFYLKPKQATKGELAIFLAFAPPSLNYWAQAEDSFNIDLTNLSTLKKTPDDLYSFKISFDLDKIKEGKIIGPDTHLRDIIIVVADVNSDFKGRMYLDNVRFTNMLFEDVTPQTTGYEAISKLYSKKIVNGISTNLFGPEKAVTRAEVAAMAVRLLDLQEESYNGEFVDVSKNSWYANEVSTAYKAGIILGDGKYIKPEKAVTREEMAVFAMRIYRILTDEKAEATEEIAISDKNSISSWARQDVNAAISLGLMDVFTDGSFGPKVKVTRAEATQIIYKILELTGKMHHHHHH, SEQ ID NO. 2) with specific enzyme activity of 0.051U/mg protein at 37deg.C; a mutant enzyme with a specific enzyme activity of 0.060U/mg was selected at 37℃and the transformant strain No. SXT2.
Sequencing of coding sequences and coding amino acid sequences
Strain SXT2 was inoculated in 6mL LB medium (containing 50mg/L kanamycin sulfate) and cultured overnight at 37℃at 220rpm for 14h.
Respectively taking a proper amount of bacterial liquid, extracting recombinant plasmids according to the specification of a plasmid extraction kit, and sequencing the extracted plasmids by a sequencing company to obtain the mutant enzyme with the coding sequence SQ2-1 #GGATCCATGAAACGTAACCTGTTCCGCATCGTTTCTCGTGTTCTGCTGATTGCTTTCATCGCATCCATCTCTCTGGTGGGTGCTATGAGCTATTTCCCGGTAGAAACCCAGGCGGCCCCGGATTGGTCTATTCCGTCCCTGTGCGAATCTTATAAAGATGACTTTATGATCGGTGTTGCGATCCCTGCCCGTTGTCTGAGCAACGATACCGACAAACGCATGGTTCTGAAACATTTCAACTCTATCACCGCCGAAAACGAAATGAAGCCAGAAAGCCTGCTGGCCGGTCAGACTAGCACCGGCCTGTCTTACCGCTTTTCCACTGCAGACGCTTTTGTGGATTTCGCGAGCACTAACAAAATTGGCATCCGCGGCCATACCCTGGTTTGGCACAACCAGACCCCGGATTGGTTCTTCAAAGATTCTAACGGTCAGCGTCTGTCCAAGGACGCACTGCTGGCTCGTCTGCTGCAGTACATCTATGATGTTGTGGGCCGTTACAAGGGCAAAGTGTATGCGTGGGATGTAGTGAACGAGGCAATCGACGAAAACCAACCGGACAGCTACCGTCGCTCTACCTGGTACGAGATCTGTGGTCCGGAATACATTGAGAAAGCCTTCATCTGGGCCCACGAGGCTGACCCGAATGCGAAACTGTTTTACAACGATTATAATACCGAGATCAGCAAGAAACGTGACTTTATCTACAACATGGTTAAAAACCTGAAATCCAAGGGCATCCCTATTCACGGCATCGGTATGCAGTGTCATATTAATGTGAACTGGCCGTCCGTTAGCGAAATTGAAAACTCTATCAAACTGTTCTCCAGCATCCCGGGTATCGAAATTCATATCACTGAACTGGACATGTCTCTGTATAACTACGGCTCTAGCGAAAACTACTCCACCCCGCCGCAGGACCTGCTGCAGAAGCAAAGCCAGAAATACAAGGAAATTTTTACTATGCTGAAAAAATACAAAAACGTTGTTAAGAGCGTTACCTTCTGGGGTCTGAAAGACGACTACTCTTGGCTGCGCAGCTTCTACGGCAAAAACGACTGGCCGCTGCTGTTCTTCGAAGACTACTCTGCTAAACCGGCTTATTGGGCGGTCATCGAAGCTAGCGGCGTGACTACCTCCTCCCCGACCCCGACTCCGACCCCGACCGTAACCGTTACTCCGACCCCGACTCCGACTCCGACCCCAACTGTGACCGCGACTCCTACTCCGACCCCTACCCCGGTCTCCACTCCGGCAACTGGCGGCCAAATCAAAGTGCTGTACGCTAATAAGGAAACCAACTCCACCACGAATACCATCCGTCCGTGGCTGAAGGTTGTTAACAGCGGCTCCAGCAGCATCGACCTGTCTCGCGTTACTATCCGCTATTGGTATACCGTTGACGGTGAACGTGCCCAGTCCGCAGTCTCTGATTGGGCGCAGATCGGTGCTTCCAACGTCACTTTCAAATTTGTAAAACTGAGCTCTAGCGTGAGCGGTGCTTACTACTACCTGGAACTGGGTTTCAAGAGCGGTGCTGGTCAGCTGCAGCCGGGTAAAGACACTGGCGAGATCCAGATCCGTTTCAACAAATCTGACTGGTCTAACTATAACCAGGGCAACGACTGGTCTTGGCTGCAGTCCATGACCTCTTACGGTGAAAACGAGAAAGTTACTGCGTACATTGATGGTGTTCTGGTGTGGGGCCAGGAACCGTCTTGGGACATTAGCGAACTGTCTATTTCTGGTGAATACGTTCGCAGCCGTATCAAAGGTATCCCATACCAGCCGATCGAACGTACCCTGAAAATCTCCCAGGACCAGGTGGCATGTGCACCGATTGGCCAGCCGATCCTGCCGTCTGACTTCGAGGACGGTACCCGTCAGGGCTGGGATTGGGACGGCCCGTCCGGTGTTAAAGGTGCTCTGACCATTGAAGAAGCGAATGGCTCCAACGCTCTGAGCTGGGAAGTTGAGTACCCGGAAAAAAAACTGCAGGATGGCTGGGCATCTGCTCCTCGCCTGATCCTGCGTAATATTAACACCACGCGTGGTGATTGTAAATACCTGTGCTTCGACTTCTATCTGAAGCCGAAGCAGGCGACCAAAGGCGAACTGGCGATCTTCCTGGCGTTTGCTCCTCCGAGCCTGAACTACTGGGCTCAAGCTGAAGACTCCTTCAACATCGACCTGACTAATCTGTCTACTCTGAAGAAAACCCCGGATGATCTGTACTCTTTCGTTATTTCTTTCGATCTGAAAAAGATCAAAGAAGGCAAAATCATCGGCCCTGACACCCACCTGCGTGACATCATCATCGTTGTAGCAGACGTGAACTCTGACTTCAAAGGTCGTATGTATCTGGACAATGTGCGTTTCACGAACATGCTGTTCGAAGATGTAACGCCGCAGACTACGGGTTACGAAGCGATCTCTAAACTGTACTCTAAGAAAATTGTTAACGGCATCAGCACCAACCTGTTCGGCCCGGAGAAAGCTGTTACTCGCGCAGAAGTGGCGGCTATGGCAGTCCGTCTGCTGGACCTGCAAGAAGAATCTTACAACGGTGAGTTTGTAGACGTATCTAAAAACTCCTGGTACGCGAACGAAGTATCTACTGCGTACAAAGCAGGTATCATTCTGGGCGACGGTAAGTACATCAAACCGGAGAAAGCCGTTACTCGCGAGGAAATGGCGGTCTTCGCGATGCGTATCTACCGTATCCTGACTGACGAAAAAGCAGAAGCGACCGAAGAAATTGCAATTTCTGACAAAAACAGCATTTCTTCCTGGGCGCGTCAAGATGTAAACGCTGCTATCTCTCTGGGCCTGATGGACGTCTTCACTGACGGTTCTTTCGGCCCGAAGGTGAAAGTGACTCGTGCCGAAGCGACTCAGATTATTTATAAGATCCTGGAACTGACTGGTAAAATGCACCACCACCACCACCATTAAGAATTCAs shown in SEQ ID NO. 3), includes a restriction enzyme site and a terminator TAA, the amino acid sequence of which is SQ2 (MKRNLFRIVSRVLLIAFIASISLVGAMSYFPVETQAAPDWSIPSLCESYKDDFMIGVAIPARCLSNDTDKRMVLKHFNSITAENEMKPESLLAGQTSTGLSYRFSTADAFVDFASTNKIGIRGHTLVWHNQTPDWFFKDSNGQRLSKDALLARLLQYIYDVVGRYKGKVYAWDVVNEAIDENQPDSYRRSTWYEICGPEYIEKAFIWAHEADPNAKLFYNDYNTEISKKRDFIYNMVKNLKSKGIPIHGIGMQCHINVNWPSVSEIENSIKLFSSIPGIEIHITELDMSLYNYGSSENYSTPPQDLLQKQSQKYKEIFTMLKKYKNVVKSVTFWGLKDDYSWLRSFYGKNDWPLLFFEDYSAKPAYWAVIEASGVTTSSPTPTPTPTVTVTPTPTPTPTPTVTATPTPTPTPVSTPATGGQIKVLYANKETNSTTNTIRPWLKVVNSGSSSIDLSRVTIRYWYTVDGERAQSAVSDWAQIGASNVTFKFVKLSSSVSGAYYYLELGFKSGAGQLQPGKDTGEIQIRFNKSDWSNYNQGNDWSWLQSMTSYGENEKVTAYIDGVLVWGQEPSWDISELSISGEYVRSRIKGIPYQPIERTLKISQDQVACAPIGQPILPSDFEDGTRQGWDWDGPSGVKGALTIEEANGSNALSWEVEYPEKKLQDGWASAPRLILRNINTTRGDCKYLCFDFYLKPKQATKGELAIFLAFAPPSLNYWAQAEDSFNIDLTNLSTLKKTPDDLYSFVISFDLKKIKEGKIIGPDTHLRDIIIVVADVNSDFKGRMYLDNVRFTNMLFEDVTPQTTGYEAISKLYSKKIVNGISTNLFGPEKAVTRAEVAAMAVRLLDLQEESYNGEFVDVSKNSWYANEVSTAYKAGIILGDGKYIKPEKAVTREEMAVFAMRIYRILTDEKAEATEEIAISDKNSISSWARQDVNAAISLGLMDVFTDGSFGPKVKVTRAEATQIIYKILELTGKMHHHHHH, as shown in SEQ ID NO. 4).
In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Sequence listing
<110> North China biological pharmacy (Hubei) Co., ltd
<120> construction method of cellulase adapting to traditional Chinese medicine feed additive
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2961
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
ggatccatga aacgtaacct gttccgcatc gtttctcgtg ttgtactgat tgctttcatc 60
gcatccatct ctctggtggg tgctatgagc tatttcccgg tagaaaccca ggcggccccg 120
gattggtcta ttccgtccct gtgcgaatct tataaagatg actttatgat cggtgttgcg 180
atccctgccc gttgtctgag caacgatacc gacaaacgca tggttctgaa acatttcaac 240
tctatcaccg ccgaaaacga aatgaagcca gaaagcctgc tggccggtca gactagcacc 300
ggcctgtctt accgcttttc cactgcagac gcttttgtgg atttcgcgag cactaacaaa 360
attggcatcc gcggccatac cctggtttgg cacaaccaga ccccggattg gttcttcaaa 420
gattctaacg gtcagcgtct gtccaaggac gcactgctgg ctcgtctgaa acagtacatc 480
tatgatgttg tgggccgtta caagggcaaa gtgtatgcgt gggatgtagt gaacgaggca 540
atcgacgaaa accaaccgga cagctaccgt cgctctacct ggtacgagat ctgtggtccg 600
gaatacattg agaaagcctt catctgggcc cacgaggctg acccgaatgc gaaactgttt 660
tacaacgatt ataataccga gatcagcaag aaacgtgact ttatctacaa catggttaaa 720
aacctgaaat ccaagggcat ccctattcac ggcatcggta tgcagtgtca tattaatgtg 780
aactggccgt ccgttagcga aattgaaaac tctatcaaac tgttctccag catcccgggt 840
atcgaaattc atatcactga actggacatg tctctgtata actacggctc tagcgaaaac 900
tactccaccc cgccgcagga cctgctgcag aagcaaagcc agaaatacaa ggaaattttt 960
actatgctga aaaaatacaa aaacgttgtt aagagcgtta ccttctgggg tctgaaagac 1020
gactactctt ggctgcgcag cttctacggc aaaaacgact ggccgctgct gttcttcgaa 1080
gactactctg ctaaaccggc ttattgggcg gtcatcgaag ctagcggcgt gactacctcc 1140
tccccgaccc cgactccgac cccgaccgta accgttactc cgaccccgac tccgactccg 1200
accccaactg tgaccgcgac tcctactccg acccctaccc cggtctccac tccggcaact 1260
ggcggccaaa tcaaagtgct gtacgctaat aaggaaacca actccaccac gaataccatc 1320
cgtccgtggc tgaaggttgt taacagcggc tccagcagca tcgacctgtc tcgcgttact 1380
atccgctatt ggtataccgt tgacggtgaa cgtgcccagt ccgcagtctc tgattgggcg 1440
cagatcggtg cttccaacgt cactttcaaa tttgtaaaac tgagctctag cgtgagcggt 1500
gctgactact acctggaaat cggtttcaag agcggtgctg gtcagctgca gccgggtaaa 1560
gacactggcg agatccagat ccgtttcaac aaatctgact ggtctaacta taaccagggc 1620
aacgactggt cttggctgca gtccatgacc tcttacggtg aaaacgagaa agttactgcg 1680
tacattgatg gtgttctggt gtggggccag gaaccgtctt gggacattag cgaactgtct 1740
atttctggtg aatacgttcg cagccgtatc aaaggtatcc cataccagcc gatcgaacgt 1800
accctgaaaa tctcccagga ccaggtggca tgtgcaccga ttggccagcc gatcctgccg 1860
tctgacttcg aggacggtac ccgtcagggc tgggattggg acggcccgtc cggtgttaaa 1920
ggtgctctga ccattgaaga agcgaatggc tccaacgctc tgagctggga agttgagtac 1980
ccggaaaaaa aactgcagga tggctgggca tctgctcctc gcctgatcct gcgtaatatt 2040
aacaccacgc gtggtgattg taaatacctg tgcttcgact tctatctgaa gccgaagcag 2100
gcgaccaaag gcgaactggc gatcttcctg gcgtttgctc ctccgagcct gaactactgg 2160
gctcaagctg aagactcctt caacatcgac ctgactaatc tgtctactct gaagaaaacc 2220
ccggatgatc tgtactcttt caaaatttct ttcgatctgg ataagatcaa agaaggcaaa 2280
atcatcggcc ctgacaccca cctgcgtgac atcatcatcg ttgtagcaga cgtgaactct 2340
gacttcaaag gtcgtatgta tctggacaat gtgcgtttca cgaacatgct gttcgaagat 2400
gtaacgccgc agactacggg ttacgaagcg atctctaaac tgtactctaa gaaaattgtt 2460
aacggcatca gcaccaacct gttcggcccg gagaaagctg ttactcgcgc agaagtggcg 2520
gctatggcag tccgtctgct ggacctgcaa gaagaatctt acaacggtga gtttgtagac 2580
gtatctaaaa actcctggta cgcgaacgaa gtatctactg cgtacaaagc aggtatcatt 2640
ctgggcgacg gtaagtacat caaaccggag aaagccgtta ctcgcgagga aatggcggtc 2700
ttcgcgatgc gtatctaccg tatcctgact gacgaaaaag cagaagcgac cgaagaaatt 2760
gcaatttctg acaaaaacag catttcttcc tgggcgcgtc aagatgtaaa cgctgctatc 2820
tctctgggcc tgatggacgt cttcactgac ggttctttcg gcccgaaggt gaaagtgact 2880
cgtgccgaag cgactcagat tatttataag atcctggaac tgactggtaa aatgcaccac 2940
caccaccacc attaagaatt c 2961
<210> 2
<211> 982
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 2
Met Lys Arg Asn Leu Phe Arg Ile Val Ser Arg Val Val Leu Ile Ala
1 5 10 15
Phe Ile Ala Ser Ile Ser Leu Val Gly Ala Met Ser Tyr Phe Pro Val
20 25 30
Glu Thr Gln Ala Ala Pro Asp Trp Ser Ile Pro Ser Leu Cys Glu Ser
35 40 45
Tyr Lys Asp Asp Phe Met Ile Gly Val Ala Ile Pro Ala Arg Cys Leu
50 55 60
Ser Asn Asp Thr Asp Lys Arg Met Val Leu Lys His Phe Asn Ser Ile
65 70 75 80
Thr Ala Glu Asn Glu Met Lys Pro Glu Ser Leu Leu Ala Gly Gln Thr
85 90 95
Ser Thr Gly Leu Ser Tyr Arg Phe Ser Thr Ala Asp Ala Phe Val Asp
100 105 110
Phe Ala Ser Thr Asn Lys Ile Gly Ile Arg Gly His Thr Leu Val Trp
115 120 125
His Asn Gln Thr Pro Asp Trp Phe Phe Lys Asp Ser Asn Gly Gln Arg
130 135 140
Leu Ser Lys Asp Ala Leu Leu Ala Arg Leu Lys Gln Tyr Ile Tyr Asp
145 150 155 160
Val Val Gly Arg Tyr Lys Gly Lys Val Tyr Ala Trp Asp Val Val Asn
165 170 175
Glu Ala Ile Asp Glu Asn Gln Pro Asp Ser Tyr Arg Arg Ser Thr Trp
180 185 190
Tyr Glu Ile Cys Gly Pro Glu Tyr Ile Glu Lys Ala Phe Ile Trp Ala
195 200 205
His Glu Ala Asp Pro Asn Ala Lys Leu Phe Tyr Asn Asp Tyr Asn Thr
210 215 220
Glu Ile Ser Lys Lys Arg Asp Phe Ile Tyr Asn Met Val Lys Asn Leu
225 230 235 240
Lys Ser Lys Gly Ile Pro Ile His Gly Ile Gly Met Gln Cys His Ile
245 250 255
Asn Val Asn Trp Pro Ser Val Ser Glu Ile Glu Asn Ser Ile Lys Leu
260 265 270
Phe Ser Ser Ile Pro Gly Ile Glu Ile His Ile Thr Glu Leu Asp Met
275 280 285
Ser Leu Tyr Asn Tyr Gly Ser Ser Glu Asn Tyr Ser Thr Pro Pro Gln
290 295 300
Asp Leu Leu Gln Lys Gln Ser Gln Lys Tyr Lys Glu Ile Phe Thr Met
305 310 315 320
Leu Lys Lys Tyr Lys Asn Val Val Lys Ser Val Thr Phe Trp Gly Leu
325 330 335
Lys Asp Asp Tyr Ser Trp Leu Arg Ser Phe Tyr Gly Lys Asn Asp Trp
340 345 350
Pro Leu Leu Phe Phe Glu Asp Tyr Ser Ala Lys Pro Ala Tyr Trp Ala
355 360 365
Val Ile Glu Ala Ser Gly Val Thr Thr Ser Ser Pro Thr Pro Thr Pro
370 375 380
Thr Pro Thr Val Thr Val Thr Pro Thr Pro Thr Pro Thr Pro Thr Pro
385 390 395 400
Thr Val Thr Ala Thr Pro Thr Pro Thr Pro Thr Pro Val Ser Thr Pro
405 410 415
Ala Thr Gly Gly Gln Ile Lys Val Leu Tyr Ala Asn Lys Glu Thr Asn
420 425 430
Ser Thr Thr Asn Thr Ile Arg Pro Trp Leu Lys Val Val Asn Ser Gly
435 440 445
Ser Ser Ser Ile Asp Leu Ser Arg Val Thr Ile Arg Tyr Trp Tyr Thr
450 455 460
Val Asp Gly Glu Arg Ala Gln Ser Ala Val Ser Asp Trp Ala Gln Ile
465 470 475 480
Gly Ala Ser Asn Val Thr Phe Lys Phe Val Lys Leu Ser Ser Ser Val
485 490 495
Ser Gly Ala Asp Tyr Tyr Leu Glu Ile Gly Phe Lys Ser Gly Ala Gly
500 505 510
Gln Leu Gln Pro Gly Lys Asp Thr Gly Glu Ile Gln Ile Arg Phe Asn
515 520 525
Lys Ser Asp Trp Ser Asn Tyr Asn Gln Gly Asn Asp Trp Ser Trp Leu
530 535 540
Gln Ser Met Thr Ser Tyr Gly Glu Asn Glu Lys Val Thr Ala Tyr Ile
545 550 555 560
Asp Gly Val Leu Val Trp Gly Gln Glu Pro Ser Trp Asp Ile Ser Glu
565 570 575
Leu Ser Ile Ser Gly Glu Tyr Val Arg Ser Arg Ile Lys Gly Ile Pro
580 585 590
Tyr Gln Pro Ile Glu Arg Thr Leu Lys Ile Ser Gln Asp Gln Val Ala
595 600 605
Cys Ala Pro Ile Gly Gln Pro Ile Leu Pro Ser Asp Phe Glu Asp Gly
610 615 620
Thr Arg Gln Gly Trp Asp Trp Asp Gly Pro Ser Gly Val Lys Gly Ala
625 630 635 640
Leu Thr Ile Glu Glu Ala Asn Gly Ser Asn Ala Leu Ser Trp Glu Val
645 650 655
Glu Tyr Pro Glu Lys Lys Leu Gln Asp Gly Trp Ala Ser Ala Pro Arg
660 665 670
Leu Ile Leu Arg Asn Ile Asn Thr Thr Arg Gly Asp Cys Lys Tyr Leu
675 680 685
Cys Phe Asp Phe Tyr Leu Lys Pro Lys Gln Ala Thr Lys Gly Glu Leu
690 695 700
Ala Ile Phe Leu Ala Phe Ala Pro Pro Ser Leu Asn Tyr Trp Ala Gln
705 710 715 720
Ala Glu Asp Ser Phe Asn Ile Asp Leu Thr Asn Leu Ser Thr Leu Lys
725 730 735
Lys Thr Pro Asp Asp Leu Tyr Ser Phe Lys Ile Ser Phe Asp Leu Asp
740 745 750
Lys Ile Lys Glu Gly Lys Ile Ile Gly Pro Asp Thr His Leu Arg Asp
755 760 765
Ile Ile Ile Val Val Ala Asp Val Asn Ser Asp Phe Lys Gly Arg Met
770 775 780
Tyr Leu Asp Asn Val Arg Phe Thr Asn Met Leu Phe Glu Asp Val Thr
785 790 795 800
Pro Gln Thr Thr Gly Tyr Glu Ala Ile Ser Lys Leu Tyr Ser Lys Lys
805 810 815
Ile Val Asn Gly Ile Ser Thr Asn Leu Phe Gly Pro Glu Lys Ala Val
820 825 830
Thr Arg Ala Glu Val Ala Ala Met Ala Val Arg Leu Leu Asp Leu Gln
835 840 845
Glu Glu Ser Tyr Asn Gly Glu Phe Val Asp Val Ser Lys Asn Ser Trp
850 855 860
Tyr Ala Asn Glu Val Ser Thr Ala Tyr Lys Ala Gly Ile Ile Leu Gly
865 870 875 880
Asp Gly Lys Tyr Ile Lys Pro Glu Lys Ala Val Thr Arg Glu Glu Met
885 890 895
Ala Val Phe Ala Met Arg Ile Tyr Arg Ile Leu Thr Asp Glu Lys Ala
900 905 910
Glu Ala Thr Glu Glu Ile Ala Ile Ser Asp Lys Asn Ser Ile Ser Ser
915 920 925
Trp Ala Arg Gln Asp Val Asn Ala Ala Ile Ser Leu Gly Leu Met Asp
930 935 940
Val Phe Thr Asp Gly Ser Phe Gly Pro Lys Val Lys Val Thr Arg Ala
945 950 955 960
Glu Ala Thr Gln Ile Ile Tyr Lys Ile Leu Glu Leu Thr Gly Lys Met
965 970 975
His His His His His His
980
<210> 3
<211> 2961
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
ggatccatga aacgtaacct gttccgcatc gtttctcgtg ttctgctgat tgctttcatc 60
gcatccatct ctctggtggg tgctatgagc tatttcccgg tagaaaccca ggcggccccg 120
gattggtcta ttccgtccct gtgcgaatct tataaagatg actttatgat cggtgttgcg 180
atccctgccc gttgtctgag caacgatacc gacaaacgca tggttctgaa acatttcaac 240
tctatcaccg ccgaaaacga aatgaagcca gaaagcctgc tggccggtca gactagcacc 300
ggcctgtctt accgcttttc cactgcagac gcttttgtgg atttcgcgag cactaacaaa 360
attggcatcc gcggccatac cctggtttgg cacaaccaga ccccggattg gttcttcaaa 420
gattctaacg gtcagcgtct gtccaaggac gcactgctgg ctcgtctgct gcagtacatc 480
tatgatgttg tgggccgtta caagggcaaa gtgtatgcgt gggatgtagt gaacgaggca 540
atcgacgaaa accaaccgga cagctaccgt cgctctacct ggtacgagat ctgtggtccg 600
gaatacattg agaaagcctt catctgggcc cacgaggctg acccgaatgc gaaactgttt 660
tacaacgatt ataataccga gatcagcaag aaacgtgact ttatctacaa catggttaaa 720
aacctgaaat ccaagggcat ccctattcac ggcatcggta tgcagtgtca tattaatgtg 780
aactggccgt ccgttagcga aattgaaaac tctatcaaac tgttctccag catcccgggt 840
atcgaaattc atatcactga actggacatg tctctgtata actacggctc tagcgaaaac 900
tactccaccc cgccgcagga cctgctgcag aagcaaagcc agaaatacaa ggaaattttt 960
actatgctga aaaaatacaa aaacgttgtt aagagcgtta ccttctgggg tctgaaagac 1020
gactactctt ggctgcgcag cttctacggc aaaaacgact ggccgctgct gttcttcgaa 1080
gactactctg ctaaaccggc ttattgggcg gtcatcgaag ctagcggcgt gactacctcc 1140
tccccgaccc cgactccgac cccgaccgta accgttactc cgaccccgac tccgactccg 1200
accccaactg tgaccgcgac tcctactccg acccctaccc cggtctccac tccggcaact 1260
ggcggccaaa tcaaagtgct gtacgctaat aaggaaacca actccaccac gaataccatc 1320
cgtccgtggc tgaaggttgt taacagcggc tccagcagca tcgacctgtc tcgcgttact 1380
atccgctatt ggtataccgt tgacggtgaa cgtgcccagt ccgcagtctc tgattgggcg 1440
cagatcggtg cttccaacgt cactttcaaa tttgtaaaac tgagctctag cgtgagcggt 1500
gcttactact acctggaact gggtttcaag agcggtgctg gtcagctgca gccgggtaaa 1560
gacactggcg agatccagat ccgtttcaac aaatctgact ggtctaacta taaccagggc 1620
aacgactggt cttggctgca gtccatgacc tcttacggtg aaaacgagaa agttactgcg 1680
tacattgatg gtgttctggt gtggggccag gaaccgtctt gggacattag cgaactgtct 1740
atttctggtg aatacgttcg cagccgtatc aaaggtatcc cataccagcc gatcgaacgt 1800
accctgaaaa tctcccagga ccaggtggca tgtgcaccga ttggccagcc gatcctgccg 1860
tctgacttcg aggacggtac ccgtcagggc tgggattggg acggcccgtc cggtgttaaa 1920
ggtgctctga ccattgaaga agcgaatggc tccaacgctc tgagctggga agttgagtac 1980
ccggaaaaaa aactgcagga tggctgggca tctgctcctc gcctgatcct gcgtaatatt 2040
aacaccacgc gtggtgattg taaatacctg tgcttcgact tctatctgaa gccgaagcag 2100
gcgaccaaag gcgaactggc gatcttcctg gcgtttgctc ctccgagcct gaactactgg 2160
gctcaagctg aagactcctt caacatcgac ctgactaatc tgtctactct gaagaaaacc 2220
ccggatgatc tgtactcttt cgttatttct ttcgatctga aaaagatcaa agaaggcaaa 2280
atcatcggcc ctgacaccca cctgcgtgac atcatcatcg ttgtagcaga cgtgaactct 2340
gacttcaaag gtcgtatgta tctggacaat gtgcgtttca cgaacatgct gttcgaagat 2400
gtaacgccgc agactacggg ttacgaagcg atctctaaac tgtactctaa gaaaattgtt 2460
aacggcatca gcaccaacct gttcggcccg gagaaagctg ttactcgcgc agaagtggcg 2520
gctatggcag tccgtctgct ggacctgcaa gaagaatctt acaacggtga gtttgtagac 2580
gtatctaaaa actcctggta cgcgaacgaa gtatctactg cgtacaaagc aggtatcatt 2640
ctgggcgacg gtaagtacat caaaccggag aaagccgtta ctcgcgagga aatggcggtc 2700
ttcgcgatgc gtatctaccg tatcctgact gacgaaaaag cagaagcgac cgaagaaatt 2760
gcaatttctg acaaaaacag catttcttcc tgggcgcgtc aagatgtaaa cgctgctatc 2820
tctctgggcc tgatggacgt cttcactgac ggttctttcg gcccgaaggt gaaagtgact 2880
cgtgccgaag cgactcagat tatttataag atcctggaac tgactggtaa aatgcaccac 2940
caccaccacc attaagaatt c 2961
<210> 4
<211> 982
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 4
Met Lys Arg Asn Leu Phe Arg Ile Val Ser Arg Val Leu Leu Ile Ala
1 5 10 15
Phe Ile Ala Ser Ile Ser Leu Val Gly Ala Met Ser Tyr Phe Pro Val
20 25 30
Glu Thr Gln Ala Ala Pro Asp Trp Ser Ile Pro Ser Leu Cys Glu Ser
35 40 45
Tyr Lys Asp Asp Phe Met Ile Gly Val Ala Ile Pro Ala Arg Cys Leu
50 55 60
Ser Asn Asp Thr Asp Lys Arg Met Val Leu Lys His Phe Asn Ser Ile
65 70 75 80
Thr Ala Glu Asn Glu Met Lys Pro Glu Ser Leu Leu Ala Gly Gln Thr
85 90 95
Ser Thr Gly Leu Ser Tyr Arg Phe Ser Thr Ala Asp Ala Phe Val Asp
100 105 110
Phe Ala Ser Thr Asn Lys Ile Gly Ile Arg Gly His Thr Leu Val Trp
115 120 125
His Asn Gln Thr Pro Asp Trp Phe Phe Lys Asp Ser Asn Gly Gln Arg
130 135 140
Leu Ser Lys Asp Ala Leu Leu Ala Arg Leu Leu Gln Tyr Ile Tyr Asp
145 150 155 160
Val Val Gly Arg Tyr Lys Gly Lys Val Tyr Ala Trp Asp Val Val Asn
165 170 175
Glu Ala Ile Asp Glu Asn Gln Pro Asp Ser Tyr Arg Arg Ser Thr Trp
180 185 190
Tyr Glu Ile Cys Gly Pro Glu Tyr Ile Glu Lys Ala Phe Ile Trp Ala
195 200 205
His Glu Ala Asp Pro Asn Ala Lys Leu Phe Tyr Asn Asp Tyr Asn Thr
210 215 220
Glu Ile Ser Lys Lys Arg Asp Phe Ile Tyr Asn Met Val Lys Asn Leu
225 230 235 240
Lys Ser Lys Gly Ile Pro Ile His Gly Ile Gly Met Gln Cys His Ile
245 250 255
Asn Val Asn Trp Pro Ser Val Ser Glu Ile Glu Asn Ser Ile Lys Leu
260 265 270
Phe Ser Ser Ile Pro Gly Ile Glu Ile His Ile Thr Glu Leu Asp Met
275 280 285
Ser Leu Tyr Asn Tyr Gly Ser Ser Glu Asn Tyr Ser Thr Pro Pro Gln
290 295 300
Asp Leu Leu Gln Lys Gln Ser Gln Lys Tyr Lys Glu Ile Phe Thr Met
305 310 315 320
Leu Lys Lys Tyr Lys Asn Val Val Lys Ser Val Thr Phe Trp Gly Leu
325 330 335
Lys Asp Asp Tyr Ser Trp Leu Arg Ser Phe Tyr Gly Lys Asn Asp Trp
340 345 350
Pro Leu Leu Phe Phe Glu Asp Tyr Ser Ala Lys Pro Ala Tyr Trp Ala
355 360 365
Val Ile Glu Ala Ser Gly Val Thr Thr Ser Ser Pro Thr Pro Thr Pro
370 375 380
Thr Pro Thr Val Thr Val Thr Pro Thr Pro Thr Pro Thr Pro Thr Pro
385 390 395 400
Thr Val Thr Ala Thr Pro Thr Pro Thr Pro Thr Pro Val Ser Thr Pro
405 410 415
Ala Thr Gly Gly Gln Ile Lys Val Leu Tyr Ala Asn Lys Glu Thr Asn
420 425 430
Ser Thr Thr Asn Thr Ile Arg Pro Trp Leu Lys Val Val Asn Ser Gly
435 440 445
Ser Ser Ser Ile Asp Leu Ser Arg Val Thr Ile Arg Tyr Trp Tyr Thr
450 455 460
Val Asp Gly Glu Arg Ala Gln Ser Ala Val Ser Asp Trp Ala Gln Ile
465 470 475 480
Gly Ala Ser Asn Val Thr Phe Lys Phe Val Lys Leu Ser Ser Ser Val
485 490 495
Ser Gly Ala Tyr Tyr Tyr Leu Glu Leu Gly Phe Lys Ser Gly Ala Gly
500 505 510
Gln Leu Gln Pro Gly Lys Asp Thr Gly Glu Ile Gln Ile Arg Phe Asn
515 520 525
Lys Ser Asp Trp Ser Asn Tyr Asn Gln Gly Asn Asp Trp Ser Trp Leu
530 535 540
Gln Ser Met Thr Ser Tyr Gly Glu Asn Glu Lys Val Thr Ala Tyr Ile
545 550 555 560
Asp Gly Val Leu Val Trp Gly Gln Glu Pro Ser Trp Asp Ile Ser Glu
565 570 575
Leu Ser Ile Ser Gly Glu Tyr Val Arg Ser Arg Ile Lys Gly Ile Pro
580 585 590
Tyr Gln Pro Ile Glu Arg Thr Leu Lys Ile Ser Gln Asp Gln Val Ala
595 600 605
Cys Ala Pro Ile Gly Gln Pro Ile Leu Pro Ser Asp Phe Glu Asp Gly
610 615 620
Thr Arg Gln Gly Trp Asp Trp Asp Gly Pro Ser Gly Val Lys Gly Ala
625 630 635 640
Leu Thr Ile Glu Glu Ala Asn Gly Ser Asn Ala Leu Ser Trp Glu Val
645 650 655
Glu Tyr Pro Glu Lys Lys Leu Gln Asp Gly Trp Ala Ser Ala Pro Arg
660 665 670
Leu Ile Leu Arg Asn Ile Asn Thr Thr Arg Gly Asp Cys Lys Tyr Leu
675 680 685
Cys Phe Asp Phe Tyr Leu Lys Pro Lys Gln Ala Thr Lys Gly Glu Leu
690 695 700
Ala Ile Phe Leu Ala Phe Ala Pro Pro Ser Leu Asn Tyr Trp Ala Gln
705 710 715 720
Ala Glu Asp Ser Phe Asn Ile Asp Leu Thr Asn Leu Ser Thr Leu Lys
725 730 735
Lys Thr Pro Asp Asp Leu Tyr Ser Phe Val Ile Ser Phe Asp Leu Lys
740 745 750
Lys Ile Lys Glu Gly Lys Ile Ile Gly Pro Asp Thr His Leu Arg Asp
755 760 765
Ile Ile Ile Val Val Ala Asp Val Asn Ser Asp Phe Lys Gly Arg Met
770 775 780
Tyr Leu Asp Asn Val Arg Phe Thr Asn Met Leu Phe Glu Asp Val Thr
785 790 795 800
Pro Gln Thr Thr Gly Tyr Glu Ala Ile Ser Lys Leu Tyr Ser Lys Lys
805 810 815
Ile Val Asn Gly Ile Ser Thr Asn Leu Phe Gly Pro Glu Lys Ala Val
820 825 830
Thr Arg Ala Glu Val Ala Ala Met Ala Val Arg Leu Leu Asp Leu Gln
835 840 845
Glu Glu Ser Tyr Asn Gly Glu Phe Val Asp Val Ser Lys Asn Ser Trp
850 855 860
Tyr Ala Asn Glu Val Ser Thr Ala Tyr Lys Ala Gly Ile Ile Leu Gly
865 870 875 880
Asp Gly Lys Tyr Ile Lys Pro Glu Lys Ala Val Thr Arg Glu Glu Met
885 890 895
Ala Val Phe Ala Met Arg Ile Tyr Arg Ile Leu Thr Asp Glu Lys Ala
900 905 910
Glu Ala Thr Glu Glu Ile Ala Ile Ser Asp Lys Asn Ser Ile Ser Ser
915 920 925
Trp Ala Arg Gln Asp Val Asn Ala Ala Ile Ser Leu Gly Leu Met Asp
930 935 940
Val Phe Thr Asp Gly Ser Phe Gly Pro Lys Val Lys Val Thr Arg Ala
945 950 955 960
Glu Ala Thr Gln Ile Ile Tyr Lys Ile Leu Glu Leu Thr Gly Lys Met
965 970 975
His His His His His His
980
Claims (2)
1. A cellulase adapting to a traditional Chinese medicine feed additive is characterized in that the nucleotide sequence of the coding enzyme is shown as SEQ ID NO. 3; the amino acid sequence is shown as SEQ ID NO. 4.
2. Use of a cellulase according to claim 1 adapted to a chinese herbal feed additive in fermentation.
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| CN114196695A (en) * | 2021-11-26 | 2022-03-18 | 中农华威生物制药(湖北)有限公司 | Construction method of high-activity traditional Chinese medicine feed additive trypsin |
| CN114015677A (en) * | 2021-11-26 | 2022-02-08 | 中农华威生物制药(湖北)有限公司 | Cellulase for promoting release of traditional Chinese medicine feed additive in intestinal tract and production method thereof |
| CN114958809A (en) * | 2022-06-14 | 2022-08-30 | 中农华威生物制药(湖北)有限公司 | Construction method of endo-beta-glucanase suitable for high-temperature granulation of feed |
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