CN1699576A - Endo beta-1,3 glucanase gene and process for cloning the same - Google Patents
Endo beta-1,3 glucanase gene and process for cloning the same Download PDFInfo
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- CN1699576A CN1699576A CN 200510080820 CN200510080820A CN1699576A CN 1699576 A CN1699576 A CN 1699576A CN 200510080820 CN200510080820 CN 200510080820 CN 200510080820 A CN200510080820 A CN 200510080820A CN 1699576 A CN1699576 A CN 1699576A
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- gly
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- leu
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- 101000866354 Cryptopygus antarcticus Glucan endo-1,3-beta-glucosidase Proteins 0.000 title claims abstract description 26
- 238000000034 method Methods 0.000 title claims abstract description 16
- 238000010367 cloning Methods 0.000 title claims abstract description 9
- 230000008569 process Effects 0.000 title claims abstract description 7
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 35
- 241000894006 Bacteria Species 0.000 claims abstract description 12
- 241000588724 Escherichia coli Species 0.000 claims abstract description 11
- 241000221961 Neurospora crassa Species 0.000 claims abstract description 8
- 239000013612 plasmid Substances 0.000 claims description 17
- 238000012408 PCR amplification Methods 0.000 claims description 14
- 101150021650 gluA gene Proteins 0.000 claims description 10
- 239000002773 nucleotide Substances 0.000 claims description 10
- 125000003729 nucleotide group Chemical group 0.000 claims description 10
- 108010047857 aspartylglycine Proteins 0.000 claims description 7
- 108091092195 Intron Proteins 0.000 claims description 6
- 239000012634 fragment Substances 0.000 claims description 6
- 108010050848 glycylleucine Proteins 0.000 claims description 6
- 108090000790 Enzymes Proteins 0.000 claims description 5
- 102000004190 Enzymes Human genes 0.000 claims description 5
- TZOVVRJYUDETQG-RCOVLWMOSA-N Gly-Asp-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)CN TZOVVRJYUDETQG-RCOVLWMOSA-N 0.000 claims description 5
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 5
- AEJSNWMRPXAKCW-WHFBIAKZSA-N Cys-Ala-Gly Chemical compound SC[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O AEJSNWMRPXAKCW-WHFBIAKZSA-N 0.000 claims description 4
- XUORRGAFUQIMLC-STQMWFEESA-N Gly-Arg-Tyr Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)CN)O XUORRGAFUQIMLC-STQMWFEESA-N 0.000 claims description 4
- JBCLFWXMTIKCCB-UHFFFAOYSA-N H-Gly-Phe-OH Natural products NCC(=O)NC(C(O)=O)CC1=CC=CC=C1 JBCLFWXMTIKCCB-UHFFFAOYSA-N 0.000 claims description 4
- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 claims description 4
- NADLKBTYNKUJEP-KATARQTJSA-N Ser-Thr-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O NADLKBTYNKUJEP-KATARQTJSA-N 0.000 claims description 4
- 108010005233 alanylglutamic acid Proteins 0.000 claims description 4
- KOSRFJWDECSPRO-UHFFFAOYSA-N alpha-L-glutamyl-L-glutamic acid Natural products OC(=O)CCC(N)C(=O)NC(CCC(O)=O)C(O)=O KOSRFJWDECSPRO-UHFFFAOYSA-N 0.000 claims description 4
- 108010060035 arginylproline Proteins 0.000 claims description 4
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 claims description 4
- YMAWOPBAYDPSLA-UHFFFAOYSA-N glycylglycine Chemical compound [NH3+]CC(=O)NCC([O-])=O YMAWOPBAYDPSLA-UHFFFAOYSA-N 0.000 claims description 4
- 108010081551 glycylphenylalanine Proteins 0.000 claims description 4
- 108010077515 glycylproline Proteins 0.000 claims description 4
- 108010057821 leucylproline Proteins 0.000 claims description 4
- 108010029020 prolylglycine Proteins 0.000 claims description 4
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- MHHAWNPHDLCPLF-ULQDDVLXSA-N Val-Phe-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)CC1=CC=CC=C1 MHHAWNPHDLCPLF-ULQDDVLXSA-N 0.000 claims description 3
- 230000000295 complement effect Effects 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 238000011084 recovery Methods 0.000 claims description 3
- COEXAQSTZUWMRI-STQMWFEESA-N (2s)-1-[2-[[(2s)-2-amino-3-(4-hydroxyphenyl)propanoyl]amino]acetyl]pyrrolidine-2-carboxylic acid Chemical compound C([C@H](N)C(=O)NCC(=O)N1[C@@H](CCC1)C(O)=O)C1=CC=C(O)C=C1 COEXAQSTZUWMRI-STQMWFEESA-N 0.000 claims description 2
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- IFTVANMRTIHKML-WDSKDSINSA-N Ala-Gln-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O IFTVANMRTIHKML-WDSKDSINSA-N 0.000 claims description 2
- BLGHHPHXVJWCNK-GUBZILKMSA-N Ala-Gln-Leu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(O)=O BLGHHPHXVJWCNK-GUBZILKMSA-N 0.000 claims description 2
- CZPAHAKGPDUIPJ-CIUDSAMLSA-N Ala-Gln-Pro Chemical compound C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N1CCC[C@H]1C(O)=O CZPAHAKGPDUIPJ-CIUDSAMLSA-N 0.000 claims description 2
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- ZBLQIYPCUWZSRZ-QEJZJMRPSA-N Ala-Phe-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](C)N)CC1=CC=CC=C1 ZBLQIYPCUWZSRZ-QEJZJMRPSA-N 0.000 claims description 2
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- DCVYRWFAMZFSDA-ZLUOBGJFSA-N Ala-Ser-Ala Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O DCVYRWFAMZFSDA-ZLUOBGJFSA-N 0.000 claims description 2
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Abstract
The invention relates to an endo beta-1 glucanase gene and process for cloning, which belongs to the field of biological genetic engineering. the invention provides an endo-beta-1,3-glucanase glu originated from Neurospora crassa AS 3.1604, the nucleic acid sequence of the gene glu and the amino acid sequence of the corresponding protein, bacillus coli expression carrier pET28a-glu containing gene glu and the highly effective expression in bacillus coli, the invention can be applied to the construction of genetic engineering bacterium for the industrial production of the endo-beta-1,3-glucanase, and the increase of level and quality for endo-beta-1,3-glucanase by means of biofermentation.
Description
Technical Field
An endo beta-1, 3 glucanase gene and a cloning method thereof, belonging to the field of microbial genetic engineering.
Background
The beta-1, 3-glucanases are divided into endo-beta-1, 3-glucanases (EC3.2.1.39) and exo-beta-1, 3-glucanases (EC3.2.1.58), which are widely distributed in bacteria, fungi and higher plants. The endo-beta-1, 3-glucanase specifically hydrolyzes macromolecular glucan polymerized by beta-1, 3-glycosidic bonds in an internal random cutting mode, and the product is oligosaccharide or glucose.
The physiological function of the beta-1, 3-glucanases varies from source to source: in plants, it is mainly a defense system against pathogenic fungi, although studies have shown that it plays a role in cell differentiation; among the bacteria, it plays a nutritional role, since most bacteria do not contain β -1, 3-glucan; in fungi, β -1, 3-glucanase has many different functions, and first, it has been shown to have some physiological role in the formation and disruption of morphology during fungal development and differentiation; secondly, as a lyase, β -1, 3-glucanase has a relationship with the transfer of β -1, 3-glucan when carbon and energy sources are depleted.
Beta-1, 3-glucosidic bonds are present in the non-starch polysaccharides of hemicellulose of the cell walls of gramineous plants (maize, rice, sorghum, etc.) and are generally branched, and are one of the important causes for the viscosity of the solution to increase. The addition of endo-beta-1, 3-glucanase can disintegrate the network structure formed by beta-1, 3-glycosidic bond branches, increase the water solubility of the polysaccharide and be further effectively utilized. For example, in the beverage industry, glucan is one of the factors that contribute to beer and juice turbidity, and the presence of glucan in particular during wort filtration severely impedes filtration, greatly slowing down the filtration rate. The addition of highly active and stable dextranase clearly greatly facilitates filtration and clarification. In addition, in the feed industry, on one hand, the viscosity of chyme is increased after the beta-glucan is dissolved, and digestion and absorption of other nutrient components are not facilitated; on the other hand, the undigested and absorbed glucan has strong water absorption, greatly increases the defecation amount of animals, and is extremely unfriendly to the environment. The addition of the endo-beta-1, 3-glucanase can reduce the viscosity of the feed, increase the feed intake of livestock and fish, and reduce the defecation volume of the livestock and fish, thereby reducing the pollution to the environment. Therefore, the development and research of the endo-beta-1, 3-glucanase have important significance.
The study work of scholars at home and abroad on the aspect of heterologous expression of endo-beta-1, 3-glucanase is little. Vladimir V.Zverlov et al cloned from Thermomyces (Thermotoga neocolitana) and successfully expressed a thermophilic beta-1, 3-glucanase (LminA) in E.coli. Hong et al from Streptomyces
(Streptomyces sioyaensis) and expresses endo-. beta. -1, 3-glucanase in E.coli. The beta-1, 3-glucanase from tobacco is cloned by cDNA and successfully expressed in colibacillus.
Disclosure of Invention
The invention aims to find a new beta-1, 3-glucanase and a coding gene thereof, clone a gene of the enzyme by means of gene amplification and the like, and finally express and prepare the beta-1, 3-glucanase in escherichia coli.
The invention provides a beta-1, 3-glucanase gene sequence from neurospora crassa and a corresponding amino acid sequence thereof. Provides an expression vector pET28a-Glu containing beta-1, 3-glucanase gene of Neurospora crassa expressed in Escherichia coli and a gene recombination strain EC-Glu thereof. The achievement of the invention can be used for the construction and the large-scale preparation of the genetic engineering bacteria for the industrial production of the beta-1, 3-glucanase.
The technical scheme of the invention is as follows: the amino acid sequence of the endo-beta-1, 3-glucanase was searched for microbial genome sequences using BLAST software, and the product encoded by open reading frame B23B10.170 in the rough Neurospora genome sequence was probably endo-beta-1, 3-glucanase. The sequence was extracted from the genome sequence, the gene was found to contain two introns by analysis using the GenScan software, and the encoded product of the gene was analyzed using the SignalP V2.0 program. It was determined to be a secreted protein and the cleavage site for the signal peptide was determined. On the basis of the analysis, primers P1 and P2 are designed, and 2432bp nucleotide fragment gluA without a coding signal peptide sequence is amplified by PCR (polymerase chain reaction) by taking the chromosomal DNA of the neurospora crassa AS3.1604 AS a template, wherein the PCR amplification condition is 95 ℃ for 5 min; 30s at 94 ℃, 50s at 54 ℃, 2min at 72 ℃ for 30s, and 35 cycles; 10min at 72 ℃; the amplified target fragment gluA is cut by EcoRI and connected with pUC18 cut by the same enzyme to obtain a recombinant plasmid pUC-gluA. The nucleotide sequence determination confirmed that the endo-beta-1, 3-glucanase gene gluA was obtained, in which 61bp after 267 th base was the first intron and 34bp after 448 th base was the second intron.
And (3) carrying out exon splicing by adopting a PCR method, and removing two introns in the target gene. Primers P3, P4, P5 and P6 were first designed, wherein the 5' ends of the two pairs of primers P3 and P4, P5 and P6 are complementary to 20 and 23 bases, respectively, to facilitate splicing. PCR was carried out using pUC-gluA as a template and P1 and P3, P4 and P2 as primers to obtain PCR products P1P3 and P4P2, respectively. The PCR amplification conditions to obtain product P1P3 was 95 ℃ for 5 min; 30s at 94 ℃, 50s at 54 ℃, 30s at 72 ℃ and 35 cycles; 10min at 72 ℃; the PCR amplification conditions to obtain the PCR product P4P2 were 95 ℃ for 5 min; 30s at 94 ℃, 50s at 54 ℃, 2min at 72 ℃ and 35 cycles; 10min at 72 ℃. Then, the mixture of P1P3 and P4P2 is used as a template, PCR splicing is carried out through primers P1 and P2, and the first intron intr1 is removed. And (3) taking the obtained product with the first intron removed as a template, combining the product with P1, P2, P5 and P6, repeating the process under the same conditions to perform PCR amplification, obtaining products P1P5 and P6P2, performing PCR splicing by using primers P1 and P2, removing the second intron intr2, cloning the glu with the intron removed into pUC18, obtaining a recombinant plasmid pUC-glu, and performing sequence determination to confirm that the intron is correctly removed. The nucleotide sequence of the gene glu is as follows
atgtctccat tgctggacgt ctccgcttca gtctccttga actggaccag cgctcaaggt 60
gtttccgacc gaccgaccag gttttggtac tccagtatcg accacagcac tccccttgtg 120
cgcggcttcg ctcccgacct tgacggagat gtcaactatg ccgtcttcaa ggcagtgaaa 180
cccggcgatg gggcgagtat ccaaacagcg atcaactcgg ggaccaacgg tgctaagaga 240
cacggtctat ggtttgcttc ccagccacga gttgtgtaca taccgccggg aacatacgag 300
atctctgaga ccatcttcat gaacactgac acagttctga tgggcgacgc aacagatgta 360
aggacgatgg cctctcccat gcaagcggaa ccacccatca tcaaagcatc ttcgaacttc 420
tccgggaatc aaacgttgat ctctggccaa gaccccgcaa ctggtatttc cggtgagcta 480
tcgttcgccg tctccttgaa gaacttaatc ctcgacacca ccaatatccc aggagaccaa 540
gccttcacag ctctctggtg gggtgttgct caaggagctc agttgcagaa tgtgaagatc 600
cgtatggcgc ctgccatcga tggtgaggga cacagtggta ttcgcctcgg ccgtggctcg 660
actctcggag tttcggatgt tcgtatcgaa tacgggcaaa acggtatctg gtataacggc 720
catcagcaag cagttttcaa gagcatctac ttcttcaaaa atgctgtggg aatgttcatt 780
gacggtggcg ccacgatcag catcgtcaac ccgacctttg acggctgtgg cttgggcgtc 840
taccacgtcg cgggcaaccc ttggattggt ctaatcgatg ccatctctat caactccggt 900
acgacgctga agacgacaga ctggccaaac tacctggtcg agaaccttcg tgtcatcagc 960
ggaaaaaccg agaacgcggt cgaagggccc ggcgactttg ttctggcaac caagccaaac 1020
gtagcccagc tctcgtacgc caacactgtt ggccatgatc ccatctatgg ccccattgaa 1080
gcagcgcagt tgaaccgtcc atcatcgctg gcgcctggac ctgatgggcg ttatgcatac 1140
cttccagcac cgaactatgc cgagctcagc gtccaagact ttctcaacgt caaagatcct 1200
cttcagaacg gaggctgcct cgtctttggc gacaacaccc gagacgagtc ctccaccctc 1260
aacgccatcc ttcgtctggc cgctcgtcag aacaagatcg cttactttcc cttcggcaag 1320
taccgcgtcg actcgactct tttcgttccc tccggctccc gtattgtcgg cgaagcgtgg 1380
gccacaatca cgggatatgg ccccttcttc acagacagcg ctcatcccca accgatcatc 1440
aaggtcggca accccggcga tattggcacc gcgcacatcc aggatatgcg cttcaccgta 1500
tcggacgtgc ttcccggagc catcatcctg cagttcaacc tcgccggtgc gcagccgggg 1560
gacgtggcaa tctggaactc gcttgtcaca gtcggaggaa cgcggggtgc gaaggcgtta 1620
acggacaagt gcgtcaatcc ggagacggac gaggcgtgca aggctgcttt cttgggtatc 1680
catctcgctt cgacgtcgtc cgtgtatctc gagaacgtat ggaactgggt agccgaccac 1740
atcgccgaag aaccgatatc gccgggcggg agcaacatcg ccggaaaggg cggagtgctc 1800
gtcgaagcga ccaaggggac ctggctgcac gcgctgggat cggagcactg gtggctgtac 1860
cagcttaatc tgcgaaaggc gtcgaatgtg ttagtgacga tgttgcaaag cgagaccaac 1920
tatgaccagg gagacaacgc ggtgcaggtg gtgccgcatc cgtggacgcc ggacgtggag 1980
ggatggggcg atccggactt tggctggtgc gcggggcagg cgaacgagaa gaggtgtcga 2040
atggggttcg cgaactacat caatggcgga agcaacattc ggacctacgc cagtgcctcg 2100
tgggcgttct tcagcgggcc gggataccaa gggtgcgcgg ggcagtatca gtgccaacgg 2160
tatatgcatt gggtggagga gacaccggcc aatttgcagg cgtttgggtt gtgctcgaag 2220
gatacgtggg cgacgttgag gttggagaat ggaaccgaga ttgtcacgaa cgaggggttc 2280
accgggtcgt ggtctgggtc gggaggcgat gtcggcaggt acactccgga ggcatcg tga 2340
Wherein,atgis a start codon for the gene encoding the polypeptide,tgais a stop codon.
According to the nucleotide sequence, the amino acid sequence of the beta-1, 3-glucanase is as follows:
Met Ser Pro Leu Leu Asp Val Ser Ala Ser Val Ser Leu Asn Trp
1 5 10 15
Thr Ser Ala Gln Gly Val Ser Asp Arg Pro Thr Arg Phe Trp Tyr
20 25 30
Ser Ser Ile Asp His Ser Thr Pro Leu Val Arg Gly Phe Ala Pro
35 40 45
Asp Leu Asp Gly Asp Val Asn Tyr Ala Val Phe Lys Ala Val Lys
50 55 60
Pro Gly Asp Gly Ala Ser Ile Gln Thr Ala Ile Asn Ser Gly Thr
65 70 75
Asn Gly Ala Lys Arg His Gly Leu Trp Phe Ala Ser Gln Pro Arg
80 85 90
Val Val Tyr Ile Pro Pro Gly Thr Tyr Glu Ile Ser Glu Thr Ile
95 100 105
Phe Met Asn Thr Asp Thr Val Leu Met Gly Asp Ala Thr Asp Val
110 115 120
Arg Thr Met Ala Ser Pro Met Gln Ala Glu Pro Pro Ile Ile Lys
125 130 135
Ala Ser Ser Asn Phe Ser Gly Asn Gln Thr Leu Ile Ser Gly Gln
140 145 150
Asp Pro Ala Thr Gly Ile Ser Gly Glu Leu Ser Phe Ala Val Ser
155 160 165
Leu Lys Asn Leu Ile Leu Asp Thr Thr Asn Ile Pro Gly Asp Gln
170 175 180
Ala Phe Thr Ala Leu Trp Trp Gly Val Ala Gln Gly Ala Gln Leu
185 190 195
Gln Asn Val Lys Ile Arg Met Ala Pro Ala Ile Asp Gly Glu Gly
200 205 210
His Ser Gly Ile Arg Leu Gly Arg Gly Ser Thr Leu Gly Val Ser
215 220 225
Asp Val Arg Ile Glu Tyr Gly Gln Asn Gly Ile Trp Tyr Asn Gly
230 235 240
His Gln Gln Ala Val Phe Lys Ser Ile Tyr Phe Phe Lys Asn Ala
245 250 255
Val Gly Met Phe Ile Asp Gly Gly Ala Thr Ile Ser Ile Val Asn
260 265 270
Pro Thr Phe Asp Gly Cys Gly Leu Gly Val Tyr His Val Ala Gly
275 280 285
Asn Pro Trp Ile Gly Leu Ile Asp Ala Ile Ser Ile Asn Ser Gly
290 295 300
Thr Thr Leu Lys Thr Thr Asp Trp Pro Asn Tyr Leu Val Glu Asn
305 310 315
Leu Arg Val Ile Ser Gly Lys Thr Glu Asn Ala Val Glu Gly Pro
320 325 330
Gly Asp Phe Val Leu Ala Thr Lys Pro Asn Val Ala Gln Leu Ser
335 340 345
Tyr Ala Asn Thr Val Gly His Asp Pro Ile Tyr Gly Pro Ile Glu
350 355 360
Ala Ala Gln Leu Asn Arg Pro Ser Ser Leu Ala Pro Gly Pro Asp
365 370 375
Gly Arg Tyr Ala Tyr Leu Pro Ala Pro Asn Tyr Ala Glu Leu Ser
380 385 390
Val Gln Asp Phe Leu Asn Val Lys Asp Pro Leu Gln Asn Gly Gly
395 400 405
Cys Leu Val Phe Gly Asp Asn Thr Arg Asp Glu Ser Ser Thr Leu
410 415 420
Asn Ala Ile Leu Arg Leu Ala Ala Arg Gln Asn Lys Ile Ala Tyr
425 430 435
Phe Pro Phe Gly Lys Tyr Arg Val Asp Ser Thr Leu Phe Val Pro
440 445 450
Ser Gly Ser Arg Ile Val Gly Glu Ala Trp Ala Thr Ile Thr Gly
455 460 465
Tyr Gly Pro Phe Phe Thr Asp Ser Ala His Pro Gln Pro Ile Ile
470 475 480
Lys Val Gly Asn Pro Gly Asp Ile Gly Thr Ala His Ile Gln Asp
485 490 495
Met Arg Phe Thr Val Ser Asp Val Leu Pro Gly Ala Ile Ile Leu
500 505 510
Gln Phe Asn Leu Ala Gly Ala Gln Pro Gly Asp Val Ala Ile Trp
515 520 525
Asn Ser Leu Val Thr Val Gly Gly Thr Arg Gly Ala Lys Ala Leu
530 535 540
Thr Asp Lys Cys Val Asn Pro Glu Thr Asp Glu Ala Cys Lys Ala
545 550 555
Ala Phe Leu Gly Ile His Leu Ala Ser Thr Ser Ser Val Tyr Leu
560 565 570
Glu Asn Val Trp Asn Trp Val Ala Asp His Ile Ala Glu Glu Pro
575 580 585
Ile Ser Pro Gly Gly Ser Asn Ile Ala Gly Lys Gly Gly Val Leu
590 595 600
Val Glu Ala Thr Lys Gly Thr Trp Leu His Ala Leu Gly Ser Glu
605 610 615
His Trp Trp Leu Tyr Gln Leu Asn Leu Arg Lys Ala Ser Asn Val
620 625 630
Leu Val Thr Met Leu Gln Ser Glu Thr Asn Tyr Asp Gln Gly Asp
635 640 645
Asn Ala Val Gln Val Val Pro His Pro Trp Thr Pro Asp Val Glu
650 655 660
Gly Trp Gly Asp Pro Asp Phe Gly Trp Cys Ala Gly Gln Ala Asn
665 670 675
Glu Lys Arg Cys Arg Met Gly Phe Ala Asn Tyr Ile Asn Gly Gly
680 685 690
Ser Asn Ile Arg Thr Tyr Ala Ser Ala Ser Trp Ala Phe Phe Ser
695 700 705
Gly Pro Gly Tyr Gln Gly Cys Ala Gly Gln Tyr Gln Cys Gln Arg
710 715 720
Tyr Met His Trp Val Glu Glu Thr Pro Ala Asn Leu Gln Ala Phe
725 730 735
Gly Leu Cys Ser Lys Asp Thr Trp Ala Thr Leu Arg Leu Glu Asn
740 745 750
Gly Thr Glu Ile Val Thr Asn Glu Gly Phe Thr Gly Ser Trp Ser
755 760 765
Gly Ser Gly Gly Asp Val Gly Arg Tyr Thr Pro Glu Ala Ser
770 775 779
the beta-mannanase consists of 779 amino acid residues.
The recombinant plasmid pUC-glu is cut by EcoRI, the glu fragment is obtained by gel recovery, and the glu fragment is cloned into the EcoRI site of the plasmid pET28a to obtain the recombinant plasmid pET28a-glu with the glu inserted in the same direction. The recombinant plasmid pET28a-Glu is transformed into Escherichia coli DE3(RILplus) to obtain a gene recombinant bacterium EC-Glu.
The invention has the beneficial effects that: the invention provides an endo beta-1, 3 glucanase gene glu from Neurospora crassa AS3.1604, provides a nucleotide sequence of the gene glu and an amino acid sequence of corresponding protein, and provides an Escherichia coli expression vector pET28a-glu containing the gene glu and high-efficiency expression in Escherichia coli. The invention can be used for constructing the genetic engineering bacteria for the industrial production of the endo-beta-1, 3-glucanase, and improving the level and the quality of the endo-beta-1, 3-glucanase produced by a microbial fermentation method.
Drawings
FIG. 1 Process for removal of introns by splicing. P1-P6 are primers, and P1P3, P4P2, P1P5 and P6P2 are corresponding PCR amplification products.
FIG. 2 physical map of recombinant expression plasmid pET28 a-glu.
FIG. 3 SDS-PAGE analysis of recombinant bacteria expressing beta-1, 3-glucanase. 1. Protein molecular weight standards; 2. empty vector control; EC-Glu.
Detailed Description
Example 1 cloning and engineering of endo-beta-1, 3-glucanase Gene glu
P1 accggaattcatgtctccattgctggacgt
P2 cgtgaattcacgatgcctccggagtgt
P3 cccggcggtatgtacacaactcgtggctgggaagcaaacc
P4 gttgtgtacataccgccgggaacat
P5 gatgctttgatgatgggtggttccgcttgcatgggagagg
P6 ccacccatcatcaaagcatctcg
The amino acid sequence of the endo-beta-1, 3-glucanase was searched for microbial genome sequences using BLAST software, and the product encoded by open reading frame B23B10.170 in the rough Neurospora genome sequence was probably endo-beta-1, 3-glucanase. The sequence was extracted from the genome sequence, the gene was found to contain two introns by analysis using the GenScan software, and the encoded product of the gene was analyzed by the Signa1PV2.0 program. It was determined to be a secreted protein and the cleavage site for the signal peptide was determined. On the basis of the analysis, primers P1 and P2 are designed, and 2432bp nucleotide fragment gluA without a coding signal peptide sequence is amplified by PCR (polymerase chain reaction) by taking the chromosomal DNA of the neurospora crassa AS3.1604 AS a template, wherein the PCR amplification condition is 95 ℃ for 5 min; 30s at 94 ℃, 50s at 54 ℃, 2min at 72 ℃ for 30s, and 35 cycles; 10min at 72 ℃; the amplified target fragment gluA is cut by EcoRI and connected with pUC18 cut by the same enzyme to obtain a recombinant plasmid pUC-gluA. The nucleotide sequence determination confirms that the gene gluA of the endo-beta-1, 3-glucanase is obtained, wherein 61bp after 267 th base is a first intron, and 34bp after 448 th base is a second intron. Exon splicing is performed by using a PCR method, and two introns in a target gene are removed (figure 1). Primers P3, P4, P5 and P6 were first designed, wherein the 5' ends of the two pairs of primers P3 and P4, P5 and P6 are complementary to 20 and 23 bases, respectively, to facilitate splicing. PCR was carried out using pUC-gluA as a template and P1 and P3, P4 and P2 as primers to obtain PCR products P1P3 and P4P2, respectively. The PCR amplification conditions to obtain product P1P3 was 95 ℃ for 5 min; 30s at 94 ℃, 50s at 54 ℃, 30s at 72 ℃ and 35 cycles; 10min at 72 ℃; the PCR amplification conditions to obtain the PCR product P4P2 were 95 ℃ for 5 min; 30s at 94 ℃, 50s at 54 ℃, 2min at 72 ℃ and 35 cycles; 10min at 72 ℃. And (3) taking the obtained product with the first intron removed as a template, then properly combining P1, P2, P5 and P6, repeating the process and carrying out PCR amplification under the same conditions, firstly obtaining products P1P5 and P6P2, then carrying out PCR splicing by using primers P1 and P2, removing a second intron, cloning the glu with the intron removed into pUC18, obtaining a recombinant plasmid pUC-glu, carrying out sequence determination, and confirming that the intron is correctly removed.
Example 2 expression of endo-. beta.1, 3-glucanase Gene glu
The recombinant plasmid pUC-glu was digested with EcoRI, the gene glu fragment was recovered by gel recovery, and the gene glu was cloned into the EcoRI site of plasmid pET28a to obtain a recombinant plasmid pET28a-glu into which the gene glu was inserted in the forward direction (FIG. 2). The recombinant plasmid pET28a-Glu is transformed into Escherichia coli DE3(RILplus) to obtain a gene recombinant bacterium EC-Glu. The recombinant bacterium EC-Glu is inoculated in 35mL LB culture medium, is subjected to shaking culture at 37 ℃ and 200r/min until the OD value is about 0.6, and is added with isopropyl-beta-D-galactoside with the final concentration of 0.5mmol/L for induced culture for 4 h. And (3) centrifuging to collect thalli, adding lysozyme with the final concentration of 12.5 mu g/mL to carry out cell disruption, and obtaining crude enzyme liquid of endo-beta-1, 3-glucanase. The expression product is detected by an SDS-PAGE method (figure 3), and the recombinant bacterium EC-Glu expresses a remarkable protein band at 80 kD. The recombinant beta-1, 3 glucanase showed the best capacity to liquefy barley glucans at 45-55 ℃.
Claims (3)
1. An endo beta-1, 3 glucanase gene glu from Neurospora crassa AS3.1604, the nucleotide sequence of which is:
atgtctccat tgctggacgt ctccgcttca gtctccttga actggaccag cgctcaaggt 60
gtttccgacc gaccgaccag gttttggtac tccagtatcg accacagcac tccccttgtg 120
cgcggcttcg ctcccgacct tgacggagat gtcaactatg ccgtcttcaa ggcagtgaaa 180
cccggcgatg gggcgagtat ccaaacagcg atcaactcgg ggaccaacgg tgctaagaga 240
cacggtctat ggtttgcttc ccagccacga gttgtgtaca taccgccggg aacatacgag 300
atctctgaga ccatcttcat gaacactgac acagttctga tgggcgacgc aacagatgta 360
aggacgatgg cctctcccat gcaagcggaa ccacccatca tcaaagcatc ttcgaacttc 420
tccgggaatc aaacgttgat ctctggccaa gaccccgcaa ctggtatttc cggtgagcta 480
tcgttcgccg tctccttgaa gaacttaatc ctcgacacca ccaatatccc aggagaccaa 540
gccttcacag ctctctggtg gggtgttgct caaggagctc agttgcagaa tgtgaagatc 600
cgtatggcgc ctgccatcga tggtgaggga cacagtggta ttcgcctcgg ccgtggctcg 660
actctcggag tttcggatgt tcgtatcgaa tacgggcaaa acggtatctg gtataacggc 720
catcagcaag cagttttcaa gagcatctac ttcttcaaaa atgctgtggg aatgttcatt 780
gacggtggcg ccacgatcag catcgtcaac ccgacctttg acggctgtgg cttgggcgtc 840
taccacgtcg cgggcaaccc ttggattggt ctaatcgatg ccatctctat caactccggt 900
acgacgctga agacgacaga ctggccaaac tacctggtcg agaaccttcg tgtcatcagc 960
ggaaaaaccg agaacgcggt cgaagggccc ggcgactttg ttctggcaac caagccaaac 1020
gtagcccagc tctcgtacgc caacactgtt ggccatgatc ccatctatgg ccccattgaa 1080
gcagcgcagt tgaaccgtcc atcatcgctg gcgcctggac ctgatgggcg ttatgcatac 1140
cttccagcac cgaactatgc cgagctcagc gtccaagact ttctcaacgt caaagatcct 1200
cttcagaacg gaggctgcct cgtctttggc gacaacaccc gagacgagtc ctccaccctc 1260
aacgccatcc ttcgtctggc cgctcgtcag aacaagatcg cttactttcc cttcggcaag 1320
taccgcgtcg actcgactct tttcgttccc tccggctccc gtattgtcgg cgaagcgtgg 1380
gccacaatca cgggatatgg ccccttcttc acagacagcg ctcatcccca accgatcatc 1440
aaggtcggca accccggcga tattggcacc gcgcacatcc aggatatgcg cttcaccgta 1500
tcggacgtgc ttcccggagc catcatcctg cagttcaacc tcgccggtgc gcagccgggg 1560
gacgtggcaa tctggaactc gcttgtcaca gtcggaggaa cgcggggtgc gaaggcgtta 1620
acggacaagt gcgtcaatcc ggagacggac gaggcgtgca aggctgcttt cttgggtatc 1680
catctcgctt cgacgtcgtc cgtgtatctc gagaacgtat ggaactgggt agccgaccac 1740
atcgccgaag aaccgatatc gccgggcggg agcaacatcg ccggaaaggg cggagtgctc 1800
gtcgaagcga ccaaggggac ctggctgcac gcgctgggat cggagcactg gtggctgtac 1860
cagcttaatc tgcgaaaggc gtcgaatgtg ttagtgacga tgttgcaaag cgagaccaac 1920
tatgaccagg gagacaacgc ggtgcaggtg gtgccgcatc cgtggacgcc ggacgtggag 1980
ggatggggcg atccggactt tggctggtgc gcggggcagg cgaacgagaa gaggtgtcga 2040
atggggttcg cgaactacat caatggcgga agcaacattc ggacctacgc cagtgcctcg 2100
tgggcgttct tcagcgggcc gggataccaa gggtgcgcgg ggcagtatca gtgccaacgg 2160
tatatgcatt gggtggagga gacaccggcc aatttgcagg cgtttgggtt gtgctcgaag 2220
gatacgtggg cgacgttgag gttggagaat ggaaccgaga ttgtcacgaa cgaggggttc 2280
accgggtcgt ggtctgggtc gggaggcgat gtcggcaggt acactccgga ggcatcg tga 2340
wherein,atgis a start codon for the gene encoding the polypeptide,tgais a stop codon.
2. The gene glu according to claim 1, which has an amino acid composition of:
Met Ser Pro Leu Leu Asp Val Ser Ala Ser Val Ser Leu Asn Trp
1 5 10 15
Thr Ser Ala Gln Gly Val Ser Asp Arg Pro Thr Arg Phe Trp Tyr
20 25 30
Ser Ser Ile Asp His Ser Thr Pro Leu Val Arg Gly Phe Ala Pro
35 40 45
Asp Leu Asp Gly Asp Val Asn Tyr Ala Val Phe Lys Ala Val Lys
50 55 60
Pro Gly Asp Gly Ala Ser Ile Gln Thr Ala Ile Asn Ser Gly Thr
65 70 75
Asn Gly Ala Lys Arg His Gly Leu Trp Phe Ala Ser Gln Pro Arg
80 85 90
Val Val Tyr Ile Pro Pro Gly Thr Tyr Glu Ile Ser Glu Thr Ile
95 100 105
Phe Met Asn Thr Asp Thr Val Leu Met Gly Asp Ala Thr Asp Val
110 115 120
Arg Thr Met Ala Ser Pro Met Gln Ala Glu Pro Pro Ile Ile Lys
125 130 135
Ala Ser Ser Asn Phe Ser Gly Asn Gln Thr Leu Ile Ser Gly Gln
140 145 150
Asp Pro Ala Thr Gly Ile Ser Gly Glu Leu Ser Phe Ala Val Ser
155 160 165
Leu Lys Asn Leu Ile Leu Asp Thr Thr Asn Ile Pro Gly Asp Gln
170 175 180
Ala Phe Thr Ala Leu Trp Trp Gly Val Ala Gln Gly Ala Gln Leu
185 190 195
Gln Asn Val Lys Ile Arg Met Ala Pro Ala Ile Asp Gly Glu Gly
200 205 210
His Ser Gly Ile Arg Leu Gly Arg Gly Ser Thr Leu Gly Val Ser
215 220 225
Asp Val Arg Ile Glu Tyr Gly Gln Asn Gly Ile Trp Tyr Asn Gly
230 235 240
His Gln Gln Ala Val Phe Lys Ser Ile Tyr Phe Phe Lys Asn Ala
245 250 255
Val Gly Met Phe Ile Asp Gly Gly Ala Thr Ile Ser Ile Val Asn
260 265 270
Pro Thr Phe Asp Gly Cys Gly Leu Gly Val Tyr His Val Ala Gly
275 280 285
Asn Pro Trp Ile Gly Leu Ile Asp Ala Ile Ser Ile Asn Ser Gly
290 295 300
Thr Thr Leu Lys Thr Thr Asp Trp Pro Asn Tyr Leu Val Glu Asn
305 310 315
Leu Arg Val Ile Ser Gly Lys Thr Glu Asn Ala Val Glu Gly Pro
320 325 330
Gly Asp Phe Val Leu Ala Thr Lys Pro Asn Val Ala Gln Leu Ser
335 340 345
Tyr Ala Asn Thr Val Gly His Asp Pro Ile Tyr Gly Pro Ile Glu
350 355 360
Ala Ala Gln Leu Asn Arg Pro Ser Ser Leu Ala Pro Gly Pro Asp
365 370 375
Gly Arg Tyr Ala Tyr Leu Pro Ala Pro Asn Tyr Ala Glu Leu Ser
380 385 390
Val Gln Asp Phe Leu Asn Val Lys Asp Pro Leu Gln Asn Gly Gly
395 400 405
Cys Leu Val Phe Gly Asp Asn Thr Arg Asp Glu Ser Ser Thr Leu
410 415 420
Asn Ala Ile Leu Arg Leu Ala Ala Arg Gln Asn Lys Ile Ala Tyr
425 430 435
Phe Pro Phe Gly Lys Tyr Arg Val Asp Ser Thr Leu Phe Val Pro
440 445 450
Ser Gly Ser Arg Ile Val Gly Glu Ala Trp Ala Thr Ile Thr Gly
455 460 465
Tyr Gly Pro Phe Phe Thr Asp Ser Ala His Pro Gln Pro Ile Ile
470 475 480
Lys Val Gly Asn Pro Gly Asp Ile Gly Thr Ala His Ile Gln Asp
485 490 495
Met Arg Phe Thr Val Ser Asp Val Leu Pro Gly Ala Ile Ile Leu
500 505 510
Gln Phe Asn Leu Ala Gly Ala Gln Pro Gly Asp Val Ala Ile Trp
515 520 525
Asn Ser Leu Val Thr Val Gly Gly Thr Arg Gly Ala Lys Ala Leu
530 535 540
Thr Asp Lys Cys Val Asn Pro Glu Thr Asp Glu Ala Cys Lys Ala
545 550 555
Ala Phe Leu Gly Ile His Leu Ala Ser Thr Ser Ser Val Tyr Leu
560 565 570
Glu Asn Val Trp Asn Trp Val Ala Asp His Ile Ala Glu Glu Pro
575 580 585
Ile Ser Pro Gly Gly Ser Asn Ile Ala Gly Lys Gly Gly Val Leu
590 595 600
Val Glu Ala Thr Lys Gly Thr Trp Leu His Ala Leu Gly Ser Glu
605 610 615
His Trp Trp Leu Tyr Gln Leu Asn Leu Arg Lys Ala Ser Asn Val
620 625 630
Leu Val Thr Met Leu Gln Ser Glu Thr Asn Tyr Asp Gln Gly Asp
635 640 645
Asn Ala Val Gln Val Val Pro His Pro Trp Thr Pro Asp Val Glu
650 655 660
Gly Trp Gly Asp Pro Asp Phe Gly Trp Cys Ala Gly Gln Ala Asn
665 670 675
Glu Lys Arg Cys Arg Met Gly Phe Ala Asn Tyr Ile Asn Gly Gly
680 685 690
Ser Asn Ile Arg Thr Tyr Ala Ser Ala Ser Trp Ala Phe Phe Ser
695 700 705
Gly Pro Gly Tyr Gln Gly Cys Ala Gly Gln Tyr Gln Cys Gln Arg
710 715 720
Tyr Met His Trp Val Glu Glu Thr Pro Ala Asn Leu Gln Ala Phe
725 730 735
Gly Leu Cys Ser Lys Asp Thr Trp Ala Thr Leu Arg Leu Glu Asn
740 745 750
Gly Thr Glu Ile Val Thr Asn Glu Gly Phe Thr Gly Ser Trp Ser
755 760 765
Gly Ser Gly Gly Asp Val Gly Arg Tyr Thr Pro Glu Ala Ser
770 775 779。
3. the method for cloning the endo-beta-1, 3-glucanase gene glu according to claim 1, wherein the method is characterized in that
A) The sequences of the primers used are as follows
P1 accggaattcatgtctccattgctggacgt
P2 cgtgaattcacgatgcctccggagtgt
P3 cccggcggtatgtacacaactcgtggctgggaagcaaacc
P4 gttgtgtacataccgccgggaacat
P5 gatgctttgatgatgggtggttccgcttgcatgggagagg
P6 ccacccatcatcaaagcatctcg
B) Using neurospora crassa AS3.1604 chromosome DNA AS a template, and performing PCR amplification on 2432bp nucleotide fragment gluA without a coding signal peptide sequence through a primer P1 and a primer P2 under the condition of 95 ℃ for 5 min; 30s at 94 ℃, 50s at 54 ℃, 2min at 72 ℃ for 30s, and 35 cycles; 10min at 72 ℃;
C) connecting the amplified fragment gluA with pUC18 cut by the same enzyme through EcoR I enzyme digestion to obtain a recombinant plasmid pUC-gluA, and confirming to obtain an endo-beta-1, 3 glucanase gene gluA through nucleotide sequence determination, wherein 61bp behind 267 base is a first intron intr1, and 34bp behind 448 base is a second intron intr 2;
D) carrying out exon splicing by adopting a PGR method, removing two introns in gluA of the gene, and designing primers P3, P4, P5 and P6, wherein 20 and 23 bases at the 5' ends of two pairs of primers P5 and P6 are complementary for splicing P3 and P4;
E) using pUC-gluA as a template and P1 and P3 as primers as PGR, and obtaining a PCR product P1P3 by amplification, wherein the PCR amplification condition is 95 ℃ for 5 min; 30s at 94 ℃, 50s at 54 ℃, 30s at 72 ℃ and 35 cycles; 10min at 72 ℃;
F) using pUC-gluA as a template and P4 and P2 as primers as PGR, and obtaining a PCR product P4P2 by amplification, wherein the PCR amplification condition is 95 ℃ for 5 min; 30s at 94 ℃, 50s at 54 ℃, 2min at 72 ℃ and 35 cycles; 10min at 72 ℃;
G) performing PCR splicing by using a mixture of P1P3 and P4P2 as a template through primers P1 and P2, wherein the PCR amplification condition is 95 ℃ for 5 min; 30s at 94 ℃, 50s at 54 ℃, 2min at 72 ℃ and 30s for 35 cycles; removing the first inclusion intr1 at 72 ℃ for 10 min;
H) taking the product obtained in the step G) after the first intron is removed as a template, then combining the product with P1, P2, P5 and P6 under the same conditions as the processes of E) and F), firstly obtaining products P1P5 and P6P2 by PCR amplification, then carrying out PCR splicing by primers P1 and P2 under the same conditions as the processes of G), and removing the second intron intr 2;
I) cloning the glu with the intron removed into pUCl8 to obtain a recombinant plasmid pUC-glu, and performing sequence determination to confirm that the intron is correctly removed;
J) expression of glu: the recombinant plasmid pUC-Glu is cut by EcoR I, the gene Glu fragment is obtained by glue recovery, the gene Glu is cloned into the EcoR I site of the plasmid pET28a to obtain the recombinant plasmid pET28a-Glu with the gene Glu inserted in the forward direction, and the recombinant expression plasmid pET28a-Glu is transformed into escherichia coli DE3 to obtain the gene recombinant bacterium EC-Glu.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102168059A (en) * | 2011-01-06 | 2011-08-31 | 江苏锐阳生物科技有限公司 | Method for efficiently preparing beta-glucanase |
| CN105831428A (en) * | 2005-12-15 | 2016-08-10 | 美国礼来公司 | Enzymes for reduced immunological stress |
| CN117625507A (en) * | 2023-11-30 | 2024-03-01 | 江南大学 | Rhizobia genetic engineering modification and double-stage fermentation method for high-yield soluble beta-1, 3-glucan |
-
2005
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105831428A (en) * | 2005-12-15 | 2016-08-10 | 美国礼来公司 | Enzymes for reduced immunological stress |
| CN105831428B (en) * | 2005-12-15 | 2020-12-01 | 伊兰科美国公司 | Enzymes for reducing immune stress |
| CN102168059A (en) * | 2011-01-06 | 2011-08-31 | 江苏锐阳生物科技有限公司 | Method for efficiently preparing beta-glucanase |
| CN117625507A (en) * | 2023-11-30 | 2024-03-01 | 江南大学 | Rhizobia genetic engineering modification and double-stage fermentation method for high-yield soluble beta-1, 3-glucan |
| CN117625507B (en) * | 2023-11-30 | 2024-09-17 | 江南大学 | Rhizobia genetic engineering modification and double-stage fermentation method for high-yield soluble beta-1, 3-glucan |
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