CN101671686A - 一种甘油脱水酶基因、载体、工程菌及其应用 - Google Patents
一种甘油脱水酶基因、载体、工程菌及其应用 Download PDFInfo
- Publication number
- CN101671686A CN101671686A CN200910153309A CN200910153309A CN101671686A CN 101671686 A CN101671686 A CN 101671686A CN 200910153309 A CN200910153309 A CN 200910153309A CN 200910153309 A CN200910153309 A CN 200910153309A CN 101671686 A CN101671686 A CN 101671686A
- Authority
- CN
- China
- Prior art keywords
- glycerol
- gene
- recombinant
- glycerol dehydratase
- ala
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108010025885 Glycerol dehydratase Proteins 0.000 title claims abstract description 31
- 241000894006 Bacteria Species 0.000 title claims abstract description 17
- 239000013598 vector Substances 0.000 title claims abstract description 15
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 103
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 37
- 102000004190 Enzymes Human genes 0.000 claims abstract description 21
- 108090000790 Enzymes Proteins 0.000 claims abstract description 21
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 13
- AKXKFZDCRYJKTF-UHFFFAOYSA-N 3-Hydroxypropionaldehyde Chemical compound OCCC=O AKXKFZDCRYJKTF-UHFFFAOYSA-N 0.000 claims abstract description 10
- NBBJYMSMWIIQGU-UHFFFAOYSA-N Propionic aldehyde Chemical compound CCC=O NBBJYMSMWIIQGU-UHFFFAOYSA-N 0.000 claims abstract description 10
- 235000011187 glycerol Nutrition 0.000 claims description 35
- 238000005215 recombination Methods 0.000 claims description 16
- 230000006798 recombination Effects 0.000 claims description 16
- 241000186660 Lactobacillus Species 0.000 claims description 10
- 229940039696 lactobacillus Drugs 0.000 claims description 10
- 230000008521 reorganization Effects 0.000 claims description 10
- IKHGUXGNUITLKF-UHFFFAOYSA-N Acetaldehyde Chemical compound CC=O IKHGUXGNUITLKF-UHFFFAOYSA-N 0.000 claims description 8
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 claims description 8
- 230000001939 inductive effect Effects 0.000 claims description 7
- 125000003729 nucleotide group Chemical group 0.000 claims description 6
- 241000186604 Lactobacillus reuteri Species 0.000 claims description 5
- 229940001882 lactobacillus reuteri Drugs 0.000 claims description 5
- 239000002773 nucleotide Substances 0.000 claims description 5
- 235000015097 nutrients Nutrition 0.000 claims description 5
- 239000000758 substrate Substances 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 4
- 210000001082 somatic cell Anatomy 0.000 claims description 4
- 230000001131 transforming effect Effects 0.000 claims description 4
- 230000000968 intestinal effect Effects 0.000 claims description 2
- 238000004321 preservation Methods 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 241000588724 Escherichia coli Species 0.000 abstract description 24
- 239000013612 plasmid Substances 0.000 abstract description 15
- 238000006243 chemical reaction Methods 0.000 abstract description 9
- 239000013604 expression vector Substances 0.000 abstract description 6
- 230000009466 transformation Effects 0.000 abstract description 5
- 230000003248 secreting effect Effects 0.000 abstract description 4
- 238000010276 construction Methods 0.000 abstract description 2
- 230000003834 intracellular effect Effects 0.000 abstract 2
- -1 1, 2-propylene Chemical group 0.000 abstract 1
- 150000001299 aldehydes Chemical class 0.000 abstract 1
- ZTQSAGDEMFDKMZ-UHFFFAOYSA-N butyric aldehyde Natural products CCCC=O ZTQSAGDEMFDKMZ-UHFFFAOYSA-N 0.000 abstract 1
- 108700026220 vif Genes Proteins 0.000 abstract 1
- 239000012634 fragment Substances 0.000 description 10
- 238000003259 recombinant expression Methods 0.000 description 8
- 230000001580 bacterial effect Effects 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 241000672609 Escherichia coli BL21 Species 0.000 description 4
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 238000012408 PCR amplification Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 108091008146 restriction endonucleases Proteins 0.000 description 3
- 102220023257 rs387907546 Human genes 0.000 description 3
- 102000012410 DNA Ligases Human genes 0.000 description 2
- 108010061982 DNA Ligases Proteins 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 2
- 102220369447 c.1352G>A Human genes 0.000 description 2
- 102220369445 c.668T>C Human genes 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 239000013599 cloning vector Substances 0.000 description 2
- ZIHHMGTYZOSFRC-UWWAPWIJSA-M cobamamide Chemical compound C1(/[C@](C)(CCC(=O)NC[C@H](C)OP(O)(=O)OC2[C@H]([C@H](O[C@@H]2CO)N2C3=CC(C)=C(C)C=C3N=C2)O)[C@@H](CC(N)=O)[C@]2(N1[Co+]C[C@@H]1[C@H]([C@@H](O)[C@@H](O1)N1C3=NC=NC(N)=C3N=C1)O)[H])=C(C)\C([C@H](C/1(C)C)CCC(N)=O)=N\C\1=C/C([C@H]([C@@]\1(CC(N)=O)C)CCC(N)=O)=N/C/1=C(C)\C1=N[C@]2(C)[C@@](C)(CC(N)=O)[C@@H]1CCC(N)=O ZIHHMGTYZOSFRC-UWWAPWIJSA-M 0.000 description 2
- 229960005452 cobamamide Drugs 0.000 description 2
- 235000006279 cobamamide Nutrition 0.000 description 2
- 239000011789 cobamamide Substances 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 230000005611 electricity Effects 0.000 description 2
- XUWPJKDMEZSVTP-LTYMHZPRSA-N kalafungina Chemical compound O=C1C2=C(O)C=CC=C2C(=O)C2=C1[C@@H](C)O[C@H]1[C@@H]2OC(=O)C1 XUWPJKDMEZSVTP-LTYMHZPRSA-N 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 102220023258 rs387907548 Human genes 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 244000248349 Citrus limon Species 0.000 description 1
- 235000005979 Citrus limon Nutrition 0.000 description 1
- 241000193403 Clostridium Species 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 108010002747 Pfu DNA polymerase Proteins 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 150000001413 amino acids Chemical group 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 102220369446 c.1274G>A Human genes 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 1
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 1
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 1
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 1
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 150000002314 glycerols Chemical class 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 102220023256 rs387907547 Human genes 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 229960004799 tryptophan Drugs 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
本发明提供了及一种编码甘油脱水酶的甘油脱水酶基因、含有该基因的重组载体、该重组载体转化得到的重组基因工程菌,以及其在制备重组甘油脱水酶中的应用。该甘油脱水酶基因可与表达载体连接构建得到含该基因的胞内表达重组质粒或分泌表达重组质粒,再分别对应转化至大肠杆菌菌株中,获得重组大肠杆菌,该重组大肠杆菌含有重组甘油脱水酶,可以重组大肠杆菌为酶源进行生物催化与转化。重组甘油脱水酶作为转化用酶,可以分别以甘油、1,2-丙二醇或乙醇为底物,进行转化反应制备相应的3-羟基丙醛、丙醛或乙醛等。
Description
(一)技术领域
本发明涉及一种编码甘油脱水酶的甘油脱水酶基因、含有该基因的重组载体、该重组载体转化得到的重组基因工程菌,以及其在制备重组甘油脱水酶中的应用。
(二)背景技术
甘油脱水酶能催化甘油、1,2-丙二醇和乙醇分别生成3-羟基丙醛、丙醛和乙醛,在化学合成工业中具有广泛的应用前景。甘油脱水酶主要存在于克雷伯氏杆菌、乳酸杆菌、弗氏柠檬菌、巴氏梭状芽孢杆菌中。甘油脱水酶是由αβγ三个亚基通过非共价键的疏水作用缔合而成的异形六聚体,只有在辅酶维生素B12存在时才能发挥其催化作用。
目前不同来源的甘油脱水酶基因已经被克隆和测序,并且实现了在不同宿主中的表达。然而现有的甘油脱水酶均存在酶活力较低,且辅酶维生素B12成本较高等问题,已成为限制甘油脱水酶进行大规模工业化生产的瓶颈,因此,构建具有工业用途的甘油脱水酶基因工程菌意义重大。另外,利用基因工程技术来改善野生型甘油脱水酶菌株的不足,为甘油脱水酶的工业化应用提供了新思路。
(三)发明内容
本发明目的是提供一种甘油脱水酶基因、含有该基因的重组载体、该重组载体转化得到的重组基因工程菌,以及其在制备重组甘油脱水酶中的应用。
本发明采用的技术方案是:
一种甘油脱水酶基因,具有SEQ ID NO:1所示的核苷酸序列。
该甘油脱水酶基因由如下方法得到:利用PCR技术,在引物1(CAGTTCCAGGTGTTCCGG)、引物2(ATGAAACGCCAGAAACGCT)的作用下,以来源于乳酸杆菌(Lactobacillus reuteri)菌株CCTCC NO:M 209213中的总基因组DNA为模板,克隆得到约1.6kb的甘油脱水酶的基因片段。并将该片段连接到pMD18-T载体上获得克隆载体pMD18-T-dhaB和转化了pMD18-T-dhaB的重组大肠杆菌JM109/pMD18-T-dhaB。对重组质粒进行测序,并利用软件对测序结果进行分析,分析表明该序列含有一个长为1674bp的开发阅读框。该基因核苷酸序列为:
1 ATGAAACGCC AGAAACGCTT TGAAGAACTG GAAAAACGTC CGATCCACCA GGACACCTTC
61 GTAAAAGAGT GGCCGGAAGA AGGCTTTGTG GCAATGATGG GCCCGAACGA TCCGAAACCG
121 AGCGTTAAAG TGGAAAACGG CAAAATTGTG GAAATGGACG GCAAAAAGCT GGAAGATTTC
181 GATCTGATCG ATCTGTACAT TGCAAAATAC GGCATTAACA TTGATAACGT TGAAAAAGTA
241 ATGAACATGG ATTCCACCAA AATCGCGCGT ATGCTGGTAG ACCCGAACGT GTCCCGTGAC
301 GAAATTATTG AAATCACCTC TGCTCTGACT CCGGCTAAAG CGGAAGAGAT CATTAGCAAA
361 CTGGATTTTG GTGAAATGAT CATGGCAGTA AAAAAGATGC GTCCGCGTCG TAAGCCAGAC
421 AACCAGTGCC ACGTTACCAA CACCGTAGAC AATCCTGTAC AGATCGCTGC GGATGCTGCG
481 GACGCGGCTC TGCGTGGCTT TCCGGAACAG GAAACCACGA CCGCTGTTGC ACGCTACGCT
541 CCATTCAACG CCATTTCCAT CCTGATCGGT GCTCAGACCG GTCGCCCGGG TGTTCTGACT
601 CAGTGCTCTG TCGAAGAGGC CACCGAACTG CAGCTGGGTA TGCGCGGTTT TACTGCCTAC
661 GCAGAAACCA TTAGCGTGTA CGGCACCGAC CGCGTTTTCA CCGATGGTGA CGACACCCCG
721 TGGAGCAAGG GCTTTCTGGC GAGCTGTTAT GCCTCCCGTG GTCTGAAAAT GCGTTTTACC
781 TCTGGTGCGG GTTCCGAAGT CCTGATGGGC TACCCGGAAG GTAAATCCAT GCTGTACCTG
841 GAAGCCCGTT GTATCCTGCT GACCAAAGCA AGCGGTGTTC AAGGCCTGCA GAATGGTGCG
901 GTGTCCTGTA TTGAAATCCC TGGCGCTGTT CCTAACGGTA TCCGTGAAGT GCTGGGCGAA
961 AACCTGCTGT GCATGATGTG TGACATCGAA TGTGCATCCG GTTGTGATCA GGCTTATTCT
1021 CACTCTGATA TGCGTCGCAC CGAGCGTTTT ATCGGTCAGT TTATCGCGGG TACCGACTAC
1081 ATCAACTCCG GCTATTCTTC TACTCCGAAT TACGACAACA CTTTCGCAGG CAGCAACACG
1141 GATGCTATGG ACTACGATGA CATGTACGTA ATGGAGCGTG ACCTGGGTCA ATACTATGGT
1201 ATTCATCCGG TTAAAGAGGA GACTATTATC AAAGCACGTA ACAAAGCTGC GAAAGCTCTG
1261 CAGGCTGTTT TCGAGGACCT GGGCCTGCCA AAAATCACCG ATGAAGAAGT AGAAGCTGCT
1321 ACGTACGCGA ACACCCACGA TGACATGCCG AAACGTGATA TGGTTGCTGA TATGAAAGCA
1381 GCGCAGGATA TGATGGATCG TGGCATCACC GCCATCGATA TCATCAAGGC TCTGTACAAT
1441 CACGGTTTCA AAGACGTTGC CGAAGCAATC CTGAACCTGC AGAAACAGAA AGTGGTTGGC
1501 GATTATCTGC AGACCTCTAG CATCTTCGAT AAGGACTGGA ACGTTACTTC CGCCGTGAAC
1561 GACGGTAACG ATTACCAGGG TCCGGGTACC GGCTACCGTC TGTATGAAGA CAAAGAAGAA
1621 TGGGACCGTA TTAAAGACCT GCCGTTCGCT CTGGATCCGG AACACCTGGA ACTG
利用软件对该基因序列进行分析,推知所述甘油脱水酶基因编码SEQ ID NO:2所示的氨基酸序列:
1 MKRQKRFEEL EKRPIHQDTF
21 VKEWPEEGFV AMMGPNDPKP
41 SVKVENGKIV EMDGKKLEDF
61 DLIDLYIAKY GINIDNVEKV
81 MNMDSTKIAR MLVDPNVSRD
101 EIIEITSALT PAKAEEIISK
121 LDFGEMIMAV KKMRPRRKPD
141 NQCHVTNTVD NPVQIAADAA
161 DAALRGFPEQ ETTTAVARYA
181 PFNAISILIG AQTGRPGVLT
201 QCSVEEATEL QLGMRGFTAY
221 AETISVYGTD RVFTDGDDTP
241 WSKGFLASCY ASRGLKMRFT
261 SGAGSEVLMG YPEGKSMLYL
281 EARCILLTKA SGVQGLQNGA
301 VSCIEIPGAV PNGIREVLGE
321 NLLCMMCDIE CASGCDQAYS
341 HSDMRRTERF IGQFIAGTDY
361 INSGYSSTPN YDNTFAGSNT
381 DAMDYDDMYV MERDLGQYYG
401 IHPVKEETII KARNKAAKAL
421 QAVFEDLGLP KITDEEVEAA
441 TYANTHDDMP KRDMVADMKA
461 AQDMMDRGIT AIDIIKALYN
481 HGFKDVAEAI LNLQKQKVVG
501 DYLQTSSIFD KDWNVTSAVN
521 DGNDYQGPGT GYRLYEDKEE
541 WDRIKDLPFA LDPEHLEL
产甘油脱水酶的乳酸杆菌(Lactobacillus reuteri)ZJB-09105,保藏于中国典型培养物保藏中心,地址:中国武汉武汉大学,430072,保藏日期:2009年09月27日,保藏编号:CCTCC NO:M 209213。
本发明还涉及一种含有所述甘油脱水酶基因的重组载体,以及用所述重组载体转化得到的重组基因工程菌。
本申请发明人根据测序结果设计表达引物3(GCCGAATTCATGAAACGCCAGAAACGCTTTGAAG)和引物4(AATAAGCTTCAGTTCCAGGTGTTCCGGATCCAGAG),以重组质粒pMD18-T-dhaB为模板,通过PCR扩增得到了用于表达的甘油脱水酶基因。本发明将甘油脱水酶基因同表达载体pET28b连接,构建了含有甘油脱水酶基因的表达重组质粒pET28b-dhaB。
将表达重组质粒pET28b-dhaB转化至大肠杆菌BL21菌株中,获得含有表达重组质粒pET28b-dhaB的重组大肠杆菌BL21/pET28b-dhaB,以重组菌为酶源,进行生物催化与转化。
将胞内表达重组质粒pTrc99a-NIT转化至大肠杆菌JM109菌株中,获得含有胞内表达重组质粒pTrc99a-NIT的重组大肠杆菌JM109/pTrc99a-NIT,或将分泌表达重组质粒pET20b-NIT转化至大肠杆菌BL21菌株中,获得含有分泌表达重组质粒pET20b-NIT的重组大肠杆菌BL21/pET20b-NIT。以重组菌为酶源,进行生物催化与转化。
本发明还涉及所述的甘油脱水酶基因在制备重组甘油脱水酶中的应用。
具体的,所述的应用为:构建含有所述甘油脱水酶基因的重组载体,将所述重组载体转化至大肠杆菌中,获得的重组基因工程菌进行诱导培养,培养液分离得到含有重组甘油脱水酶的菌体细胞。
本发明的有益效果主要体现在:提供了一种来源于乳酸杆菌(Lactobacillus reuteri)菌株中的甘油脱水酶基因核苷酸序列;并将该基因与表达载体连接构建得到含该基因的表达重组质粒pET28b-dhaB,再转化至大肠杆菌BL21中,获得含有表达重组质粒pET28b-dhaB的重组大肠杆菌BL21/pET28b-dhaB。可应用本发明构建的重组大肠杆菌为酶源进行生物催化与转化。重组甘油脱水酶作为转化用酶,分别以甘油、1,2-丙二醇或乙醇为底物,进行转化反应制备相应的3-羟基丙醛、丙醛或乙醛。
(四)附图说明
图1为克隆载体pMD18-T-dhaB物理图谱;
图2为pET28b-dhaB重组质粒物理图谱;
图3为甘油脱水酶基因PCR扩增argrose电泳图;1:DL2000DNAMarker;2~5:利用引物1和引物2扩增得到的甘油脱水酶基因片段;
图4为阳性重组质粒pET28b-dhaB的酶切结构图;1:DL2000 DNAMarker;2:甘油脱水酶基因片段;3:pET28b-dhaB/EcoR I样品;4:pET28b-dhaB/Hind III样品;5:pET28b-dhaB/EcoR I and Hind III样品;6:λDNA/Hind III DNA Marker;
图5为甘油脱水酶SDS-PAGE图;1:蛋白分子量Marker;2:E.coli BL21;3:未诱导的E.coli BL21/pET28b-dhaB;4:0.5mM IPTG诱导的E.coliBL21/pET28b-dhaB;5:0.1mM IPTG诱导的E.coli BL21/pET28b-dhaB;6:1mM IPTG诱导的E.coli BL21/pET28b-dhaB;
(五)具体实施方式
下面结合具体实施例对本发明进行进一步描述,但本发明的保护范围并不仅限于此:
实施例1:
用核酸快速提取仪提取乳酸杆菌ZJB-09105(Lactobacillus reuteriZJB-09105)菌株(CCTCC NO:M 209213)的总基因组DNA,以该基因组DNA为模板,在引物1(CAGTTCCAGGTGTTCCGG)、引物2(ATGAAACGCCAGAAACGCT)的作用下进行PCR扩增。
PCR反应体系各组分加入量(总体积50μL):10×Pfu DNA PolymeraseBuffer(TaKaRa)5μL,10mM dNTP mixture(dATP、dCTP、dGTP和dTTP各2.5mM)1μL,浓度为50μM的克隆引物1、引物2各1μL,基因组DNA1μL,Pfu DNA Polymerase(TaKaRa)1μL,无核酸水40μL。
采用Biorad的PCR仪,PCR反应条件为:预变性94℃5min,然后进入温度循环94℃30s,55℃1.5min,72℃2min,共35个循环,最后72℃延伸10min,终止温度为8℃。
取5μL PCR反应液用0.9%琼脂糖凝胶电泳检测。切胶回收该片段并纯化,利用Taq DNA聚合酶向片段5’端引入碱基A。在T4DNA连接酶作用下将该片段同T载体进行连接,得到克隆重组质粒pMD18-T-dhaB见图1。将该重组质粒电转化至大肠杆菌JM109中,利用篮白斑筛选系统进行筛选,随机挑取白色克隆测序,利用软件分析测序结果,结果表明:经引物1和引物2扩增到的核苷酸序列长度为1674bp(其核苷酸序列如SEQ ID NO:1所示),该序列编码一个完整的开放阅读框。
实施例2:
根据实施例1分析结果设计表达引物3(GCCGAATTCATGAAACGCCAGAAACGCTTTGAAG)和引物4(AATAAGCTTCAGTTCCAGGTGTTCCGGATCCAGAG),并分别在引物3和引物4中引入了EcoRI和HindIII限制性酶切位点。在引物3和引物4的引发下,利用高保真Pyrobest DNA聚合酶进行扩增,获得长为1674bp的甘油脱水酶基因片段,测序后利用EcoRI和HindIII限制性内切酶对扩增片段进行处理,并利用T4DNA连接酶将该片段同用相同的限制性内切酶处理的表达载体pET28b进行连接,构建表达载体pET28b-dhaB。将构建的表达载体pET28b-dhaB电转化至大肠杆菌BL21中,涂平板37℃下培养过夜,随机挑取克隆抽提质粒进行酶切鉴定,鉴定结果见图4。
实施例3:
将实施例2验证后的含有表达重组质粒pET28b-dhaB的重组大肠杆菌BL21/pET28b-dhaB用含有50μg/ml卡那霉素抗性的LB液体培养基培养12h,再以体积比1%接种量接种到新鲜的含有50μg/ml卡那霉素抗性的LB液体培养基中,培养至菌体浓度OD600约0.6左右,再向LB液体培养基加入终浓度为0.5mM的IPTG,诱导培养8h后,4℃、5000rpm离心10min,收集含有重组甘油脱水酶的菌体细胞。
实施例4:
以实施例3中获得的含有表达重组质粒pET28b-dhaB的重组大肠杆菌BL21/pET28b-dhaB湿菌体作为转化用酶,以甘油为底物,进行转化反应制备3-羟基丙醛。转化体系及转化操作如下:在50ml转化瓶中加入1g湿菌体和底物浓度为1%的10ml反应液,37℃下,150r/min转化30mn,离心去除菌体,上清液中含有3-羟基丙醛。
采用显色法来检测产物3-羟基丙醛的浓度,取1ml上清液与0.75mlD,L-色氨酸试剂、3ml浓HCl(37.2%)混匀,37℃水浴20min后,在560nm测定OD值。酶活单位(U)定义为:在37℃条件下,1min内催化甘油生成1μmol 3-羟基丙醛所需要的酶量定义为1U。甘油脱水酶的酶活以每mg干菌体所含有的酶活力单位表示(U/mg)。根据体系中3-羟基丙醛的生成量推知重组菌酶活。测定结果见表1。
表1:以重组大肠杆菌BL21/pET28b-dhaB为酶源测定的甘油脱水酶酶活力测定结果
菌株/质粒 | 酶活(U/mg(wet cells)) |
大肠杆菌BL21 | 0 |
大肠杆菌BL21/pET28b | 0 |
大肠杆菌BL21/pET28b-dhaB-1 | 1.46 |
大肠杆菌BL21/pET28b-dhaB-2 | 1.39 |
结论:由上述实验结果可知,将本发明甘油脱水酶基因转化大肠杆菌得到重组大肠杆菌具有较强产甘油脱水酶能力,可直接以含酶的菌体细胞为酶源进行生物催化或转化反应。重组甘油脱水酶作为转化用酶,可以分别以以甘油、1,2-丙二醇或乙醇为底物,进行转化反应制备相应的3-羟基丙醛、丙醛或乙醛等。
SEQUENCE LISTING
<110>浙江工业大学
<120>一种甘油脱水酶基因、载体、工程菌及其应用
<130>
<160>6
<170>PatentIn version 3.4
<210>1
<211>1674
<212>DNA
<213>Lactobacillus reuteri
<400>1
atgaaacgcc agaaacgctt tgaagaactg gaaaaacgtc cgatccacca ggacaccttc 60
gtaaaagagt ggccggaaga aggctttgtg gcaatgatgg gcccgaacga tccgaaaccg 120
agcgttaaag tggaaaacgg caaaattgtg gaaatggacg gcaaaaagct ggaagatttc 180
gatctgatcg atctgtacat tgcaaaatac ggcattaaca ttgataacgt tgaaaaagta 240
atgaacatgg attccaccaa aatcgcgcgt atgctggtag acccgaacgt gtcccgtgac 300
gaaattattg aaatcacctc tgctctgact ccggctaaag cggaagagat cattagcaaa 360
ctggattttg gtgaaatgat catggcagta aaaaagatgc gtccgcgtcg taagccagac 420
aaccagtgcc acgttaccaa caccgtagac aatcctgtac agatcgctgc ggatgctgcg 480
gacgcggctc tgcgtggctt tccggaacag gaaaccacga ccgctgttgc acgctacgct 540
ccattcaacg ccatttccat cctgatcggt gctcagaccg gtcgcccggg tgttctgact 600
cagtgctctg tcgaagaggc caccgaactg cagctgggta tgcgcggttt tactgcctac 660
gcagaaacca ttagcgtgta cggcaccgac cgcgttttca ccgatggtga cgacaccccg 720
tggagcaagg gctttctggc gagctgttat gcctcccgtg gtctgaaaat gcgttttacc 780
tctggtgcgg gttccgaagt cctgatgggc tacccggaag gtaaatccat gctgtacctg 840
gaagcccgtt gtatcctgct gaccaaagca agcggtgttc aaggcctgca gaatggtgcg 900
gtgtcctgta ttgaaatccc tggcgctgtt cctaacggta tccgtgaagt gctgggcgaa 960
aacctgctgt gcatgatgtg tgacatcgaa tgtgcatccg gttgtgatca ggcttattct 1020
cactctgata tgcgtcgcac cgagcgtttt atcggtcagt ttatcgcggg taccgactac 1080
atcaactccg gctattcttc tactccgaat tacgacaaca ctttcgcagg cagcaacacg 1140
gatgctatgg actacgatga catgtacgta atggagcgtg acctgggtca atactatggt 1200
attcatccgg ttaaagagga gactattatc aaagcacgta acaaagctgc gaaagctctg 1260
caggctgttt tcgaggacct gggcctgcca aaaatcaccg atgaagaagt agaagctgct 1320
acgtacgcga acacccacga tgacatgccg aaacgtgata tggttgctga tatgaaagca 1380
gcgcaggata tgatggatcg tggcatcacc gccatcgata tcatcaaggc tctgtacaat 1440
cacggtttca aagacgttgc cgaagcaatc ctgaacctgc agaaacagaa agtggttggc 1500
gattatctgc agacctctag catcttcgat aaggactgga acgttacttc cgccgtgaac 1560
gacggtaacg attaccaggg tccgggtacc ggctaccgtc tgtatgaaga caaagaagaa 1620
tgggaccgta ttaaagacct gccgttcgct ctggatccgg aacacctgga actg 1674
<210>2
<211>558
<212>PRT
<213>Lactobacillus reuteri
<400>2
Met Lys Arg Gln Lys Arg Phe Glu Glu Leu Glu Lys Arg Pro Ile His
1 5 10 15
Gln Asp Thr Phe Val Lys Glu Trp Pro Glu Glu Gly Phe Val Ala Met
20 25 30
Met Gly Pro Asn Asp Pro Lys Pro Ser Val Lys Val Glu Asn Gly Lys
35 40 45
Ile Val Glu Met Asp Gly Lys Lys Leu Glu Asp Phe Asp Leu Ile Asp
50 55 60
Leu Tyr Ile Ala Lys Tyr Gly Ile Asn Ile Asp Asn Val Glu Lys Val
65 70 75 80
Met Asn Met Asp Ser Thr Lys Ile Ala Arg Met Leu Val Asp Pro Asn
85 90 95
Val Ser Arg Asp Glu Ile Ile Glu Ile Thr Ser Ala Leu Thr Pro Ala
100 105 110
Lys Ala Glu Glu Ile Ile Ser Lys Leu Asp Phe Gly Glu Met Ile Met
115 120 125
Ala Val Lys Lys Met Arg Pro Arg Arg Lys Pro Asp Asn Gln Cys His
130 135 140
Val Thr Asn Thr Val Asp Asn Pro Val Gln Ile Ala Ala Asp Ala Ala
145 150 155 160
Asp Ala Ala Leu Arg Gly Phe Pro Glu Gln Glu Thr Thr Thr Ala Val
165 170 175
Ala Arg Tyr Ala Pro Phe Asn Ala Ile Ser Ile Leu Ile Gly Ala Gln
180 185 190
Thr Gly Arg Pro Gly Val Leu Thr Gln Cys Ser Val Glu Glu Ala Thr
195 200 205
Glu Leu Gln Leu Gly Met Arg Gly Phe Thr Ala Tyr Ala Glu Thr Ile
210 215 220
Ser Val Tyr Gly Thr Asp Arg Val Phe Thr Asp Gly Asp Asp Thr Pro
225 230 235 240
Trp Ser Lys Gly Phe Leu Ala Ser Cys Tyr Ala Ser Arg Gly Leu Lys
245 250 255
Met Arg Phe Thr Ser Gly Ala Gly Ser Glu Val Leu Met Gly Tyr Pro
260 265 270
Glu Gly Lys Ser Met Leu Tyr Leu Glu Ala Arg Cys Ile Leu Leu Thr
275 280 285
Lys Ala Ser Gly Val Gln Gly Leu Gln Asn Gly Ala Val Ser Cys Ile
290 295 300
Glu Ile Pro Gly Ala Val Pro Asn Gly Ile Arg Glu Val Leu Gly Glu
305 310 315 320
Asn Leu Leu Cys Met Met Cys Asp Ile Glu Cys Ala Ser Gly Cys Asp
325 330 335
Gln Ala Tyr Ser His Ser Asp Met Arg Arg Thr Glu Arg Phe Ile Gly
340 345 350
Gln Phe Ile Ala Gly Thr Asp Tyr Ile Asn Ser Gly Tyr Ser Ser Thr
355 360 365
Pro Asn Tyr Asp Asn Thr Phe Ala Gly Ser Asn Thr Asp Ala Met Asp
370 375 380
Tyr Asp Asp Met Tyr Val Met Glu Arg Asp Leu Gly Gln Tyr Tyr Gly
385 390 395 400
Ile His Pro Val Lys Glu Glu Thr Ile Ile Lys Ala Arg Asn Lys Ala
405 410 415
Ala Lys Ala Leu Gln Ala Val Phe Glu Asp Leu Gly Leu Pro Lys Ile
420 425 430
Thr Asp Glu Glu Val Glu Ala Ala Thr Tyr Ala Asn Thr His Asp Asp
435 440 445
Met Pro Lys Arg Asp Met Val Ala Asp Met Lys Ala Ala Gln Asp Met
450 455 460
Met Asp Arg Gly Ile Thr Ala Ile Asp Ile Ile Lys Ala Leu Tyr Asn
465 470 475 480
His Gly Phe Lys Asp Val Ala Glu Ala Ile Leu Asn Leu Gln Lys Gln
485 490 495
Lys Val Val Gly Asp Tyr Leu Gln Thr Ser Ser Ile Phe Asp Lys Asp
500 505 510
Trp Asn Val Thr Ser Ala Val Asn Asp Gly Asn Asp Tyr Gln Gly Pro
515 520 525
Gly Thr Gly Tyr Arg Leu Tyr Glu Asp Lys Glu Glu Trp Asp Arg Ile
530 535 540
Lys Asp Leu Pro Phe Ala Leu Asp Pro Glu His Leu Glu Leu
545 550 555
<210>3
<211>18
<212>DNA
<213>Unknown
<220>
<223>人工序列
<400>3
<210>4
<211>19
<212>DNA
<213>Unknown
<220>
<223>人工序列
<400>4
atgaaacgcc agaaacgct 19
<210>5
<211>34
<212>DNA
<213>Unknown
<220>
<223>人工序列
<400>5
gccgaattca tgaaacgcca gaaacgcttt gaag 34
<210>6
<211>35
<212>DNA
<213>Unknown
<220>
<223>人工序列
<400>6
aataagcttc agttccaggt gttccggatc cagag 35
Claims (9)
1.一种甘油脱水酶基因,具有SEQ ID NO:1所示的核苷酸序列。
2.如权利要求1所述的甘油脱水酶基因基因,其特征在于所述甘油脱水酶基因基因编码SEQ ID NO:2所示的氨基酸序列。
3.如权利要求1所述的甘油脱水酶基因,其特征在于所述甘油脱水酶基因基因源自乳酸杆菌(Lactobacillus reuteri)CCTCC NO:M 209213。
4.乳酸杆菌(Lactobacillus reuteri)ZJB-09105,保藏于中国典型培养物保藏中心,地址:中国武汉武汉大学,430072,保藏日期:2009年09月27日,保藏编号:CCTCC NO:M 209213。
5.一种含有权利要求1所述甘油脱水酶基因的重组载体。
6.一种用权利要求5所述重组载体转化得到的重组基因工程菌。
7.如权利要求1所述的甘油脱水酶基因在制备重组甘油脱水酶中的应用。
8.如权利要求7所述的应用,其特征在于所述的应用为:构建含有所述甘油脱水酶基因的重组载体,将所述重组载体转化至大肠杆菌中,获得的重组基因工程菌进行诱导培养,培养液分离得到含有重组甘油脱水酶的菌体细胞。
9.如权利要求8所述的应用,其特征在于所述重组甘油脱水酶作为转化用酶,分别以甘油、1,2-丙二醇或乙醇为底物,进行转化反应制备相应的3-羟基丙醛、丙醛或乙醛。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN2009101533093A CN101671686B (zh) | 2009-10-15 | 2009-10-15 | 一种甘油脱水酶基因、载体、工程菌及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN2009101533093A CN101671686B (zh) | 2009-10-15 | 2009-10-15 | 一种甘油脱水酶基因、载体、工程菌及其应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN101671686A true CN101671686A (zh) | 2010-03-17 |
CN101671686B CN101671686B (zh) | 2012-06-27 |
Family
ID=42019105
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN2009101533093A Active CN101671686B (zh) | 2009-10-15 | 2009-10-15 | 一种甘油脱水酶基因、载体、工程菌及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN101671686B (zh) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101921785A (zh) * | 2010-07-05 | 2010-12-22 | 浙江工业大学 | 一种醛脱氢酶基因、载体、工程菌及其应用 |
CN109207500A (zh) * | 2018-09-29 | 2019-01-15 | 清华大学 | 一种不依赖辅酶b12的甘油脱水酶基因及其应用 |
CN110656076A (zh) * | 2019-09-25 | 2020-01-07 | 中国农业科学院油料作物研究所 | 一种原核工程菌株及其制备方法和应用 |
CN112625994A (zh) * | 2021-01-05 | 2021-04-09 | 清华大学 | 一种重组需钠弧菌及其应用 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1865431A (zh) * | 2006-04-07 | 2006-11-22 | 江南大学 | 产1,3-丙二醇工程菌及其构建和用该菌生产1,3-丙二醇的方法 |
-
2009
- 2009-10-15 CN CN2009101533093A patent/CN101671686B/zh active Active
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101921785A (zh) * | 2010-07-05 | 2010-12-22 | 浙江工业大学 | 一种醛脱氢酶基因、载体、工程菌及其应用 |
CN109207500A (zh) * | 2018-09-29 | 2019-01-15 | 清华大学 | 一种不依赖辅酶b12的甘油脱水酶基因及其应用 |
CN110656076A (zh) * | 2019-09-25 | 2020-01-07 | 中国农业科学院油料作物研究所 | 一种原核工程菌株及其制备方法和应用 |
CN112625994A (zh) * | 2021-01-05 | 2021-04-09 | 清华大学 | 一种重组需钠弧菌及其应用 |
CN112625994B (zh) * | 2021-01-05 | 2022-04-15 | 清华大学 | 一种重组需钠弧菌及其应用 |
Also Published As
Publication number | Publication date |
---|---|
CN101671686B (zh) | 2012-06-27 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN101921785A (zh) | 一种醛脱氢酶基因、载体、工程菌及其应用 | |
Montero-Barrientos et al. | Overexpression of a Trichoderma HSP70 gene increases fungal resistance to heat and other abiotic stresses | |
Elleuche et al. | Evolution of carbonic anhydrases in fungi | |
CN102978193B (zh) | 卤醇脱卤酶、编码基因、载体、菌株及应用 | |
CN101671686B (zh) | 一种甘油脱水酶基因、载体、工程菌及其应用 | |
Parta et al. | HYP1, a hydrophobin gene from Aspergillus fumigatus, complements the rodletless phenotype in Aspergillus nidulans | |
CN109576244B (zh) | 一种新型脂肪酶及其制备与应用 | |
Montero-Barrientos et al. | The heterologous overexpression of hsp23, a small heat-shock protein gene from Trichoderma virens, confers thermotolerance to T. harzianum | |
CN103614349A (zh) | 一种烯醇脱氢酶、编码基因、载体、工程菌及其应用 | |
CN104928310B (zh) | 甲硫氨酸裂解酶及其编码基因和生物合成方法 | |
Bowen et al. | Candidate effector gene identification in the ascomycete fungal phytopathogen Venturia inaequalis by expressed sequence tag analysis | |
CN109385412A (zh) | 一种高表达高活性多形拟杆菌肝素酶i融合蛋白及其编码基因和应用 | |
CN103468660A (zh) | 高活性中性植酸酶突变体及其基因和用途 | |
CN107365755A (zh) | 一种新型脂肪酶及其基因、工程菌和制备方法 | |
CN106591253B (zh) | 一种4-羟苯甲酸-聚异戊二烯转移酶突变体及其制备方法和应用 | |
CN104152472A (zh) | 一种双鸟苷酸环化酶基因、载体、工程菌及其应用 | |
CN108129556A (zh) | 水稻来源的金属镉结合蛋白及其编码基因和应用 | |
CN100355893C (zh) | 一种表达肝素酶的方法及其专用表达载体 | |
CN102086455B (zh) | 絮凝酵母絮凝基因、其表达产物及其用途 | |
Rafeeq et al. | Characterisation and comparative analysis of hydrophobin isolated from Pleurotus floridanus (PfH) | |
CN107841490B (zh) | 双功能亚甲基四氢叶酸脱氢酶/环化酶及其多克隆抗体 | |
CN102899330A (zh) | 栉孔扇贝TGF-β I型受体基因及其SNP位点 | |
CN102649950B (zh) | 一种突变的中性植酸酶及其基因和用途 | |
CN112359051A (zh) | 一种来源于三叶青的苯丙氨酸解氨酶基因ThPAL及其应用 | |
CN109161539A (zh) | 一种有机溶剂耐受性氨肽酶LapA及其制备方法和应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C14 | Grant of patent or utility model | ||
GR01 | Patent grant |