CN101709286B - Nitrile hydration enzyme gene engineering bacterium and application - Google Patents
Nitrile hydration enzyme gene engineering bacterium and application Download PDFInfo
- Publication number
- CN101709286B CN101709286B CN2009102101773A CN200910210177A CN101709286B CN 101709286 B CN101709286 B CN 101709286B CN 2009102101773 A CN2009102101773 A CN 2009102101773A CN 200910210177 A CN200910210177 A CN 200910210177A CN 101709286 B CN101709286 B CN 101709286B
- Authority
- CN
- China
- Prior art keywords
- nitrile hydratase
- gene engineering
- hydratase gene
- engineering bacterium
- liquid
- 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.)
- Expired - Fee Related
Links
- 238000006703 hydration reaction Methods 0.000 title claims abstract description 19
- 230000036571 hydration Effects 0.000 title claims abstract description 12
- 241000894006 Bacteria Species 0.000 title claims description 59
- 108090000790 Enzymes Proteins 0.000 title abstract description 32
- 150000002825 nitriles Chemical class 0.000 title abstract description 4
- 238000000034 method Methods 0.000 claims abstract description 67
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 claims abstract description 50
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 47
- NLHHRLWOUZZQLW-UHFFFAOYSA-N Acrylonitrile Chemical compound C=CC#N NLHHRLWOUZZQLW-UHFFFAOYSA-N 0.000 claims abstract description 18
- 101100152731 Arabidopsis thaliana TH2 gene Proteins 0.000 claims abstract description 11
- 241000187693 Rhodococcus rhodochrous Species 0.000 claims abstract description 8
- 108010024026 Nitrile hydratase Proteins 0.000 claims description 97
- 239000007788 liquid Substances 0.000 claims description 29
- 241001478240 Coccus Species 0.000 claims description 17
- 238000005406 washing Methods 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 10
- 229940041514 candida albicans extract Drugs 0.000 claims description 10
- 239000008103 glucose Substances 0.000 claims description 10
- 239000012138 yeast extract Substances 0.000 claims description 10
- 239000012528 membrane Substances 0.000 claims description 8
- 102000004169 proteins and genes Human genes 0.000 claims description 8
- 239000001888 Peptone Substances 0.000 claims description 6
- 108010080698 Peptones Proteins 0.000 claims description 6
- 239000012510 hollow fiber Substances 0.000 claims description 6
- 238000001471 micro-filtration Methods 0.000 claims description 6
- 244000005700 microbiome Species 0.000 claims description 6
- 235000019319 peptone Nutrition 0.000 claims description 6
- 238000000926 separation method Methods 0.000 claims description 6
- 238000000108 ultra-filtration Methods 0.000 claims description 6
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 4
- 101100400378 Mus musculus Marveld2 gene Proteins 0.000 claims description 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 4
- 230000001133 acceleration Effects 0.000 claims description 4
- 239000004202 carbamide Substances 0.000 claims description 4
- 238000004321 preservation Methods 0.000 claims description 4
- 230000004913 activation Effects 0.000 claims description 3
- 238000004140 cleaning Methods 0.000 claims description 3
- LPUQAYUQRXPFSQ-DFWYDOINSA-M monosodium L-glutamate Chemical compound [Na+].[O-]C(=O)[C@@H](N)CCC(O)=O LPUQAYUQRXPFSQ-DFWYDOINSA-M 0.000 claims description 3
- 235000013923 monosodium glutamate Nutrition 0.000 claims description 3
- 239000004223 monosodium glutamate Substances 0.000 claims description 3
- 238000010899 nucleation Methods 0.000 claims description 3
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 2
- 229910021529 ammonia Inorganic materials 0.000 claims description 2
- 238000011001 backwashing Methods 0.000 claims description 2
- 239000012560 cell impurity Substances 0.000 claims description 2
- 238000010790 dilution Methods 0.000 claims description 2
- 239000012895 dilution Substances 0.000 claims description 2
- 230000008030 elimination Effects 0.000 claims description 2
- 238000003379 elimination reaction Methods 0.000 claims description 2
- 102000004190 Enzymes Human genes 0.000 abstract description 30
- 238000004519 manufacturing process Methods 0.000 abstract description 17
- 230000000694 effects Effects 0.000 abstract description 5
- 239000013612 plasmid Substances 0.000 description 48
- 241000187654 Nocardia Species 0.000 description 45
- 210000004027 cell Anatomy 0.000 description 25
- 230000008859 change Effects 0.000 description 24
- 238000010276 construction Methods 0.000 description 18
- 101710143259 Nitrile hydratase subunit alpha Proteins 0.000 description 15
- 101710139480 Nitrile hydratase subunit beta Proteins 0.000 description 15
- 241000316848 Rhodococcus <scale insect> Species 0.000 description 14
- 239000000047 product Substances 0.000 description 14
- 230000006798 recombination Effects 0.000 description 13
- 238000005215 recombination Methods 0.000 description 13
- 241000588724 Escherichia coli Species 0.000 description 12
- 108010020856 N-terminal nucleophile hydrolase Proteins 0.000 description 12
- 230000008569 process Effects 0.000 description 11
- 230000008521 reorganization Effects 0.000 description 10
- 230000000813 microbial effect Effects 0.000 description 9
- 238000012360 testing method Methods 0.000 description 9
- 230000001580 bacterial effect Effects 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 8
- 108020004414 DNA Proteins 0.000 description 7
- 230000029087 digestion Effects 0.000 description 7
- 102000012410 DNA Ligases Human genes 0.000 description 6
- 108010061982 DNA Ligases Proteins 0.000 description 6
- 108091028043 Nucleic acid sequence Proteins 0.000 description 6
- 238000012408 PCR amplification Methods 0.000 description 6
- 108091081024 Start codon Proteins 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 239000000284 extract Substances 0.000 description 5
- 238000010353 genetic engineering Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 238000011144 upstream manufacturing Methods 0.000 description 5
- 238000001962 electrophoresis Methods 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 230000000968 intestinal effect Effects 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 231100000350 mutagenesis Toxicity 0.000 description 4
- 238000002703 mutagenesis Methods 0.000 description 4
- 231100000219 mutagenic Toxicity 0.000 description 4
- 230000003505 mutagenic effect Effects 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 108091008146 restriction endonucleases Proteins 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 238000012807 shake-flask culturing Methods 0.000 description 4
- 230000009466 transformation Effects 0.000 description 4
- NIXOWILDQLNWCW-UHFFFAOYSA-N Acrylic acid Chemical compound OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 3
- 241000233866 Fungi Species 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 239000006227 byproduct Substances 0.000 description 3
- 238000006555 catalytic reaction Methods 0.000 description 3
- 238000004520 electroporation Methods 0.000 description 3
- 238000000855 fermentation Methods 0.000 description 3
- 230000004151 fermentation Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000010369 molecular cloning Methods 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000011218 seed culture Methods 0.000 description 3
- 101100412856 Mus musculus Rhod gene Proteins 0.000 description 2
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 101100242191 Tetraodon nigroviridis rho gene Proteins 0.000 description 2
- 241001052560 Thallis Species 0.000 description 2
- 150000001408 amides Chemical class 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 241000186063 Arthrobacter Species 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000588807 Bordetella Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 241001006344 Microbacterium flavum Species 0.000 description 1
- 102000005431 Molecular Chaperones Human genes 0.000 description 1
- 108010006519 Molecular Chaperones Proteins 0.000 description 1
- 241000589180 Rhizobium Species 0.000 description 1
- 241000109365 Rosa arkansana Species 0.000 description 1
- 235000005066 Rosa arkansana Nutrition 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 239000003905 agrochemical Substances 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000005354 coacervation Methods 0.000 description 1
- 239000003245 coal Substances 0.000 description 1
- 229910001429 cobalt ion Inorganic materials 0.000 description 1
- XLJKHNWPARRRJB-UHFFFAOYSA-N cobalt(2+) Chemical compound [Co+2] XLJKHNWPARRRJB-UHFFFAOYSA-N 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- RTZKZFJDLAIYFH-UHFFFAOYSA-N ether Substances CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 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 1
- 239000003550 marker Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 238000005272 metallurgy Methods 0.000 description 1
- FQPSGWSUVKBHSU-UHFFFAOYSA-N methacrylamide Chemical compound CC(=C)C(N)=O FQPSGWSUVKBHSU-UHFFFAOYSA-N 0.000 description 1
- 238000005065 mining Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 229920002503 polyoxyethylene-polyoxypropylene Polymers 0.000 description 1
- 235000019260 propionic acid Nutrition 0.000 description 1
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000008707 rearrangement Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 239000009671 shengli Substances 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Images
Landscapes
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a nitrile hydration enzyme gene engineering strain of R.rhodochrous TH-2. The strain can effectively express a nitrile hydration enzyme and has high level of enzyme activity, good thermal stability and good tolerance to acrylonitrile and acrylamide; furthermore, the invention also discloses a method for producing the acrylamide by the gene engineering strain; and the obtained acrylamide produced by the method has high quality.
Description
The application is to be that September 20, application number in 2007 are the applying date: 200710122047.5, denomination of invention dividing an application for " a kind of construction process of nitrile hydratase gene engineering bacterium and engineering strain and application " application.
Technical field
The invention belongs to the industrial microbial technology field, relate in particular to a kind of method and resulting engineering strain and application that utilizes Nocardia bacteria-bacillus coli shuttle plasmid construction to efficiently express nitrile hydratase gene engineering bacterium.
Background technology
(Acrylamide, molecular formula is C to utilize acrylic amide
3H
5NO, structural formula are H
2C=CHCONH
2) the various types of SEPIGEL 305s of synthetic (PAM) have in industrial production such as TOR, water treatment, papermaking, mining, metallurgy, coal washing and manufacturing super absorbent resin very widely and to use.
The production of acrylic amide generally is to be raw material with the vinyl cyanide, carries out catalytic hydration and forms.Sulfuric acid hydration process, copper catalysis method, three developmental stage of microbial method have been experienced in the production of acrylic amide.Because microbial method has that reaction is carried out at normal temperatures and pressures, energy consumption is low, simple to operate, safety, vinyl cyanide transformation efficiency are high, production concentration and purity advantages of higher, with data by MoM and MEI, microbial method has remarkable advantages.
In the initial period of utilizing microbial method, mainly be that screening and domestication have the active bacterial strain of Nitrile hydratase.Major progress aspect mikrobe has: Japan; Study group of Hideaki Yamada discloses a kind of wild rose look rhodococcus J-1 in patent " method of biological production of amides " (patent No.: ZL88106735)); Nippon Chemical Ind is in patent " utilizing mikrobe to prepare the method for the acid amides " (patent No.: ZL86100062) disclose a kind of rhodococcus S-6, oxidation Arthrobacter and microbacterium flavum; Britain Ciba Special Chemical Water Treatment Co., Ltd. is at patent " prunosus red coccus bacterial strain NCIMB 41164 and the purposes of producing Nitrile hydratase thereof " (application number: disclose 200480035487.1) prunosus red coccus NCIMB 41164 bacterial strains or its two mutants a kind of; U.S. Ashland Licensing And Intellectual Property Ltd patent " cultural method of nitrile hydratase-producing strain rodococcus rhodochrous M 33 " (application number: 200480043243.8) " in a kind of rodococcus rhodochrous M 33 is disclosed, Shanghai City agricultural chemicals place patent " the microorganism catalysis method is produced acrylic amide " (application number: disclose different mikrobes such as wild propionic acid rod bacillus and naturalized strain thereof 03115536.7) and carried out the microbial method production of acrylic amide.Aspect working method; Chemical industry system of Tsing-Hua University discloses " method that acrylic amide is produced in a kind of microbial transformation of using membrane technique " (patent No.: ZL 03109806.1), " improving the directive breeding method of Nitrile hydratase product tolerance " (patent No.: ZL 03130658.6) and " glucose-Co
2+The process method of fermentative prepn Nitrile hydratase is added in coupling " (patent No.: ZL 03121944.6); Henan Jiaozuo Duoshengduo Chemical Co., Ltd. discloses " the mikrobe coacervation is produced the method for acrylic amide " (application number: 200410010364.4); Germany Stockhausen GmbH & Co. KG discloses " method for preparing a kind of acrylamide solution with a kind of biological catalyst " (application number: 02808905.7); " Fourier transform infrared on-line measurement vinyl cyanide and acrylic amide " or the like.Aspect the gene studies that transforms the acrylonitrile process acrylic amide, Mitsubishi change into Co., Ltd. to from the Nitrile hydratase gene of rhizobium with the albumen application patent " having the active novel protein of Nitrile hydratase and this proteic gene of encoding " (application number: 93106122.9); Mitsui Chemicals, Inc to from the albumen of the Nitrile hydratase of thermophilic false Nocardia bacteria JCM3095 and encode it the gene application patent " albumen of participating in activation of nitrile hydratase and its gene of the encoding " (patent No.: ZL99106291.4); " novel nitrile hydratase " (patent No.: 02156180.X), and studied the expression of this gene in recombination bacillus coli; Germany Degussa has applied for " Nitrile hydratase of Rhod " (application number: 200580008206.8); Mitsubishi Li Yang Co., Ltd. discloses " improved nitrile hydratase " (application number: 200580016665.0), provide and the Nitrile hydratase gene is carried out rite-directed mutagenesis improved stable on heating method; Tsing-Hua University clones from Nocardia bacteria and has obtained the Nitrile hydratase gene and applied for patent " a kind of Nitrile hydratase and encoding sox thereof and application " (patent No.: ZL 200410042576.0), further sudden change and the high expression level of this gene in recombination bacillus coli is studied.
Nocardia bacteria and Rhod have thicker cell walls in gram positive bacterial strain.Simultaneously; Also possibly there is some Chaperones Molecular in natural expression strain as nitrile metabolic enzyme system in Nocardia bacteria and the rhodococcus, or good redox environment; Make the interior Nitrile hydratase of expressing of born of the same parents compare, have higher thermostability and substrate, product tolerance with recombination bacillus coli.For this reason, the mistake that adopts Nocardia bacteria and rhodococcus host to carry out the Nitrile hydratase gene is expressed in has more wide prospect in the industrial production.
Summary of the invention
One of the object of the invention provides a kind of construction process that efficiently expresses the genetic engineering bacterium of Nitrile hydratase.
Two of the object of the invention provides the engineering strain that efficiently expresses Nitrile hydratase.
Three of the object of the invention provides the method for utilizing the nitrile hydratase gene engineering bacterium strain to produce acrylic amide.
A kind of construction process that efficiently expresses nitrile hydratase gene engineering bacterium of the present invention; Mainly be to utilize Nocardia bacteria-bacillus coli shuttle plasmid pNV18 and pNV19; The replication orgin pAL5000 that had both contained Nocardia bacteria/rhodococcus; Contain colibacillary replication orgin again, therefore both can in Nocardia bacteria/rhodococcus, express, again can be in the characteristics of expression in escherichia coli; Can in recombination bacillus coli, realize modified recombinant easily, be transferred to the expression of carrying out target gene in reorganization Nocardia bacteria/rhodococcus then foreign gene.
A kind of construction process that efficiently expresses nitrile hydratase gene engineering bacterium of the present invention, carry out according to following steps:
(1) according to the dna sequence dna design primer of purpose promoter gene, is that template is carried out the PCR method amplification, obtains promoter gene with the DNA that contains the purpose promoter gene;
(2) promoter gene and Nocardia bacteria-bacillus coli shuttle plasmid are carried out double digestion respectively, 36～38 ℃ of reactions are spent the night; Then enzyme is cut product purification, carry out ligation 14～16h with the T4DNA ligase enzyme at 3～5 ℃ again, get the ligation thing;
(3) the ligation thing is transformed host bacterium e.coli JM109 competent cell; Coating LB dull and stereotyped (kalamycin resistance gene kan+); Select positive colony; And cultivate, the plasmid of extraction a small amount of then carries out enzyme and cuts checking, can have the Nocardia bacteria-bacillus coli shuttle plasmid of purpose promoter gene;
(4), and be that template is carried out pcr amplification with total DNA of Nocardia bacteria Nocardia YS-2002 with original Nitrile hydratase gene according to Nitrile hydratase gene order design upstream and downstream primer, the dna sequence dna of Nitrile hydratase; Carry out double digestion with EcoRI and BamHI then, with the T4 ligase enzyme Nitrile hydratase dna sequence dna is inserted escherichia coli plasmid pET28 again, obtain recombinant plasmid pET28-NHase; With test kit rite-directed mutagenesis method the α subunit initiator codon gtg of Nitrile hydratase gene is sported atg again, obtain sudden change Nitrile hydratase gene NHase
M
(5) the sudden change Nitrile hydratase gene NHase that obtains with step (4)
MDna sequence dna be that template is carried out pcr amplification, obtain sudden change Nitrile hydratase gene NHase
MDna sequence dna, then with BamHI and HindIII to sudden change Nitrile hydratase gene NHase
MCarry out double digestion with Nocardia bacteria-bacillus coli shuttle plasmid, 36～38 ℃ of reactions are spent the night; Then enzyme is cut product purification, carry out ligation 14-16h with the T4DNA ligase enzyme at 3～5 ℃ again, get the ligation thing;
(6) step (5) ligation thing is transformed host bacterium E.coli JM109 competent cell; Be coated on then on LB flat board (Kan+) substratum; Select positive colony; And in the LB substratum 37 ℃ of incubated overnight, extract in a small amount plasmid then and carry out enzyme and cut checking, can have purpose promoter gene and sudden change Nitrile hydratase gene NHase
MShuttle plasmid;
(7) with step (6) gained shuttle plasmid with electroporation conversion method transformed host cell, promptly obtain to have purpose promoter gene and sudden change Nitrile hydratase gene NHase
MGenetic engineering bacterium.
Purpose promoter gene described in the above-mentioned construction process step (1) is Ntn hydrolase promoter gene or intestinal bacteria tac promoter gene.
Ntn hydrolase promoter gene described in the above-mentioned construction process, its-35 district is respectively with-10 region sequences: TTGTGG and TAACGT; It is from Nocardia bacteria Nocardia YS-2002; With upstream primer CGGAATTCTGCGGACGGCGGATACGT and downstream primer CGGGATCCCTAGGACTCCTTAGTGACT, be that template is carried out pcr amplification and can be got the Ntn hydrolase promoter gene with the DNA of Nocardia bacteria Nocardia YS-2002.
Intestinal bacteria tac promoter gene described in the above-mentioned construction process, its-35 district is respectively with-10 region sequences: TTGACA and TATAAT.Intestinal bacteria tac promoter gene is from plasmid pMAL-p2x (U.S. NEB company); With upstream primer CGGAATTCATTCTCATGTTTGACAGCT and downstream primer CGGGATCCGGTCCTTG TTGGTGAAGT is amplimer, is that template is carried out pcr amplification and can be got intestinal bacteria tac promoter gene with the DNA of plasmid pMAL-p2x.
Nocardia bacteria described in the above-mentioned construction process step (2)-bacillus coli shuttle plasmid is Nocardia bacteria-rhodococcus shuttle plasmid pNV18 or pNV19 (pNV18:DDBJ DB, AB267085; The pNV19:DDBJ DB, AB267086) and the plasmid of deriving.
The described plasmid of deriving can be pNV18.1 etc.
The purifying that the middle enzyme of above-mentioned construction process step (2) is cut product can adopt the PCR product to reclaim purification process commonly used in test kit or other molecular cloning.
Nitrile hydratase gene described in the above-mentioned construction process step (4) be meant come from nocardial original Nitrile hydratase gene (Genbank:AY168347) or obtain through methods such as sudden changes, rearrangement, disappearance with from the consensus dna sequence property of nocardial Nitrile hydratase gene more than or equal to 70%, the protein sequence consistence is more than or equal to 70% homologous gene.
Test kit rite-directed mutagenesis method (ExSite described in the above-mentioned construction process step (4)
TMPCR-basedsite-directed mutagenesis kit, Stratagene, USA) see Yue SHI etc. (Enzyme andMicrobial Technology, 2004,35 (6-7), 557-562); Can the α subunit initiator codon gtg of Nitrile hydratase gene be sported atg, thereby obtain sudden change Nitrile hydratase gene NHase
M
Sudden change Nitrile hydratase base described in the above-mentioned construction process step (4) is meant that α subunit initiator codon gtg sports the Nitrile hydratase gene of atg.
The Nocardia bacteria of the purpose promoter gene described in the above-mentioned construction process step (3)-bacillus coli shuttle plasmid is to have the pNV18 plasmid of Ntn hydrolase promoter gene or tac promoter gene and the plasmid of deriving thereof.
Shuttle plasmid described in the above-mentioned construction process step (6) is for having Ntn hydrolase promoter gene or tac promoter gene and sudden change Nitrile hydratase gene NHase
MThe pNV18 plasmid and the plasmid of deriving thereof.
Host described in the above-mentioned construction process step (7) is Nocardia bacteria or prunosus red coccus.
The described Nocardia bacteria of above-mentioned host is Nocardia bacteria Nocardia YS-2002 or its mutagenic fungi; Wherein Nocardia bacteria Nocardia YS-2002 is that the contriver obtains through screening from the bacterial strain from the Shengli Oil Field collection; The mutagenic fungi of Nocardia YS-2002 can adopt Nocardia YS-2002 under uv lamp, to add LiCl
2Method is carried out mutagenic obtained.
The described prunosus red coccus of above-mentioned host is prunosus red coccus Rhodococcus rhodochrous ATCC33278 (in American Type Culture Collecti's preservation) or its domestication strain or mutagenic fungi.
The genetic engineering bacterium that acquisition described in the above-mentioned construction process step (7) has purpose promotor and sudden change Nitrile hydratase gene can be promise card bordetella gene engineering bacteria or prunosus red coccus genetic engineering bacterium.
Utilize the nitrile hydratase gene engineering bacterium strain of above-mentioned construction process gained, carry tac promoter gene or Ntn hydrolase promoter gene and sudden change Nitrile hydratase gene NHase
M
Sudden change Nitrile hydratase gene described in the above-mentioned nitrile hydratase gene engineering bacterium strain is meant that α subunit initiator codon gtg sports the Nitrile hydratase gene of atg.
Tac promoter gene and sudden change Nitrile hydratase gene NHase are carried in above-mentioned nitrile hydratase gene engineering bacterium strain
M
Above-mentioned nitrile hydratase gene engineering bacterium strain is Nocardia bacteria TH-1; Nocardia; Be deposited in Chinese microorganism strain management committee common micro-organisms preservation center on July 12nd, 2007; The culture presevation registration number is CGMCC No.2104, and it has tac promotor and sudden change Nitrile hydratase gene NHase
M
Above-mentioned nitrile hydratase gene engineering bacterium strain is prunosus red coccus TH-2; R.rhodochrous; Be deposited in Chinese microorganism strain management committee common micro-organisms preservation center on July 12nd, 2007; The culture presevation registration number is CGMCC No.2105, and it has tac promotor and sudden change Nitrile hydratase gene NHase
M
Ntn hydrolase promoter gene and sudden change Nitrile hydratase gene NHase are carried in above-mentioned nitrile hydratase gene engineering bacterium strain
M
Above-mentioned nitrile hydratase gene engineering bacterium strain is Nocardia bacteria Nocardia/pNV-Pa-NH
M, it has Ntn hydrolase promotor and sudden change Nitrile hydratase gene NHase
M
Above-mentioned nitrile hydratase gene engineering bacterium strain is prunosus red coccus rhodochrous/pNV-Pa-NH
M, it has Ntn hydrolase promotor and sudden change Nitrile hydratase gene NHase
M
Utilize above-mentioned nitrile hydratase gene engineering bacterium to produce the method for acrylic amide, carry out according to following steps:
(1) will insert in the activation medium at the nitrile hydratase gene engineering bacterium of 4 ℃ of preservations, be to cultivate 32～56 hours under the condition of 150～250 commentaries on classics/min at 28～30 ℃, shaking speed, obtains the bacterium liquid of activatory nitrile hydratase gene engineering bacterium; The proportion of composing of wherein said substratum is: glucose 20～30g/L, yeast extract paste 1～5g/L, peptone 7～10g/L, KH
2PO
40.5～0.75g/L, K
2HPO
40.5～0.75g/L, MgSO
47H
2O 0.5～1.0g/L, pH7.5, all the other are water;
(2) be inoculated in the seeding tank that substratum is housed according to 0.5～1.0% volume percent nitrile hydratase gene engineering bacterium liquid step (1); Be to cultivate 32～48 hours under the condition of 200～300 commentaries on classics/min at 28～30 ℃, shaking speed, the seed liquor of nitrile hydratase gene engineering bacterium; The proportion of composing of described substratum is: glucose 20～30g/L, yeast extract paste 1～5g/L, peptone 7～10g/L, KH
2PO
40.5～0.75g/L, K
2HPO
40.5～0.75g/L, MgSO
47H
2O 0.5～1.0g/L, bubble enemy 0.2～0.3g/L, pH7.5, all the other are water;
(3) according to 5.0～10.0% volume percent step (2) gained seed liquor is changeed and is inoculated in the fermentor tank that substratum is housed, under 28～30 ℃, pH7.5～8.5 conditions, cultivated 36～48 hours, fermented liquid; The proportion of composing of said substratum is: glucose 20～40g/L, yeast extract paste 5.0～10.0g/L, urea 7.5～10.0g/L, KH
2PO
40.5～1.0g/L, K
2HPO
40.5～1.0g/L, MgSO
47H
2O 0.5～1.5g/L, monosodium glutamate 0.5～1.5g/L, CoCl
20.04～0.12mM, acrylic amide 2～6mM, bubble enemy 0.2～0.3g/L, pH 7.5～8.5, and all the other are water;
(4) be the bacterium liquid of 5～8g/L (dry weight) with the fermented liquid dilution; With hollow fiber microfiltration membrane the thalline of gathering in the crops is carried out the micro-filtration washing then; Adopt during cleaning just washing with the two-way repeatable operation of backwash be 5～20 until the colourity of washings, protein concn is 3～10ppm; Just washing pressure-controlling is 0.06～0.1MPa, and backwash pressure is controlled to be 0.01～0.1MPa; Again bacterium liquid is made into 10～20g/L (dry weight), volume(tric)fraction 5～8% is squeezed into the hydration reaction still, regulates vinyl cyanide flow to 0.5～1.0m
3/ h, stream adds vinyl cyanide in the hydration still then, and flow acceleration is progressively turned down from high to low; Reacting kettle jacketing leads to-5～-9 ℃ of chilled brines or liquefied ammonia, at 16～26 ℃, carries out hydration reaction 1～3 hour under pH6.7～7.5 conditions; Then aqua liquid is squeezed into the hollow fiber ultrafiltration membrane separation system with recycle pump; With just washing with back-washing method repeatable operation elimination aqua liquid in cell impurity and foreign protein; Colourity until aqua liquid is 5～15, and protein concn is 5～15ppm, obtains the acrylic amide aqua liquid.
Nitrile hydratase gene engineering bacterium described in the method steps of above-mentioned production acrylic amide (1) is meant Nocardia bacteria Nocardia/pNV-Pa-NH
M, Nocardia bacteria TH-1, prunosus red coccus R.rhodochrous/pNV-Pa-NH
MOr prunosus red coccus TH-2.
Bubble enemy described in the method for above-mentioned production acrylic amide is the polyoxyethylene polyoxypropylene glyceryl ether, can buy from market.
In the step in the method for above-mentioned production acrylic amide (2) when a large amount of seed cell forms be chain when spherical, finish seed culture, living weight is about 3～6g/L.
Be single coccoid to thalli morphology in the step in the method for above-mentioned production acrylic amide (3), living weight >=8g/L, Nitrile hydratase enzyme work >=1850U/ml.
Nitrile hydratase gene engineering bacterium cell described in the method steps of above-mentioned production acrylic amide (4) can be reused 3～8 times.
Adopting when cleaning in the method for above-mentioned production acrylic amide and just washing and the two-way repeatable operation of backwash, is to stop up in order to prevent.
Work as acrylamide concentration in the method steps of above-mentioned production acrylic amide (4) and reach 25%～40% (W%V), acrylonitrile concentration during vinylformic acid by product≤0.5% (W%V), can stop hydration reaction less than 0.1% (W%V).
It is host strain that the present invention adopts Nocardia bacteria or rhodococcus; Adopt Nocardia bacteria/rhodococcus-bacillus coli shuttle plasmid high expression level Nitrile hydratase gene; The sudden change transformation of screening and optimizing and Nitrile hydratase gene through promoter gene on the shuttle plasmid improves activity, thermostability and product, the substrate tolerance of recombinant bacterial strain Nitrile hydratase; Adopting recombination Nocardia bacteria or recombination rhodococcus free cell is catalyzer, can realize the microbial method production of bulk chemical acrylic amide.
Advantage of the present invention and beneficial effect: (1) gained nitrile hydratase gene engineering bacterium of the present invention strain Nitrile hydratase expression level is high; (2) Nitrile hydratase enzyme running water of the present invention is flat high, and Heat stability is good is to vinyl cyanide and acrylic amide better tolerance; (3) the acrylic amide quality of the inventive method production is high, is suitable for industrialized production.
Description of drawings
Fig. 1 carries the Nitrile hydratase of tac promotor (Ptac) and expresses shuttle plasmid pNV-Ptac-NH
MSynoptic diagram.
Fig. 2 carries the Nitrile hydratase of Ntn hydrolase promotor (Pa) and expresses shuttle plasmid pNV-Pa-NH
MSynoptic diagram.
Embodiment
Carry the shuttle plasmid pNV18-Ptac-NH of tac promotor
MStructure
With plasmid pMAL-p2x (U.S. NEB company) is that template is carried out pcr amplification, and upstream primer is CG
GAATTCATTCTCATGTTTGACAGCT (underscore partly is the EcoRI restriction enzyme site) and downstream primer are CG
GGATCCGGTCCTTG TTGGTGAAGT (underscore partly is the BamHI restriction enzyme site) obtains containing the dna sequence dna of tac promotor (35 districts with-10 region sequences are: TTGACA and TATAAT); With EcoR I and BamH I double digestion tac promoter gene and shuttle plasmid pNV18,37 ℃ of reactions are spent the night; Enzyme is cut product reclaim the test kit purifying, adopt the T4DNA ligase enzyme to carry out ligation 14h then at 4 ℃ with the PCR product; The ligation product is transformed the competent cell of host bacterium E.coli JM109, select positive colony, in the LB substratum, extract in a small amount plasmid after 37 ℃ of incubated overnight and carry out that enzyme is cut and the electrophoresis checking, obtain containing the shuttle plasmid of tac promotor; The shuttle plasmid that contains the tac promotor with BamHI and HindIII double digestion gained; Connect inserting α subunit initiator codon gtg with the T4DNA ligase enzyme sports the Nitrile hydratase gene of atg (mutation method is seen Yue SHI etc., Enzyme and MicrobialTechnology, 2004; 35 (6-7); 557-562), the competent cell of Transformed E .coli JM109 is selected positive colony; In the LB substratum, extract in a small amount plasmid after 37 ℃ of incubated overnight and carry out that enzyme is cut and the electrophoresis checking, verify that correct shuttle plasmid is the sudden change Nitrile hydratase genetic expression shuttle plasmid pNV-Ptac-NH that carries the tac promotor
M(see figure 1).
Embodiment 2
Carry the shuttle plasmid pNV18-Pa-NH of Ntn hydrolase promotor (Pa)
MStructure
Total DNA with the Nocardia bacteria Nocardia YS-2002 of contriver laboratory acclimation and screening (being kept at Tsing-Hua University's chemical industry is public institute's bioinformation of bioid and biocatalysis engineering experiment chamber) is that template is carried out pcr amplification, upstream primer CG
GAATTCTGCGGACGGCGGATACGT (underscore partly is the EcoRI restriction enzyme site) and downstream primer CG
GGATCCCTAGGACTCCTTAGTGACT (underscore partly is the BamHI restriction enzyme site), the dna sequence dna of acquisition Ntn hydrolase promoter gene Pa; With EcoR I and BamH I double digestion Pa and shuttle plasmid pNV18,37 ℃ of reactions are spent the night; After enzyme cut product and reclaim the test kit purifying with the PCR product, adopt the T4 dna ligases to carry out ligation 14h at 4 ℃.The ligation thing is transformed host bacterium E.coli JM109 competent cell, and coating LB dull and stereotyped (Kan+) selects positive colony, carries out shake-flask culture 12h.Harvested cell extracts in a small amount plasmid and carries out that enzyme is cut and the electrophoresis checking, must contain the plasmid of pa; Contain the plasmid of pa, be connected with the T4DNA ligase enzyme and insert α subunit initiator codon gtg and sport the Nitrile hydratase gene of atg (mutation method is seen Yue SHI etc. with BamHI and HindIII double digestion; Enzyme and Microbial Technology, 2004,35 (6-7); 557-562); The competent cell of Transformed E .coli JM109 is selected positive colony, extracts in a small amount plasmid behind the shake-flask culture and carries out enzyme once more and cut with electrophoresis and verify.Verify that correct shuttle plasmid is the sudden change Nitrile hydratase genetic expression shuttle plasmid pNV-Pa-NH that carries the Pa promotor
M(see figure 2).
Embodiment 3
The structure of recombination Nocardia bacteria TH-1
Have the tac promotor and suddenly change Nitrile hydratase gene high expression shuttle plasmid pNV-Ptac-NH what embodiment 1 made up
MCompetent cell with electroporation conversion Nocardia bacteria Nocardia YS-2002 can get recombination Nocardia bacteria TH-1.Wherein, the preparation method of Gram-positive bacterium competence cell in " molecular cloning guide " (J. Sa nurse Brooker, D.W. La Saier work) is adopted in the preparation of Nocardia bacteria competent cell.Get plasmid behind the 1 μ l purifying in the centrifuge tube of a 1.5ml, the electric revolving cup of itself and 0.1CM is placed precooling on ice together; The competent cell that 50 μ l are prepared shifts in the centrifuge tube of 1.5ml so far, and careful mixing is placed 10min on ice; Open electroporation, regulating voltage is 1250V; The mixture of shuttle plasmid and competent cell is transferred in the electric revolving cup of precooling, knocks gently and make mixture evenly get into the bottom of electric revolving cup, note in the mixture bubble being arranged simultaneously.Electric revolving cup is put into electric conversion instrument, presses shock button, hear buzzer after, in cup, add rapidly the SOC liquid nutrient medium (prescription is seen " molecular cloning guide ") of 800 μ l, behind the re-suspended cell, transfer in the centrifuge tube of 1.5ml.Place 28 ℃, the 220rpm shaking table is cultivated 2h; Get 200 μ l bacterium liquid and coat that to contain that antibiotic LB solid medium of 20 μ g/ml cards dull and stereotyped, put into 28 ℃ of incubators and cultivate and occur the nocardial single bacterium colony of reorganization after 60～72 hours, recombination Nocardia bacteria Nocardia/pNV-Ptac-NH
M, bacterial strain is by name: TH-1.
Embodiment 4
The structure of recombination rhodococcus TH-2
With embodiment 1 gained pNV-Ptac-NH
MShuttle plasmid is with fax hole method transformed host cell rhodococcus R.rhodochrous ATCC 33278, and wherein electric method for transformation obtains recombination rhodococcus R.rhodochrous/pNV-Ptac-NH with embodiment 3
M, bacterial strain is by name: TH-2.
Embodiment 5:
The expression test of Nocardia bacteria TH-1 Nitrile hydratase
Adopt the triangle of 500ml band baffle plate to shake bottle; The amount of dress substratum is 10%; 28 ℃ of shaking table batch culture, shaking speed is 200rpm with embodiment 3 gained Nocardia bacteria TH-1 and Nocardia bacteria Nocardia YS-2002 (being called for short " wild bacterium "), cultivates 92 hours.The proportion of composing of said substratum is: glucose 20g/L, yeast extract paste 5g/L, urea 7.5g/L, KH
2PO
40.5g/L, K
2HPO
40.5g/L, MgSO
47H
2O0.5g/L, bubble enemy 0.2g/L, the pH value is 7.5, divalent cobalt ion 40ppm, all the other are water; The result shows, adopts the nocardial growing state of reorganization and the original strain basically identical of tac promoter expression Nitrile hydratase, and enzyme expression alive is then different.Fermentation culture 48 hours, the enzyme work of reorganization bacterium reached 2939U/ml, and wild bacterium enzyme is lived and is 2711U/ml, and the work of reorganization bacterium enzyme has improved 8.4% than wild bacterium; Fermentation culture 72 hours, reorganization bacterium enzyme is lived (3331U/ml) than wild bacterium (2838U/ml) raising 17.4%.Continue fermentation culture to 92 hour, the work of reorganization bacterium enzyme is up to 4015U/ml.Wherein, the gc marker method of employing standard is surveyed in the enzyme biopsy of Nitrile hydratase, and the iu that enzyme is lived is defined as: PM catalysis generates the required enzyme amount of 1 μ mol acrylic amide; 1 μ mol acrylic amide/(min.ml bacterium liquid)=1U/ml.
Embodiment 6
The test of prunosus red coccus TH-2 Nitrile hydratase expression of gene
Embodiment 4 gained recombination prunosus red coccus TH-2 are carried out the 72h shake-flask culture; And be contrast with original wild strain R.rhodochrous ATCC 33278; Under 28 ℃, shaking speed 200 commentaries on classics/min conditions, cultivated 48 hours, the proportion of composing of substratum is: glucose 20g/L, yeast extract paste 5g/L; Peptone 7g/L, KH
2PO
40.5g/L, K
2HPO
40.5g/L, MgSO
47H
2O 0.5g/L, pH7.5, USAF RH-1 0.2g/L, all the other are water.The result shows that the Nitrile hydratase activity of wild prunosus red coccus is very low, and total enzyme work is merely 49U/ml, but 352U/ml is brought up in total enzyme work of reorganization prunosus red coccus TH-2, has improved about 7.2 times than wild strain.
Embodiment 7,
Utilize Nocardia bacteria TH-1 to produce the test of acrylic amide
Smooth, moistening single bacterium colony on the picking Nocardia bacteria TH-1 solid plate is seeded in the 50ml seed culture medium in (500ml shakes bottle), cultivates 48h for 28 ℃; (proportion of composing of said substratum is shaking speed 200 commentaries on classics/min: glucose 20g/L; Yeast extract paste 5g/L, peptone 10g/L, KH
2PO
40.5g/L, K
2HPO
40.5g/L, MgSO
47H
2O 0.5g/L, pH7.5, all the other are water), get activatory Nocardia bacteria TH-1; Be that 0.5% inoculum size is inoculated in the 500L seeding tank with volume ratio then; Mixing speed 250 commentaries on classics/min cultivate 40hr for 28 ℃, and it is spherical that the seed cell form becomes chain; Finishing seed culture (substratum is formed the same), is that 10% inoculum size switching liquid amount is 3 tons 5m with volume ratio
3(proportion of composing of fermention medium is fermentor tank: glucose 30g/L, yeast extract paste 5.0g/L, urea 7.5g/L, KH
2PO
40.75g/L, K
2HPO
40.75g/L, MgSO
47H
2O 1.0g/L, monosodium glutamate 0.75g/L, CoCl
20.06mM, acrylic amide 4mM, bubble enemy 0.2g/L, pH 7.5, all the other are water), when being cultured to 40h under 28 ℃ of conditions, thalli morphology is single coccoid, and living weight is 10g/L, and the Nitrile hydratase enzyme is lived and is 2780U/ml.
Stop to cultivate, 3 tons of fermented liquids are diluted to 4 tons, adopt the microfiltration membrane washed cell, just washing the about 0.08MPa of pressure pressure, the about 0.09Mpa of backwash pressure; According to 5% volume(tric)fraction preparation cell suspending liquid, the enzyme activity of resuspended liquid is about 2300U/ml.Regulate initial vinyl cyanide flow 1.0m
3/ h, stream adds vinyl cyanide in the hydration still, and flow acceleration is progressively turned down from high to low; Reacting kettle jacketing leads to-5 ℃ of chilled brines, guarantees that hydration temperature maintains about 20 ℃, and pH7.5 measures acrylamide concentration and reaches 30% (W%V) after 2 hours, finish hydration reaction.Adopt recycle pump that acrylamide solution is squeezed into the hollow fiber ultrafiltration membrane separation system and carry out ultra-filtration and separation, the result obtains 30% acrylamide solution, and outward appearance is as clear as crystal; Through detecting foreign protein content is 8ppm, and colourity is 10, acrylonitrile concentration 0.01% (W%V), and vinylformic acid by-product concentration 0.1% is high-quality acrylic amide aqua product.
After the genetic engineering bacterium free cell used 6 times repeatedly, the remarkable inactivation of Nitrile hydratase was with carrying out waste treatment after cell centrifugation, the collection.
Embodiment 8
Utilize prunosus red coccus TH-2 to produce the test of acrylic amide
According to embodiment 7 described methods, single bacterium colony of picking recombination prunosus red coccus TH-2 carries out shake-flask culture, seed tank culture and fermentor cultivation, obtains the reorganization rhodococcus of 12g/L, and the Nitrile hydratase enzyme is lived and is 690U/ml.Results reorganization rhodococcus free cell, according to embodiment 7 described methods, after the micro-filtration washing, catalytic hydration acrylonitrile process acrylic amide.According to 10% volume(tric)fraction preparation cell suspending liquid, the enzyme activity of resuspended liquid is about 1000U/ml.Regulate initial vinyl cyanide flow 1.0m
3/ h, stream adds vinyl cyanide in the hydration still, and flow acceleration is progressively turned down from high to low.Reacting kettle jacketing leads to-5 ℃ of chilled brines, guarantees that hydration temperature maintains about 20 ℃, and pH7.5 measures acrylamide concentration and reaches 20% (W%V) after 2 hours, finish hydration reaction.Adopt recycle pump that acrylamide solution is squeezed into the hollow fiber ultrafiltration membrane separation system and carry out ultra-filtration and separation; The result obtains 20% acrylamide solution, and outward appearance is as clear as crystal, and foreign protein content is 15ppm; Colourity is 20; Acrylonitrile concentration 0.08% (W%V), vinylformic acid by-product concentration 0.4% is qualified acrylic amide aqua product.
Claims (2)
1. nitrile hydratase gene engineering bacterium strain; It is characterized in that described engineering strain is prunosus red coccus (Rhodococcus rhodochrous) TH-2; Be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, preservation registration number is CGMCC No.2105.
2. the method for utilizing the described nitrile hydratase gene engineering bacterium strain of claim 1 to produce acrylic amide, carry out according to following steps:
(1) will insert in the activation medium in the nitrile hydratase gene engineering bacterium strain of 4 ℃ of preservations, be to cultivate 32～56 hours under the condition of 150～250 commentaries on classics/min at 28～30 ℃, shaking speed, obtains the bacterium liquid of activatory nitrile hydratase gene engineering bacterium; The proportion of composing of wherein said substratum is: glucose 20～30g/L, yeast extract paste 1～5g/L, peptone 7～10g/L, KH
2PO
40.5～0.75g/L, K
2HPO
40.5～0.75g/L, MgSO
47H
2O 0.5～1.0g/L, pH7.5, all the other are water;
(2) be inoculated in the seeding tank that substratum is housed according to 0.5～1.0% volume percent nitrile hydratase gene engineering bacterium liquid step (1); Be to cultivate 32～48 hours under the condition of 200～300 commentaries on classics/min at 28～30 ℃, shaking speed, the seed liquor of nitrile hydratase gene engineering bacterium; The proportion of composing of described substratum is: glucose 20～30g/L, yeast extract paste 1～5g/L, peptone 7～10g/L, KH
2PO
40.5～0.75g/L, K
2HPO
40.5～0.75g/L, MgSO
47H
2O 0.5～1.0g/L, pH7.5, all the other are water;
(3) according to 5.0～10.0% volume percent step (2) gained seed liquor is changeed and is inoculated in the fermentor tank that substratum is housed, under 28～30 ℃, pH7.5～8.5 conditions, cultivated 36～48 hours, fermented liquid; The proportion of composing of said substratum is: glucose 20～40g/L, yeast extract paste 5.0～10.0g/L, urea 7.5～10.0g/L, KH
2PO
40.5～1.0g/L, K
2HPO
40.5～1.0g/L, MgSO
47H
2O 0.5～1.5g/L, monosodium glutamate 0.5～1.5g/L, CoCl
20.04～0.12mM, acrylic amide 2～6mM, bubble enemy 0.2～0.3g/L, pH 7.5～8.5, and all the other are water;
(4) be the bacterium liquid of 5～8g/L with fermented liquid dilution, with hollow fiber microfiltration membrane the thalline of results carried out the micro-filtration washing then, adopt during cleaning just washing with the two-way repeatable operation of backwash be 5～20 until the colourity of washings, protein concn is 3～10ppm; Just washing pressure-controlling is 0.06～0.1MPa, and backwash pressure is controlled to be 0.01～0.1MPa; Again bacterium liquid is made into 10～20g/L, volume(tric)fraction 5～8% is squeezed into the hydration reaction still, regulates vinyl cyanide flow to 0.5～1.0m
3/ h, stream adds vinyl cyanide in the hydration still then, and flow acceleration is progressively turned down from high to low; Reacting kettle jacketing leads to-5～-9 ℃ of chilled brines or liquefied ammonia, at 16～26 ℃, carries out hydration reaction 1～3 hour under pH6.7～7.5 conditions; Then aqua liquid is squeezed into the hollow fiber ultrafiltration membrane separation system with recycle pump; With just washing with back-washing method repeatable operation elimination aqua liquid in cell impurity and foreign protein; Colourity until aqua liquid is 5～15, and protein concn is 5～15ppm, obtains the acrylic amide aqua liquid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009102101773A CN101709286B (en) | 2007-09-20 | 2007-09-20 | Nitrile hydration enzyme gene engineering bacterium and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009102101773A CN101709286B (en) | 2007-09-20 | 2007-09-20 | Nitrile hydration enzyme gene engineering bacterium and application |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2007101220475A Division CN101186911B (en) | 2007-09-20 | 2007-09-20 | Method for constructing nitrile hydratase gene engineering bacterium, genetic engineering strain and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101709286A CN101709286A (en) | 2010-05-19 |
| CN101709286B true CN101709286B (en) | 2012-05-23 |
Family
ID=42402091
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2009102101773A Expired - Fee Related CN101709286B (en) | 2007-09-20 | 2007-09-20 | Nitrile hydration enzyme gene engineering bacterium and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101709286B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102329744B (en) * | 2010-07-13 | 2014-04-23 | 中国环境科学研究院 | Heterotrophs nitrobacteria, biosensor comprising heterotrophs nitrobacteria and method for detecting water body toxicity |
| CN104498466B (en) * | 2014-12-11 | 2017-07-07 | 浙江大学 | nitrile hydratase and its application |
| CN106148312A (en) * | 2015-04-15 | 2016-11-23 | 四川光亚聚合物化工有限公司 | The stably method of storage Production of Acrylamide by Microbial Method fermentation broth enzyme activity |
| ITUA20163572A1 (en) * | 2016-05-18 | 2017-11-18 | Columbia S R L | BIOTECHNOLOGICAL METHOD FOR THE PRODUCTION OF ACRYLAMIDE AND ITS NEW BACTERIAL STRAIN |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1886502A (en) * | 2003-12-02 | 2006-12-27 | 西巴特殊化学水处理有限公司 | Strain of rhodococcus rhodochrous ncimb 41164 and its use asproducer of nitrile hydratase |
-
2007
- 2007-09-20 CN CN2009102101773A patent/CN101709286B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1886502A (en) * | 2003-12-02 | 2006-12-27 | 西巴特殊化学水处理有限公司 | Strain of rhodococcus rhodochrous ncimb 41164 and its use asproducer of nitrile hydratase |
Non-Patent Citations (1)
| Title |
|---|
| 史悦等.微生物法生产丙烯酰胺的研究(I)———腈水合酶产生菌株的培养和高活力的表达.《生物工程学报》.2002,第17卷(第1期),55-58. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101709286A (en) | 2010-05-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101663389B (en) | An amidase gene knock-out engineered strain for nitrile hydratase production, its construction and application | |
| CN106119322B (en) | A kind of zymotechnique improving recombination human source collagen production level | |
| CN101186911B (en) | Method for constructing nitrile hydratase gene engineering bacterium, genetic engineering strain and application thereof | |
| CN105296456B (en) | A kind of stability-enhanced glutamic acid decarboxylase enzyme mutant of pH and its application | |
| CN107354188A (en) | The technique of ETEC JL GlcN fermenting and producing N acetylglucosamines | |
| CN102643770B (en) | Escherichia coli for producing succinic acid by utilizing pure anaerobic growth of synthetic culture medium and application thereof | |
| CN105368766B (en) | One plant of method for producing the genetic engineering bacterium of pentanediamine and its preparing pentanediamine | |
| CN109988739A (en) | The method that one-step method efficiently prepares small molecule chondroitin sulfate and micromolecule hyaluronic acid | |
| CN100334200C (en) | Glutamic acid capable of having high-yield glutamine | |
| CN110373359A (en) | A kind of streptomyces albus X-18 and the method using bacterium production epsilon-polylysine | |
| CN112322556B (en) | High-salt environment-resistant staphylococcus nepalensis and culture method | |
| CN106434510A (en) | Genetically engineered bacterium for producing L-aspartic acid through fermentation | |
| CN101709286B (en) | Nitrile hydration enzyme gene engineering bacterium and application | |
| CN110484471A (en) | The method that one plant height produces the acidproof bacterial strain of bacteria cellulose and its produces bacteria cellulose | |
| CN102994430B (en) | Bacterial cellulose production strain and application thereof | |
| CN105177084B (en) | A kind of method of inulin enzyme mutant fermenting and producing oligofructose | |
| CN101463358A (en) | Nitrile hydratase gene cluster and use thereof | |
| CN103642854A (en) | Method for producing succinic acid by immobilized corynebacterium glutamicum and repeated batch fermentation | |
| CN103060244B (en) | Bacillus marinus and method for producing catalase by using same | |
| CN104805144A (en) | Method for producing L-citrulline with high efficiency | |
| CN110066757B (en) | Pseudomonas capable of producing feruloyl esterase and application thereof | |
| CN105175275B (en) | A kind of isolation and purification method of L ornithine | |
| CN104630195A (en) | Marine microorganism fermentation production method for low temperature gamma-lactamase | |
| CN101643712A (en) | Escherichia coli strain for efficiently converting glutamine to synthesize L-theanine and application thereof | |
| CN110423711B (en) | Low-temperature chitinase-producing strain from Antarctic and fermentation method thereof |
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 | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120523 Termination date: 20150920 |
|
| EXPY | Termination of patent right or utility model |