CN101054570A - Preparation for L-homophenylalanine by immobilization recombination of E.coliBL21-pET/aspC cell - Google Patents
Preparation for L-homophenylalanine by immobilization recombination of E.coliBL21-pET/aspC cell Download PDFInfo
- Publication number
- CN101054570A CN101054570A CN200710021346.XA CN200710021346A CN101054570A CN 101054570 A CN101054570 A CN 101054570A CN 200710021346 A CN200710021346 A CN 200710021346A CN 101054570 A CN101054570 A CN 101054570A
- Authority
- CN
- China
- Prior art keywords
- cell
- aspc
- hyperphenylalaninemia
- pet
- acid
- 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
- 101150005925 aspC gene Proteins 0.000 title claims abstract description 38
- 101100492609 Talaromyces wortmannii astC gene Proteins 0.000 title claims abstract description 36
- 101150116772 aatA gene Proteins 0.000 title claims abstract description 36
- 101150100742 dapL gene Proteins 0.000 title claims abstract description 36
- 238000002360 preparation method Methods 0.000 title claims description 13
- JTTHKOPSMAVJFE-VIFPVBQESA-N L-homophenylalanine Chemical compound OC(=O)[C@@H](N)CCC1=CC=CC=C1 JTTHKOPSMAVJFE-VIFPVBQESA-N 0.000 title 1
- 230000006798 recombination Effects 0.000 title 1
- 238000005215 recombination Methods 0.000 title 1
- 210000001822 immobilized cell Anatomy 0.000 claims abstract description 35
- 238000000034 method Methods 0.000 claims abstract description 35
- 241000588724 Escherichia coli Species 0.000 claims abstract description 32
- 210000004027 cell Anatomy 0.000 claims abstract description 21
- 229960002989 glutamic acid Drugs 0.000 claims abstract description 18
- 238000002156 mixing Methods 0.000 claims abstract description 18
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims abstract description 6
- 239000011261 inert gas Substances 0.000 claims abstract description 3
- 238000006243 chemical reaction Methods 0.000 claims description 58
- 239000004372 Polyvinyl alcohol Substances 0.000 claims description 33
- 229920002451 polyvinyl alcohol Polymers 0.000 claims description 33
- 238000003756 stirring Methods 0.000 claims description 30
- MIPFGRYMMYSKKF-UHFFFAOYSA-N C(=O)=C(C(=O)O)CCC1=CC=CC=C1 Chemical compound C(=O)=C(C(=O)O)CCC1=CC=CC=C1 MIPFGRYMMYSKKF-UHFFFAOYSA-N 0.000 claims description 20
- 239000000725 suspension Substances 0.000 claims description 20
- 241000894006 Bacteria Species 0.000 claims description 17
- 239000003431 cross linking reagent Substances 0.000 claims description 17
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 16
- 235000010413 sodium alginate Nutrition 0.000 claims description 16
- 239000000661 sodium alginate Substances 0.000 claims description 16
- 229940005550 sodium alginate Drugs 0.000 claims description 16
- 238000001816 cooling Methods 0.000 claims description 15
- 238000010438 heat treatment Methods 0.000 claims description 15
- 230000008961 swelling Effects 0.000 claims description 15
- 230000008521 reorganization Effects 0.000 claims description 13
- 239000004094 surface-active agent Substances 0.000 claims description 9
- CKLJMWTZIZZHCS-UHFFFAOYSA-N D-OH-Asp Natural products OC(=O)C(N)CC(O)=O CKLJMWTZIZZHCS-UHFFFAOYSA-N 0.000 claims description 4
- CKLJMWTZIZZHCS-UWTATZPHSA-N L-Aspartic acid Natural products OC(=O)[C@H](N)CC(O)=O CKLJMWTZIZZHCS-UWTATZPHSA-N 0.000 claims description 4
- 229960005261 aspartic acid Drugs 0.000 claims description 4
- 229920000136 polysorbate Polymers 0.000 claims description 4
- 230000008569 process Effects 0.000 claims description 4
- UKVHQGHRJIEIML-UHFFFAOYSA-L calcium boric acid dichloride Chemical compound [Cl-].[Cl-].[Ca+2].OB(O)O UKVHQGHRJIEIML-UHFFFAOYSA-L 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 2
- 238000005406 washing Methods 0.000 claims description 2
- 239000002994 raw material Substances 0.000 claims 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 abstract description 28
- 239000005515 coenzyme Substances 0.000 abstract description 7
- 102000004190 Enzymes Human genes 0.000 abstract description 4
- 108090000790 Enzymes Proteins 0.000 abstract description 4
- 239000012295 chemical reaction liquid Substances 0.000 abstract description 4
- 238000000926 separation method Methods 0.000 abstract description 2
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 abstract 3
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 abstract 3
- 230000003834 intracellular effect Effects 0.000 abstract 2
- AXLLOSUYAVXOIN-UHFFFAOYSA-N 2-oxo-3-phenylbutanoic acid Chemical compound OC(=O)C(=O)C(C)C1=CC=CC=C1 AXLLOSUYAVXOIN-UHFFFAOYSA-N 0.000 abstract 1
- 238000001914 filtration Methods 0.000 abstract 1
- 239000007788 liquid Substances 0.000 abstract 1
- 239000002736 nonionic surfactant Substances 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 37
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 28
- 229910052757 nitrogen Inorganic materials 0.000 description 14
- 239000002504 physiological saline solution Substances 0.000 description 14
- 230000001105 regulatory effect Effects 0.000 description 14
- 239000011324 bead Substances 0.000 description 13
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 13
- 229920000053 polysorbate 80 Polymers 0.000 description 13
- 230000009466 transformation Effects 0.000 description 10
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 239000001110 calcium chloride Substances 0.000 description 9
- 229910001628 calcium chloride Inorganic materials 0.000 description 9
- 230000000052 comparative effect Effects 0.000 description 7
- 108010003415 Aspartate Aminotransferases Proteins 0.000 description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 239000000654 additive Substances 0.000 description 4
- 230000000996 additive effect Effects 0.000 description 4
- -1 nitrite compound Chemical class 0.000 description 4
- NGVDGCNFYWLIFO-UHFFFAOYSA-N pyridoxal 5'-phosphate Chemical compound CC1=NC=C(COP(O)(O)=O)C(C=O)=C1O NGVDGCNFYWLIFO-UHFFFAOYSA-N 0.000 description 4
- 102000004625 Aspartate Aminotransferases Human genes 0.000 description 3
- XPCFTKFZXHTYIP-PMACEKPBSA-N Benazepril Chemical compound C([C@@H](C(=O)OCC)N[C@@H]1C(N(CC(O)=O)C2=CC=CC=C2CC1)=O)CC1=CC=CC=C1 XPCFTKFZXHTYIP-PMACEKPBSA-N 0.000 description 3
- 108010061435 Enalapril Proteins 0.000 description 3
- 239000002220 antihypertensive agent Substances 0.000 description 3
- 229960004530 benazepril Drugs 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 229960000873 enalapril Drugs 0.000 description 3
- GBXSMTUPTTWBMN-XIRDDKMYSA-N enalapril Chemical compound C([C@@H](C(=O)OCC)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(O)=O)CC1=CC=CC=C1 GBXSMTUPTTWBMN-XIRDDKMYSA-N 0.000 description 3
- 150000002148 esters Chemical class 0.000 description 3
- 238000001179 sorption measurement Methods 0.000 description 3
- WEQPBCSPRXFQQS-UHFFFAOYSA-N 4,5-dihydro-1,2-oxazole Chemical compound C1CC=NO1 WEQPBCSPRXFQQS-UHFFFAOYSA-N 0.000 description 2
- 239000005541 ACE inhibitor Substances 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-M Aminoacetate Chemical compound NCC([O-])=O DHMQDGOQFOQNFH-UHFFFAOYSA-M 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 241000447394 Desmos Species 0.000 description 2
- 206010020772 Hypertension Diseases 0.000 description 2
- 108010007859 Lisinopril Proteins 0.000 description 2
- 229920001214 Polysorbate 60 Polymers 0.000 description 2
- 229940044094 angiotensin-converting-enzyme inhibitor Drugs 0.000 description 2
- 229940127088 antihypertensive drug Drugs 0.000 description 2
- 229960000830 captopril Drugs 0.000 description 2
- FAKRSMQSSFJEIM-RQJHMYQMSA-N captopril Chemical compound SC[C@@H](C)C(=O)N1CCC[C@H]1C(O)=O FAKRSMQSSFJEIM-RQJHMYQMSA-N 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 229960002394 lisinopril Drugs 0.000 description 2
- RLAWWYSOJDYHDC-BZSNNMDCSA-N lisinopril Chemical compound C([C@H](N[C@@H](CCCCN)C(=O)N1[C@@H](CCC1)C(O)=O)C(O)=O)CC1=CC=CC=C1 RLAWWYSOJDYHDC-BZSNNMDCSA-N 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 235000007682 pyridoxal 5'-phosphate Nutrition 0.000 description 2
- 239000011589 pyridoxal 5'-phosphate Substances 0.000 description 2
- 229960001327 pyridoxal phosphate Drugs 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000010257 thawing Methods 0.000 description 2
- 102000015427 Angiotensins Human genes 0.000 description 1
- 108010064733 Angiotensins Proteins 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 1
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 229940030600 antihypertensive agent Drugs 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 229960005025 cilazapril Drugs 0.000 description 1
- HHHKFGXWKKUNCY-FHWLQOOXSA-N cilazapril Chemical compound C([C@@H](C(=O)OCC)N[C@@H]1C(N2[C@@H](CCCN2CCC1)C(O)=O)=O)CC1=CC=CC=C1 HHHKFGXWKKUNCY-FHWLQOOXSA-N 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000010924 continuous production Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000006352 cycloaddition reaction Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 239000003168 generic drug Substances 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 230000001631 hypertensive effect Effects 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 150000007530 organic bases Chemical class 0.000 description 1
- 238000006146 oximation reaction Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 229960004084 temocapril Drugs 0.000 description 1
- FIQOFIRCTOWDOW-BJLQDIEVSA-N temocapril Chemical compound C([C@@H](C(=O)OCC)N[C@@H]1C(N(CC(O)=O)C[C@H](SC1)C=1SC=CC=1)=O)CC1=CC=CC=C1 FIQOFIRCTOWDOW-BJLQDIEVSA-N 0.000 description 1
- 238000005891 transamination reaction Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
Images
Abstract
The present invention discloses a method for preparing L-high phenylalanine using immobilized recombinant E.coli BL21-pET/aspC cell. The method is: mixing evenly of alpha-oxo-phenylbutyric acid and L-glutamic acid or L-aspartate, adjusting pH to 7.0-9.5, adding nonionic surfactant to prepare reaction liquid, adding immmobilized cell in reaction liquid and reacting 12-24hours under 30-50 degree and inert gas protection, suspending liquid being obtained, adjusting pH, filtering to obtain L-high phenylalanine. The inventive immobilization style promotes the stability of intracellular enzyme, reduces the intracellular coenzyme loss, simplifies the separation process. The immobilized cell has good mechanical intensity, tenacity. The water-absorbing dilatability is prominently improved and easily operable. The yield of L-high phenylalanine is promoted dramatically.
Description
Technical field
The present invention relates to the amino acid whose method of immobilization reorganization E.coli BL21-pET/aspC cell preparation, relate to specifically and will contain the reorganization E.coli BL21-pET/aspC cell fixation of aspartate aminotransferase, substrate alpha-carbonyl benzenebutanoic acid is changed into the method for L-hyperphenylalaninemia by transamination.
Background technology
L-hyperphenylalaninemia and ester thereof are the important source material that is used for preparing Angiotensin (ACE) inhibitor class medicine.It is the common intermediate of present about in the world 20 kinds of antihypertensive agents, for example Enalapril (enalapril), Benazepril (benazepril), Lisinopril (lisinopril), Captopril (captopril), Temocapril, Cilazapril (Yipingshu) etc.Whole world hypertensive patient to 2006 year can surpass 6.5 hundred million.Huge patient colony and high growth rate make that the antihypertensive drug market growth impetus is very obvious.Global hypertension drug market will reach 38,500,000,000 dollars in 2006, will be above 52,000,000,000 dollars to market capacity expectation in 2007.The patent aspect, enalapril (Merk company) expired in 2000, and benazepril (Ciba-Geigy company) expired in 2002, and other patent also expired before 2005.ACE inhibitor is as generic drugs in 5 years from now on, and ratio shared in antihypertensive drug market is bigger, accounts for 25% in dollar.Domestic a lot of pharmacy corporations are all copied ACE inhibitor class medicine in exploitation at present, thereby the demand of L-hyperphenylalaninemia and ester thereof will sharply increase.
The method of the existing L-of preparation hyperphenylalaninemia mainly is chemical synthesis (CN1361098A), step is as follows: (a) will carry out oximation reaction under the additive salt of glycinate or glycinate and the nitrite compound low temperature, produce N-hydroxyl imide base chloracetic acid ester (Cloroximidoacetate Ester); (b) in the presence of organic bases, N-hydroxyl imide base chloracetic acid ester and vinylbenzene are carried out cycloaddition reaction generation isoxazoline (Isoxazoline); (c) in the presence of chiral catalyst, pressure hydration is obtained chipal compounds L-hyperphenylalaninemia.
Summary of the invention
The purpose of this invention is to provide a kind of method with immobilization reorganization E.coli BL21-pET/aspC cell continuous production L-hyperphenylalaninemia, improved the stability of desmo enzyme, reduced the loss of internal cell coenzyme, simplified separating technology, make the L-hyperphenylalaninemia prepare productive rate and significantly improve, reduce production costs.
Purpose of the present invention can reach by following measure:
A kind of method with immobilization reorganization E.coli BL21-pET/aspC cell preparation L-hyperphenylalaninemia; with alpha-carbonyl benzenebutanoic acid and L-L-glutamic acid or L-aspartic acid mixing; regulate pH to 7.0~9.5; add nonionic surface active agent and be made into reaction solution; the immobilization E.coli BL21-pET/aspC cell of recombinating is added in the reaction solution; 30~50 ℃ of reaction 4~24h under protection of inert gas, white suspension, regulate pH filter the L-hyperphenylalaninemia.
Alpha-carbonyl benzenebutanoic acid and L-L-glutamic acid are 1: 1~1.6 mixed in molar ratio, immobilized cell (being immobilization reorganization E.coli BL21-pET/aspC cell) is 1%~5% with the mass volume ratio of reaction solution, and nonionic surface active agent and reaction solution mass volume ratio are 0.1%~0.6%.
Above-mentioned nonionic surface active agent is preferably polysorbate--and tween (Tween) series of surfactants, optimum is Tween-20, Tween-40, Tween-60, Tween-65, Tween8-0, Tween-85.
In the said fixing cell immobilized microorganism cells be the reorganization E.coli BL21-pET/aspC that contains aspartate aminotransferase.
The preparation method of reorganization E.coli BL21-pET/aspC sees patent CN1757737A.
The concrete preparation method of reorganization E.coli BL21-pET/aspC is:
Aspartate aminotransferase aspC gene is transferred from intestinal bacteria K12 (reference culture).By the NCBI retrieval, as follows through VectorNTI8.0 software design primer according to e. coli k12 (reference culture) the aspC sequence of report:
Forward primer: 5`ATGTTTGAGAACATTACCGC3`
Anti-narrow-necked earthen jar primer: 5`GTTTGTCATCAGTCTCAGCC3`.
Adopt PCR to angle the aspartate aminotransferase gene aspC that gets e. coli k12.
Aspartate aminotransferase gene aspC links to each other with following constitutive promoter (being designated as P123), and (ruling as follows in the change base position of this promotor) obtains P123-aspC.
TGTTGTGTGGAATTGTGAGCGGATTGCAATTTCACACA
ACAACACACCTTAACACTCGCCTA
ACGTTAAAGTGTGTP
And P123-aspC is inserted in the pMD-18T of the TaKaRa company carrier, identify angling the aspC gene of getting to carry out order-checking.Utilize the HindIII and the EcoR I site double digestion of T carrier to obtain the new segment P123-aspC that has restriction enzyme site.Be inserted among the carrier PET22b after same enzyme is cut.Obtain recombinant plasmid pET/aspC.Preparation E.coli BL21 competent cell imports recombinant plasmid pET/aspC among the E.coli BL21, selects recon E.coli BL21-pET/aspC.
The present invention uses hydrochloric acid and NaOH solution to regulate the pH value.
The process for fixation of immobilized cell is: with polyvinyl alcohol swelling 24h, add sodium alginate, the Autoclave heating is fully dissolved it, stirs while hot.Bacteria suspension is mixed with gac, treat that polyvinyl alcohol cooling back stirs with it.Be added drop-wise in the cross-linking agent solution with syringe, form the bead of certain diameter, and soak 3-12h (refrigerator cold-storage spends the night) therein.With the physiological saline washing, refrigerator is preserved, and is used for transforming.
In immobilized cell by mass ratio, polyvinyl alcohol: sodium alginate: microorganism cells (weight in wet base): gac=8~12: 0.1~1: 10~30: 5~20; Cross-linking agent solution is borax-calcium chloride solution, boric acid-calcium chloride solution or boric acid-borax-calcium chloride solution.
Preferred method for transformation is: get a certain amount of alpha-carbonyl benzenebutanoic acid, add L-Glu in molar ratio, mixing is regulated pH to 8.5, adds 0.4% tween-80.Add immobilized cell.The inflated with nitrogen protection places 40 ℃, 120rpm shaking table, reaction 20h.Get white suspension.
With the white suspension that obtains, acidifying removes by filter insolubles, and re-adjustment pH to 5.5 obtains the L-hyperphenylalaninemia solid of white.Be specially immobilized cell is taken out from reaction solution, add hydrochloric acid at reaction solution and regulate below the pH to 1, remove by filter the insolubles in the reaction solution, add NaOH and regulate pH to 5.5, separate out white insolubles in the reaction solution and be the L-hyperphenylalaninemia, filter promptly.
The invention is characterized in the immobilization reorganization E.coli BL21-pET/aspC cell processes, mix with bacteria suspension with gac earlier.With the polyvinyl alcohol mixing embedding of adding sodium alginate, drop to borax-calcium chloride solution and solidify again, because gac has certain adsorption to the coenzyme pyridoxal phosphate, adopt this method, the stability of coenzyme is significantly improved than additive method.In L-hyperphenylalaninemia preparation process, adopting cheap L-L-glutamic acid is amino donor, adds the Tweens tensio-active agent, makes that the product form is trickle crystal, and productive rate significantly improves.
The present invention adopts immobilized mode, has improved the physical strength of immobilized cell, has improved the stability of desmo enzyme, reduce the loss of internal cell coenzyme, simplified separating technology, considered immobilized cell balling-up effect, physical strength, the transformation period of immobilized cell can reach 16 days (batch).The immobilized cell of the present invention's preparation, physical strength is good, and toughness is big, and water-swelling significantly improves, easy handling.L-hyperphenylalaninemia productive rate improves significantly.
The present invention has following advantage:
1. contrast chemical synthesis, the enzyme reaction mild condition only needs normal temperature and pressure.
2.L-hyperphenylalaninemia solubleness under reaction conditions is low, adopts the immobilized cell preparation, the separation of product is simple.
3. the present invention adds activated carbon in immobilization process, because gac adopts this method to the adsorption of coenzyme pyridoxal phosphate, the immobilized stability of coenzyme is significantly improved than additive method, batch significantly increases than additive method.
4. to adopt cheap L-L-glutamic acid be amino donor in the present invention, L-aspartic acid relatively, and cost will reduce.
5. the present invention adds 0.4% tween-80 in reaction process, makes product improve the adsorption effect of immobilized cell particle, and mass transfer effect strengthens, and productive rate is original 135.28%.
Description of drawings
Fig. 1 is various embodiments of the present invention and Comparative Examples immobilization transformation period and average conversion comparison diagram.
Embodiment
Embodiment 1:
Get 2g polyvinyl alcohol swelling 24h, add the 0.04g sodium alginate, the Autoclave heating is fully dissolved it, stirs while hot.The 6g E.coli BL21-pET/aspC bacteria suspension of recombinating is mixed with the gac of 1g, treat that polyvinyl alcohol cooling back stirs with it.Be added drop-wise in borax-calcium chloride cross-linking agent solution with syringe, form the bead of certain diameter, soak 3h, refrigerator is freezing.Wash with physiological saline after thawing.
Get the alpha-carbonyl benzenebutanoic acid of 30mL 10g/L, add 0.3g (1: 1.2 in molar ratio) L-Glu (L-L-glutamic acid), mixing is regulated pH to 8.5, and the tween-80 that adds 0.4% (with total reaction liquid mass volume ratio) is made into reaction solution.The immobilized cell that adds 3% (w/v).The inflated with nitrogen protection places 37 ℃, 120rpm shaking table, reaction 20h.Immobilized cell is taken out from reaction solution, add hcl acidifying to pH to 1, remove by filter the insolubles in the reaction solution, add NaOH and regulate pH to 5.5, separate out white insolubles in the reaction solution and be the L-hyperphenylalaninemia, filter promptly at reaction solution.The transformation period of immobilized cell be 13 days (batch), average conversion (the alpha-carbonyl benzenebutanoic acid is converted into the ratio of L-hyperphenylalaninemia) is 73.2%.
Embodiment 2:
Get 2g polyvinyl alcohol swelling 24h, add the 0.04g sodium alginate, the Autoclave heating is fully dissolved it, stirs while hot.The 6g E.coli BL21-pET/aspC bacteria suspension of recombinating is mixed with the gac of 1g, treat that polyvinyl alcohol cooling back stirs with it.Be added drop-wise in borax-calcium chloride cross-linking agent solution with syringe, form the bead of certain diameter, and soak 12h therein.Wash with physiological saline.
Get the alpha-carbonyl benzenebutanoic acid of 30mL 10g/L, add 0.4g (1: 1.6 in molar ratio) L-Glu (L-L-glutamic acid), mixing is regulated pH to 8.5, and the Tween-60 that adds 0.5% (w/v) is made into reaction solution.The immobilized cell that adds 4% (w/v).The inflated with nitrogen protection places 37 ℃, 120rpm shaking table, reaction 20h.Press the method for embodiment 1 and extract the L-hyperphenylalaninemia.The transformation period of immobilized cell be 16 days (batch), average conversion is 76.5%.
Embodiment 3:
Get 2g polyvinyl alcohol swelling 24h, add the 0.04g sodium alginate, the Autoclave heating is fully dissolved it, stirs while hot.The 6g E.coli BL21-pET/aspC bacteria suspension of recombinating is mixed with the gac of 1g, treat that polyvinyl alcohol cooling back stirs with it.Be added drop-wise in borax-calcium chloride cross-linking agent solution with syringe, form the bead of certain diameter, and soak 12h therein.Wash with physiological saline.
Get the alpha-carbonyl benzenebutanoic acid of 30mL10g/L, add 0.3g (1: 1.2 in molar ratio) L-Glu (L-L-glutamic acid), mixing is regulated pH to 8.5, and the tween-80 that adds 0.4% (w/v) is made into reaction solution.The immobilized cell that adds 3% (w/v).The inflated with nitrogen protection places 40 ℃, 120rpm shaking table, reaction 20h.Press the method for embodiment 1 and extract the L-hyperphenylalaninemia.Average conversion is 90.1%.
Embodiment 4:
Get 8g polyvinyl alcohol swelling, add the 1g sodium alginate, the Autoclave heating is fully dissolved it, stirs while hot.The 30g E.coli BL21-pET/aspC bacteria suspension of recombinating is mixed with the gac of 15g, treat that polyvinyl alcohol cooling back stirs with it.Be added drop-wise in boric acid-borax-calcium chloride cross-linking agent solution with syringe, form the bead of certain diameter, and soak 12h therein.Wash with physiological saline.
Get the alpha-carbonyl benzenebutanoic acid of 30mL10g/L, add 0.3g (1: 1.2 in molar ratio) L-Glu (L-L-glutamic acid), mixing is regulated pH to 7.0, and the tween-80 that adds 0.4% (w/v) is made into reaction solution.The immobilized cell that adds 3% (w/v).The inflated with nitrogen protection places 40 ℃, 120rpm shaking table, reaction 24h.Press the method for embodiment 1 and extract the L-hyperphenylalaninemia.Average conversion is 83.2%.
Embodiment 5:
Get 12g polyvinyl alcohol swelling, add the 0.1g sodium alginate, the Autoclave heating is fully dissolved it, stirs while hot.The 10g E.coli BL21-pET/aspC bacteria suspension of recombinating is mixed with the gac of 5g, treat that polyvinyl alcohol cooling back stirs with it.Be added drop-wise in borax-calcium chloride cross-linking agent solution with syringe, form the bead of certain diameter, and soak 12h therein.Wash with physiological saline.
Get the alpha-carbonyl benzenebutanoic acid of 30mL10g/L, add 0.3g (1: 1.2 in molar ratio) L-Glu (L-L-glutamic acid), mixing is regulated pH to 8.5, and the tween-80 that adds 0.2% (w/v) is made into reaction solution.The immobilized cell that adds 4% (w/v).The inflated with nitrogen protection places 40 ℃, 120rpm shaking table, reaction 20h.Press the method for embodiment 1 and extract the L-hyperphenylalaninemia.Average conversion is 88.3%.
Embodiment 6:
Get 2g polyvinyl alcohol swelling 24h, add the 0.04g sodium alginate, the Autoclave heating is fully dissolved it, stirs while hot.The 6g E.coli BL21-pET/aspC bacteria suspension of recombinating is mixed with the gac of 1g, treat that polyvinyl alcohol cooling back stirs with it.Be added drop-wise in borax-calcium chloride cross-linking agent solution with syringe, form the bead of certain diameter, and soak 12h therein.Wash with physiological saline.
Get the alpha-carbonyl benzenebutanoic acid of 30mL10g/L, add 0.27g (1: 1.2 in molar ratio) L-Asp (L-aspartic acid), mixing is regulated pH to 8.5, and the tween-80 that adds 0.4% (w/v) is made into reaction solution.The immobilized cell cell that adds 3% (w/v).The inflated with nitrogen protection places 40 ℃, 120rpm shaking table, reaction 20h.Press the method for embodiment 1 and extract the L-hyperphenylalaninemia.Average conversion is 80.3%.
Embodiment 7:
Get 2g polyvinyl alcohol swelling 24h, add the 0.04g sodium alginate, the Autoclave heating is fully dissolved it, stirs while hot.The 6g E.coli BL21-pET/aspC bacteria suspension of recombinating is mixed with the gac of 1g, treat that polyvinyl alcohol cooling back stirs with it.Be added drop-wise in borax-calcium chloride cross-linking agent solution with syringe, form the bead of certain diameter, and soak 10h therein.Wash with physiological saline.
Get the alpha-carbonyl benzenebutanoic acid of 30mL10g/L, add 0.3g (1: 1.2 in molar ratio) L-Glu, mixing is regulated pH to 8.5, and the tween-80 that adds 0.4% (w/v) is made into reaction solution.The immobilized cell that adds 3% (w/v).The inflated with nitrogen protection places 40 ℃, 120rpm shaking table, reaction 14h.Press the method for embodiment 1 and extract the L-hyperphenylalaninemia.Average conversion is 85.6%.
Embodiment 8:
Get 2g polyvinyl alcohol swelling 24h, add the 0.04g sodium alginate, the Autoclave heating is fully dissolved it, stirs while hot.The 6g E.coli BL21-pETC/aspC bacteria suspension of recombinating is mixed with the gac of 1g, treat that polyvinyl alcohol cooling back stirs with it.Be added drop-wise in boric acid-calcium chloride cross-linking agent solution with syringe, form the bead of certain diameter, and soak 12h therein.Wash with physiological saline.
Get the alpha-carbonyl benzenebutanoic acid of 30mL10g/L, add 0.3g (1: 1.2 in molar ratio) L-Glu (L-L-glutamic acid), mixing is regulated pH to 9.5, and the tween-80 that adds 0.4% (w/v) is made into reaction solution.The immobilized cell that adds 3% (w/v).The inflated with nitrogen protection places 35 ℃, 120rpm shaking table, reaction 20h.Press the method for embodiment 1 and extract the L-hyperphenylalaninemia.Average conversion is 87.1%.
Embodiment 9:
Get 2g polyvinyl alcohol swelling 24h, add the 0.04g sodium alginate, the Autoclave heating is fully dissolved it, stirs while hot.The 6g E.coli BL21-pET/aspC bacteria suspension of recombinating is mixed with the gac of 1g, treat that polyvinyl alcohol cooling back stirs with it.Be added drop-wise in borax-calcium chloride cross-linking agent solution with syringe, form the bead of certain diameter, and soak 12h therein.Wash with physiological saline.
Get the alpha-carbonyl benzenebutanoic acid of 30mL10g/L, add 0.3g (1: 1.2 in molar ratio) L-Glu (L-L-glutamic acid), mixing is regulated pH to 8.5, and the tween-80 that adds 0.6% (with total reaction liquid mass volume ratio) is made into reaction solution.The immobilized cell that adds 3% (w/v).The inflated with nitrogen protection places 40 ℃, 120rpm shaking table, reaction 20h.Press the method for embodiment 1 and extract the L-hyperphenylalaninemia.Average conversion is 84.7%.
Comparative Examples 1:
With 2g polyvinyl alcohol swelling 24h, the Autoclave heating is fully dissolved it, stirs while hot.Treat that polyvinyl alcohol cooling back and 6g reorganization E.coli BL21-pET/aspC bacteria suspension stir.Add linking agent and stir, refrigerator is freezing.Be cut into the fritter of 3 * 3 * 3mm after thawing.Wash with physiological saline.
Get the alpha-carbonyl benzenebutanoic acid of 30mL10g/L, add 0.3g (1: 1.2 in molar ratio) L-Glu (L-L-glutamic acid), mixing is regulated pH to 8.5, adds the tween-80 of 0.4% (mass volume ratio).The immobilized cell that adds 3% (w/v).The inflated with nitrogen protection places 37 ℃, 120rpm shaking table, reaction 20h.Press the method for embodiment 1 and extract the L-hyperphenylalaninemia.The transformation period of immobilized cell be 6 days (batch), average conversion is 64.9%.
Comparative Examples 2:
With 2g polyvinyl alcohol swelling 24h, the Autoclave heating is fully dissolved it, stirs while hot.Treat that polyvinyl alcohol cooling back and E.coli BL21-pET/aspC bacteria suspension that 6g is recombinated stir.Be added drop-wise in the cross-linking agent solution with syringe, form the bead of certain diameter, and soak 12h therein.Wash with physiological saline.
Get the alpha-carbonyl benzenebutanoic acid of 30mL10g/L, add 0.3g (1: 1.2 in molar ratio) L-Glu (L-L-glutamic acid), mixing is regulated pH to 8.5, adds the tween-80 of 0.4% (mass volume ratio).The immobilized cell that adds 3% (w/v).The inflated with nitrogen protection places 37 ℃, 120rpm shaking table, reaction 20h.Press the method for embodiment 1 and extract the L-hyperphenylalaninemia.The transformation period of immobilized cell be 7 days (batch), average conversion is 75.2%.
Comparative Examples 3:
With 2g polyvinyl alcohol swelling 24h, add the sodium alginate of 0.04g, the Autoclave heating is fully dissolved it, stirs while hot.Treat that polyvinyl alcohol cooling back and E.coli BL21-pET/aspC bacteria suspension that 6g is recombinated stir.Be added drop-wise in the cross-linking agent solution with syringe, form the bead of certain diameter, the balling-up of immobilization gel is effective, and soaks 12h therein.Wash with physiological saline.
Get the alpha-carbonyl benzenebutanoic acid of 30mL10g/L, add 0.3g (1: 1.2 in molar ratio) L-Glu (L-L-glutamic acid), mixing is regulated pH to 8.5, adds the tween-80 of 0.4% (mass volume ratio).The immobilized cell that adds 3% (w/v).The inflated with nitrogen protection places 37 ℃, 120rpm shaking table, reaction 20h.Press the method for embodiment 1 and extract the L-hyperphenylalaninemia.The transformation period of immobilized cell be 8 days (batch), average conversion is 77.3%.
Comparative Examples 4:
With 2g polyvinyl alcohol swelling 24h, add the sodium alginate of 0.04g, the Autoclave heating is fully dissolved it, stirs while hot.The 6g E.coli BL21-pET/aspC bacteria suspension of recombinating is mixed with the gac of 1g, treat that polyvinyl alcohol cooling back stirs with it.Be added drop-wise in borax-calcium chloride cross-linking agent solution with syringe, form the bead of certain diameter, and soak 12h therein.Wash with physiological saline.
Get the alpha-carbonyl benzenebutanoic acid of 30mL10g/L, add 0.3g (1: 1.2 in molar ratio) L-Glu (L-L-glutamic acid), mixing is regulated pH to 8.5.The immobilized cell that adds 3% (w/v).The inflated with nitrogen protection places 37 ℃, 120rpm shaking table, reaction 20h.Press the method for embodiment 1 and extract the L-hyperphenylalaninemia.The transformation period of immobilized cell be 5 days (batch), average conversion 46.6%.
Can find out that by Comparative Examples 2 and 3 in the immobilization of cell, gac has vital role for the transformation period that promotes immobilized cell.Can find out that by Comparative Examples 4 nonionic surface active agent is also significant for the lifting of average conversion.
Claims (6)
1, a kind of method of immobilization reorganization E.coli BL21-pET/aspC cell preparation L-hyperphenylalaninemia; it is characterized in that alpha-carbonyl benzenebutanoic acid and L-L-glutamic acid or L-aspartic acid mixing; regulate pH to 7.0~9.5; add nonionic surface active agent and be made into reaction solution; the immobilization E.coliBL21-pET/aspC cell of recombinating is added in the reaction solution; 30~50 ℃ of reaction 4~24h under protection of inert gas, white suspension, regulate pH filter the L-hyperphenylalaninemia.
2, the method for preparing the L-hyperphenylalaninemia according to claim 1, it is characterized in that alpha-carbonyl benzenebutanoic acid and L-L-glutamic acid are 1: 1~1.6 mixed in molar ratio, the mass volume ratio of immobilized cell and reaction solution is 1%~5%, and the mass volume ratio of nonionic surface active agent and reaction solution is 0.1%~0.6%.
3, the method for preparing the L-hyperphenylalaninemia according to claim 1 and 2, the process for fixation of E.coli BL21-pET/aspC cell of it is characterized in that recombinating is: after the polyvinyl alcohol swelling, add sodium alginate, the Autoclave heating is dissolved it, after treating the polyvinyl alcohol cooling, add the mixture that contains reorganization E.coliBL21-pET/aspC bacteria suspension and gac, be added drop-wise in the cross-linking agent solution after stirring, form coccoid, and in cross-linking agent solution, soak 3~12h, being fixed cell after the washing.
4, the method for preparing the L-hyperphenylalaninemia according to claim 3 is characterized in that in the preparation of immobilized cell each raw material by mass ratio, polyvinyl alcohol: sodium alginate: gac=8~12: 0.1~1: 5~20.
5, the method for preparing the L-hyperphenylalaninemia according to claim 3 is characterized in that cross-linking agent solution is borax-calcium chloride solution, boric acid-calcium chloride solution or boric acid-borax-calcium chloride solution.
6, the method for preparing the L-hyperphenylalaninemia according to claim 1 and 2 is characterized in that described nonionic surface active agent is polysorbate-tween series of surfactants.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CNB200710021346XA CN100513556C (en) | 2007-04-09 | 2007-04-09 | Preparation for L-homophenylalanine by immobilization recombination of E.coliBL21-pET/aspC cell |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CNB200710021346XA CN100513556C (en) | 2007-04-09 | 2007-04-09 | Preparation for L-homophenylalanine by immobilization recombination of E.coliBL21-pET/aspC cell |
Publications (2)
Publication Number | Publication Date |
---|---|
CN101054570A true CN101054570A (en) | 2007-10-17 |
CN100513556C CN100513556C (en) | 2009-07-15 |
Family
ID=38794627
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CNB200710021346XA Expired - Fee Related CN100513556C (en) | 2007-04-09 | 2007-04-09 | Preparation for L-homophenylalanine by immobilization recombination of E.coliBL21-pET/aspC cell |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN100513556C (en) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101831467A (en) * | 2010-04-21 | 2010-09-15 | 广西大学 | Method for preparing L-alanine by adding laurate alcohol ester phenylacetate |
CN103276025A (en) * | 2013-06-27 | 2013-09-04 | 凯莱英医药集团(天津)股份有限公司 | Synthetic method of L-heterocyclic amino acid and pharmaceutical composition with L-heterocyclic amino acid |
CN107421796A (en) * | 2011-06-17 | 2017-12-01 | 罗氏血液诊断股份有限公司 | Solution for the tissue processing of biological sample |
WO2023245933A1 (en) * | 2022-06-23 | 2023-12-28 | 江南大学 | Method for efficiently producing l-homophenylalanine and strain for producing l-homophenylalanine |
-
2007
- 2007-04-09 CN CNB200710021346XA patent/CN100513556C/en not_active Expired - Fee Related
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101831467A (en) * | 2010-04-21 | 2010-09-15 | 广西大学 | Method for preparing L-alanine by adding laurate alcohol ester phenylacetate |
CN107421796A (en) * | 2011-06-17 | 2017-12-01 | 罗氏血液诊断股份有限公司 | Solution for the tissue processing of biological sample |
CN107421796B (en) * | 2011-06-17 | 2021-05-04 | 罗氏血液诊断股份有限公司 | Solution for tissue processing of biological samples |
CN103276025A (en) * | 2013-06-27 | 2013-09-04 | 凯莱英医药集团(天津)股份有限公司 | Synthetic method of L-heterocyclic amino acid and pharmaceutical composition with L-heterocyclic amino acid |
CN103276025B (en) * | 2013-06-27 | 2015-01-07 | 凯莱英医药集团(天津)股份有限公司 | Synthetic method of L-heterocyclic amino acid and pharmaceutical composition with L-heterocyclic amino acid |
WO2023245933A1 (en) * | 2022-06-23 | 2023-12-28 | 江南大学 | Method for efficiently producing l-homophenylalanine and strain for producing l-homophenylalanine |
Also Published As
Publication number | Publication date |
---|---|
CN100513556C (en) | 2009-07-15 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN101054570A (en) | Preparation for L-homophenylalanine by immobilization recombination of E.coliBL21-pET/aspC cell | |
CN102352387A (en) | Method for synthesizing non-natural amino acid by utilizing immobilized whole cell catalyst | |
CN111172140B (en) | Nitrilase mutant and application thereof in preparation of anti-epileptic drug intermediate | |
CN1101355A (en) | Method for production of acrylate and acrylate containing polymer | |
CN101050147A (en) | Water retaining slow-release / controled fertilizer, and preparation method | |
CN106801043A (en) | One kind restructuring transaminase and its preparation method and application | |
CN104805069A (en) | Immobilized transaminase and applications thereof in synthesizing of Sitagliptin intermediate | |
CN106995808B (en) | A kind of recombination transaminase and its application | |
CN100384923C (en) | Process for preparing hyaluronic acid-chitosan crosslinked biocompatible materials | |
CN103484419A (en) | Glutamic acid decarboxylase recombinant bacterium, and construction method and application thereof | |
CN1305843C (en) | Process for preparing 3-chloro-2-methyl thiobenzoxide | |
CN102604899B (en) | Genetic engineering bacteria containing L-alanine racemase genes and application of genetic engineering bacteria | |
CN102517351A (en) | Method for producing L-2-aminobutyric acid by double immobilized multi-enzyme systems | |
WO2023186186A1 (en) | Method for preparing immobilized enzyme having high stability | |
CN101649334B (en) | Method for preparing optically active theanine by enzymatic method | |
CN105504112B (en) | A kind of polymer and its application | |
CN100418900C (en) | High molecular weight, hydrophilic and oleophilic cation type flocculant and its preparing method | |
CN1948473A (en) | Method of extracting and purifying trypase in pancrease slag | |
CN109485148A (en) | A kind of biomass solid carbon source and its preparation method and application | |
CN1066773C (en) | Enzymatic coupling of L-phenylalanine methyl ester and N-benzyloxycarbonyl-aspartic acid | |
CN103992486B (en) | Click chemical-synthesis preparation method of light-operated tetrazole-alkene for polypeptide hydrogel | |
CN1130338C (en) | Fixed-bed process for preparing pentachlorophenyl nitrile by catalytic chloration of phenyl nitrile | |
CN103289083A (en) | Method for rapidly degrading high-molecular-weight polyamino acid and application of method | |
CN110818834B (en) | Room-temperature phosphorescent material based on polymer hydrogen bonds and preparation method thereof | |
CN110791534B (en) | Method for improving exogenous synthesis yield of water-soluble phycocyanin |
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 | ||
C17 | Cessation of patent right | ||
CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20090715 Termination date: 20120409 |