CN102352387A - Method for synthesizing non-natural amino acid by utilizing immobilized whole cell catalyst - Google Patents
Method for synthesizing non-natural amino acid by utilizing immobilized whole cell catalyst Download PDFInfo
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- CN102352387A CN102352387A CN2011102770303A CN201110277030A CN102352387A CN 102352387 A CN102352387 A CN 102352387A CN 2011102770303 A CN2011102770303 A CN 2011102770303A CN 201110277030 A CN201110277030 A CN 201110277030A CN 102352387 A CN102352387 A CN 102352387A
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- Prior art keywords
- cell
- natural amino
- immobilized whole
- amino acid
- alpha
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- 238000000034 method Methods 0.000 title claims abstract description 30
- 150000001413 amino acids Chemical class 0.000 title claims abstract description 22
- 239000003054 catalyst Substances 0.000 title claims abstract description 10
- 230000002194 synthesizing effect Effects 0.000 title abstract 3
- 108010028658 Leucine Dehydrogenase Proteins 0.000 claims abstract description 30
- 108090000698 Formate Dehydrogenases Proteins 0.000 claims abstract description 13
- 238000006243 chemical reaction Methods 0.000 claims abstract description 13
- 241000588724 Escherichia coli Species 0.000 claims abstract description 9
- DSSYKIVIOFKYAU-XCBNKYQSSA-N (R)-camphor Chemical compound C1C[C@@]2(C)C(=O)C[C@@H]1C2(C)C DSSYKIVIOFKYAU-XCBNKYQSSA-N 0.000 claims description 17
- 229960000846 camphor Drugs 0.000 claims description 17
- 239000013612 plasmid Substances 0.000 claims description 17
- -1 alpha-ketoacid salt Chemical class 0.000 claims description 16
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 12
- 238000002360 preparation method Methods 0.000 claims description 12
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- 241000193386 Lysinibacillus sphaericus Species 0.000 claims description 10
- 239000000243 solution Substances 0.000 claims description 10
- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical compound [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 claims description 8
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- 238000000605 extraction Methods 0.000 description 3
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- HFBCHNRFRYLZNV-GUBZILKMSA-N Leu-Glu-Asp Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O HFBCHNRFRYLZNV-GUBZILKMSA-N 0.000 description 2
- KXODZBLFVFSLAI-AVGNSLFASA-N Leu-His-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC(C)C)CC1=CN=CN1 KXODZBLFVFSLAI-AVGNSLFASA-N 0.000 description 2
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/584—Recycling of catalysts
Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
The invention relates to the fields of pharmacy and biotechnology and provides a method for synthesizing non-natural amino acid by utilizing an immobilized whole cell catalyst. The non-natural amino acid is prepared by reducing alpha-ketonic acid in the presence of the whole cell catalyst comprising leucine dehydrogenase and formate dehydrogenase capable of regenerating cofactors. In particular, the method comprises the following steps of: synthesizing L-tertiary leucine by using trimethyl pyruvic acid as a substrate and by an enzyme method; cloning the leucine dehydrogenase and the formate dehydrogenase into recombinant escherichia coli to perform coexpression and immobilize cells; and transforming the substrate through immobilized whole cell catalysis, without adding 'adscititious' cofactors. The immobilized whole cell catalysis has the advantages of low cost and convenience for operation. The method belongs to a biological synthesis method. By the method, the reaction condition is mild; effect is special; and byproducts are avoided. The method has application value in the industries such as medicines, food and the like.
Description
Technical field
The invention belongs to the pharmaceutical industry biological technical field, relate to the method for the synthetic alpha-non-natural amino acid of a kind of immobilized whole-cell catalyzer.
Background technology
The alpha-non-natural amino acid 26S Proteasome Structure and Function is various, is midbody commonly used, for example L-Terleu during a lot of medicines synthesize.
Alpha-ketoacid (compound 1) can generate alpha-non-natural amino acid (compound 2 and compound 3) through biocatalysis.
R represents aliphatic side chains like-(CH
2)
nCH
3Or-C (CH
3)
3Deng; Or aromatic side chain is like Ph (phenyl) or Bn (benzyl).
The coenzyme NAD that uses in the biological catalysis
+Can be used for substrate conversion, but non-renewable, need replenish new coenzyme.Therefore, if can make regenerating coenzyme in the reaction process, then can reduce cost greatly.With the trimethylammonium pyruvic acid is that the synthetic L-Terleu of substrate enzyme process needs leucine dehydrogenase and hydrogenlyase, and wherein hydrogenlyase is used to the coenzyme of regenerating.
Summary of the invention
The present invention aims to provide the method for the synthetic alpha-non-natural amino acid of a kind of immobilized whole-cell catalyzer.
Technical scheme does, may further comprise the steps:
(1) immobilized whole-cell catalyzer, alpha-ketoacid salt, ammonium formiate and phosphoric acid buffer are mixed, under ℃ condition of pH6.0～9.0,25～40, react 5～20 hours (preferably ph=8.0,30 ℃ of temperature); Immobilized whole-cell catalyzer weight in wet base concentration is 10～200g/L in the reaction mixture, and the alpha-ketoacid salt concn is 100～1000mmol/L, and ammonium formiate concentration is 500～1500mmol/L;
Contain reconstitution cell in the described immobilized whole-cell catalyzer, contain formate dehydrogenase gene and leucine dehydrogenase gene in the reconstitution cell.
(2) reclaim the immobilized whole-cell catalyzer, reaction solution is cooled to 5～15 ℃, and ph is adjusted to 13～14; Under 5～15 ℃, drip methyl-chloroformate, stirring reaction 8～16 hours; Ph is adjusted to 1.5～3, and under 5～15 ℃, extracting and washing is dry.
Step also adds NAD in (1)
+, concentration is 0.05～0.5g/L.
Said alpha-ketoacid salt structure is suc as formula shown in 1,
R is aliphatic side chains, phenyl or benzyl.
Be preferably the trimethylammonium Pyruvic acid sodium salt.
Said leucine dehydrogenase gene order and formate dehydrogenase gene sequence are respectively shown in SEQID No.1 and SEQ ID No.2; The leucine dehydrogenase gene is selected from Bacillus sphaericus, bacillus cereus or bacstearothermophilus; Formate dehydrogenase gene is selected from rood yeast or Candida boidinii.
Shown in leucine dehydrogenase and hydrogenlyase aminoacid sequence SEQ ID No.3 and the SEQ IDNo.4.
The immobilized whole-cell catalyzer comprises leucine dehydrogenase and can make the hydrogenlyase of cofactor regeneration, and the preparation method is:
The recombinant plasmid that (1) will contain leucine dehydrogenase gene and formate dehydrogenase gene changes e. coli host bacteria over to, obtains reconstitution cell;
(2) reconstitution cell is mixed with fixative solution, be laid on the plane, 25～35 ℃ are incubated 1～1.5 hour down, and stablize after scouring with salts solution;
Fixing agent is an at least a mixture in polyoxyethylene glycol and SEPIGEL 305, Z 150PH or the alginate calcium;
The concentration of polyoxyethylene glycol is 0.08～0.1g/ml; SEPIGEL 305, Z 150PH or alginate calcium concentration are 0.1～0.2g/ml.
Utilization of the present invention comprises leucine dehydrogenase and can make whole-cell catalyst reduction alpha-ketoacid (compound 1) the preparation alpha-non-natural amino acid (compound 2 and compound 3) of the hydrogenlyase of cofactor regeneration.
Preferably; With the trimethylammonium pyruvic acid is that the synthetic L-Terleu of substrate enzyme process needs leucine dehydrogenase and hydrogenlyase (being used for regenerating coenzyme); With the leucine dehydrogenase of different sources (Leucine dehydrogenase, LeuDH) and hydrogenlyase (Formate dehydrogenase, FDH; Regenerating coenzyme with) be cloned into coexpression in the recombination bacillus coli, and with cell fixation.
The present invention utilizes the synthetic alpha-non-natural amino acid of immobilization reorganization whole-cell catalytic, like the L-Terleu, need not add " external " cofactor and substrate is transformed.And immobilized whole-cell catalysis has low, the easy to operate advantage of cost,
The compound method of this alpha-non-natural amino acid belongs to biological synthesis process, and reaction conditions is gentle, acts on single-mindedly, and no coupling product all has using value in industries such as medicine and food.
Description of drawings
Fig. 1 is the collection of illustrative plates of plasmid pACYCDuet-1-BsLeuDH.
Fig. 2 is the collection of illustrative plates of plasmid pET21a-LeFDH.
Fig. 3 is embodiment 7 magnetic resonance detection results.
Embodiment
Below in conjunction with embodiment the present invention is done further in detail, intactly explains:
The preparation of embodiment 1 recombinant plasmid pACYCDuet-1-BsLeuDH:
Comprise from the leucine dehydrogenase (Leucine dehydrogenase) of Bacillus sphaericus (Bacillus sphaericus IFO 3525) with from the preparation of the whole-cell catalyst of the hydrogenlyase of rood yeast (Lodderomyceselongisporus NRRL YB-4239):
(1) preparation of recombinant plasmid pACYCDuet-1-BsLeuDH:
According to this area method in common (" molecular cloning experiment guide "; Science Press; The 3rd edition) extract the genomic dna of Bacillus sphaericus, utilize primer amplified to go out the leucine dehydrogenase gene fragment, this fragment is done double digestion with restriction enzyme NcoI and BamHI; With agarose gel electrophoresis purified genes fragment, be connected and transformed into escherichia coli host bacterium Top10 cell with pACYCDuet-1 through the same restrictions enzymic digestion.Picking transformant inoculation LB liquid nutrient medium was cultivated 20 hours for 37 ℃, extracted recombinant plasmid with plasmid extraction kit (vast Tyke).The recombinant plasmid collection of illustrative plates is as shown in Figure 1.
(2) preparation of recombinant plasmid pET21a-LeFDH:
Extract rood zymic genomic dna according to the method described above; Utilize primer amplified to go out the formate dehydrogenase gene fragment; This fragment is done double digestion with restriction enzyme NdeI and BamHI; With agarose gel electrophoresis purified genes fragment, be connected and transformed into escherichia coli host bacterium Top10 cell with pET-21a through the same restrictions enzymic digestion.Picking transformant inoculation LB liquid nutrient medium was cultivated 20 hours for 37 ℃, extracted recombinant plasmid with plasmid extraction kit (vast Tyke).The recombinant plasmid collection of illustrative plates is as shown in Figure 2.
(3) preparation of recombinant bacterial strain:
Chemoreception attitude cell with recombinant plasmid pET21a-LeFDH transformed into escherichia coli expressive host BL21 (DE3).This plasmid contains the gene of coding from rood zymic hydrogenlyase.This recombination bacillus coli BL21 (DE3) (pET21a-LeFDH) is processed chemoreception attitude cell, transform above-mentioned reorganization bacterium with recombinant plasmid pACYCDuet-1-BsLeuDH.This plasmid contains the leucine dehydrogenase gene from Bacillus sphaericus.These two genes are all expressed under the control of T7 promotor, and leucine dehydrogenase and formate dehydrogenase gene nucleotide sequence are respectively shown in sequence table SEQ ID No.1 and SEQ ID No.2.Leucine dehydrogenase and hydrogenlyase aminoacid sequence are respectively shown in sequence table SEQ ID No.3 and SEQ ID No.4.
(4) preparation of active cells:
With e. coli bl21 (DE3) (pACYCDuet-1-BsLeuDH, bacterium colony pET21a-LeFDH) in the 5ml LB substratum that has added microbiotic (100 μ g/ml penbritins and 34 μ g/ml paraxin) in 37 ℃ of vibrations (160rpm) overnight cultures., to the fresh LB substratum of 100ml (containing 100 μ g/ml penbritins and 34 μ g/ml paraxin) in, cultivate OD600 for 37 ℃ and arrive about 0.8 with this bacterium of 1: 100 inoculum size switching, adding 0.1mM IPTG inductor was 20 ℃ of shaking culture 20 hours.Through centrifugal (10,000g, 10 minutes, 4 ℃) harvested cell, supernatant discarded is used for cell the bio-transformation experiment or is stored in-20 ℃.
The preparation of embodiment 2 immobilized whole-cells
3.0 gram polyoxyethylene glycol are dissolved in the water of 35ml, add 5.0 gram Z 150PH (PVA) and temperature is raised to 90-95 ℃ of dissolving.After treating that Z 150PH dissolves fully, cool the temperature to 25 ℃-30 ℃, add 4 grammes per square metre group cells (weight in wet base), mix, draw mixed solution, and annotate and to form the disk shape in the plane, and bathed 1 to 1.5 hour 30 ℃ of temperature with Dispette.Move into the 0.1M metabisulfite solution and stablized 2 hours, the filter dry doubling is used the clear water washed twice, places the 10mM sodium phosphate buffer for use.
Cell fixation reagent Z 150PH also can replace with SEPIGEL 305 or alginate calcium, and effect is identical.
Embodiment 3
Use comprises the synthetic L-Terleu of whole-cell catalyst of leucine dehydrogenase and hydrogenlyase:
In reaction vessel, add 100mM phosphoric acid buffer (the pH value is adjusted to 8.0) at 30 ℃; The adding final concentration is the whole-cell catalyst BL21 (DE3) (pET21a-LeFDH, pACYCDuet-1-BsLDH) (comprising leucine dehydrogenase and rood zymic hydrogenlyase from Bacillus sphaericus) of 100g/L (wet cell weight) embodiment 2 preparations, and final concentration is that trimethylammonium Pyruvic acid sodium salt and the final concentration of 300mM is the ammonium formiate of 750mM.
Reaction mixture was stirred 16 hours at 30 ℃.In the certain time interval sampling, the growing amount of product is confirmed with HPLC.Transformation efficiency is greater than 99% after reacting 16 hours.Product adds N-methoxycarbonyl blocking group through the method for embodiment 7, and purifies, and its structure is confirmed (Fig. 3) through magnetic resonance detection.
Embodiment 4: use the synthetic L-Terleu of the whole-cell catalyst that comprises leucine dehydrogenase and hydrogenlyase (to add NAD
+)
In reaction vessel, add 100mM phosphoric acid buffer (pH value is adjusted to 8.0) at 30 ℃, final concentration is that whole-cell catalyst BL21 (DE3) (pET21a-LeFDH, pACYCDuet-1-BsLDH) (comprising leucine dehydrogenase and rood zymic hydrogenlyase from Bacillus sphaericus), the final concentration that 100g/L (wet cell weight) embodiment 2 prepares is the NAD of 0.4g/L
+, final concentration is that trimethylammonium Pyruvic acid sodium salt and the final concentration of 800mM is the ammonium formiate of 1200mM.Reaction mixture was stirred 16 hours at 30 ℃.In the certain time interval sampling, the growing amount of product is confirmed with HPLC.Transformation efficiency is greater than 99% after reacting 16 hours.Product adds N-methoxycarbonyl blocking group through the method for embodiment 7, and purifies, and its structure is confirmed through magnetic resonance detection.
Embodiment 5: use the synthetic L-Terleu of the immobilized whole-cell catalyzer that comprises leucine dehydrogenase and hydrogenlyase
In reaction vessel, add 100mM phosphoric acid buffer (the pH value is adjusted to 8.0) at 30 ℃, final concentration is the trimethylammonium Pyruvic acid sodium salt of 600mM, final concentration is that ammonium formiate and the final concentration of 1500mM is the NAD of 0.4g/L
+, make that total liquid volume is 5ml).The 4 gram immobilization reconstitution cell catalyzer afterreactions that in system, add embodiment 2 preparations begin to carry out, with reaction mixture 30 ℃ of stirrings.In the certain time interval sampling, the growing amount of product is confirmed with HPLC.Transformation efficiency is greater than 98% after reacting 6 hours.Product adds N-methoxycarbonyl blocking group through the method for embodiment 7, and purifies, and its structure is confirmed through magnetic resonance detection.
Embodiment 6: recycling comprises the synthetic L-Terleu of immobilized whole-cell catalyzer of leucine dehydrogenase and hydrogenlyase
The immobilized whole-cell catalyzer of in embodiment 5, finishing using is through centrifugal recovery.In reaction vessel, add 100mM phosphoric acid buffer (the pH value is adjusted to 8.0) at 30 ℃, final concentration is the trimethylammonium Pyruvic acid sodium salt of 600mM, final concentration is that ammonium formiate and the final concentration of 1500mM is the NAD of 0.4g/L
+, make that total liquid volume is 5ml).Begin to carry out adding 4 gram immobilization reconstitution cell catalyzer afterreactions, with reaction mixture 30 ℃ of stirrings.In the certain time interval sampling, the growing amount of product is confirmed with HPLC.Transformation efficiency is greater than 98% after reacting 6 hours.Product adds N-methoxycarbonyl blocking group through the method for embodiment 7, and purifies, and its structure is confirmed through magnetic resonance detection.The immobilized whole-cell catalyzer can be reused more than 6 times at least.
Embodiment 7: synthetic L-Terleu enzyme reaction solution aftertreatment
After the catalyzed reaction of synthetic embodiment 3～6 is accomplished, treat that enzyme reaction solution (1260ml) is cooled to 10 ℃, add aqueous sodium hydroxide solution (temperature is lower than 30 ℃) and transfer to pH greater than 13, continue cooling.Treat that temperature reduces to 10 ℃, begin to drip methyl-chloroformate, 6 hours time, 10～15 ℃ of temperature, pH in the system＞13.Stirred overnight (8～15 hours) adds concentrated hydrochloric acid and is adjusted to pH=2 after HPLC detection raw material reaction is complete, 10～15 ℃ of system temperatures are with dichloromethane extraction 3 times (1000ml * 3); After standing demix, methylene dichloride passed through the filtering enzyme mutually, with saturated sodium sulfite anhy 96 300ml washing once, saturated aqueous common salt 300ml washed once; Standing demix, organic phase is in 45 ℃ of outer temperature, vacuum tightness 0.09MPa; Evaporate to dryness gets bullion 115g, purity 94.5%, maximum single assorted 1.5%.Add entry 260ml, be warming up to 75 ℃, after observing solid and dissolving fully, stir 15min, remove oil bath, the stirred overnight crystallization of lowering the temperature naturally, mistake filters product.Product drying: 50 ℃, vacuum tightness＞0.09MPa dried by the fire 4 hours, and 85g weighs.Filtrating is with twice of ethyl acetate extraction (500ml * 2), and solvent evaporated gets thick product, can carry out crystallization once more, to reclaim product.Magnetic resonance detection result such as Fig. 3.
Sequence table
SEQUENCE LISTING
< 110>section's biological medicine (Shanghai) Co., Ltd. still
< 120>method of the synthetic alpha-non-natural amino acid of immobilized whole-cell catalyzer
< 130>method of the synthetic alpha-non-natural amino acid of immobilized whole-cell catalyzer
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 1095
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< 213>Bacillus sphaericus, bacillus cereus or bacstearothermophilus
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<212> DNA
< 213>rood yeast or Candida boidinii
<400> 2
atgggtaagc caaaggtttt attagttctt tacgctggtg gtgagcatgc caaacaagaa 60
aagaaattgt tgggtgcaat tgagaatgag ttgggccttc gtcaatttat tgaggaccat 120
ggctacgact tggttgcaac aaccgacaaa gaaggtgaaa actcggcttt tgacaagaac 180
ttggaagatg cagaggttgt tattaccacg ccattttacc cagcttatct taccaaggag 240
agaatcgaaa aagcacctaa attgaagatt gccattaccg ctggtgttgg ttccgaccat 300
gtcaacttgg atgctgctaa tgccagagat atttcagttt tggaggttac tggttccaac 360
gtccagtcgg ttgcagagca tgctgttatg accatgcttg tgttgattag aaactacaac 420
attggccact tgcaagcaga aagtggtggc tgggatgtcg cagcagttgc caaggaggag 480
tttgatcttg aaggtaaggt gattgcaact gttggtgctg gtagaattgg ttacagaatt 540
ttggaaagat tagtcccatt caacccaaag aaattattgt actatgacta ccaacctttg 600
cccgctgctg ctgaagaaaa gttgaacaag gcttcccaat tgtacaatga cgttgacact 660
attgttgaaa aagttgacca attagaagac ttggttgccg aggctgatat cgtgactatc 720
aactgcccat tgcacgaaaa aactaaaggt ttgtttgaca aggccttgat ttcgagaatg 780
aagaaaggtt catacttggt caacaccgct agaggcgcca tttgtgatgc cgatgcagtt 840
gttgatgcat tgtcctctgg ccaccttgcc ggatacggtg gtgatgtttg gaatgtgcaa 900
ccagctccaa aagaccatcc atggagaaaa atgcacaacc catacggtcc agagtatggt 960
aatgcaatga ccatccacgt ttccggtacc tcattagatg cccaagccag atacgctgaa 1020
ggggtaaagc aaatcttgac tcaatacttt gacaagactt acaactacag accacaagat 1080
atcatctgta ttgacggtga ctatgcaact aaagcttatg gtcaacgtgc taagaaggag 1140
caaacagagg ctggaaatgt ttataagtaa 1170
<210> 3
<211> 364
<212> PRT
< 213>Bacillus sphaericus, bacillus cereus or bacstearothermophilus
<400> 3
Met Glu Ile Phe Lys Tyr Met Glu Lys Tyr Asp Tyr Glu Gln Leu Val
1 5 10 15
Phe Cys Gln Asp Glu Ala Ser Gly Leu Lys Ala Ile Ile Ala Ile His
20 25 30
Asp Thr Thr Leu Gly Pro Ala Leu Gly Gly Ala Arg Met Trp Thr Tyr
35 40 45
Ala Thr Glu Glu Asn Ala Ile Glu Asp Ala Leu Arg Leu Ala Arg Gly
50 55 60
Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys
65 70 75 80
Thr Val Ile Ile Gly Asp Pro Phe Lys Asp Lys Asn Glu Glu Met Phe
85 90 95
Arg Ala Leu Gly Arg Phe Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr
100 105 110
Ala Glu Asp Val Gly Thr Thr Val Thr Asp Met Asp Leu Ile His Glu
115 120 125
Glu Thr Asn Tyr Val Thr Gly Ile Ser Pro Ala Phe Gly Ser Ser Gly
130 135 140
Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala
145 150 155 160
Ala Ala Lys Glu Ala Phe Gly Thr Asp Met Leu Glu Gly Arg Thr Ile
165 170 175
Ser Val Gln Gly Leu Gly Asn Val Ala Tyr Lys Leu Cys Glu Tyr Leu
180 185 190
His Asn Glu Gly Ala Lys Leu Val Val Thr Asp Ile Asn Gln Ala Ala
195 200 205
Ile Asp Arg Val Val Asn Asp Phe Gly Ala Thr Ala Val Ala Pro Asp
210 215 220
Glu Ile Tyr Ser Gln Glu Val Asp Ile Phe Ser Pro Cys Ala Leu Gly
225 230 235 240
Ala Ile Leu Asn Asp Glu Thr Ile Pro Gln Leu Lys Ala Lys Val Ile
245 250 255
Ala Gly Ser Ala Asn Asn Gln Leu Gln Asp Ser Arg His Gly Asp Tyr
260 265 270
Leu His Glu Leu Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala
275 280 285
Gly Gly Val Ile Asn Val Ala Asp Glu Leu Tyr Gly Tyr Asn Arg Glu
290 295 300
Arg Ala Leu Lys Arg Val Asp Gly Ile Tyr Asp Ser Ile Glu Lys Ile
305 310 315 320
Phe Glu Ile Ser Lys Arg Asp Ser Ile Pro Thr Tyr Val Ala Ala Asn
325 330 335
Arg Leu Ala Glu Glu Arg Ile Ala Arg Val Ala Lys Ser Arg Ser Gln
340 345 350
Phe Leu Lys Asn Glu Lys Asn Ile Leu Asn Gly Arg
355 360
<210> 4
<211> 389
<212> PRT
< 213>rood yeast or Candida boidinii
<400> 4
Met Gly Lys Pro Lys Val Leu Leu Val Leu Tyr Ala Gly Gly Glu His
1 5 10 15
Ala Lys Gln Glu Lys Lys Leu Leu Gly Ala Ile Glu Asn Glu Leu Gly
20 25 30
Leu Arg Gln Phe Ile Glu Asp His Gly Tyr Asp Leu Val Ala Thr Thr
35 40 45
Asp Lys Glu Gly Glu Asn Ser Ala Phe Asp Lys Asn Leu Glu Asp Ala
50 55 60
Glu Val Val Ile Thr Thr Pro Phe Tyr Pro Ala Tyr Leu Thr Lys Glu
65 70 75 80
Arg Ile Glu Lys Ala Pro Lys Leu Lys Ile Ala Ile Thr Ala Gly Val
85 90 95
Gly Ser Asp His Val Asn Leu Asp Ala Ala Asn Ala Arg Asp Ile Ser
100 105 110
Val Leu Glu Val Thr Gly Ser Asn Val Gln Ser Val Ala Glu His Ala
115 120 125
Val Met Thr Met Leu Val Leu Ile Arg Asn Tyr Asn Ile Gly His Leu
130 135 140
Gln Ala Glu Ser Gly Gly Trp Asp Val Ala Ala Val Ala Lys Glu Glu
145 150 155 160
Phe Asp Leu Glu Gly Lys Val Ile Ala Thr Val Gly Ala Gly Arg Ile
165 170 175
Gly Tyr Arg Ile Leu Glu Arg Leu Val Pro Phe Asn Pro Lys Lys Leu
180 185 190
Leu Tyr Tyr Asp Tyr Gln Pro Leu Pro Ala Ala Ala Glu Glu Lys Leu
195 200 205
Asn Lys Ala Ser Gln Leu Tyr Asn Asp Val Asp Thr Ile Val Glu Lys
210 215 220
Val Asp Gln Leu Glu Asp Leu Val Ala Glu Ala Asp Ile Val Thr Ile
225 230 235 240
Asn Cys Pro Leu His Glu Lys Thr Lys Gly Leu Phe Asp Lys Ala Leu
245 250 255
Ile Ser Arg Met Lys Lys Gly Ser Tyr Leu Val Asn Thr Ala Arg Gly
260 265 270
Ala Ile Cys Asp Ala Asp Ala Val Val Asp Ala Leu Ser Ser Gly His
275 280 285
Leu Ala Gly Tyr Gly Gly Asp Val Trp Asn Val Gln Pro Ala Pro Lys
290 295 300
Asp His Pro Trp Arg Lys Met His Asn Pro Tyr Gly Pro Glu Tyr Gly
305 310 315 320
Asn Ala Met Thr Ile His Val Ser Gly Thr Ser Leu Asp Ala Gln Ala
325 330 335
Arg Tyr Ala Glu Gly Val Lys Gln Ile Leu Thr Gln Tyr Phe Asp Lys
340 345 350
Thr Tyr Asn Tyr Arg Pro Gln Asp Ile Ile Cys Ile Asp Gly Asp Tyr
355 360 365
Ala Thr Lys Ala Tyr Gly Gln Arg Ala Lys Lys Glu Gln Thr Glu Ala
370 375 380
Gly Asn Val Tyr Lys
385
Claims (7)
1. the method for the synthetic alpha-non-natural amino acid of immobilized whole-cell catalyzer is characterized in that, may further comprise the steps:
(1) immobilized whole-cell catalyzer, alpha-ketoacid salt, ammonium formiate and phosphoric acid buffer are mixed, under ℃ condition of pH6.0～9.0,25～40, reacted 5～20 hours; Immobilized whole-cell catalyzer weight in wet base concentration is 10～200g/L in the reaction mixture, and the alpha-ketoacid salt concn is 100～1000mmol/L, and ammonium formiate concentration is 500～1500mmol/L;
Contain reconstitution cell in the described immobilized whole-cell catalyzer, contain formate dehydrogenase gene and leucine dehydrogenase gene in the reconstitution cell;
(2) reclaim the immobilized whole-cell catalyzer, reaction solution is cooled to 5～15 ℃, and ph is adjusted to 13～14; Under 5～15 ℃, drip methyl-chloroformate, stirring reaction 8～16 hours; Ph is adjusted to 1.5～3, and under 5～15 ℃, extracting and washing is dry.
2. the method for the synthetic alpha-non-natural amino acid of the said immobilized whole-cell catalyzer of claim 1 is characterized in that step also adds NAD in (1)
+, concentration is 0.05～0.5g/L.
3. the said immobilized whole-cell catalyzer of claim 1 synthesizes the method for alpha-non-natural amino acid, it is characterized in that said immobilized whole-cell Preparation of catalysts method is:
The recombinant plasmid that (1) will contain leucine dehydrogenase gene and formate dehydrogenase gene changes e. coli host bacteria over to, obtains reconstitution cell;
(2) reconstitution cell is mixed with fixative solution, be laid on the plane, 25～35 ℃ are incubated 1～1.5 hour down, and stablize after scouring with salts solution;
Fixing agent is an at least a mixture in polyoxyethylene glycol and SEPIGEL 305, Z 150PH or the alginate calcium;
The concentration of polyoxyethylene glycol is 0.08～0.1g/ml; SEPIGEL 305, Z 150PH or alginate calcium concentration are 0.1～0.2g/ml.
4. the method for the synthetic alpha-non-natural amino acid of the said immobilized whole-cell catalyzer of claim 1 is characterized in that, said alpha-ketoacid salt structure is suc as formula shown in 1,
R is aliphatic side chains, phenyl or benzyl.
5. the method for claim 1 or 4 said synthetic alpha-non-natural amino acids is characterized in that, the alpha-ketoacid salt in the step (1) is the trimethylammonium Pyruvic acid sodium salt.
6. the method for the synthetic alpha-non-natural amino acid of the said immobilized whole-cell catalyzer of claim 1 is characterized in that said leucine dehydrogenase gene order and formate dehydrogenase gene sequence are respectively shown in SEQ ID No.1 and SEQ ID No.2.
7. the method for the synthetic alpha-non-natural amino acid of claim 1 or 6 said immobilized whole-cell catalyzer is characterized in that said leucine dehydrogenase gene is selected from Bacillus sphaericus, bacillus cereus or bacstearothermophilus; Formate dehydrogenase gene is selected from rood yeast or Candida boidinii.
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| CN103966275A (en) * | 2013-02-05 | 2014-08-06 | 山东斯递尔化工科技有限公司 | Method for preparing highly pure L-tertiary leucine through biological process |
| CN104099383A (en) * | 2014-07-29 | 2014-10-15 | 尚科生物医药（上海）有限公司 | Biological preparation method for (S)-N-t-butyloxycarboryl-3-hydroxide radical piperidine |
| CN104561161A (en) * | 2014-12-22 | 2015-04-29 | 厦门大学 | Method of preparing chiral tert-leucine by virtue of marine enzyme catalysis asymmetric reduction and enzyme |
| CN104946694A (en) * | 2015-07-24 | 2015-09-30 | 雅本化学股份有限公司 | Method for preparing L-2-aminobutyric acid through biocatalysis |
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| CN105154488A (en) * | 2015-10-22 | 2015-12-16 | 厦门大学 | Method for preparing L-tertiary leucine based on biological brick tandem double enzymes |
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| CN110669753A (en) * | 2019-11-05 | 2020-01-10 | 杭州师范大学 | A multi-point immobilization method based on unnatural amino acid modifying enzymes |
| CN111686809A (en) * | 2020-06-21 | 2020-09-22 | 复旦大学 | Carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst and preparation method and application thereof |
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| CN107043754B (en) * | 2012-03-30 | 2021-07-06 | 味之素株式会社 | Modified leucine dehydrogenase |
| CN102978251A (en) * | 2012-12-03 | 2013-03-20 | 苏州汉酶生物技术有限公司 | Method for producing L-tert-leucine |
| CN103966275A (en) * | 2013-02-05 | 2014-08-06 | 山东斯递尔化工科技有限公司 | Method for preparing highly pure L-tertiary leucine through biological process |
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| CN104099383A (en) * | 2014-07-29 | 2014-10-15 | 尚科生物医药（上海）有限公司 | Biological preparation method for (S)-N-t-butyloxycarboryl-3-hydroxide radical piperidine |
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