CN113355366A - Method for preparing 2-phenethyl alcohol by multi-enzyme cascade - Google Patents
Method for preparing 2-phenethyl alcohol by multi-enzyme cascade Download PDFInfo
- Publication number
- CN113355366A CN113355366A CN202110496774.8A CN202110496774A CN113355366A CN 113355366 A CN113355366 A CN 113355366A CN 202110496774 A CN202110496774 A CN 202110496774A CN 113355366 A CN113355366 A CN 113355366A
- Authority
- CN
- China
- Prior art keywords
- enzyme
- carbonyl reductase
- reaction
- ala
- ser
- 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
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01184—Carbonyl reductase (NADPH) (1.1.1.184)
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the field of genetic engineering and enzyme engineering, and the invention utilizes a genetic engineering and site-directed mutagenesis method to express carbonyl reductase derived from candida parapsilosis (with the accession number of ATCC 7330), and carries out site-directed mutagenesis to obtain a mutant A121N with the enzyme activity of the carbonyl reductase improved by 6 times, thereby providing an enzyme source for preparing 2-phenethyl alcohol by reducing phenylacetaldehyde by utilizing the carbonyl reductase or mutant enzyme thereof. On the basis, the method takes phenylacetaldehyde as a raw material, utilizes coupling of glucose dehydrogenase and carbonyl reductase, regenerates coenzyme NADPH in situ, adopts ultrafiltration equipment to separate, concentrate and recover enzyme after catalytic reaction, can realize enzyme recovery for more than 5 times, greatly reduces the preparation cost of 2-phenethyl alcohol, and has tubular reaction conversion rate of more than 90 percent and intermittent reaction conversion rate of more than 80 percent under the optimized process.
Description
Technical Field
The invention belongs to the field of genetic engineering and enzyme engineering, and relates to a method for preparing 2-phenethyl alcohol by multi-enzyme cascade.
Background
2-phenylethyl alcohol (also known as beta-phenylethyl alcohol) is a rose-flavored substance, and the world produces about 10000 tons of ethanol every year. It is a second major category of important food or cosmetic or perfume additives, second only to vanillin, for modulating the flavor of beverages and fragrance in cosmetics. Can inhibit gram-negative bacteria, coccus, bacillus and partial fungi; it is also a substrate for synthesizing some high value-added drugs such as phenylethanoid glycosides, and has the effects of resisting bacteria, resisting tumors, strengthening heart and the like, and the phenylethanol can also be used as an insecticide and a novel biofuel. Therefore, the low-cost and high-efficiency synthesis of the 2-phenethyl alcohol is very important.
At present, three methods of plant extraction, chemical synthesis and biological synthesis are mainly used for synthesizing the 2-phenethyl alcohol. (1) The extraction method comprises the following steps: the 2-phenethyl alcohol can be extracted from rose petals, but the cost is too high, and the yield is lower; (2) the chemical synthesis method mainly uses benzene, cyclohexane or benzyl alcohol, CO and H2Synthesized under the action of a catalyst. But the chemical synthesis process contains benzene and ethylene toxic and harmful substances, and the addition of the benzene and ethylene toxic and harmful substances into food can threaten the food safety to a certain extent, so that the mode of biologically synthesizing 2-phenethyl alcohol becomes a hotspot of current research; (3) biological synthesis is currently reported to be mainly synthesized by microbial fermentation. The microorganisms mainly used are Cicerinus angustifolia (CN202011414356.1), Saccharomyces cerevisiae (CN201910098372.5, CN201310243384.5, CN200910049170.8 and CN201710378178.3), Candida glycerinogenes (CN201610256845.6), recombinant Saccharomyces cerevisiae (CN202010933723.2, CN201910688005.0, recombinant Escherichia coli (CN201710256900.6, CN201010276491.3) and Trichoderma reesei (CN201310482330.4), and the like, but the research shows that the high-concentration 2-phenylethyl alcohol has certain toxicity to microbial cellsWhen the 2-phenylethyl alcohol content exceeds 2g/L, the growth of the microorganism is inhibited to different degrees, thereby restricting the application of the method for directly converting by using the microorganism. Therefore, with the development of enzyme engineering and protein engineering technologies, methods for performing catalytic conversion directly using enzymes have received much attention.
At present, no patent for directly utilizing enzyme to catalyze and synthesize 2-phenethyl alcohol is reported. The enzymatic synthesis has many advantages, cannot be subject to the technical bottleneck of inhibition of the product on microorganisms, and simultaneously, the coenzyme NADPH is regenerated in situ by utilizing parallel reaction, and the enzyme is recycled by utilizing an ultrafiltration technology, so that the cost of the enzyme is greatly reduced, and the guarantee is provided for the enzymatic synthesis of the 2-phenethyl alcohol.
Disclosure of Invention
In order to solve the technical problem, a method for preparing 2-phenethyl alcohol by multi-enzyme cascade is provided.
The scheme of the invention is as follows:
the technical route is as follows:
the main method of the invention is shown in a route, takes phenylacetaldehyde as a raw material, adds enzyme source 1 carbonyl reductase or enzyme source 2 mutant enzyme A121N, reacts at 25-35 ℃ and pH6-8, adds glucose dehydrogenase to regenerate reduced coenzyme NADPH, adopts tubular reaction continuous production or batch preparation of intermittent reaction, selects an organic membrane material with a certain aperture to carry out ultrafiltration, separates a product and the enzyme, and concentrates the enzyme for recycling. The penetrating fluid is subjected to reduced pressure distillation, and 2-phenethyl alcohol is collected.
The technical scheme of the invention is as follows:
1. enzyme source 1: by constructing recombinant bacteria E.coli BL21(DE3)/pACYCDuet-1-CpCR, the carbonyl reductase CpCR gene of Candida parapsilosis (number ATCC 7330) with a histidine tag is expressed, and the recombinant enzyme CpCR is separated, purified and enzymatically characterized by Ni-Agarose affinity chromatography. The gene sequence of recombinase CpCR has the full length of 1107 bp, contains 368 amino acids, has the molecular weight of about 41kD, and has the specific enzyme activity of 20U/mg. The enzyme activity of the liquid enzyme preparation for catalytic reaction is 10000U/L.
2. Enzyme source 2: the mutant enzyme A121N was obtained,
the self-designed primer A121n 2-f was synthesized by Biotechnology Ltd of Shanghai: GCAAACAAGG TGCAaatACATACAACTCCAAGGATGTCAGATC and A121n 2-r: attTGCACCTTG TTT GCA GTA TTGCTCATTGT, site-directed Mutagenesis is carried out by adopting a site-directed Mutagenesis Kit Mut Express II Fast Mutagenesis Kit V2 of Nanjing Novozam Biotech GmbH, to construct a mutase A121N, and the enzyme activity of the liquid enzyme preparation is improved to 6000U/L.
3. The glucose dehydrogenase is provided by the enzyme preparation division of Angel Yeast GmbH, granted patent CN 107779459A, and the strain is Escherichia coli Escherichia coli.A149-170, and is deposited in China center for type culture Collection with the deposit number of CCTCC M2016102. The molecular weight is about 30 kD. The enzyme activity of the provided liquid enzyme preparation is 5000U/L.
4. An enzyme catalysis reaction system: aqueous two-phase systems of polyethylene glycol or aqueous and organic solvents, such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate buffer solutions and methyl tert-butyl ether, potassium dihydrogen phosphate and dipotassium hydrogen phosphate buffer solutions and n-hexane.
5. Reaction temperature: 10-33 ℃ and pH 6-8.
6. Continuous tubular reaction or batch reaction;
7. the addition amount of carbonyl reductase is 1-5% of the mass of phenylacetaldehyde substrate, the addition amount of glucose dehydrogenase is 2-6% of the mass of glucose, the optimal reaction temperature is 25 ℃, and the pH value is 7.
8. And (3) ultrafiltration concentration: after the tubular reaction of carbonyl reductase and glucose dehydrogenase, separating enzyme and micromolecule products by selecting ultrafiltration equipment with the membrane aperture of 5-20kD, leading the products to enter a reduced pressure distillation process in permeate liquid, recycling enzyme liquid, recovering general enzyme for more than 5 times, keeping the enzyme activity more than 80 percent, and supplementing liquid enzyme preparation about 20 percent for a second continuous reaction system.
9. The 2-phenethyl alcohol is obtained by reduced pressure distillation, the yield of the tubular reactor reaches over 90 percent, the yield of the batch reaction reaches over 80 percent, and the product purity is over 99 percent.
The invention has the beneficial effects that:
the invention utilizes carbonyl reductase from candida parapsilosis and a mutant A121N thereof to carry out catalytic synthesis on 2-phenethyl alcohol in a tubular reactor or a batch reactor, wherein coenzyme NADPH is regenerated by glucose dehydrogenase, and the reacted enzyme is concentrated and recycled by adopting an ultrafiltration technology.
Drawings
FIG. 1 is a schematic structural diagram of a recombinant expression vector pACYCDuet-1-cpcr;
Detailed Description
The invention is further illustrated by the following examples, but the scope of the invention as claimed is not limited to the scope of the examples.
Example 1
Scheme 1: expression and purification of wild strain carbonyl reductase and amino acid sequence SEQID NO. 1.
The Candida parapsilosis ATCC7330 whole genome is taken as a template, and the primer: pACYC-F: 5' -GCCTGCAG (Pst I)ATGACTAAAGCAGTACCAGACA-3′；pACYC-R2：5′-TGTCGAC(Sal I)TAAGCTTTGAATGCTTTGTCG-3', amplifying the CpCR gene. The reaction system is as follows: 0.5. mu.L of genomic DNA template, 0.5. mu.L of Phusion DNA polymerase, 10. mu.L of Phusion GC Buffer (5X), 2.5. mu.L of pACYC-F (10. mu. mol/L), 2.5. mu.L of pACYC-R2 (10. mu. mol/L), 1. mu.L of dNTP (10. mu. mol/L), 1.5. mu.L of DMSO, 1.5. mu.L of Mg2+，30μL ddH2And O. Amplification conditions: 98-30 s, 98-10 s, 53-20 s, 72-45 s, 75-5 min and 4-infinity.
The CpCR amplification product was directionally cloned into the expression vector pACYC Duet-1 after double digestion with restriction enzymes Pst I and Sal I, and E.coli BL21(DE3) competent cells were transformed. After transformation, a proper amount of the bacterial liquid is taken and coated on an LB plate containing 100 mu g/mL chloramphenicol, and is cultured in the dark at 37 ℃, and positive transformants are screened. After the plasmids were cultured and verified by sequencing, a cloning vector was obtained and named pACYCDuet-1-cpcr (see FIG. 1).
Single colonies containing the recombinant expression plasmid were picked and cultured overnight in LB medium containing 100. mu.g/mL chloramphenicol. By OD600The resulting mixture was inoculated into 100mL of induction medium (100. mu.g/mL chloramphenicol, 1mmol/L Mg) at a ratio of 42+、1mmol/L Zn2+) Culturing at 23 deg.C and 200rpm to OD600At 0.6, IPTG was added to a final concentration of 0.4mmol/L and induction was carried out at 23 ℃ for 16 h. At the same time, empty vector and uninduced Escherichia coli were used as controls. The cells were collected by centrifugation, resuspended in an appropriate volume of Buffer A (20mmol/L Tris, 500mmol/L NaCl, 5% glycerol, 0.5mmol/L PMSF, pH 7.5), sonicated (2 s work, 6s stop, 25min), and centrifuged (12000 rpm, 4 ℃, 10min) to obtain the supernatant. And respectively detecting the supernatant and the precipitate as protein samples by SDS-PAGE polyacrylamide gel electrophoresis, and recording and analyzing the results.
The crude protein was isolated and purified using Ni-Agarose column, the supernatant of the target protein with histidine tag was filtered through 0.45 μm filter and loaded onto the column, washed with 10 column volumes Binding Buffer (20mmol/L Tris-HCl, 10mmol/L imidazole, 500mmol/L NaCl, pH 8.0) and 20mL resolution Buffer (20mmol/L Tris-HCl, 500mmol/L imidazole, 500mmol/L NaCl, pH 8.0), and fractionated into 1mL fractions and stored at 4 ℃ for use. And detecting the protein sample by SDS-PAGE, and recording and analyzing the result to obtain the recombinase CpCR with high specific enzyme activity. The amino acid sequence of the polypeptide is SEQID NO. 1.
Example 2
The mutant enzyme is obtained by site-directed mutagenesis of alanine A at position 121 to asparagine by site-directed mutagenesis technology, and has an amino acid sequence shown as SEQID NO.2, and the specific steps are as follows:
(1) primer design
Primer design was carried out using SnapGene software and synthesized by Bioengineering, Inc. (Shanghai).
A121N-f:GCAAACAAGGTGCAaatACATACAACTCCAAGGATGTCAGATC
A121N-r：attTGCACCTTGTTTGCAGTATTGCTCATTGT:
2) Mutant plasmid amplification
pACYCDuet-1-cpcr was amplified using Phanta Max Super-Fidelity DNApolymerase as follows: 1ng of genomic DNA template, 1. mu.L of Phanta Max Super-Fidelity DNApolymerase, 25. mu.L of Max Buffer (2X), 2. mu. L F/R primer (10. mu. mol/L), 1. mu.L of dNTP (10. mu. mol/L), ddH2O to 50. mu.L. Amplification conditions: pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 15s, annealing at 61 ℃ for 15s, extension at 72 ℃ for 60s, 30 cycles, and final heat preservation at 75 ℃ for 5 min.
(3) Digestion of the amplification product Dpnl
To prevent the formation of false positive transformants after transformation of the original template plasmid, a Dpnl digestion is required prior to the recombinant circularization. The reaction system is as follows: mu.L of Dpnl, 50. mu.L of amplified mutant plasmid. The treatment conditions are as follows: incubate at 37 ℃ for 1 h.
(4) Recombination reactions
The amplified mutant plasmid is linear, and homologous recombination of the mutant plasmid is carried out under the catalysis of the Exnase II enzyme to complete the cyclization process.
(5) Construction of the mutant enzyme A121N
Site-directed Mutagenesis was performed using a site-directed Mutagenesis Kit Mut Express II Fast Mutagenesis Kit V2 of Nanjing Novozam Biotechnology Ltd to obtain a mutation point plasmid, and the mutation point plasmid was transformed into E.coli BL21(DE3) competent cells to construct a mutant enzyme A121N genetically engineered bacterium. Storing at-80 deg.C.
The colony of the mutant enzyme A121N gene engineering bacteria containing the recombinant expression plasmid is picked up and cultured in LB culture medium containing 100 mug/mL chloramphenicol overnight. By OD600The resulting mixture was inoculated into 100mL of induction medium (100. mu.g/mL chloramphenicol, 1mmol/L Mg) at a ratio of 42+、1mmol/L Zn2+) Culturing at 23 deg.C and 200rpm to OD600At 0.6, IPTG was added to a final concentration of 0.4mmol/L and induction was carried out at 23 ℃ for 16 h. At the same time, empty vector and uninduced Escherichia coli were used as controls. The cells were collected by centrifugation, resuspended in an appropriate volume of Buffer A (20mmol/L Tris, 500mmol/L NaCl, 5% glycerol, 0.5mmol/L PMSF, pH 7.5), sonicated (2 s work, 6s stop, 25min), and centrifuged (12000 rpm, 4 ℃, 10min) to obtain the supernatant. And respectively detecting the supernatant and the precipitate as protein samples by SDS-PAGE polyacrylamide gel electrophoresis, and recording and analyzing the results.
The crude protein was isolated and purified using Ni-Agarose column, the supernatant of the target protein with histidine tag was filtered through 0.45 μm filter and loaded onto the column, washed with 10 column volumes Binding Buffer (20mmol/L Tris-HCl, 10mmol/L imidazole, 500mmol/L NaCl, pH 8.0) and 20mL resolution Buffer (20mmol/L Tris-HCl, 500mmol/L imidazole, 500mmol/L NaCl, pH 8.0), and fractionated into 1mL fractions and stored at 4 ℃ for further use. And detecting the protein sample by SDS-PAGE, and recording and analyzing the result to obtain the recombinase CpCR with high specific enzyme activity. The amino acid sequence of the polypeptide is SEQID NO. 2.
Example 3
Under the conditions of 25 ℃ and pH 7, 50kg/h of phenylacetaldehyde methyl tert-butyl ether solution, 50kg/h of glucose, 1.5kg/h of carbonyl reductase liquid enzyme preparation (carbonyl reductase constructed by the method of example 2) and 1.5kg/h of glucose dehydrogenase monopotassium phosphate and dipotassium phosphate buffer solution are respectively conveyed, the reaction retention time is 30 minutes, then the mixture is subjected to ultrafiltration by an organic membrane with 15kD of membrane pore size and water flux of 60kg/h, 85 percent of ketoreductase and glucose dehydrogenase are recovered and returned to a tubular reactor, the recovery and utilization times are 7 times, the tubular reaction conversion rate reaches 95 percent, and the product purity is 99.6 percent.
Example 4
Under the conditions that the temperature is 30 ℃ and the pH value is 7.5, in a two-phase system consisting of potassium dihydrogen phosphate, dipotassium hydrogen phosphate buffer solution and normal hexane, 40kg of phenylacetaldehyde, 40kg of glucose, 1kg of carbonyl reductase liquid enzyme preparation (carbonyl reductase constructed by the method of example 2) and 1kg of glucose dehydrogenase are respectively added into a 300L reaction kettle, when the reaction is finished, an organic membrane ultrafiltration device with the pore diameter of 20kD and the water flux of 80kg/h is selected, 75 percent of the ketoreductase and the glucose dehydrogenase are recovered and returned to a batch reactor, the reaction times are 5 times, the conversion rate of the batch reaction kettle can reach more than 80 percent, 2-phenethyl alcohol is obtained through reduced pressure distillation, and the purity of the product is 99.6 percent.
The above examples are merely representative of preferred embodiments of the present invention, and the description thereof is more specific and detailed, but not to be construed as limiting the scope of the present invention. It should be noted that, for those skilled in the art, various changes, modifications and substitutions can be made without departing from the spirit of the present invention, and these are all within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
SEQUENCE LISTING
<110> university of three gorges
<120> method for preparing 2-phenethyl alcohol by multi-enzyme cascade
<130> 2021
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 368
<212> PRT
<213> Artificial sequence
<400> 1
Met Thr Lys Ala Val Pro Asp Lys Phe Gln Gly Phe Ala Val Ser Asp
1 5 10 15
Pro Lys Asn Trp Asn Arg Pro Lys Leu Ala Ser Tyr Glu Arg Lys Gln
20 25 30
Ile Asn Pro His Asp Val Val Leu Lys Asn Glu Val Cys Gly Leu Cys
35 40 45
Tyr Ser Asp Ile His Thr Leu Ser Ala Gly Trp Gln Pro Leu Gln Arg
50 55 60
Asp Asn Leu Val Val Gly His Glu Ile Ile Gly Glu Val Ile Ala Val
65 70 75 80
Gly Asp Glu Val Thr Glu Phe Lys Val Gly Asp Arg Val Gly Ile Gly
85 90 95
Ala Ala Ser Ser Ser Cys Arg Ser Cys Gln Arg Cys Asp Ser Asp Asn
100 105 110
Glu Gln Tyr Cys Lys Gln Gly Ala Ala Thr Tyr Asn Ser Lys Asp Val
115 120 125
Arg Ser Asn Asn Tyr Val Thr Gln Gly Gly Tyr Ser Ser His Ser Ile
130 135 140
Ala Asp Glu Lys Phe Val Phe Ala Ile Pro Glu Asp Leu Pro Ser Ser
145 150 155 160
Tyr Gly Ala Pro Leu Met Cys Ala Gly Ile Thr Val Phe Ser Pro Leu
165 170 175
Ile Arg Asn Leu Gly Leu Asp Ala Arg Gly Lys Asn Val Gly Ile Ile
180 185 190
Gly Ile Gly Gly Leu Gly His Leu Ala Leu Gln Phe Ala Asn Ala Met
195 200 205
Gly Ala Asn Val Thr Ala Phe Ser Arg Ser Ser Ser Lys Lys Glu Gln
210 215 220
Ala Met Lys Leu Gly Ala His Asp Phe Val Ala Thr Gly Glu Asp Lys
225 230 235 240
Thr Trp Tyr Lys Asn Tyr Asp Asp His Phe Asp Phe Ile Leu Asn Cys
245 250 255
Ala Ser Gly Ile Asp Gly Leu Asn Leu Ser Glu Tyr Leu Ser Thr Leu
260 265 270
Lys Val Asp Lys Lys Phe Val Ser Val Gly Leu Pro Pro Ser Glu Asp
275 280 285
Lys Phe Glu Val Ser Pro Phe Thr Phe Leu Gln Gln Gly Ala Ser Phe
290 295 300
Gly Ser Ser Leu Leu Gly Ser Lys Thr Glu Val Lys Glu Met Leu Asn
305 310 315 320
Leu Ala Ala Lys His Asn Val Arg Pro Met Ile Glu Glu Val Pro Ile
325 330 335
Ser Glu Glu Asn Cys Ala Lys Ala Leu Asp Arg Cys His Ala Gly Asp
340 345 350
Val Arg Tyr Arg Phe Val Phe Thr Asp Phe Asp Lys Ala Phe Lys Ala
355 360 365
<210> 2
<211> 368
<212> PRT
<213> Artificial sequence
<400> 2
Met Thr Lys Ala Val Pro Asp Lys Phe Gln Gly Phe Ala Val Ser Asp
1 5 10 15
Pro Lys Asn Trp Asn Arg Pro Lys Leu Ala Ser Tyr Glu Arg Lys Gln
20 25 30
Ile Asn Pro His Asp Val Val Leu Lys Asn Glu Val Cys Gly Leu Cys
35 40 45
Tyr Ser Asp Ile His Thr Leu Ser Ala Gly Trp Gln Pro Leu Gln Arg
50 55 60
Asp Asn Leu Val Val Gly His Glu Ile Ile Gly Glu Val Ile Ala Val
65 70 75 80
Gly Asp Glu Val Thr Glu Phe Lys Val Gly Asp Arg Val Gly Ile Gly
85 90 95
Ala Ala Ser Ser Ser Cys Arg Ser Cys Gln Arg Cys Asp Ser Asp Asn
100 105 110
Glu Gln Tyr Cys Lys Gln Gly Ala Asn Thr Tyr Asn Ser Lys Asp Val
115 120 125
Arg Ser Asn Asn Tyr Val Thr Gln Gly Gly Tyr Ser Ser His Ser Ile
130 135 140
Ala Asp Glu Lys Phe Val Phe Ala Ile Pro Glu Asp Leu Pro Ser Ser
145 150 155 160
Tyr Gly Ala Pro Leu Met Cys Ala Gly Ile Thr Val Phe Ser Pro Leu
165 170 175
Ile Arg Asn Leu Gly Leu Asp Ala Arg Gly Lys Asn Val Gly Ile Ile
180 185 190
Gly Ile Gly Gly Leu Gly His Leu Ala Leu Gln Phe Ala Asn Ala Met
195 200 205
Gly Ala Asn Val Thr Ala Phe Ser Arg Ser Ser Ser Lys Lys Glu Gln
210 215 220
Ala Met Lys Leu Gly Ala His Asp Phe Val Ala Thr Gly Glu Asp Lys
225 230 235 240
Thr Trp Tyr Lys Asn Tyr Asp Asp His Phe Asp Phe Ile Leu Asn Cys
245 250 255
Ala Ser Gly Ile Asp Gly Leu Asn Leu Ser Glu Tyr Leu Ser Thr Leu
260 265 270
Lys Val Asp Lys Lys Phe Val Ser Val Gly Leu Pro Pro Ser Glu Asp
275 280 285
Lys Phe Glu Val Ser Pro Phe Thr Phe Leu Gln Gln Gly Ala Ser Phe
290 295 300
Gly Ser Ser Leu Leu Gly Ser Lys Thr Glu Val Lys Glu Met Leu Asn
305 310 315 320
Leu Ala Ala Lys His Asn Val Arg Pro Met Ile Glu Glu Val Pro Ile
325 330 335
Ser Glu Glu Asn Cys Ala Lys Ala Leu Asp Arg Cys His Ala Gly Asp
340 345 350
Val Arg Tyr Arg Phe Val Phe Thr Asp Phe Asp Lys Ala Phe Lys Ala
355 360 365
Claims (9)
1. A method for preparing 2-phenethyl alcohol by multi-enzyme cascade is characterized in that: the method is for preparing 2-phenylethyl alcohol by catalytic reduction of phenylacetaldehyde by carbonyl reductase which is ATCC7330 carbonyl reductase or mutant enzyme A121N.
2. The method of claim 1, wherein: the amino acid sequence of ATCC7330 carbonyl reductase is shown as SEQ ID NO.1, and the amino acid sequence of mutant enzyme A121N is shown as SEQ ID NO. 2.
3. The method of claim 1, wherein the catalytic reduction reaction comprises the steps of:
s1: adding phenylacetaldehyde, glucose, carbonyl reductase and glucose dehydrogenase into an enzyme catalytic reaction system for reaction;
s2: after the reaction is finished, performing ultrafiltration by an organic membrane, recovering carbonyl reductase and glucose dehydrogenase, returning the carbonyl reductase and the glucose dehydrogenase to the reactor, and recycling;
s3: reduced pressure distillation to obtain 2-phenethyl alcohol.
4. The method as claimed in claim 3, wherein the reaction conditions of step S1 are 25-35 ℃ and pH 6-8.
5. The method of claim 3, wherein the step S1 reaction is a continuous tubular reaction or a batch reaction.
6. The method according to claim 3, wherein the carbonyl reductase is added in an amount of 1 to 5% by mass based on the phenylacetaldehyde substrate in step S1, and the glucose dehydrogenase is added in an amount of 2 to 6% by mass based on the glucose.
7. The method of claim 3, wherein the step S1 enzyme-catalyzed reaction system is a two-phase aqueous system of polyethylene glycol or a two-phase system of water and an organic solvent.
8. The method of claim 3, wherein the step S1 enzyme-catalyzed reaction system is potassium dihydrogen phosphate and dipotassium hydrogen phosphate buffer solution, methyl tert-butyl ether, potassium dihydrogen phosphate and dipotassium hydrogen phosphate buffer solution, and n-hexane.
9. The method according to claim 3, wherein the step S2: the pore size of the ultrafiltration membrane of the organic membrane is 5-20 kD.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110496774.8A CN113355366B (en) | 2021-05-07 | 2021-05-07 | Method for preparing 2-phenethyl alcohol by multi-enzyme cascade |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110496774.8A CN113355366B (en) | 2021-05-07 | 2021-05-07 | Method for preparing 2-phenethyl alcohol by multi-enzyme cascade |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113355366A true CN113355366A (en) | 2021-09-07 |
| CN113355366B CN113355366B (en) | 2022-12-20 |
Family
ID=77525909
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110496774.8A Active CN113355366B (en) | 2021-05-07 | 2021-05-07 | Method for preparing 2-phenethyl alcohol by multi-enzyme cascade |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113355366B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112266937A (en) * | 2020-12-07 | 2021-01-26 | 福建农林大学 | Culture medium suitable for synthesizing 2-phenethyl alcohol by virtue of biological fermentation of grifola frondosa |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102876733A (en) * | 2012-10-23 | 2013-01-16 | 凯莱英医药集团（天津）股份有限公司 | Method for preparing alcohol compound with high optical purity |
| CN106701698A (en) * | 2016-11-15 | 2017-05-24 | 华东理工大学 | Carbonyl reductase, mutant and application thereof in preparation of antifungal drug intermediates |
| CN109943597A (en) * | 2019-03-06 | 2019-06-28 | 泰州市惠利生物科技有限公司 | A method of extraction preparation s-4- chloro-3-hydroxyl ethyl butyrate is coupled using enzyme mebrane reactor |
| CN110452861A (en) * | 2019-07-10 | 2019-11-15 | 杭州师范大学 | A kind of genetic recombination engineering bacteria and its catalyzing and synthesizing the application in D- pantoyl internal ester |
| CN111073912A (en) * | 2018-10-18 | 2020-04-28 | 上海医药工业研究院 | Biological preparation method of (S) -2-chloro-1- (2,4-dichlorophenyl) ethanol |
-
2021
- 2021-05-07 CN CN202110496774.8A patent/CN113355366B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102876733A (en) * | 2012-10-23 | 2013-01-16 | 凯莱英医药集团（天津）股份有限公司 | Method for preparing alcohol compound with high optical purity |
| CN106701698A (en) * | 2016-11-15 | 2017-05-24 | 华东理工大学 | Carbonyl reductase, mutant and application thereof in preparation of antifungal drug intermediates |
| CN111073912A (en) * | 2018-10-18 | 2020-04-28 | 上海医药工业研究院 | Biological preparation method of (S) -2-chloro-1- (2,4-dichlorophenyl) ethanol |
| CN109943597A (en) * | 2019-03-06 | 2019-06-28 | 泰州市惠利生物科技有限公司 | A method of extraction preparation s-4- chloro-3-hydroxyl ethyl butyrate is coupled using enzyme mebrane reactor |
| CN110452861A (en) * | 2019-07-10 | 2019-11-15 | 杭州师范大学 | A kind of genetic recombination engineering bacteria and its catalyzing and synthesizing the application in D- pantoyl internal ester |
Non-Patent Citations (3)
| Title |
|---|
| NIDHI AGGARWAL等: "Understanding substrate specificity and enantioselectivity of carbonyl reductase from Candida parapsilosis ATCC 7330 (CpCR): Experimental and modeling studies", 《MOLECULAR CATALYSIS》 * |
| 丁东栋等: "生物转化法合成2-苯乙醇的研究进展", 《工业微生物》 * |
| 宋婷等: "近平滑假丝酵母ATCC 7330羰基还原酶CpCR的表达及酶学性质研究", 《食品与发酵工业》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112266937A (en) * | 2020-12-07 | 2021-01-26 | 福建农林大学 | Culture medium suitable for synthesizing 2-phenethyl alcohol by virtue of biological fermentation of grifola frondosa |
| CN112266937B (en) * | 2020-12-07 | 2022-07-05 | 福建农林大学 | Culture medium suitable for synthesizing 2-phenethyl alcohol by virtue of biological fermentation of grifola frondosa |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113355366B (en) | 2022-12-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108467860B (en) | Method for high yield of gamma-aminobutyric acid | |
| CN106929521B (en) | Aldehyde ketone reductase gene recombination co-expression vector, engineering bacterium and application thereof | |
| CN111218467B (en) | Construction and application of MTHase and MTSase synchronously secreted recombinant bacillus subtilis | |
| CN112143764B (en) | Method for preparing intermediate compound of brivaracetam by using biological enzyme catalysis | |
| CN109055324B (en) | Improved ketoreductase and application thereof | |
| CN113308443B (en) | Monascus monooxygenase mutant and application thereof | |
| CN109468291B (en) | Carbonyl reductase EbSDR8 mutant and construction method and application thereof | |
| CN114480512B (en) | Application of oxidoreductase and mutant thereof in biosynthesis of nootkatone | |
| CN109371070B (en) | Method for high yield of alpha-ketoisovaleric acid | |
| CN113355366B (en) | Method for preparing 2-phenethyl alcohol by multi-enzyme cascade | |
| CN106399398A (en) | Bio-preparation method for (R)-3,5-bis(trifluoromethyl) phenyl ethanol | |
| CN111454918B (en) | Enol reductase mutant and application thereof in preparation of (R) -citronellal | |
| CN101302503B (en) | Mutated plant lactobacillus plantarum D-lactate dehydrogenases, genes, recombinase and use thereof | |
| CN112143688B (en) | Construction and application of recombinant escherichia coli | |
| CN110592035B (en) | Carbonyl reductase mutant, recombinant expression vector and application of carbonyl reductase mutant in production of chiral alcohol | |
| CN108103049B (en) | Thermophilic L-asparaginase mutant and screening and fermenting method thereof | |
| CN113151131B (en) | Self-induction culture medium for producing isoeugenol monooxygenase and application thereof | |
| CN109943542A (en) | A kind of alcohol dehydrogenase for the production of atazanavir intermediate | |
| CN115433721A (en) | Carbonyl reductase mutant and application thereof | |
| CN113930457A (en) | Method for synthesizing (S) -citronellol by double-enzyme coupling | |
| CN107312787B (en) | Fusion protein gene, engineering bacterium and application of fusion protein gene and engineering bacterium in preparation of 9 α -hydroxy-androstenedione | |
| WO2005123921A1 (en) | Novel glycerol dehydrogenase, gene therefor, and method of utilizing the same | |
| CN113151209B (en) | Short-chain dehydrogenase BLSDR8 and encoding gene and application thereof | |
| CN106047826B (en) | Aldehyde dehydrogenase, its recombinant expression transformants and the application in the synthesis of statin precursor | |
| CN116024184B (en) | Aldehyde dehydrogenase mutant and application thereof in preparation of 5-hydroxymethyl-2-furancarboxylic acid |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |