CN113355366B

CN113355366B - Method for preparing 2-phenethyl alcohol by multi-enzyme cascade

Info

Publication number: CN113355366B
Application number: CN202110496774.8A
Authority: CN
Inventors: 龚大春; 万里; 张书银; 王德林; 叶顺; 陈孟军; 宋婷; 吕育财; 任立伟
Original assignee: China Three Gorges University CTGU
Current assignee: China Three Gorges University CTGU
Priority date: 2021-05-07
Filing date: 2021-05-07
Publication date: 2022-12-20
Anticipated expiration: 2041-05-07
Also published as: CN113355366A

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 (deposit number 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

Method for preparing 2-phenethyl alcohol by multi-enzyme cascade

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 aromas 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.

Currently 2-phenylethyl alcoholThe synthesis mainly comprises three methods of plant extraction, chemical synthesis and biological synthesis. (1) extraction method: 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 H ₂ Synthesized 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 reported mainly by microbial fermentation synthesis. The used microorganisms mainly comprise Gracilaria verrucosa (CN 202011414356.1), saccharomyces cerevisiae (CN 201910098372.5, CN201310243384.5, CN200910049170.8, CN 201710378178.3), candida glycerinogenes (CN 201610256845.6), recombinant Saccharomyces cerevisiae (CN 202010933933723.8802, CN 201910605.0, recombinant Escherichia coli (CN 201710256900.6, CN 201010276491.3), trichoderma reesei (CN 201310482330.4) and the like, but researches show that high-concentration 2-phenylethyl alcohol has certain toxicity on microbial cells, and when the content of the 2-phenylethyl alcohol exceeds 2g/L, the growth of the microorganisms is inhibited to different degrees, so that the application of the method for directly transforming the microorganisms is restricted. 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 as shown in the route, taking phenylacetaldehyde as raw material, adding enzyme source 1 carbonyl reductase or enzyme source 2 mutant enzyme A121N, reacting at 25-35 ℃ and pH6-8, adding glucose dehydrogenase to regenerate reduced coenzyme NADPH, adopting tubular reaction continuous production or batch preparation of intermittent reaction, selecting organic membrane material with certain aperture to carry out ultrafiltration, separating product and enzyme, concentrating enzyme and recycling. And distilling the penetrating fluid under reduced pressure, and collecting the 2-phenethyl alcohol.

The technical scheme of the invention is as follows:

1. enzyme source 1: the carbonyl reductase CpCR gene of Candida parapsilosis (number ATCC 7330) with a histidine tag is expressed by constructing a recombinant bacterium E.coli BL21 (DE 3)/pACYCDuet-1-CpCR, and the recombinant enzyme CpCR is separated, purified and enzymatically characterized by adopting Ni-Agarose affinity chromatography. The gene sequence of recombinase CpCR has the full length of 1 107bp, 368 amino acids, the molecular weight of about 41kD and 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 used as a mutant enzyme,

the primers A121n 2-f were synthesized by Biotechnology Ltd of Shanghai: GCAAACAAGG TGCAaatacATACAACCAACTCAAGGATGTCAGATC 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 Novowed Biotech Co., ltd to construct a mutase A121N, and the enzyme activity of a liquid enzyme preparation is improved to 6000U/L.

3. The glucose dehydrogenase is provided by the enzyme preparation division of Angel Yeast GmbH, and the granted patent is CN 107779459A, and the strain is Escherichia coli Escherichia coli.A149-170, and is preserved in China center for type culture Collection with the preservation 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 small molecular products by selecting ultrafiltration equipment with the membrane aperture of 5-20kD, leading the products to enter a reduced pressure distillation process in a permeate liquid, recycling enzyme liquid, recovering general enzyme for at least more than 5 times, keeping the enzyme activity above 80 percent, and supplementing a liquid enzyme preparation of about 20 percent for a second continuous reaction system.

9. And (3) carrying out reduced pressure distillation to obtain the 2-phenethyl alcohol, wherein 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 an intermittent reactor, wherein, coenzyme NADPH is regenerated by adopting glucose dehydrogenase, and the enzyme after the reaction 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 present 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.

Candida parapsilosis ATCC7330 whole genome is used 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 Mg ²⁺ ，30μL ddH ₂ And O. Amplification conditions: 98-30s, 98-10s, 53-20s, 72-45s, 75-5min, and 4-infinity.

The CpCR amplification product is directionally cloned to an expression vector pACYC Duet-1 after double digestion by restriction enzymes Pst I and Sal I, and E.coli BL21 (DE 3) competent cells are 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 OD ₆₀₀ The resulting mixture was inoculated into 100mL of induction medium (100. Mu.g/mL chloramphenicol, 1mmol/L Mg) at a ratio of 4 ²⁺ 、1mmol/L Zn ²⁺ ) Culturing at 23 deg.C and 200rpm to OD ₆₀₀ At 0.6, IPTG was added to a final concentration of 0.4mmol/L and induced at 23 ℃ for 16h. At the same time, the control was made with the empty vector and uninduced E.coli. The cells were collected by centrifugation, resuspended in an appropriate volume of Buffer A (20 mmol/L Tris,500mmol/L NaCl,5% glycerol, 0.5mmol/L PMSF, pH 7.5), sonicated (2 s work, 6s stop, 25min), and centrifuged (12 000rpm,4 ℃,10 min) to take 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 (20 mmol/L Tris-HCl,10mmol/L imidazole, 500mmol/L NaCl, pH 8.0) and 20mL resolution Buffer (20 mmol/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

Amplification of pACYCDuet-1-cpcr using Phanta Max Super-Fidelity DNApolymerase was performed 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 of 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 finally heat preservation at 75 ℃ for 5min.

(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 1h.

(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 is carried out by adopting a site-directed Mutagenesis Kit Mut Express II Fast Mutagenesis Kit V2 of Nanjing Kenzuyin Biotechnology Limited to obtain a mutation point plasmid, and the mutation point plasmid is transformed into E.coli BL21 (DE 3) competent cells to construct a mutant enzyme A121N genetic engineering strain. 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 OD ₆₀₀ The resulting mixture was inoculated into 100mL of induction medium (100. Mu.g/mL chloramphenicol, 1mmol/L Mg) at a ratio of 4 ²⁺ 、1mmol/L Zn ²⁺ ) Culturing at 23 deg.C and 200rpm to OD ₆₀₀ At 0.6, IPTG was added to a final concentration of 0.4mmol/L and induction was carried out at 23 ℃ for 16h. At the same time, the control was made with the empty vector and uninduced E.coli. The cells were collected by centrifugation, resuspended in an appropriate volume of Buffer A (20 mmol/L Tris,500mmol/L NaCl,5% glycerol, 0.5mmol/L PMSF, pH 7.5), sonicated (2 s work, 6s stop, 25min), and centrifuged (12 000rpm,4 ℃,10 min) to take 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 (20 mmol/L Tris-HCl,10mmol/L imidazole, 500mmol/L NaCl, pH 8.0) and 20mL resolution Buffer (20 mmol/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 recombinase CpCR with higher 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, 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 in a two-phase system consisting of monopotassium phosphate, dipotassium phosphate buffer solution and normal hexane, the reaction is carried out to the end point, an organic membrane ultrafiltration device with 20kD membrane aperture and water flux of 80kg/h is selected, 75 percent of the ketoreductase and the glucose dehydrogenase are recovered and returned to a batch reactor for 5 times, the conversion rate of the batch reaction kettle can reach more than 80 percent, 2-phenethyl alcohol is obtained by reduced pressure distillation, and the product purity 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 various changes, modifications and substitutions may be made by those skilled in the art without departing from the spirit of the invention, and all are intended to be included within the scope of the invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

SEQUENCE LISTING

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Ser Glu Glu Asn Cys Ala Lys Ala Leu Asp Arg Cys His Ala Gly Asp

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