CN116083381A - Alcohol dehydrogenase and application thereof in preparation of duloxetine key intermediate - Google Patents

Abstract

The invention discloses novel alcohol dehydrogenase derived from lactobacillus (Lactobacillus parafarraginis), recombinant bacteria and application thereof in biocatalysis for preparing duloxetine key intermediates. The alcohol dehydrogenase can asymmetrically reduce N, N-dimethyl-3-ketone-3- (2-thiophene) -1-propylamine or salt thereof and N-methyl-3-ketone-3- (2-thiophene) -1-propylamine or salt thereof to prepare (S) -N, N-dimethyl-3-hydroxy-3- (2-thiophene) -1-propylamine and (S) -N-methyl-3-hydroxy-3- (2-thiophene) -1-propylamine, and has the advantages of mild reaction conditions, environmental friendliness, strict selectivity and the like compared with a chemical method; the alcohol dehydrogenase can catalyze 100g/L substrate conversion reaction, the conversion rate is more than 99%, the ee value of the product is more than 99.5%, the reaction rate is fast, the control is easy, the catalysis level is obviously improved compared with the existing report, and the alcohol dehydrogenase has great industrialized application potential.

Description

Alcohol dehydrogenase and application thereof in preparation of duloxetine key intermediate

Technical Field

The invention relates to novel alcohol dehydrogenase and application thereof, in particular to recombinant alcohol dehydrogenase, coding gene, recombinant expression vector and recombinant expression transformant containing the gene, and application of the enzyme or recombinant cells containing the enzyme as a catalyst in preparing duloxetine key intermediates.

Background

Duloxetine hydrochloride (trade name)

) Is a 5-hydroxytryptamine (5-HT) and Norepinephrine (NE) reuptake inhibitor developed by Gift corporation in the United states, and has remarkable therapeutic effects on major depression, diabetic peripheral neuralgia, fibromyalgia syndrome, etc. (Bioorganic)&Medicinal Chemistry Letters 13(2003)4477-4480；Journal of Clinical Psychopharmacology 34(2014)9-16)。

Duloxetine is a chiral drug, only the (S) -type of enantiomer has obvious pharmacological action, and the key step for synthesizing (S) -duloxetine is the construction of chiral hydroxyl. Chiral thiophenols, including gamma-amino alcohols, gamma-chlorohydrins, beta-hydroxy esters, and beta-hydroxynitriles, are reported to date as important intermediates for introducing chiral hydroxyl groups (US 8673607B2, US9228213B2, US7790436B2, bioscience Biotechnology and Biochemistry (2004) 1481-1488, cn 102643879B), among which (S) -N, N-dimethyl-3-hydroxy-3- (2-thiophene) -1-propylamine ((S) -DMAA) and (S) -N-methyl-3-hydroxy-3- (2-thiophene) -1-propylamine ((S) -MMAA) are of most industrial value.

The current methods for synthesizing (S) -DMAA mainly comprise a chemical method and a biological enzyme method. The chemical method comprises a resolution method and an asymmetric reduction method at present, wherein (S) -mandelic acid is utilized to resolve racemic DMAA to obtain (S) -DMAA, and then (R) -DMAA is subjected to racemization to realize substrate recycling, but the method has long reaction steps, high energy consumption and less than 70 percent of yield. The latter uses Li (ent-Chirald) ₂ AlH ₂ The asymmetric reduction of N, N-dimethyl-3-keto-3- (2-thiophene) -1-propanamine hydrochloride (DMAK) by using borane or metallic ruthenium complex as catalyst produces (S) -DMAA, but the selectivity is poor, the by-products are more, and the separation is difficult (Bioorganic Chemistry (2016) 82-89). In contrast, the biological enzyme method has the advantages of strong specificity, mild reaction condition, environmental protection and the like, and the main application mode is to prepare the biological enzyme by asymmetrically reducing DMAK by carbonyl reductase(S) -DMAA. Patent CN103013898B discloses a carbonyl reductase from Kluyveromyce marxianus, the concentration of a substrate is 3.3g/L, the conversion rate is 41.7% in 48 hours of reaction, and the ee value is 97.5%; patent CN104830924B discloses a carbonyl reductase ChKRED10 from Chryseobacterium sp.CA49, substrate concentration 1g/L, conversion rate 39% in 24h, ee value>99 percent; patent CN105803013B discloses a carbonyl reductase CR2 from Candida macedoniensis AKU4588, substrate concentration 1g/L, conversion rate 92.1% for 6h, ee value>99 percent; patent CN110229796A discloses a ketoreductase RKRED from Rhodococcus ruber, substrate concentration of 5-10g/L, conversion rate of 85-95% in 24h reaction, ee value>99%, the above patent has the problems of low substrate concentration and long reaction time, and lacks the basis of industrial production. Patent US8673607B2 discloses a ketoreductase from Lactobacillus kefiri and mutants thereof, substrate concentration 100g/L, conversion rate>99, ee value>99 percent, the reaction process can be effectively carried out only by negative pressure control to remove the byproduct acetone, the operation is difficult, the requirements on bioreactor equipment are high, the reaction time is long, and the productivity is low. Patent CN105039361B discloses a carbonyl reductase from Rhodosporidium toruloides, substrate concentration 219g/L, conversion rate>99, ee value>99%, but coenzyme NADP in the reaction process ⁺ The consumption amount is up to 0.75mmol/L, the consumption amount of the auxiliary substrate glucose is up to 6mol/L, the cost of the coenzyme raw material is high, and the industrialization requirement cannot be met.

The report on the synthesis of (S) -MMAA is less, patent CN110229796A discloses a ketoreductase RKRED from Rhodococcus ruber, the concentration of a substrate is 5-10g/L, the conversion rate of the reaction for 24 hours is 95-99%, and the ee value>99 percent; patent CN105039361B discloses a carbonyl reductase from Rhodosporidium toruloides, substrate concentration 50g/L, conversion 75%, ee value>99 percent; patent US9228213B2 discloses a ketoreductase from Lactobacillus kefiri and mutants thereof, substrate concentration 146g/L, conversion rate in 24h of reaction>99, ee value>99%, but the reaction process needs negative pressure control, and auxiliary substrate isopropanol and coenzyme NADP are added ⁺ Can react completely with fresh enzyme liquid, has great difficulty in process control and needs to be applied to bioreactor equipmentAnd (5) carrying out height finding.

Therefore, the method has important significance in improving the industrial level of duloxetine enzymatic process by excavating and screening alcohol dehydrogenase industrial enzyme with good catalytic performance and constructing a high-efficiency and easily-controlled catalytic process.

Disclosure of Invention

The invention aims to provide a novel alcohol dehydrogenase derived from lactobacillus (Lactobacillus parafarraginis), which can catalyze asymmetric reduction of DMAK and MMAK to generate (S) -DMAA and (S) -MMAA, and provides an industrial enzyme source for a biological enzyme process of a duloxetine key intermediate.

The technical scheme adopted by the invention is as follows:

the invention provides an alcohol dehydrogenase (LpADH) derived from lactobacillus (Lactobacillus parafarraginis), wherein the amino acid sequence of the LpADH is shown as SEQ ID NO: 1.

The LpADH is obtained by a gene database mining method, the selected LpADH is subjected to total gene synthesis, a recombinant vector and a recombinant escherichia coli cell are constructed, and activity and stereoselectivity are verified.

Due to the specificity of the amino acid sequence, any polypeptide comprising SEQ NO:1 or a variant thereof, such as a conservative variant, a biologically active fragment or a derivative thereof, as long as the polypeptide fragment or polypeptide variant has a homology of more than 90% with the aforementioned amino acid sequence. The alteration may comprise a deletion, insertion or substitution of an amino acid in the amino acid sequence.

The invention also relates to a coding gene of the LpADH. The nucleotide sequence of the gene is shown in SEQ ID NO: 2.

Due to the specificity of the nucleotide sequence, any of SEQ ID NOs: 2, and all variants of the polynucleotide having a homology of 90% or more to the polynucleotide are within the scope of the present invention. A variant of the polynucleotide refers to a polynucleotide sequence having one or more nucleotide changes.

The invention also relates to a recombinant vector containing the coding gene and recombinant genetic engineering bacteria obtained by utilizing the recombinant vector to transform. The recombinant vector is constructed by connecting the nucleotide sequence of the LpADH encoding gene of the invention to various vectors by a conventional method. The vector may be any of a variety of vectors conventional in the art, such as various plasmids, phage or viral vectors, and the like, with pET42a being preferred. Preferably, the recombinant expression vector of the present invention can be obtained by the following method: the LpADH gene product obtained by PCR amplification was ligated with pMD-18T to form a cloning vector. The cloning vector is subjected to double restriction enzyme digestion by restriction enzyme Nde I/Xho I, recovered by gel digestion and connected with pET42a recovered by the same enzyme digestion treatment, and the LpADH recombinant expression plasmid pET42a-LpADH of the invention is constructed.

The invention also provides a genetically engineered bacterium containing the coding gene or the recombinant vector. The genetically engineered bacterium can be obtained by transforming the recombinant expression vector of the invention into a host microorganism. The host microorganism may be any of various host microorganisms conventionally used in the art as long as it is satisfied that the recombinant expression vector can stably self-replicate and that the carried LpADH gene of the present invention can be efficiently expressed. The invention is preferably E.coli, more preferably E.coli BL21 (DE 3). The recombinant plasmid pET42a-LpADH is transformed into E.coli BL21 (DE 3) to obtain recombinant escherichia coli, and the recombinant bacteria, enzyme solution, cells or immobilized forms of the enzyme solution are used as enzyme sources for biocatalysis.

The invention also relates to application of the LpADH in biocatalysis of DMAK and MMAK for asymmetric reduction preparation of (S) -DMAA and (S) -MMAA. The application is to make substrate DMAK or MMAK, enzyme and NADP ⁺ Adding auxiliary substrate and buffer solution into a reactor for catalytic reaction; the concentration of the substrate is 1-100g/L; the buffer solution is citric acid-sodium citrate with pH of 4.0-9.0, phosphate, tris-HCl buffer solution and the like; the reaction temperature of the application is 10-40 ℃; the auxiliary substrate is glucose monohydrate, and Glucose Dehydrogenase (GDH) is added to form a coenzyme circulation system. The reaction formula is shown below.

Advantageous effects of the inventionThe main effects are as follows: obtaining a novel alcohol dehydrogenase which can catalyze the asymmetric reduction of DMAK and MMAK to prepare (S) -DMAA and (S) -MMAA, wherein the substrate concentration is 100g/L, and the conversion rate is 100g/L>99.9%, ee value>99.5% coenzyme NADP ⁺ The amount was only 0.1mmol/L. The reaction condition is mild, the reaction rate is high, the specificity is strong, the optical purity of the product is high, and the method has great industrial application potential.

Drawings

FIG. 1 is a map of the expression vector pET42 a-LpADH;

FIG. 2 is a SDS-PAGE map of the induced expression of engineering bacteria; lane M is protein molecular weight Marker, lane 1 is IPTG-induced e.coli BL21 (DE 3)/pET 42 a-lpadih cell disruption supernatant, and lane 2 is IPTG-induced e.coli BL21 (DE 3)/pET 42 a-lpadih cell disruption inclusion body pellet;

Detailed Description

The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:

example 1: construction of recombinant expression vector and recombinant engineering bacteria

The alcohol dehydrogenase is obtained by a gene database mining method, optimized according to the amino acid sequence of the enzyme and the codon preference of escherichia coli, restriction enzyme cutting sites Nde I and Xho I are designed according to the characteristics of an expression vector pET42a, and the alcohol dehydrogenase gene LpADH (shown as SEQ ID NO: 2) is synthesized by a total gene synthesis method.

The LpADH gene fragment was subjected to double digestion and recovery treatment using Nde I and Xho I restriction enzymes, and the fragment was ligated with commercial vector pET42a treated with the same restriction enzymes overnight at 16℃using T4DNA ligase, thereby constructing recombinant expression vector pET42a-LpADH. The constructed recombinant expression vector pET42a-LpADH is transformed into E.coli BL21 (DE 3) competent cells, coated on LB plate containing 50 mug/mL kanamycin with final concentration, and cultured overnight at 37 ℃; colony PCR identification is carried out by randomly picking clones from colonies growing on the flat plate, and positive clone sequencing verification shows that the recombinant expression vector pET42a-LpADH is successfully transformed into an expression host E.coli BL21 (DE 3), and the LpADH gene is successfully cloned into Nde I and Xho I sites of pET42a (figure 1).

Example 2: preparation and expression verification of recombinant LpADH-containing bacterial cells

Inoculating the genetically engineered bacterium E.coli BL21 (DE 3)/pET 42a-LpADH constructed in example 1 into LB medium containing 50 mug/mL kanamycin, and culturing at 37 ℃ until the concentration of thalli OD ₆₀₀ The value is 0.4-0.6, IPTG with the final concentration of 0.1mmol/L is added into LB liquid culture medium, after induced culture is carried out at 22 ℃ for overnight, the culture solution is centrifuged at 4 ℃ for 10min at 10000 Xg, the supernatant is discarded, and the wet bacterial cells containing recombinant LpADH are collected.

1g of wet thalli is weighed, suspended in 10mL of phosphate buffer solution (pH 7.0), and subjected to ultrasonic crushing, and the size and the expression quantity of the soluble recombinant protein are verified by SDS-PAGE electrophoresis (figure 2), wherein the significant overexpression exists at the target molecular weight of the recombinant protein, and most of the recombinant protein is expressed in a soluble way and a small quantity is inclusion body.

Example 3: enzyme activity and stereoselectivity verification of alcohol dehydrogenase

The crushed supernatant containing LpADH prepared in example 2 was used as a catalyst and DMAK was used as a substrate.

The catalytic system comprises the following components and reaction conditions: 10mL of phosphate buffer (100 mmol/L, pH 7.0) containing 10g/L DMAK,15g/L glucose monohydrate, 1mM NADP ⁺ 1mL of the disrupted supernatant containing LpADH and 0.2mL of the glucose dehydrogenase (Bacillus megaterium as a source of gene, disruption solution preparation was the same as in example 2). The reaction was carried out at 30℃and at a rotational speed of 150rpm, after 3 hours of reaction, the conversion of the substrate DMAK was checked by HPLC (for detection methods see patent U.S. Pat. No. 8673607B 2)>99%, ee value of product>99.5％。

Example 4: alcohol dehydrogenase freeze-dried powder preparation

100g of the recombinant cells prepared in example 2 and containing LpADH were added to 400mL of water to prepare a bacterial suspension. Adding the bacterial suspension into a precooled high-pressure homogenizer, homogenizing under 650-900bar, centrifuging at 4deg.C and 10000 Xg for 20min, collecting supernatant, pre-freezing at-20deg.C, vacuum lyophilizing for 48 hr, and grinding to obtain LpADH lyophilized powder.

Example 5: catalytic preparation of (S) -DMAA by LpADH

The LpADH lyophilized enzyme powder obtained in the method of example 4 was used as a catalyst and DMAK was used as a substrate.

The catalytic system comprises the following components and reaction conditions: 50mL of phosphate buffer (100 mmol/L, pH 6.5) was added 0.5g of LpADH lyophilized enzyme powder, 0.1g of glucose dehydrogenase lyophilized enzyme powder (preparation method same as in example 4), 5g of DMAK,7g of glucose monohydrate and 1mmol/L of NADP ⁺ The reaction system is formed. The reaction is carried out at 30 ℃, the pH is controlled to be 6.5 during the process, and the rotating speed is 200rpm; after 2h of reaction, conversion of substrate DMAK>99%, ee value of product>99.5％。

Example 6: batch fed preparation of (S) -DMAA

The LpADH lyophilized enzyme powder obtained in the method of example 4 was used as a catalyst and DMAK was used as a substrate.

The catalytic system comprises the following components and reaction conditions: 40mL of phosphate buffer (100 mmol/L, pH 6.5) was added 0.5g of LpADH lyophilized enzyme powder, 0.1g of glucose dehydrogenase lyophilized enzyme powder (preparation method same as in example 4), 1g of DMAK,1.4g of glucose monohydrate and 0.4mmol/L of NADP ⁺ An initial reaction system was constituted, and 1g of DMAK and 1.4g of glucose monohydrate were fed every 0.5 hour, for a total of 4 times. The reaction is carried out at 30 ℃, the pH is controlled to be 6.5 during the process, and the rotating speed is 200rpm; after 5h of reaction, conversion of substrate DMAK>99%, ee value of product>99.5％。

Example 7: batch fed preparation of (S) -DMAA

The LpADH lyophilized enzyme powder obtained in the method of example 4 was used as a catalyst and DMAK was used as a substrate.

The catalytic system comprises the following components and reaction conditions: 40mL of phosphate buffer (100 mmol/L, pH 6.5) was added 0.3g of LpADH lyophilized enzyme powder, 0.1g of glucose dehydrogenase lyophilized enzyme powder (preparation method same as in example 4), 1g of DMAK,1.12g of glucose monohydrate and 0.1mmol/L of NADP ⁺ An initial reaction system was constituted, 1g of DMAK and 1.12g of glucose monohydrate were fed every 1 hour, and a total of 4 times were fed. The reaction is carried out at 30 ℃, the pH is controlled to be 6.5 during the process, and the rotating speed is 200rpm; after 6h of reaction, conversion of substrate DMAK>99%, ee value of product>99.5％。

Example 8: batch fed preparation of (S) -MMAA

The LpADH lyophilized enzyme powder obtained in the method of example 4 was used as a catalyst and MMAK was used as a substrate.

The catalytic system comprises the following components and reaction conditions: 40mL of phosphate buffer (100 mmol/L, pH 6.5) was added 0.5g of LpADH lyophilized enzyme powder, 0.1g of glucose dehydrogenase lyophilized enzyme powder (preparation method same as in example 4), 1g of MMAK,1.2g of glucose monohydrate and 0.1mmol/L of NADP ⁺ An initial reaction system was constituted, 1g of MMAK and 1.2g of glucose monohydrate were fed every 1 hour, and a total of 4 times were fed. The reaction is carried out at 30 ℃, the pH is controlled to be 6.5 during the process, and the rotating speed is 200rpm; after 7h of reaction, the MMAK conversion of the substrate was checked by HPLC (for detection methods see patent US9228213B 2)>99%, ee value of product>99.5％。

Sequence listing

<110> Shanghai Oibo biological medicine Co., ltd

<120> an alcohol dehydrogenase and its use in the preparation of duloxetine key intermediates

<130> 2021.11.05

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 252

<212> PRT

<213> Lactobacillus parafarraginis

<400> 1

Met Ser Asn Arg Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Thr

1 5 10 15

Leu Gly Ile Gly Leu Ala Val Ala His Arg Phe Val Asp Glu Gly Ala

20 25 30

Lys Val Val Ile Thr Gly Arg Arg Ala Asp Ile Gly Glu Arg Ala Ala

35 40 45

Lys Ser Ile Gly Gly Pro Asp Val Ile Arg Phe Met Arg Gln Asp Ala

50 55 60

Ser Asp Glu Asp Gly Trp Val Asp Thr Trp Asp Lys Thr Glu Glu Ala

65 70 75 80

Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Asp Val Val

85 90 95

Lys Ser Val Glu Asn Thr Thr Thr Glu Glu Trp Arg Asn Val Leu Ala

100 105 110

Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg

115 120 125

Met Lys Asn Lys Asn Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile

130 135 140

Phe Gly Met Val Gly Asp Pro Thr Val Gly Ala Tyr Asn Ala Thr Lys

145 150 155 160

Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Val Asp Cys Ala Leu

165 170 175

Lys Asp Tyr Gly Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys

180 185 190

Thr Pro Met Leu Asp Asn Val Glu Gly Ala Glu Ala Ala Trp Ser Ala

195 200 205

Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asp Asp Ile Ala

210 215 220

Trp Val Cys Val Tyr Leu Ala Ser Gly Glu Ser Lys Phe Ala Thr Gly

225 230 235 240

Ser Glu Phe Thr Ile Asp Gly Gly Trp Thr Ala Gln

245 250

<210> 2

<211> 759

<212> DNA

<213> Lactobacillus parafarraginis

<400> 2

atgagcaacc gtctgaaagg taaagttgcg attgttacgg gtggtaccct gggcattggc 60

ctggcagttg cacatcgttt tgtggatgaa ggcgcgaaag tggttattac cggccgtcgt 120

gcagatattg gtgaacgtgc ggcgaaaagc attggtggtc cggatgttat tcgttttatg 180

cgtcaggatg caagcgatga agatggttgg gttgatacgt gggataaaac agaagaagca 240

tttggtccgg tgaccacagt tgtgaataac gcaggtattg atgtggtgaa aagcgttgaa 300

aataccacca cagaagaatg gcgtaatgtg ctggcagtga atctggatgg tgtgtttttt 360

ggtacacgtc tgggcattca gcgtatgaaa aataaaaatc tgggtgcatc aatcatcaat 420

atgagcagca tttttggtat ggttggtgat ccgaccgtgg gtgcgtataa tgcgaccaaa 480

ggcgcagttc gtattatgag caaaagcgca gcagtggatt gtgcactgaa agattatggt 540

gttcgtgtta atacagttca tccgggtcct attaaaacac cgatgctgga taatgttgaa 600

ggtgcagaag cggcatggtc agcacgtaca aaaaccccga tgggccatat tggtgaaccg 660

gatgatatcg catgggtttg tgtttatctg gcaagcggtg aaagcaaatt tgcgacaggt 720

agcgaattta caattgatgg tggttggaca gcgcagtaa 759

Claims (5)

1. An alcohol dehydrogenase characterized in that the amino acid sequence of said alcohol dehydrogenase has at least 90% homology with SEQ ID NO. 1.

2. A coding gene encoding the alcohol dehydrogenase according to claim 1.

3. A recombinant vector constructed from the coding gene of claim 2.

4. A recombinant genetically engineered bacterium transformed by the recombinant vector of claim 3, wherein the genetically engineered bacterium comprises, but is not limited to, hosts such as E.coli, pichia pastoris and bacillus subtilis.

5. Use of a recombinant genetically engineered bacterium according to claim 4 for the preparation of (S) -N, N-dimethyl-3-hydroxy-3- (2-thiophene) -1-propylamine ((S) -DMAA) and (S) -N-methyl-3-hydroxy-3- (2-thiophene) -1-propylamine ((S) -MMAA), characterized in that the use is based on N, N-dimethyl-3-keto-3- (2-thiophene) -1-propylamine or a salt thereof (DMAK) and N-methyl-3-keto-3- (2-thiophene) -1-propylamine or a salt thereof (MMAK) as substrates in the presence of coenzyme NADP ⁺ And a coenzyme regeneration system, under the catalysis of alcohol dehydrogenase, recombinant cells containing alcohol dehydrogenase or immobilized cells (enzymes).

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