CN110283799B - Aldehyde ketone reductase BsAKR (YvgN) and mutant and application thereof - Google Patents
Aldehyde ketone reductase BsAKR (YvgN) and mutant and application thereof Download PDFInfo
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- CN110283799B CN110283799B CN201910375432.3A CN201910375432A CN110283799B CN 110283799 B CN110283799 B CN 110283799B CN 201910375432 A CN201910375432 A CN 201910375432A CN 110283799 B CN110283799 B CN 110283799B
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- 108010084469 Aldo-Keto Reductases Proteins 0.000 claims abstract description 23
- 239000000758 substrate Substances 0.000 claims abstract description 23
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- 238000006243 chemical reaction Methods 0.000 claims description 8
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Abstract
The invention relates to the technical field of biology, and relates to a plurality of mutants of ketoreductase and application thereof, wherein the mutants are obtained by carrying out mutation on wild Bacillus subtilis aldo-ketoreductase (BsAKR (YvgN)), and in particular relates to a plurality of mutants of ketoreductase and a preparation method thereof. Also relates to a method for obtaining an S or R configuration secondary alcohol compound with optical activity by catalyzing the reduction of alpha-keto acid ester compounds and 1-acetophenone compounds by using the aldehyde ketone reductase BsAKR (YvgN) and the mutant thereof. Compared with a wild type, the stereoselectivity of the mutant is improved or reversed, the substrate is catalyzed to obtain chiral alcohols with two configurations (R or S type), such as optically pure (R) -o-chloromandelic acid methyl ester, (S) -2-hydroxy-4-phenyl ethyl butyrate and (R) -2-hydroxy-4-phenyl ethyl butyrate, and the mutant has good application value in the field of chiral alcohol preparation.
Description
Technical Field
The invention relates to the technical field of biology, and relates to a plurality of mutants of ketoreductase and application thereof, wherein the mutants are obtained by carrying out mutation on wild Bacillus subtilis aldo-ketoreductase (BsAKR (YvgN)), and in particular relates to a plurality of mutants of ketoreductase and a preparation method thereof. Also relates to a method for obtaining an S or R configuration secondary alcohol compound with optical activity by catalyzing the reduction of alpha-keto acid ester compounds and 1-acetophenone compounds by using the aldehyde ketone reductase BsAKR (YvgN) and the mutant thereof.
Background
The chiral carbon atom of the chiral alcohol is connected with an active hydroxyl functional group, so that the chiral alcohol becomes a key synthetic intermediate for fine chemical engineering, medicine and agricultural application. For example, ethyl chiral alcohol (3R, 5R) -6-cyano-3,5-dihydroxyhexanoate (ethyl (3R, 5R) -6-cyanoo-3, 5-dihydrohexanoate) is a hypolipidemic agent developed by the company Peucedani, atorvastatin calcium (Lipitor)TM) Key chiral intermediates in the synthesis process. In addition, chiral alcohols (such as chiral halohydrins) containing other active functional groups can also be converted into other chiral modules with wider application values, such as chiral epoxy compounds, chiral diols, chiral hydroxy nitriles, chiral hydroxy azides, chiral amino alcohols and the like.
The alpha-keto ester compound is widely applied in the fields of chemical industry and medicine as an important synthetic intermediate, and the R-and S-forms of the alcohol have important application values. (R) -O-chloromandelic acid methyl ester (molecular formula is o-Cl-C)6H4CH(OH)COOCH3Molecular weight 200.62, cas number: 32345-59-8) is an important chiral intermediate for synthesizing clopidogrel, a platelet aggregation inhibitor. Clopidogrel is an Adenosine Diphosphate (ADP) receptor blocker, can be combined with an ADP receptor on the surface of a platelet membrane to prevent fibrinogen from being treatedBinds with glycoprotein GPIIb/IIIa receptor, thereby inhibiting platelet aggregation, is mainly used for treating acute myocardial infarction, and has wide market at home. (R) -2-hydroxy-4-phenylbutyric acid Ethyl ester [ Ethyl (R) -2-hydroxy-4-phenylbutyrate, (R) -HPBE]Are important precursors for the manufacture of various Angiotensin Converting Enzyme (ACE) inhibitors, such as benazepril, enalapril and ramipril, which are widely used in the treatment of hypertension and congestive heart failure. ACE inhibitors interfere with the conversion of angiotensin I to angiotensin II, and are beneficial in reducing cardiac preload. And meanwhile, the ACE inhibitor can prevent or reverse cardiovascular remodeling and inhibit hypertrophy and hyperplasia of cardiac muscle and blood vessels. Occupies a large share in the domestic antihypertensive drug market. In addition, (R) -phenylacetylcarbinol is a precursor of (-) -ephedrine, a drug used to prevent hypotension during spinal anesthesia, and is also used to treat asthma, lethargy.
Optically pure 1-phenyl ethanol compounds, in particular alpha halogen substituted 1-phenyl ethanol compounds are important chiral intermediates in the synthesis process of medicaments and other fine chemicals. At present, various methods for obtaining optically pure chiral purity are developed, including two major classes of kinetic resolution and asymmetric synthesis. The theoretical yield of the asymmetric synthesis method of the prochiral carbonyl compound can reach 100 percent, and the method is an important way for obtaining the optically pure 1-phenyl ethanol compound. Researchers have developed chemical synthesis methods for synthesizing optically pure chiral alcohols by using chiral metal ligands as catalysts, and some of them are applied to industrial production, however, their application in synthesis of drugs and the like is limited due to their harsh conditions and heavy metal residue problems. Biocatalysis has received increasing attention in the synthesis of chiral alcohols due to its advantages of being environmentally friendly, mild in reaction conditions, high in regio-and stereoselectivity, and the like.
Aldo Ketoreductases (AKRs) are a type that depends on NAD (P)+The oxidoreductase of (1), comprising more than 190 members. The structure is composed of (alpha/beta)8The structure comprises a barrel-shaped structure and a loop region, an active center consists of loop4, loop7 and loop C, and catalytic active sites comprise Tyr, asp, lys and His. Aldehyde ketonesThe substrate of the proenzyme is prochiral ketone, and the type is wide, such as alpha-ketoester, beta-ketoester, aliphatic chain ketone, cyclic ketone and the like. In order to improve the practicability of the aldehyde ketone reductase in the process of asymmetrically catalyzing and reducing alpha-keto ester and 1-phenyl ethyl ketone compounds, a method for changing the catalytic property of the enzyme by rational protein design and directed evolution is used for modifying the stereoselectivity of the aldehyde ketone reductase by researchers. It has been proposed in the prior art that the substrate profile of an aldehyde-ketone reductase can be influenced by Protein modifications of the loop region of the aldehyde-ketone reductase (Protein Eng, des Sel 2014 27. Furthermore, zhang et al semi-rational design of a thermostable aldoketoreductase from thermatopaa maritima for the synthesis of enantiomerically pure ethyl-2-hydroxy-4-phenylbutyric acid showed that W21 and W86 of this AKR play an important role in determining enantioselectivity (Sci Rep 2017.
The aldehyde ketone reductase BsAKR (YvgN) which is obtained from the bacillus subtilis by gene mining in the laboratory shows poor activity and stereoselectivity on alpha-keto ester and 1-acetophenone in a wild state. The stereoselectivity of the enzyme is improved and reversed through rational design and protein directed evolution, the biocatalytic preparation of the optically pure chiral alcohol is realized, and the application value is very important.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides the aldehyde ketone reductase BsAKR (YvgN) and also provides a reductase mutant obtained by mutating the aldehyde ketone reductase, and the aldehyde ketone reductase BsAKR (YvgN) and the mutant can improve the enantioselectivity and the activity of the aldehyde ketone reductase BsAKR (YvgN) and the mutant to alpha-keto acid ester compounds or 1-acetophenone compounds, so that the corresponding chiral alcohol route obtained by reducing the aldehyde ketone reductase is efficient and feasible.
The invention provides aldehyde ketone reductase BsAKR (YvgN), which is determined to be wild type bacillus subtilis aldehyde ketone reductase by sequencing, and the sequence of the aldehyde ketone reductase BsAKR is shown in SEQ ID NO. 1.
The invention also provides a mutant of the aldehyde ketone reductase with improved property, which is obtained by mutation of a wild type bacillus subtilis aldehyde ketone reductase BsAKR (YvgN) gene (SEQ ID NO. 1), the aldehyde ketone reductase is expressed in escherichia coli engineering bacteria, and the aldehyde ketone reductase and the mutant thereof can reduce alpha-keto acid ester compounds to obtain S or R configuration secondary alcohols with optical activity.
Further, the aldehyde ketone reductase gene is connected to an expression plasmid to obtain the recombinant aldehyde ketone reductase. The expression plasmid is:
the recombinant aldone reductase comprises an amino acid sequence with at least 75 percent of identity with SEQ ID NO. 02.
Further, the present invention provides mutants of aldehyde ketone reductase BsaKR (YvgN) obtained by mutation at position 25 or 113, or mutation at both positions 25 and 113 of recombinant aldehyde ketone reductase (i.e., SEQ ID NO. 2).
The mutant of the aldehyde ketone reductase BsAKR (YvgN) corresponds to the 25 th residue of SEQ ID NO.2 as a smaller amino acid residue, and the 113 th residue as a larger amino acid residue, and the amino acids are important for improving the activity and the enantioselectivity of the aldehyde ketone reductase.
The smaller amino acids involved in the present invention are Ala, ser, thr, val, ile and Leu, preferably serine (Ser); the larger amino acid residues involved are Pro, phe, try, leu, preferably phenylalanine (Phe).
In some embodiments of the invention, the aldone reductase mutants containing mutations exhibit higher activity and stereoselectivity for alpha-keto acid ester compounds and 1-acetophenone compounds, and comprise one or two mutations, namely, a mutation at position 25 or position 113, or a mutation at positions 25 and 113 simultaneously, and a mutation at position 25 of an amino acid sequence to Ser; 113 th amino acid sequence is mutated to Phe.
The amino acid sequence of the aldehyde ketone reductase mutant is preferably shown in SEQ ID NO.4,6 and 8.
Embodiments of the invention include nucleic acids encoding the aldoketoreductase mutants having at least 75% sequence identity to the nucleic acid sequence encoding the BsaKR (YvgN) mutant of the invention.
The sequence of the nucleic acid capable of coding the mutant is shown in SEQ ID NO.3,5 and 7.
Related embodiments of the invention also include vectors comprising these nucleic acids and host cells comprising such vectors.
The invention also provides application of the aldone reductase and the mutant thereof in reducing alpha-keto acid esters and 1-acetophenone compounds. The wild-type aldo-keto reductase BsAKR (YvgN) and the mutant thereof, or the cell containing the wild-type or mutant enzyme can be used as a catalyst to catalyze and asymmetrically reduce alpha-keto ester or 1-acetophenone compounds to obtain corresponding optical pure chiral products.
The wild-type aldone reductase BsAKR (YvgN) and the mutant thereof, or cells containing the wild-type or mutant enzyme can be used as a catalyst to catalyze and asymmetrically reduce alpha-keto acid esters and 1-acetophenone compounds to obtain corresponding optical homochiral products with different configurations (S and R configurations).
The alpha-ketonic acid ester compound is shown as a formula I, and the 1-phenyl ethyl ketone compound is shown as a formula II:
wherein,
R1is C1-C4 alkyl, phenyl or phenyl with substituent, and the substituent of the phenyl is halogen or C1-C4 alkyl;
R2is C1-C4 alkyl;
R3is a halogenated C1-C4 alkyl group.
R4Is halogen, halogenated C1-C4 alkyl;
preferably, the first and second air flow paths are arranged in parallel,
R1is phenyl or phenyl with substituent, and the substituent is halogen;
R2is C1-C2 alkyl;
R3is halogenated C1-C4 alkyl, and the halogen of the halogenated alkyl is F or Cl.
R4Is chlorine, halogenated C1-C4 alkyl;
some typical structures are described below:
one embodiment of the invention provides a method for asymmetrically reducing alpha-keto acid esters and 1-acetophenone compounds, which comprises the following steps:
in a phosphate buffer solution of pH5-7, in the presence of glucose dehydrogenase, glucose and NADP+In the presence of aldehyde ketone reductase or its mutant, alpha-keto acid ester or 1-phenyl ethyl ketone compound is reduced to produce optically active chiral secondary alcohol.
The dosage of aldone reductase mutant is 0.02-40g/L, the dosage of glucose dehydrogenase is 0.01-5g/L, the dosage of glucose is 6-200g/L, and NADP+The dosage is 0.1-0.5mmol, the concentration of the alpha-ketonic acid ester or the 1-phenyl ethyl ketone compound is 3-100g/L, the buffer solution is a phosphate buffer solution with the pH value of 5-7, and the reaction temperature is 30-40 ℃.
The aldehyde ketone reductase or the mutant thereof can exist in various forms such as cells, crude enzyme powder, enzyme solution, immobilized enzyme and the like in various feasible ways.
The invention has the beneficial technical effects that: compared with a naturally-occurring ketoreductase wild type, the activity and enantioselectivity of the aldehyde ketone reductase mutant on alpha-ketonic acid esters and 1-acetophenone compounds are reversed and improved by mutating bacillus subtilis aldehyde ketone reductase BsAKR (YvgN).
Drawings
FIG. 1 shows the construction of genes of wild-type aldo-keto reductase BsAKR (YvgN) and its mutant enzyme, and recombinant expression vector containing the genes.
FIG. 2 shows the HPLC detection results of the reduction of substrate Sub1 catalyzed by wild-type aldo-keto reductase BsaKR (YvgN) (SEQ ID NO. 2) and its mutant (SEQ ID NO.4,6, 8);
sub1: sub1 substrate control; rac-1: product racemate control
FIG. 3 shows the HPLC results of the reduction of substrate Sub2 catalyzed by wild-type aldoketoreductase BsAKR (YvgN) (SEQ ID NO. 2) and its mutant (SEQ ID NO.4,6, 8).
FIG. 4 shows the HPLC detection results of the reduction of substrate Sub3 catalyzed by wild-type aldoketoreductase BsaKR (YvgN) (SEQ ID NO. 2) and its mutant (SEQ ID NO.4,6, 8).
FIG. 5 shows the HPLC detection results of the wild-type aldoketoreductase BsAKR (YvgN) mutant (SEQ ID NO.4, 8) enzyme catalyzing the reduction of the substrate Sub 4.
FIG. 6 shows the HPLC detection results of the wild-type aldoketoreductase BsAKR (YvgN) mutant (SEQ ID NO.4, 8) enzyme catalyzing the reduction of the substrate Sub 5.
FIG. 7 shows the HPLC results of the reduction of substrate Sub6 catalyzed by wild-type aldoketoreductase BsaKR (YvgN) (SEQ ID NO. 2) and its mutant (SEQ ID NO.4,6, 8).
FIG. 8 shows the HPLC results of the reduction of the substrate Sub7 catalyzed by wild-type aldoketoreductase BsAKR (YvgN) (SEQ ID NO. 2) and its mutant (SEQ ID NO.4,6, 8).
FIG. 9 shows the HPLC results of the reduction of substrate Sub8 catalyzed by wild-type aldoketoreductase BsaKR (YvgN) (SEQ ID NO. 2) and its mutant (SEQ ID NO.4,6, 8).
FIG. 10 shows the HPLC results of the reduction of substrate Sub9 catalyzed by wild-type aldoketoreductase BsaKR (YvgN) (SEQ ID NO. 2) and its mutant (SEQ ID NO.4,6, 8).
FIG. 11 shows the HPLC results of the reduction of the substrate Sub10 catalyzed by wild-type aldone reductase BsAKR (YvgN) (SEQ ID NO. 2) and its mutant (SEQ ID NO. 4).
FIG. 12 shows the HPLC results of the reduction of substrate Sub11 catalyzed by wild-type aldoketoreductase BsaKR (YvgN) (SEQ ID NO. 2) and its mutant (SEQ ID NO.4,6, 8).
FIG. 13 shows the HPLC detection results of the wild-type aldoketoreductase BsAKR (YvgN) mutant (SEQ ID NO.4, 6) enzyme catalyzing the reduction of the substrate Sub 12.
FIG. 14 shows the HPLC results of the reduction of substrate Sub13 catalyzed by wild-type aldoketoreductase BsaKR (YvgN) (SEQ ID NO. 2) and its mutant (SEQ ID NO.4,6, 8).
Detailed Description
The aldehyde ketone reductase mutant of the present invention, and the reduction of α -keto acid esters and 1-phenylacetones using the enzyme are described below by way of specific embodiments. Unless otherwise specified, the protocols used in the present invention are well known to those skilled in the art. Furthermore, the examples are to be construed as illustrative, and not restrictive.
Definitions of certain terms.
Asymmetric reduction is a method for obtaining an optically pure corresponding reduction product by reducing a prochiral compound, and in the invention, the aldehyde ketone reductase catalyzes an alpha-keto acid ester compound or a 1-phenyl acetophenone compound to selectively obtain corresponding alcohol with S or R configuration.
Enantiomeric excess is defined as the amount of one isomer a in an enantiomeric mixture which is more abundant than the other isomer B in the total amount, abbreviated ee, and is expressed by the formula (a-B)/(a + B) × 100%, and the enantiomeric excess is used to indicate the optical purity of a chiral compound. The higher the ee value, the higher the optical purity.
The 20 amino acids are abbreviated as follows
| ASP | D | Aspartic acid | Ile | I | Isoleucine |
| Thr | T | Threonine | Leu | L | Leucine |
| Ser | S | Serine | Thr | T | Tyrosine |
| Glu | E | Glutamic acid | Phe | F | Phenylalanine |
| Pro | P | Proline | His | H | Histidine |
| Gly | G | Glycine | Lys | L | Lysine |
| Ala | A | Alanine | Arg | R | Arginine |
| Cys | C | Cysteine | Trp | W | Tryptophan |
| Val | V | Valine | Gln | Q | Glutamine |
| Met | M | Methionine | Asn | N | Asparagine |
The amino acids are classified into larger and smaller amino acids according to the steric hindrance of amino acid side chain groups, the smaller amino acids involved in the invention are Ala, ser, thr, val, ile and Leu, and the larger amino acid residues are Pro, phe, try and Leu.
The examples relate to the formulation of the culture medium.
LB liquid medium: 0.5% yeast extract, 1% peptone and 1% sodium chloride (for example, preparing solid medium, adding 1.5% agar before sterilization), and autoclaving at 115 deg.C for 30min.
Example 1 extraction of Bacillus subtilis genomic DNA
a) After bacillus subtilis is cultured by LB liquid overnight, the fermentation liquor is centrifuged for 5min by 3000r/min in a 1mL centrifuge tube, the supernatant is discarded to collect the bacteria, and the steps can be repeated for several times to obtain enough cells; b) Extracting the genome of the bacillus cereus according to the operation instruction of the bacterial genome DNA extraction kit.
Example 2 cloning of the wild-type BsAKR (YvgN) Gene
The Bacillus subtilis genomic DNA obtained in example 1 was used as a template for PCR reaction in the following reaction system:
and (3) amplification procedure: 94 ℃ below zero: 10min (30s at 94 ℃, 30s at 45 ℃,30 s at 72 ℃) and 10min at 72 ℃.
Primer 1: bsaKR (YvgN) -EcoR I-F: CCGGAATTCGATGCCAACAAGTTTAAAA；
Primer 2: bsaKR (YvgN) -Xho I-R: CCGCTCGAGAAACAGAAGCTCATCAGG；
Restriction sites are underlined;
the DNA fragment obtained by PCR amplification is purified by a gel recovery kit. E.coli DH 5. Alpha. Containing pET28b MBP Plasmid was cultured overnight in LB broth at 37 ℃ and 220r/min, and Plasmid extraction was performed using the reference TIAnprep Mini Plasmid Kit.
The target fragment and the plasmid pET28b MBP plasmid are subjected to double restriction enzyme digestion, and the restriction enzyme digestion system is as follows:
and recovering fragments of the enzyme digestion product by using a gel recovery kit, and connecting the fragments by using T4 ligase.
Example 3 E.coli Rosetta (DE 3) preparation and transformation of competent cells
a) 0.4mL of the seed culture medium is inoculated into 20mL of LB liquid culture medium for culture for 3h; b) 2mL of thalli are enriched in a 1.5mL EP tube twice at 3000r/min for 5min, and the supernatant is discarded; c) Adding 100 μ l ice-cold TSS solution, re-suspending thallus, ice-cooling for 30min; d) Adding 20 μ L of the connecting solution, gently rotating and mixing, and performing ice bath for 30min; e) Heat shock was performed at 42 ℃ for 60s, ice bath was performed for 2min, and 600. Mu.L of LB liquid medium was added. Culturing at 37 ℃, and performing shaking culture at 150r/min for 1h; f) mu.L of each was plated on LB-resistant plates.
EXAMPLE 4 construction of mutants
The construction of the mutant pools was performed by overlap extension PCR using the mutant and flanking primers. (designed mutation primers are as follows:.)
PCR amplification conditions: 94 ℃ below zero: 10Min (94 ℃ 30s,45 ℃ 30s,72 ℃ 30 s) 35 cycles, 72 ℃: and (5) 10min. The resulting gene fragment was purified and amplified by PCR as follows
The desired fragment was obtained by PCR as described in example 2 and ligated into the pET28b MBP vector and transformed into E.coli Rosetta (DE 3) as described in example 3. Respectively obtaining wild aldehyde ketone reductase BsAKR (YvgN) and mutants thereof.
Example 5 mutant expression
a) Inoculating a single colony into a 4ml LB liquid culture medium with kanamycin resistance, and culturing at 37 ℃ and 200rpm for 6 hours to obtain a seed solution; b) Inoculating 20 μ l seed solution into 20ml LB liquid culture medium with kanamycin resistance, culturing at 37 deg.C and 200rpm until OD600 of the culture solution reaches 0.8-1.0, adding 0.5mM IPTG, and cooling to 20 deg.C for inducing expression for 20 hr; c) Collecting thallus by centrifuging culture solution at 4000rpm × 15min, washing twice with normal saline, reselecting thallus with 0.1M pH6.0 sodium phosphate buffer solution, adding lysozyme with final concentration of 1mg/ml, crushing at 30 deg.C and 200rpm for 1h, centrifuging at 4 deg.C and 10000rpm, collecting supernatant, and screening.
Example 6 expression of glucose dehydrogenase
Coli Rosetta (DE 3) containing the pET22b-GDH plasmid was expressed as described in example 5 to obtain glucose dehydrogenase.
Example 7 catalytic reduction of crude enzyme solution
Typical substrates referred to in this example are shown below:
reaction system: the supernatants were mixed together in 450. Mu.l portions each as described in examples 5 and 6 to a final concentration of 0.3mM NADP+8mg of glucose and 4mg of substrate (100 mu.l of methanol for dissolution) and reacting for 6 hours at 30 ℃; extracted three times with equal volumes of ethyl acetate.
Example 8 high Performance liquid phase analysis of substrates and products
The substrate of the high performance liquid phase analysis comprises Sub1-13, and the structure of Sub1-13 is as follows:
the conditions for detecting Sub1, 2 and the product are as follows: a chromatographic column: AD-H, mobile phase: n-hexane: isopropanol =90, flow rate: 0.8ml/min, wavelength: 254nm, column temperature: 25 ℃, detector: and a UV detector.
The conditions for detecting Sub3, 4, 5 and the product are as follows: a chromatographic column: OD-H, mobile phase: n-hexane: isopropanol =90, flow rate: 0.8ml/min, wavelength: 254nm, column temperature: 25 ℃, detector: and a UV detector.
The conditions for detecting Sub6, 8 and the product were: a chromatographic column: OD-H, mobile phase: n-hexane: isopropanol =98, flow rate: 0.8ml/min, wavelength: 254nm, column temperature: 25 ℃, detector: and a UV detector.
The conditions for detecting Sub7, 9 and the product are as follows: a chromatographic column: OB-H, mobile phase: n-hexane: isopropanol =95, flow rate: 0.8ml/min, wavelength: 254nm, column temperature: 25 ℃, detector: and a UV detector.
The conditions for detecting Sub10 and the product were: a chromatographic column: OJ-H, mobile phase: n-hexane: isopropanol =95, flow rate: 0.8ml/min, wavelength: 254nm, column temperature: 25 ℃, detector: and a UV detector.
The conditions for detecting Sub11 and the product were: a chromatographic column: OJ-H, mobile phase: n-hexane: isopropanol =90, flow rate: 0.8ml/min, wavelength: 254nm, column temperature: 25 ℃, detector: and a UV detector.
The conditions for detecting Sub12 and the product were: a chromatographic column: OD-H, mobile phase: n-hexane: isopropanol =90, flow rate: 0.8ml/min, wavelength: 254nm, column temperature: 25 ℃, detector: and a UV detector.
The conditions for detecting Sub13 and the product were: a chromatographic column: AD-H, mobile phase: n-hexane: isopropanol =95, flow rate: 0.8ml/min, wavelength: 254nm, column temperature: 25 ℃, detector: and a UV detector.
The detection results of the wild-type aldo-keto reductase (SEQ ID NO. 2) and its mutant (SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO. 8) catalyzing the above-mentioned substrate are shown in Table 1:
TABLE 1 detection results of aldehyde ketone reductase BsAKR (YvgN) wild type and its mutant for catalytic reduction of alpha-keto acid esters and 1-acetophenone compounds
a) Conversion [% ]; b) Reduction product alcohol enantiomer excess (ee%); c) Absolute configuration of the reduction product alcohol; NA No activity was detected.
Sequence listing
<110> Shenyang university of pharmacy
<120> aldehyde ketone reductase BsAKR (YvgN) and mutant and application thereof
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 828
<212> DNA
<213> Bacillus subtilis
<400> 1
atgccaacaa gtttaaaaga tactgtaaag ttacataacg gagtggaaat gccttggttc 60
ggtcttggtg tttttaaagt agaaaatgga agtgaagcga ctgaatcagt gaaagcggca 120
attaaaaacg gctaccgcag cattgataca gcagctgtct acaaaaatga agaaggtgtt 180
ggcatcggga tcaaggaatc cggtgtggca agagaagagc tctttattac atcaaaagta 240
tggaatgaag atcaaggcta cgaaacaacg cttgctgcct ttgaaaaaag cttggaaaga 300
cttcagcttg attacttaga tctatatttg atccactggc ctggcaagga taaatataaa 360
gatacatggc gtgcgcttga aaagctgtat aaagacggca aaatccgcgc aatcggtgtc 420
agcaacttcc aagttcatca tttagaagaa ctgctgaaag acgcagaaat caaaccgatg 480
gtgaaccaag tagaatttca ccctcgtttg acgcagaaag aattaagaga ttactgcaaa 540
gcgcaaggca ttcagttgga agcgtggtct ccgctcatgc agggacagct tttggataac 600
gaagtgcttg cacagattgc tgaaaaacac aataaatctg tggctcaagt cattttgcgc 660
tgggatcttc agcatgaagt tgtaacaatt ccaaaatcca tcaaagaaca ccgtatcatc 720
gaaaacgctg atatttttga tttcgaattg tctcaggaag acatggacaa aattgacgcg 780
ttaaacaaag atgagcgtgt cggtccaaat cctgatgagc ttctgttt 828
<210> 2
<211> 276
<212> PRT
<213> Bacillus subtilis
<400> 2
Met Pro Thr Ser Leu Lys Asp Thr Val Lys Leu His Asn Gly Val Glu
1 5 10 15
Met Pro Trp Phe Gly Leu Gly Val Phe Lys Val Glu Asn Gly Ser Glu
20 25 30
Ala Thr Glu Ser Val Lys Ala Ala Ile Lys Asn Gly Tyr Arg Ser Ile
35 40 45
Asp Thr Ala Ala Val Tyr Lys Asn Glu Glu Gly Val Gly Ile Gly Ile
50 55 60
Lys Glu Ser Gly Val Ala Arg Glu Glu Leu Phe Ile Thr Ser Lys Val
65 70 75 80
Trp Asn Glu Asp Gln Gly Tyr Glu Thr Thr Leu Ala Ala Phe Glu Lys
85 90 95
Ser Leu Glu Arg Leu Gln Leu Asp Tyr Leu Asp Leu Tyr Leu Ile His
100 105 110
Trp Pro Gly Lys Asp Lys Tyr Lys Asp Thr Trp Arg Ala Leu Glu Lys
115 120 125
Leu Tyr Lys Asp Gly Lys Ile Arg Ala Ile Gly Val Ser Asn Phe Gln
130 135 140
Val His His Leu Glu Glu Leu Leu Lys Asp Ala Glu Ile Lys Pro Met
145 150 155 160
Val Asn Gln Val Glu Phe His Pro Arg Leu Thr Gln Lys Glu Leu Arg
165 170 175
Asp Tyr Cys Lys Ala Gln Gly Ile Gln Leu Glu Ala Trp Ser Pro Leu
180 185 190
Met Gln Gly Gln Leu Leu Asp Asn Glu Val Leu Ala Gln Ile Ala Glu
195 200 205
Lys His Asn Lys Ser Val Ala Gln Val Ile Leu Arg Trp Asp Leu Gln
210 215 220
His Glu Val Val Thr Ile Pro Lys Ser Ile Lys Glu His Arg Ile Ile
225 230 235 240
Glu Asn Ala Asp Ile Phe Asp Phe Glu Leu Ser Gln Glu Asp Met Asp
245 250 255
Lys Ile Asp Ala Leu Asn Lys Asp Glu Arg Val Gly Pro Asn Pro Asp
260 265 270
Glu Leu Leu Phe
275
<210> 3
<211> 828
<212> DNA
<213> Bacillus subtilis
<400> 3
atgccaacaa gtttaaaaga tactgtaaag ttacataacg gagtggaaat gccttggttc 60
ggtcttggtg ttagcaaagt agaaaatgga agtgaagcga ctgaatcagt gaaagcggca 120
attaaaaacg gctaccgcag cattgataca gcagctgtct acaaaaatga agaaggtgtt 180
ggcatcggga tcaaggaatc cggtgtggca agagaagagc tctttattac atcaaaagta 240
tggaatgaag atcaaggcta cgaaacaacg cttgctgcct ttgaaaaaag cttggaaaga 300
cttcagcttg attacttaga tctatatttg atccactggc ctggcaagga taaatataaa 360
gatacatggc gtgcgcttga aaagctgtat aaagacggca aaatccgcgc aatcggtgtc 420
agcaacttcc aagttcatca tttagaagaa ctgctgaaag acgcagaaat caaaccgatg 480
gtgaaccaag tagaatttca ccctcgtttg acgcagaaag aattaagaga ttactgcaaa 540
gcgcaaggca ttcagttgga agcgtggtct ccgctcatgc agggacagct tttggataac 600
gaagtgcttg cacagattgc tgaaaaacac aataaatctg tggctcaagt cattttgcgc 660
tgggatcttc agcatgaagt tgtaacaatt ccaaaatcca tcaaagaaca ccgtatcatc 720
gaaaacgctg atatttttga tttcgaattg tctcaggaag acatggacaa aattgacgcg 780
ttaaacaaag atgagcgtgt cggtccaaat cctgatgagc ttctgttt 828
<210> 4
<211> 276
<212> PRT
<213> Bacillus subtilis
<400> 4
Met Pro Thr Ser Leu Lys Asp Thr Val Lys Leu His Asn Gly Val Glu
1 5 10 15
Met Pro Trp Phe Gly Leu Gly Val Ser Lys Val Glu Asn Gly Ser Glu
20 25 30
Ala Thr Glu Ser Val Lys Ala Ala Ile Lys Asn Gly Tyr Arg Ser Ile
35 40 45
Asp Thr Ala Ala Val Tyr Lys Asn Glu Glu Gly Val Gly Ile Gly Ile
50 55 60
Lys Glu Ser Gly Val Ala Arg Glu Glu Leu Phe Ile Thr Ser Lys Val
65 70 75 80
Trp Asn Glu Asp Gln Gly Tyr Glu Thr Thr Leu Ala Ala Phe Glu Lys
85 90 95
Ser Leu Glu Arg Leu Gln Leu Asp Tyr Leu Asp Leu Tyr Leu Ile His
100 105 110
Trp Pro Gly Lys Asp Lys Tyr Lys Asp Thr Trp Arg Ala Leu Glu Lys
115 120 125
Leu Tyr Lys Asp Gly Lys Ile Arg Ala Ile Gly Val Ser Asn Phe Gln
130 135 140
Val His His Leu Glu Glu Leu Leu Lys Asp Ala Glu Ile Lys Pro Met
145 150 155 160
Val Asn Gln Val Glu Phe His Pro Arg Leu Thr Gln Lys Glu Leu Arg
165 170 175
Asp Tyr Cys Lys Ala Gln Gly Ile Gln Leu Glu Ala Trp Ser Pro Leu
180 185 190
Met Gln Gly Gln Leu Leu Asp Asn Glu Val Leu Ala Gln Ile Ala Glu
195 200 205
Lys His Asn Lys Ser Val Ala Gln Val Ile Leu Arg Trp Asp Leu Gln
210 215 220
His Glu Val Val Thr Ile Pro Lys Ser Ile Lys Glu His Arg Ile Ile
225 230 235 240
Glu Asn Ala Asp Ile Phe Asp Phe Glu Leu Ser Gln Glu Asp Met Asp
245 250 255
Lys Ile Asp Ala Leu Asn Lys Asp Glu Arg Val Gly Pro Asn Pro Asp
260 265 270
Glu Leu Leu Phe
275
<210> 5
<211> 828
<212> DNA
<213> Bacillus subtilis
<400> 5
atgccaacaa gtttaaaaga tactgtaaag ttacataacg gagtggaaat gccttggttc 60
ggtcttggtg tttttaaagt agaaaatgga agtgaagcga ctgaatcagt gaaagcggca 120
attaaaaacg gctaccgcag cattgataca gcagctgtct acaaaaatga agaaggtgtt 180
ggcatcggga tcaaggaatc cggtgtggca agagaagagc tctttattac atcaaaagta 240
tggaatgaag atcaaggcta cgaaacaacg cttgctgcct ttgaaaaaag cttggaaaga 300
cttcagcttg attacttaga tctatatttg atccactttc ctggcaagga taaatataaa 360
gatacatggc gtgcgcttga aaagctgtat aaagacggca aaatccgcgc aatcggtgtc 420
agcaacttcc aagttcatca tttagaagaa ctgctgaaag acgcagaaat caaaccgatg 480
gtgaaccaag tagaatttca ccctcgtttg acgcagaaag aattaagaga ttactgcaaa 540
gcgcaaggca ttcagttgga agcgtggtct ccgctcatgc agggacagct tttggataac 600
gaagtgcttg cacagattgc tgaaaaacac aataaatctg tggctcaagt cattttgcgc 660
tgggatcttc agcatgaagt tgtaacaatt ccaaaatcca tcaaagaaca ccgtatcatc 720
gaaaacgctg atatttttga tttcgaattg tctcaggaag acatggacaa aattgacgcg 780
ttaaacaaag atgagcgtgt cggtccaaat cctgatgagc ttctgttt 828
<210> 6
<211> 276
<212> PRT
<213> Bacillus subtilis
<400> 6
Met Pro Thr Ser Leu Lys Asp Thr Val Lys Leu His Asn Gly Val Glu
1 5 10 15
Met Pro Trp Phe Gly Leu Gly Val Phe Lys Val Glu Asn Gly Ser Glu
20 25 30
Ala Thr Glu Ser Val Lys Ala Ala Ile Lys Asn Gly Tyr Arg Ser Ile
35 40 45
Asp Thr Ala Ala Val Tyr Lys Asn Glu Glu Gly Val Gly Ile Gly Ile
50 55 60
Lys Glu Ser Gly Val Ala Arg Glu Glu Leu Phe Ile Thr Ser Lys Val
65 70 75 80
Trp Asn Glu Asp Gln Gly Tyr Glu Thr Thr Leu Ala Ala Phe Glu Lys
85 90 95
Ser Leu Glu Arg Leu Gln Leu Asp Tyr Leu Asp Leu Tyr Leu Ile His
100 105 110
Phe Pro Gly Lys Asp Lys Tyr Lys Asp Thr Trp Arg Ala Leu Glu Lys
115 120 125
Leu Tyr Lys Asp Gly Lys Ile Arg Ala Ile Gly Val Ser Asn Phe Gln
130 135 140
Val His His Leu Glu Glu Leu Leu Lys Asp Ala Glu Ile Lys Pro Met
145 150 155 160
Val Asn Gln Val Glu Phe His Pro Arg Leu Thr Gln Lys Glu Leu Arg
165 170 175
Asp Tyr Cys Lys Ala Gln Gly Ile Gln Leu Glu Ala Trp Ser Pro Leu
180 185 190
Met Gln Gly Gln Leu Leu Asp Asn Glu Val Leu Ala Gln Ile Ala Glu
195 200 205
Lys His Asn Lys Ser Val Ala Gln Val Ile Leu Arg Trp Asp Leu Gln
210 215 220
His Glu Val Val Thr Ile Pro Lys Ser Ile Lys Glu His Arg Ile Ile
225 230 235 240
Glu Asn Ala Asp Ile Phe Asp Phe Glu Leu Ser Gln Glu Asp Met Asp
245 250 255
Lys Ile Asp Ala Leu Asn Lys Asp Glu Arg Val Gly Pro Asn Pro Asp
260 265 270
Glu Leu Leu Phe
275
<210> 7
<211> 828
<212> DNA
<213> Bacillus subtilis
<400> 7
atgccaacaa gtttaaaaga tactgtaaag ttacataacg gagtggaaat gccttggttc 60
ggtcttggtg ttagcaaagt agaaaatgga agtgaagcga ctgaatcagt gaaagcggca 120
attaaaaacg gctaccgcag cattgataca gcagctgtct acaaaaatga agaaggtgtt 180
ggcatcggga tcaaggaatc cggtgtggca agagaagagc tctttattac atcaaaagta 240
tggaatgaag atcaaggcta cgaaacaacg cttgctgcct ttgaaaaaag cttggaaaga 300
cttcagcttg attacttaga tctatatttg atccactttc ctggcaagga taaatataaa 360
gatacatggc gtgcgcttga aaagctgtat aaagacggca aaatccgcgc aatcggtgtc 420
agcaacttcc aagttcatca tttagaagaa ctgctgaaag acgcagaaat caaaccgatg 480
gtgaaccaag tagaatttca ccctcgtttg acgcagaaag aattaagaga ttactgcaaa 540
gcgcaaggca ttcagttgga agcgtggtct ccgctcatgc agggacagct tttggataac 600
gaagtgcttg cacagattgc tgaaaaacac aataaatctg tggctcaagt cattttgcgc 660
tgggatcttc agcatgaagt tgtaacaatt ccaaaatcca tcaaagaaca ccgtatcatc 720
gaaaacgctg atatttttga tttcgaattg tctcaggaag acatggacaa aattgacgcg 780
ttaaacaaag atgagcgtgt cggtccaaat cctgatgagc ttctgttt 828
<210> 8
<211> 276
<212> PRT
<213> Bacillus subtilis
<400> 8
Met Pro Thr Ser Leu Lys Asp Thr Val Lys Leu His Asn Gly Val Glu
1 5 10 15
Met Pro Trp Phe Gly Leu Gly Val Ser Lys Val Glu Asn Gly Ser Glu
20 25 30
Ala Thr Glu Ser Val Lys Ala Ala Ile Lys Asn Gly Tyr Arg Ser Ile
35 40 45
Asp Thr Ala Ala Val Tyr Lys Asn Glu Glu Gly Val Gly Ile Gly Ile
50 55 60
Lys Glu Ser Gly Val Ala Arg Glu Glu Leu Phe Ile Thr Ser Lys Val
65 70 75 80
Trp Asn Glu Asp Gln Gly Tyr Glu Thr Thr Leu Ala Ala Phe Glu Lys
85 90 95
Ser Leu Glu Arg Leu Gln Leu Asp Tyr Leu Asp Leu Tyr Leu Ile His
100 105 110
Phe Pro Gly Lys Asp Lys Tyr Lys Asp Thr Trp Arg Ala Leu Glu Lys
115 120 125
Leu Tyr Lys Asp Gly Lys Ile Arg Ala Ile Gly Val Ser Asn Phe Gln
130 135 140
Val His His Leu Glu Glu Leu Leu Lys Asp Ala Glu Ile Lys Pro Met
145 150 155 160
Val Asn Gln Val Glu Phe His Pro Arg Leu Thr Gln Lys Glu Leu Arg
165 170 175
Asp Tyr Cys Lys Ala Gln Gly Ile Gln Leu Glu Ala Trp Ser Pro Leu
180 185 190
Met Gln Gly Gln Leu Leu Asp Asn Glu Val Leu Ala Gln Ile Ala Glu
195 200 205
Lys His Asn Lys Ser Val Ala Gln Val Ile Leu Arg Trp Asp Leu Gln
210 215 220
His Glu Val Val Thr Ile Pro Lys Ser Ile Lys Glu His Arg Ile Ile
225 230 235 240
Glu Asn Ala Asp Ile Phe Asp Phe Glu Leu Ser Gln Glu Asp Met Asp
245 250 255
Lys Ile Asp Ala Leu Asn Lys Asp Glu Arg Val Gly Pro Asn Pro Asp
260 265 270
Glu Leu Leu Phe
275
Claims (10)
1. The mutant of aldehyde ketone reductase is characterized in that the amino acid sequence of the mutant is shown in SEQ ID NO.4,6 and 8.
2. A nucleic acid encoding the aldoketoreductase mutant of claim 1.
3. The nucleic acid of claim 2, having the nucleic acid sequence set forth in SEQ ID No.3,5,7.
4. An expression vector comprising the nucleic acid of claim 2 or 3 and capable of expression in a host cell.
5. A host cell comprising the nucleic acid of claim 2 or 3 or the expression vector of claim 4.
6. The host cell of claim 5, wherein the host cell is E.coli.
7. The use of the aldo-keto reductase mutant of claim 1 in the reduction of alpha-keto acid esters or 1-acetophenone compounds.
8. The use of claim 7, wherein the reduction process comprises: in a phosphate buffer solution of pH5-7, in the presence of glucose dehydrogenase, glucose and NADP+In the presence of the aldehyde ketone reductase mutant as described in claim 1, reducing the alpha-keto acid ester or 1-acetophenone compound to produce an optically active chiral secondary alcohol.
9. The use according to claim 8, wherein the aldone reductase is present in an amount of 0.02-40g/L, the glucose dehydrogenase is present in an amount of 0.01-5g/L, the glucose is present in an amount of 6-200g/L, and the NADP is present+The dosage is 0.1-0.5mmol, the substrate concentration is 3-100g/L, the buffer solution is phosphate buffer solution with pH of 5-7, and the reaction temperature is 30-40 ℃.
10. The use of claim 7, wherein the α -ketonate compound is of formula I, and the 1-acetophenone compound is of formula II:
wherein:
R1is C1-C4 alkyl, phenyl or phenyl with substituent, and the substituent of the phenyl is halogen or C1-C4 alkyl;
R2is C1-C4 alkyl;
R3is a halogenated C1-C4 alkyl group;
R4is halogen, halogenated C1-C4 alkyl.
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| CN105624125A (en) * | 2014-11-26 | 2016-06-01 | 南京博优康远生物医药科技有限公司 | Aldo-keto reductase and application thereof in synthesis of (2S,3R)-2-benzoylaminomethyl-3-hydroxybutyrate |
| CN107058248A (en) * | 2017-04-26 | 2017-08-18 | 浙江工业大学 | One kind restructuring aldehyde Ketoreductase mutant, gene, carrier, engineering bacteria and its application |
| CN109055327A (en) * | 2018-07-23 | 2018-12-21 | 浙江工业大学 | Aldehyde Ketoreductase mutant and its application |
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| CN107058248A (en) * | 2017-04-26 | 2017-08-18 | 浙江工业大学 | One kind restructuring aldehyde Ketoreductase mutant, gene, carrier, engineering bacteria and its application |
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