CN109055327B - Aldehyde ketone reductase mutant and application thereof - Google Patents
Aldehyde ketone reductase mutant and application thereof Download PDFInfo
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- CN109055327B CN109055327B CN201810812118.2A CN201810812118A CN109055327B CN 109055327 B CN109055327 B CN 109055327B CN 201810812118 A CN201810812118 A CN 201810812118A CN 109055327 B CN109055327 B CN 109055327B
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Abstract
The invention discloses an aldehyde ketone reductase mutant and application thereof in asymmetric reduction of tert-butyl 6-cyano- (5R) -hydroxy-3-carbonyl hexanoate to prepare tert-butyl 6-cyano- (3R,5R) -dihydroxyhexanoate, wherein the aldehyde ketone reductase mutant is obtained by carrying out single mutation or multiple mutations on 125 th, 30 th, 212 th and 63 th positions of amino acid shown in SEQ ID No. 2. Compared with the parent aldoketoreductase, the specific enzyme activity of the prepared aldoketoreductase mutant pKLAKR-I125V-S30P-Q212R-I63W is improved by 1.31 times, the maximum substrate of 6-cyano- (5R) -hydroxy-3-carbonyl hexanoate tert-butyl ester is 80g/L, and the aldoketoreductase mutant has better industrial application prospect.
Description
Technical Field
The invention relates to construction of an aldehyde-ketone reductase KlAKR mutant, and development of an aldehyde-ketone reductase recombinant strain and application of the aldehyde-ketone reductase recombinant strain in chiral biosynthesis of atorvastatin side chain 6-cyano- (3R,5R) -dihydroxy tert-butyl hexanoate.
Background
Cardiovascular and cerebrovascular diseases become one of the main diseases harmful to human health, and the incidence of the cardiovascular and cerebrovascular diseases is closely related to the cholesterol level in human blood. Atorvastatin calcium can competitively inhibit the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase which is a key rate-limiting enzyme for cholesterol synthesis in liver, reduce the synthesis of cholesterol, and reduce the concentration of low-density lipoprotein cholesterol (LDL-C) and triglyceride in blood, and is the only single lipid-lowering drug variety with the global accumulated sales of over 1000 hundred million dollars.
The 6-cyano- (3R,5R) -dihydroxy-hexanoic acid tert-butyl ester is an important two-chiral diol intermediate for the synthesis of atorvastatin calcium and is also a key pharmacophore. Because each national drug administration sets strict limits on the chiral purity of the drug (e.e. value > 99%, d.e. value > 99%), the synthesis technology of the 6-cyano- (3R,5R) -dihydroxy-hexanoic acid tert-butyl ester becomes a key core technology for synthesizing atorvastatin calcium. The traditional chemical synthesis process of 6-cyano- (3R,5R) -tert-butyl dihydroxyhexanoate starts from ketonic acid (ester), a chiral center is constructed by asymmetric hydrogenation reduction of borane, the reaction route is complex, reagents such as inflammable and explosive borane and the like and harsh reaction conditions are required, the optical purity of the product is low, the yield is low, the process energy consumption is large, the reaction waste is difficult to treat, and the process belongs to a non-environment-friendly production mode. The synthesis of chiral alcohol by using the oxidoreductase to asymmetrically reduce the prochiral ketone has the technical advantages of high selectivity, mild reaction conditions, environmental friendliness and the like, and the preparation process has high economy and meets the requirement of green chemistry. Therefore, the technology for synthesizing the tbutyl 6-cyano- (3R,5R) -dihydroxyhexanoate by developing the technique for biologically and asymmetrically reducing the tbutyl 6-cyano- (5R) -hydroxy-3-carbonyl hexanoate has great economic and social benefits.
The aldoketoreductase superfamily is a class of NAD (P) H-dependent oxidoreductases, which are typically single subunit proteins of about 320 amino acids, 34-37kDa in size, and (. alpha./. beta.)8The catalytic tetrad of the cylindrical structure is composed of tyrosine, histidine, aspartic acid and lysine, and has wide substrate spectrum, including aliphatic and aromatic aldehyde, ketone, monosaccharide and the like.
We cloned the aldone reductase KlAKR from Kluyveromyces lactis CCTCC M2014380 and realized heterologous over-expression in Escherichia coli (Escherichia coli), the enzyme can catalyze the asymmetric reduction of tbutyl 6-cyano- (5R) -hydroxy-3-carbonyl hexanoate to synthesize tbutyl 6-cyano- (3R,5R) -dihydroxyhexanoate with de value of more than 99%, but the activity of the enzyme on tbutyl 6-cyano- (5R) -hydroxy-3-carbonyl hexanoate is not high enough, and the site-directed mutagenesis technology obtains a dominant mutant KlAKR-Y295W-W296L which is disclosed in patent application (201610124451.5) and can completely convert 50g/L of substrate in 80 minutes without adding exogenous coenzyme NADPH, but the activity of the enzyme is found, The patent refers to the field of 'chemical engineering techniques'.
Disclosure of Invention
The invention aims to solve the problems of low asymmetric reduction activity and low substrate concentration of the existing aldehyde-ketone reductase on the tert-butyl 6-cyano- (5R) -hydroxy-3-carbonyl hexanoate, and provides a stereoselective aldehyde-ketone reductase mutant, a gene recombinant bacterium using the aldehyde-ketone reductase mutant and a crude enzyme solution thereof as a biocatalyst for chiral biosynthesis of the tert-butyl 6-cyano- (3R,5R) -dihydroxyhexanoate, wherein the activity of the catalyst is improved by 1.31 times, and the substrate concentration is improved by 60%.
The technical scheme adopted by the invention is as follows:
the invention provides an aldehyde ketone reductase mutant which is obtained by carrying out single mutation or multiple mutation on 125 th, 30 th, 212 th and 63 th positions of amino acid shown in SEQ ID No. 2. The amino acid shown in SEQ ID No.2 is the parent aldehyde ketone reductase KlAKR, and the nucleotide sequence is shown in SEQ ID No. 1.
Further, it is preferred that the aldoketoreductase mutant is one of the following: (1) isoleucine at the 125 th site of the amino acid shown in SEQ ID No.2 is mutated into leucine (pKLAKR-I125L) or valine (pKLAKR-I125V); (2) mutation of isoleucine at position 125 of amino acid shown in SEQ ID No.2 into valine and mutation of serine at position 30 into alanine (pKLAKR-I125V-S30A), histidine (pKLAKR-I125V-S30H) or proline (pKLAKR-I125V-S30P); (3) mutation of isoleucine at position 125 to valine and glutamine at position 212 to arginine (pKLAKR-I125V-Q212R) or asparagine (pKLAKR-I125V-Q212N) of the amino acid shown in SEQ ID No. 2; (4) the 125 th isoleucine of the amino acid shown in SEQ ID No.2 is mutated into valine, the 30 th serine is mutated into proline, and the 212 th glutamine is mutated into arginine (pKLAKR-I125V-S30P-Q212R) or asparagine (pKLAKR-I125V-S30P-Q212N); (5) mutation of 125 th isoleucine to valine, mutation of 30 th serine to alanine, and mutation of 212 th glutamine to arginine (pKLAKR-I125V-S30A-Q212R) of amino acid shown in SEQ ID No. 2; (6) the 125 th isoleucine of the amino acid shown in SEQ ID No.2 is mutated into valine, the 30 th serine is mutated into proline, the 212 th glutamine is mutated into arginine, and the 63 th isoleucine is mutated into tryptophan (pKLAKR-I125V-S30P-Q212R-I63W).
Furthermore, the aldehyde ketone reductase mutant is obtained by mutating isoleucine at position 125 of the amino acid shown in SEQ ID No.2 into valine, simultaneously mutating serine at position 30 into proline, mutating glutamine at position 212 into arginine, and mutating isoleucine at position 63 into tryptophan.
The invention also relates to a coding gene, a recombinant vector and engineering bacteria of the aldehyde ketone reductase mutant. Preferably, the recombinant expression vector pET-28b (+); coli BL21(DE3) is selected as the host cell, crude enzyme liquid is obtained through protein induction expression and cell disruption, and the catalytic property is superior to that of the parent aldehyde ketone reductase.
The invention also provides an application of the aldone reductase mutant in asymmetric reduction of tert-butyl 6-cyano- (5R) -hydroxy-3-carbonyl hexanoate to prepare tert-butyl 6-cyano- (3R,5R) -dihydroxyhexanoate, and the application method specifically comprises the following steps: mixing wet thalli obtained by induced culture of recombinant genetic engineering bacteria containing aldone reductase mutant genes and wet thalli obtained by induced culture of engineering bacteria containing glucose dehydrogenase genes, then carrying out resuspension by using phosphate buffer solution with pH 7.5 and 100mM, carrying out ultrasonic crushing, taking crushed mixed liquor as a catalyst, taking 6-cyano- (5R) -hydroxyl-3-carbonyl tert-butyl hexanoate as a substrate and glucose as an auxiliary substrate to form a reaction system, carrying out reaction at the temperature of 30 ℃ and under the condition of 300 revolutions per minute of 150 materials, finishing the reaction, separating and purifying reaction liquid, and obtaining 6-cyano- (3R,5R) -dihydroxy tert-butyl hexanoate; the nucleotide sequence of the glucose dehydrogenase gene is SEQ ID No.3 (the amino acid sequence of the coding protein is SEQ ID No. 4).
Further, in the reaction system, the final concentration of the 6-cyano- (5R) -hydroxyl-3-carbonyl tert-butyl hexanoate is 50-100g/L, the final concentration of glucose is 75-150 g/L, the dosage of the catalyst is 50-100g/L (preferably 75-80g/L) based on the total amount of wet bacteria before crushing, and wet bacteria obtained by induced culture of recombinant genetic engineering bacteria containing aldone reductase mutant genes and wet bacteria obtained by induced culture of engineering bacteria containing glucose dehydrogenase genes are mixed in a mass ratio of 3: 1.
Further, the wet cells were prepared as follows: inoculating recombinant genetic engineering bacteria containing aldone reductase mutant genes into LB liquid culture medium containing 50 mug/mL kanamycin at the final concentration, culturing at 37 ℃ for 9 hours, inoculating the recombinant genetic engineering bacteria into fresh LB liquid culture medium containing 50 mug/mL kanamycin at the final concentration by the inoculation amount of 2% of volume concentration, culturing at 37 ℃ for 1.5 hours at 180 r/min, adding IPTG (0.1 mM) at the final concentration into the culture solution, culturing at 28 ℃ for 10 hours, and centrifuging at 4 ℃ for 8000 r/min for 10 minutes to obtain wet thalli containing aldone reductase; the preparation method of the wet thallus obtained by induced culture of the engineering bacteria containing the glucose dehydrogenase gene is the same as that of the wet thallus containing the aldone reductase gene.
Further, the ultrasonication conditions are as follows: mixing wet thalli obtained by induced culture of recombinant gene engineering bacteria containing aldone reductase mutant genes and wet thalli obtained by induced culture of engineering bacteria containing glucose dehydrogenase genes, then resuspending the mixture by using a phosphate buffer solution with the pH value of 7.5 and the concentration of 100mM, carrying out ultrasonic crushing on an ice-water mixture for 10 minutes, wherein the ultrasonic crushing conditions are as follows: the power was 400W, crushing for 1 second, and pausing for 1 second.
The base sequence total length of the parent KlAKR of the aldehyde ketone reductase and the aldehyde ketone reductase mutant is 930bp, starting from the first base to the 930 th base, the initiation codon is ATG, and the termination codon is TAA.
The invention relates to an aldehyde ketone reductase mutant, which is obtained by adopting a site-specific saturation mutation technology, mutating the aldehyde ketone reductase gene of SEQ ID No.1 by using the technology, transferring the obtained mutant plasmid into an E.coliBL21(DE3) competent cell in a heat shock mode, inoculating, transferring, inducing and recovering thalli of the obtained strain, catalyzing 6-cyano- (5R) -hydroxy-3-carbonyl tert-butyl hexanoate by using crude enzyme liquid to asymmetrically reduce the 6-cyano- (3R,5R) -dihydroxy-hexanoate to prepare optically pure 6-cyano- (3R,5R) -dihydroxy-hexanoate tert-butyl ester, and the specific method comprises the following steps: the original strain is activated in the first step, a female parent E.coli BL21(DE3) pET-28b (+) -klakr is obtained, and a plasmid pET-28b (+) -klakr (pET-28b (+) -klakr) is extracted and stored for later use. And secondly, comparing SWISS-MODEL with pKLAKR to obtain a template protein crystal structure of homologous modeling, utilizing Modeller 9.14 to carry out homologous modeling, carrying out molecular docking, selecting a proper mutation site, mainly obtaining an amino acid residue near an active channel to obtain an active pocket accessory, designing a mutation primer, carrying out mutation PCR by taking pET-28b (+) -pKlAKR as a template plasmid, obtaining a mutation plasmid, transforming, carrying out screening of dominant mutant bacteria, and carrying out sequencing detection and storage on the dominant mutant.
The aldehyde ketone reductase mutant and the glucose dehydrogenase gene engineering bacteria are inoculated, transferred, induced and recovered, and the culture medium can be any culture medium which can enable the bacteria to grow and produce the invention in the field, preferably an LB culture medium: 10g/L of peptone, 5g/L of yeast powder, 10g/L of NaCl and distilled water for dissolving, wherein the pH value is 7.0. The culture method and the culture conditions are not particularly limited, and may be appropriately selected according to the type of host and factors such as the culture method, and the like, according to ordinary knowledge in the art. The glucose dehydrogenase is derived from Exiguobacterium sibiricum, a glucose dehydrogenase gene sequence number (GenBank: No. KM817194.1) and a used vector pET-28b (+), and a recombinant expression vector pET-28b (+) -esgdh is constructed.
Compared with the prior art, the invention has the main beneficial effects that: compared with the parent aldoketoreductase, the specific enzyme activity of the prepared aldoketoreductase mutant pKLAKR-I125V-S30P-Q212R-I63W is improved by 1.31 times, the maximum substrate of 6-cyano- (5R) -hydroxy-3-carbonyl hexanoate tert-butyl ester is 80g/L, and the aldoketoreductase mutant has better industrial application prospect.
Drawings
FIG. 1 is a schematic diagram of the reaction of the aldone reductase mutant pKLAKR-I125V-S30P-Q212R-I63W coupled with glucose dehydrogenase to catalyze the asymmetric reduction of tbutyl 6-cyano- (5R) -hydroxy-3-carbonylhexanoate to tbutyl 6-cyano- (3R,5R) -dihydroxyhexanoate.
FIG. 2 is a nucleic acid electrophoresis of site-directed saturation mutagenesis of aldone reductase. M: standard nucleic acid molecular weight; lane 1: pET-28b (+) -pklakr; lane 2: pET-28b (+) -pklakr-I125V; lane 3: pET-28b (+) -pklakr-S30P; lane 4: pET-28b (+) -pklakr-Q212R; lane 5: pET-28b (+) -pklakr-I63W; lane 6: pET-28b (+) -pklakr-I125V-S30P; lane 7: pET-28b (+) -pklakr-I125V-S30P-Q212R; lane 8: pET-28b (+) -pklakr-I125V-S30P-Q212R-I63W.
FIG. 3 shows SDS-PAGE of crude enzyme solution (A) and pure enzyme solution (B) of the aldone reductase mutant. M: standard protein molecular weight; lane 1: a parent aldone reductase; lane 2: pKLAKR-I125V; lane 3: pKLAKR-S30P; lane 4: pKLAKR-Q212R; lane 5: pKLAKR-I63W; lane 6: pKLAKR-I125V-S30P; lane 7: pKLAKR-I125V-S30P-Q212R; lane 8: pKLAKR-I125V-S30P-Q212R-I63W.
FIG. 4 is a time course diagram of the preparation of tert-butyl 6-cyano- (3R,5R) -dihydroxyhexanoate by asymmetric reduction of tert-butyl 6-cyano- (5R) -hydroxy-3-carbonylhexanoate using the aldo-keto reductase mutant pKLAKR-I125V-S30P-Q212R-I63W coupled to EsGDH.
Detailed Description
The invention is further described below with reference to specific examples.
Example 1: construction and screening of aldehyde ketone reductase mutant library
An expression vector pET-28b (+) -klakr is constructed by the aldone reductase gene (shown in an amino acid sequence SEQ ID No.2 and a nucleotide sequence SEQ ID No. 1), and escherichia coli is transformed to obtain an original strain E.coli BL21(DE3)/pET28 b-pklakr.
The preparation of the aldoketoreductase mutant library is realized by 4 rounds of site-directed saturation mutagenesis, the design of primers is shown in table 1, the first round is that the 125 th isoleucine of the amino acid sequence of the aldoketoreductase shown in SEQ ID No.2 is mutated into the rest 19 amino acids by saturation mutagenesis PCR with a vector pET-28b (+) -klakr as a template and I125F and I125R in table 1 as primers, and the aldoketoreductase mutant pKLAKR-I125V is obtained by screening dominant strains. In the second round, the mutant pKLAKR-I125V corresponding to the amino acid sequence SEQ ID No.3 is used as a template, S30F and S30R in Table 1 are used as primers, and the aldehyde ketone reductase mutant pKLAKR-I125V-S30P is obtained by performing saturation mutation PCR, transformation and plate coating and screening dominant strains. The third round takes the mutant pKLAKR-I125V-S30P as a template, takes Q212F and Q212R in Table 1 as primers, and obtains the aldehyde ketone reductase mutant pKLAKR-I125V-S30P-Q212R by saturated mutation PCR, transformation, plate coating and dominant strain screening. The fourth round takes the mutant pKLAKR-I125V-S30P-Q212R as a template, takes I63F and I63R in Table 1 as primers, and is subjected to saturation mutation PCR, transformation and plate coating, and the aldehyde ketone reductase mutant pKLAKR-I125V-S30P-Q212R-I63W is obtained by screening dominant strains, and the other dominant single mutants pKLAKR-S30P, pKLAKR-Q212R and pKLAKR-I63W in the later experiment are constructed by the same method.
The mutant PCR system (100. mu.L) was: 25. mu.L of 2-fold Phanta Max buffer, 1. mu.L of dNTPs, 1. mu.L of mutation upper and lower primers, 1. mu.L of template, 0.5. mu.L of Phanta Super-Fidelity DNA polymerase, and complement ddH2O to 50. mu.L. The PCR conditions were: pre-denaturation at 95 ℃ for 5 min, after 25 cycles: 95 ℃ for 15 seconds, 56 ℃ for 15 seconds, 72 ℃ for 6 minutes, and finally 72 ℃ for a final extension of 10 minutes. And (3) carrying out positive verification of DNA agarose gel electrophoresis on PCR results respectively, carrying out Dpn I enzyme digestion on a template of a PCR product, inactivating the template at 37 ℃, 3 hours, 160 revolutions per minute and 65 ℃ for 1 minute, carrying out heat shock transformation on the PCR product, activating Escherichia coli E.coli BL21(DE3), placing the template at 37 ℃ and 160 revolutions per minute, culturing the template for 1 hour, coating the template on an LB plate containing 50 mu g/mL kanamycin resistance, carrying out inverted culture at 37 ℃ overnight, and carrying out dominant mutant screening on the obtained mutants under the following screening conditions: 10g DCW/L cell (mass ratio of aldone reductase mutant to glucose dehydrogenase thallus is 3:1), adding PBS (100mM) with pH 7.0 to resuspend the cell, crushing the cell for 10min (ultrasonic crushing condition: power 400W, crushing for 1s, stopping for 1s) on an ice-water mixture to obtain a crude enzyme solution, adding 6-cyano- (5R) -hydroxy-3-carbonyl hexanoate tert-butyl ester with final concentration of 75g/L and 112.5g/L glucose, reacting at 30 ℃ and 150R/min, sampling and detecting the concentration of 6-cyano- (3R,5R) -dihydroxy hexanoate after the reaction is finished, and screening to obtain the dominant strain. The obtained dominant strains are sent to Hangzhou Ongke biotechnology limited company for sequencing and storage. Glucose dehydrogenase cells were prepared in the same manner as in example 2.
TABLE 1 aldehyde ketone reductase site-directed saturation mutagenesis primer design
Example 2: aldehyde ketone reductase female parent, mutant and induced expression of glucose dehydrogenase
Glucose dehydrogenase gene esgdh (nucleotide sequence is shown as SEQ ID No.3, amino acid sequence is shown as SEQ ID No.4) is cloned from Exiguobacterium sibiricum, and is connected to pET-28b (+) vector through double enzyme digestion, the recombinant plasmid is introduced into Escherichia coli E.coli BL21(DE3), and recombinant glucose dehydrogenase strain E.coli BL21(DE3)/pET28b-esgdh is obtained.
The starting strain E.coli BL21(DE3)/pET28b-pklakr and the aldone reductase mutant strain and the recombinant glucose dehydrogenase strain E.coli BL21(DE3)/pET28b-esgdh of example 1 were inoculated into LB liquid medium containing 50. mu.g/mL kanamycin, respectively, and cultured at 37 ℃ for 9 hours, and inoculated into fresh LB liquid medium containing 50. mu.g/mL kanamycin at a volume fraction of 2% (v/v), cultured at 37 ℃ for 1.5 hours at 180 rpm, and then added with IPTG at a final concentration of 0.1mM, cultured at 28 ℃ for 10 hours, and centrifuged at 4 ℃ for 10 minutes at 8000 rpm to obtain wet cells. The cell obtained in the above way produces corresponding protein, can be used for preparing protein pure enzyme liquid, and can also be used for preparing 6-cyano- (3R,5R) -dihydroxyhexanoic acid tert-butyl ester by catalyzing asymmetric reduction of 6-cyano- (5R) -hydroxy-3-carbonyl hexanoic acid tert-butyl ester by crude enzyme liquid.
Example 3: mutant library screening
The wet bacterial cells of the mutant strain induced to express in example 2 and the wet bacterial cells of glucose dehydrogenase were mixed at a mass ratio of 3:1, and the mixture was resuspended in a phosphate buffer solution of 100mM and pH 7.5 at a concentration of 50g/L of the total bacterial cells, and then sonicated in an ice-water mixture for 10 minutes under the sonication conditions: the power is 400W, the crushing is carried out for 1 second, the suspension is carried out for 1 second, and the crude enzyme liquid of the mutant strain is obtained. Under the same conditions, the original strain E.coli BL21(DE3)/pET28b-pklakr is used to replace the wet thallus of the mutant strain to prepare the crude enzyme solution of the original strain.
Using crude enzyme solution of mutant strain or crude enzyme solution of original strain as catalyst, 6-cyano- (5R) -hydroxy-3-carbonyl tert-butyl hexanoate as substrate, glucose as auxiliary substrate, and no exogenous NADPH or NADP+The endogenous NADPH of the strain is used to establishAnd a coenzyme circulating system is formed. The reaction system is selected to be 10mL, the dosage of the catalyst is 50g/L of the total concentration of wet thalli before crushing, the final concentration of a substrate is 75g/L, the final concentration of glucose is 112.5g/L, the reaction is carried out for 1 hour at 30 ℃ and 150R/min, a sample is taken, 100 mu L and 900 mu L of absolute ethyl alcohol precipitation protein are taken, namely the reaction solution is diluted by 10 times, the temperature is kept overnight at-20 ℃, the reaction solution is centrifuged for 3 minutes at 12000R/min, the supernatant is taken and is filtered by a 0.22 mu M microfiltration membrane to be taken as a liquid phase sample, and HPLC (high performance liquid chromatography) is used for detecting the tert-butyl 6-cyano- (5R) -hydroxy-3-carbonyl hexanoate, the tert-butyl 6-cyano- (3R,5R) -dihydroxyhexanoate, the tert-butylpThe value is obtained. With the products 6-cyano- (3R,5R) -dihydroxyhexanoic acid tert-butyl ester and depDominant mutants were screened as indicators and the results are shown in table 2.
Liquid phase detection conditions: chromatographic column
C18 (4.6X 250mm, Acchrom, China) column, mobile phase acetonitrile/water volume ratio of 1:3, flow rate of 1.0mL/min, detection wavelength of 210nm, sample introduction of 20. mu.L, column temperature of 40 ℃. Tbutyl 6-cyano- (5R) -hydroxy-3-carbonylhexanoate, tbutyl 6-cyano- (3R,5R) -dihydroxyhexanoate, tbutyl 6-cyano- (3S,5R) -dihydroxyhexanoate retention times were, respectively: 15.6 min, 10.1 min, 9.3 min.
TABLE 2 catalytic Performance and stereoselectivity of pKLAKR and its mutants
Example 4: purification of aldehyde ketone reductase female parent and mutant thereof
The dominant mutants obtained in example 3 (pKLAKR-I125V, pKLAKR-S30P, pKLAKR-Q212R, pKLAKR-I63W, pKLAKR-I125V-S30P, pKLAKR-I125V-S30P-Q212R in Table 2,
pKLAKR-I125V-S30P-Q212R-I63W), wet cells of the aldoketoreductase mutant were obtained according to the method described in example 2, suspended in buffer A (0.3M NaCl, 30mM imidazole in pH 8.0, 50mM sodium phosphate buffer), sonicated for 20 minutes (ice bath, power 400W, disruption for 1 second, suspension for 1 second), centrifuged at 12000 rpm for 20 minutes at 4 ℃ and the supernatant was collected.
The mutant protein was purified using a Ni affinity column (1.6X 10cm, Bio-Rad, USA) by the following procedure: firstly, a binding buffer solution (pH 8.0 containing 0.3M NaCl and 50mM sodium phosphate buffer solution) with 5 times of column volume is used for balancing the Ni column until the baseline is stable; sample loading, wherein the flow rate is 1mL/min, and the sample loading amount is 25-40mg/mL protein, so that the target protein is adsorbed on the Ni column; ③ flushing the hybrid protein by using buffer solution A (containing 0.3M NaCl, pH 8.0 of 30mM imidazole and 50mM sodium phosphate buffer solution) with 6 times of column volume, wherein the flow rate is 1mL/min until the base line is stable; and fourthly, eluting with a buffer solution B (pH 8.0 containing 0.3M NaCl and 500mM imidazole and 50mM sodium phosphate buffer solution) at the flow rate of 1mL/min, and collecting the target protein. Putting the target protein into a phosphate buffer solution with pH 7.5 and 20mM for dialysis overnight to obtain purified enzyme; fifthly, washing the Ni column by 5 times of column volume of binding buffer solution (pH 8.0 containing 0.3M NaCl and 50mM sodium phosphate buffer solution) until the base line is stable, and preserving the Ni column by 5 times of column volume of ultrapure water containing 20 percent of ethanol.
Pure aldoketoreductase enzyme of the starting strain E.coli BL21(DE3)/pET28b-pklakr was collected under the same conditions.
Example 5: determination of parent aldehyde ketone reductase and its mutant enzyme specific activity
The enzyme activity unit (U) is defined as: the amount of enzyme required per minute for the formation of 1. mu. mol of tbutyl 6-cyano- (3R,5R) -dihydroxyhexanoate at 30 ℃ and pH 7.5 is defined as one enzyme activity unit, U. Specific enzyme activity is defined as the number of units of activity per mg of enzyme protein, U/mg.
Enzyme activity detection standard conditions: 30mM tbutyl 6-cyano- (5R) -hydroxy-3-carbonylhexanoate, 3mM NADPH, appropriate amounts of enzyme solution, reaction at 30 ℃ at pH 7.5 at 300rpm for 5 minutes, sample processing and HPLC assay.
The protein concentration was measured using a bisquinolinecarboxylic acid protein assay kit (Nanjing Kaikyi Biotech development Co., Ltd., Nanjing).
The specific enzyme activities of the parent aldoketoreductase and its mutant are shown in Table 3.
TABLE 3 relative enzyme Activity and epimeric selectivity (de) of the mutantsp) Value of
a: under standard conditions, the initial enzyme activity of pKLAKR was assigned to 100%
Example 6: determination of female parent aldehyde ketone reductase and kinetic parameters of mutant thereof
Looking at kinetic parameters of aldone reductase and its mutation, tbutyl 6-cyano- (5R) -hydroxy-3-carbonyl hexanoate was used as a substrate, the concentration was set to 2-10mM (2, 4, 6, 8, 10mM), the concentration of exogenous coenzyme NADPH was set to 1-5mM (1, 2, 3, 4, 5mM), and a certain amount of pure enzyme solution was added (see example 3).
The reaction system was selected to be 500. mu.L, 100. mu.L of the pure enzyme solution collected in example 3, the substrate and the exogenous coenzyme NADPH were added, 100mM phosphate buffer solution at pH 7.5 was used as a reaction medium, a sample was taken at 30 ℃ for 1 hour at 150 rpm, and the reaction solution was subjected to HPLC to determine the concentration of tert-butyl 6-cyano- (3R,5R) -dihydroxyhexanoate (see example 3).
V can be calculated by double reciprocal mapping according to the reaction mechanism of aldehyde ketone reductase catalysis and the sequence forced reaction mechanismmax、Km A、Km BThe results are shown in Table 4 by comparing kcatAnd KmIt was found that K of pKLAKR for tbutyl 6-cyano- (5R) -hydroxy-3-carbonylhexanoate and NADPHmThe values were 3.45mM and 0.11mM, respectively, except for the increase in the mutant pKLAKR-S30P, the mutants all had a decrease in affinity for tbutyl 6-cyano- (5R) -hydroxy-3-carbonylhexanoate and NADPH. Catalytic efficiency k of mutant pKLAKR-I125V-S30P-Q212R-I63W on tbutyl 6-cyano- (5R) -hydroxy-3-carbonyl hexanoatecat/Km BReaches 36.31s-1·mM-1Comparison parent (k)cat/Km B＝12.41s-1·mM-1) The catalytic efficiency of the catalyst to coenzyme NADPH reaches 1438.23s by 2.93 times-1·mM-1Comparison parent (k)cat/Km A＝385.67s-1·mM-1) The improvement is 3.73 times.
TABLE 4 comparison of kinetic parameters of the parent pKLAKR and its mutants
Example 7: acaldoketoreductase mutant pKLAKR-I125V-S30P-Q212R-I63W asymmetric reduction of tbutyl 6-cyano- (5R) -hydroxy-3-carbonylhexanoate
According to the description of example 2, 3g of the aldone reductase mutant pKLAKR-I125V-S30P-Q212R-I63W strain obtained by fermentation was mixed with 1g of glucose dehydrogenase EsGDH strain, resuspended in 40mL of phosphate buffer (100mM), disrupted on ice (sonication conditions: power 400W, disruption for 1S, and stop for 1S), the whole disrupted mixture (i.e., crude enzyme solution) was taken, reduced tbutyl 6-cyano- (5R) -hydroxy-3-carbonyl hexanoate was added to a final concentration of 80g/L, glucose was added to a final concentration of 120g/L to constitute a reaction system, the reaction was carried out at 30 ℃ under a magnetic stirring speed of 300rpm, and 2M Na was fed2CO3The aqueous solution maintained the reaction solution pH at 7.5. The liquid phase method shown in example 3 was used to detect the formation of the product tbutyl 6-cyano- (3R,5R) -dihydroxyhexanoate and the change in the de value during the reaction, and the reaction progress curve is shown in FIG. 4. The figure shows that the product concentration gradually increases with the time, the reaction is completed within 90 minutes, the substrate conversion rate is more than 99%, and the de value of the product is always kept above 99.5%.
Sequence listing
<110> Zhejiang industrial university
<120> aldehyde ketone reductase mutant and application thereof
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atcctgctgt cttgggttta cgaacgtggt atcctgccgg ttaccacctc ttctaaaatc 780
gaacgtatcc agcaggctca ggacatcttc tctttctctc tggctaacga agaagttcag 840
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20 25 30
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35 40 45
Leu Asp Thr Val Pro Gly Val Val His Ile Asp Ala Ala Glu Ile Tyr
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Ser Glu Asn Pro Lys Val Ala Leu Glu Thr Ser Leu Lys Lys Leu Gly
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Ile Leu Leu Ser Trp Val Tyr Glu Arg Gly Ile Leu Pro Val Thr Thr
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Claims (7)
1. An aldehyde-ketone reductase mutant, wherein the aldehyde-ketone reductase mutant is one of: (1) isoleucine at the 125 th site of the amino acid shown in SEQ ID No.2 is mutated into valine; (2) isoleucine at the 125 th site of the amino acid shown in SEQ ID No.2 is mutated into valine, and simultaneously serine at the 30 th site is mutated into alanine, histidine or proline; (3) isoleucine at position 125 of amino acid shown in SEQ ID No.2 is mutated into valine, and glutamine at position 212 is mutated into arginine or asparagine; (4) mutating isoleucine at position 125 of amino acid shown in SEQ ID No.2 to valine, simultaneously mutating serine at position 30 to proline, and mutating glutamine at position 212 to arginine or asparagine; (5) mutating isoleucine at position 125 of amino acid shown in SEQ ID No.2 to valine, simultaneously mutating serine at position 30 to alanine, and mutating glutamine at position 212 to arginine; (6) the 125 th isoleucine of the amino acid shown in SEQ ID No.2 is mutated into valine, the 30 th serine is mutated into proline, the 212 th glutamine is mutated into arginine, and the 63 th isoleucine is mutated into tryptophan.
2. The aldoketoreductase mutant of claim 1, wherein the aldoketoreductase mutant is obtained by mutating isoleucine at position 125 of amino acid shown in SEQ ID No.2 to valine, simultaneously mutating serine at position 30 to proline, mutating glutamine at position 212 to arginine, and mutating isoleucine at position 63 to tryptophan.
3. An application of the aldone reductase mutant of claim 1 in asymmetric reduction of tbutyl 6-cyano- (5R) -hydroxy-3-carbonyl hexanoate to prepare tbutyl 6-cyano- (3R,5R) -dihydroxyhexanoate.
4. The use according to claim 3, characterized in that the method of application is: mixing wet thalli obtained by induced culture of recombinant genetic engineering bacteria containing aldone reductase mutant genes and wet thalli obtained by induced culture of engineering bacteria containing glucose dehydrogenase genes, then carrying out resuspension by using phosphate buffer solution with pH 7.5 and 100mM, carrying out ultrasonic crushing, taking crushed mixed liquor as a catalyst, taking 6-cyano- (5R) -hydroxyl-3-carbonyl tert-butyl hexanoate as a substrate and glucose as an auxiliary substrate to form a reaction system, carrying out reaction at the temperature of 30 ℃ and under the condition of 300 revolutions per minute of 150 materials, finishing the reaction, separating and purifying reaction liquid, and obtaining 6-cyano- (3R,5R) -dihydroxy tert-butyl hexanoate; the nucleotide sequence of the glucose dehydrogenase gene is shown in SEQ ID No. 3.
5. The use according to claim 4, wherein in the reaction system, the final concentration of tert-butyl 6-cyano- (5R) -hydroxy-3-carbonyl hexanoate is 50-100g/L, the final concentration of glucose is 75-150 g/L, the dosage of the catalyst is 50-100g/L based on the total amount of wet bacteria before crushing, and wet bacteria obtained by induced culture of recombinant genetic engineering bacteria containing an aldone reductase mutant gene and wet bacteria obtained by induced culture of engineering bacteria containing a glucose dehydrogenase gene are mixed in a mass ratio of 3: 1.
6. The use according to claim 4, wherein the wet biomass is prepared by: inoculating recombinant genetic engineering bacteria containing an aldone reductase mutant gene into an LB liquid culture medium containing 50 mu g/mL kanamycin at the final concentration, culturing at 37 ℃ for 9 hours, inoculating the recombinant genetic engineering bacteria into a fresh LB liquid culture medium containing 50 mu g/mL kanamycin at the final concentration by an inoculation amount of 2% of volume concentration, culturing at 37 ℃ for 1.5 hours at 180 r/min, adding IPTG (isopropyl thiogalactoside) at the final concentration of 0.1mM into a culture solution, culturing at 28 ℃ for 10 hours, and centrifuging at 4 ℃ for 8000 r/min for 10 minutes to obtain wet thalli containing the aldone reductase mutant; the preparation method of the wet thallus obtained by induced culture of the engineering bacteria containing the glucose dehydrogenase gene is the same as that of the wet thallus containing the aldone reductase mutant gene.
7. Use according to claim 4, characterized in that the ultrasonication conditions are: mixing wet thalli obtained by induced culture of recombinant gene engineering bacteria containing aldone reductase mutant genes and wet thalli obtained by induced culture of engineering bacteria containing glucose dehydrogenase genes, then resuspending the mixture by using a phosphate buffer solution with the pH value of 7.5 and the concentration of 100mM, carrying out ultrasonic crushing on an ice-water mixture for 10 minutes, wherein the ultrasonic crushing conditions are as follows: the power was 400W, crushing for 1 second, and pausing for 1 second.
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| CN110004120B (en) * | 2019-01-25 | 2021-02-02 | 浙江工业大学 | Recombinant aldone reductase mutant and application thereof |
| CN110283799B (en) * | 2019-05-07 | 2022-11-01 | 沈阳药科大学 | Aldehyde ketone reductase BsAKR (YvgN) and mutant and application thereof |
| CN110272879B (en) * | 2019-05-07 | 2022-11-18 | 沈阳药科大学 | Aldehyde ketone reductase BcAKR and mutant and application thereof |
| CN110577940B (en) * | 2019-09-29 | 2021-06-08 | 浙江工业大学 | Kluyveromyces marxianus aldehyde ketone reductase KmAKR mutant and application thereof |
| CN110938608A (en) * | 2019-12-20 | 2020-03-31 | 台州酶易生物技术有限公司 | Aldehyde ketone reductase mutant, encoding gene and application of aldehyde ketone reductase mutant in synthesis of (S) -TCPE |
| CN111172140B (en) * | 2020-01-21 | 2022-04-19 | 浙江工业大学 | Nitrilase mutant and application thereof in preparation of anti-epileptic drug intermediate |
| CN115232800B (en) * | 2022-05-13 | 2023-09-05 | 浙江工业大学 | 298-site mutant aldehyde ketone reductase mutant and application thereof |
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| CN104498510A (en) * | 2014-11-13 | 2015-04-08 | 浙江工业大学 | Aldehyde ketoreductase bacterial strain, aldehyde ketoreductase gene, vector, engineering bacteria and application thereof |
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