CN112852768B - Carbonyl reductase mutant and application thereof - Google Patents
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Abstract
The invention provides a carbonyl reductase mutant and application thereof in S-3-cyclopentyl-3-hydroxypropionitrile synthesis, and particularly provides a carbonyl reductase mutant for improving stereoselectivity of catalytic synthesis of S-3-cyclopentyl-3-hydroxypropionitrile, wherein the mutant protein is an unnatural protein, has the characteristic of remarkably improving stereoselectivity of catalyzing 3-cyclopentyl-3-oxopropanenitrile to generate S-3-cyclopentyl-3-hydroxypropionitrile, and is mutated in one or more core amino acids related to enzyme catalytic activity of wild-type carbonyl reductase. The carbonyl reductase mutant can obviously improve the optical purity of the product.
Description
Technical Field
The invention belongs to the field of bioengineering, and particularly relates to a carbonyl reductase mutant and application thereof in preparation of chiral beta-hydroxy nitrile.
Background
Chiral beta-hydroxynitriles are key intermediates for the synthesis of many natural products and biologically active substances, although chemical asymmetric synthesis methods have been greatly developed in recent years, and can be prepared by asymmetric catalytic hydrogenation reduction reactions (a synthetic method of ruxotinib intermediate, royal, liu, chenjie, yangshijong, li qian, conliang, CN 104496904a), oxamicheal Addition reactions (Oxa-Michael Addition to alpha, beta-unreacted Nitriles: An experiential Route to gamma-Amino Alcohols and Derivatives guest reactions, zijstra Douwe s., De Vries Johannes, Otten Edwin chem 2018,10, 2868-sand 2872), enzymatic resolution (catalytic Enhancement of biological conversion of beta-Hydroxy chemical conversion, luminescence-synthesis of dye-dye conversion of dye-reaction, dye-reaction of dye-reaction, wang Mei-Xiang Organic Letters 2006,8,3231-3234) and the like, but the asymmetric synthesis method utilizing the catalysis of a biological system and an enzyme system draws more attention by virtue of the advantages of mild conditions, environmental friendliness, high chemical and stereoselectivity, simple and convenient operation and the like. The synthesis of chiral beta-hydroxy nitrile can be effectively realized by the dynamic resolution, the de-symmetry, the de-racemization, the alpha-carbonyl nitrile reduction and the like of the biocatalytic beta-hydroxy acid derivative, and the formula 1 shows.
Reduction of formula 13-cyclopentyl-3-oxopropanenitrile to prepare 3-cyclopentyl-3-hydroxypropanenitrile
Disclosure of Invention
In order to synthesize chiral beta-hydroxy nitrile under mild reaction conditions, the invention takes alpha-carbonyl nitrile as a substrate, and carbonyl reductase and a mutant thereof are used as catalysts to reduce carbonyl so as to prepare the beta-hydroxy nitrile with high chiral purity.
The invention has the beneficial effects that: the stereoselectivity of the beta-carbonyl nitrile is greatly improved by modifying carbonyl reductase, and the S-beta-hydroxyl nitrile is synthesized with high purity.
Detailed Description
The following examples further illustrate the present invention but are not to be construed as limiting the invention.
The invention provides a method for synthesizing beta-hydroxy nitrile with high chiral purity, which uses carbonyl reductase after engineering modification as a catalyst, the modified carbonyl reductase has at least 90% of identity with SEQ ID NO.1, and the ee value of the mutant transformation product S-beta-hydroxy nitrile is more than 98%.
In another aspect, there is provided a carbonyl reductase mutein, wherein the mutein is a non-native protein and wherein the mutein is a carbonyl reductase mutant mutated at one or more positions corresponding to positions 93,138,139, and 144 in SEQ ID NO.1: 1-249:
in another preferred embodiment, the histidine (H) at position 93 is mutated to phenylalanine (F), valine (V), tryptophan (W), preferably phenylalanine (F).
In another preferred embodiment, the valine at position 138 is mutated to phenylalanine (F), asparagine (N), and preferably phenylalanine (F).
In another preferred embodiment, alanine (a) at position 139 is mutated to valine (V), leucine (L) and isoleucine (I), preferably valine (V).
In another preferred embodiment, leucine (L) at position 144 is mutated to phenylalanine (F), asparagine (N), and preferably phenylalanine (F).
In another preferred embodiment, the carbonyl nitrile substrate is 3-cyclopentyl-3-oxopropanenitrile.
In another preferred embodiment, the carbonyl reductase mutant catalyzes the following reaction: specifically, the catalytic reaction takes wet thalli obtained by fermentation culture of carbonyl reductase mutant encoding genes and glucose dehydrogenase/acid dehydrogenase encoding gene engineering bacteria as a catalyst, takes 3-cyclopentyl-3-oxopropanenitrile as a substrate, adds a certain amount of glucose/sodium formate, takes a buffer solution with the pH of 6.0-10.0 as a reaction medium, and carries out reduction reaction at the temperature of 25-50 ℃ under the stirring condition;
the reaction has one or more characteristics selected from the group consisting of:
(i) the reaction system contains 10-100g/L of bacteria, preferably 10-30 g/L;
(ii) the pH of the reaction system is 6.0 to 10.0, preferably 6 to 8, more preferably 6.5;
(iii) the temperature of the reaction system is 25-50 ℃, preferably 25-35 ℃, more preferably 30 ℃.
(iv) The cosolvent of the reaction system is cosolvent, acetonitrile, acetone, methanol, ethanol, dimethyl sulfoxide, N-dimethylformamide, tetrahydrofuran, ethyl acetate, methyl tert-butyl ether, dichloromethane and 1, 4-dioxane, preferably cosolvent, methanol, ethanol, N-dimethylformamide and acetone, more preferably methanol.
(v) The concentration of the catalytic substrate is 10-200g/L, and preferably, the concentration of the substrate is 30-180 g/L;
(vi) the ee value of the S-beta-hydroxy nitrile obtained by catalysis is more than or equal to 90 percent, preferably more than or equal to 95 percent, and more preferably more than or equal to 99.0 percent.
The third aspect of the present invention provides an application of carbonyl reductase mutant in preparing S-beta-hydroxy nitrile compound, comprising the steps of:
(i) contacting the mutant of the second aspect of the invention with a reaction substrate to perform a catalytic reaction, thereby obtaining S- β -hydroxynitrile; and
(ii) optionally, isolating and purifying the S- β -hydroxy nitrile compound.
Drawings
FIG. 1 shows the results of the induced expression of BuADH and mutant proteins. Wherein, M represents Marker, 1 is BuADH soluble protein expression, 2 is BuADH insoluble protein expression, 3, 5 and 7 are representative mutant soluble protein expression, and 4, 6 and 8 are representative mutant insoluble protein expression.
FIG. 2 the product 3-cyclopentyl-3-hydroxypropionitrile racemate.
FIG. 3S-3-cyclopentyl-3-hydroxypropionitrile.
Detailed Description
Term(s) for
As used herein, the term "indicates that amino acid a at position xx is changed to amino acid B, e.g., H93F indicates that amino acid H at position 93 is mutated to F, and so on.
In a preferred embodiment of the present invention, the carbonyl reductase mutant of the present invention is prepared as follows: coli as an expression host.
Specifically, the preparation method comprises the following steps: (1) the gene of the corresponding mutation site of carbonyl reductase BuADH is constructed on a pET21a expression vector to obtain a recombinant plasmid with the target enzyme gene. (2) The recombinant plasmid is transferred into host bacterial cell, preferably Escherichia coli BL21(DE3), to obtain corresponding engineering strain. (3) The engineering strain is inoculated into LB culture medium, cultured for 3 hours at 37 ℃, added with 0.1mM isopropyl thiogalactoside and cultured for 12 hours at 25 ℃. (4) The cells were collected by centrifugation.
Example 1 construction of a pool of carbonyl reductase BuADH mutants
According to the known carbonyl reductase structure, the structure of BuADH is simulated, non-conserved residues in a substrate binding pocket are selected to carry out saturation mutation respectively, and a mutation primer is designed by adopting a degenerate codon NNK, and pET21a-BuADH is used as a template. And (3) picking the obtained monoclonal colony into a 96-hole deep-hole plate for culturing, and carrying out high-throughput activity screening on the expressed protein, wherein the screening method is to detect the decrease of NADPH at 340 nm.
The site of the library construction mutation is 93, 137, 138,139, 144 and 205 respectively, beneficial mutation sites 93,138,139 and 144 with improved stereoselectivity are obtained, the sites are mutants 1, 2, 3 and 4 respectively, and the protein sequence is shown in SEQ ID NO. 2-5.
The result of the experiment determination of the conversion of the 3-cyclopentyl-3-oxopropanenitrile shows that the mutant 3 has the best activity in single-point mutation, the stereoselectivity is obviously improved, and the protein sequence is shown in SEQ ID No. 4.
Example 2 construction of a pool of carbonyl reductase BuADH combinatorial mutants
And according to the saturated mutation result, selecting mutants with improved activity and optimal stereoselectivity as templates, and respectively constructing a combined mutant library. The results of the experimental determination on the conversion of 3-cyclopentyl-3-oxopropanenitrile show that the mutants obtained by two-site combined mutation in single-site mutation are mutants 5 and 6, the protein sequences are SEQ ID NO. 6 and 7, the mutant 5 is used as a template, the L144 mutation site is introduced to obtain the mutant 6, the protein sequence is SEQ ID NO. 8, the mutant 6 is used as a template, and V138 is introduced, and the results are shown in Table 1.
Example 3: inducible expression of carbonyl reductase BuADH mutant
Preparing 50mL of seed liquid, wherein the culture medium is LB liquid culture medium (peptone 10g/L, yeast powder 5g/L, NaCl10g/L), picking single colony of the genetically engineered bacteria by using an inoculating loop, inoculating into the culture medium, and culturing at 37 ℃ and 200rpm overnight. The seed liquid for overnight culture was transferred to a fermentation medium (LB medium) at an inoculum size of 1%, cultured at 37 ℃ and 200rpm to OD600About 0.6-1.0 mM IPTG was added and the mixture was induced at 25 ℃ and 200rpm for 10-12 hours. The cells were collected by centrifugation at 6000rpm at 4 ℃ and then washed with sodium phosphate buffer (100 mM)pH 7.0) was washed once. SDS-PAGE electrophorograms showed the induced expression of BuADH and the mutants, as shown in FIG. 1. The results show that both the mutant protein obtained by the method of the present example and BuADH contain soluble protein expression.
Example 4: method for catalyzing 3-cyclopentyl-3-oxopropanenitrile by carbonyl reductase BuADH mutant 3 recombinant bacteria
The BuADH mutant 3 of the present invention (protein sequence shown in SEQ ID NO: 4) was induced to express by the method of example 3, cells were collected by centrifugation (6000rpm), and the cells were used as biocatalysts to regenerate the carbonyl reductase cofactor by co-expressing the GDH (glucose dehydrogenase) gene with the mutant gene or adding glucose dehydrogenase.
(1) The thalli is taken and resuspended in 100mL sodium phosphate buffer solution (pH,6.5, 100mM), the concentration of the thalli (co-expressing GDH gene) is 20g/L, substrate 3-cyclopentyl-3-oxopropanenitrile is added to the final concentration of 20g/L, glucose is 50g/L, the reaction pH is adjusted to 6.5 by NaOH in the reaction process, shaking table reaction is carried out at 30 ℃ and 200r/min, and the reaction is stopped after 10 h. After the reaction, the reaction solution was extracted with ethyl acetate several times, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. GC detection shows that the yield is more than 99%, the product content is more than or equal to 95%, and the ee value is more than or equal to 99% (S).
(2) Taking the thalli to be resuspended in 100mL of sodium phosphate buffer solution (pH,6.5 and 100mM), wherein the thalli concentration is 20g/L, adding a substrate of 3-cyclopentyl-3-oxopropionitrile to a final concentration of 40g/L, 80g/L glucose and 0.1g/L GDH, adjusting the reaction pH to 6.5 by using NaOH during the reaction, carrying out shaking table reaction at 30 ℃ and 200r/min, and stopping the reaction after 16 h. After the reaction, the reaction solution was extracted with ethyl acetate several times, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. GC detection shows that the yield is more than 99%, the product content is more than or equal to 95%, and the ee value is more than or equal to 99% (S).
(3) The thalli is taken and resuspended in 100mL sodium phosphate buffer solution (pH 6.5, 100mM), the concentration of the thalli (co-expressing GDH gene) is 30g/L, substrate 3-cyclopentyl-3-oxopropanenitrile is added to the final concentration of 150g/L, glucose is 400g/L, NaOH is used for adjusting the reaction pH to be 6.5 in the reaction process, the shaking table reaction is carried out at 30 ℃ and 200r/min, and the reaction is stopped after 24 h. After the reaction, the reaction solution was extracted with ethyl acetate several times, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. GC detection shows that the yield is more than 99%, the product content is more than or equal to 95%, and the ee value is more than or equal to 99% (S).
(4) The cells were resuspended in 100mL of sodium phosphate buffer (pH 6.5, 100mM) at a cell (co-expressed GDH gene) concentration of 30g/L, and substrate 3-cyclopentyl-3-oxopropanenitrile was added to a final concentration of 200g/L, glucose 500g/L, NADP+In the reaction process of 0.1g/L, NaOH is used for adjusting the pH value of the reaction to be 6.5, the shaking table reaction is carried out at 30 ℃ and 200r/min, and the reaction is stopped after 24 hours. After the reaction, the reaction solution was extracted with ethyl acetate several times, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. GC assay, yield>99 percent, the product content is more than or equal to 95 percent, and the ee value is more than or equal to 99 percent (S).
(5) The cells were resuspended in 100mL of sodium phosphate buffer (pH 6.5, 100mM) at a cell concentration of 30g/L (co-expressed GDH gene), and 10mL of DMF solution containing substrate 3-cyclopentyl-3-oxopropanenitrile (final concentration of 200 g/L), glucose 500g/L, NADP+In the reaction process of 0.1g/L, NaOH is used for adjusting the pH value of the reaction to be 6.5, the shaking table reaction is carried out at 30 ℃ and 200r/min, and the reaction is stopped after 24 hours. After the reaction, the reaction solution was extracted with ethyl acetate several times, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. GC assay, yield>99 percent, the product content is more than or equal to 95 percent, and the ee value is more than or equal to 99 percent (S).
(6) The cells were resuspended in 100mL of a sodium phosphate buffer (pH 6.5, 100mM) at a cell concentration of 30g/L (co-expressed GDH gene), and 10mL of a DMSO solution containing a substrate of 3-cyclopentyl-3-oxopropanenitrile (final concentration of 200 g/L), glucose 500g/L, and NADP+In the reaction process of 0.1g/L, NaOH is used for adjusting the pH value of the reaction to be 6.5, the shaking table reaction is carried out at 30 ℃ and 200r/min, and the reaction is stopped after 24 hours. After the reaction, the reaction solution was extracted with ethyl acetate several times, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. GC assay, yield>99 percent, the product content is more than or equal to 95 percent, and the ee value is more than or equal to 99 percent (S).
(7) The cells were resuspended in 100mL of a sodium phosphate buffer (pH 6.5, 100mM) at a concentration of 30g/L, and the substrate 3-cyclopentyl-10mL of methanol solution with a final concentration of 3-oxopropanenitrile of 200g/L, glucose of 500g/L, NADP+In the reaction process of 0.1g/L, NaOH is used for adjusting the pH value of the reaction to be 6.5, the shaking table reaction is carried out at 30 ℃ and 200r/min, and the reaction is stopped after 24 hours. After the reaction, the reaction solution was extracted with ethyl acetate several times, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. GC assay, yield>99 percent, the product content is more than or equal to 95 percent, and the ee value is more than or equal to 99 percent (S).
(8) The cells were resuspended in 100mL of sodium phosphate buffer (pH 6.5, 100mM) at a cell concentration of 30g/L (co-expressed GDH gene), and 10mL of ethyl acetate solution containing substrate 3-cyclopentyl-3-oxopropanenitrile (final concentration of 200 g/L), glucose 500g/L, NADP+In the reaction process of 0.1g/L, NaOH is used for adjusting the pH value of the reaction to be 6.5, the shaking table reaction is carried out at 30 ℃ and 200r/min, and the reaction is stopped after 24 hours. After the reaction, the reaction solution was extracted with ethyl acetate several times, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. GC assay, yield>99 percent, the product content is more than or equal to 95 percent, and the ee value is more than or equal to 99 percent (S).
(9) The cells were resuspended in 100mL of sodium phosphate buffer (pH 6.5, 100mM) at a cell concentration of 30g/L (co-expressed GDH gene), and 10mL of 1, 4-dioxane solution containing 200g/L of substrate 3-cyclopentyl-3-oxopropanenitrile, 500g/L of glucose, NADP+In the reaction process of 0.1g/L, NaOH is used for adjusting the pH value of the reaction to be 6.5, the shaking table reaction is carried out at 30 ℃ and 200r/min, and the reaction is stopped after 24 hours. After the reaction, the reaction solution was extracted with ethyl acetate several times, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. GC assay, yield>99 percent, the product content is more than or equal to 95 percent, and the ee value is more than or equal to 99 percent (S).
Example 5: method for catalyzing 3-cyclopentyl-3-oxopropanenitrile by carbonyl reductase BuADH mutant 5 recombinant bacteria
The BuADH mutant 5 of the present invention (protein sequence shown in SEQ ID NO: 6) was induced to express by the method of example 3, and the cells were collected by centrifugation (6000rpm) and used as biocatalysts to regenerate the carbonyl reductase cofactor by co-expressing FDH (formate dehydrogenase) gene with the mutant gene or adding formate dehydrogenase.
(1) The thalli is taken and resuspended in 100mL sodium phosphate buffer solution (pH,6.5, 100mM), the concentration of the thalli (co-expressing FDH gene) is 20g/L, substrate 3-cyclopentyl-3-oxopropanenitrile is added to the final concentration of 20g/L, glucose is 50g/L, the shaking table reaction is carried out at 30 ℃ and 200r/min, and the reaction is stopped after 10 h. After the reaction, the reaction solution was extracted with ethyl acetate several times, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. GC detection shows that the yield is more than 99%, the product content is more than or equal to 95%, and the ee value is more than or equal to 99% (S).
(2) The thalli is taken and resuspended in 100mL sodium phosphate buffer solution (pH,6.5, 100mM), the thalli concentration is 20g/L, a substrate of 3-cyclopentyl-3-oxopropanenitrile is added until the final concentration is 40g/L, sodium formate is 40g/L, FDH is 0.1g/L, shaking table reaction is carried out at 30 ℃ and 200r/min, and the reaction is stopped after 16 h. After the reaction, the reaction solution was extracted with ethyl acetate several times, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. GC detection shows that the yield is more than 99%, the product content is more than or equal to 95%, and the ee value is more than or equal to 99% (S).
(3) The thalli is taken and resuspended in 100mL sodium phosphate buffer solution (pH 6.5, 100mM), the concentration of the thalli (co-expressing FDH gene) is 30g/L, substrate 3-cyclopentyl-3-oxopropanenitrile is added to the final concentration of 150g/L, sodium formate is 200g/L, the shaking table reaction is carried out at 30 ℃ and 200r/min, and the reaction is stopped after 24 h. After the reaction, the reaction solution was extracted with ethyl acetate several times, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. GC detection shows that the yield is more than 99%, the product content is more than or equal to 95%, and the ee value is more than or equal to 99% (S).
(4) The thalli is taken and resuspended in 100mL sodium phosphate buffer solution (pH 6.5, 100mM), the concentration of the thalli (co-expressed FDH gene) is 30g/L, substrate 3-cyclopentyl-3-oxopropanenitrile is added to the final concentration of 200g/L, sodium formate is 300g/L, NADP+The reaction was carried out at 30 ℃ and 200r/min in a shaker at 0.1g/L and stopped after 24 h. After the reaction, the reaction solution was extracted with ethyl acetate several times, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. GC assay, yield>99 percent, the product content is more than or equal to 95 percent, and the ee value is more than or equal to 99 percent (S). FIG. 2 the product 3-cyclopentyl-3-hydroxypropionitrile racemate; FIG. 3S-3-cyclopentyl-3-hydroxypropionitrile.
Sequence listing
<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences
<120> carbonyl reductase mutant and use thereof
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Glu Glu Ile Thr Pro Gln His Tyr Asp Ala Thr Phe Asp Val Asn Val
100 105 110
Arg Gly Leu Ile Phe Thr Val Gln Lys Ala Leu Pro Leu Met Gly Asn
115 120 125
Gly Ser Ser Ile Ile Leu Thr Ser Ser Val Val Gly Val Lys Gly Leu
130 135 140
Pro Ala His Asp Thr Tyr Ser Ala Ala Lys Ala Ala Val Arg Ser Leu
145 150 155 160
Ala Arg Thr Trp Thr Val Glu Leu Lys Gly Arg Gly Ile Arg Val Asn
165 170 175
Ala Ile Ser Pro Gly Ala Ile Asp Thr Pro Ile Ile Asp Ser Gln Val
180 185 190
Ala Thr Ala Ala Glu Ala Asp Glu Leu Arg Ala Arg Phe Ala Ala Ala
195 200 205
Thr Pro Leu Gly Arg Ile Gly Arg Pro Glu Glu Ile Ala Ser Ala Ala
210 215 220
Leu Phe Leu Ala Ser Asp Glu Ser Ser Phe Val Ala Gly Ile Asp Leu
225 230 235 240
Phe Val Asp Gly Gly Leu Ala Gln Val
245
<210> 5
<211> 249
<212> PRT
<213> Burkholderia sp. BT03
<400> 5
Met Asn Arg Leu Ala Ser Arg Thr Ala Val Ile Thr Gly Gly Ser Ser
1 5 10 15
Gly Ile Gly Leu Ala Thr Ala Gln Arg Phe Val Asp Glu Gly Ala Tyr
20 25 30
Val Phe Ile Val Gly Arg Arg Gln Leu Glu Leu Asp Lys Ala Val Gln
35 40 45
Gln Ile Gly Arg Asn Val Thr Ala Val Gln Ala Asp Val Thr Lys Leu
50 55 60
Asp Glu Leu Asp Arg Leu Phe Ser Ile Val Gly Glu Gln Arg Gly Lys
65 70 75 80
Ile Asp Val Leu Phe Ala Asn Ser Gly Ala Val Glu His Arg Thr Leu
85 90 95
Glu Glu Ile Thr Pro Gln His Tyr Asp Ala Thr Phe Asp Val Asn Val
100 105 110
Arg Gly Leu Ile Phe Thr Val Gln Lys Ala Leu Pro Leu Met Gly Asn
115 120 125
Gly Ser Ser Ile Ile Leu Thr Ser Ser Val Ala Gly Val Lys Gly Asn
130 135 140
Pro Ala His Asp Thr Tyr Ser Ala Ala Lys Ala Ala Val Arg Ser Leu
145 150 155 160
Ala Arg Thr Trp Thr Val Glu Leu Lys Gly Arg Gly Ile Arg Val Asn
165 170 175
Ala Ile Ser Pro Gly Ala Ile Asp Thr Pro Ile Ile Asp Ser Gln Val
180 185 190
Ala Thr Ala Ala Glu Ala Asp Glu Leu Arg Ala Arg Phe Ala Ala Ala
195 200 205
Thr Pro Leu Gly Arg Ile Gly Arg Pro Glu Glu Ile Ala Ser Ala Ala
210 215 220
Leu Phe Leu Ala Ser Asp Glu Ser Ser Phe Val Ala Gly Ile Asp Leu
225 230 235 240
Phe Val Asp Gly Gly Leu Ala Gln Val
245
<210> 6
<211> 249
<212> PRT
<213> Burkholderia sp. BT03
<400> 6
Met Asn Arg Leu Ala Ser Arg Thr Ala Val Ile Thr Gly Gly Ser Ser
1 5 10 15
Gly Ile Gly Leu Ala Thr Ala Gln Arg Phe Val Asp Glu Gly Ala Tyr
20 25 30
Val Phe Ile Val Gly Arg Arg Gln Leu Glu Leu Asp Lys Ala Val Gln
35 40 45
Gln Ile Gly Arg Asn Val Thr Ala Val Gln Ala Asp Val Thr Lys Leu
50 55 60
Asp Glu Leu Asp Arg Leu Phe Ser Ile Val Gly Glu Gln Arg Gly Lys
65 70 75 80
Ile Asp Val Leu Phe Ala Asn Ser Gly Ala Val Glu Val Arg Thr Leu
85 90 95
Glu Glu Ile Thr Pro Gln His Tyr Asp Ala Thr Phe Asp Val Asn Val
100 105 110
Arg Gly Leu Ile Phe Thr Val Gln Lys Ala Leu Pro Leu Met Gly Asn
115 120 125
Gly Ser Ser Ile Ile Leu Thr Ser Ser Val Val Gly Val Lys Gly Leu
130 135 140
Pro Ala His Asp Thr Tyr Ser Ala Ala Lys Ala Ala Val Arg Ser Leu
145 150 155 160
Ala Arg Thr Trp Thr Val Glu Leu Lys Gly Arg Gly Ile Arg Val Asn
165 170 175
Ala Ile Ser Pro Gly Ala Ile Asp Thr Pro Ile Ile Asp Ser Gln Val
180 185 190
Ala Thr Ala Ala Glu Ala Asp Glu Leu Arg Ala Arg Phe Ala Ala Ala
195 200 205
Thr Pro Leu Gly Arg Ile Gly Arg Pro Glu Glu Ile Ala Ser Ala Ala
210 215 220
Leu Phe Leu Ala Ser Asp Glu Ser Ser Phe Val Ala Gly Ile Asp Leu
225 230 235 240
Phe Val Asp Gly Gly Leu Ala Gln Val
245
<210> 7
<211> 249
<212> PRT
<213> Burkholderia sp. BT03
<400> 7
Met Asn Arg Leu Ala Ser Arg Thr Ala Val Ile Thr Gly Gly Ser Ser
1 5 10 15
Gly Ile Gly Leu Ala Thr Ala Gln Arg Phe Val Asp Glu Gly Ala Tyr
20 25 30
Val Phe Ile Val Gly Arg Arg Gln Leu Glu Leu Asp Lys Ala Val Gln
35 40 45
Gln Ile Gly Arg Asn Val Thr Ala Val Gln Ala Asp Val Thr Lys Leu
50 55 60
Asp Glu Leu Asp Arg Leu Phe Ser Ile Val Gly Glu Gln Arg Gly Lys
65 70 75 80
Ile Asp Val Leu Phe Ala Asn Ser Gly Ala Val Glu His Arg Thr Leu
85 90 95
Glu Glu Ile Thr Pro Gln His Tyr Asp Ala Thr Phe Asp Val Asn Val
100 105 110
Arg Gly Leu Ile Phe Thr Val Gln Lys Ala Leu Pro Leu Met Gly Asn
115 120 125
Gly Ser Ser Ile Ile Leu Thr Ser Ser Val Val Gly Val Lys Gly Asn
130 135 140
Pro Ala His Asp Thr Tyr Ser Ala Ala Lys Ala Ala Val Arg Ser Leu
145 150 155 160
Ala Arg Thr Trp Thr Val Glu Leu Lys Gly Arg Gly Ile Arg Val Asn
165 170 175
Ala Ile Ser Pro Gly Ala Ile Asp Thr Pro Ile Ile Asp Ser Gln Val
180 185 190
Ala Thr Ala Ala Glu Ala Asp Glu Leu Arg Ala Arg Phe Ala Ala Ala
195 200 205
Thr Pro Leu Gly Arg Ile Gly Arg Pro Glu Glu Ile Ala Ser Ala Ala
210 215 220
Leu Phe Leu Ala Ser Asp Glu Ser Ser Phe Val Ala Gly Ile Asp Leu
225 230 235 240
Phe Val Asp Gly Gly Leu Ala Gln Val
245
<210> 8
<211> 249
<212> PRT
<213> Burkholderia sp. BT03
<400> 8
Met Asn Arg Leu Ala Ser Arg Thr Ala Val Ile Thr Gly Gly Ser Ser
1 5 10 15
Gly Ile Gly Leu Ala Thr Ala Gln Arg Phe Val Asp Glu Gly Ala Tyr
20 25 30
Val Phe Ile Val Gly Arg Arg Gln Leu Glu Leu Asp Lys Ala Val Gln
35 40 45
Gln Ile Gly Arg Asn Val Thr Ala Val Gln Ala Asp Val Thr Lys Leu
50 55 60
Asp Glu Leu Asp Arg Leu Phe Ser Ile Val Gly Glu Gln Arg Gly Lys
65 70 75 80
Ile Asp Val Leu Phe Ala Asn Ser Gly Ala Val Glu Val Arg Thr Leu
85 90 95
Glu Glu Ile Thr Pro Gln His Tyr Asp Ala Thr Phe Asp Val Asn Val
100 105 110
Arg Gly Leu Ile Phe Thr Val Gln Lys Ala Leu Pro Leu Met Gly Asn
115 120 125
Gly Ser Ser Ile Ile Leu Thr Ser Ser Val Val Gly Val Lys Gly Asn
130 135 140
Pro Ala His Asp Thr Tyr Ser Ala Ala Lys Ala Ala Val Arg Ser Leu
145 150 155 160
Ala Arg Thr Trp Thr Val Glu Leu Lys Gly Arg Gly Ile Arg Val Asn
165 170 175
Ala Ile Ser Pro Gly Ala Ile Asp Thr Pro Ile Ile Asp Ser Gln Val
180 185 190
Ala Thr Ala Ala Glu Ala Asp Glu Leu Arg Ala Arg Phe Ala Ala Ala
195 200 205
Thr Pro Leu Gly Arg Ile Gly Arg Pro Glu Glu Ile Ala Ser Ala Ala
210 215 220
Leu Phe Leu Ala Ser Asp Glu Ser Ser Phe Val Ala Gly Ile Asp Leu
225 230 235 240
Phe Val Asp Gly Gly Leu Ala Gln Val
245
Claims (2)
1. The carbonyl reductase is characterized in that the amino acid sequence of the carbonyl reductase is the mutation of any one of the following sites on SEQ ID NO. 1;
histidine 93 (H) to phenylalanine (F);
or valine (V) at position 138 is mutated to phenylalanine (F);
or alanine (a) at position 139 to valine (V);
or leucine (L) at position 144 is mutated to phenylalanine (F);
or the 93 th histidine (H) is mutated into the phenylalanine (F), and the 139 th alanine (A) is mutated into the valine (V);
or alanine (a) at position 139 to valine (V); leucine (L) at position 144 was mutated to phenylalanine (F);
or histidine 93 (H) to phenylalanine (F); alanine (a) at position 139 was mutated to valine (V); leucine (L) at position 144 was mutated to phenylalanine (F);
or the 93 rd histidine (H) is mutated into valine (V)
Or the 93 rd histidine (H) is mutated to tryptophan (W);
or valine (V) at position 138 is mutated to asparagine (N);
or alanine (a) at position 139 to leucine (L);
or alanine (A) at position 139 is mutated to isoleucine (I);
or leucine (L) at position 144 is mutated to asparagine (N);
or histidine (H) at position 93 is mutated to valine (V); alanine (a) at position 139 was mutated to valine (V);
or the 93 rd histidine (H) is mutated to tryptophan (W); alanine (a) at position 139 was mutated to valine (V);
or histidine (H) at position 93 is mutated to valine (V); alanine (a) at position 139 was mutated to isoleucine (I);
or alanine (a) at position 139 to valine (V); leucine (L) at position 144 was mutated to asparagine (N);
or histidine 93 (H) to phenylalanine (F); alanine (a) at position 139 was mutated to valine (V); leucine (L) at position 144 was mutated to asparagine (N);
or histidine 93 (H) to phenylalanine (F); valine (V) at position 138 was mutated to asparagine (N); alanine (a) at position 139 was mutated to valine (V); leucine (L) at position 144 was mutated to phenylalanine (F);
or histidine 93 (H) to phenylalanine (F); valine (V) at position 138 was mutated to phenylalanine (F); alanine (a) at position 139 was mutated to valine (V); leucine (L) at position 144 was mutated to phenylalanine (F).
2. A process for stereoselective reduction of an α -carbonyl nitrile comprising the steps of:
(i) contacting the carbonyl reductase of claim 1 with a reaction substrate, 3-cyclopentyl-3-oxopropanenitrile, to perform a catalytic reaction, thereby obtaining S-3-cyclopentyl-3-hydroxypropionitrile; and
(ii) optionally, isolating and purifying said S-3-cyclopentyl-3-hydroxypropionitrile.
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| CN202010788330.7A CN112852768B (en) | 2020-08-07 | 2020-08-07 | Carbonyl reductase mutant and application thereof |
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| CN112852768B true CN112852768B (en) | 2021-10-08 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2000236883A (en) * | 1998-12-21 | 2000-09-05 | Daicel Chem Ind Ltd | New carbonyl reductase, production of the enzyme, dna coding for the enzyme, and production of alcohol using the same |
| CN115725524A (en) * | 2018-05-31 | 2023-03-03 | 中国科学院天津工业生物技术研究所 | Carbonyl reductase mutant and application thereof in reduction of cyclopentadione compounds |
| CN110713991B (en) * | 2018-07-13 | 2022-01-25 | 中国科学院天津工业生物技术研究所 | Application of carbonyl reductase and mutant thereof in synthesis of indacaterol drug intermediate |
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