CN112831532B - Method for enzymatic synthesis of D-leucine - Google Patents
Method for enzymatic synthesis of D-leucine Download PDFInfo
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- CN112831532B CN112831532B CN202110333271.9A CN202110333271A CN112831532B CN 112831532 B CN112831532 B CN 112831532B CN 202110333271 A CN202110333271 A CN 202110333271A CN 112831532 B CN112831532 B CN 112831532B
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- val
- gly
- ala
- glu
- amino acid
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/99—Oxidoreductases acting on the CH-OH group of donors (1.1) with other acceptors (1.1.99)
- C12Y101/9901—Glucose dehydrogenase (acceptor) (1.1.99.10)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
- C12Y104/99—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with other acceptors (1.4.99)
- C12Y104/99001—D-Amino-acid dehydrogenase (1.4.99.1)
Abstract
The invention discloses a method for enzymatically synthesizing D-leucine, which takes alpha-ketoisocaproic acid as a substrate, uses D-amino acid dehydrogenase to catalyze the substrate to perform dehydrogenation reaction and amino transfer reaction, and obtains the D-leucine, wherein the ee value of the product is more than 99%, and the method has wide industrial application prospect.
Description
Technical Field
The invention belongs to the technical field of enzyme catalysis, and particularly relates to a method for enzymatically synthesizing D-leucine and a D-amino acid dehydrogenase mutant used for the method.
Background
D-leucine is an unnatural amino acid, also known as D-2-amino-4-methylpentanoic acid, with CAS number 328-38-1, and is a white scale powder. The amino acid is a branched chain amino acid, can be used as a nutritional supplement, is helpful for muscle recovery after training, controls blood sugar, provides energy for body tissues, and enhances immunity. D-leucine has many applications in the fields of medicine and chemical industry.
D-leucine can be obtained by fermentation (CN 110330441A); or by chiral resolution of DL-leucine racemate (CN 103981248A), both of which have advantages and disadvantages, and which are characterized by relatively high cost. The fermentation method has long production period and complicated steps of extraction and purification of the fermented product, and the chiral resolution is not economically feasible because leucine with higher price is used as a raw material in the chemical resolution method, the enzymatic method or the induced crystallization method.
Disclosure of Invention
In order to reduce the production cost of D-leucine, the present invention has developed a novel pathway for the preparation of D-leucine by synthesis, the route of which is shown below.
The inventor has conducted extensive screening for an amino acid dehydrogenase capable of catalyzing alpha-ketoisohexide to produce D-leucine, and screened an amino acid dehydrogenase with high stereoselectivity and relatively high enzyme activity, which is NADP+ cofactor dependent D-type amino acid dehydrogenase (called utDAADH for short) from Bacillus thermoglobus (Ureibacillus thermosphaericus) (SEQ ID NO: 1). Furthermore, in order to improve the enzyme activity, the wild enzyme is modified by genetic engineering to construct a mutant capable of catalyzing the alpha-ketoisohexide reaction with high efficiency, so that the mutant is used for synthesizing D-leucine with high optical purity. Specifically, the present invention includes the following technical matters.
A method for enzymatic synthesis of D-leucine uses alpha-ketoisocaproic acid as substrate, and D-amino acid dehydrogenase is used for catalyzing the dehydrogenation reaction and the amino transfer reaction of the substrate to obtain the D-leucine.
In the above method, glucose dehydrogenase and coenzyme NADPH may be added to the reaction system. Since D-amino acid dehydrogenase is an NADP+ cofactor-dependent enzyme, the reaction system contains a glucose dehydrogenase-dependent NADP+ cofactor regeneration system, for example, to which glucose dehydrogenase and coenzyme NADPH (i.e., NADP+), are added. By means of the NADP+ cofactor regeneration system, the production costs of D-leucine can be further reduced, which is economically advantageous.
Preferably, ammonium salt or aqueous ammonia may be added as an ammonia donor to the reaction system.
In one embodiment, the D-amino acid dehydrogenase used in the above method is a D-amino acid dehydrogenase of Bacillus thermocellum (Ureibacillus thermosphaericus) origin SEQ ID NO 1 or a mutant having 90% or more homology, preferably 95% or more homology, preferably 98% or more homology, more preferably 99% or more homology thereto.
MSKIRIGIVGYGNLGRGVEAAIQQNPDMELVAVFTRRDPKTVAVKSNVKVLHVDDAQSYKDEIDVMILCGGSATDLPEQGPYFAQYFNTIDSFDTHARIPDYFDAVNAAAEQSGKVAIISVGWDPGLFSLNRLLGEVVLPVGNTYTFWGKGVSQGHSDAIRRIQGVKNAVQYTIPIDEAVNRVRSGENPELSTREKHARECFVVLEEGADPAKVEHEIKTMPNYFDEYDTTVHFISEEELKQNHSGMPHGGFVIRSGKSDEGHKQIIEFSLNLESNPMFTSSALVAYARAAYRLSQNGDKGAKTVFDIPFGLLSPKSPEDLRKELL(SEQ ID NO:1)。
Preferably, the amino acid sequence of the mutant is SEQ ID NO. 3 or SEQ ID NO. 4. However, the D-amino acid dehydrogenase mutant of the present invention is not limited thereto. Among them, the D-amino acid dehydrogenase mutant SEQ ID NO. 3 has been reported in patent document CN202110108232.9, and it has been found that it can catalyze the reaction of alpha-ketoisohexide to produce D-leucine.
MSKIRIGIVGYGNLGRGVEAAIQQNPDMELVAVFTRRDPKTVAVKSNVKVLHVDDAQSYKDEIDVMILCGGSATDLPEQGPYFAQYFNTIDSFATHARIPDYFDAVNAAAEQSGKVAIISVGWDPGLFSLNRLLGEVVLPVGNTYTFWGKGVSQGHSDAIRRIQGVKNAVQYTIPIDEAVNRVRSGENPELSTREKHAVECFVVLEEGADPAKVEHEIKTMPNYFDEYDTTVHFISEEELKQNHSGMPTGGFVIRSGKSDEGHKQIIEFSLNLESNPMFTSSALVAYARAAYRLSQNGDKGAKTVFDIPFGLLSPKSPEDLRKELL(SEQ ID NO:3)。
The mutant is a mutant of wild D-amino acid dehydrogenase SEQ ID NO. 1, wherein D at 94 th is replaced by A, R at 199 th is replaced by V, and H at 249 th is replaced by T.
Another D-amino acid dehydrogenase mutant, SEQ ID NO. 4 differs from SEQ ID NO. 3 only in that the D at position 94 of 1 is replaced by L, the amino acid sequence of which is:
MSKIRIGIVGYGNLGRGVEAAIQQNPDMELVAVFTRRDPKTVAVKSNVKVLHVDDAQSYKDEIDVMILCGGSATDLPEQGPYFAQYFNTIDSFLTHARIPDYFDAVNAAAEQSGKVAIISVGWDPGLFSLNRLLGEVVLPVGNTYTFWGKGVSQGHSDAIRRIQGVKNAVQYTIPIDEAVNRVRSGENPELSTREKHAVECFVVLEEGADPAKVEHEIKTMPNYFDEYDTTVHFISEEELKQNHSGMPTGGFVIRSGKSDEGHKQIIEFSLNLESNPMFTSSALVAYARAAYRLSQNGDKGAKTVFDIPFGLLSPKSPEDLRKELL(SEQ ID NO:4)。
the mutant is a mutant of wild D-amino acid dehydrogenase SEQ ID NO. 1, wherein D at 94 th is replaced by L, R at 199 th is replaced by V, and H at 249 th is replaced by T.
Alternatively, the glucose dehydrogenase is derived from Bacillus cereus (Bacillus cereus), the amino acid sequence of which is SEQ ID NO. 6, and the coding gene of which is SEQ ID NO. 7 (GenBank sequence number AE 016877.1) can be obtained by expression in Escherichia coli. However, the glucose dehydrogenase used in combination with the D-amino acid dehydrogenase of the present invention or the mutant thereof is not limited thereto.
In another aspect, the invention provides a D-amino acid dehydrogenase mutant, the amino acid sequence of which is SEQ ID NO. 4.
The D-amino acid dehydrogenase mutant can be obtained by fermentation expression of genetically engineered bacteria. When the expression host bacterium of the D-amino acid dehydrogenase mutant SEQ ID NO. 4 is Escherichia coli, the nucleotide sequence of the encoding gene thereof may be SEQ ID NO. 5.
The coding gene can be cloned on a proper vector, and then the vector is transformed into competent cells of escherichia coli to obtain a transformant for expressing the D-amino acid dehydrogenase mutant SEQ ID NO. 4. Preferably, the vector is a PET series plasmid, such as, but not limited to, pET24a or pET28 a.
The D-amino acid dehydrogenase mutant SEQ ID NO. 4 can be obtained through fermentation of genetically engineered bacteria. For example, after microbial fermentation, the thalli are resuspended with buffer solution, broken by ultrasound, centrifuged, the supernatant is collected and subjected to column chromatography, and the target protein is eluted, thus obtaining the purified D-amino acid dehydrogenase mutant.
In another aspect, the invention provides the use of the above-described D-amino acid dehydrogenase mutant SEQ ID NO. 4 or SEQ ID NO. 3 for the preparation of D-leucine.
The invention provides a new idea for preparing high optical purity D-leucine, which adopts D-amino acid dehydrogenase such as SEQ ID NO. 4, takes 100mM alpha-ketoisocaproic acid as a substrate, can reach a conversion rate of 95% in 8 hours, and the ee value of the product D-leucine exceeds 99%, thus showing good industrial development and application prospects.
Detailed Description
In exploring the enzymatic synthesis of D-amino acids such as D-tert-leucine, D-leucine, etc., wild-type D-amino acid dehydrogenase derived from microbial Bacillus thermosiphon (Ureibacillus thermosphaericus), genBank accession number BAK86217.1, amino acid sequence SEQ ID NO 1 was selected. When it is expressed in E.coli, the coding gene may be SEQ ID NO. 2.
Experiments show that the catalytic activity is low, and the requirements of industrialized production of D-tertiary leucine, D-leucine and the like are difficult to meet. The wild-type enzyme SEQ ID NO. 1 is analyzed by bioinformatics technology, and the fact that some sites in the amino acid sequence play a key role in the aspects of the structure and the function of the enzyme is judged, then the wild-type enzyme SEQ ID NO. 1 is modified by site-directed saturation mutation and other technologies, and the important point is that active site amino acids (including 94 th aspartic acid site, 199 th arginine site and 249 th histidine) catalyzing substrates to perform dehydrogenation reaction and amino transfer reaction are replaced so as to obtain mutants with improved enzyme activity. A series of mutants with improved enzyme activity, such as mutant SEQ ID NO. 3 (D94A, R199V, H249T) and mutant SEQ ID NO. 4 (D94L, R199V, H249T) are screened out through screening of a plurality of mutants.
It was found that these mutants are highly selective for substrates, such as mutant SEQ ID NO 3, which is incorporated herein by reference in its part, efficiently catalyzes the reaction of trimethylpyruvic acid to D-tert-leucine as reported in patent document CN 202110108232.9. However, when the substrate is changed into alpha-ketoisocaproic acid, the catalytic activity is obviously reduced. While the other amino acid has only one site difference (the 94 th alanine A and leucine L difference) of the mutant SEQ ID NO 4 shows higher enzyme activity and high stereoselectivity to the substrate alpha-ketoisohexide, the two mutants have the reason for the difference in substrate selectivity to be further analyzed and studied.
In the present invention, the terms "wild type", "wild-type enzyme" and "wild-type enzyme" mean the same meaning, and all refer to the wild-type sequence of D-type amino acid dehydrogenase SEQ ID NO. 1. Correspondingly, the D-amino acid dehydrogenase mutants SEQ ID NO. 3 and SEQ ID NO. 4 may also be referred to simply as "mutants" or "mutant enzymes".
The D-amino acid dehydrogenase mutant SEQ ID NO. 4 of the present invention has only 326 amino acids in number and a clear structure, and thus the encoding genes thereof, expression cassettes and plasmids comprising these genes, and transformants comprising the plasmids can be easily obtained by those skilled in the art. These genes, expression cassettes, plasmids, transformants can be obtained by genetic engineering construction methods well known to those skilled in the art.
The wild-type D-amino acid dehydrogenase SEQ ID NO. 1, its mutants SEQ ID NO. 3 and SEQ ID NO. 4 are collectively referred to herein as D-amino acid dehydrogenases, based on the same catalytic function considerations.
Glucose dehydrogenase used in combination with the D-amino acid dehydrogenase of the present invention can also be expressed by E.coli.
The invention adopts a coupling reaction mode of D-amino acid dehydrogenase and glucose dehydrogenase, takes alpha-ketoisocaproic acid and glucose as substrates and takes NADP+ as cofactor, and prepares the D-leucine by a one-pot method. Wherein glucose is a substrate for glucose dehydrogenase, which catalyzes glucose oxidation while NADP is reacted + Reduced to NADPH.
When used as biocatalysts for the production of D-leucine, the D-amino acid dehydrogenase and glucose dehydrogenase used in the catalytic synthesis of the present invention may take the form of enzymes or expressed microbial fermentation tubes. The enzyme forms include free enzymes, immobilized enzymes, including purified enzymes, crude enzymes, fermentation broths, carrier immobilized enzymes, and the like.
The present invention will be described in further detail with reference to specific examples. It should be understood that the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
The amounts, amounts and concentrations of various substances are referred to herein, wherein the percentages refer to percentages by mass unless otherwise specified.
Examples
Materials and methods
The whole gene synthesis, primer synthesis and sequencing in the examples were all performed by su Jin Weizhi biotechnology, inc.
Examples of molecular biology experiments include plasmid construction, digestion, ligation, competent cell preparation, transformation, medium preparation, etc., and are mainly described in "molecular cloning Experimental guidelines (third edition), J.Sam Broker, D.W. Lassel (America) code, huang Peitang, et al, scientific Press, beijing, 2002). The specific experimental conditions can be determined by simple experiments, if necessary.
The PCR amplification experiments were performed according to the reaction conditions or kit instructions provided by the plasmid or DNA template suppliers. Can be adjusted if necessary by simple tests.
LB medium: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride, pH7.2. (LB solid Medium additionally 20g/L agar powder.)
TB medium: 24g/L yeast extract, 12g/L tryptone, 16.43g/L K 2 HPO 4 .3H 2 O、2.31g/L KH 2 PO 4 5g/L glycerol, pH7.0-7.5. (TB solid Medium additionally 20g/L agar powder.)
For convenience of description, in the examples, a certain enzyme protein number, its gene number and its expression strain number are sometimes mixed/applied, and those skilled in the art will readily understand that they refer to different organism meanings in different environments.
Expression strains of wild-type D-amino acid dehydrogenase SEQ ID NO. 1, glucose dehydrogenase SEQ ID NO. 6, D-amino acid dehydrogenase (D94A, R199V, H249T) mutant SEQ ID NO. 3 were constructed according to the methods reported in examples 1-3 of patent document CN202110108232.9, respectively.
EXAMPLE 1 construction of wild-type D-amino acid dehydrogenase-expressing Strain
And (3) carrying out codon optimization on wild type D-amino acid dehydrogenase SEQ ID NO. 1 from the bacillus thermosphere urea (Ureibacillus thermosphaericus), synthesizing a coding gene sequence SEQ ID NO. 2 through total genes, designing restriction enzyme sites Nde I and XhoI at two ends of the genes, and subcloning the restriction enzyme sites Nde I and XhoI into corresponding sites of a vector pET24a (Novagen) to obtain a recombinant plasmid pET24a-utDAADH. And transforming the recombinant plasmid pET24a-utDAADH into an expression host escherichia coli BL21 (DE 3) to obtain the recombinant escherichia coli utDAADH for expressing the wild enzyme.
EXAMPLE 2 construction of glucose dehydrogenase-expressing Strain
For glucose dehydrogenase SEQ ID NO. 6 from Bacillus cereus, the whole gene is synthesized into a coding gene sequence SEQ ID NO. 7, restriction enzyme sites Nde I and XhoI are designed at two ends of the gene, and the restriction enzyme sites Nde I and XhoI are subcloned into corresponding sites of a vector pET24a (Novagen) to obtain a recombinant plasmid pET24a-bcGDH. The recombinant plasmid pET24a-bcGDH is transformed into an expression host escherichia coli BL21 (DE 3) to obtain the recombinant escherichia coli bcGDH for expressing glucose dehydrogenase.
EXAMPLE 3 construction of (D94A, R199V, H249T) mutant strains
A mutant SEQ ID NO:3 expression strain (D94A, R199V, H249T) was constructed according to the method reported in example 3 of patent document CN202110108232.9, comprising the steps of:
3.1 plasmid pET24a-utDAADH of the strain utDAADH is taken as a template, and the three sites 94 th site, 199 th site and 249 th site in the wild type D-amino acid dehydrogenation SEQ ID NO:1 are modified into D94A, R199V, H249T by a gene site-directed mutagenesis technology, and then a mutant expression strain utDAADH-M containing the three mutations is constructed according to the method in example 1. The primers used in the construction were as follows: utDAADH-94F:5' -CAACACCATCGACTCTTTCGCCACCCACGCTCGTATC-3’，utDAADH-199F：5’-CTACCCGTGAAAAACACGCTGTTGAATGCTTCGTTGTTC-3’，utDAADH-199R：5’-GAACAACGAAGCATTCAACAGCGTGTTTTTCACGGGTAG-3’，utDAADH-249R：5’-GATAACGAAGCCGCCGGTGGGCATACCAGAGTG-3’。
Directly amplifying the P1 fragment by taking pET24a-utDAADH plasmid as a template, taking utDAADH-94F and utDAADH-199R as primer pairs, directly amplifying the P2 fragment by taking 2utDAADH-199F and utDAADH-249R as primer pairs, amplifying the large fragment P by taking over-stacking PCR of the P1 and P2 fragments as primer pairs, and then carrying out MegaPrimer PCR by taking the large fragment P as primer pairs to construct the site-directed mutant expression plasmid.
50 μl of the P1 and P2 fragments: 10ng plasmid template, 10pmol primer pair, 1 XKOD plus buffer,0.2mM dNTP,1.5mM MgSO 4 5 unitsKOD-plus DNA polymerase of (A).
PCR reaction conditions for P1 and P2 fragments: 95 ℃ for 1min;98℃for 10s,57℃for 30s,68℃for 1min/kbp,30 cycles; and at 68℃for 10min.
After the PCR is finished, the PCR product of the P1 fragment is about 353bp, the PCR product of the P2 fragment is about 188bp, and the PCR products of the P1 and P2 are respectively cut and recovered.
And (3) performing over-stacking PCR by taking the fragment P1 and the fragment P2 after gel cutting recovery as templates and taking utDAADH-94F and utDAADH-249R as primers to obtain the fragment P with the length of about 502bp, and gel cutting recovery.
Over-stacking PCR reaction system: 5 mu l P fragment, 5 mu l P fragment, 10pmol primer pair, 1 XKOD plus buffer,0.2mM dNTP,1.5mM MgSO 4 KOD-plus DNA polymerase of 5 units.
Over-stacking PCR reaction conditions: 3min at 95 ℃;98℃for 10s,60℃for 30s,68℃for 1min/kbp,25 cycles; and at 68℃for 10min.
The fragment P is used as a large primer, the pET24a-utDAADH plasmid is used as a template, KOD-plus DNA polymerase is used as MegaPrimer PCR, and the reaction system is as follows: 250ng of P fragment, 10ng of plasmid template, 1 XKOD plus buffer,0.2mM dNTP,1.5mM MgSO 4 KOD-plus DNA polymerase of 5 units.
MegaPrimer PCR reaction conditions: 94 ℃ for 5min;98℃for 10s,60℃for 30s,68℃for 2min/kbp,25 cycles; and at 68℃for 10min.
The plasmid template is digested by DpnI, competent cells of E.coli BL21 (DE 3) are chemically transformed, the positive clone strain is subjected to test tube culture, plasmids are extracted, and the construction of the mutant strain utDAADH-M is successfully determined by plasmid sequencing. The strain can express mutant enzyme SEQ ID NO. 3.
EXAMPLE 4 construction of (D94L, R199V, H249T) mutant strains
Similarly to example 3, another (D94L, R199V, H249T) mutant SEQ ID NO:4 expressing strain was constructed comprising the steps of:
plasmid pET24a-utDAADH of utDAADH strain is used as template, three sites of 94 site, 199 site and 249 site are modified into D94L, R199V and H249T by site-directed mutagenesis technology, and three kinds of mutations are constructedMutant strain utDAADH-M437. The primers used in the construction were as follows: utDAADH-94LF:5' -CAACACCATCGACTCTTTCCTGACCCACGCTCGTATC-3’utDAADH-199F：5’-CTACCCGTGAAAAACACGCTGTTGAATGCTTCGTTGTTC-3’utDAADH-199R：5’-GAACAACGAAGCATTCAACAGCGTGTTTTTCACGGGTAG-3’utDAADH-249R：5’-GATAACGAAGCCGCCGGTGGGCATACCAGAGTG-3’
Directly amplifying the P3 fragment by taking pET24a-utDAADH plasmid as a template, taking utDAADH-94LF and utDAADH-199R as primer pairs, directly amplifying the P4 fragment by taking 2utDAADH-199F and utDAADH-249R as primer pairs, amplifying the large fragment P-1 by taking over-mapping PCR of the P3 and P4 fragments and then carrying out MegaPrimer PCR by taking the P-1 large fragment as primer pairs, thereby constructing the site-directed mutant strain utDAADH-M437.
P3 and P4 fragments 50. Mu.L PCR reaction System: 10ng plasmid template, 10pmol primer pair, 1 XKOD plus buffer,0.2mM dNTP,1.5mM MgSO 4 KOD-plus DNA polymerase of 5 units.
PCR reaction conditions for P3 and P4 fragments: 95 ℃ for 1min;98℃for 10s,57℃for 30s,68℃for 1min/kbp;30 cycles; and at 68℃for 10min.
After the PCR is finished, the P3 fragment PCR product is about 353bp, the P4 fragment PCR product is about 188bp, and the P3 and P4 PCR products are respectively cut and recovered.
And (3) performing over-stacking PCR by taking the fragment P3 and the fragment P4 which are recovered by cutting the gel as templates and taking utDAADH-94LF and utDAADH-249R as primers to obtain a fragment P-1 with the length of about 502bp, and recovering the cutting the gel.
Over-stacking PCR reaction system: 5 mu L P fragment, 5 mu L P fragment, 10pmol primer pair, 1 XKOD plus buffer,0.2mM dNTP,1.5mM MgSO 4 KOD-plus DNA polymerase of 5 units.
Over-stacking PCR reaction conditions: 3min at 95 ℃;98℃for 10s,60℃for 30s,68℃for 1min/kbp;25 cycles; and at 68℃for 10min.
The fragment P-1 is used as a large primer, the pET24a-utDAADH plasmid is used as a template, KOD-plus DNA polymerase is used as MegaPrimer PCR, and the reaction system is as follows: 250ng of P-1 fragment, 10ng of plasmid template, 1 XKOD plus buffer,0.2mM dNTP,1.5mM MgSO 4 KOD-plus DNA polymerase of 5 units.
MegaPrimer PCR reaction conditions: 94 ℃ for 5min;98℃for 10s,60℃for 30s,68℃for 2min/kbp,25 cycles; and at 68℃for 10min. The plasmid template is digested by DpnI, competent cells of E.coli BL21 (DE 3) are chemically transformed, the positive clone strain is subjected to test tube culture, plasmids are extracted, and the plasmid sequencing determines that the mutant strain utDAADH-M437 is successfully constructed. The strain can express mutant enzyme SEQ ID NO. 4.
Example 5 comparison of enzyme Activity
5.1 shaking flask fermentation
Single colonies were picked from LB plates of utDAADH, utDAADH-M and utDAADH-M437, respectively, and inoculated into LB liquid medium containing 50. Mu.g/mL kanamycin sulfate, respectively, and cultured overnight at 37℃at 230 rpm. According to the volume ratio of 1:100, the overnight cultures were each transferred to 1L of TB medium containing 50. Mu.g/mL kanamycin sulfate and incubated at 37℃at 230rpm to OD 600 At=0.6-0.8, IPTG was added at a final concentration of 0.1mM, and incubated at 25 ℃ at 200rpm overnight. Then, the cells were collected by centrifugation at 8000rpm at 4℃for 10min.
5.2 extraction of pure enzymes of D-amino acid dehydrogenase expressed by utDAADH, utDAADH-M and tDAADH-M437
The cells were resuspended in 50mL of equilibration buffer (20 mM potassium phosphate buffer, 200mM NaCl, pH 7.8), then sonicated, and the disrupted cells were centrifuged at 12000rpm for 20min at 4℃to collect the supernatant. The supernatant was added to an affinity column containing 10mL of Ni-NAT matrix at a rate of 1mL/min, and then the column was washed with an equilibration buffer containing 50mM imidazole to elute the impurities. Finally, the target protein was eluted with an equilibration buffer containing 500mM imidazole, and the peak eluate was collected.
Desalting the eluate with ultrafiltration tube with molecular weight cut-off of 10kDa to obtain pure enzyme.
5.3A glucose dehydrogenase SEQ ID NO. 6 pure enzyme was prepared in a similar manner to steps 5.1 and 5.2.
5.4 determination of specific Activity of D-amino acid dehydrogenase
500 μl of reaction system was used: 200mM glycine-KOH buffer (pH 10.5), 200mM ammonium chloride, 20mM alpha-ketoisocaproic acid, 5mM NADPH, 50. Mu.l of the desalted pure enzyme of step 5.2, 450. Mu.l of pure water, 45℃water bath, and reaction for 20min were carried out, and the amount of activity was determined by measuring the change in absorbance at 340 nm.
Meanwhile, the BCA Protein Assay Kit kit of Thermo Scientific company is adopted to measure the protein concentration of the pure enzyme, so that the specific activity of the pure enzyme is obtained.
Test results show that for the reaction of catalyzing the substrate alpha-ketoisohexide to be converted into D-leucine, the unit enzyme activity of the mutant SEQ ID NO. 4 is improved by 211 times compared with that of the wild type enzyme SEQ ID NO. 1; the unit enzyme activity of the mutant SEQ ID NO. 3 is improved by 55 times compared with that of the wild type enzyme SEQ ID NO. 1.
EXAMPLE 6 mutant SEQ ID NO. 4 for the Synthesis of D-leucine
The catalytic reaction of the substrate alpha-ketoisocaproic acid was performed using the pure enzymes D-amino acid dehydrogenase SEQ ID NO 4 and glucose dehydrogenase SEQ ID NO 6 as follows.
50ml of reaction system: glycine-KOH buffer (20 mM, pH 10), 100mM of alpha-ketoisocaproic acid, 0.3M glucose, 1mM of coenzyme NADP+,150mM of ammonium chloride, 0.1mg/ml of glucose dehydrogenase, 10U/ml of D-amino acid dehydrogenase pure enzyme, and the pH of the reaction system was kept at 10.0 by correcting with ammonia water. After 4 hours of reaction at 45 ℃, 10U/ml of D-amino acid dehydrogenase pure enzyme is added, the reaction is continued for 6 to 10 hours, after sampling and centrifugation, the supernatant is directly subjected to HPLC analysis after passing through a 0.22 mu m membrane, the final reaction is confirmed for 8 hours, the substrate conversion rate is over 95%, and the ee value of the product is over 99%.
HPLC detection method: agilent 1260; kromasil 100C18 column (250X 4mm,5 μm); mobile phase a:10mM sodium acetate, pH 6.00; mobile phase B:85% acetonitrile in water; derivatization agent: 0.1372g of phthalic dicarboxaldehyde and 0.0589g N-isobutyryl-L-cysteine were taken up to 10ml in 0.1M boric acid buffer (pH 10.4); sample injection amount: 5 μl; column temperature is 30 ℃; flow rate: 1ml/min; detection wavelength: 334nm.
In summary, the D-amino acid dehydrogenase SEQ ID NO. 1 and mutants SEQ ID NOs 3-4 screened by the method can be used for catalyzing and synthesizing the D-leucine by taking the alpha-ketoisocaproic acid as a substrate, for example, the substrate concentration of 100mM can be adopted, the substrate conversion rate exceeds 95% and the ee value of the product D-leucine exceeds 99% within 8 hours, and the method has industrial development and application potential.
Sequence listing
<110> Luoyang Hua Rong Biotechnology Co., ltd
<120> a method for enzymatically synthesizing D-leucine
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<213> Ureibacillus thermosphaericus
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Met Ser Lys Ile Arg Ile Gly Ile Val Gly Tyr Gly Asn Leu Gly Arg
1 5 10 15
Gly Val Glu Ala Ala Ile Gln Gln Asn Pro Asp Met Glu Leu Val Ala
20 25 30
Val Phe Thr Arg Arg Asp Pro Lys Thr Val Ala Val Lys Ser Asn Val
35 40 45
Lys Val Leu His Val Asp Asp Ala Gln Ser Tyr Lys Asp Glu Ile Asp
50 55 60
Val Met Ile Leu Cys Gly Gly Ser Ala Thr Asp Leu Pro Glu Gln Gly
65 70 75 80
Pro Tyr Phe Ala Gln Tyr Phe Asn Thr Ile Asp Ser Phe Asp Thr His
85 90 95
Ala Arg Ile Pro Asp Tyr Phe Asp Ala Val Asn Ala Ala Ala Glu Gln
100 105 110
Ser Gly Lys Val Ala Ile Ile Ser Val Gly Trp Asp Pro Gly Leu Phe
115 120 125
Ser Leu Asn Arg Leu Leu Gly Glu Val Val Leu Pro Val Gly Asn Thr
130 135 140
Tyr Thr Phe Trp Gly Lys Gly Val Ser Gln Gly His Ser Asp Ala Ile
145 150 155 160
Arg Arg Ile Gln Gly Val Lys Asn Ala Val Gln Tyr Thr Ile Pro Ile
165 170 175
Asp Glu Ala Val Asn Arg Val Arg Ser Gly Glu Asn Pro Glu Leu Ser
180 185 190
Thr Arg Glu Lys His Ala Arg Glu Cys Phe Val Val Leu Glu Glu Gly
195 200 205
Ala Asp Pro Ala Lys Val Glu His Glu Ile Lys Thr Met Pro Asn Tyr
210 215 220
Phe Asp Glu Tyr Asp Thr Thr Val His Phe Ile Ser Glu Glu Glu Leu
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Lys Gln Asn His Ser Gly Met Pro His Gly Gly Phe Val Ile Arg Ser
245 250 255
Gly Lys Ser Asp Glu Gly His Lys Gln Ile Ile Glu Phe Ser Leu Asn
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Leu Glu Ser Asn Pro Met Phe Thr Ser Ser Ala Leu Val Ala Tyr Ala
275 280 285
Arg Ala Ala Tyr Arg Leu Ser Gln Asn Gly Asp Lys Gly Ala Lys Thr
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Val Phe Asp Ile Pro Phe Gly Leu Leu Ser Pro Lys Ser Pro Glu Asp
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Leu Arg Lys Glu Leu Leu
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atgtctaaaa tccgtatcgg tatcgttggt tacggtaacc tgggtcgtgg tgttgaagct 60
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accgttgctg ttaaatctaa cgttaaagtt ctgcacgttg acgacgctca gtcttacaaa 180
gacgaaatcg acgttatgat cctgtgcggt ggttctgcta ccgacctgcc ggaacagggt 240
ccgtacttcg ctcagtactt caacaccatc gactctttcg acacccacgc tcgtatcccg 300
gactacttcg acgctgttaa cgctgctgct gaacagtctg gtaaagttgc tatcatctct 360
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gttggtaaca cctacacctt ctggggcaag ggtgtaagcc agggtcactc tgacgctatc 480
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aaacagaacc actctggtat gccccacggc ggcttcgtta tccgttcggg taaatctgac 780
gaaggtcaca aacagatcat cgaattctct ctgaacctgg aatctaaccc gatgttcacc 840
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Met Ser Lys Ile Arg Ile Gly Ile Val Gly Tyr Gly Asn Leu Gly Arg
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Gly Val Glu Ala Ala Ile Gln Gln Asn Pro Asp Met Glu Leu Val Ala
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Val Phe Thr Arg Arg Asp Pro Lys Thr Val Ala Val Lys Ser Asn Val
35 40 45
Lys Val Leu His Val Asp Asp Ala Gln Ser Tyr Lys Asp Glu Ile Asp
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Val Met Ile Leu Cys Gly Gly Ser Ala Thr Asp Leu Pro Glu Gln Gly
65 70 75 80
Pro Tyr Phe Ala Gln Tyr Phe Asn Thr Ile Asp Ser Phe Ala Thr His
85 90 95
Ala Arg Ile Pro Asp Tyr Phe Asp Ala Val Asn Ala Ala Ala Glu Gln
100 105 110
Ser Gly Lys Val Ala Ile Ile Ser Val Gly Trp Asp Pro Gly Leu Phe
115 120 125
Ser Leu Asn Arg Leu Leu Gly Glu Val Val Leu Pro Val Gly Asn Thr
130 135 140
Tyr Thr Phe Trp Gly Lys Gly Val Ser Gln Gly His Ser Asp Ala Ile
145 150 155 160
Arg Arg Ile Gln Gly Val Lys Asn Ala Val Gln Tyr Thr Ile Pro Ile
165 170 175
Asp Glu Ala Val Asn Arg Val Arg Ser Gly Glu Asn Pro Glu Leu Ser
180 185 190
Thr Arg Glu Lys His Ala Val Glu Cys Phe Val Val Leu Glu Glu Gly
195 200 205
Ala Asp Pro Ala Lys Val Glu His Glu Ile Lys Thr Met Pro Asn Tyr
210 215 220
Phe Asp Glu Tyr Asp Thr Thr Val His Phe Ile Ser Glu Glu Glu Leu
225 230 235 240
Lys Gln Asn His Ser Gly Met Pro Thr Gly Gly Phe Val Ile Arg Ser
245 250 255
Gly Lys Ser Asp Glu Gly His Lys Gln Ile Ile Glu Phe Ser Leu Asn
260 265 270
Leu Glu Ser Asn Pro Met Phe Thr Ser Ser Ala Leu Val Ala Tyr Ala
275 280 285
Arg Ala Ala Tyr Arg Leu Ser Gln Asn Gly Asp Lys Gly Ala Lys Thr
290 295 300
Val Phe Asp Ile Pro Phe Gly Leu Leu Ser Pro Lys Ser Pro Glu Asp
305 310 315 320
Leu Arg Lys Glu Leu Leu
325
<210> 4
<211> 326
<212> PRT
<213> Artificial sequence ()
<400> 4
Met Ser Lys Ile Arg Ile Gly Ile Val Gly Tyr Gly Asn Leu Gly Arg
1 5 10 15
Gly Val Glu Ala Ala Ile Gln Gln Asn Pro Asp Met Glu Leu Val Ala
20 25 30
Val Phe Thr Arg Arg Asp Pro Lys Thr Val Ala Val Lys Ser Asn Val
35 40 45
Lys Val Leu His Val Asp Asp Ala Gln Ser Tyr Lys Asp Glu Ile Asp
50 55 60
Val Met Ile Leu Cys Gly Gly Ser Ala Thr Asp Leu Pro Glu Gln Gly
65 70 75 80
Pro Tyr Phe Ala Gln Tyr Phe Asn Thr Ile Asp Ser Phe Leu Thr His
85 90 95
Ala Arg Ile Pro Asp Tyr Phe Asp Ala Val Asn Ala Ala Ala Glu Gln
100 105 110
Ser Gly Lys Val Ala Ile Ile Ser Val Gly Trp Asp Pro Gly Leu Phe
115 120 125
Ser Leu Asn Arg Leu Leu Gly Glu Val Val Leu Pro Val Gly Asn Thr
130 135 140
Tyr Thr Phe Trp Gly Lys Gly Val Ser Gln Gly His Ser Asp Ala Ile
145 150 155 160
Arg Arg Ile Gln Gly Val Lys Asn Ala Val Gln Tyr Thr Ile Pro Ile
165 170 175
Asp Glu Ala Val Asn Arg Val Arg Ser Gly Glu Asn Pro Glu Leu Ser
180 185 190
Thr Arg Glu Lys His Ala Val Glu Cys Phe Val Val Leu Glu Glu Gly
195 200 205
Ala Asp Pro Ala Lys Val Glu His Glu Ile Lys Thr Met Pro Asn Tyr
210 215 220
Phe Asp Glu Tyr Asp Thr Thr Val His Phe Ile Ser Glu Glu Glu Leu
225 230 235 240
Lys Gln Asn His Ser Gly Met Pro Thr Gly Gly Phe Val Ile Arg Ser
245 250 255
Gly Lys Ser Asp Glu Gly His Lys Gln Ile Ile Glu Phe Ser Leu Asn
260 265 270
Leu Glu Ser Asn Pro Met Phe Thr Ser Ser Ala Leu Val Ala Tyr Ala
275 280 285
Arg Ala Ala Tyr Arg Leu Ser Gln Asn Gly Asp Lys Gly Ala Lys Thr
290 295 300
Val Phe Asp Ile Pro Phe Gly Leu Leu Ser Pro Lys Ser Pro Glu Asp
305 310 315 320
Leu Arg Lys Glu Leu Leu
325
<210> 5
<211> 981
<212> DNA
<213> Artificial sequence ()
<400> 5
atgtctaaaa tccgtatcgg tatcgttggt tacggtaacc tgggtcgtgg tgttgaagct 60
gctatccagc agaacccgga catggaactg gttgctgttt tcacccgtcg tgacccgaaa 120
accgttgctg ttaaatctaa cgttaaagtt ctgcacgttg acgacgctca gtcttacaaa 180
gacgaaatcg acgttatgat cctgtgcggt ggttctgcta ccgacctgcc ggaacagggt 240
ccgtacttcg ctcagtactt caacaccatc gactctttcc tgacccacgc tcgtatcccg 300
gactacttcg acgctgttaa cgctgctgct gaacagtctg gtaaagttgc tatcatctct 360
gttggttggg acccgggtct gttctctctg aaccgtctgc tgggtgaagt tgttctgccg 420
gttggtaaca cctacacctt ctggggcaag ggtgtaagcc agggtcactc tgacgctatc 480
cgtcgtatcc agggtgttaa aaacgctgtt cagtacacca tcccgatcga cgaagctgtt 540
aaccgtgttc gttctggtga aaacccggaa ctgtctaccc gtgaaaaaca cgctgttgaa 600
tgcttcgttg ttctggaaga aggtgctgac ccggctaaag ttgaacacga aatcaaaacc 660
atgccgaact acttcgacga atacgacacc accgttcact tcatctctga agaagaactg 720
aaacagaacc actctggtat gcccaccggc ggcttcgtta tccgttcggg taaatctgac 780
gaaggtcaca aacagatcat cgaattctct ctgaacctgg aatctaaccc gatgttcacc 840
tcttctgctc tggttgctta cgctcgtgct gcttaccgtc tgtctcagaa cggtgacaaa 900
ggtgctaaaa ccgttttcga catcccgttc ggtctgctgt ctccgaaatc tccggaagac 960
ctgcgtaaag aactgctgta a 981
<210> 6
<211> 261
<212> PRT
<213> Bacillus cereus
<400> 6
Met Tyr Ser Asp Leu Ala Gly Lys Val Val Val Ile Thr Gly Ser Ala
1 5 10 15
Thr Gly Leu Gly Arg Ala Met Gly Val Arg Phe Ala Lys Glu Lys Ala
20 25 30
Lys Val Val Ile Asn Tyr Arg Ser Arg Glu Ser Glu Ala Asn Asp Val
35 40 45
Leu Glu Glu Ile Lys Lys Val Gly Gly Glu Ala Ile Ala Val Lys Gly
50 55 60
Asp Val Thr Val Glu Ser Asp Val Val Asn Leu Ile Gln Ser Ala Val
65 70 75 80
Lys Glu Phe Gly Thr Leu Asp Val Met Ile Asn Asn Ala Gly Ile Glu
85 90 95
Asn Ala Val Pro Ser His Glu Met Pro Leu Glu Asp Trp Asn Arg Val
100 105 110
Ile Asn Thr Asn Leu Thr Gly Ala Phe Leu Gly Ser Arg Glu Ala Ile
115 120 125
Lys Tyr Phe Val Glu His Asp Ile Lys Gly Ser Val Ile Asn Met Ser
130 135 140
Ser Val His Glu Lys Ile Pro Trp Pro Leu Phe Val His Tyr Ala Ala
145 150 155 160
Ser Lys Gly Gly Ile Lys Leu Met Thr Glu Thr Leu Ala Leu Glu Tyr
165 170 175
Ala Pro Lys Gly Ile Arg Val Asn Asn Ile Gly Pro Gly Ala Ile Asn
180 185 190
Thr Pro Ile Asn Ala Glu Lys Phe Ala Asp Pro Lys Lys Arg Ala Asp
195 200 205
Val Glu Ser Met Ile Pro Met Gly Tyr Ile Gly Asn Pro Glu Glu Ile
210 215 220
Ala Ala Val Ala Thr Trp Leu Ala Ser Ser Glu Ala Ser Tyr Val Thr
225 230 235 240
Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Leu Tyr Pro Ser Phe
245 250 255
Gln Ala Gly Arg Gly
260
<210> 7
<211> 786
<212> DNA
<213> Bacillus cereus
<400> 7
atgtatagtg atttagcagg gaaagttgtc gttattacag gatcagcaac tggtcttgga 60
agagcgatgg gagtgaggtt tgctaaggaa aaagcgaaag tggttattaa ttatcgctca 120
cgagaatcag aagcgaatga tgtgttagaa gaaattaaaa aggtaggcgg cgaagcgatt 180
gctgtaaaag gtgatgtaac cgtcgaatca gatgttgtga atctcattca atctgctgtg 240
aaagagtttg gtacgcttga cgttatgatt aataatgcag ggatagaaaa cgcggtaccg 300
tcgcatgaaa tgccgcttga agattggaat agggtaatta atacaaattt aacaggtgct 360
tttttaggaa gtcgtgaagc gattaaatat tttgtagaac atgatattaa aggttctgtc 420
attaatatgt ctagtgttca tgagaaaatt ccgtggccac tatttgtgca ctatgcagcg 480
agtaagggtg gtattaaact gatgacagaa acgttagcgc tagaatatgc gccaaaaggt 540
attcgagtaa ataatattgg accaggtgca attaataccc cgattaatgc agaaaagttt 600
gctgatccta aaaaacgtgc tgacgtagaa agtatgatac cgatgggcta tattggaaac 660
cctgaagaaa ttgcagcagt agcaacttgg ctcgcttctt cagaggcgag ttatgtaacg 720
ggcattacgc tatttgcaga tggtggaatg acgttatatc catcgtttca agctgggcgt 780
gggtaa 786
Claims (8)
1. A method for enzymatically synthesizing D-leucine is characterized in that alpha-ketoisocaproic acid is used as a substrate, and D-amino acid dehydrogenase mutant is used for catalyzing the substrate to perform dehydrogenation reaction and amino transfer reaction to obtain D-leucine, wherein
The amino acid sequence of the D-amino acid dehydrogenase mutant is SEQ ID NO. 4.
2. The method according to claim 1, wherein glucose dehydrogenase and coenzyme NADPH are added to the reaction system.
3. The method according to claim 1, wherein an ammonium salt or aqueous ammonia is further added as an ammonia donor to the reaction system.
4. The method of claim 2, wherein the glucose dehydrogenase has the amino acid sequence of SEQ ID NO. 6.
5. A D-amino acid dehydrogenase mutant is characterized in that the amino acid sequence is SEQ ID NO. 4.
6. A gene encoding the D-amino acid dehydrogenase mutant according to claim 5.
7. The gene according to claim 6, wherein the nucleotide sequence is SEQ ID NO. 5.
8. Use of a D-amino acid dehydrogenase mutant as claimed in claim 5 for the preparation of D-leucine.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0315786A1 (en) * | 1987-10-14 | 1989-05-17 | Stamicarbon B.V. | Process for the preparation of a d-alfa-amino acid from the corresponding alfa-keto acid |
| CN102250976A (en) * | 2011-05-06 | 2011-11-23 | 凯莱英医药化学（天津）有限公司 | Synthesis method of chiral tert-leucine and final product obtained in method |
-
2021
- 2021-03-29 CN CN202110333271.9A patent/CN112831532B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0315786A1 (en) * | 1987-10-14 | 1989-05-17 | Stamicarbon B.V. | Process for the preparation of a d-alfa-amino acid from the corresponding alfa-keto acid |
| CN102250976A (en) * | 2011-05-06 | 2011-11-23 | 凯莱英医药化学（天津）有限公司 | Synthesis method of chiral tert-leucine and final product obtained in method |
Non-Patent Citations (5)
| Title |
|---|
| Akita, H等.Efficient synthesis of D-branched-chain amino acids and their labeled compounds with stable isotopes using D-amino acid dehydrogenase.Appl Microbiol Biotechnol.2013,(第98期),1135-1143. * |
| Artificial Biocatalytic Cascade with Three Enzymes in One Pot for Asymmetric Synthesis of Chiral Unnatural Amino Acids;Haisheng Zhou等;European Journal of Organic Chemistry(第38期);6470-6477 * |
| diaminopimelate dehydrogenase [Ureibacillus thermosphaericus];NCBI;NCBI;ACCESSION WP_016838077.1 * |
| Efficient synthesis of D-branched-chain amino acids and their labeled compounds with stable isotopes using D-amino acid dehydrogenase;Akita, H等;Appl Microbiol Biotechnol(第98期);1135-1143 * |
| Glucose 1-dehydrogenase B [Bacillus cereus BDRD-Cer4];NCBI;NCBI;ACCESSION EEL09314 * |
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