CN106701723B - D-Fructose -6- phosphate aldolase A mutant, recombinant expression carrier, genetic engineering bacterium and its application and reaction product - Google Patents

D-Fructose -6- phosphate aldolase A mutant, recombinant expression carrier, genetic engineering bacterium and its application and reaction product Download PDF

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CN106701723B
CN106701723B CN201611192703.4A CN201611192703A CN106701723B CN 106701723 B CN106701723 B CN 106701723B CN 201611192703 A CN201611192703 A CN 201611192703A CN 106701723 B CN106701723 B CN 106701723B
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phosphate aldolase
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CN106701723A (en
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于洪巍
杨小红
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Hangzhou Xinhai Enzyme Source Biotechnology Co ltd
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Zhejiang University ZJU
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Abstract

The present invention provides the FSAA mutant and its nucleotide sequence that catalytic activity significantly improves, and recombinant expression carrier and genetic engineering bacterium containing corresponding mutant gene, the aldol reaction of genetic engineering bacterium asymmetry catalysis cinnamic acid/α-bromocinnamaldehyde/4- nitro cinnamaldehyde/pyridine-2-formaldehyde and hydroxypropanone- (HA) by these FSAA mutant or containing corresponding mutant protein can prepare the chiral product of high-optical-purity, the latter can be used as valuable chiral building block, have important potential using value in field of medicaments.

Description

D-Fructose -6- phosphate aldolase A mutant, recombinant expression carrier, genetic engineering bacterium and It is applied and reaction product
Technical field
The invention belongs to genetic engineerings and enzyme engineering field, and in particular to the molecule of D-Fructose -6- phosphate aldolase A changes It makes, obtains mutant, recombinant expression carrier, genetic engineering bacterium and asymmetry catalysis cinnamic acid/α-bromocinnamaldehyde/4- nitro meat The aldol reaction of cinnamic aldehyde/pyridine-2-formaldehyde and hydroxypropanone- (HA) is to prepare the application in optical activity product.
Background technique
Asymmetric direct aldol reaction is a kind of very important C-C addition reaction.Its product is natural polyhydroxy Compound or completely new polyhydroxy small molecule.Since hydroxyl can be converted into various other functional groups, these polyhydroxys easily Base small molecule becomes one of most important chiral building block, especially has important application value in field of medicaments.Among these, (3S, 4R, E) -3,4- dihydroxy -6- phenyl -5- hexene -2- ketone (3a, formula 1) is de novo formation C- phenyl-β-L- gulose glycosides With the important chiral precursor of C- phenyl-α-L- mannoside.The two is effective inhibitor of glycosidase and glycosyl transferase, tool afterwards Standby potential antibacterium, antitumor and antimycotic ability, therefore the asymmetric syntheses of 3a has important medical value.Currently The synthesis path of 3a depends on the cinnamic acid of chemical catalysis and the aldol reaction of hydroxypropanone-.The catalyst being related to Mostly boride, titanium compound, chiral tertiary amine, NaOH and indium-nitrification mercury compound system, conversion ratio is relatively high, but three-dimensional Selectivity is insufficient.Since the reaction generates two new functional chiral centres, the absolute control to the reaction chiral chemistry Just it is particularly important.Therefore, cinnamic acid and the highly-solid selectively of hydroxypropanone- condensation are the main of the chemical catalysis reaction Obstacle.For from another point of view, high catalytic efficiency, selectivity is good, by-product is few and reaction condition is mild because having for biocatalysis The advantages that and become green chemical synthesis optimization approach.Among these, aldolase being capable of native catalytic aldol condensation as one kind The catalyst of reaction has very high catalytic efficiency and stereoselectivity, therefore can be used as asymmetric syntheses (3S, 4R) -3a's Preferred approach.
In the aldolase with catalysis asymmetric direct aldol reaction ability, D-Fructose -6- phosphate aldolase A (FSAA) can using the hydroxypropanone- (HA), dihydroxyacetone (DHA) (DHA) and hydroxy butanone (HB) of non-phosphorylating as substrate, rather than Relatively expensive and prepare relatively complicated phosphoric acid dihydroxyacetone (DHA) (DHAP), the latter is the catalysis bottom of DHAP dependent form aldolase Object.In addition, FSAA catalysate has very high optical purity, therefore the enzyme just obtains extensive concern once report.This Seminar's early period screens aldolase using cinnamic acid and HA as substrate, finds that FSAA has highly-solid selectively and is catalyzed this instead for the first time The ability answered, while FSAA, to cinnamic acid analog, α-bromocinnamaldehyde, pnitrocinnamaidehyde and pyridine-2-formaldehyde are also shown Corresponding catalysis activity.But due to cinnamic acid and cortex cinnamomi aldehyde structure may be replaced more special, there is one to be total to phenyl ring The double bond of yoke, the electron delocalization effect of generation reduce cinnamic acid and replace reaction vigor of the cinnamic acid as substrate, cause FSAA is catalyzed the vigor of above-mentioned reaction far from industrialization production requirements are met, therefore, it is necessary to which improving by the relevant technologies should The catalytic efficiency of enzyme is sufficiently to excavate its application value.
Summary of the invention
The purpose of the present invention is for the first time using enzyme molecule as the aldol reaction between the cinnamic acid of biocatalyst and HA, Furthermore strong biocatalyst, the i.e. mutant of the FSAA of enzyme activity raising and its recombination large intestine bar are provided for these reactions Bacterium.
To achieve the goals above, the invention adopts the following technical scheme:
The present invention provides the mutant of D-Fructose -6- phosphate aldolase A (FSAA) that catalytic activity significantly improves a kind of.This A little mutant are constructed on the basis of being the aldolase FSAA amino acid sequence shown in SEQ ID No:2, the aldolase nucleotide Sequence is as shown in SEQ ID No:1 in sequence table.
Specifically, the mutant is the 59th site glutamine on the amino acid sequence shown in SEQ ID No:2 (Gln59) different substitutions is carried out.
Preferably, the amino acid sequence of the D-Fructose -6- phosphate aldolase A mutant is selected from following mutation sequence:
Amino acid sequence shown in SEQ ID NO:4, the 59th sports leucine (Gln59Leu, i.e. Q59L)；
Amino acid sequence shown in SEQ ID NO:6, the 59th sports threonine (Gln59Thr, i.e. Q59T).
It is any that missing is passed through to amino acid in above-mentioned variant amino acid sequence, is inserted into or replaces one or several amino acid And with aldolase activity, still fall within protection scope of the present invention.
Mutant Q59L nucleotide sequence is as shown in SEQ ID No:3 in sequence table, encoding amino acid sequence such as sequence Shown in table SEQID No:4；Mutant Q59T nucleotide sequence encodes amino acid sequence as shown in SEQ ID No:5 in sequence table Column are as shown in sequence table SEQ ID No:6.
As it is known by the man skilled in the art, the nucleotide sequence of FSAA mutant of the invention is also possible to polynucleotide Shown in amino acid composition protein other any nucleotide sequences.
Any substitution, missing or insertion process for carrying out one or more nucleotide to shown mutant nucleotide sequence obtains The nucleotide sequence obtained all belongs to the scope of protection of the present invention as long as it has 90% or more homology with nucleotide.
The present invention also provides a kind of recombinant expression carrier, the recombinant expression carrier contains the nucleosides of FSAA mutant gene Acid sequence.FSAA mutant nucleotide sequence of the invention can be connected to respectively by these recombinant vectors by conventional method in that art It is built-up on kind carrier.The carrier can be the various carriers of this field routine, such as various plasmids, bacteriophage or viral vectors Deng preferably pET-30a.
The present invention also provides a kind of genetic engineering bacteriums, can be by converting recombinant expression carrier of the invention to the micro- life of host It is obtained in object.The host microorganism can be the various host microorganisms of this field routine, as long as meeting recombinant expression carrier Self-replacation can be stablized and entrained short-chain dehydrogenase/reductase enzyme mutant gene of the invention can be with effective expression.This Invent preferred Escherichia coli, more preferable E. coli BL21 (DE3).
The fifth aspect of the present invention provides a kind of preparation method for recombinating FSAA mutant, includes the following steps: culture originally The recombinant expression transformants of invention, induction obtain recombination FSAA mutant protein.Wherein, the culture recombinant expression transformants Culture medium used, which can be this field, can make transformants grew and generate the culture medium of FSAA mutant protein of the invention, excellent Select LB culture medium: peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, pH 7.2.Cultural method and condition of culture are without spy Different limitation, as long as enabling transformant to grow and generating FSAA mutant protein.It is preferred that following methods: will be the present invention relates to Recombination bacillus coli be seeded in the LB culture medium containing kanamycins and chloramphenicol and cultivate, as the optical density OD600 of culture solution When reaching 0.5~0.7, in final concentration of 0.1~1.0mM isopropyl-beta D-thio galactopyranoside (IPTG) and final concentration It, can high efficient expression recombination FSAA mutant protein of the invention under induction for the L-arabinose of 1mg/mL.
The sixth aspect of the present invention provides the FSAA mutant or its genetic engineering bacterium in asymmetry catalysis cinnamic acid/α- The aldol reaction of bromocinnamaldehyde/4- nitro cinnamaldehyde/pyridine-2-formaldehyde and HA are to prepare answering in optical activity product With.
Specifically, the application are as follows: with cinnamic acid (1a), α-bromocinnamaldehyde (1b), 4- nitro cinnamaldehyde (1c) and pyrrole Pyridine -2- formaldehyde (1d) and HA are substrate, and the FSAA mutant or its genetic engineering bacterium are catalyst, at 30 DEG C, in pH 6.5 Citric acid-sodium citrate buffer solution constitute conversion reaction system in react, after fully reacting, reaction solution is isolated and purified To corresponding product.
Specific reaction equation is as follows:
The reaction condition can be selected by the normal condition used in this field.
Further, aldehyde initial substrate concentration is 10~500mmol/L in the transformation system, and hydroxypropanone- initial concentration is 50~2500mmol/L.
Further, the pure enzyme of FSAA mutant preferable concentration in reaction solution is 0.3~0.6mg/ in the transformation system mL.The quality dosage of thallus is calculated as 80g/L with thallus weight in wet base in the transformation system.
Further, the reaction carries out in the citric acid-sodium citrate buffer solution of pH 6.5.
Further, reaction system can also add 1% dithiothreitol (DTT) (DTT).
Further, the cosolvent in reaction system can be DMSO or DMF.
Further, the concentration of DMSO or DMF is 10~20% in reaction system.
Further, the conversion reaction solution isolation and purification method are as follows: after reaction, 3 times are added in pure enzymatic system It is centrifuged (Whole cell catalytic system is directly centrifuged) after the methanol-ice bath 3h of volume, supernatant is taken to be extracted with isometric ethyl acetate 3 times, organic layer is the crude product containing corresponding product, and crude product purification is obtained corresponding product.The method of crude product purification is Techniques well known, usually organic solvent extraction, silica gel post separation and thin-layer chromatography etc..
The present invention also provides the aromatic diol product that above-mentioned aldol reaction generates, these product specific structures such as formulas I Shown in 3a-3d.Title is as follows:
(3S, 4R) -3a:(3S, 4R, E) -3,4- dihydroxy -6- phenyl -5- hexene -2- ketone
3b:(Z) the bromo- 3,4- dihydroxy -6- phenyl -5- hexene -2- ketone of -5-
(3S, 4R) -3b:(3S, 4S, Z) the bromo- 3,4- dihydroxy -6- phenyl -5- hexene -2- ketone of -5-
3c:(E) -3,4- dihydroxy -6- (4- nitrobenzophenone) -5- hexene -2- ketone
(3S, 4R) -3c:(3S, 4R, E) -3,4- dihydroxy -6- (4- nitrobenzophenone) -5- hexene -2- ketone
3d:3,4- dihydroxy -4- (2- pyridyl group) -2- butanone
(3S, 4R) -3d:(3S, 4R) -3,4- dihydroxy -4- (2- pyridyl group) -2- butanone
Compared with prior art, the invention has the following advantages that
The present invention provides FSAA mutant and its nucleotide sequence that catalytic activity significantly improves, and containing corresponding prominent The recombinant expression carrier and genetic engineering bacterium of variant gene.Pass through these FSAA mutant or the base containing corresponding mutant protein Because the aldol condensation of engineering bacteria asymmetry catalysis cinnamic acid/α-bromocinnamaldehyde/4- nitro cinnamaldehyde/pyridine-2-formaldehyde and HA is anti- The chiral product 3a-3d of high-optical-purity should can be prepared, the latter can be used as valuable chiral building block, have weight in field of medicaments The potential using value wanted.
Heretofore described FSAA mutant or genetic engineering bacterium containing mutant protein have high catalytic activity, can It synthesizes high-optical-purity product (ee > 99%, dr > 93:7).Catalyst is easily prepared, reaction condition is mild, substrate wide adaptability, It is environmental-friendly, and its genetic engineering bacterium can under higher concentration of substrate efficient catalytic asymmetric direct aldol reaction, tool There is good industrial applications development prospect.
Detailed description of the invention
Fig. 1 is FSAA and its mutant isolates and purifies rear SDS-PAGE figure.
Specific embodiment
The present invention is described further combined with specific embodiments below, but protection scope of the present invention is not limited in This:
Embodiment 1: the building of mutant
Using the oligonucleotide fragment containing catastrophe point as primer (table 1), using QuickChangeTM method (Stratagene, La Jolla, CA) expands the pET-30a recombinant plasmid containing FSAA gene.
1 mutation construction primer of table
aUnderscore is denoted as mutational site
PCR reaction system: 5 × PrimerSTAR buffer (Mg2+plus), 5 μ L；DNTPs (each 2.5mM), 2.0 μ L； Upstream primer (10 μM), 1.0 μ L；Downstream primer (10 μM), 1.0 μ L；Recombinant plasmid template, 15ng； PrimerSTARpolymeraseTM HS (2.5U/ μ L), 0.5 μ L；Add ddH2O to total volume be 25 μ L.
PCR program: (1) 98 DEG C, 1min；(2) 98 DEG C, 10s；(3) 55 DEG C, 10s；(4) 72 DEG C, 7min.Step (2)-(4) 4 DEG C are cooled to after circulation 20 times.
PCR product is once purged, using the restriction enzyme DpnI of specific recognition methylation sites digested with Degradation template plasmid.Endonuclease reaction system and condition: the PCR product of the 17 cleaned processing of μ L, 2.0 μ 10 × buffers of L, 1.0 μ L restriction enzyme DpnI, 37 DEG C of heat preservation 1h.
The above-mentioned PCR product through digestion processing is converted into e. coli bl21 (DE3), obtains recombinating large intestine accordingly Bacillus, is coated on the plate containing kanamycins, overnight incubation at 37 DEG C, and random picked clones carry out bacterium colony PCR identification and sequencing Verifying, the results showed that the recombinant expression carrier successful conversion containing FSAA mutant gene to expressive host E.coli BL21 (DE3) in, corresponding plasmid, conversion to the large intestine bar containing molecular chaperones pGro7 plasmid are extracted using plasmid extraction kit It is correctly folded in bacterium BL21 (DE3) for assisted mutagenesis body protein, it is final to obtain mutant Q59L and Q59T.Nucleotide sequence is surveyed Sequence result is respectively as shown in SEQ ID No:3 in sequence table and SEQ ID No:5, corresponding encoded protein amino acid sequence such as sequence In list shown in SEQID No:4 and SEQ ID No:6.
The inducing expression of embodiment 2:FSAA mutant
The engineering bacteria that embodiment 1 constructs is seeded to the LB Liquid Culture containing 50 μ g/mL kanamycins and 20 μ g/mL chloramphenicol In base, 37 DEG C of overnight incubations, then be forwarded to 2% inoculum concentration (v/v) containing 50 μ g/mL kanamycins and 20 μ g/mL chloramphenicol In 100mL LB culture medium, 37 DEG C, 220rpm is cultivated to cell concentration OD600 to 0.6 or so, is added final concentration of 0.1mM's The L-arabinose of IPTG and 1mg/mL, after 26 DEG C of Fiber differentiation 7h, 4 DEG C, 4000rpm centrifugation 10min collect thallus, in -80 DEG C storage is spare.
Embodiment 3:FSAA mutant isolates and purifies
The somatic cells that embodiment 2 is collected are suspended in 20mL Na2HPO4-NaH2PO4In buffer (100mM, pH 8.0), Oscillation shakes up and is crushed (effective time 8min) under postposition ultrasonic wave.Broken liquid is broken in 12,000rpm centrifugation 10min removal cell Piece is collected supernatant (crude enzyme liquid) and is isolated and purified for the subsequent of enzyme.Purification column is HisTrap (GE Healthcare) parent And chromatographic column, packed column volume 5mL first use loading equilibration buffer (20mM sodium phosphate, 500mM NaCl and 20mM imidazoles, pH 7.4) Ni-NTA column is balanced, with the rate loading crude enzyme liquid of 5mL/min, is eluted with loading equilibration buffer unadsorbed to remove Albumen finally collects target egg with elution buffer (20mM sodium phosphate, 500mM NaCl and 500mM imidazoles, pH 7.4) elution It is white.Enzyme solution carries out desalination with HiTrap desalting column, and desalination buffer is citric acid-sodium citrate (100mM, pH 6.5) buffering Liquid, the pure enzyme solution of gained are spare in 4 DEG C of storages.Enzyme solution after purification is analyzed with SDS-PAGE, and SDS-PAGE electrophoresis is shown in Fig. 1, The result shows that obtaining electrophoretically pure recombination FSAA and its mutant through HisTrap affinity chromatography.
The test of the enzyme activity of embodiment 4:FSAA and its mutant
Screening stage, reaction system are (0.3mL): 100mM aldehyde substrate, 500mM hydroxypropanone-, citric acid-sodium citrate Buffer (100mM, pH 6.5) and suitable pure enzyme.Substrate is respectively cinnamic acid 1a, α-bromocinnamaldehyde 1b, 4- nitro cinnamaldehyde 1c and pyridine-2-formaldehyde 1d.Wild type FSAA and its mutant catalytic phase answer substrate initial velocity (v0,nmol min-1mg-1) and turn Rate is as shown in table 2.
2 wild type FSAA of table and its mutant catalytic phase answer substrate initial velocity and conversion ratio
The kinetic parameter of embodiment 5:FSAA and its mutant
At the standard conditions, enzyme activity determination is carried out by changing the concentration of two substrates in reaction system respectively, according to Double-reciprocal plot method calculates corresponding kinetic constant.Substrate and its following (total volume of concentration used in kinetic constant calculating For 0.3mL):
1. the HA of 200mM is dissolved in citric acid-sodium citrate buffer solution (100mM, pH 6.5), and keeps HA concentration It is constant.The cinnamic acid of various concentration is added in reaction system (5~200mM), FSAA, FSAA Q59L and FSAA is then added Q59T controls conversion ratio lower than 5% to start reaction.At the end of reaction be added 0.9mL methanol terminate reaction, after centrifugation with HPLC is analyzed；
2. the cinnamic acid of 80mM is dissolved in citric acid-sodium citrate buffer solution (100mM, pH 6.5), and keeps cortex cinnamomi Aldehyde concentration is constant.The HA of various concentration is added in reaction system (20~250mM), is then added FSAA, FSAAQ59L and FSAA Q59T controls conversion ratio lower than 5% to start reaction.The methanol that 0.9mL is added at the end of reaction terminates reaction, from It is analyzed after the heart with HPLC；
Wild type FSAA and its mutant catalytic phase answer the apparent kinetics parameter of substrate as shown in table 3.
3 FSAA of table and its mutant asymmetric catalysis apparent kinetics parameter
Embodiment 5:FSAA and its mutant Q59L, Q59T conversion high concentration cinnamic acid and HA
Reaction system (10.0mL): 0.8g wet thallus cell, 250mM/500mM cinnamic acid, 1.25M/2.5M hydroxypropanone-, With 100mM, the citric acid-sodium citrate buffer solution polishing of pH 6.5 to 10.0mL.In 30 DEG C, reacted under the conditions of 220rpm.? Cortex cinnamomi aldehyde concentration is under conditions of 250mM, HA concentration are 1.25M, and the full cell of wild type FSAA is lived in starting stage of reaction catalysis Property be significantly lower than mutant Q59L and Q59T.After reaction for 24 hours, the conversion ratio of wild type FSAA and mutant Q59L reach identical water Flat (56%) and no longer extends at any time and increase, and the conversion ratio of mutant Q59T reaches highest (78%) in 4h.In meat Cinnamic aldehyde concentration is under conditions of 500mM, HA concentration are 2.5M, and the full cell of wild type FSAA reaches maximum conversion rate 51% in 36h, Mutant Q59L vigor under the concentration is greatly lowered, and reaction 2h conversion ratio is only 5% and does not extend at any time and increase, And mutant Q59T still maintains higher vigor under the concentration, conversion ratio reaches highest (90%) in 8h, even higher than its Conversion ratio under lower concentration of substrate.According to the equilibrium constant of reported aromatic aldehyde and ketone substrate aldol reaction, work as bottom When object concentration increases, in the case where substrate suppression is not present, conversion ratio also can correspondingly increase, therefore mutant Q59T is catalyzed The conversion ratio (90%) of 500mM cinnamic acid is higher than its conversion ratio (78%) for being catalyzed 250mM cinnamic acid.And Q59T is high in catalysis Concentration of substrate still shows good stereoselectivity when converting, product ee value keeps 99% or more, product dr value > 95:5.
To sum up, FSAA mutant of the present invention or genetic engineering bacterium containing corresponding mutant protein being capable of asymmetry catalysis meat The aldol reaction of cinnamic aldehyde/α-bromocinnamaldehyde/4- nitro cinnamaldehyde/pyridine-2-formaldehyde and HA are to prepare high-optical-purity Chiral product, and the full cell of mutant Q59T effective spirit catalytic of cinnamaldehyde and not urging for HA can claim directly under higher concentration of substrate Aldol reaction, and good stereoselectivity is kept, show that there is the whole-cell biocatalyst wide industrialization to answer Use prospect.
SEQUENCE LISTING
<110>Zhejiang University
<120>D-Fructose -6- phosphate aldolase A mutant, recombinant expression carrier, genetic engineering bacterium and its application and anti- Answer product
<130> 2016
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 663
<212> DNA
<213>artificial sequence
<400> 1
atggaactgt atctggatac ttcagacgtt gttgcggtga aggcgctgtc acgtattttt 60
ccgctggcgg gtgtgaccac taacccaagc attatcgccg cgggtaaaaa accgctggat 120
gttgtgcttc cgcaacttca tgaagcgatg ggcggtcagg ggcgtctgtt tgcccaggta 180
atggctacca ctgccgaagg gatggttaat gacgcgctta agctgcgttc tattattgcg 240
gatatcgtgg tgaaagttcc ggtgaccgcc gaggggctgg cagctattaa gatgttaaaa 300
gcggaaggga ttccgacgct gggaaccgcg gtatatggcg cagcacaagg gctgctgtcg 360
gcgctggcag gtgcggaata tgttgcgcct tacgttaatc gtattgatgc tcagggcggt 420
agcggcattc agactgtgac cgacttacac cagttattga aaatgcatgc gccgcaggcg 480
aaagtgctgg cagcgagttt caaaaccccg cgtcaggcgc tggactgctt actggcagga 540
tgtgaatcaa ttactctgcc actggatgtg gcacaacaga tgattagcta tccggcggtt 600
gatgccgctg tggcgaagtt tgagcaggac tggcagggag cgtttggcag aacgtcgatt 660
taa 663
<210> 2
<211> 220
<212> PRT
<213>artificial sequence
<400> 2
Met Glu Leu Tyr Leu Asp Thr Ser Asp Val Val Ala Val Lys Ala Leu
1 5 10 15
Ser Arg Ile Phe Pro Leu Ala Gly Val Thr Thr Asn Pro Ser Ile Ile
20 25 30
Ala Ala Gly Lys Lys Pro Leu Asp Val Val Leu Pro Gln Leu His Glu
35 40 45
Ala Met Gly Gly Gln Gly Arg Leu Phe Ala Gln Val Met Ala Thr Thr
50 55 60
Ala Glu Gly Met Val Asn Asp Ala Leu Lys Leu Arg Ser Ile Ile Ala
65 70 75 80
Asp Ile Val Val Lys Val Pro Val Thr Ala Glu Gly Leu Ala Ala Ile
85 90 95
Lys Met Leu Lys Ala Glu Gly Ile Pro Thr Leu Gly Thr Ala Val Tyr
100 105 110
Gly Ala Ala Gln Gly Leu Leu Ser Ala Leu Ala Gly Ala Glu Tyr Val
115 120 125
Ala Pro Tyr Val Asn Arg Ile Asp Ala Gln Gly Gly Ser Gly Ile Gln
130 135 140
Thr Val Thr Asp Leu His Gln Leu Leu Lys Met His Ala Pro Gln Ala
145 150 155 160
Lys Val Leu Ala Ala Ser Phe Lys Thr Pro Arg Gln Ala Leu Asp Cys
165 170 175
Leu Leu Ala Gly Cys Glu Ser Ile Thr Leu Pro Leu Asp Val Ala Gln
180 185 190
Gln Met Ile Ser Tyr Pro Ala Val Asp Ala Ala Val Ala Lys Phe Glu
195 200 205
Gln Asp Trp Gln Gly Ala Phe Gly Arg Thr Ser Ile
210 215 220
<210> 3
<211> 663
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<213>artificial sequence
<400> 3
atggaactgt atctggatac ttcagacgtt gttgcggtga aggcgctgtc acgtattttt 60
ccgctggcgg gtgtgaccac taacccaagc attatcgccg cgggtaaaaa accgctggat 120
gttgtgcttc cgcaacttca tgaagcgatg ggcggtcagg ggcgtctgtt tgccctggta 180
atggctacca ctgccgaagg gatggttaat gacgcgctta agctgcgttc tattattgcg 240
gatatcgtgg tgaaagttcc ggtgaccgcc gaggggctgg cagctattaa gatgttaaaa 300
gcggaaggga ttccgacgct gggaaccgcg gtatatggcg cagcacaagg gctgctgtcg 360
gcgctggcag gtgcggaata tgttgcgcct tacgttaatc gtattgatgc tcagggcggt 420
agcggcattc agactgtgac cgacttacac cagttattga aaatgcatgc gccgcaggcg 480
aaagtgctgg cagcgagttt caaaaccccg cgtcaggcgc tggactgctt actggcagga 540
tgtgaatcaa ttactctgcc actggatgtg gcacaacaga tgattagcta tccggcggtt 600
gatgccgctg tggcgaagtt tgagcaggac tggcagggag cgtttggcag aacgtcgatt 660
taa 663
<210> 4
<211> 220
<212> PRT
<213>artificial sequence
<400> 4
Met Glu Leu Tyr Leu Asp Thr Ser Asp Val Val Ala Val Lys Ala Leu
1 5 10 15
Ser Arg Ile Phe Pro Leu Ala Gly Val Thr Thr Asn Pro Ser Ile Ile
20 25 30
Ala Ala Gly Lys Lys Pro Leu Asp Val Val Leu Pro Gln Leu His Glu
35 40 45
Ala Met Gly Gly Gln Gly Arg Leu Phe Ala Leu Val Met Ala Thr Thr
50 55 60
Ala Glu Gly Met Val Asn Asp Ala Leu Lys Leu Arg Ser Ile Ile Ala
65 70 75 80
Asp Ile Val Val Lys Val Pro Val Thr Ala Glu Gly Leu Ala Ala Ile
85 90 95
Lys Met Leu Lys Ala Glu Gly Ile Pro Thr Leu Gly Thr Ala Val Tyr
100 105 110
Gly Ala Ala Gln Gly Leu Leu Ser Ala Leu Ala Gly Ala Glu Tyr Val
115 120 125
Ala Pro Tyr Val Asn Arg Ile Asp Ala Gln Gly Gly Ser Gly Ile Gln
130 135 140
Thr Val Thr Asp Leu His Gln Leu Leu Lys Met His Ala Pro Gln Ala
145 150 155 160
Lys Val Leu Ala Ala Ser Phe Lys Thr Pro Arg Gln Ala Leu Asp Cys
165 170 175
Leu Leu Ala Gly Cys Glu Ser Ile Thr Leu Pro Leu Asp Val Ala Gln
180 185 190
Gln Met Ile Ser Tyr Pro Ala Val Asp Ala Ala Val Ala Lys Phe Glu
195 200 205
Gln Asp Trp Gln Gly Ala Phe Gly Arg Thr Ser Ile
210 215 220
<210> 5
<211> 663
<212> DNA
<213>artificial sequence
<400> 5
atggaactgt atctggatac ttcagacgtt gttgcggtga aggcgctgtc acgtattttt 60
ccgctggcgg gtgtgaccac taacccaagc attatcgccg cgggtaaaaa accgctggat 120
gttgtgcttc cgcaacttca tgaagcgatg ggcggtcagg ggcgtctgtt tgccaccgta 180
atggctacca ctgccgaagg gatggttaat gacgcgctta agctgcgttc tattattgcg 240
gatatcgtgg tgaaagttcc ggtgaccgcc gaggggctgg cagctattaa gatgttaaaa 300
gcggaaggga ttccgacgct gggaaccgcg gtatatggcg cagcacaagg gctgctgtcg 360
gcgctggcag gtgcggaata tgttgcgcct tacgttaatc gtattgatgc tcagggcggt 420
agcggcattc agactgtgac cgacttacac cagttattga aaatgcatgc gccgcaggcg 480
aaagtgctgg cagcgagttt caaaaccccg cgtcaggcgc tggactgctt actggcagga 540
tgtgaatcaa ttactctgcc actggatgtg gcacaacaga tgattagcta tccggcggtt 600
gatgccgctg tggcgaagtt tgagcaggac tggcagggag cgtttggcag aacgtcgatt 660
taa 663
<210> 6
<211> 220
<212> PRT
<213>artificial sequence
<400> 6
Met Glu Leu Tyr Leu Asp Thr Ser Asp Val Val Ala Val Lys Ala Leu
1 5 10 15
Ser Arg Ile Phe Pro Leu Ala Gly Val Thr Thr Asn Pro Ser Ile Ile
20 25 30
Ala Ala Gly Lys Lys Pro Leu Asp Val Val Leu Pro Gln Leu His Glu
35 40 45
Ala Met Gly Gly Gln Gly Arg Leu Phe Ala Thr Val Met Ala Thr Thr
50 55 60
Ala Glu Gly Met Val Asn Asp Ala Leu Lys Leu Arg Ser Ile Ile Ala
65 70 75 80
Asp Ile Val Val Lys Val Pro Val Thr Ala Glu Gly Leu Ala Ala Ile
85 90 95
Lys Met Leu Lys Ala Glu Gly Ile Pro Thr Leu Gly Thr Ala Val Tyr
100 105 110
Gly Ala Ala Gln Gly Leu Leu Ser Ala Leu Ala Gly Ala Glu Tyr Val
115 120 125
Ala Pro Tyr Val Asn Arg Ile Asp Ala Gln Gly Gly Ser Gly Ile Gln
130 135 140
Thr Val Thr Asp Leu His Gln Leu Leu Lys Met His Ala Pro Gln Ala
145 150 155 160
Lys Val Leu Ala Ala Ser Phe Lys Thr Pro Arg Gln Ala Leu Asp Cys
165 170 175
Leu Leu Ala Gly Cys Glu Ser Ile Thr Leu Pro Leu Asp Val Ala Gln
180 185 190
Gln Met Ile Ser Tyr Pro Ala Val Asp Ala Ala Val Ala Lys Phe Glu
195 200 205
Gln Asp Trp Gln Gly Ala Phe Gly Arg Thr Ser Ile
210 215 220

Claims (8)

1. a kind of D-Fructose -6- phosphate aldolase A mutant, it is characterised in that: the mutant is shown in the SEQ ID No.2 Amino acid sequence on the 59th site glutamine carry out different substitutions；
The amino acid sequence of the D-Fructose -6- phosphate aldolase A mutant is selected from following mutation sequence:
Amino acid sequence shown in SEQ ID NO:4, the 59th sports leucine；
Amino acid sequence shown in SEQ ID NO:6, the 59th sports threonine.
2. D-Fructose -6- phosphate aldolase A mutant according to claim 1, which is characterized in that encode the D- fruit The nucleotide sequence of sugar -6- phosphate aldolase A mutant is selected from following sequence:
Nucleotide sequence shown in SEQ ID NO:3；
Nucleotide sequence shown in SEQ ID NO:5.
3. recombinant expression carrier, which is characterized in that the recombinant expression carrier includes to encode amino acid sequence described in claim 1 The nucleotide sequence of column.
4. genetic engineering bacterium, which is characterized in that the genetic engineering bacterium by recombinant expression carrier as claimed in claim 3 convert to It is obtained in host microorganism.
5. genetic engineering bacterium according to claim 4, which is characterized in that the host microorganism is Escherichia coli.
The preparation method of 6.D- fructose-6-phosphate aldolase A mutant, which is characterized in that gene described in culture claim 5 Engineering bacteria, induction obtain D-Fructose -6- phosphate aldolase A recombinant protein.
7. D-Fructose -6- phosphate aldolase A mutant as described in claim 1 or genetic engineering as claimed in claim 4 Bacterium any one and hydroxypropanone- in asymmetry catalysis cinnamic acid, α-bromocinnamaldehyde, 4- nitro cinnamaldehyde, pyridine-2-formaldehyde Aldol reaction to prepare the application in optical activity product.
8. applying according to claim 7, which is characterized in that cinnamic acid, α-bromocinnamaldehyde, 4- nitro cinnamaldehyde, pyridine -2- Any one in formaldehyde and hydroxypropanone- are substrate, and the D-Fructose -6- phosphate aldolase A mutant or genetic engineering bacterium are to urge Agent is reacted, wherein aldehyde initial substrate concentration be 10~500mmol/L, hydroxypropanone- initial concentration be 50~ 2500mmol/L。
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