CN106701723A - D-fructose-6-phosphate aldolase A mutant, recombinant expression vector, genetically engineered bacterium and application and reaction product thereof - Google Patents

D-fructose-6-phosphate aldolase A mutant, recombinant expression vector, genetically engineered bacterium and application and reaction product thereof Download PDF

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CN106701723A
CN106701723A CN201611192703.4A CN201611192703A CN106701723A CN 106701723 A CN106701723 A CN 106701723A CN 201611192703 A CN201611192703 A CN 201611192703A CN 106701723 A CN106701723 A CN 106701723A
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fructose
leu
dihydroxy
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CN106701723B (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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    • C12Y401/02013—Fructose-bisphosphate aldolase (4.1.2.13)

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Abstract

The invention provides an FSAA mutant with remarkably increased catalytic activity, a nucleotide sequence thereof, a recombinant expression vector containing a corresponding mutant gene and a genetically engineered bacterium. A chiral product with high optical purity can be prepared by aldol condensation reaction in which cinnamyl aldehyde/alpha-bromocinnamaldehyde/4-nitrocinnamaldehyde/pyridine-2-formaldehyde and hydroxyacetone (HA) are asymmetrically catalyzed by the FSAA mutants or the genetically engineered bacteria containing a corresponding mutant protein, the chiral product can serve as a valuable chiral building block, and important potential application value in the field of medicines is realized.

Description

D-Fructose -6- phosphate aldolase A mutant, recombinant expression carrier, genetic engineering bacterium and Its application and product
Technical field
The invention belongs to genetic engineering and enzyme engineering field, and in particular to the molecule of D-Fructose -6- phosphate aldolases A changes Make, obtain 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 preparing the application in optical activity product.
Background technology
Asymmetric direct aldol reaction is the very important C-C addition reactions of a class.Its product is natural polyhydroxy Compound or brand-new polyhydroxy small molecule.Because hydroxyl can easily be converted into various other functional groups, these polyhydroxys Base small molecule turns into 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- mannosides.Both are 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 are of a relatively high, but three-dimensional Selectivity is not enough.Because the reaction generates two new feature 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 reactions Obstacle.For from another point of view, living things catalysis is because with high catalytic efficiency, the good, accessory substance of selectivity is few and reaction condition is gentle The advantages of and turn into the optimization approach of green chemical synthesis.Among these, aldolase being capable of native catalytic aldol condensation as a class The catalyst of reaction, with catalytic efficiency and stereoselectivity very high, therefore can be used as asymmetric syntheses (3S, 4R) -3a's First-selected approach.
In the aldolase with catalysis asymmetric direct aldol reaction ability, D-Fructose -6- phosphate aldolases A (FSAA) can using the hydroxypropanone- (HA) of non-phosphorylating, dihydroxyacetone (DHA) (DHA) and hydroxy butanone (HB) 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 aldolases Thing.Additionally, FSAA catalysates have optical purity very high, therefore the enzyme just obtains extensive concern once report.This Seminar's early stage screens aldolase using cinnamic acid and HA as substrate, finds to possess highly-solid selectively by FSAA that to be catalyzed this anti-first The ability answered, while FSAA is to cinnamic acid analog, α-bromocinnamaldehyde, pnitrocinnamaidehyde and pyridine-2-formaldehyde also show Corresponding catalysis activity.But, may be more special due to cinnamic acid and substitution Chinese cassia tree aldehyde structure, it is total to phenyl ring with one The double bond of yoke, the electron delocalization effect of generation reduces cinnamic acid and substitution cinnamic acid as the reaction vigor of substrate, causes FSAA is catalyzed the vigor of above-mentioned reaction far from industrialization production requirements are met, therefore, it is necessary to improved by correlation technique should The catalytic efficiency of enzyme is fully excavating its application value.
The content of the invention
The purpose of the present invention be first using enzyme molecule as the aldol reaction between the cinnamic acid of biocatalyst and HA, The mutant and its restructuring large intestine bar of the FSAA that strong biocatalyst, i.e. enzyme activity are improved are provided for these reactions in addition Bacterium.
To achieve these goals, the present invention uses following technical scheme：
The present invention provides a kind of mutant of the D-Fructose -6- phosphate aldolases A (FSAA) that catalysis activity is significantly improved.This A little mutant are in SEQ ID No:Built on the basis of the amino acid sequence of aldolase FSAA shown in 2, the aldolase nucleotides SEQ ID No in sequence such as sequence table:Shown in 1.
Specifically, the mutant is in SEQ ID No:59th site glutamine on amino acid sequence shown in 2 (Gln59) different substitutions are carried out.
Preferably, the amino acid sequence of the D-Fructose -6- phosphate aldolases A mutant is selected from following mutation sequence：
SEQ ID NO:Amino acid sequence shown in 4, the 59th sports leucine (Gln59Leu, i.e. Q59L)；
SEQ ID NO:Amino acid sequence shown in 6, the 59th sports threonine (Gln59Thr, i.e. Q59T).
It is any to pass through missing to amino acid in above-mentioned variant amino acid sequence, insert or replace one or several amino acid And with aldolase activity, still fall within protection scope of the present invention.
SEQ ID No in mutant Q59L nucleotide sequences such as sequence table:Shown in 3, its encoding amino acid sequence such as sequence Table SEQID No:Shown in 4；SEQ ID No in mutant Q59T nucleotide sequences such as sequence table:Shown in 5, its coded amino acid sequence Row such as sequence table SEQ ID No:Shown in 6.
As it is known by the man skilled in the art, the nucleotide sequence of FSAA mutant of the invention can also be polynucleotide Shown in amino acid composition protein other any nucleotide sequences.
Any substitution that one or more nucleotides are carried out to shown mutant nucleotide sequence, missing or insertion process are obtained The nucleotide sequence for obtaining, as long as it has more than 90% homology with nucleotides, belongs to protection scope of the present invention.
The present invention also provides a kind of recombinant expression carrier, and the recombinant expression carrier contains the nucleosides of FSAA mutant genes Acid sequence.Can be connected to FSAA mutant nucleotide sequence of the invention respectively by this area conventional method by these recombinant vectors Plant built-up on carrier.The carrier can be the conventional various carriers in this area, such as various plasmids, bacteriophage or viral vectors Deng preferably pET-30a.
The present invention also provides a kind of genetic engineering bacterium, can be converted to the micro- life of host by by recombinant expression carrier of the invention Obtained in thing.Described host microorganism can be the conventional various host microorganisms in this area, 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 preferably E. coli BL21 (DE3).
The fifth aspect of the present invention provides a kind of preparation method of restructuring FSAA mutant, comprises the following steps：Culture is originally The recombinant expression transformants of invention, induction obtains restructuring FSAA mutant proteins.Wherein, described culture recombinant expression transformants Culture medium used can be that this area can make transformants grew and produce the culture medium of FSAA mutant proteins of the invention, excellent Select LB culture mediums：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 producing FSAA mutant proteins.It is preferred that following methods：Will be the present invention relates to Recombination bacillus coli be seeded in the LB culture mediums containing kanamycins and chloramphenicol cultivate, as the optical density OD600 of nutrient solution When reaching 0.5~0.7, in final concentration of 0.1~1.0mM isopropyl-beta D-thios galactopyranoside (IPTG) and final concentration For under the induction of the L-arabinose of 1mg/mL, you can high efficient expression restructuring FSAA mutant proteins of the invention.
The sixth aspect of the present invention provide the FSAA mutant or its genetic engineering bacterium asymmetry catalysis cinnamic acid/α- The aldol reaction of bromocinnamaldehyde/4- nitro cinnamaldehydes/pyridine-2-formaldehyde and HA with prepare in optical activity product should With.
Specifically, described application is：With cinnamic acid (1a), α-bromocinnamaldehyde (1b), 4- nitro cinnamaldehydes (1c) and pyrrole Pyridine -2- formaldehyde (1d) and HA is 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, reaction completely after, reaction solution is isolated and purified To corresponding product.
Specific reaction equation is as follows：
Described reaction condition can be as used by this area normal condition selected.
Further, aldehyde initial substrate concentration is 10~500mmol/L in the transformation system, and hydroxypropanone- initial concentration is 50~2500mmol/L.
Further, in the transformation system the pure enzyme of FSAA mutant in reaction solution preferably concentration be 0.3~0.6mg/ mL.The quality consumption of thalline is calculated as 80g/L with thalline weight in wet base in the transformation system.
Further, the reaction is carried 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 is：After reaction terminates, 3 times are added in pure enzymatic system (Whole cell catalytic system is directly centrifuged) is centrifuged after the methanol-ice bath 3h of volume, takes supernatant and is extracted with isometric ethyl acetate 3 times, organic layer is the crude product containing corresponding product, i.e., crude product purification is obtained into 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 of above-mentioned aldol reaction generation, these product concrete structures such as formula I Shown 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- hexenes -2- ketone of -5-
(3S,4R)-3b：The bromo- 3,4- dihydroxy -6- phenyl -5- hexenes -2- ketone of (3S, 4S, Z) -5-
3c：(E) -3,4- dihydroxy -6- (4- nitrobenzophenones) -5- hexene -2- ketone
(3S,4R)-3c：(3S, 4R, E) -3,4- dihydroxy -6- (4- nitrobenzophenones) -5- hexene -2- ketone
3d：3,4- dihydroxy -4- (2- pyridine radicals) -2- butanone
(3S,4R)-3d：(3S, 4R) -3,4- dihydroxy -4- (2- pyridine radicals) -2- butanone
Compared with prior art, the present invention has advantages below：
The invention provides FSAA mutant and its nucleotide sequence that catalysis activity is significantly improved, and containing corresponding prominent The recombinant expression carrier and genetic engineering bacterium of variant gene.Base by these FSAA mutant or containing corresponding mutant protein Because the aldol condensation of engineering bacteria asymmetry catalysis cinnamic acid/α-bromocinnamaldehyde/4- nitro cinnamaldehydes/pyridine-2-formaldehyde and HA is anti- The chiral product 3a-3d of high-optical-purity should be prepared, the latter there can be weight as valuable chiral building block in field of medicaments The potential using value wanted.
Heretofore described FSAA mutant or the genetic engineering bacterium containing mutant protein have high catalytic activity, can Synthesis high-optical-purity product (ee>99%, dr>93：7).Catalyst is easily prepared, reaction condition is gentle, substrate wide adaptability, It is environment-friendly, and its genetic engineering bacterium can under concentration of substrate higher efficient catalytic asymmetric direct aldol reaction, tool There is good industrial applications DEVELOPMENT PROSPECT.
Brief description of the drawings
Fig. 1 is FSAA and its mutant isolates and purifies rear SDS-PAGE figures.
Specific embodiment
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in This：
Embodiment 1：The structure of mutant
It is primer (table 1) with the oligonucleotide fragment containing catastrophe point, using QuickChangeTM methods (Stratagene, La Jolla, CA) expands the pET-30a recombinant plasmids containing FSAA genes.
The mutation construction primer of table 1
aUnderscore is denoted as mutational site
PCR reaction systems：5 × PrimerSTAR buffer (Mg2+plus), 5 μ L；DNTPs (each 2.5mM), 2.0 μ L； Sense primer (10 μM), 1.0 μ L；Anti-sense primer (10 μM), 1.0 μ L；Recombinant plasmid template, 15ng； PrimerSTARpolymeraseTM HS (2.5U/ μ L), 0.5 μ L；Plus ddH2O to cumulative volume be 25 μ L.
PCR programs：(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 circulating 20 times.
PCR primer is once purged, using the restriction enzyme DpnI of specific recognition methylation sites digested with Degraded template plasmid.Endonuclease reaction system and condition：The PCR primer of the 17 cleaned treatment of μ L, 2.0 μ 10 × buffer solutions of L, 1.0 μ L restriction enzymes DpnI, 37 DEG C of insulation 1h.
The above-mentioned PCR primer processed through digestion is converted into e. coli bl21 (DE3), large intestine is recombinated accordingly Bacillus, coats the flat board containing kanamycins, and overnight incubation at 37 DEG C, random picked clones carry out bacterium colony PCR identifications and sequencing Checking, as a result shows the recombinant expression carrier successful conversion containing FSAA mutant genes to expressive host E.coli BL21 (DE3) in, corresponding plasmid, conversion to the large intestine bar containing molecular chaperones pGro7 plasmids are extracted using plasmid extraction kit Correctly folded for assisted mutagenesis body protein in bacterium BL21 (DE3), it is final to obtain mutant Q59L and Q59T.Nucleotide sequence is surveyed Sequence result is respectively such as SEQ ID No in sequence table:3 and SEQ ID No:Shown in 5, corresponding encoded protein amino acid sequence such as sequence SEQID No in list:4 and SEQ ID No:Shown in 6.
Embodiment 2：The induced expression of FSAA mutant
The engineering bacteria that embodiment 1 builds is seeded to the LB Liquid Cultures containing 50 μ g/mL kanamycins and 20 μ g/mL chloramphenicol In base, 37 DEG C of overnight incubations, then be forwarded to containing 50 μ g/mL kanamycins and 20 μ g/mL chloramphenicol with 2% inoculum concentration (v/v) In 100mL LB culture mediums, 37 DEG C, 220rpm is cultivated to cell concentration OD600 to 0.6 or so, adds 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 collects thallines, in -80 DEG C storage is standby.
Embodiment 3：FSAA mutant is isolated and purified
The somatic cells that embodiment 2 is collected are suspended in 20mL Na2HPO4-NaH2PO4In buffer solution (100mM, pH 8.0), Vibration crushes (effective time 8min) under shaking up rearmounted ultrasonic wave.Broken liquid is broken in 12,000rpm centrifugation 10min removal cells Piece, collects supernatant (crude enzyme liquid) and is isolated and purified for the follow-up of enzyme.Purification column is HisTrap (GE Healthcare) parents And chromatographic column, packed column volume is 5mL, first with loading level pad (20mM sodium phosphates, 500mM NaCl and 20mM imidazoles, pH 7.4) Ni-NTA posts are balanced, with the speed loading crude enzyme liquid of 5mL/min, is eluted with loading level pad unadsorbed to remove Albumen, is finally eluted with elution buffer (20mM sodium phosphates, 500mM NaCl and 500mM imidazoles, pH 7.4) and collects target egg In vain.Enzyme liquid carries out desalination with HiTrap desalting columns, and desalination buffer solution is buffered for citric acid-sodium citrate (100mM, pH 6.5) Liquid, the pure enzyme liquid of gained is standby in 4 DEG C of storages.Enzyme liquid after purification is analyzed with SDS-PAGE, and SDS-PAGE electrophoresis is shown in Fig. 1, Result shows through HisTrap affinity chromatographys, obtains electrophoretically pure restructuring FSAA and its mutant.
Embodiment 4：The enzyme activity test of FSAA and its mutant
Screening stage, reaction system is (0.3mL):100mM aldehyde substrates, 500mM hydroxypropanone-s, citric acid-sodium citrate Buffer solution (100mM, pH 6.5) and appropriate 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.
The wild type FSAA of table 2 and its mutant catalytic phase answer substrate initial velocity and conversion ratio
Embodiment 5：The kinetic parameter of FSAA and its mutant
At the standard conditions, by changing reaction system respectively in two concentration of substrate carry out enzyme activity determination, according to Double-reciprocal plot method calculates corresponding kinetic constant.Substrate used and its following (cumulative volume of concentration in kinetic constant calculating It is 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.Add the cinnamic acid of various concentrations in reaction system (5~200mM), be subsequently adding FSAA, FSAA Q59L and FSAA Q59T controls conversion ratio to be less than 5% to start reaction.At the end of reaction add 0.9mL methyl alcohol terminating 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 Chinese cassia tree Aldehyde concentration is constant.Add the HA of various concentrations in reaction system (20~250mM), be subsequently adding FSAA, FSAAQ59L and FSAA Q59T control conversion ratio to be less than 5% to start reaction.The methyl alcohol terminating reaction of 0.9mL is added at the end of reaction, from It is analyzed with HPLC after the heart；
Wild type FSAA and its mutant catalytic phase answer the apparent kinetics parameter of substrate as shown in table 3.
The FSAA of table 3 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 cells, 250mM/500mM cinnamic acids, 1.25M/2.5M hydroxypropanone-s, 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. Chinese cassia tree aldehyde concentration is 250mM, HA concentration under conditions of 1.25M, the full cells of wild type FSAA are lived in initial action stage catalytic Property be significantly lower than mutant Q59L and Q59T.After reaction 24h, the conversion ratio of wild type FSAA and mutant Q59L reaches identical water Flat (56%) and no longer increase with time lengthening, and the conversion ratio of mutant Q59T reaches highest (78%) in 4h.In meat Cinnamic aldehyde concentration be 500mM, HA concentration be 2.5M under conditions of, the full cells of wild type FSAA reach maximum conversion rate 51% in 36h, Mutant Q59L vigor under the concentration is greatly lowered, and reaction 2h conversion ratios are only 5% and do not increase with time lengthening, And mutant Q59T remains in that vigor higher under the concentration, conversion ratio reaches highest (90%) in 8h, even above its Conversion ratio under relatively low concentration of substrate.According to the aromatic aldehyde reported and the equilibrium constant of ketone substrate aldol reaction, work as bottom When thing concentration is raised, in the case where suppressing in the absence of substrate, conversion ratio also can correspondingly increase, therefore mutant Q59T is catalyzed The conversion ratio (90%) of 500mM cinnamic acids is catalyzed the conversion ratio (78%) of 250mM cinnamic acids higher than it.And Q59T is high in catalysis Concentration of substrate still shows good stereoselectivity when converting, product ee values keep more than 99%, product dr values>95:5.
To sum up, FSAA mutant of the present invention or the genetic engineering bacterium containing corresponding mutant protein being capable of asymmetry catalysis meat The aldol reaction of cinnamic aldehyde/α-bromocinnamaldehyde/4- nitro cinnamaldehydes/pyridine-2-formaldehyde and HA is preparing high-optical-purity Chiral product, and the full cells of mutant Q59T effective spirit catalytic of cinnamaldehyde and HA can not urge the title direct under concentration of substrate higher 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 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
<212> DNA
<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 (10)

1. a kind of D-Fructose -6- phosphate aldolases A mutant, it is characterised in that：The mutant is in SEQ ID No.2 institutes The 59th site glutamine carries out different substitutions on the amino acid sequence for showing.
2. D-Fructose -6- phosphate aldolases A mutant according to claim 1, it is characterised in that the D-Fructose -6- The amino acid sequence of phosphate aldolase A mutant is selected from following mutation sequence：
SEQ ID NO:Amino acid sequence shown in 4, the 59th sports leucine；
SEQ ID NO:Amino acid sequence shown in 6, the 59th sports threonine.
3. D-Fructose -6- phosphate aldolases A mutant according to claim 2, it is characterised in that the described D- of coding is really The nucleotide sequence of sugar -6- phosphate aldolase A mutant is selected from one sequence：
SEQ ID NO:Nucleotide sequence shown in 3；
SEQ ID NO:Nucleotide sequence shown in 5.
4. recombinant expression carrier, it is characterised in that the recombinant expression carrier is comprising described in coding claim 1-2 any one Amino acid sequence nucleotide sequence.
5. genetic engineering bacterium, it is characterised in that the genetic engineering bacterium as the recombinant expression carrier described in claim 4 convert to Obtained in host microorganism.
6. genetic engineering bacterium according to claim 5, it is characterised in that the host microorganism is Escherichia coli.
The preparation method of 7.D- fructose-6-phosphate ALD-A mutant, it is characterised in that described in culture claim 5 or 6 Genetic engineering bacterium, induction obtains D-Fructose -6- phosphate aldolase A recombinant proteins.
8. D-Fructose -6- phosphate aldolase A mutant as claimed in claim 1 or the genetic engineering bacterium as described in 5 be not right Claim spirit catalytic of cinnamaldehyde, α-bromocinnamaldehyde, 4- nitro cinnamaldehydes, in pyridine-2-formaldehyde any one and hydroxypropanone- aldol contracting Reaction is closed to prepare the application in optical activity product.
9. apply according to claim 8, it is characterised in that cinnamic acid, α-bromocinnamaldehyde, 4- nitro cinnamaldehydes, pyridine -2- Any one and hydroxypropanone- are substrate in formaldehyde, 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。
10. the product that aldol reaction as described in claim 8 is obtained, it is characterised in that the product is virtue Fragrant glycol, is one of following：
(3S, 4R, E) -3,4- dihydroxy -6- phenyl -5- hexene -2- ketone
(Z) the bromo- 3,4- dihydroxy -6- phenyl -5- hexenes -2- ketone of -5-
The bromo- 3,4- dihydroxy -6- phenyl -5- hexenes -2- ketone of (3S, 4S, Z) -5-
(E) -3,4- dihydroxy -6- (4- nitrobenzophenones) -5- hexene -2- ketone
(3S, 4R, E) -3,4- dihydroxy -6- (4- nitrobenzophenones) -5- hexene -2- ketone
3,4- dihydroxy -4- (2- pyridine radicals) -2- butanone
(3S, 4R) -3,4- dihydroxy -4- (2- pyridine radicals) -2- butanone.
CN201611192703.4A 2016-12-21 2016-12-21 D-Fructose -6- phosphate aldolase A mutant, recombinant expression carrier, genetic engineering bacterium and its application and reaction product Active CN106701723B (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107739742A (en) * 2017-10-18 2018-02-27 中国科学院天津工业生物技术研究所 A kind of method of whole-cell catalytic production L erythruloses
CN107858340A (en) * 2017-12-22 2018-03-30 浙江大学 The phosphate aldolase A mutant of D fructose 6, recombinant expression carrier, genetic engineering bacterium and its application of high catalytic activity
CN109207139A (en) * 2018-10-19 2019-01-15 河南国中铭泰化工科技有限公司 A kind of non-crosslinked plant gum fracturing fluid and preparation method thereof
CN112852890A (en) * 2020-08-27 2021-05-28 中国科学院天津工业生物技术研究所 Biological synthesis method of polyhydroxy diketone and hydroxy furanone compound

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LENA STELLMACHER等: "Acid-Base Catalyst Discriminates between a Fructose 6-Phosphate Aldolase and a Transaldolase", 《CHEMCATCHEM》 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107739742A (en) * 2017-10-18 2018-02-27 中国科学院天津工业生物技术研究所 A kind of method of whole-cell catalytic production L erythruloses
CN107858340A (en) * 2017-12-22 2018-03-30 浙江大学 The phosphate aldolase A mutant of D fructose 6, recombinant expression carrier, genetic engineering bacterium and its application of high catalytic activity
CN107858340B (en) * 2017-12-22 2020-04-14 浙江大学 High-catalytic-activity D-fructose-6-phosphate aldolase A mutant, recombinant expression vector, genetically engineered bacterium and application thereof
CN109207139A (en) * 2018-10-19 2019-01-15 河南国中铭泰化工科技有限公司 A kind of non-crosslinked plant gum fracturing fluid and preparation method thereof
CN112852890A (en) * 2020-08-27 2021-05-28 中国科学院天津工业生物技术研究所 Biological synthesis method of polyhydroxy diketone and hydroxy furanone compound

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