CN110452861A - A kind of genetic recombination engineering bacteria and its catalyzing and synthesizing the application in D- pantoyl internal ester - Google Patents
A kind of genetic recombination engineering bacteria and its catalyzing and synthesizing the application in D- pantoyl internal ester Download PDFInfo
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Abstract
The invention discloses a kind of genetic recombination engineering bacteria and its application in D-pantoyl lactone is being catalyzed and synthesized, is belonging to biocatalysis research field.The genetic recombination engineering bacteria, using Escherichia coli as host strain, containing the recombinant expression plasmid of conjugation polyketone reductase gene and glucose dehydrogenase gene is contained in the host strain, the nucleotide sequence of the conjugation polyketone reductase gene is as shown in SEQ ID NO.1.The engineering bacteria is induced while expressing polyketone reductase and glucose dehydrogenase, it is applied to asymmetry catalysis synthesis D-pantoyl lactone, pass through the synergistic effect of above-mentioned double enzymes, the in-situ regeneration for realizing coenzyme NADP 11, can reach the purpose efficiently produced from ketone group pantoic acid lactone to D-pantoyl lactone.This process simplify production technologies, while avoiding the addition of additional coenzyme, reduce production cost.
Description
Technical field
The present invention relates to biocatalysis research fields, and in particular to a kind of genetic engineering bacterium and asymmetric using the engineering bacteria
The method for catalyzing and synthesizing D-pantoyl lactone.
Background technique
Pantothenic acid is also known as vitamin B5, is one kind of water-soluble (vitamin) B race, is widely present in nature animal and plant food
In, human body enteral beneficial bacterium can also be synthesized voluntarily.Pantothenic acid in vivo mainly in the form of coacetylase (Coenzyme A) participate in sugar,
Rouge, protein metabolism are nutriments necessary to brain and nerve.In addition, pantothenic acid has lipoid peroxidization resistant.Nature
Pantothenic acid there are two kinds of configurations of D- and L-, but only D- type have physiological activity.Since the stability of D-VB5 is poor, usually with D-
Calcium pantothenate is its commercial form, is widely used in the fields such as medicine, food, feed and daily use chemicals.With mentioning for people's quality of life
It rises, the demand of D-VB5 and D-VB5 calcium will be further increased.The inverse synthetic reaction formula of D-VB5 calcium are as follows:
D- pantoyl internal ester (D-PL) is the key that synthesis D-VB5 calcium chiral intermediate.Currently, in industrial production D- pantoyl
Ester mainly uses enzymatic hydrolysis Kinetic Resolution (such as ZL 200510123566.4；ZL201610361405.7 etc.).Firstly,
By the aldol reaction of isobutylaldehyde and formaldehyde, in acid condition with hydrogen cyanide addition, further lactate synthesis racemic
DL- pantoyl internal ester.DL- pantoyl internal ester passes through hydrolase selective catalysis again, obtains the D- pantoyl internal ester of high-optical-purity.It should
The major defect of method is embodied in high toxicity compound hydrogen cyanide, and the use of a large amount of strong acid and strong bases, does not meet green chemical industry
The requirement of development.
Oxidoreducing enzyme asymmetric reduction prochiral carbonyl compounds synthesis of chiral alcohol is considered as most efficient method.Altogether
Yoke polyketone reductase (Conjugated polyketone reductases, CPRs) asymmetry catalysis ketopantoic acid lactone, i.e.,
Dihydro -4,4- dimethyl -2,3- furasndione (KPL) synthesizes D- pantoyl internal ester, embodies with hydrolase resolution process comparative advantages
: (1) CPRs have very high catalysis stereoselectivity, guarantees that product has high antimer optical purity, enantiomeric excess value
(enantiomeric excess, e.e.) > 99.9%；(2) it with isobutylaldehyde and diethy-aceto oxalate etc. for synthesis material, avoids passing
A large amount of uses of deadly poisonous compound hydrogen cyanide and strong acid and strong base in technique of uniting.However, conjugation polyketone reductase gene resource phase
To scarcity, at present only from the CPR-C1 and CPR-2 of Candida parapsilosis IFO 0708
(Appl.Microbiol.Biotechnol.2004,64:359-366), from Candida orthopsilosis's
CorCPR (J.Biotechnol.2019,291:26-34) and CduCPR from Candida dubliniensis
The report such as (Enzyme Microb.Tech.2019,126:77-85) genes.Moreover, the genes such as CPR-C1 and CPR-C2 are in large intestine
Expression is lower in bacillus, and the catalytic efficiency for directly resulting in recombinant cell is lower.
Summary of the invention
The purpose of the present invention is to provide a kind of genetic recombination engineering bacterias, while expressing conjugation polyketone reductase and glucose
Dehydrogenase is applied to catalysis ketopantoic acid lactone (KPL) asymmetric reduction production D- pantoyl internal ester.
To achieve the above object, the present invention adopts the following technical scheme:
A kind of genetic recombination engineering bacteria contains in the host strain using Escherichia coli as host strain and contains conjugation polyketone
The recombinant expression plasmid of reductase gene and glucose dehydrogenase gene, the nucleotide sequence of the conjugation polyketone reductase gene
As shown in SEQ ID NO.1.
The present invention is based on the sequence signatures for having reported conjugation polyketone reductase, tactful from GenBank number using gene mine locating
The conjugation polyketone reductase gene of Candida albicans, nucleotide sequence such as SEQ ID NO.1 are derived from according to screening in library
It is shown, and construct recombinant expression plasmid.Research expression, the gene can have in E. coli and catalyze and synthesize D-
The vigor and selectivity of pantoyl internal ester.Meanwhile the present invention clones glucose dehydrogenase gene into recombinant expression plasmid, realizes auxiliary
The in-situ regeneration of enzyme NADPH.
Specific steps are as follows: by gene cloning, obtain conjugation polyketone reductase gene sequence and glucose dehydrogenase respectively
Two sections of sequences are connected on double expression plasmid carrier by gene order, obtain recombinant expression plasmid.Recombinant expression plasmid is converted
Into host cell E. coli, genetic recombination engineering bacteria is obtained.
The genetic recombination engineering bacteria expresses simultaneously through induction and is conjugated polyketone reductase and glucose dehydrogenase, applied to urging
Change the asymmetric reduction of ketopantoic acid lactone, the pure D- pantoyl internal ester of synthesizing optical, reaction equation are as follows:
As above-mentioned reaction equation is conjugated polyketone reductase in asymmetry catalysis substrate ketopantoic acid lactone during the reaction
D- pantoyl internal ester is continuously generated, needs to consume reproducibility enzyme the enzyme II, NADPH of chemical equivalent.It is de- that recombination glucose is expressed simultaneously
Hydrogen enzyme realizes the regeneration of coenzyme NADP 11 using glucose as substrate.
Preferably, the nucleotide sequence of the glucose dehydrogenase gene is as shown in SEQ ID NO.2.The glucose
Dehydrogenase gene derives from 168 bacterium of Bacillus subtilis, but is not limited to the bacterium.
Preferably, the initial carrier of the recombinant expression plasmid is double expression plasmid carrier pACYCDuet-1.
Specifically, the conjugation polyketone reductase gene is located at restriction enzyme site in double expression plasmid carrier pACYCDuet-1
Between BamH I and Hind III, glucose dehydrogenase gene is located in pACYCDuet-1 between Nde I and Xho I.
Preferably, the host strain is e. coli bl21 (DE3) bacterial strain.
The present invention provides application of the genetic recombination engineering bacteria in asymmetry catalysis synthesis D- pantoyl internal ester.
The application, comprising the following steps: the wet thallus with the fermented culture acquisition of the genetic recombination engineering bacteria is to urge
Agent, using ketopantoic acid lactone as substrate, the phosphate buffer for being 5.0~8.0 using pH adds in reaction system as reaction medium
Glucose is reacted under the conditions of 100~300rpm at 25~40 DEG C, obtains the D- pantoyl internal ester.
The method of the genetic recombination engineering bacterium fermentation culture includes: that the genetic recombination engineering bacteria is inoculated in containing antibiosis
In the LB culture medium of element, 35~40 DEG C, revolving speed is cultivated under the conditions of being 180~220rpm to OD600Value reaches 0.6~0.8, is added
0.1~0.5mM isopropylthiogalactoside (IPTG) continues 10~12h of culture in 18 DEG C, under the conditions of 200rpm, is centrifuged, and receives
Collect thallus and obtains the wet thallus.
Genetic engineering bacterium cell is resuspended with phosphate buffer, obtains resting cell suspension；Add into resting cell suspension
Ketone group pantoic acid lactone, glucose is added to be reacted, after the reaction was completed, from isolating and purifying to obtain in Pantothenic acid in reaction solution
Ester.
Preferably, the dosage of catalyst is calculated as 80~120g/L, the general solution of substrate ketone with wet thallus weight in reaction system
Acid lactone is continuously added into the flow velocity of 0.55~0.75mmol/h into reaction system, mole of glucose and ketopantoic acid lactone
Than >=1.25:1.
In catalytic reaction process, continue current adding substrate KPL, accumulate it not in the reaction system, is turned by catalyst rapidly
Change forms product, reduces the spontaneous hydrolysis of substrate KPL, is conducive to the asymmetric syntheses of D- pantoyl internal ester.
Suitable reaction temperature and reacting solution pH value is conducive to the progress of reaction, and the temperature of the reaction is 25~35
DEG C, pH is 6.5~8.0.If temperature is excessively high, pH value meta-acid or meta-alkali be easy to cause the inactivation of enzyme in reaction process.It is more excellent
Choosing, reaction temperature are 35 DEG C, pH 6.5, and enzyme catalytic effect is best under this reaction condition, and the yield of D-pantoyl lactone is most
It is high.
Genetic engineering bacterium internal cell coenzyme can be self-sufficient, does not need that NADP is added in reaction system+。
It is that the present invention has the utility model has the advantages that
The present invention will be conjugated polyketone reductase gene and glucose dehydrogenase gene is sequentially inserted into expression vector, constructs
Recombinant expression plasmid；It is conducted into constructing host cell genetic engineering bacterium again, it can be achieved that polyketone reductase and glucose dehydrogenase
Efficient coexpression.By the synergistic effect of above-mentioned double enzymes, the in-situ regeneration of coenzyme NADP 11 is realized, can reach from the general solution of ketone group
The purpose that acid lactone is efficiently produced to D-pantoyl lactone, the optical selective (e.e. value) > 99% of target product, conversion ratio is big
In 95%, space-time yield reaches 157gL-1·d-1.The genetic engineering bacterium shows good catalytic stability, high catalysis is lived
The advantages that power, being expected to, which becomes bioanalysis, prepares the good industrial catalyst of D- pantoyl internal ester, and promotes the upgrading of its synthesis technology.
Using the engineering bacteria for catalyzing and synthesizing high optical voidness D- pantoyl internal ester, high poison examination is relatively avoided compared with traditional handicraft
The use of agent hydrogen cyanide simplifies production technology, while avoiding the addition of additional coenzyme, reduces production cost.
Detailed description of the invention
Fig. 1 is the nucleic acid electrophoresis figure for being conjugated polyketone reductase gene CPR, wherein M: nucleic acid Marker；1: gene C PR sample
Product.
Fig. 2 is the SDS-PAGE electrophoresis of genetically engineered E.coli LP01 induction expression protein, wherein M: protein
Marker；1:pACYCDuet-1 empty control plasmid；2: genetically engineered E.coli LP01 induction thallus breaks born of the same parents' supernatant；3: gene
Engineering bacteria E.coli LP01 induces thallus to break born of the same parents' precipitating；The sample pair that 4:CPR enzyme is expressed on carrier pACYCDuet-1 carrier
According to；The sample controls that 5:GDH enzyme is expressed on carrier pACYCDuet-1 carrier.
Fig. 3 is the influence that reaction temperature prepares D-PL to genetic engineering bacterium catalysis.
Fig. 4 is the influence reacted pH and prepare D-PL to genetic engineering bacterium catalysis.
Fig. 5 is the influence that glucose and KPL molar ratio prepare D-PL to genetic engineering bacterium catalysis.
Fig. 6 is the influence that coenzyme additive amount prepares D-PL to genetic engineering bacterium catalysis.
Fig. 7 is the catalysis reaction process that genetic engineering bacterium continuous feeding strategy prepares D-PL.
Fig. 8 is the gas phase analysis standard diagram of substrate ketone group pantoic acid lactone (KPL).
Fig. 9 is the gas phase analysis standard diagram of DL- pantoyl internal ester (DL-PL).
Figure 10 is the D-PLDE gas phase analysis map that genetically engineered E.coli PL01 is catalyzed and synthesized.
Figure 11 is synthetic product D-PL's1H NMR spectra.
Figure 12 is synthetic product D-PL's13C NMR spectra.
Specific embodiment
The present invention is further described combined with specific embodiments below, but given embodiment should not be understood as to this hair
The limitation of bright protection scope, protection scope of the present invention are not limited to that.
The building of 1 recombinant expression plasmid pACYC-CPR-GDH of embodiment
Glucose dehydrogenase GDH base from 168 bacterium of Bacillus subtilis is cloned using primers F _ GDH/R_GDH
Cause.The nucleic acid sequence of primers F _ GDH and R_GDH is respectively as follows:
5'-GGAATTCCATATGTACCCGGACCTGAAAGG-3'；
5’-CCGCTCGAGTTAACCACGACCAGCCTGGA-3’。
Genetic fragment using Nde I and Xho I double digestion GDH gene, after recycling digestion；Nde I and Xho are used simultaneously
I double digestion expression plasmid pACYCDuet-1, the plasmid fragments after recycling digestion.It is attached using T4 ligase, is produced after connection
Object transformed clone host E.coli DH5 α.With the LB solid plate screening containing chlorampenicol resistant, positive transformant is selected, is sequenced
The result shows that the recombinant plasmid after gene order is errorless, as recombinant expression plasmid pACYC-GDH, save backup in -20 DEG C.
Using F_CPR/R_CPR Clone Origin in the conjugation polyketone reductase gene of Candida albicans, such as Fig. 1 institute
Show.The sequence of primers F _ CPR and R_CPR is respectively as follows:
5’-CGGGATCCATGACAAGTCATACACATCCAGTTAAA-3'；
5’-CCCAAGCTTTAAATCTTTAAAGGCTTCATGAAAAAA-3’。
Polyketone reductase CPR gene is conjugated using BamH I and Hind III double digestion, the genetic fragment after recycling digestion；
BamH I and Hind III double digestion recombinant plasmid pACYC-GDH are used simultaneously, the recombinant plasmid segment after recycling digestion.Using
T4 ligase is attached, enzyme-linked product transformed clone host E.coli DH5 α.It is sieved with the LB solid plate containing chlorampenicol resistant
Choosing, selects positive transformant, and sequencing result shows the recombinant plasmid after gene order is errorless, as recombinant plasmid pACYC-CPR-
GDH is saved backup in -20 DEG C of refrigerators.
The building and catalyst preparation of 2 genetically engineered E.coli PL01 of embodiment
The recombinant expression plasmid pACYC-CPR-GDH constructed in embodiment 1 is converted into expressive host E.coli BL21
(DE3) to get genetically engineered E.coli PL01.E.coli PL01 is inoculated in 10mL cuvette cartridge LB liquid medium (38 μ
G/mL chloramphenicol) in, at 37 DEG C, 12~16h of shaken cultivation under the conditions of 200rpm.The culture solution is connect by 1~5% inoculum concentration
Kind is in the bottled liquid 50mL LB culture medium of 250mL triangular pyramidal (38 μ g/mL chloramphenicol), in 37 DEG C, under the conditions of 200rpm
2~3h of shaken cultivation, as inoculum density OD600When value reaches 0.6~0.8,0.1~0.5mM isopropylthiogalactoside is added
(IPTG), continue to cultivate 12h in 18 DEG C, under the conditions of 200rpm.
It is centrifuged 5~10min under the conditions of 5000~10000 × g and collects cell, i.e., as asymmetric syntheses D- pantoyl
The catalyst of lactone.In ice bath, using sonicated cells, 20~40min is centrifuged under the conditions of 12000~15000 × g,
Clasmatosis supernatant and broken precipitating are received respectively, using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
It is detected, SDS-PAGE electrophoresis is as shown in Figure 2.
3 genetically engineered E.coli PL01 of embodiment catalyzes and synthesizes the optimization of D- pantoyl internal ester reaction condition
In 5mL 0.1M kaliumphosphate buffer (pH 6.5) reaction system, contain 0.5g genetically engineered E.coli
PL01 resting cell, 100mM KPL, 125mM glucose carry out reaction 20min under 35 DEG C and 200rpm.
The optimum pH that E.coli PL01 catalyzes and synthesizes D-PL is 6.5 (Fig. 3), and optimal reactive temperature is 35 DEG C (Fig. 4),
The molar ratio that glucose and substrate KPL are added in reaction solution is 1.25:1 (Fig. 5), and genetic engineering bacterium internal cell coenzyme can be self-supporting
It is self-sustaining, it does not need that NADP is added in reaction system+(Fig. 6).
The synthesis of 4 genetically engineered E.coli PL01 of embodiment catalysis D- pantoyl internal ester
It collects and genetically engineered E.coli PL01 fermentation liquid, 8000~12000rpm, is centrifuged 5~10min by 4~20 DEG C
Thallus is obtained, supernatant to the greatest extent is abandoned, uses H2O is cleaned twice, then phosphate buffer (0.2ML-1, pH 6.5) be resuspended thallus to get
Genetically engineered E.coli PL01 resting cell bacteria suspension.
In 5mL 0.1M kaliumphosphate buffer (pH 6.5) reaction system, contain 0.5g genetically engineered E.coli
PL01 cell, 1.13g glucose are reacted under 35 DEG C and 200rpm.By the way that 0.640g substrate KPL is dissolved in five times
Substrate stock solution is prepared in 5mL 0.1M dipotassium hydrogen phosphate-citric acid (pH 2.5), the concentration of each substrate KPL is 1M.
It is slowly flowed by syringe pump and adds liquid storage, flow velocity 0.67mL/h.Every time after preceding 1mL Substrate stock solution stream adds, then carry out
The configuration of Substrate stock solution next time.In reaction process, by with 1M Na2CO3PH value in reaction is maintained into pH 6.5.Instead
It should be carried out under 35 DEG C and 200rpm, terminate reaction in stream plus after terminating 0.5h.
In reaction process, every 1 hour, it is taken out four aliquots (each 100 μ L).Two of them sample is through lactone
Change (lactonize by mixing reaction mixture with isometric 1M HCl, and acquired solution is boiled 10 minutes) to lead to afterwards
The hydrolysis situation of GC analysis substrate is crossed, other two sample is directly analyzed.
The yield and optical purity analysis of reaction product D- pantoyl internal ester:
The e.e. value of reaction product D-pantoyl lactone be by using equipped with RT- β dextrorotation column (internal diameter 30m,
Agilent 7890A gas chromatograph 0.25mm) is analyzed.Analysis condition: chromatographic column: RT- β dextrorotation column (internal diameter 30m,
0.25mm)；Gas phase condition: gasification temperature is set as 230 DEG C；Detector temperature is set as 250 DEG C；Column oven uses temperature programming journey
Sequence, initial temperature are set as 60 DEG C, and 10 DEG C/min is heated to 150 DEG C, keeps 5min；It the use of nitrogen is carrier gas, flow is constant current
2.0mL/min.Yield and e.e. are worth calculation formula:
Products collection efficiency (%)=production concentration/theoretical product maximum concentration × 100%；
E.e. value (%)=[(A of D-pantoyl lactoneR-AS)/(AR+AS)] × 100%.
As a result as shown in fig. 7, after 8 hours, reaction total volume is 12.5mL, and the concentration of D- pantoyl internal ester is 379mM, mapping
Body excessive value > 99%.The conversion ratio of product is 95% simultaneously, and space-time yield reaches 157gL-1·d-1。
In addition, do not checked in catalytic process KPL show established process be efficient and substrate continuously into
Material strategy significantly reduces the influence of asymmetric syntheses of the spontaneous hydrolysis to D- pantoyl internal ester.
Finally, after evaporation and drying, obtaining white solid D- pantoyl internal ester by extraction with 68% separation yield.
The gas phase map of substrate KPL, DL-PL and catalytic reaction products as shown in figs. 8-10, enantiomeric excess value
(e.e.) > 99%.Catalysate1H NMR spectra and13C NMR spectra is as shown in FIG. 11 and 12, further proves catalysis institute
Obtaining product is high optically pure D- pantoyl internal ester.
Sequence table
<110>Hangzhou Pedagogic University
<120>a kind of genetic recombination engineering bacteria and its application in D- pantoyl internal ester is being catalyzed and synthesized
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 927
<212> DNA
<213>Candida albicans (Candida albicans)
<400> 1
atgacaagtc atacacatcc agttaaactg tcacgcacct ttaaaaccaa agcgggcgat 60
gaactgagca tcggcaccgg tacgggcacg aagtggaaaa aagatagcaa actggatgaa 120
atcaatcagg aactggtgga tcagatctta ctggccatta aactgggtta tcgtcatatt 180
gataccgcgg aagtgtataa tacacaggcc gaagttggcg aagcaattaa aaaatcaggt 240
atccctcggg aacagctgtg gattacgacc aaatataatc cgggttgggg cgacataaaa 300
gcgtctagtg cgagtccgca ggaatctatc gataaagccc tgaaacagtt aggaacagat 360
tatattgatc tgtatctgat tcatcagccg ttttttacgg aagaaaatac gcatggctat 420
agcctgatcg atacatggaa aattctgatc gaagctaaaa aacagggcaa aattcgcgaa 480
attggcgtgt ctaattttgc catcaaacat ctagaagctc tgaaaaaagt gagcgaacca 540
gaatattatc cagttgttaa tcagatcgaa tctcatccgt ttctgcagga tcagagtaaa 600
aatatcacca aatattcaca ggaaaatggc attctggtgg aagccttttc tcctctgacg 660
ccggctagtc gcttagatgc caatccgtta accgattatt tggaagaact ggtgaaaaaa 720
tataataaaa cgccgggtca gctgctgctg cgctataccc tgcagcgcgg cattctgcct 780
attacaacga gtgctaaaga atctcgcatc aaagaatctt tagatgtgtt tgattttgaa 840
ctgaccaaag aagaatttga taaaattacg gaaattggta atgccaatcc tcatcgtgtg 900
ttttttcatg aagcctttaa agattta 927
<210> 2
<211> 786
<212> DNA
<213>bacillus subtilis (Bacillus subtilis)
<400> 2
atgtacccgg acctgaaagg taaagttgtt gctatcaccg gtgctgcttc tggtctgggt 60
aaagctatgg ctatccgttt cggtaaagaa caggctaaag ttgttatcaa ctactactct 120
aacaaacagg acccgaacga agttaaagaa gaagttatca aagctggtgg tgaagctgtt 180
gttgttcagg gtgacgttac caaagaagaa gacgttaaaa acatcgttca gaccgctatc 240
aaagaatttg gtaccctgga catcatgata aacaacgctg gtctggaaaa cccggttccg 300
tctcacgaaa tgccgctgaa agactgggac aaagttatcg gtaccaacct gaccggtgct 360
ttcctgggtt ctcgtgaagc tatcaaatac ttcgttgaaa acgacatcaa aggtaacgtt 420
atcaacatgt cttctgttca cgaagttatc ccgtggccgc tgttcgttca ctacgctgct 480
tctaaaggtg gtatcaaact gatgaccgaa accctggctc tggaatacgc tccgaaaggt 540
atccgtgtta acaacatcgg tccgggtgct atcaacaccc cgataaacgc tgaaaaattc 600
gctgacccga aacagaaagc tgacgttgaa tctatgatac cgatgggtta catcggtgaa 660
ccggaagaaa tcgctgctgt tgctgcttgg ctggcttcta aagaagctag ttacgttacc 720
ggtatcaccc tgttcgctga cggtggtatg acccagtacc cgtctttcca ggctggtcgt 780
ggttaa 786
Claims (9)
1. a kind of genetic recombination engineering bacteria, using Escherichia coli as host strain, which is characterized in that contain in the host strain and contain
It is conjugated the recombinant expression plasmid of polyketone reductase gene and glucose dehydrogenase gene, the core of the conjugation polyketone reductase gene
Nucleotide sequence is as shown in SEQ ID NO.1.
2. genetic recombination engineering bacteria as described in claim 1, which is characterized in that the nucleotide of the glucose dehydrogenase gene
Sequence is as shown in SEQ ID NO.2.
3. genetic recombination engineering bacteria as described in claim 1, which is characterized in that the initial carrier of the recombinant expression plasmid is
Double expression plasmid carrier pACYCDuet-1.
4. genetic recombination engineering bacteria as claimed in claim 3, which is characterized in that the conjugation polyketone reductase gene is located at double
In expression plasmid carrier pACYCDuet-1 between restriction enzyme site BamH I and Hind III, glucose dehydrogenase gene is located at
In pACYCDuet-1 between Nde I and Xho I.
5. genetic recombination engineering bacteria according to any one of claims 1-4 answering in asymmetry catalysis synthesis D- pantoyl internal ester
With.
6. application as claimed in claim 5, which comprises the following steps: with the genetic recombination engineering bacteria through sending out
The wet thallus that ferment culture obtains is that catalyst is with the phosphate buffer that pH is 5.0~8.0 using ketopantoic acid lactone as substrate
Reaction medium adds glucose in reaction system, at 25~40 DEG C, is reacted under the conditions of 100~300rpm, obtain the D-
Pantoyl internal ester.
7. application as claimed in claim 6, which is characterized in that the method for the genetic recombination engineering bacterium fermentation culture includes:
The genetic recombination engineering bacteria is inoculated in antibiotic LB culture medium, culture to OD600Value reaches 0.6~0.8, is added
0.1~0.5mM isopropylthiogalactoside continues 10~12h of culture in 18 DEG C, under the conditions of 200rpm, and thallus is collected in centrifugation
Obtain the wet thallus.
8. application as claimed in claim 6, which is characterized in that the dosage of catalyst is calculated as in reaction system with wet thallus weight
80~120g/L, substrate ketopantoic acid lactone are continuously added into the flow velocity of 0.55~0.75mmol/h into reaction system, grape
Molar ratio >=1.25:1 of sugar and ketopantoic acid lactone.
9. application as claimed in claim 6, which is characterized in that the pH value of reaction medium is 6.5, catalytic reaction condition 35
DEG C, 200rpm.
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