CN107267474A - A kind of dihydrolipoamide dehydrogenase mutant protein and its preparation method and application - Google Patents
A kind of dihydrolipoamide dehydrogenase mutant protein and its preparation method and application Download PDFInfo
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- CN107267474A CN107267474A CN201710650938.1A CN201710650938A CN107267474A CN 107267474 A CN107267474 A CN 107267474A CN 201710650938 A CN201710650938 A CN 201710650938A CN 107267474 A CN107267474 A CN 107267474A
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- C07K2319/43—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a FLAG-tag
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Abstract
The invention discloses a kind of dihydrolipoamide dehydrogenase mutant protein and its preparation method and application.The invention provides a kind of protein (IAA350/351/358VVV mutant proteins), for dihydrolipoamide dehydrogenase is carried out into the protein that point mutation first and point mutation second and point mutation third are obtained；Point mutation first is that by I the amino acids residue of dihydrolipoamide dehydrogenase the 350th is sported into V；Point mutation second is that by A the amino acids residue of dihydrolipoamide dehydrogenase the 351st is sported into V；Point mutation third is that by A the amino acids residue of dihydrolipoamide dehydrogenase the 358th is sported into V.The mutant protein that the present invention is provided, is reduced to the sensitiveness that NADH suppresses so that tolerance increases of the PDH in anaerobism culture of Escherichia coli to NADH, can effectively improve PDH activity.The present invention is with a wide range of applications for the reduction such as Production of Succinic Acid by Escherichia coli salt, ethanol, butanol, 1,3 propane diols chemical substance.
Description
Technical field
The present invention relates to a kind of dihydrolipoamide dehydrogenase mutant protein and its preparation method and application.
Background technology
Escherichia coli, facultative heterotrophism can grow under aerobic and oxygen free condition.Pyruvic dehydrogenase (PDH) is also known as third
Ketoacid dehydrogenase complex (PDHC), in mitochondria, the committed step for controlling carbohydrate to utilize, i.e. pyruvic acid turn
Turn to acetyl coenzyme A and NADH.PDH three components are encoded by single operator, and the operator includes regulatory gene (pdhR)
With three structural genes, three structural genes are aceE genes (encoding pyruvate acidohydrogenase E1), aceF genes (coding dihydro sulphur
Caprylamide transacetylase E2) and lpd genes (coding dihydrolipoamide dehydrogenase E3).Under anaerobic, Escherichia coli
Cell extract in can detect PDH activity, however, the PDH in Escherichia coli body is typically inactive.There is oxide growth
The NADH produced in period, glycolysis is finally oxidized, and the organic compound produced in glycolytic cycle by glucose is used
Make electron acceptor, to maintain redox equilibrium, and the growth of persistent bacterial under anaerobic.Due to two kinds of growth patterns
Between electron acceptor difference, [NADH]/[NAD that anaerobic cell is reported+] ratioFar above aerobic cellRatio.Due to NADH suppression, the PDH activity in anaerobism culture of Escherichia coli is very low or can't detect
Its activity.Further research has confirmed that PDH NADH sensitiveness is located at dihydrolipoamide dehydrogenase
In (Dihydrolipoamide dehydrogenase, Lpd), because only that this enzyme and NAD+It is used as substrate interaction.
Industrial fermentation is intended under conditions of aerobic or anaerobism, and microorganism utilizes cheap raw material production value product.
In anaerobic fermentation process, reducing power NADH focuses primarily upon the synthesis of product rather than is oxidized, so as to cause target product
Yield is higher.For example, the organic acid such as lactic acid and butanedioic acid, the two can effectively be produced by anaerobic fermentation.However, lactic acid and
The theoretical maximum yield of butanedioic acid is supplied by NADH to be limited.Because：Under anaerobic, due to PDH vigor by
To suppression, 1 mole of glucose is only capable of providing 2 moles of NADH through glycolytic pathway；But under aerobic condition, 1 mole
Glucose can produce 4 moles of NADH.It can be seen that, the supplies of enough reducing powers is for obtaining the theoretical maximum yield of target product
It is vital.PDH catalysis conversion of pyruvate is acetyl coenzyme A, while with the generation of a molecule NADH.If PDH enzymes
It is activated, NADH's can be obtained by improvement using degree, causing the yield of the product of NADH dependent forms in metabolic pathway increases.
The content of the invention
It is an object of the invention to provide a kind of dihydrolipoamide dehydrogenase mutant protein and its preparation method and application.
It is following (a1) or (a2) the invention provides a kind of protein (IAA350/351/358VVV mutant proteins)：
(a1) dihydrolipoamide dehydrogenase is subjected to the protein that point mutation first and point mutation second and point mutation third are obtained；
The point mutation first is that by I the amino acids residue of dihydrolipoamide dehydrogenase the 350th is sported into V；The point mutation second is
The amino acids residue of dihydrolipoamide dehydrogenase the 351st is sported into V by A；The point mutation third is by dihydrolipoamide
The amino acids residue of dehydrogenase the 358th sports V by A；
(a2) fused protein obtained in N-terminal or/and C-terminal the connection label of (a1).
The label is shown in Table 1.
The sequence of the label of table 1
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (being usually 5) | RRRRR |
| Poly-His | 2-10 (being usually 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
The dihydrolipoamide dehydrogenase is following (b1) or (b2) or (b3)：
(b1) protein shown in the sequence 1 of sequence table；
(b2) there is the egg of more than 90% homogeneity from Escherichia coli and with the protein shown in the sequence 1 of sequence table
White matter；
(b3) from Escherichia coli and with the protein shown in the sequence 1 of sequence table with more than 90% homogeneity and tool
There is the protein of dihydrolipoamide dehydrogenase function.
The Escherichia coli concretely Escherichia coli MG1655.
The gene of coding IAA350/351/358VVV mutant proteins falls within protection scope of the present invention.
The gene is that DNA molecular shown in the sequence 2 of sequence table be mutated as follows to obtained DNA molecular：1048-
1053 nucleotides are by " ATCGCC " mutation in order to which " GTTGTT ", also, 1072-1074 nucleotides are sported by " GCA "
" GTT ".
Expression cassette, recombinant vector, transgenic cell line or recombinant bacterium containing the gene belong to the protection of the present invention
Scope.
The recombinant vector is concretely in multiple cloning sites (such as BamHI and XhoI digestions of pET-28a (+) plasmid
Between site) the obtained recombinant plasmid of the DNA molecular shown in the sequence 2 of insetion sequence table.
Recombinant vector described in any of the above is concretely imported out the recombinant bacterium that bacterium germination is obtained by the recombinant bacterium.It is described go out
Bacterium germination can be Escherichia coli, concretely e. coli bl21 (DE3).
The present invention also protects IAA350/351/358VVV mutant proteins as the application of dihydrolipoamide dehydrogenase.
The present invention also protects a kind of pyruvate dehydrogenase complex, it is characterised in that：It is mutated with IAA350/351/358VVV
Body protein instead of the dihydrolipoamide dehydrogenase in pyruvate dehydrogenase complex.
The present invention also protects the pyruvate dehydrogenase complex as the application of pyruvate dehydrogenase complex.
The present invention also protects a kind of method for preparing IAA350/351/358VVV mutant proteins, including ferments described heavy
The step of group bacterium.
The present invention also protects a kind of method that releasing dihydrolipoamide dehydrogenase is suppressed by NADH, comprises the following steps：
Dihydrolipoamide dehydrogenase is subjected to point mutation first and point mutation second and point mutation third；The point mutation first is that dihydro sulphur is pungent
The amino acids residue of acid amides dehydrogenase the 350th sports V by I；The point mutation second is by dihydrolipoamide dehydrogenase the 351st
Amino acids residue sports V by A；The point mutation third is by A by the amino acids residue of dihydrolipoamide dehydrogenase the 358th
Sport V.
In order to obtain the maximum production of target product under anaerobic condition, microorganism must provide enough reducing equivalents.Third
Ketoacid dehydrogenase catalysis conversion of pyruvate is acetyl coenzyme A, while with the generation of a molecule NADH.But in anaerobic condition
Under, because dihydrolipoamide dehydrogenase is suppressed by NADH, the PDH of Escherichia coli is typically inactive.The present invention's
Inventor establishes the huge mutain storehouse of capacity by a large amount of sequence analyses, structural analysis, to multiple simple point mutation albumen
It is compared with the enzyme activity of multipoint mutation albumen, NADH can be eliminated and (and then eliminate NADH pairs to LPD inhibitory action by finding
PDH suppression) crucial catastrophe point, it was found that the mutant protein (IAA350/351/358VVV) of three point mutation.
Compared with wild-type protein and other mutant proteins, mutant protein (IAA350/351/358VVV) can be more
The effective vigor for improving PDH under anaerobic condition, the sensitiveness that reduction PDH suppresses to NADH.Even if there is higher level
NADH, mutant protein IAA350/351/358VVV is also active.Mutant protein IAA350/351/358VVV PDH
Activity is in NADH]/[NAD+] than being 0.15 when be more than 6 times of A358V mutant.It is reported that grow under aerobic conditions
Intracellular [NADH]/[NAD of Escherichia coli+] ratio about 0.03 to 0.05.Under this ratio, PDH compounds are remained most
Almost the 90% of big activity.It is expected that [NADH]/[NAD of anaerobic cell+] ratio will be above the concentration of aerobic cellIt is worth noting that, in addition [NADH]/[NAD+] than be 0.15 under conditions of, mutant protein
IAA350/351/358VVV PDH activity is still higher than the activity without any NADH wild-type protein PDH added.
The mutant protein (IAA350/351/358VVV) that the present invention is provided, is reduced to the sensitiveness that NADH suppresses so that
Tolerance increases of the PDH in anaerobism culture of Escherichia coli to NADH, can effectively improve PDH activity.The present invention is for big
The reduction chemical substances such as enterobacteria production succinate, ethanol, butanol, 1,3- propane diols are with a wide range of applications.
Brief description of the drawings
Fig. 1 is the result of embodiment 2.
Fig. 2 is the result of embodiment 3.
Embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method, is conventional method unless otherwise specified.Test material used in following embodiments, is certainly unless otherwise specified
What routine biochemistry reagent shop was commercially available.Quantitative test in following examples, is respectively provided with three repetition experiments, as a result makes even
Average.
E.coli BL21(DE3)：Tiangen.pET-28a(+)：Novagan, Lausanne, Switzerland.
Each primer used is shown in Table 2 in embodiment 1.
Table 2
The framework of embodiment 1, recombinant bacterium
First, the structure of recombinant plasmid
1st, using Escherichia coli MG1655 genome as template, performing PCR amplification is entered using the F and R primer pairs constituted, obtained
Pcr amplification product.
2nd, the pcr amplification product that step 1 is obtained is taken, double digestion is carried out with restriction enzyme BamHI and XhoI, enzyme is reclaimed
Cut product.
3rd, pET-28a (+) plasmid is taken, double digestion is carried out with restriction enzyme BamHI and XhoI, carrier framework is reclaimed.
4th, the digestion products of step 2 and the carrier framework of step 3 are connected, obtains recombinant plasmid pET-28a-LPD.According to
Sequencing result, carries out structure to recombinant plasmid pET-28a-LPD and is described as follows：In the BamHI and XhoI of pET-28a (+) plasmid
The DNA molecular shown in the sequence 2 of sequence table is inserted between restriction enzyme site.DNA molecular shown in the sequence 2 of sequence table, coding
Wild-type protein shown in the sequence 1 of sequence table.
5th, using recombinant plasmid pET-28a-LPD as template, single-point is carried out using the A358V+ and A358V- primer pairs constituted
Mutation, obtains recombinant plasmid pET-28a-LPD (A358V).Recombinant plasmid pET-28a-LPD (A358V) is sequenced, is sequenced
As a result show：Compared with recombinant plasmid pET-28a-LPD, recombinant plasmid pET-28a-LPD (A358V) difference is only that sequence
1072-1074 nucleotides of DNA molecular shown in row 2 is mutated for " GTT " by " GCA ".DNA molecular coding after mutation
A358V mutant proteins.
6th, using recombinant plasmid pET-28a-LPD as template, using drawing that IA350/351VV+ and IA350/351VV- is constituted
Thing obtains recombinant plasmid pET-28a-LPD (IA350/351VV) to carrying out two point mutation.By recombinant plasmid pET-28a-LPD
(IA350/351VV) it is sequenced, sequencing result shows：Compared with recombinant plasmid pET-28a-LPD, recombinant plasmid pET-28a-
LPD (IA350/351VV) difference is only that 1048-1053 nucleotides of DNA molecular shown in sequence 2 is prominent by " ATCGCC "
Become for " GTTGTT ".DNA molecular coding IA350/351VV mutant proteins after mutation.
7th, using recombinant plasmid pET-28a-LPD as template, using drawing that EA354/358KV+ and EA354/358KV- is constituted
Thing obtains recombinant plasmid pET-28a-LPD (EA354/358KV) to carrying out two point mutation.By recombinant plasmid pET-28a-LPD
(EA354/358KV) it is sequenced, sequencing result shows：Compared with recombinant plasmid pET-28a-LPD, recombinant plasmid pET-28a-
LPD (EA354/358KV) difference, which is only that, is replaced 1060-1062 nucleotides of DNA molecular shown in sequence 2 by " GAA "
For " AAA ", and 1072-1074 nucleotides of DNA molecular shown in sequence 2 is mutated for " GTT " by " GCA ".Mutation
DNA molecular coding EA354/358KV mutant proteins afterwards.
8th, with recombinant plasmid pET-28a-LPD (A358V) for template, using IA350/351VV+ and IA350/351VV- groups
Into primer pair carry out two point mutation, obtain recombinant plasmid pET-28a-LPD (IAA350/351/358VVV).By recombinant plasmid
PET-28a-LPD (IAA350/351/358VVV) is sequenced, and sequencing result shows：With recombinant plasmid pET-28a-LPD phases
Than recombinant plasmid pET-28a-LPD (IAA350/351/358VVV) difference is only that DNA molecular shown in sequence 2
1048-1053 nucleotides are mutated for " GTTGTT " by " ATCGCC ", and by DNA molecular 1072-1074 shown in sequence 2
Position nucleotides is mutated for " GTT " by " GCA ".DNA molecular coding IAA350/351/358VVV mutant proteins after mutation.
9th, with recombinant plasmid pET-28a-LPD (EA354/358KV) for template, using IA350/351VV+ and IA350/
The primer pair of 351VV- compositions carries out two point mutation, obtains recombinant plasmid pET-28a-LPD (IAEA350/351/354/
358VVKV).Recombinant plasmid pET-28a-LPD (IAEA350/351/354/358VVKV) is sequenced, sequencing result shows,
Compared with recombinant plasmid pET-28a-LPD, recombinant plasmid pET-28a-LPD (IAEA350/351/354/358VVKV) difference
It is only that and is mutated 1048-1053 nucleotides of DNA molecular shown in sequence 2 for " GTTGTT " by " ATCGCC ", and will
1060-1062 nucleotides of DNA molecular shown in sequence 2 is replaced for " AAA " by " GAA ", and by DNA shown in sequence 2 points
Sub- 1072-1074 nucleotides are mutated for " GTT " by " GCA ".DNA molecular coding IAEA350/351/354/ after mutation
358VVKV mutant proteins.
2nd, the structure of recombinant bacterium
Recombinant plasmid pET-28a-LPD is imported into e. coli bl21 (DE3), recombinant bacterium WT is obtained.
Recombinant plasmid pET-28a-LPD (A358V) is imported into e. coli bl21 (DE3), recombinant bacterium A358V is obtained.
Recombinant plasmid pET-28a-LPD (IA350/351VV) is imported into e. coli bl21 (DE3), recombinant bacterium is obtained
IA350/351VV。
Recombinant plasmid pET-28a-LPD (EA354/358KV) is imported into e. coli bl21 (DE3), recombinant bacterium is obtained
EA354/358KV。
Recombinant plasmid pET-28a-LPD (IAA350/351/358VVV) is imported into e. coli bl21 (DE3), weight is obtained
Group bacterium IAA350/351/358VVV.
Recombinant plasmid pET-28a-LPD (IAEA350/351/354/358VVKV) is imported into e. coli bl21 (DE3),
Obtain recombinant bacterium IAEA350/351/354/358VVKV.
Embodiment 2, the preparation of wild-type protein and each mutant protein and Enzyme activity assay
First, the expression and purifying of albumen
Be respectively adopted embodiment 1 preparation each recombinant bacterium prepare N-terminal fusion have His6Each protein of label, specifically
Step is as follows：
1st, recombinant bacterium is seeded to the LB liquid medium containing 30 μ g/mL kanamycins, 37 DEG C, 170rpm shaken cultivations extremely
OD600nmValue=0.6-0.8.
2nd, complete after step 1, isopropyl-beta D-thio galactopyranoside (IPTG) is added in system and makes its concentration
For 1mM, 25 DEG C, 120rpm shaken cultivations 8 hours.
3rd, complete after step 2, cell is collected by centrifugation.
4th, the cell that step 3 is obtained is taken, is suspended with combination buffer, then carrying out ultrasonication, (10% power breaks 3
Second stops 5 seconds, 10 minutes total times), then 12000g is centrifuged 20 minutes, collects supernatant.
Combination buffer (pH7.8)：Tris-HCl containing 20mM, 500mM sodium chloride and 10mM imidazoles, surplus is water.
5th, the supernatant that step 4 is obtained is taken, destination protein is purified using nickel affinity chromatography.
The pillar of nickel affinity chromatography is：Ni-Agarose affinity columns (15mL).
Purge process：1. loading 2mL supernatants；2. 3 column volumes are washed with combination buffer；3. eluted with 2.5mL slow
Fliud flushing is eluted, and collects solution after post.
Elution buffer (pH7.8)：Tris-HCl containing 20mM, 500mM sodium chloride and 300mM imidazoles, surplus is water.
6th, take what step 5 obtained to cross solution after post, using the desalting column of GE Healthcare companies, by the solvent of albumen
System replaces with pH7.0,0.01M PBS, obtains protein solution.
The protein solution that recombinant bacterium WT progress above-mentioned steps are obtained is named as WT protein solutions.
The protein solution that recombinant bacterium A358V progress above-mentioned steps are obtained is named as A358V protein solutions.
The protein solution that recombinant bacterium IA350/351VV progress above-mentioned steps are obtained is named as IA350/351VV protein solutions.
The protein solution that recombinant bacterium EA354/358KV progress above-mentioned steps are obtained is named as EA354/358KV protein solutions.
The protein solution that recombinant bacterium IAA350/351/358VVV progress above-mentioned steps are obtained is named as IAA350/351/
358VVV protein solutions.
The protein solution that recombinant bacterium IAEA350/351/354/358VVKV progress above-mentioned steps are obtained is named as IAEA350/
351/354/358VVKV protein solutions.
Using bovine serum albumin(BSA) as standard, the protein compression in each protein solution is determined by Bradford methods
Degree.
2nd, Enzyme activity assay
Each protein solution for taking step one to prepare, respectively as solution to be measured, detects that it is de- as dihydrolipoamide
The enzyme activity of hydrogen enzyme.
Reaction system (1.0ml)：KH containing 0.1M2PO4、3mM NAD+, NADH, 3mM DL- dihydrolipoic acids, 1.5mM
EDTA and solution to be measured, surplus are pH8.0,0.01M PBS.
In reaction system, different NADH concentration are set, and 0 concentration is to be added without NADH.
In reaction system, protein concentration is 3mg/ml (in terms of total concentration), and albumen source is solution to be measured.
Reaction condition：30 DEG C stand reaction.
Persistently detect absorbance (A at 340nm340nm), every 10 seconds one sub-values of note.
△A340nmThe A of=the 30 second340nmThe A of-the 10 second340nm。
By " NADH and NAD+Mol ratio be used as solution to be measured for 0, WT protein solutions " △ that obtains under the conditions of this
A340nmIt is used as reference point.Calculate the △ A obtained under other conditions340nmWith the ratio of reference point, as LPD is with respect to enzyme activity.
As a result Fig. 1 is seen.In Fig. 1, abscissa is NADH and NAD in initial reaction system+Mol ratio.With NADH concentration
Increase, LPD enzyme activity is gradually suppressed.The activity of IAA350/351/358VVV mutant proteins far above wild-type protein and
Every other mutant protein.It is worth noting that, in [NADH]/[NAD+] than for 0.15 " harshness " under the conditions of, IAA350/
The activity of 351/358VVV mutant proteins is still higher than the activity without any NADH wild-type proteins added.IAEA350/
The activity of 351/354/358VVKV mutant proteins is less than wild-type protein.
One unit definition of enzymatic activity is that 1mg albumen generates 1 μm of ol NADH in 1min.As " NADH and NAD+Rub
You for 0, WT protein solutions than being used as solution to be measured " when, the enzyme activity of wild-type protein is 1.51 ± 0.03U/mg.
The sensitiveness that embodiment 3, each pyruvate dehydrogenase complex suppress to NADH
In order to confirm that IAA350/351/358VVV mutant proteins are converted into whole third to the relatively hyposensitivity that NADH suppresses
The relatively low sensitivity that ketoacid dehydrogenase complex suppresses to NADH, activity is measured using full cell extract.
First, full cell extract is prepared
Each recombinant bacterium that the preparation of embodiment 1 is respectively adopted prepares full cell extract, and specific steps are as follows：
1st, recombinant bacterium is seeded to the LB liquid medium containing 30 μ g/mL kanamycins, 37 DEG C, 170rpm shaken cultivations extremely
OD600nmValue=0.6-0.8.
2nd, complete after step 1, isopropyl-beta D-thio galactopyranoside (IPTG) is added in system and makes its concentration
For 1mM, 25 DEG C, 120rpm shaken cultivations 8 hours.
3rd, complete after step 2, cell is collected by centrifugation.
4th, the cell that step 3 is obtained is taken, is suspended with combination buffer, then carrying out ultrasonication, (10% power breaks 3
Second stops 5 seconds, 10 minutes total times), then 12000g is centrifuged 20 minutes, collects supernatant, as full cell extract.
The full cell extract that recombinant bacterium WT progress above-mentioned steps are obtained is named as the full cell extracts of WT.
The full cell extract that recombinant bacterium A358V progress above-mentioned steps are obtained is named as the full cell extracts of A358V.
The full cell extract that recombinant bacterium IAA350/351/358VVV progress above-mentioned steps are obtained is named as IAA350/
The full cell extracts of 351/358VVV.
2nd, Enzyme activity assay
Each the full cell extract for taking step one to prepare, respectively as solution to be measured, detects it as pyruvate dehydrogenase
The enzyme activity of multienzyme complex.
Initial system (1ml)：Diphosphothiamine containing 0.2mM, 0.1mM CoA, 10mM MgCl2、1mM DTT、2mM
NAD+, NADH and solution to be measured, surplus is pH7.5,100mM Tris-HCl buffer solutions.
In initial system, different NADH concentration are set, and 0 concentration is to be added without NADH.
In reaction system, protein concentration is 3mg/ml (in terms of total concentration), and albumen source is solution to be measured.
Pyruvic acid is added into initial system and makes its concentration for 5mM to start reaction.
Reaction condition：30 DEG C stand reaction.
Persistently detect absorbance (A at 340nm340nm), every 10 seconds one sub-values of note.
△A340nmThe A of=the 30 second340nmThe A of-the 10 second340nm。
By " NADH and NAD+Mol ratio be used as solution to be measured for 0, WT protein solutions " △ that obtains under the conditions of this
A340nmIt is used as reference point.Calculate the △ A obtained under other conditions340nmIt is relative enzyme activity with the ratio of reference point.
As a result Fig. 2 is seen.In Fig. 2, the picture left above is NADH and NAD in initial reaction system+Mol ratio be 0 under the conditions of phase
To enzyme activity, top right plot is NADH and NAD in initial reaction system+Mol ratio be 0.08 under the conditions of relative enzyme activity, lower-left figure
For NADH and NAD in initial reaction system+Mol ratio be 0.1 under the conditions of relative enzyme activity, bottom-right graph be initial reaction system
Middle NADH and NAD+Mol ratio be 0.15 under the conditions of relative enzyme activity.With IAA350/351/358VVV mutant proteins
The activity of pyruvate dehydrogenase complex is higher than the pyruvate dehydrogenase complex with wild-type protein and dashed forward with A358V
The pyruvate dehydrogenase complex of misfolded proteins.IAA350/351/358VVV mutant proteins can effectively release PDH to NADH's
Suppress sensitiveness.Natural PDH is in NADH]/[NAD+] than being 0.15 when be totally constrained, and with IAA350/351/358VVV
The PDH of mutant protein still retains about the 30% of reference point.
One unit definition of enzymatic activity is that 1mg albumen generates 1 μm of ol NADH in 1min.As " NADH and NAD+Rub
You for 0, WT protein solutions than being used as solution to be measured " when, enzyme activity is 2.63 ± 0.02U/mg.
SEQUENCE LISTING
<110>China Agricultural University
<120>A kind of dihydrolipoamide dehydrogenase mutant protein and its preparation method and application
<130> GNCYX171347
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 474
<212> PRT
<213>Escherichia coli
<400> 1
Met Ser Thr Glu Ile Lys Thr Gln Val Val Val Leu Gly Ala Gly Pro
1 5 10 15
Ala Gly Tyr Ser Ala Ala Phe Arg Cys Ala Asp Leu Gly Leu Glu Thr
20 25 30
Val Ile Val Glu Arg Tyr Asn Thr Leu Gly Gly Val Cys Leu Asn Val
35 40 45
Gly Cys Ile Pro Ser Lys Ala Leu Leu His Val Ala Lys Val Ile Glu
50 55 60
Glu Ala Lys Ala Leu Ala Glu His Gly Ile Val Phe Gly Glu Pro Lys
65 70 75 80
Thr Asp Ile Asp Lys Ile Arg Thr Trp Lys Glu Lys Val Ile Asn Gln
85 90 95
Leu Thr Gly Gly Leu Ala Gly Met Ala Lys Gly Arg Lys Val Lys Val
100 105 110
Val Asn Gly Leu Gly Lys Phe Thr Gly Ala Asn Thr Leu Glu Val Glu
115 120 125
Gly Glu Asn Gly Lys Thr Val Ile Asn Phe Asp Asn Ala Ile Ile Ala
130 135 140
Ala Gly Ser Arg Pro Ile Gln Leu Pro Phe Ile Pro His Glu Asp Pro
145 150 155 160
Arg Ile Trp Asp Ser Thr Asp Ala Leu Glu Leu Lys Glu Val Pro Glu
165 170 175
Arg Leu Leu Val Met Gly Gly Gly Ile Ile Gly Leu Glu Met Gly Thr
180 185 190
Val Tyr His Ala Leu Gly Ser Gln Ile Asp Val Val Glu Met Phe Asp
195 200 205
Gln Val Ile Pro Ala Ala Asp Lys Asp Ile Val Lys Val Phe Thr Lys
210 215 220
Arg Ile Ser Lys Lys Phe Asn Leu Met Leu Glu Thr Lys Val Thr Ala
225 230 235 240
Val Glu Ala Lys Glu Asp Gly Ile Tyr Val Thr Met Glu Gly Lys Lys
245 250 255
Ala Pro Ala Glu Pro Gln Arg Tyr Asp Ala Val Leu Val Ala Ile Gly
260 265 270
Arg Val Pro Asn Gly Lys Asn Leu Asp Ala Gly Lys Ala Gly Val Glu
275 280 285
Val Asp Asp Arg Gly Phe Ile Arg Val Asp Lys Gln Leu Arg Thr Asn
290 295 300
Val Pro His Ile Phe Ala Ile Gly Asp Ile Val Gly Gln Pro Met Leu
305 310 315 320
Ala His Lys Gly Val His Glu Gly His Val Ala Ala Glu Val Ile Ala
325 330 335
Gly Lys Lys His Tyr Phe Asp Pro Lys Val Ile Pro Ser Ile Ala Tyr
340 345 350
Thr Glu Pro Glu Val Ala Trp Val Gly Leu Thr Glu Lys Glu Ala Lys
355 360 365
Glu Lys Gly Ile Ser Tyr Glu Thr Ala Thr Phe Pro Trp Ala Ala Ser
370 375 380
Gly Arg Ala Ile Ala Ser Asp Cys Ala Asp Gly Met Thr Lys Leu Ile
385 390 395 400
Phe Asp Lys Glu Ser His Arg Val Ile Gly Gly Ala Ile Val Gly Thr
405 410 415
Asn Gly Gly Glu Leu Leu Gly Glu Ile Gly Leu Ala Ile Glu Met Gly
420 425 430
Cys Asp Ala Glu Asp Ile Ala Leu Thr Ile His Ala His Pro Thr Leu
435 440 445
His Glu Ser Val Gly Leu Ala Ala Glu Val Phe Glu Gly Ser Ile Thr
450 455 460
Asp Leu Pro Asn Pro Lys Ala Lys Lys Lys
465 470
<210> 2
<211> 1425
<212> DNA
<213>Escherichia coli
<400> 2
atgagtactg aaatcaaaac tcaggtcgtg gtacttgggg caggccccgc aggttactcc 60
gctgccttcc gttgcgctga tttaggtctg gaaaccgtaa tcgtagaacg ttacaacacc 120
cttggcggtg tttgcctgaa cgtcggctgt atcccttcta aagcactgct gcacgtagca 180
aaagttatcg aagaagccaa agcgctggct gaacacggta tcgtcttcgg cgaaccgaaa 240
accgatatcg acaagattcg tacctggaaa gagaaagtga tcaatcagct gaccggtggt 300
ctggctggta tggcgaaagg ccgcaaagtc aaagtggtca acggtctggg taaattcacc 360
ggggctaaca ccctggaagt tgaaggtgag aacggcaaaa ccgtgatcaa cttcgacaac 420
gcgatcattg cagcgggttc tcgcccgatc caactgccgt ttattccgca tgaagatccg 480
cgtatctggg actccactga cgcgctggaa ctgaaagaag taccagaacg cctgctggta 540
atgggtggcg gtatcatcgg tctggaaatg ggcaccgttt accacgcgct gggttcacag 600
attgacgtgg ttgaaatgtt cgaccaggtt atcccggcag ctgacaaaga catcgttaaa 660
gtcttcacca agcgtatcag caagaaattc aacctgatgc tggaaaccaa agttaccgcc 720
gttgaagcga aagaagacgg catttatgtg acgatggaag gcaaaaaagc acccgctgaa 780
ccgcagcgtt acgacgccgt gctggtagcg attggtcgtg tgccgaacgg taaaaacctc 840
gacgcaggca aagcaggcgt ggaagttgac gaccgtggtt tcatccgcgt tgacaaacag 900
ctgcgtacca acgtaccgca catctttgct atcggcgata tcgtcggtca accgatgctg 960
gcacacaaag gtgttcacga aggtcacgtt gccgctgaag ttatcgccgg taagaaacac 1020
tacttcgatc cgaaagttat cccgtccatc gcctataccg aaccagaagt tgcatgggtg 1080
ggtctgactg agaaagaagc gaaagagaaa ggcatcagct atgaaaccgc caccttcccg 1140
tgggctgctt ctggtcgtgc tatcgcttcc gactgcgcag acggtatgac caagctgatt 1200
ttcgacaaag aatctcaccg tgtgatcggt ggtgcgattg tcggtactaa cggcggcgag 1260
ctgctgggtg aaatcggcct ggcaatcgaa atgggttgtg atgctgaaga catcgcactg 1320
accatccacg cgcacccgac tctgcacgag tctgtgggcc tggcggcaga agtgttcgaa 1380
ggtagcatta ccgacctgcc gaacccgaaa gcgaagaaga agtaa 1425
Claims (10)
1. a kind of protein, is following (a1) or (a2)：
(a1) dihydrolipoamide dehydrogenase is subjected to the protein that point mutation first and point mutation second and point mutation third are obtained；It is described
Point mutation first is that by I the amino acids residue of dihydrolipoamide dehydrogenase the 350th is sported into V；The point mutation second is by two
The amino acids residue of hydrogen diaphorase the 351st sports V by A；The point mutation third is by dihydrolipoamide dehydrogenation
The amino acids residue of enzyme the 358th sports V by A；
(a2) fused protein obtained in N-terminal or/and C-terminal the connection label of (a1).
2. protein as claimed in claim 1, it is characterised in that：The dihydrolipoamide dehydrogenase be following (b1) or
Or (b3) (b2)：
(b1) protein shown in the sequence 1 of sequence table；
(b2) there is the albumen of more than 90% homogeneity from Escherichia coli and with the protein shown in the sequence 1 of sequence table
Matter；
(b3) from Escherichia coli and with the protein shown in the sequence 1 of sequence table with more than 90% homogeneity and with two
The protein of hydrogen diaphorase function.
3. encode the gene of protein described in claim 1 or 2.
4. gene as claimed in claim 3, it is characterised in that：The gene is to enter DNA molecular shown in the sequence 2 of sequence table
Row is mutated obtained DNA molecular as follows：1048-1053 nucleotides are mutated for " GTTGTT " by " ATCGCC ", also, the
1072-1074 nucleotides are mutated for " GTT " by " GCA ".
5. expression cassette, recombinant vector, transgenic cell line or recombinant bacterium containing gene described in claim 3 or 4.
6. protein described in claim 1 or 2 is used as the application of dihydrolipoamide dehydrogenase.
7. a kind of pyruvate dehydrogenase complex, it is characterised in that：Third is instead of with IAA350/351/358VVV mutant proteins
Dihydrolipoamide dehydrogenase in ketoacid dehydrogenase complex.
8. pyruvate dehydrogenase complex described in claim 7 is used as the application of pyruvate dehydrogenase complex.
9. one kind prepares method of protein described in claim 1 or 2, including recombinant bacterium described in the fermentation claim 5
Step.
10. a kind of release the method that dihydrolipoamide dehydrogenase is suppressed by NADH, comprise the following steps：By dihydrolipoamide
Dehydrogenase carries out point mutation first and point mutation second and point mutation third；The point mutation first is by dihydrolipoamide dehydrogenase the
350 amino acids residues sport V by I；The point mutation second is by the amino acids residue of dihydrolipoamide dehydrogenase the 351st
V is sported by A；The point mutation third is that by A the amino acids residue of dihydrolipoamide dehydrogenase the 358th is sported into V.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110577941A (en) * | 2019-08-30 | 2019-12-17 | 厦门大学 | amine dehydrogenase and application thereof |
| CN110628739A (en) * | 2019-08-14 | 2019-12-31 | 华东理工大学 | Amine dehydrogenase mutant and application thereof in synthesis of chiral amine and amino alcohol |
| CN111662888A (en) * | 2020-06-15 | 2020-09-15 | 北京达成生物科技有限公司 | Diaphorase mutant with high thermal stability, gene and preparation method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101657541A (en) * | 2006-05-01 | 2010-02-24 | 佛罗里达大学研究基金会 | Alcohol production in the non-recombinant hosts |
| EP3196312A1 (en) * | 2016-01-25 | 2017-07-26 | Metabolic Explorer | Efficient conversion of methylglyoxal into hydroxyacetone using novel enzymes and appli cations thereof |
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2017
- 2017-08-02 CN CN201710650938.1A patent/CN107267474B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101657541A (en) * | 2006-05-01 | 2010-02-24 | 佛罗里达大学研究基金会 | Alcohol production in the non-recombinant hosts |
| EP3196312A1 (en) * | 2016-01-25 | 2017-07-26 | Metabolic Explorer | Efficient conversion of methylglyoxal into hydroxyacetone using novel enzymes and appli cations thereof |
Non-Patent Citations (1)
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| XIUWEN WANG等: "Altering the sensitivity of Escherichia coli pyruvate dehydrogenase complex to NADH inhibition by structure-guided design", 《ENZYME AND MICROBIAL TECHNOLOGY》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110628739A (en) * | 2019-08-14 | 2019-12-31 | 华东理工大学 | Amine dehydrogenase mutant and application thereof in synthesis of chiral amine and amino alcohol |
| CN110577941A (en) * | 2019-08-30 | 2019-12-17 | 厦门大学 | amine dehydrogenase and application thereof |
| CN111662888A (en) * | 2020-06-15 | 2020-09-15 | 北京达成生物科技有限公司 | Diaphorase mutant with high thermal stability, gene and preparation method thereof |
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