CN106497895B - Leucine dehydrogenase mutant, encoding gene, carrier, engineering bacteria and its application - Google Patents
Leucine dehydrogenase mutant, encoding gene, carrier, engineering bacteria and its application Download PDFInfo
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Abstract
The invention discloses a kind of leucine dehydrogenase mutant, encoding gene, recombinant vector, genetic engineering bacterium and its applications；The recombination bacillus coli of high efficient expression leucine dehydrogenase provided by the invention has many advantages, such as yield height, simple process, convenient for industrial applications, which is used directly for the production of C4H9NO2；Wherein LeuDH-Q358T mutant reaches 965.7U/g to the total enzyme activity of 2- batanone acid under the conditions of 35 DEG C after fermentation, and the conversion ratio of 2- batanone acid reaches 99%, this provides good technical support for the large-scale production of C4H9NO2.
Description
(1) technical field
The present invention relates to leucine dehydrogenase mutant, and produce leucine dehydrogenase gene engineering bacteria and its construction method and
Using belonging to genetic engineering field.
(2) technical background
Leucine dehydrogenase (Leucine dehydrogenase, abbreviation Leu DH, EC 1.4.1.9), can be catalyzed α-
Batanone acid is converted into C4H9NO2, is important one of the biocatalyst of field of medicine production, especially in bioconversion
Preparing in the technique of C4H9NO2 has the function of key.
C4H9NO2 (L-ABA) is a kind of non-natural chiralα-aminoacid, is important industrial chemicals and medicine
Intermediate.For example, being used for the production of antiepileptic Levetiracetam and antituberculotic ebutol.
There is leucine dehydrogenase to come for producing the report of C4H9NO2, and from current result at present
It sees, leucine dehydrogenase is also the limiting factor in the production technology.Therefore the leucine dehydrogenation of novel high vigor is mutated
Enzyme gene, and highly expressed genetic engineering bacterium is constructed in the preparation of L-ABA by technique for gene engineering and is had great importance.
(3) summary of the invention
It is an object of the present invention to provide a kind of leucine dehydrogenase mutant, encoding gene, recombinant vector, genetic engineering bacterium with
And the application in C4H9NO2 is prepared in catalysis α-batanone acid.
The technical solution adopted by the present invention is that:
The present invention provides a kind of leucine dehydrogenase mutant, and the mutant is by amino acid shown in SEQ ID NO.12
The 41st, 61,77,347 and 358 progress list of sequence (encoding gene nucleotide sequence is as shown in SEQ ID NO.1),
Double or three site mutations form.
Further, the single mutation is that the 41st leucine is sported to glycine (L41G), the 77th leucine mutation
It is that threonine (A61T), the 347th methionine sport glycine for cysteine (L77C), the 61st alanine mutation
(M347G) or the 358th glutamine sports threonine (Q358T).Described pair sports M347G/Q358T, M347G/
L41G,M347G/L77C；Three site mutation is M347G/Q358T/L41G, M347G/Q358T/L77C.
Further, the variant amino acid sequence is one of following: SEQ ID NO.13, SEQ ID NO.14, SEQ
ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20, SEQ
ID NO.21 and SEQ ID NO.22.
It is any to contain SEQ ID NO.12, SEQ ID NO.13, SEQ ID due to the particularity of amino acid sequence
NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID
The segment of the polypeptide of amino acid sequence shown in NO.20 or its variant, such as its examples of conservative variations, bioactive fragment or derivative,
As long as the segment or polypeptide variants of the polypeptide and aforementioned amino acid sequences homology belong to present invention protection model 95% or more
The column enclosed.Specifically, described change may include the missing of amino acid, insertion or replacement in amino acid sequence；Wherein, for variant
It is conservative sexually revise, the amino acid replaced have structure similar with original acid or chemical property, such as replaced with leucine
Isoleucine, variant can also have non-conservation change, such as replace glycine with tryptophan.
The present invention also provides a kind of leucine dehydrogenase mutant code genes, under the gene nucleotide series are
One of column: SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID
NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10 and SEQ ID NO.11.
Due to the particularity of nucleotide sequence, any SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID
NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID
The variant of polynucleotides shown in NO.11 belongs to protection of the present invention as long as it has 90% or more homology with the polynucleotides
The column of range.The variant of the polynucleotides refers to a kind of polynucleotide sequence changed with one or more nucleotide.This
The variant of polynucleotides can make the variant of raw allelic variant or non-life, including substitution variants, Deletion variants and
It is inserted into variant.As known in the art, allelic variant is the alternative forms of a polynucleotides, it may be one multiple
Substitution, missing or the insertion of nucleotide, but not from substantially change its encode amino acid function.
The invention further relates to a kind of recombinant vectors gene constructed by the leucine dehydrogenase mutant code.
The invention further relates to a kind of recombination engineerings by recombinant vector conversion preparation.
The present invention provides a kind of leucine dehydrogenase mutant in catalysis 2- butanone acid isomerization preparation L-2- amino
Application in butyric acid, the specific application are as follows: with the recombination engineering of the gene of mutant code containing leucine dehydrogenase through luring
Leading the pure enzyme that the wet thallus that culture obtains isolates and purifies is enzyme source, using 2- batanone acid as substrate, using NADH and ammonium hydroxide as auxiliary agent, with
The phosphate buffer of pH=7.5,0.02M are that reaction medium constitutes reaction system, are reacted under the conditions of 10-50 DEG C, 150r/min,
After fully reacting, reaction solution is isolated and purified, obtains C4H9NO2.
Further, in the reaction system, substrate 2- batanone acid initial concentration is 5-200g/L, and preferably 10g/L, enzyme source is used
Amount is 500~1000U/L, preferably 818.7U/L, and the NADH mass dosage is 5~100g/L, preferably 70g/L, the ammonium hydroxide
Volume final concentration of 1~10%, preferably 1%.
Further, the enzyme source prepares as follows: by the recombination work of the gene of mutant code containing leucine dehydrogenase
Journey bacterium is seeded to the LB liquid medium of the 40 μ g/mL kanamycins containing final concentration, and 37 DEG C, 150r/min cultivates to OD600=0.8
~1.0, obtain seed liquor；Seed liquor is transferred to the LB of the 40 μ g/mL kanamycins containing final concentration with the inoculum concentration of volumetric concentration 2%
In fluid nutrient medium, OD is cultivated under the conditions of 37 DEG C, 150r/min600=0.4~0.8, add final concentration 0.5mM IPTG, 28
DEG C, 150r/min Fiber differentiation 10h, obtain induction broth, induction broth be centrifuged, collect wet thallus, it is raw with 0.85%
After managing salt water rinse, by 100g thallus/L physiological saline ratio dissolution thallus, after shaken well, ultrasonication, 12000r/
Min, 20min centrifugation, supernatant is crude enzyme liquid, and by ni-sepharose purification, it is enzyme source that collection, which obtains pure enzyme solution,；The LB Liquid Culture
Base composition: tryptone 10g/L, yeast powder 5g/L, NaCl 10g/L, solvent are water, pH value 7.0.
The present invention also provides a kind of leucine dehydrogenase mutant code genes in preparing leucine dehydrogenase
Using the application are as follows: building contains the recombinant vector of the leucine dehydrogenase mutant gene, by the recombinant vector
Into Escherichia coli, the recombination engineering bacteria of acquisition carries out Fiber differentiation for conversion, takes culture solution isolated de- containing leucine
The somatic cells of hydrogen enzyme mutant.
Further, leucine dehydrogenase mutant of the present invention is prepared in C4H9NO2 in catalysis α-batanone acid
Application method are as follows:
(1) screening of recombinant bacterium: having kalamycin resistance gene on the recombinant expression plasmid pET-28b-leudh,
If recombinant plasmid has been transformed into Escherichia coli, recombinant bacterial strain have kalamycin resistance, can containing 50 μ g/mL cards that
It is grown on the plate of mycin；Picking positive transformant, as leucine dehydrogenase recombinant bacterium BL21/pET-28b-leudh.
(2) building of leucine dehydrogenase mutant expression bacterial strain
Using the plasmid of recombinant bacterium BL21/pET-28b-leudh as template, it is carried out continuously two-wheeled mutation PCR, by the 347th
Methionine has been mutated into glycine, the 358th glutamine has been mutated into threonine, mutant plasmid is through Dpn I digestion mould
It is transferred in Escherichia coli after plate, recombinant bacterial strain has kalamycin resistance, can give birth on the plate containing 40 μ g/mL kanamycins
Long, the sequencing of picking positive transformant, sequence, which is shown, is successfully mutated 347 sites and 358 sites, as leucine dehydrogenase mutant
Bacterium BL21/pET-28b-mut-leudh.
(3) recombinant mutant bacterium expresses leucine dehydrogenase through culture
LB liquid medium (g/L) composition: peptone 10, yeast powder 5, NaCl 10, solvent are water, with 1M NaOH by pH
It is adjusted to 7.0；LB solid medium adds 20g/L agar；High pressure sterilization；Use preceding 50 μ g/mL kanamycins of addition final concentration.
Leucine dehydrogenase mutant bacterium is seeded to the LB liquid medium of 50 μ g/mL kanamycins of final concentration, 250mL
Triangular flask, liquid amount 50mL, 37 DEG C of cultivation temperature, shaking speed 150r/min, culture to OD600=0.8~1.0, it is planted
Sub- liquid；Seed liquor is transferred to the training of the fermentation equipped with 50 μ g/mL kanamycins of 100mL final concentration with the inoculum concentration of volumetric concentration 2%
In the 500mL triangular flask for supporting base (LB liquid medium), 2~3h (OD is cultivated under the conditions of 37 DEG C, 150r/min600=0.4~
0.8) final concentration 24 μ g/mL IPTG, 28 DEG C, 150r/min Fiber differentiation 10h, are added, induction broth is obtained.
(4) leucine dehydrogenase enzyme activity determination
Reaction system are as follows: 50mM batanone acid, 50mM ammonia (add, 10mL system adds 33 μ L) 0.4mM in the form of ammonium hydroxide
The enzyme solution of NADH, dehy after ni-sepharose purification, the phosphate buffer of pH=7.5,0.02M；By substrate, ammonium hydroxide and NADH at 35 DEG C
10min is preheated, then plus 3mL is in quartz colorimetric utensil, and is rapidly added 10 μ L of the enzyme solution after ni-sepharose purification, uses divide immediately
Light photometric determination OD340It is worth and is set to the OD at 0 moment of reaction340Value.Its OD of real-time monitoring340Drop-out value.1U enzyme activity is defined as 35
Enzyme amount needed for 1 μm of oL NADH of catalysis consumption per minute at DEG C.
Leucine dehydrogenase mutant of the present invention can concentration of substrate be 360g/L, 35 DEG C of reaction temperature when enzyme activity
Reach 414.3U/g (wild type leucine dehydrogenase enzyme activity is 104.4U/g), conversion ratio reaches 99%.
Ammonium hydroxide used in the present invention is often referred to the ammonia aqueous solution of mass concentration 25-28%.
Compared with prior art, beneficial effect of the present invention is mainly reflected in:
The present invention constructs the recombination bacillus coli of one plant of high efficient expression leucine dehydrogenase mutant, improves leucine
The enzyme activity of dehydrogenase；There is yield height using strain production leucine dehydrogenase, simple process, be convenient for industrial applications etc. excellent
Point, the bacterial strain can be used for the production of C4H9NO2, after fermentation to α-batanone acid total enzyme activity under the conditions of 35 DEG C
Reach 414.3U/g, conversion ratio reaches 99%, this large-scale production for C4H9NO2 as well as important medicine
Intermediate provides good technical support.
(4) Detailed description of the invention
Fig. 1 leudh genetic fragment nucleic acid electrophoresis figure, swimming lane 2mark, swimming lane 1,3leudhPCR amplified production.
Fig. 2 plasmid pET-28b-leudh physical map.
Fig. 3 wild-type bacteria SDS-PAGE electrophoresis, swimming lane 1, albumen in 2 broken liquid precipitates, swimming lane 3, in 4 broken liquid supernatants
Albumen.
Influence of Fig. 4 temperature to M347G/Q358T vigor.
Influence of Fig. 5 metal ion to M347G/Q358T vigor.
(5) specific embodiment
The present invention is described further combined with specific embodiments below, but protection scope of the present invention is not limited in
This:
Embodiment 1: the gene chemical synthesis of leucine dehydrogenase
The gene source of leucine dehydrogenase is in Thermoactinomyces intermedius (Thermoactinomyces
Intermedius whole genome sequence).In order to express with His-tag after so that the gene is connected to carrier pET-28b
Albumen, cut off its terminator codon, and be referring to its sequence and common with the codon preference of B.subtilis 168
Restriction enzyme enzyme recognition site BamH I, Xho I, Pst I, Hind III and Nco I carry out sequence optimisation, newly-designed bright
The sequence of propylhomoserin dehydrogenase gene (leudh) encodes the sequence of amino acid as shown in SEQ ID NO.1 as SEQ ID NO.12 institute
Show, gene chemical synthesis working delegation Shanghai Xu Guan biotechnology Development Co., Ltd completes, and is connected to cloning vector after gene chemical synthesis
In pET28b.
Embodiment 2: the building of leucine dehydrogenase recombination bacillus coli
On the basis of embodiment 1, using round pcr, with the nucleotide sequence (shown in SEQ ID NO.1) of synthesis for mould
Plate, with leudh-F:5 '-CGTACGCTTCCGAANNNGAGGCGATTGAAG-3 ' and leudh-R:5 '-
CTTCAATCGCCTCNNNTTCGGAAGCGTACG-3 ' is primer, is expanded to leucine dehydrogenase gene (leudh), and
Introduce Nco I and Xho I restriction enzyme site respectively with 3' at its end 5'.PCR reaction system (50 μ L) are as follows: 10 × Taq
5 μ L, dNTP Mixture of polymerase buffer, 4 μ L；1 μ L of template DNA；Each 0.5 μ L of upstream and downstream primer；Taq
0.5 μ L of polymerase archaeal dna polymerase；38.5 μ L of sterile water.The program of PCR reaction is 94 DEG C of initial denaturation 5min；94 DEG C of denaturation
45s, 50 DEG C of annealing 45s, 72 DEG C of extension 1min (30 circulations)；72 DEG C of extension 10min.With the verifying of 1% agarose gel electrophoresis
And pcr amplification product is recycled with 0.8% agarose gel electrophoresis, it as a result expands to being consistent with SEQ ID No.1 sequence size
Leudh genetic fragment (swimming lane 1,3 in Fig. 1).
With the leudh gene of restriction enzyme NdeI and Bgl II double digestion after purification, (PCR amplification recycled is produced
Object) and carrier pET-28b (+), with T4DNA ligase in 16 DEG C of connections overnight.Coupled reaction system is (20 μ L): target gene
2 μ L, T4 DNA ligase of 12 μ L of segment, 5 μ L, 10 × T4DNA ligase buffer of carrier DNA, 1 μ L.Connection product conversion is big
Enterobacteria BL21 competent cell, method for transformation are as follows: take 5~10 μ L of connection product, be added to the impression of 100 μ L e. coli bl21s
In state cell, after mixing well, ice bath 30min；Eppendorf pipe equipped with mixture is placed in 42 DEG C of water-bath heat shock 90s,
It is transferred to cooled on ice 2min immediately after；600 μ L LB liquid mediums are added in Xiang Guanzhong, are placed in 37 DEG C, 150r/min constant temperature
2~4h is cultivated on shaking table, is then coated on the LB plate containing 50 μ g/mL kanamycins, 37 DEG C of 12~18h of culture.Picking
Positive colony, 37 DEG C, 150r/min shaken cultivation stay overnight, extract plasmid, after sequence verification, the prokaryotic expression that is correctly constructed
Plasmid pET-28b-leudh, physical map are shown in Fig. 2.Positive colony containing plasmid pET-28b-leudh correctly constructs
Recombination bacillus coli BL21/pET-28b-leudh, is named as LEUDH.
Embodiment 3: the building of leucine dehydrogenase single-site mutant body
The recombination bacillus coli BL21/pET-28b-leudh constructed in embodiment 2 is extracted into plasmid, with plasmid pET-
28b-leudh designs the mutant primer of six pairs of rite-directed mutagenesis according to parental array as template.
L41G-F:5′-CCCTGGGTCCGGCGGTTGGTGGTATGCGTATG-3 ' and
L41G-R:5′-CATACGCATACCACCACCAGCCGGACCCAGGG-3′；
A61T-F:5′-GATTGAAGATGCTCTACTCTGGGTCGTGGCATGAC-3 ' and
A61T-R:5′-GTCATGCCACGACCCAGAGTCAGAGCATCTTCAATC-3′；
L77C-F:5 '-GCTGCAGGTCTGAACTGTGGTGGCGGCAAAACC-3 ' and
L77C-R:5 '-GGTTTTGCCGCCACCACAGTTCAGACCTGCAGG-3'；
M347G-F:5 '-GAACGTATCGAAATGGGTCGTAAGACCCGCAGAAAGGTG-3 ' and
M347G-R:5 '-GTGCTGCGGGTCTTACGNNNCAACCCGATACGATACGTTC-3'；
Q358T-F:5 '-CACCTTTCTGCAGGACACTCGTAACCTGATCAAC-3 ' and
Q358T-R:5 '-GTGCTGCGGGTCTTACGAGTCATTTCGATACGTTC-3 ' (underscore is mutating alkali yl) benefit
It is mutated with round pcr.PCR reaction system are as follows: 2 ×GC Buffer 25 μ L, dNTPs are (each
2.5mmol/L) 4 μ L, 1 μ L of forward primer (10 μM), reverse primer (10 μM) 1 μ L, 1 μ L of template DNA,HS
Distilled water is added to 50 μ L in DNA Polymerase (2.5U/ μ L) 0.5 μ L.PCR amplification program are as follows: 98 DEG C of initial denaturation 3min；With
30 circulations (98 DEG C of 10s, 58 DEG C of 15s, 72 DEG C of 7min) are carried out afterwards；72 DEG C of extension 10min；Last 4 DEG C of heat preservations.PCR product
It is verified through 0.8% agarose gel electrophoresis, as a result amplification to the genetic fragment being consistent with purpose carrier size.
PCR product converts e. coli bl21 (DE3) competent cell (detailed process after Dpn I digestion template 3h
See embodiment 2), competent cell after LB solid medium (40 μ g/mL containing kanamycin) overnight incubation, choose monoclonal into
Row bacterium colony PCR verifying, the bacterium for being verified as positive colony are inoculated in culture in LB liquid medium (40 μ g/mL containing kanamycin)
After 8h, plasmid is extracted, serves extra large Sani Biotechnology Co., Ltd sequencing, sequencing result is shown correctly, the single mutant of building
Leucine dehydrogenase is respectively designated as LeuDH-L41G, LeuDH-L77C, LeuDH-A61T, LeuDH-M347G, LeuDH-
Q358T。
Embodiment 4: the building of leucine dehydrogenase double-site mutant body
The recombination bacillus coli BL21/pET-28b-leudh constructed in embodiment 2 is extracted into plasmid, with plasmid pET-
28b-leudh designs mutant primer the M347G-F:5 '-GAACGTATCGAAA of rite-directed mutagenesis according to parental array as template
TGGGTCGTAAGACCCGCAGAAAGGTG-3 ' and M347G-R:5 '-GTGCTGCGGGTCTTACGNNNCAACCCGATACGAT
ACGTTC-3 ', (underscore is mutating alkali yl) carry out first round mutation using fast PCR technology.Again with introduce Q358T, L41G,
L77C catastrophe point is that template carries out the second wheel mutation, primer Q358T-F:5 '-CACCTTTCTGCAGGACACTCGTAACCTG
ATCAAC-3 ' and Q358T-R:5 '-GTGCTGCGGGTCTTACGAGTCATTTCGATACGTTC-3'；L41G-F:5′-CCCTG
GGTCCGGCGGTTGGTGGTATGCGTATG-3 ' and L41G-R:5 '-CATACGCATACCACCACCAGCCGGACCCAGGG-
3′；L77C-F:5 '-GCTGCAGGTCTGAACTGTGGTGGCGGCAAAACC-3 ' and L77C-R:5 '-GGTTTTGCCGCCACCACAGTTCAGACCTGCAGG-3'.PCR reaction system are as follows: 2 ×GC Buffer 25 μ L, dNTPs are (each
2.5mmol/L) 4 μ L, 1 μ L of forward primer (10 μM), reverse primer (10 μM) 1 μ L, 1 μ L of template DNA,HS
Distilled water is added to 50 μ L in DNA Polymerase (2.5U/ μ L) 0.5 μ L.PCR amplification condition are as follows: 98 DEG C of initial denaturation 3min；With
30 circulations (98 DEG C of 10s, 58 DEG C of 15s, 72 DEG C of 7min) are carried out afterwards；72 DEG C of extension 10min；Last 4 DEG C of heat preservations.PCR product
It is verified through 0.8% agarose gel electrophoresis, as a result amplification to the genetic fragment being consistent with purpose carrier size.
PCR product converts e. coli bl21 (DE3) competent cell (detailed process after Dpn I digestion template 3h
See embodiment 2), competent cell after LB solid medium (40 μ g/mL containing kanamycin) overnight incubation, choose monoclonal into
Row bacterium colony PCR verifying, the bacterium for being verified as positive colony are inoculated in culture in LB liquid medium (40 μ g/mL containing kanamycin)
After 8h, plasmid is extracted, serves extra large Sani Biotechnology Co., Ltd sequencing, sequencing result is shown correctly, the double-mutant of building
Leucine dehydrogenase is named as LeuDH-M347G/Q358T, LeuDH-M347G/L41G, LeuDH-M347G/L77C.
Embodiment 5: the building of three site mutant of leucine dehydrogenase
The recombination bacillus coli BL21/pET-28b-leudh constructed in embodiment 2 is extracted into plasmid, with plasmid pET-
28b-leudh designs mutant primer the M347G-F:5 '-GAACGTATCGAAA of rite-directed mutagenesis according to parental array as template
TGGGTCGTAAGACCCGCAGAAAGGTG-3 ' and M347G-R:5 '-GTGCTGCGGGTCTTACGNNNCAACCCGATACGAT
ACGTTC-3 ', (underscore is mutating alkali yl) carry out first round mutation using fast PCR technology.Again to introduce Q358T catastrophe point
The second wheel mutation, primer Q358T-F:5 '-CACCTTTCTGCAGGAC are carried out for templateACTCGTAACCTGATCAAC-3 ' and
Q358T-R:5 '-GTGCTGCGGGTCTTACGAGTCATTTCGATACGTTC-3'.Again to introduce L41G, L77C catastrophe point as mould
Plate carries out third round mutation, and primer is L41G-F:5 '-CCCTGGGTCCGGCGGTTGGTGGTATGCGTATG-3 ' and L41G-R:
5′-CATACGCATACCACCACCAGCCGGACCCAGGG-3′；L77C-F:5 '-GCTGCAGGTCTGAACTGTGGTGGCGGC
AAAACC-3 ' and L77C-R:5 '-GGTTTTGCCGCCACCACAGTTCAGACCTGCAGG-3'.PCR reaction system are as follows: 2 ×GC Buffer 25 μ L, dNTPs (each 2.5mmol/L) 4 μ L, 1 μ L of forward primer (10 μM), reverse primer
(10 μM) 1 μ L, 1 μ L of template DNA,Distilled water is added in HS DNA Polymerase (2.5U/ μ L) 0.5 μ L
To 50 μ L.PCR amplification condition are as follows: 98 DEG C of initial denaturation 3min；30 circulations of subsequent progress (98 DEG C of 10s, 58 DEG C of 15s, 72 DEG C
7min)；72 DEG C of extension 10min；Last 4 DEG C of heat preservations.PCR product is verified through 0.8% agarose gel electrophoresis, as a result amplification to
The genetic fragment that purpose carrier size is consistent.
PCR product converts e. coli bl21 (DE3) competent cell (detailed process after Dpn I digestion template 3h
See embodiment 4), competent cell after LB solid medium (40 μ g/mL containing kanamycin) overnight incubation, choose monoclonal into
Row bacterium colony PCR verifying, the bacterium for being verified as positive colony are inoculated in culture in LB liquid medium (40 μ g/mL containing kanamycin)
After 8h, plasmid is extracted, serves extra large Sani Biotechnology Co., Ltd sequencing, sequencing result is shown correctly, the Trimutant of building
Leucine dehydrogenase is named as LeuDH-M347G/Q358T/L41G, LeuDH-M347G/Q358T/L77C.
Embodiment 6: the expression of wild type recombination bacillus coli
By the wild type leucine dehydrogenase LeuDH strain inoculated constructed in embodiment 2 into 50mL LB liquid medium
(40 μ g/mL containing kanamycin), 37 DEG C, 150r/min shaken cultivation to OD600=0.8~1.0；Culture solution is with 2% (v/v)
Inoculum concentration is inoculated into the fresh 100mL LB liquid medium containing 40 μ g/mL kanamycins, 37 DEG C, 150r/min oscillation
It cultivates to cell concentration OD600=0.6~0.8, final concentration 0.5mM IPTG, 28 DEG C, 150r/min Fiber differentiation 10h is added, receives
Collect somatic cells, obtains wild type leucine dehydrogenase wet thallus LeuDH.A certain amount of thallus is taken, with 0.85% (w/v) physiology salt
After time thallus of water rinse, thallus is dissolved in 100g thallus/L physiological saline ratio, after shaken well, ultrasonication,
12000r/min, 20min centrifugation, supernatant is crude enzyme liquid, and by ni-sepharose purification, collection obtains pure enzyme solution.
After Fiber differentiation 10h, take 20 μ L bacterium solutions, add 20 μ L SDS buffers mix, boiling water bath heat 5min, take 8 μ L into
Row SDS-PAGE electrophoretic analysis obtains the protein band that a molecular weight is about 40kDa (see Fig. 3).
The building of 7 leucine dehydrogenase mutant engineering bacteria of embodiment
The leucine dehydrogenase mutant strain constructed in embodiment 3-5 is seeded to 50mL LB liquid medium respectively
In (40 μ g/mL containing kanamycin), 37 DEG C, 150r/min shaken cultivation to OD600=0.8~1.0；Culture solution is with 2% (v/
V) inoculum concentration is inoculated into the fresh 100mL LB liquid medium containing 40 μ g/mL kanamycins, 37 DEG C, 150r/min vibration
Culture is swung to cell concentration OD600=0.6~0.8, final concentration 0.5mM IPTG, 28 DEG C, 150r/min Fiber differentiation 10h is added,
It is centrifuged 10min in 4 DEG C, 9000r/min, wet thallus is collected, a certain amount of thallus is taken, with 0.85% (w/v) physiological saline rinse one
After the thallus, by 100g thallus/L physiological saline ratio dissolution thallus, after shaken well, ultrasonication, 12000r/min,
20min centrifugation, supernatant is crude enzyme liquid, and by ni-sepharose purification, collection obtains pure enzyme solution, respectively leucine dehydrogenase mutant work
Journey bacterium LeuDH-L41G, LeuDH-L77C, LeuDH-A61T, LeuDH-M347G, LeuDH-Q358T, LeuDH-M347G/
Q358T, LeuDH-M347G/L41G, LeuDH-M347G/L77C, LeuDH-M347G/Q358T/L41G, LeuDH-M347G/
Q358T/L77C。
Embodiment 8: the enzyme activity determination of leucine dehydrogenase and its mutant
The pure enzyme solution of wild type leucine dehydrogenase LeuDH in Example 6, single mutant leucine dehydrogenation in embodiment 7
Enzyme LeuDH-L41G, LeuDH-L77C, LeuDH-A61T, LeuDH-M347G, LeuDH-Q358T, double-mutant in embodiment 7
Leucine dehydrogenase LeuDH-M347G/Q358T, LeuDH-M347G/L41G, LeuDH-M347G/L77C, three is prominent in embodiment 7
The pure enzyme solution of variant leucine dehydrogenase LeuDH-M347G/Q358T/L41G, LeuDH-M347G/Q358T/L77C, it is heavy to measure this
Group bacterium and mutant bacterium are to α-batanone acid enzyme activity.
Enzyme activity determination method are as follows: it reacts in 1.5mL Ep pipe and carries out, 1mL reaction system: 0.01g batanone acid,
0.07gNADH, 10 μ L ammonium hydroxide add the phosphate buffer (pH=7.5) of 0.02M to 900 μ L, the 100 pure enzyme solutions of μ L, and 35 DEG C of temperature,
Revolution 200rpm reacts 1h, and 20 μ L 6M HCl is added to terminate reaction, and 8 000rpm are centrifuged 10min, use after 0.22 μm of membrane filtration
The content of amino-acid analyzer method detection L-ABA.
Enzyme-activity unit definition: under the conditions of 35 DEG C, pH 7.5, enzyme needed for generating 1 μm of ol C4H9NO2 per minute
Protein content, as a leucine dehydrogenase unit of activity, are indicated with U.The specific enzyme activity of leucine dehydrogenase is with every g wet thallus institute
The enzyme activity unit contained indicates (U/g), the results are shown in Table 1.Leucine dehydrogenase enzyme mutant LeuDH-Q358T vigor is wild
2 times of type LeuDH, reach 965.7U/g.
1 leucine dehydrogenase enzyme of table and its mutant enzyme vigor
Embodiment 9: the kinetic constant of leucine dehydrogenase and its mutant measurement
The 6 pure enzyme of wild type leucine dehydrogenase LeuDH of Example, single mutant leucine dehydrogenase in embodiment 7
LeuDH-L41G, LeuDH-L77C, LeuDH-A61T, LeuDH-M347G, LeuDH-Q358T, double-mutant is bright in embodiment 7
Propylhomoserin dehydrogenase LeuDH-M347G/Q358T, LeuDH-M347G/L41G, LeuDH-M347G/L77C, three mutation in embodiment 7
The pure enzyme solution of body leucine dehydrogenase LeuDH-M347G/Q358T/L41G, LeuDH-M347G/Q358T/L77C is used for catalysis substrate
Butanone acidity test kinetic constant.Determining instrument selects microplate reader, and reaction carries out in 96 black holes plates, therefore system is smaller is
100 μ L systems, fixed NADH/ ammonia spirit concentration is 0.5M, is with concentration with the phosphate buffer of pH=7.5,0.02M
0.25, the butanone acid solution of 0.2,0.1,0.05,0.04,0.025,0.02,0.01M.Various concentration is separately added into each hole
Butanone acid solution and fixed concentration 50 μ L of NADH/ ammonia spirit, add in rear microplate reader shake uniformly, be rapidly added 5 μ L
Leucine dehydrogenase or the pure enzyme solution of its mutant, open survey after concussion.Program has been set in advance, has surveyed 10 minutes fluorescent values.
2 leucine dehydrogenase of table and its mutant enzyme kinetic constant
Embodiment 10: leucine dehydrogenase and its mutant catalysis prepare the measurement of C4H9NO2 optimum temperature
Using wild type LeuDH as control, prepared by C4H9NO2 to double-mutant LeuDH-M347G/Q358T catalysis
It is explored.By the 6 pure enzyme of wild type leucine dehydrogenase LeuDH in embodiment 6, double-mutant LeuDH- in embodiment 7
The pure enzyme solution of M347G/Q358T is as conversion enzyme, using batanone acid as substrate, carries out conversion reaction and prepares C4H9NO2.Tool
Gymnastics make it is as follows: under the conditions of 1mL reaction system, 0.01g batanone acid, 0.07gNADH, 10 μ L ammonium hydroxide, add 0.02M phosphoric acid delay
Fliud flushing (pH=7.5) is preheated 5 minutes in 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C of water-baths to 900 μ L, then
The enzyme solution of 100 μ L (8178U/L) wild-type enzymes or mutant enzyme is added to the inside.20 DEG C of differential responses temperature, 25 DEG C, 30 DEG C,
At 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 150rpm reacts 8h, measures the vigor of enzyme.Enzyme activity under optimal reactive temperature is set as
100%.
Embodiment 11: leucine dehydrogenase and its mutant thermal stability determination
Using wild type LeuDH as control, prepared by C4H9NO2 to double-mutant LeuDH-M347G/Q358T catalysis
It is explored.By the pure enzyme of wild type leucine dehydrogenase LeuDH in embodiment 6, double-mutant LeuDH-M347G/ in embodiment 7
The pure enzyme solution of Q358T is as conversion enzyme, using batanone acid as substrate, carries out conversion reaction and prepares C4H9NO2.Concrete operations
As follows: under the conditions of 1mL reaction system, 0.01g batanone acid, 0.07gNADH, 10 μ L ammonium hydroxide add the phosphate buffer of 0.02M
(pH=7.5) is kept the temperature the pure enzyme solution (8178U/L) of protoenzyme and mutant enzyme respectively in 30 DEG C, 40 DEG C, 50 DEG C of water to 900 μ L
In bath, and every taking 100 μ L enzyme solutions to measure its remnant enzyme activity for 24 hours.Leucine dehydrogenase original strain is obtained by calculation to exist
Half-life period under 30 DEG C, 40 DEG C and 50 DEG C preservation conditions is respectively 12.83d, 16.00d and 1.76d；Mutant strain M347G/
Half-life period of the Q358T under 30 DEG C, 40 DEG C and 50 DEG C preservation conditions is respectively 17.02d, 19.69d and 2.01d.Experimental result table
It is bright under the conditions of 40 DEG C, the thermal stability of leucine dehydrogenase is better than 30 DEG C of preservation condition, but in 50 DEG C of preservation condition
It is lower to become unusual unstable again.By this experiment it has also been found that the thermal stability of mutant is improved compared to original strain
32%.
Embodiment 12: influence of the bivalent metal ion to leucine dehydrogenase and its mutant
Using wild type LeuDH as control, prepared by C4H9NO2 to double-mutant LeuDH-M347G/Q358T catalysis
It is explored.By the 6 pure enzyme of wild type leucine dehydrogenase LeuDH of embodiment, 7 double-mutant LeuDH-M347G/ of embodiment
The pure enzyme solution of Q358T is as conversion enzyme, using batanone acid as substrate, carries out conversion reaction and prepares C4H9NO2.Concrete operations
It is as follows: the 1mL reaction system in the phosphate buffer of 0.02M, pH 7.5: 0.01g batanone acid, 0.07gNADH, 10 μ L ammonium hydroxide,
Add buffer to 880 μ L, then plus 100 μ L protoenzymes or mutant enzyme enzyme solution (8178U/L), the concentration that 20 μ L have been prepared is
The Cu of 100mM2+, Co2+, Ni2+, Mn2+, Ca2+, Mg2+, Ba2+, Zn2+, Fe3+, Fe2+, EDTA aqueous solution, in 35 DEG C of shaking baths
In, 8h is reacted, the vigor of enzyme is measured.The obtained enzyme activity of metal ion will be not added with and be set as 100%.Metal ion and surface are living
Property agent the active influence of leucine dehydrogenase enzyme activity is changed less, either before mutation or after being mutated.Cu2+To protoenzyme and dash forward
The enzyme activity for becoming enzyme has a slighter inhibiting effect, and Zn2+It is inhibited to the enzyme activity after mutation.
By above-mentioned experimental result it is found that by leucine dehydrogenase mutant LeuDH-M347G, TLeuDH- of the present invention
The recombination bacillus coli that Q358T, LeuDH-M347G/Q358T genetic transformation Escherichia coli obtain all has stronger production L-2- ammonia
Base butyric acid ability.
Claims (7)
1. a kind of leucine dehydrogenase mutant, it is characterised in that the mutant is by amino acid sequence shown in SEQ ID NO.12
Arrange the 347th methionine sports glycine, the 358th glutamine sports threonine, the 347th methionine sport it is sweet
Propylhomoserin and the 358th glutamine sport threonine, the 347th methionine sports glycine and the 41st leucine mutation
Glycine is sported for glycine, the 347th methionine and the 77th leucine sports cysteine, the 347th methionine
It sports glycine and the 358th glutamine sports threonine and the 41st leucine sports glycine or the 347th
Methionine sports glycine and the 358th glutamine sports threonine and the 77th leucine sports cysteine.
2. leucine dehydrogenase mutant code gene described in a kind of claim 1, it is characterised in that the gene nucleotide sequence
It is classified as one of following: SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ
ID NO.10 and SEQ ID NO.11.
3. a kind of gene constructed recombinant vector of leucine dehydrogenase mutant code as described in claim 2.
4. a kind of recombination engineering of the conversion of the recombinant vector as described in claim 3 preparation.
5. leucine dehydrogenase mutant described in a kind of claim 1 prepares C4H9NO2 in catalysis 2- butanone acid isomerization
In application, it is characterised in that the application are as follows: passed through with the recombination engineering of the gene of mutant code containing leucine dehydrogenase
The pure enzyme that the wet thallus that Fiber differentiation obtains isolates and purifies is enzyme source, using 2- batanone acid as substrate, using NADH and ammonium hydroxide as auxiliary agent,
Reaction system is constituted as reaction medium using the phosphate buffer of pH=7.5,0.02M, it is anti-under the conditions of 10-50 DEG C, 150r/min
It answers, after fully reacting, reaction solution is isolated and purified, obtain C4H9NO2.
6. application as claimed in claim 5, it is characterised in that in the reaction system, substrate 2- batanone acid initial concentration is 50-
200g/L, enzyme source dosage are 500~1000U/L, and the NADH mass dosage is 5~100g/L, the ammonium hydroxide volume final concentration
It is 1~10%.
7. application as claimed in claim 5, it is characterised in that the enzyme source prepares as follows: will contain leucine dehydrogenase and dash forward
The recombination engineering of variant encoding gene is seeded to the LB liquid medium of the 40 μ g/mL kanamycins containing final concentration, 37 DEG C,
150r/min is cultivated to OD600=0.8~1.0, obtain seed liquor；Seed liquor is transferred to the inoculum concentration of volumetric concentration 2% containing eventually
In the LB liquid medium of 40 μ g/mL kanamycins of concentration, OD is cultivated under the conditions of 37 DEG C, 150r/min600=0.4~0.8,
Add final concentration 0.5mM IPTG, 28 DEG C, 150r/min Fiber differentiation 10h, obtain induction broth, by induction broth from
The heart collects wet thallus, after 0.85% physiological saline rinse, dissolves thallus, oscillation in 100g thallus/L physiological saline ratio
After uniformly, ultrasonication, 12000r/min, 20min centrifugation, supernatant is crude enzyme liquid, and by ni-sepharose purification, collection obtains pure enzyme solution
As enzyme source；The LB liquid medium composition: tryptone 10g/L, yeast powder 5g/L, NaCl 10g/L, solvent is water, pH
Value is 7.0.
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| CN109777788B (en) * | 2019-03-19 | 2020-12-01 | 江南大学 | Leucine dehydrogenase mutant and application thereof |
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| CN111849933B (en) * | 2020-08-11 | 2022-01-14 | 厦门大学 | Leucine dehydrogenase mutant and construction method and application thereof |
| CN114456995B (en) * | 2022-01-26 | 2024-03-15 | 浙江工业大学 | Genetically engineered bacterium for high yield of L-2-aminobutyric acid, construction method and application |
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