CN109321538A - A kind of leucine dehydrogenase obtained based on database gene excavating method - Google Patents
A kind of leucine dehydrogenase obtained based on database gene excavating method Download PDFInfo
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Abstract
The invention discloses a kind of leucine dehydrogenase obtained based on database gene excavating method, the amino acid sequence of the leucine dehydrogenase is SEQ ID NO.1, and the nucleotides sequence of the gene of the leucine dehydrogenase is classified as SEQ ID NO.2.The present invention with genetic engineering means using pET-28a as expression vector the heterogenous expression in e. coli bl21 (DE3), and leucine dehydrogenase is obtained by further screening, the enzyme has enzyme activity higher, substrate tolerance is preferable, the higher characteristic of thermal stability, half-life period can effectively be extended in the industrial production, achieve the purpose that reduce production cost.
Description
Technical field
The present invention relates to genetic engineering and microorganisms technical field, more particularly, to a kind of using database gene excavating
Method prepares the process of leucine dehydrogenase.
Background technique
Chiral amino acid is a kind of medicine intermediate with important value, participates in oxidation, reduction, hydrolysis in vivo
And the formation of C-C key, there is different physiological functions, therefore many new drugs (such as protease inhibitors class, AIDS drugs)
Exploitation used artificial synthesized chiral amino acid, in all chiral amino acids, S-Leucine is due to its tertiary fourth
The steric hindrance of base side chain is big, and with height hydrophobicity, therefore with its synthesize chiral drug have in vivo it is good
Biological stability is widely used in antitumor inhibitor such as treating cancer, rheumatic arthritis and anti-HIV protease inhibitors
Chirality synthesis.
Leucine dehydrogenase (Leucine Dehydrogenase, LeuDH, EC1.4.1.9) be it is a kind of with NADH and
Its oxidation state is the oxidoreducing enzyme of cofactors.The enzyme can be catalyzed the oxidation of L-Leu and other branch L-type amino acid, together
When also some ketone acids reversibly can be reduced into corresponding L-type amino acid.The enzyme is at first by bacillus subtilis (Bacillus
Subtilis screening in), be gradually found to be present in later Bacillus, Sporosarcina, corynebacterium and
Thermoactinomyces etc..
Based on, there is no absolute corresponding relationship, sequence identity is high between albumen primary structure and the function of protein expression
Do not represent that catalytic performance is also similar, if crucial catalytic site mutates, then catalytic property will differ widely.So I
Can use the convenience of gene excavating, find that enzyme activity is higher, and substrate tolerance is preferable, the leucine of better heat stability is de-
Hydrogen enzyme, and S-Leucine is used to prepare to it and is studied, reaction condition is optimized and amplifies preparation system, to be industrialized
Production and application are ready.
Summary of the invention
In view of the above-mentioned problems existing in the prior art, the applicant provides one kind to be based on database gene excavating side
The leucine dehydrogenase that method obtains.Leucine dehydrogenase of the present invention have enzyme activity it is higher, substrate tolerance is preferable, thermal stability compared with
High characteristic can effectively extend half-life period in the industrial production, achieve the purpose that reduce production cost.
Technical scheme is as follows:
A kind of leucine dehydrogenase obtained based on database gene excavating method, the amino acid of the leucine dehydrogenase
Sequence is SEQ ID NO.1.
A kind of gene encoding the leucine dehydrogenase, nucleotides sequence are classified as SEQ ID NO.2.
A kind of expression vector containing the leucine dehydrogenase gene.
A kind of recombinant bacterium containing the expression vector, the recombinant bacterium are made by expression vector conversion host cell.
A kind of leucine dehydrogenase obtained based on database gene excavating method, the preparation method comprises the following steps:
(1) recombinant bacterium is constructed, leucine dehydrogenase gene is connected on expression plasmid carrier pET-28a (+), is obtained
Recombinant plasmid pET-28a (+)-LeuDH comprising leucine dehydrogenase gene, then by recombinant plasmid transformed to Escherichia coli
In BL21 (DE3), recombinant bacterium BL21 (DE3)/pET-28a (+)-LeuDH is obtained；
(2) recombinant bacterium is cultivated, recombinant bacterium is inoculated in the LB liquid medium after 50mL sterilizing, 37 DEG C, 180rpm item
It is incubated overnight under part, seed culture fluid is connected to containing 50 μ gmL according to 1% inoculum concentration-1The LB liquid of the 50mL of kanamycins
In body culture medium culture to OD600 be 0.4~0.6 when, 25 DEG C of induction 15h of IPTG of final concentration of 0.3mM are added；
(3) leucine dehydrogenase, the recombinant Bacillus coli cells refrigerated centrifuge after culture are collected, and is cleaned with physiological saline
Twice, with the abundant suspension cell of buffer of measurement enzyme activity, ultrasonic disruption cell, the thallus 12000rpm freezing after broken wall
It is centrifuged 10min, supernatant is collected, obtains leucine dehydrogenase enzyme solution.
10min is centrifuged under the revolving speed that centrifugal condition described in step (3) is 8800rpm；The buffer is 1M's
NH4Cl-NH3·H2O buffer, pH value 8.5；The ultrasound condition is 200W, ultrasonic 1s, interval 2s, total 10min.
The present invention obtains leucine dehydrogenase gene by database gene excavating technology screening, to have compared with homoleucine
The amino acid sequence of dehydrogenase activity is template, and BLAST is carried out in ncbi database and compares analysis, according to the screening mark of setting
Standard obtains target gene, is expanded according to gene order design primer by round pcr and obtains coding gene sequence, electroresis appraisal
Recovery purifying PCR product and it is cloned into pMD18-T vector construction recombinant plasmid afterwards, by recombinant plasmid transformed to E.coliJM109,
The multiple positive transformants of picking are into LB liquid medium, and 37 DEG C, 180rpm cultivates 12h~16h, carry out double enzymes after extracting plasmid
It cuts and is verified with PCR.Correct recombinant plasmid and expression vector will be verified to be attached after same restriction enzyme double digestion, connected
Product is converted into host cell E. coli BL21 (DE3) competent cell, realizes leucine dehydrogenase through Fiber differentiation
High efficient expression.
(1) database gene excavating technology screening leucine dehydrogenase gene
According to reported leucine dehydrogenase heterogenous expression document, the ammonia for having compared with homoleucine dehydrogenase activity is selected
Base acid sequence be template, in ncbi database carry out BLAST compare analysis, according to the screening criteria of setting obtain it is a series of with
The nucleotide sequence of the amino acid sequence of the different leucine dehydrogenase of known amino acid homology and its coding.
(2) PCR amplification leucine dehydrogenase gene
With the nucleotide sequence design primer (P1, P2) through synthesizing, pass through PCR amplification leucine dehydrogenase gene.
Pcr amplification reaction carries out in 20 μ L systems, and 10 μ L2xEs Taq MasterMix, 8 μ L are added in reaction system
DdH2O, the template DNA of 0.4 μ L, each 0.8 μ L of upstream and downstream primer.Reaction condition is to start to follow after 94 DEG C of initial denaturation 5min
Ring: 94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 1min, totally 30 recycle；72 DEG C extend 10min eventually.PCR product into
Recovery purifying and it is cloned into pMD18-T vector construction recombinant plasmid after row electroresis appraisal, extremely by recombinant plasmid transformed
E.coliJM109, the multiple positive transformants of picking are into LB liquid medium, and 37 DEG C, 180rpm cultivates 12h~16h, extract matter
Double digestion verifying is carried out after grain, and verifying is correctly subjected to sequencing.
(3) building of recombinant expression plasmid
The building of recombinant expression plasmid is carried out in this research by taking pET-28a (+) as an example, and expression plasmid and verifying are correctly contained
There is the recombinant clone plasmid of purpose gene while carrying out double digestion with identical two restriction enzymes, carries out nucleic acid after 37 DEG C of digestion 4h
Target gene and purpose plasmid are verified and be tapped and recovered to electrophoresis, and recovery product is crossed at 16 DEG C with T4DNA ligase and connected, connection
Product is converted into host cell E. coli BL21 (DE3) competent cell, is coated on containing kalamycin resistance (50mg
mL-1) LB solid medium in cultivate 10h~12h, picking positive clone molecule is to containing kanamycins (50mgmL-1) LB liquid
It is cultivated in body culture medium, extract plasmid after 12h~16h is cultivated at 37 DEG C, under the conditions of 180rpm and is named as pET-28a (+)-
LeuDH。
(4) building and screening of recombinant strains
This research is converted through 42 DEG C of thermal shock 90s to place by taking Escherichia coli as an example, by recombinant plasmid pET-28a (+)-LeuDH
In chief cell e. coli bl21 (DE3) competent cell, it is coated on containing kalamycin resistance (50mgmL-1) LB solid
10h~12h is cultivated in culture medium, picking positive clone molecule is to containing kanamycins (50mgmL-1) LB liquid medium in train
It supports overnight, next day is inoculated in by 1% inoculum concentration containing kanamycins (50mgmL-1) the fresh LB liquid medium of 50mL in train
The IPTG for supporting addition 0.3mM when about 2h to OD600 is 0.5-0.6 is induced, and inductive condition is 25 DEG C, 180rpm, 15h.Training
Recombinant Bacillus coli cells refrigerated centrifuge (8800rpm, 10min) after supporting, and cleaned twice with physiological saline, with measurement enzyme activity
The abundant suspension cell of the buffer of power (1M NH4Cl-NH3·H2O, pH value 8.5), ultrasonic disruption cell (200W, ultrasonic 1s,
Interval 2s, total 10min), the thallus 12000rpm refrigerated centrifuge 10min after broken wall obtains supernatant crude enzyme liquid.
(5) the SDS-PAGE analysis of leucine dehydrogenase is recombinated
Illustrate preparative separation glue and concentration glue according to protein adhesive kit, takes the supernatant crude enzyme liquid of the 100 above-mentioned acquisitions of μ L
Then 25 μ L 5x loading buffer are added, placing 10min after mixing well and in boiling water bath makes albuminous degeneration.Then into
Simultaneously Marker is added in row loading.Glue 80V voltage is concentrated in upper layer, and when sample is moved to separation gel, (about 30min) is electric with 120V
Pressure terminates when sample strip is moved to closing power supply, electrophoresis at the about 1cm of separation gel bottom.Separation gel is taken out, is used in culture dish
Coomassie brilliant blue R250 is dyed, and is sufficiently decolourized after dyeing with destainer, and general every 4h changes a destainer until having decolourized
At, finally observe albumen expression and record molecular size range.
(6) zymologic property research of leucine dehydrogenase is recombinated
A. the optimum temperature and temperature stability of leucine dehydrogenase are recombinated
Reaction solution is respectively placed under the conditions of 20 DEG C~60 DEG C and keeps the temperature 10min, measures enzyme activity, exploratory response temperature is to bright
The influence of propylhomoserin dehydrogenase activity.And size by probing into half-life period of the enzyme at 4 DEG C, 30 DEG C, 40 DEG C, 50 DEG C judges it
Stability at such a temperature.
B. the optimal pH and pH stability of leucine dehydrogenase are recombinated
The buffer solution of pH 7.0~11.0 is prepared, leucine dehydrogenase vigor is measured, probes into pH to the shadow of enzymatic reaction
It rings, determines optimal reaction pH.Some crude enzyme liquids are separately taken to be placed in the buffer solution that pH is respectively pH 7.0~11.0,4 DEG C of placements
Measurement remains enzyme activity afterwards for 24 hours, determines the stability of the pH of enzyme.
C. the influence of metal ion and EDTA to leucine dehydrogenase enzyme activity
Under the conditions of optimal reactive temperature and pH, added into reaction solution the different metal ion of final concentration of 1mM and
Its enzyme activity is measured again after EDTA, 40 DEG C of heat preservation 15min, not add the enzyme activity of metal ion and EDTA as control.
D. influence of the organic solvent to leucine dehydrogenase enzyme activity
Crude enzyme liquid is uniformly mixed with organic solvent by 1: 1 respectively, its enzyme activity is measured again after 4 DEG C of placement 2h, not add
Enzyme activity added with solvent is control.
E. kinetics constant KmAndMaxMeasurement
Similar with enzyme activity determination method, substrate trimethyl concentrations of pyruvate is separately added into fixed reaction system for 40mM
The NADH (0.025mM~0.4mM) of various concentration measures leucine dehydrogenase vigor.By monitor NADH concentration variation with
And first rate is reacted with corresponding, enzyme is calculated using Lineweaver-Burk graphing method with Michaelis-Menten equation
Reactive kinetics parameters.Equally, in order to measure LeuDH to the K of TMPm, then the concentration for fixing NADH is 0.3mM, is separately added into
The TMP (2 mM~50mM) of various concentration.Same method measure EsiLeuDH to natural substrate L-Leu (0.25mM~
20mM, NAD+Concentration is 1mM) and NAD+The kinetic constant of (0.1mM~20mM, L-Leu concentration are 2mM).
(7) recombination leucine dehydrogenase catalyzes and synthesizes S-Leucine
Reaction system 10mL is separately added into ammonium formate 0.8M, NAD+0.25g·L-1, the TMP of different concentration of substrate
(0.1M, 0.3M, 0.4M, 0.5M), the leucine dehydrogenase crude enzyme liquid and hydrogenlyase of appropriate bacterial cell disruption as catalyst,
Finally 10mL is complemented to the phosphate buffer of 0.1M (pH value 8.5).Reaction condition is 30 DEG C, 180rpm, is being reacted respectively
It is sampled inactivation after 0h and 12h, passes through the conversion ratio of HPLC detection substrate and product.
The present invention is beneficial to be had the technical effect that
The method that the present invention utilizes gene excavating, by constructing the leucine dehydrogenase of separate sources, with genetic engineering
Means using pET-28a as expression vector the heterogenous expression in e. coli bl21 (DE3), and by further screening obtain it is novel
Leucine dehydrogenase LeuDH from Exiguobactericum Sibiricum 255-15, the enzyme are derived from known
The sequence homology of the LeuDH of Bacillus sphaericus is 70.33%, and recombination leucine dehydrogenase shares 374 ammonia
Base acid, molecular size range is about 40.4kDa, and the enzyme activity high thermal stability of the substrate for enzymatic activity trimethyl pyruvic acid is preferable, and 40
DEG C long half time reach 54.6h, also there is 14.7h in 50 DEG C of half-life period, while the pH tolerance range of the enzyme is wider, in pH 8.0
It saves comparatively stable in buffer between~10.0, is able to maintain 90% or more of original enzyme activity afterwards for 24 hours.It is raw in industry
Half-life period is isolated and purified and extended conducive to enzyme in production, reduces production cost.
Recombinant bacterial strain BL21 (DE3)/pET-28a-EsiLeuDH shows substrate trimethyl pyruvic acid preferable affine
Power can be coupled formate dehydrogenase enzymatic trimethyl pyruvic acid and generate S-Leucine, realize that biocatalysis prepares chiral amino
The target of acid.
The present invention is conducive to the knot for further studying leucine dehydrogenase to the work of the leucine dehydrogenase zymologic property
Connection between structure and function.
Detailed description of the invention
Fig. 1 is that electrophoretogram is identified in the recombinant plasmid digestion containing leucine dehydrogenase gene.
Fig. 2 is leucine dehydrogenase enzyme solution SDS-PAGE electrophoresis result figure after inducing expression.
Fig. 3 is the optimum temperature experimental result picture of leucine dehydrogenase.
Fig. 4 is the optimal pH and pH stability experiment result figure of leucine dehydrogenase.
Fig. 5 is the influence experimental result picture of metal ion and EDTA to leucine dehydrogenase vigor.
Fig. 6 is influence experimental result picture of the organic solvent to leucine dehydrogenase vigor.
Specific embodiment
With reference to the accompanying drawings and examples, the present invention is specifically described.
Embodiment 1
This example illustrates the method based on database gene excavating technology screening leucine dehydrogenase.
According to reported leucine dehydrogenase heterogenous expression document, the ammonia for having compared with homoleucine dehydrogenase activity is selected
Base acid sequence be template, in ncbi database carry out BLAST compare analysis, according to the screening criteria of setting obtain it is a series of with
The nucleotide sequence of the amino acid sequence of the different leucine dehydrogenase of known amino acid homology and its coding.Leucine dehydrogenation
The screening of enzyme is as shown in table 1.
Table 1
| Serial number | Enzyme | Source bacterial strain | Identity to BspLeuDH |
| 1 | BspLeuDH | Bacillus sphaericus | 100% |
| 2 | LspLeuDH | Lysinibacillus sphaericus | 95.05% |
| 3 | BsuLeuDH | Bacillus subtilis | 79.95% |
| 4 | BliLeuDH | Bacillus licheniformis | 79.67% |
| 5 | BceLeuDH | Bacillus cereus | 79.12% |
| 6 | BmeLeuDH | Bacillus megaterium | 78.57% |
| 7 | EsiLeuDH | Exiguobacterium sibiricum | 70.33% |
| 8 | SmaLeuDH | Stenotrophomonas maltophilia | 50.96% |
| 9 | HlaLeuDH | Halobiforma lacisalsi | 44.48% |
| 10 | ClaLeuDH | Caldisphaera lagunensis | 22.86% |
The cloning process of 2 leucine dehydrogenase encoding gene of embodiment
(1) PCR amplification leucine dehydrogenase gene
With the nucleotide sequence design primer (P1, P2) through synthesizing, pass through PCR amplification leucine dehydrogenase gene.PCR expands
Increase reaction to carry out in 20 μ L systems, 10 μ L2x Es Taq MasterMix, the ddH2O of 8 μ L, 0.4 μ L are added in reaction system
Template DNA, each 0.8 μ L of upstream and downstream primer.Reaction condition is to start the cycle over after 94 DEG C of initial denaturation 5min: 94 DEG C of denaturation 30s,
58 DEG C of annealing 30s, 72 DEG C of extension 1min, totally 30 recycle；72 DEG C extend 10min eventually.PCR product is returned after carrying out electroresis appraisal
It receives and purifies and be cloned into pMD18-T vector construction recombinant plasmid, by recombinant plasmid transformed to E.coliJM109, the multiple sun of picking
Property transformant into LB liquid medium, 37 DEG C, 180rpm cultivate 12h~16h, extract plasmid after carry out double digestion verifying, will
Verifying correctly carries out sequencing.Nucleic acid electrophoresis result as shown in Figure 1,
In Fig. 1: M:5000bp DNA Marker；Swimming lane 1: recombinant plasmid pET-28a-BspLeuDH；Swimming lane 2:pET-
28a-LspLeuDH；Swimming lane 3:pET-28a-BsuLeuDH；Swimming lane 4:pET-28a-BliLeuDH；Swimming lane 5:pET-28a-
BceLeuDH；Swimming lane 6:pET-28a-BmeLeuDH；Swimming lane 7:pET-28a-EsiLeuDH；Swimming lane 8:pET-28a-
SmaLeuDH；Swimming lane 9:pET-28a-ClaLeuDH；Leucine dehydrogenase gene sequence is effectively expanded as seen from Figure 1
Increase.
The building of 3 recombinant expression plasmid of embodiment
By expression plasmid and verifying, correctly the recombinant clone plasmid containing target gene is limited with identical two simultaneously
Enzyme carries out double digestion, carries out nucleic acid electrophoresis verifying after 37 DEG C of digestion 4h and is tapped and recovered target gene and purpose plasmid, recycling produces
Object is crossed at 16 DEG C with T4DNA ligase and is connected, and connection product is converted to host cell E. coli BL21 (DE3) competent cell
In, it is coated on containing kalamycin resistance (50mgmL-1) LB solid medium in cultivate 10h~12h, picking positive colony
Son extremely (the 50mgmL containing kanamycins-1) LB liquid medium in cultivate, at 37 DEG C, under the conditions of 180rpm cultivate 12h~
Plasmid is extracted after 16h and is named as pET-28a (+)-LeuDH.
The building of 4 recombinant strains of embodiment and screening technique
Recombinant plasmid pET-28a (+)-LeuDH is converted through 42 DEG C of thermal shock 90s to host cell E. coli BL21
(DE3) it in competent cell, is coated on containing kalamycin resistance (50mgmL-1) LB solid medium in cultivate 10h~
12h, picking positive clone molecule is to containing kanamycins (50mgmL-1) LB liquid medium in overnight incubation, next day connects by 1%
Kind amount is inoculated in containing kanamycins (50mgmL-1) the fresh LB liquid medium of 50mL in cultivate about 2h to OD600 and be
The IPTG that 0.3mM is added when 0.5-0.6 is induced, and inductive condition is 25 DEG C, 180rpm, 15h.Recombination large intestine after culture
Bacilli-cell refrigerated centrifuge (8800rpm, 10min), and cleaned twice with physiological saline, the buffer with measurement enzyme activity is abundant
Suspension cell (1M NH4Cl-NH3·H2O, pH value 8.5), and ultrasonic disruption cell (200W, ultrasound 1 s, interval 2s, altogether
10min), the thallus 12000rpm refrigerated centrifuge 10min after broken wall, clear enzyme solution in acquisition.
The SDS-PAGE analytic process of 5 leucine dehydrogenase of embodiment.
Illustrate preparative separation glue and concentration glue according to protein adhesive kit, takes the supernatant crude enzyme liquid of the 100 above-mentioned acquisitions of μ L
Then 25 μ L 5x loading buffer are added, placing 10min after mixing well and in boiling water bath makes albuminous degeneration.Then into
Simultaneously Marker is added in row loading.Glue 80V voltage is concentrated in upper layer, when sample is moved to separation gel (about 30min), with 120V electricity
Pressure terminates when sample strip is moved to closing power supply, electrophoresis at the about 1cm of separation gel bottom.Separation gel is taken out, in culture dish
It is dyed with coomassie brilliant blue R250, is sufficiently decolourized after dyeing with destainer, general every 4h changes a destainer until decoloration
It completes, finally observe the expression of albumen and records molecular weight of albumen size.Fig. 2 is leucine dehydrogenase crude enzyme liquid SDS-
PAGE electrophoresis result figure, in figure: (A) M:Protein Marker swimming lane 1: BL21 (DE3)/pET-28a- of inducing expression
BspLeuDH supernatant, swimming lane 2: the BL21 (DE3) of inducing expression/pET-28a-BspLeuDH precipitating；Swimming lane 3 and 4:
LspLeuDH；Swimming lane 5 and 6:BsuLeuDH；Swimming lane 7 and 8:BliLeuDH；Swimming lane 9 and 10:BceLeuDH；Swimming lane 11 and 12:
BmeLeuDH；Swimming lane 13: BL21 (DE3)/pET-28a supernatant of inducing expression；Swimming lane 14: the BL21 (DE3) of inducing expression/
PET-28a precipitating.(B) M:Protein Marker swimming lane 1: on BL21 (DE3)/pET-28a-EsiLeuDH of inducing expression
Clearly, swimming lane 2: the BL21 (DE3) of inducing expression/pET-28a-EsiLeuDH precipitating；Swimming lane 3 and 4:SmaLeuDH；5 He of swimming lane
6:ClaLeuDH；Swimming lane 7: BL21 (DE3)/pET-28a supernatant of inducing expression；Swimming lane 8: the BL21 (DE3) of inducing expression/
PET-28a precipitating.As seen from Figure 2, part leucine dehydrogenase obtains high efficient expression and molecular weight of albumen size is about
40kDa。
Test case:
The enzyme activity size of 1 leucine dehydrogenase of test case
Overall reaction system 1.5mL is separately added into substrate trimethyl pyruvic acid 5mM, 0.2mM NADH, 1M NH4Cl-NH3·
H2O (pH value 8.5) buffer adds appropriate crude enzyme liquid after (30 DEG C) heat preservation 2min of water-bath and starts to react and measure and inhales at 340nm
The variation of luminosity.
The definition of enzyme activity unit (U): under the above conditions, enzyme amount or every needed for 1 μM of NADH oxidation of catalysis per minute
1 μM of NAD of minute catalysis+Enzyme amount needed for reduction is defined as an enzyme activity unit.The catalysis of leucine dehydrogenase of the present invention is living
Power is as shown in table 2.
Table 2
| Enzyme | Vigor (U/ml) | Rate activity (U/mg) | It is soluble |
| EsiLeuDH | 99.45 | 28.0 | Soluble-expression |
| BspLeuDH | 82.88 | 27.35 | Soluble-expression |
| LspLeuDH | 61.50 | 15.69 | Soluble-expression |
| BceLeuDH | 99.13 | 23.89 | Soluble-expression |
| BsuLeuDH | 11.54 | 14.43 | Most of inclusion body expression |
| BmeLeuDH | 9.04 | 11.50 | Most of inclusion body expression |
| BliLeuDH | 6.50 | 7.87 | Most of inclusion body expression |
| ClaLeuDH | 0.38 | 0.36 | Most of inclusion body expression |
| SmaLeuDH | 0.14 | 0.15 | Most of inclusion body expression |
2 thermal stability of test case
EsiLeuDH, BspLeuDH, LspLeuDH and BceLeuDH higher for enzyme activity, go out from the angle of thermal stability
Hair carries out secondary screening again, selects the half-life period that 4 enzymes are measured under the conditions of 50 DEG C, and half-life period is 19.1h respectively,
The half-life period longest of 14.4h, 11h, 10h, EsiLeuDH measure the thermostabilization of leucine dehydrogenase EsiLeuDH under different temperatures
Property it is as shown in table 3, the enzyme activity of EsiLeuDH is higher it can be seen from table 2 and table 3 and thermal stability is relatively preferable, thus choose
The research of EsiLeuDH progress zymologic property and industrial application.
Table 3
| Temperature(℃) | EsiLeuDH kD(h-1) | EsiLeuDH t1/2(h) |
| 4 | 0.00197 | 351.8 |
| 30 | 0.00255 | 271.8 |
| 40 | 0.01269 | 54.6 |
| 50 | 0.04717 | 14.7 |
3 catalysis characteristics of test case
(1) reaction solution is respectively placed under the conditions of 20 DEG C~60 DEG C and keeps the temperature 10min, measure enzyme activity, the optimal reaction of enzyme
Temperature is 40 DEG C, and when temperature is higher than 60 DEG C, enzyme activity declines rapidly (Fig. 3).
(2) buffer solution for preparing pH7.0~11.0, measures leucine dehydrogenase vigor, when reaction system pH is 7.0
In~8.0 ranges, enzyme activity is lower；When reaction system pH is 8.0~10.0 or so, enzyme activity is higher, illustrates that leucine is de-
Enzyme activity is higher under alkaline condition for hydrogen enzyme.With the increase of pH, leucine dehydrogenase vigor is all first to increase to decline afterwards, most suitable
Reacting pH is 9.0.And the pH stability range of leucine dehydrogenase is larger, saves in the buffer between pH8.0~10.0
It is comparatively stable, it is able to maintain 90% or more (Fig. 4) of original enzyme activity afterwards for 24 hours.
(3) NaCl, CaCl are chosen2、MgSO4·7H2O、MnSO4·H2O、CuSO4·5H2O、 ZnSO4·7H2O、
FeSO4·7H2O、Fe2(SO4)3, EDTA etc. be configured to the mother liquor of 500mM, be separately added into the mother liquor prepared in the reaction system, eventually
Concentration is 1mM.4 DEG C of heat preservations measure remnant enzyme activity for 24 hours.Fig. 5 as the result is shown has obviously the enzyme activity of EsiLeuDH without discovery
The metal ion of facilitation, Ca2+, Mg2+Minimum is influenced on the enzyme activity of EsiLeuDH, is still had in 30 DEG C of water-baths heat preservation 2h
90.0% or more vigor, in addition Fe3+It is affected to the enzyme activity of EsiLeuDH, enzyme activity loss is serious, relative residual vigor
50% hereinafter, in addition enzyme is containing Mn2+Buffer in it is unstable, 4 DEG C heat preservation for 24 hours afterwards residual enzyme activity only have 29% a left side
It is right.
(4) ethyl alcohol, methanol, DMSO and the SDS etc. for choosing various concentration respectively probe into influence of the organic solvent to enzyme activity,
Fig. 6 illustrates that SDS is minimum to the toxicity of the enzyme, more excellent as cosolvent, followed by ethyl alcohol.
Kinetic parameter size of 4 leucine dehydrogenase of test case to substrate and coenzyme
According to Michaelis-Menten equation, EsiLeuDH can be derived to NADH, TMP, NAD+And the apparent kinetics ginseng of L-Leu
Number KmAnd Vmax。
Michaelis-Menten equation:
Double reciprocal expressions are as follows:
According to formula 2 with Lineweaver-Burk double-reciprocal plot method, obtain straight line, this straight line horizontal axis intercept be-
1/Km, vertical axis intercept l/Vmax, therefore obtain KmAnd VmaxValue such as table 4.
Table 4
| Substrate | Km(mM) | Vmax(μmol·min-1·mL-1) |
| NADH | 0.624 | 684.93 |
| TMP | 5.96 | 404.86 |
| NAD | 1.46 | 74.91 |
| L-Leucine | 0.88 | 38.42 |
The data from table 4 it is found that EsiLeuDH to the K of L-LeumVery little, L-Leu are its natural substrates；And its
To the K of TMPmAlso smaller, illustrate that TMP is one proper substrate of the enzyme.In addition, EsiLeuDH is to NAD+And the K of NADHm
Be worth also very small, it is good enough to its affinity illustrate, in actually catalysis reaction only need to a small amount of coenzyme can go on smoothly reaction.
5 leucine dehydrogenase catalysis substrate trimethyl Pyruvate production S-Leucine of test case
It is found through the content of HPLC detection substrate and product, the substrate transformation rate when concentration of substrate is 0.1M, 0.3M, 0.4M
99% or so can be reached, and it is 42% or so that conversion ratio is lower when concentration of substrate is 0.5M, and produced under low concentration of substrate in
The yield of object is 85% or so, and efficiency of pcr product is up to 90% when wherein BspLeuDH catalysis substrate concentration is 0.4M, equally
It is 42% or so that yield is smaller under high concentration of substrate.Thus knowing leucine dehydrogenase, there are substrate suppressions under high concentration of substrate
Phenomenon processed.
SEQUENCE LISTING
<110>Southern Yangtze University
<120>a kind of leucine dehydrogenase obtained based on database gene excavating method
<130> 1
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 374
<212> PRT
<213>leucine dehydrogenase (Leucine Dehydrogenase)
<400> 1
Met Val Glu Thr Asn Val Glu Ala Arg Phe Ser Ile Phe Glu Thr Met
1 5 10 15
Ala Met Glu Asp Tyr Glu Gln Val Val Phe Cys His Asp Lys Val Ser
20 25 30
Gly Leu Lys Ala Ile Ile Ala Ile His Asp Thr Thr Leu Gly Pro Ala
35 40 45
Leu Gly Gly Leu Arg Met Trp Asn Tyr Ala Ser Asp Glu Glu Ala Leu
50 55 60
Ile Asp Ala Leu Arg Leu Ala Lys Gly Met Thr Tyr Lys Asn Ala Ala
65 70 75 80
Ala Gly Leu Asn Leu Gly Gly Gly Lys Ala Val Ile Ile Gly Asp Ala
85 90 95
Lys Thr Gln Lys Ser Glu Ala Leu Phe Arg Ala Phe Gly Arg Tyr Val
100 105 110
Gln Ser Leu Asn Gly Arg Tyr Ile Thr Ala Glu Asp Val Asn Thr Thr
115 120 125
Val Ala Asp Met Asp Tyr Ile His Met Glu Thr Asp Phe Val Thr Gly
130 135 140
Val Ser Pro Ala Phe Gly Ser Ser Gly Asn Pro Ser Pro Val Thr Ala
145 150 155 160
Tyr Gly Val Tyr Arg Gly Met Lys Ala Ala Ala Lys Glu Val Tyr Gly
165 170 175
Thr Asp Ser Leu Gly Gly Lys Thr Val Ala Ile Gln Gly Val Gly Asn
180 185 190
Val Ala Phe Asn Leu Cys Arg His Leu His Glu Glu Gly Ala Lys Leu
195 200 205
Ile Val Thr Asp Ile Asn Gln Asp Ala Leu Arg Arg Ala Glu Glu Ala
210 215 220
Phe Gly Ala Leu Val Val Gly Pro Asp Glu Ile Tyr Ser Val Asp Ala
225 230 235 240
Asp Ile Phe Ala Pro Cys Ala Leu Gly Ala Thr Leu Asn Asp Glu Thr
245 250 255
Ile Pro Gln Leu Lys Val Lys Ile Ile Ala Gly Ala Ala Asn Asn Gln
260 265 270
Leu Lys Glu Asp Arg His Gly Asp Met Leu Gln Glu Arg Gly Ile Leu
275 280 285
Tyr Thr Pro Asp Phe Val Ile Asn Ala Gly Gly Val Ile Asn Val Ala
290 295 300
Asp Glu Leu Asp Gly Tyr Asn Arg Glu Arg Ala Met Lys Lys Val Glu
305 310 315 320
Leu Val Tyr Asp Ala Val Ala Lys Val Ile Glu Ile Ala Lys Arg Asp
325 330 335
His Leu Pro Thr Tyr Arg Ala Ala Glu Lys Met Ala Glu Glu Arg Ile
340 345 350
Ala Thr Met Gly Ser Ala Arg Ser Gln Phe Leu Arg Arg Asp Lys Asn
355 360 365
Ile Leu Gly Ser Arg Gly
370
<210> 2
<211> 1125
<212> DNA
<213>leucine dehydrogenase (Leucine Dehydrogenase)
<400> 2
atggttgaaa caaacgtaga agcacgattc agtattttcg aaacgatggc aatggaagat 60
tacgaacaag tcgtattttg tcacgataaa gtctcaggat taaaggcgat tatcgcgatt 120
catgatacga cactcggacc agcactcggc ggactccgta tgtggaacta tgcgtctgac 180
gaggaagcat tgatcgacgc gcttcgtttg gcaaaaggca tgacgtataa aaatgcggca 240
gccggtctga accttggcgg cgggaaagcg gtcatcatcg gtgatgcgaa aacgcaaaaa 300
tcagaagctc tgttccgtgc attcggtcgt tacgtacagt cgttaaacgg acgttacatc 360
actgcggaag acgtcaacac aacagtcgcc gacatggatt atatccacat ggaaacagat 420
ttcgtaaccg gtgtcagccc ggcattcgga tcaagcggca atccgtcacc agtcacggct 480
tatggcgttt accgcggaat gaaggcagcc gctaaagaag tatatggcac agattcactc 540
ggaggaaaaa cagttgcgat tcaaggtgtt ggtaacgttg ctttcaacct atgccgtcac 600
ttgcatgaag aaggcgcaaa attgattgtc acagacatca atcaagatgc attacgccgt 660
gcagaagaag cgtttggcgc tctcgtcgtc ggaccggatg aaatttacag cgtcgatgcc 720
gatatctttg cgccgtgtgc cttaggtgcg acattgaacg atgagacgat tccacaactg 780
aaagtgaaaa tcattgccgg agcagcaaac aaccaactca aagaagatcg tcacggagat 840
atgctccagg aacgcggtat tttatataca ccggacttcg tcatcaacgc aggaggtgtc 900
atcaatgtgg ccgacgaact cgacgggtac aaccgtgagc gggcgatgaa aaaagtcgaa 960
ctcgtctatg atgcggtagc aaaagtcatc gaaattgcca aacgtgacca tctgccgact 1020
taccgggcag cagagaagat ggcagaagaa cggatcgcga caatgggcag tgcccgcagc 1080
cagttcttac gccgggataa aaacatttta ggatcacgcg gttaa 1125
Claims (6)
1. a kind of leucine dehydrogenase obtained based on database gene excavating method, which is characterized in that the leucine dehydrogenation
The amino acid sequence of enzyme is SEQ ID NO.1.
2. a kind of gene of leucine dehydrogenase described in coding claim 1, which is characterized in that the leucine dehydrogenase gene
Nucleotides sequence be classified as SEQ ID NO.2.
3. a kind of expression vector containing leucine dehydrogenase gene described in claim 2.
4. a kind of recombinant bacterium containing expression vector described in claim 3, which is characterized in that the recombinant bacterium is turned by expression vector
Change host cell to be made.
5. according to claim 1 leucine dehydrogenase, which is characterized in that the leucine dehydrogenase the preparation method comprises the following steps:
(1) recombinant bacterium is constructed, leucine dehydrogenase gene is connected on expression plasmid carrier pET-28a (+), is obtained comprising bright
Recombinant plasmid pET-28a (+)-LeuDH of propylhomoserin dehydrogenase gene, then by recombinant plasmid transformed to e. coli bl21
(DE3) in, recombinant bacterium BL21 (DE3)/pET-28a (+)-LeuDH is obtained；
(2) recombinant bacterium is cultivated, recombinant bacterium is inoculated in the LB liquid medium after 50mL sterilizing, 37 DEG C, under the conditions of 180rpm
It is incubated overnight, seed culture fluid is connected to containing 50 μ gmL according to 1% inoculum concentration-1The LB liquid of the 50mL of kanamycins is trained
Support base in culture to OD600 be 0.4~0.6 when, 25 DEG C of induction 15h of IPTG of final concentration of 0.3mM are added；
(3) leucine dehydrogenase, the recombinant Bacillus coli cells refrigerated centrifuge after culture are collected, and cleans two with physiological saline
It is secondary, with the abundant suspension cell of buffer of measurement enzyme activity, ultrasonic disruption cell, the thallus 12000rpm after broken wall freeze from
Heart 10min collects supernatant, obtains leucine dehydrogenase enzyme solution.
6. leucine dehydrogenase according to claim 5, which is characterized in that centrifugal condition described in step (3) is
10min is centrifuged under the revolving speed of 8800rpm；The buffer is the NH of 1M4Cl-NH3·H2O buffer, pH value 8.5；It is described
Ultrasound condition is 200W, ultrasonic 1s, interval 2s, total 10min.
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