CN110577961B - Construction method of heat-stable malic acid dehydrogenase gene, encoded protein and application thereof - Google Patents
Construction method of heat-stable malic acid dehydrogenase gene, encoded protein and application thereof Download PDFInfo
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Abstract
The invention discloses a construction method of a heat-stable malate dehydrogenase gene, a coding protein and application thereof, wherein the malate dehydrogenase gene takes a primary expression plasmid gene sequence as a template, adopts a designed mutation primer to carry out rapid point mutation PCR amplification mutation plasmid, screens the resistance of a transforming bacterium, constructs a gene sequence capable of expressing malate dehydrogenase, and transfers the constructed mutated malate dehydrogenase gene into an escherichia coli DE3 strain for expression.
Description
Technical Field
The invention relates to the technical field of biological gene engineering and protein engineering, in particular to a construction method of a heat-stable malic acid dehydrogenase gene, a coded protein and application thereof.
Background
With the development of gene engineering and protein engineering technology, the enzyme with improved physical and chemical properties can be obtained by means of computer aided design, site-directed mutation, structure extension mutation, heterozygosis enzyme, directed molecular evolution and other methods. The enzyme structure extension mutation is a new method for evolving protein engineering research, and the protein structure is expanded by adding a peptide segment at the C terminal of the enzyme protein, so that the higher structure of the enzyme protein is changed, and the aim of improving the physicochemical property of the enzyme is fulfilled.
In a natural state, malic acid (2-hydroxysuccinic acid) is a mixture of 3 existing forms due to the existence of chiral carbon atoms in a molecular structure. Wherein, D-malic acid is an extremely important 4-carbon organic chiral source, and is widely used in the fields of chiral drugs, chiral additives, chiral auxiliary agents and the like, but in the production process of the D-malic acid, because the malic acid has other two forms: the mixing of L-malic acid and D, L-malic acid results in low purification rate of D-malic acid, and in order to improve the purification rate of D-malic acid, Malate Dehydrogenase (MDH) (EC 1.1.1.37) is widely used for resolving D, L-malic acid to obtain D-malic acid due to its wide range of existence and high substrate specificity, and can realize reversible conversion between malic acid and oxaloacetic acid.
The applicant finds that the existing wild-type malate dehydrogenases for separating D, L-malic acid have poor heat resistance, long screening time in a natural state and complicated working procedures, and are not beneficial to the separation of the malate dehydrogenase from the D, L-malic acid and the application of other methods for synthesizing the purified D-malic acid
Disclosure of Invention
In view of this, the present invention aims to provide a method for constructing a thermostable malate dehydrogenase gene, a coding protein and applications thereof, so as to simplify the screening steps and improve the thermostability and activity of malate dehydrogenase.
The invention provides a construction method of a thermostable malate dehydrogenase gene based on the above objects, which is constructed by adopting a method comprising the steps of plasmid preparation, mutant primer design, PCR amplification of mutant plasmids, digestion of template plasmids and transformation screening, wherein the constructed gene has a base sequence of SEQ ID NO. 1.
Optionally, the plasmid preparation is to use a malate dehydrogenase gene sequence of microcystis aeruginosa as a template A, perform double enzyme digestion on the template A and a vector pET-28b (+) at the same time, and then perform T-mediated digestion on the template A and the vector pET-28b (+)4And (3) connecting the DNA with ligase to obtain a primary expression plasmid pET-28 b-MDH.
Optionally, the design of the mutation primer is based on the sequence of the malic acid dehydrogenase gene of the microcystis aeruginosa and the characteristics of pET-28b (+), and the mutation primer is designed by software.
Optionally, the mutation primer comprises an upstream primer and a downstream primer, wherein the upstream primer has a base sequence of SEQ ID NO.2, and the downstream primer has a base sequence of SEQ ID NO. 3.
Optionally, the PCR amplification mutant plasmid uses the gene sequence of the primary expression plasmid as a template, and the mutant primer is used for performing PCR amplification reaction to obtain a mutant recombinant plasmid.
Optionally, the PCR amplification reaction conditions are: pre-denaturation at 94 ℃ for 7min, at 94 ℃ for 30s, at 56 ℃ for 30s, at 72 ℃ for 3min, and for 35 cycles; extension at 72 ℃ for 5 min.
Optionally, the digestion template plasmid is obtained by carrying out enzyme digestion on 10-30U/. mu.L Dpn I at 30-40 ℃ for 50-70 min.
Optionally, the transformation screening is to transform the mutant plasmid into a competent cell, and extract the plasmid after kanamycin resistance screening to obtain the mutant plasmid of the thermostable malate dehydrogenase.
Optionally, the microcystis aeruginosa is microcystis aeruginosa PCC 7806.
Optionally, the competent cell is Escherichia coli DH5 α.
The application of a heat-stable malate dehydrogenase gene, wherein the malate dehydrogenase expressed by the gene is applied to the industrial production of D-malate.
From the above, the invention provides a construction method of a thermostable malate dehydrogenase gene, a coding protein and applications thereof, the malate dehydrogenase gene provided by the invention takes a primary expression plasmid gene sequence as a template, adopts a designed mutation primer to carry out rapid point mutation PCR amplification mutation plasmid, transforms bacteria resistance screening, constructs a gene sequence capable of expressing malate dehydrogenase, and transfers the constructed mutated malate dehydrogenase gene into an Escherichia coli Rosetta (DE3) strain for expression.
Drawings
FIG. 1 is a diagram showing the result of PCR amplification of a mutant recombinant plasmid according to an embodiment of the present invention;
m: 5000bp Marker, 1: amplifying the fragments;
FIG. 2 shows the mutated plasmid pET-28b-MDH according to an embodiment of the present inventionmThe double enzyme digestion identification result chart of (1);
m: 2000bp Marker, 1: carrying out double enzyme digestion on mutant plasmids;
FIG. 3 is a SDS-PAGE analysis of mutant MaMDH proteins according to the example of the invention;
m: protein molecular weight standards; 2: crude cell extract of MT induced by 0.5mM IPTG; 1: purified MT protein;
FIG. 4 is a graph showing the thermal stability analysis of MaMDH according to example of the present invention;
FIG. 5 is the process of constructing mutation of mutant MaMDH recombinant plasmid.
Detailed Description
In the following description of the embodiments, the detailed description of the present invention, such as the manufacturing processes and the operation and use methods, will be further described in detail to help those skilled in the art to more fully, accurately and deeply understand the inventive concept and technical solutions of the present invention.
Note that, WT in the examples: a wild type; MT represents a mutant; pET-28b-MDHmTo representA mutant plasmid.
In order to solve the problems that wild malate dehydrogenases for splitting D and L-malic acid in the prior art have poor heat resistance, long screening time in a natural state, complicated working procedures and are not beneficial to the realization of the splitting of the D and L-malic acid by the malate dehydrogenase and the application of purifying the synthesized D-malic acid by other methods, the construction method of the heat-stable malate dehydrogenase gene provided by the invention is constructed by adopting the methods comprising the steps of plasmid preparation, mutant primer design, PCR amplification of mutant plasmids, digestion of template plasmids and transformation screening, wherein the constructed gene has a base sequence of SEQ ID No. 1.
The amino acid composition is an important factor of enzyme heat resistance, proline and alanine residues can better stabilize a protein skeleton structure, and ion-ion interaction formed by some charged amino acids between dimers is helpful for improving the heat stability and the catalytic activity of the enzyme. The gene sequence of the primary expression plasmid is used as a template, a designed mutation primer is adopted to carry out rapid point mutation PCR amplification mutation plasmid, the resistance of transforming bacteria is screened, a gene sequence capable of expressing malate dehydrogenase is constructed, and the constructed mutant malate dehydrogenase gene is transferred into an Escherichia coli E.coli Rosetta (DE3) strain to be expressed.
The specific construction method of the heat-stable malic acid dehydrogenase gene provided by the embodiment of the invention comprises the following steps:
selection of mutation sites: through computer software aided prediction, the mutant plasmid can be read through adding one base, so that the sequence in the Escherichia coli expression vector PET-28b (+) is translated into 13 amino acid residues Ala-Arg-Ala-Pro-Pro-Pro-Pro-Leu-Arg-Ser-Gly-Cys, Pro and Ala can better stabilize the protein skeleton structure, and the thermal stability of the enzyme protein is improved. In addition, the included polar amino acid not only can enhance the solubility of the enzyme protein but also can increase the surface charge interaction of the enzyme protein if the polar amino acid is on the surface of the molecule, and salt bonds are formed to improve the thermal stability of the enzyme protein. Therefore, the present invention example adds a G base at position 969 to make extension mutation so as to lengthen the peptide chain and obtain the 13 amino acid residues on Escherichia coli. The amino acid residue after 323 of MaMDH in MT is replaced, and 14 amino acids are added to the C terminal, wherein the 13 amino acid residues on Escherichia coli are included. As shown in table 1.
TABLE 1 mutation site selection for MaMDH
Plasmid preparation: according to the GenBank, the gene sequence of Microcystis aeruginosa PCC7806 MDH is taken as a template A, the template A and a carrier pET-28b (+) are subjected to double enzyme digestion at the same time and then pass through T4And (3) connecting the DNA with ligase to obtain a primary expression plasmid pET-28 b-MDH.
Design of mutation primer: according to the gene sequence and pET-28b (+) characteristics of Microcystis aeruginosa PCC7806 MDH in GenBank, a Primer 5.0 software is used for designing an MDH mutation Primer, and the designed mutation Primer comprises:
(1) an upstream primer: 5'-TGACAGTCTCAAGGGTTTTGATGTA-3', respectively;
(2) a downstream primer: 5'-CCCTTGAGACTGTCAAGAGCTAAAT-3' are provided.
PCR amplification of mutant plasmids: taking the gene sequence of the primary expression plasmid pET-28b-MDH as a template to carry out PCR amplification under the action of designed upstream and downstream primers, wherein the PCR amplification conditions are as follows: pre-denaturation at 94 ℃ for 7 min; 30s at 94 ℃, 30s at 56 ℃, 3min at 72 ℃ and 35 cycles; and (3) extending for 5min at 72 ℃ to obtain mutant recombinant plasmids containing mutant genes.
And performing gel detection on the PCR amplification result, namely the mutant recombinant plasmid, wherein the result is shown in figure 1, a lane M in figure 1 is a 5000bp Marker, a lane 1 is the amplified mutant recombinant plasmid, the PCR amplification uses fast high-fidelity FastAlternation DNA polymerase, and the reaction system refers to an enzyme specification. A bright band above 5000bp is shown in FIG. 1, indicating that the recombinant plasmid containing mutant MaMDH was successfully cloned.
Digesting a template plasmid:carrying out enzyme digestion on the product amplified by PCR by using Dpn I, and configuring an enzyme digestion system: mu.L of PCR product and 1. mu.L of digestive enzyme (20U/. mu.L) are mixed well, digested for 1h at 37 ℃, methylated template plasmid is digested with Dpn I, and amplified mutant plasmid pET-28b-MDH is leftm。
The resulting mutant plasmid pET-28b-MDHmNdeI and XhoI double enzyme digestion identification is carried out, the enzyme digestion result is shown in figure 2, a lane M in figure 2 is 2000bp Marker, a lane 1 is mutant plasmid double enzyme digestion, and compared with Mark, a bright band in figure 2 is consistent with an expected result, and a mutant gene fragment is expected to be obtained. The mutant plasmid is verified to be accurate in mutation site through sequencing, and a G base is added to the site 969. The sequencing result is shown in SEQ ID NO. 1.
Transformation and screening: and transforming the mutant plasmid into competent cell escherichia coli E.coli DH5 alpha, screening kanamycin resistance, and extracting the plasmid to obtain the mutant plasmid of the thermostable malate dehydrogenase.
Protein expression: inducing mutant plasmids expressing thermostable malate dehydrogenase in E.coli Rosetta (DE3), inoculating successfully transformed E.coli Rosetta bacterial liquid into 5mL LB liquid culture medium containing kanamycin (30 μ g/mL) and chloramphenicol (30 μ g/mL), culturing overnight at 225rpm and 37 ℃, sucking 1mL of the culture bacterial liquid, adding into 200mL LB culture medium, culturing at 225rpm and 37 ℃ until the bacterial liquid concentration OD600When the pH value reaches 0.5, adding 0.5-0.7 mM IPTG for induction, inducing at the rotating speed of 180rpm and the low temperature of 20 ℃ for 18-20 h, centrifuging at the rotating speed of 5500rpm and the temperature of 4 ℃ for 5min, separating to obtain thalli, and adding a phosphate buffer solution with the pH value of 8.0 into the thalli;
cell disruption solution prepared by Clontech
x vector Buffer disrupted cells;
centrifuging the crushed solution at 10000rpm and 4 ℃ for 20min, and transferring the supernatant into resin;
using a metal ion affinity column (Clontech corporation) ((
purification Kit) to collect the mutant fusion protein;
the purified protein was electrophoretically detected by SDS-PAGE, and the results are shown in FIG. 3.
Lane M in figure 3 is a protein molecular weight standard; lane 2 is crude cell extract after induction of MT with 0.5mM IPTG; lane 1 is MT purified protein. As can be seen by lane comparison, the molecular weight standard band of the protein in the figure is around 40kDa, which proves that the induced expression is successful and the heterologously expressed protein is completely purified.
Specifically, the flow chart of the method for constructing the thermostable malate dehydrogenase gene provided by the embodiment of the present invention is shown in fig. 5.
In order to verify the thermostability and enzyme activity of the gene-expressed malate dehydrogenase constructed as described above, the expressed malate dehydrogenase was subjected to the following performance test in the examples of the present invention. Enzyme activity test principle: malate dehydrogenase catalyzes NAD (P) H and NAD (P) while catalyzing reversible conversion between oxaloacetate and malate+To be transformed in between. Since NAD (P) H has respective maximum absorption peaks at 340nm, the activity of the enzyme can be determined by monitoring the change of the light absorption value of the reaction at 340 nm. Reaction system: Tris-HCL (pH 8.0), oxaloacetate, NADH, L-malic acid, NAD+. Adding 1 μ L of purified enzyme solution into 999 μ L of the reaction solution according to the above reaction system (1mL), and mixing by inversion; measured at 340nm using a Cary 300Bio UV-Visible spectrophotometer. Each experiment is independently repeated for 3-4 times; 1 enzyme activity unit (U) is defined as: the amount of enzyme required to consume 1. mu.M NAD (P) H per minute.
(1) Enzyme thermostability assay
Fixing the concentration of NADH in the reaction system to be 0.16mM and the concentration of OAA to be 0.25mM, placing the enzyme in water baths at different temperatures (20-78 ℃) to heat for 20min, taking out the enzyme, carrying out ice bath for 5min, and detecting the change of the slope under the absorbance of 340 nm. The degree of influence of the reaction was determined by the ratio to the reaction having the highest enzymatic reaction rate as a standard reaction, and the results are shown in FIG. 4. The mutant MT of the experiment maintains the activity at 70 ℃ to be more than 80%, the WT activity is reduced to be less than 70%, the MT still has 10% activity when reaching 78 ℃, and the MT is improved in thermal stability particularly at 60 ℃ compared with the WT.
(2) Enzyme kinetic assay
Kinetic parameters may measure the catalytic efficiency and natural properties of the enzyme.
KmThe value allows to judge the specificity of the enzyme and the natural substrate: some enzymes act on several substrates, KmThe value varies from substrate to substrate, which helps to determine the specificity of the enzyme, 1/KmCan approximately represent the magnitude of the affinity of the enzyme for the substrate, 1/KmLarger indicates greater affinity; kmThe smallest substrate is referred to as the optimal substrate for the enzyme, i.e.the natural substrate.
kcatDenotes the number of molecules of substrate per molecule of enzyme (or per active site) per second that are converted when the enzyme is saturated with substrate, this constant, also called the conversion number (abbreviated TN), and is commonly referred to as the catalytic constant, expressed in s-1，kcatLarger means higher catalytic efficiency of the enzyme.
And k iscat/KmThe ratio can be compared with the catalytic efficiency of different enzymes or the same enzyme catalyzing different substrates. The larger the ratio, the higher the efficiency. The catalytic properties of an enzyme can be reflected by measuring kinetic parameters of the enzyme.
The NADH concentration was varied at 25 ℃ in 100mM Tris-HCl (pH 8.0) and 0.3mM OAA, and A was detected340The concentration of each NADH is parallelly and independently detected for 3-4 times, an average value is taken, and the catalytic efficiency of the mutant type and wild type recombinant MaMDH is analyzed; meanwhile, K of NADH is calculated by using a Mie equation nonlinear fitting method in Graph pad 5.0 softwaremAnd kcatThe value is obtained. The specific data of the test are shown in Table 2.
TABLE 2 enzyme kinetic parameters for wild type and mutant MaMDH
The data show the affinity of the MT mutants constructed in the examples of the invention for NADH after mutagenesis ((K)mNADH) 1.6 fold enhancement over WT and preference for NADH (k)cat/Km) The activity of the catalyst is increased to 2.6 times of the original activity, and the catalytic activity of the catalyst is increased to 1.6 times of that of the WT.
Those of ordinary skill in the art will understand that: the discussion of any embodiment above is meant to be exemplary only, and is not intended to intimate that the scope of the disclosure, including the claims, is limited to these examples; within the idea of the invention, also features in the above embodiments or in different embodiments may be combined, steps may be implemented in any order, and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity.
The embodiments of the invention are intended to embrace all such alternatives, modifications and variances that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, substitutions, improvements and the like that may be made without departing from the spirit and principles of the invention are intended to be included within the scope of the invention.
SEQUENCE LISTING
<110> university of teacher's university in Anhui
<120> construction method of heat-stable malic acid dehydrogenase gene, encoded protein and application thereof
<130> 2019-9-12
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 1020
<212> DNA
<213> Microcystis aeruginosa
<400> 1
atggttgact tctacgatac actcatcccc tgtcaatcgc cccgagtctc ggttattggt 60
gccggcaacg tcggacgcac cctggcccaa cgcatcgccg aaaaaaacct cgctgatgtg 120
gttctcctcg atatcgtcca tggtttgccc cagggtattg ccctagattt gatggaagct 180
cagggcatag aactgcacga cagcgagatt attggcacta ataactacga agacacggca 240
ggctcggata ttgtcgtgat tacggctgga ttagccagaa aacctggcat gagtcgcgat 300
gatctcatga atgtcaatgc taaaattgtc gtcgaggccg ccactaaatg cctcaagtat 360
tctccggaag ccatttttat tgtcatcact aatcccctgg atgtgatgac ctatctagtc 420
tggcaagcaa ccggtttacc cccccaaaga gtgatgggaa tggcgggagt cctcgattct 480
tcccgtttac aaacctttat cgccatggaa ttgggggtta gtaccgccga cgttcatgct 540
atggttttgg gtggtcacgg tgatttaatg ttgcccctac cccgttactg taccgtcagt 600
ggtgtgccga ttaccgaatt aatggacgag ataacaatta accgtttagt ggagagaact 660
cgcaacggtg gggctgaaat cgtcaaatta ctgcaaactg gcggagctta ttatgcccct 720
gcttcctctg cctgtaccat ggtcgagacg atattgagaa atcaatcccg tttactgccg 780
gcggcagcct atcttaaggg tgaatacggt ttacaggatg tctatctggg ggttccctgt 840
cgcctaggat gtcggggtgt ggaaagtatt ctggaagtgc gtttgaccga tgctgaacgt 900
ctggatctac acacttccgc tgcctcagtt cgtcaaaatg ttcatttagc tcttgacagt 960
ctcaagggtt ttgatgtagc tcgagcacca ccaccaccac cactgagatc cggctgctaa 1020
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<213> upstream primer (DNA)
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tgacagtctc aagggttttg atgta 25
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cccttgagac tgtcaagagc taaat 25
Claims (9)
1. A method for constructing a thermostable malic dehydrogenase gene is characterized by comprising the steps of plasmid preparation, mutant primer design, PCR amplification of mutant plasmids, digestion of template plasmids and transformation screening, wherein the base sequence of the constructed thermostable malic dehydrogenase gene is SEQ ID NO. 1.
2. The method for constructing the thermostable malate dehydrogenase gene according to claim 1, wherein the plasmid preparation is prepared by using the malate dehydrogenase gene sequence of Microcystis aeruginosa as the template A, performing double enzyme digestion on the template A and the vector pET-28b (+) at the same time, and passing through T4And (3) connecting the DNA with ligase to obtain a primary expression plasmid pET-28 b-MDH.
3. The method for constructing the heat-stable malate dehydrogenase gene according to claim 1, wherein the design of the mutant primer is based on the malate dehydrogenase gene sequence of Microcystis aeruginosa and the characteristics of pET-28b (+), and the mutant primer is designed by software.
4. The method for constructing the thermostable malate dehydrogenase gene according to claim 3, wherein the mutation primer comprises an upstream primer and a downstream primer, the base sequence of the upstream primer is SEQ ID No.2, and the base sequence of the downstream primer is SEQ ID No. 3.
5. The method for constructing the thermostable malate dehydrogenase gene according to claim 1, wherein the PCR amplification mutant plasmid is obtained by performing PCR amplification reaction by using a mutant primer and a primary expression plasmid gene sequence as a template.
6. The method for constructing the thermostable malate dehydrogenase gene according to claim 5, wherein the PCR amplification reaction conditions are as follows: pre-denaturation at 94 ℃ for 7min, at 94 ℃ for 30s, at 56 ℃ for 30s, at 72 ℃ for 3min, and for 35 cycles; extension at 72 ℃ for 5 min.
7. According toThe method for constructing a thermostable malate dehydrogenase gene according to claim 1, wherein said digestion template plasmid is prepared by using 10-30U/. mu.L DpnAnd I, carrying out enzyme digestion at 30-40 ℃ for 50-70 min to obtain mutant plasmids.
8. The method for constructing a thermostable malate dehydrogenase gene according to claim 1, wherein said transformation selection is to transform a mutant plasmid into a competent cell, and extract the plasmid after kanamycin resistance selection to obtain the mutant plasmid of the thermostable malate dehydrogenase.
9. The method for constructing the heat-stable malate dehydrogenase gene according to claim 2 or 3, wherein the microcystis aeruginosa is microcystis aeruginosa PCC 7806.
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