CN108034644B - L-lactate dehydrogenase mutant with improved catalytic efficiency and construction method thereof - Google Patents
L-lactate dehydrogenase mutant with improved catalytic efficiency and construction method thereof Download PDFInfo
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Abstract
The invention discloses an L-lactate dehydrogenase mutant with improved catalytic efficiency and a construction method thereof, belonging to the technical field of genetic engineering. According to the invention, on the basis of L-lactate dehydrogenase, the molecular structure of the L-lactate dehydrogenase is modified by site-directed mutagenesis biotechnology to obtain an L-lactate dehydrogenase escherichia coli engineering bacterium, the specific enzyme activity of the mutant enzyme is improved by 6.1 times, and the catalytic activity is improved by 4.2 times. The invention solves the problem of limitation of low catalytic activity of the L-lactate dehydrogenase and provides a new idea for molecular modification of the L-lactate dehydrogenase.
Description
Technical Field
The invention relates to an L-lactate dehydrogenase mutant with improved catalytic efficiency and a construction method thereof, belonging to the technical field of genetic engineering.
Background
For example, L-phenyllactic acid (L-phenyllactic acid) is a natural bacteriostatic agent, can also be generated by the catalysis of L-LDH (phenylpyruvic acid), can replace bacteriostatic agents in food additives and animal feeds, can also be used as a precursor for synthesizing various medicines, and can also be used as a potential polylactic acid substitute for synthesizing a novel polymer material of the polylactic acid.
While most of the wild-type L/D-LDHs now found exhibit lower activity to substrates with long aliphatic or aromatic side chains (benzoylformic acid, PPA, oxaloacetic acid, etc.), the widespread use of L/D-LDHs is limited to some extent, with the insights on of the structure, function and mechanism of action of L/D-LDHs, studies on engineering enzyme molecules to obtain L/D-LDHs with excellent enzymatic properties have rapidly progressed, Jiang et al employ a mutagenesis approach to replace Pseudomonas stzeri SDM L-LDH with Val108 Ala, to obtain a new mutase whose catalytic efficiency (kKcat/Km) for L-mandelic acid is increased by 50.1 fold compared to wild-type enzyme Bsresung et al, and the mutation of Geilstressful L-LDH is significantly increased from Bsriphilg et al site-Bsriphery site, Bsriphery strain Zhang et al, Bsributyr-LDH, Bsriphery strain L-LDH, Geilyard L-LDH, and Geiltyr-LDH-A mutation is significantly increased by 50.101, a mutation, a.
The invention adopts single-point mutation technology, based on a homologous modeling method, optimizes the molecular structure of the L-lactate dehydrogenase by mutating specific amino acid and improves the catalytic activity of the L-lactate dehydrogenase.
Disclosure of Invention
The invention aims to provide an L-lactate dehydrogenase mutant with improved catalytic activity and a construction method thereof.
The invention provides an L-lactate dehydrogenase mutant with improved catalytic activity, which is characterized in that the amino acid sequence of the mutant is a sequence shown in SEQ ID NO. 1.
The nucleotide sequence of the mutant is a sequence shown in SEQ ID NO. 3.
The mutant is obtained by mutating isoleucine at position 229 to alanine on the basis of the amino acid shown in sequence SEQ ID NO.2 (I229A).
The nucleotide sequence for coding the amino acid sequence shown in SEQ ID NO.2 is the sequence shown in SEQ ID NO. 4.
The invention also provides a genetic engineering bacterium for expressing the L-lactate dehydrogenase mutant.
The preparation method of the genetic engineering bacteria comprises the steps of taking recombinant plasmids carrying coding L-lactate dehydrogenase genes as templates, designing and synthesizing primers, carrying out site-directed mutagenesis through PCR to obtain recombinant plasmids carrying coding mutant genes, and transforming the recombinant plasmids into escherichia coli BL21 to obtain the recombinant escherichia coli genetic engineering bacteria.
The expression vector is any one of the following: pET-22b (+), pET-28a (+), pET-32a (+), and pET-41a (+).
The expression vector, in one embodiment of the invention, is pET-22b (+).
The Escherichia coli host bacteria is any one of E.coli BL21, E.coli JM109, E.coli DH5 α, or E.coli TOP 10.
Coli BL21 in one embodiment of the invention.
The preparation method specifically comprises the following steps:
(1) carrying out mutation PCR by taking a recombinant plasmid pET-22b (+) -Lvldh carrying an Lvldh gene with a sequence shown as SEQ ID NO.4 as a template, an F1primer with a sequence shown as SEQ ID NO.5 and an R1primer with a sequence shown as SEQ ID NO.6 as primers to obtain the recombinant plasmid pET-22b (+) -Lvldh encoding amino acid with the 229 th amino acid mutated from isoleucine to alanineI229A；
(2) And (3) transforming the recombinant plasmid obtained in the last step into E.coli BL21(DE3) to obtain recombinant Escherichia coli genetic engineering bacteria LcLdhBL 21.
The invention has the beneficial effects that: according to the invention, on the basis of L-lactate dehydrogenase, the molecular structure of the L-lactate dehydrogenase is modified by site-directed mutagenesis biotechnology to obtain an L-lactate dehydrogenase escherichia coli engineering bacterium, the specific enzyme activity of the mutant enzyme is improved by 6.1 times, and the catalytic activity is improved by 4.2 times. The invention solves the problem of limitation of low catalytic activity of the L-lactate dehydrogenase and provides a new idea for molecular modification of the L-lactate dehydrogenase.
Description of the drawings:
FIG. 1 shows a recombinant lactate dehydrogenase LcLDHI229ASDS-PAGE patterns of (5).
Detailed Description
In order to clearly understand the technical contents of the present invention, the following examples are given in detail for the purpose of better understanding the contents of the present invention and are not intended to limit the scope of the present invention.
The method for measuring the enzyme activity of the L-lactate dehydrogenase comprises the following steps:
the total reaction system included 50mmol/L sodium acetate buffer (pH 5.5), 0.2mmol/L ADH, 5mmol/L LPPA, and the control contained no NADH and was otherwise identical. Mixing, keeping the temperature at 35 deg.C for 5min, and adding appropriate amount of enzyme solution. The change in absorbance of NADH at 340nm was detected. The enzyme activity unit (U) is defined as: the amount of enzyme required to catalytically oxidize 1. mu. mol NADH per minute under the above conditions. The specific activity is defined as the number of units of enzyme activity per mg of enzyme protein (U/mg).
Example 1: construction of mutant enzyme gene and expression plasmid thereof
And connecting the amplified product with pUCldh, converting E.coli JM109, and performing blue-white spot screening, bacterial solution PCR verification and DNA sequencing on the gene Lldh with the nucleotide sequence shown as SEQ ID No. 4. The recombinant plasmid with the correct sequencing was designated as pUCm-T-Lldh. pUCM-T-Lldh was double-digested with Nde I and Xho I, Lldh was recovered, and ligated with pET-28a (+) by the same double-digestion to obtain a recombinant plasmid pET-28a (+) -Lldh.
Using pET-28a (+) -Lvldh recombinant plasmid as a template, F1primer (shown in SEQ ID NO. 5) and R1primer (shown in SEQ ID NO. 6) as primers, and performing site-specific mutagenesis by PCR to obtain a gene carrying a coding mutantThe recombinant plasmid is named as pET-22b (+) -LvldhI229A。
Example 2: construction of engineering bacterium for producing L-lactate dehydrogenase escherichia coli
The recombinant plasmid pET-22b (+) -Lvldh obtained in example 1 was usedI229AColi BL21 competent cells were transformed by the following method:
1) inoculating LB plate activated E.coli BL21 in 2mL LB medium, culturing at 37 deg.C and 220r/min overnight; inoculating 2% of the culture solution into 5mL of LB culture medium, and culturing at 37 ℃ at 220r/min for 4 h;
2) putting 1.4mL of the bacterial liquid into a 1.5mLEP tube, carrying out ice bath for 10min, centrifuging at 4000r/min for 2min, and collecting thalli;
3) 1mL of precooled 0.1M CaCl was added2Resuspending the cells in the solution, carrying out ice bath for 10min at 4000r/min, centrifuging for 2min, and collecting thalli;
4) add 100. mu.L of pre-cooled 0.1M CaCl2Suspending the cells in the solution, and storing at 4 deg.C for 30min for transformation;
5) completely thawing 100 μ L of competent cells on ice, gently suspending the cells, adding 5 μ L of the connecting solution, gently mixing, and standing on ice for 30 min;
6) performing water bath heat shock at 42 ℃ for 90s, and performing ice bath for 15-20 min;
7) adding 400 μ L LB culture medium, shaking and culturing at 37 deg.C and 220r/min for 1 h;
8) centrifuging at 4000r/min for 5min at room temperature, removing 450 μ L of supernatant, blowing the residual bacterial liquid, mixing uniformly, and coating on Kan plate;
8) and selecting the white bacterial colony in the flat plate, carrying out bacterial liquid PCR verification, sending the recombinant bacterium verified to be correct to a sequencing company for sequencing, and naming the recombinant bacterium with correct sequencing as LcdhBL 21.
Example 3: recombinant Escherichia coli induced expression
Activating the recombinant bacterium LcLdhBL21 constructed in example 2 on an LB plate, selecting a single colony to 2mL of LB culture medium, culturing at 37 ℃ and 220r/min for 12h, inoculating the single colony to 100mL/500mL of LB culture medium according to the inoculum size of 2%, culturing at 37 ℃ and 220r/min for 2h, adding IPTG (isopropyl-beta-thiogalactoside) to the final concentration of 0.4mM, performing induced expression at 20 ℃ for 8h, centrifuging at 8000r/min for 5min, collecting thalli, performing ultrasonic disruption to obtain a crude enzyme solution, and purifying the crude enzyme solution by using a nickel column, wherein the specific method is as follows:
1) washing the column with ultrapure water for more than 3 times;
2) washing the column with newly prepared binding solution for 3 times;
3) loading: filtering the fermentation liquid with water film, and repeatedly passing through column if the amount is large; the sample loading amount is 8mL, and is about 4mL generally; blocking the lower leak hole with a cover, combining at 4 deg.C for 30min or overnight, collecting effluent, and using for SDS-PAGE control;
4) washing the column with binding solution, collecting the permeate (containing impurity proteins or unadsorbed proteins), and comparing with SDS-PAGE;
5) eluting with the eluent, and collecting an eluted sample;
6) and (4) column washing and storage: 5mL of the binding solution, 10mL of ultrapure water, 10mL of 20% ethanol, 5mL of 20% ethanol, and storing at 4 ℃.
Analysis of purified recombinant L-lactate dehydrogenase (LcLDH)I229A) Enzymological Properties, as shown in Table 1, LcLDHI229AThe catalytic efficiency is improved by 4.2 times, and the specific enzyme activity is improved by 6.1 times. The mutant LcLDH is increased due to the improvement of substrate affinity and catalytic efficiencyI229AThe specific enzyme activity of (3). This indicates that the invention improves the enzymatic properties of L-lactate dehydrogenase by an innovative way of mutation.
TABLE 1 LcLDHI229AKinetic parameters of mutant reaction
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
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Claims (7)
1. An L-lactate dehydrogenase mutant with improved catalytic activity, which is characterized in that the amino acid sequence of the mutant is a sequence shown as SEQ ID NO. 1.
2. The mutant according to claim 1, wherein isoleucine at position 229 is mutated to alanine on the basis of the amino acid sequence shown in SEQ ID No. 2.
3. The mutant according to claim 1, wherein the nucleotide sequence of the mutant is the sequence shown in SEQ ID No. 3.
4. A recombinant expression vector comprising the mutant of claim 1.
5. A genetically engineered bacterium expressing the L-lactate dehydrogenase mutant according to claim 1.
6. A preparation method of the genetically engineered bacterium of claim 5, which is specifically characterized in that: (1) carrying out mutation PCR by using a recombinant plasmid pET-22b (+) -Lvldh carrying a lactate dehydrogenase gene Lvldh with a sequence shown as SEQ ID NO.4 as a template, an F1primer with a sequence shown as SEQ ID NO.5 and an R1primer with a sequence shown as SEQ ID NO.6 as primers to obtain the recombinant plasmid pET-22b (+) -Lvldh encoding alanine with the 229 th amino acid mutated from isoleucineI229A(ii) a (2) Transforming the recombinant plasmid obtained in the last step into E.coli BL21(DE3), obtaining the recombinant Escherichia coli genetic engineering bacteria LcLdhBL 21.
7. Use of the mutant of claim 1 in biopharmaceuticals, materials or food products.
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| MULTISPECIES:L-lactate dehydrogenase[Lactobacillus];WP_003567646;《GenBank DataBase》;20170319;1 * |
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