CN103031322A - Glutamate decarboxylase (GAD) thermally-stable variant G311P gene and application thereof - Google Patents
Glutamate decarboxylase (GAD) thermally-stable variant G311P gene and application thereof Download PDFInfo
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Abstract
The invention relates to a glutamate decarboxylase (GAD) thermally-stable variant G311P gene. Site-specific mutagenesis occurs at the 311th site of the nucleotide sequence of the GAD thermally-stable variant G311P gene. According to researches, the thermal stability of GAD coded through the variant gene obtained after mutagenesis at the site is enhanced. Through the site-specific mutagenesis on the basis of wild GAD gene coding, the thermal stability of the corresponding variant enzyme at 60-70DEG C is improved. With the improvement of the thermal stability of the enzyme, higher operating temperature can be adopted during industrial production to improve reaction speed and increase substrate solubility, and the production of GABA (gamma-aminobutyric acid) through biological transformation by using the enzyme is facilitated.
Description
Technical field
The present invention relates to technical field of molecular biology, relate in particular to a kind of L-Glutamic decarboxylase variant G311P gene and uses thereof.
Background technology
Rite-directed mutagenesis (site-directed mutagenesis or site-specific mutagenesis) refers to introduce the technology that specific base pair changes in the appointment site of target DNA fragment.It is the powerful of the complex relationship between Study on Protein structure and the function, also is the conventional means of modifying gene in the laboratory.The particular bases of certain known is fixed a point to change, lacks or inserted, can change corresponding aminoacid sequence and the structure and function feature of protein, help to understand the relation of protein structure and function.Particularly in the design and rational of enzyme, or after utilizing the orthogenesis technology screenings such as random mutation to obtain the potential important amino acid of tool site, carry out the rite-directed mutagenesis analysis, will be conducive to further understand the effect that bring into play in this site in the relation of protein structure and function.
(glutamate decarboxylase is called for short GAD to L-Glutamic decarboxylase; EC4.1.1.15) be a kind of amino acid decarboxylase that is present in widely in plant, animal and the microorganism, it can single-minded ground catalysis Pidolidone α-carboxyl decarboxylic reaction generate γ-aminobutyric acid (γ-aminobutyric acid is called for short GABA).GABA is the neurotransmitter inhibitor of a kind of key of mammalian nervous system, has the central nervous system of inhibition and is overexcited, removes the physiological functions such as nervous.Because the body and mind anxiety can cause human blood-pressure rising, immune dysfunction, metabolism disorder, therefore GABA has tranquilizing and allaying excitement, promotes sleep, brain tonic and intelligence development, reduces blood pressure human body, improve immunologic function, the effect such as delay senility, be a kind of natural amino acid that has widespread use in the food and medicine field, be approved as by health ministry " new resource food ".At present, the cell that utilizes L-Glutamic decarboxylase or have this enzyme activity carries out the biology preparation of GABA and is just more and more paid attention to.
As the key enzyme of biotechnology enrichment production GABA, the thermostability of bacterium GAD is to limit one of its factor of using in extensive bio-reactor always.The optimum temperuture of bacterium GAD is generally between 30～50 ℃, yet it is very fast to descend at enzyme activity more than 60 ℃.And in Industrial processes, often wish to adopt higher service temperature, with raising speed of reaction, increase substrate solubleness, reduction system viscosity, thereby improve the efficient of biocatalysis and conversion.
Chinese patent application 200510049189.4 and Chinese patent application 200510049187.5 disclose a kind of short lactobacillus (Lactobacillus brevis) CGMCC NO.1306 that produces γ-aminobutyric acid, GAD gene to this bacterial strain is cloned, construction recombination plasmid, be transformed in the e. coli bl21 (DE3), can realize soluble-expression.But the GAD of this genetic expression is very fast inactivation in 60 ℃ and above environment, is unfavorable for the application of this enzyme in the biological preparation of GABA.
Summary of the invention
Technical problem solved by the invention at first provides a kind of variant gene of L-Glutamic decarboxylase, the thermostability of variant enzyme under 60 ℃ and 70 ℃ that its coding is corresponding is significantly improved, thereby in Industrial processes, can adopt higher service temperature, to improve speed of reaction, to increase substrate solubleness, be conducive to utilize this enzyme to produce GABA by bio-transformation.
Further aim of the present invention provides the L-Glutamic decarboxylase of above-mentioned variant gene coding or comprises expression unit, recombinant plasmid or the transformant of above-mentioned variant gene.
In order to achieve the above object, the technical solution used in the present invention is:
The thermally-stabilised variant G311P of a kind of L-Glutamic decarboxylase gene, its nucleotides sequence are listed in the 311st rite-directed mutagenesis.Research finds that the thermostability of the L-Glutamic decarboxylase of the variant gene coding that obtains behind this site mutation strengthens to some extent.The basis of the present invention's research is from the GAD gene among short lactobacillus (Lactobacillus brevis) the CGMCC NO.1306.Wherein short lactobacillus (Lactobacillus brevis) CGMCC NO.1306 is open in Chinese patent application 200510049189.4 and Chinese patent application 200510049187.5, is deposited in Chinese common micro-organisms preservation administrative center (address: the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City) by Zhejiang University.Used in this application is Zhejiang University's present.
Find after deliberation, the nucleotide sequence of the GAD gene among short lactobacillus (Lactobacillus brevis) the CGMCC NO.1306 is shown in SEQ ID NO.3, the application sports the codon CCC of proline(Pro) (Pro) with the codon GGT of the 311st glycine of this gene (Gly), and sequence length does not change.The nucleotide sequence of the thermally-stabilised variant G311P of the L-Glutamic decarboxylase that obtains gene is shown in SEQ ID NO.1.
A kind of L-Glutamic decarboxylase by the thermally-stabilised variant G311P of above-mentioned L-Glutamic decarboxylase genes encoding.Its aminoacid sequence is preferably shown in SEQ ID NO.2.
A kind of expression of GAD unit, recombinant plasmid or transformant that comprises the thermally-stabilised variant G311P of above-mentioned L-Glutamic decarboxylase gene.The promotor of expressing the unit can be T7 promotor, lac promotor or araBAD promotor commonly used.Under the effect of these promotors, mutant enzyme can directly be realized soluble-expression in the born of the same parents in e. coli host cell (such as BL21 (DE3)).
The present invention has improved the thermostability of its corresponding variant enzyme under 60 ℃ and 70 ℃ by carrying out rite-directed mutagenesis at the wild GAD enzyme gene basis of coding.Specificity of the present invention is selected the codon in coding GAD the 311st amino acids residue site is replaced, and the codon that adopts the coding proline(Pro) is replaced the codon of coding L-glutamic acid, the 311st amino acids residue that makes GAD sports proline(Pro) by the L-glutamic acid of wild-type, be because adopt molecular design method that upper each amino-acid residue of GAD is analyzed the impact of GAD thermostability, find that the 311st amino acids residue site is the amino acid that a thermostability to GAD has material impact.Based on the relation between amino acid character and protein structure and character, the research discovery adopts proline(Pro) to replace glycine in this site can increase the rigidity of GAD protein structure, improve GAD to the stability of heat.Therefore, design primer, and made up the heat stable mutants G311P of L-Glutamic decarboxylase by the rite-directed mutagenesis method.Along with the raising of L-Glutamic decarboxylase thermostability, in Industrial processes, can adopt higher service temperature, to improve speed of reaction, to increase substrate solubleness, be conducive to utilize this enzyme to produce GABA by bio-transformation.
Description of drawings
Fig. 1 is the gene mapping of plasmid pET28a (+)-gad;
Fig. 2 is the site plan (with ball represent) of mutational site Gly311 in the GAD tertiary structure;
Fig. 3 is the rite-directed mutagenesis principle schematic;
Fig. 4 is wild-type GAD enzyme gene PCR amplified production gel electrophoresis spectrum;
Fig. 5 is mutant enzyme and wild-type GAD 60 ℃ thermostability;
Fig. 6 is mutant enzyme and wild-type GAD 70 ℃ thermostability.
Embodiment
For further specifying the present invention, specify with the following Examples:
One, construction recombination plasmid
Short lactobacillus (Lactobacillus brevis) CGMCC NO.1306 is cultured to logarithmic growth mid-term, gets 4.5mL bacterium liquid 10000rpm supernatant discarded after centrifugal a minute, utilize test kit (worker is given birth in Shanghai) by specification to extract genomic dna.
Design following degenerated primer:
The upstream degenerated primer: 5 '-cg
GgatccAtggcWatgttRtaYggWaaac-3 ' (shown in SEQ ID NO.5);
The downstream degenerated primer: 5 '-gg
GaattcTtagtgHgtgaaYccgtattt-3 ' (shown in SEQ ID NO.6);
Wherein, " ggatcc " in the upstream primer is BamH I restriction enzyme site, and " gaattc " in the downstream primer is EcoR I restriction enzyme site.
Respectively with upstream primer and downstream primer pairing, the different template of choice and optimization/Pfu/ primer ratio, annealing temperature, cycle index, carry out PCR take the gained genomic dna as template, the PCR result suitable with the clip size that is expanded, that abundance is suitable and non-specific band is few finally by the optimization of condition, has obtained the DNA band that a length is about 1400bp.
Determined PCR composing system is: ddH
2O, 38.5 μ L; 10 * Pfu buffer, 5 μ L; Upstream primer: 0.5 μ L; Downstream primer, 0.5 μ L; DNTP, 4 μ L; Template DNA, 1 μ L; Pfu, 0.5 μ L; The reaction cumulative volume is 50 μ L.
Used PCR condition is: enter amplification cycles behind 94 ℃ of sex change 5min, i.e. 94 ℃ of sex change 1.5min, 57 ℃ of annealing 1min, 72 ℃ are extended 3.5min, circulate altogether 32 times, at last again 72 ℃ extend 8min.Amplified production is checked through gel electrophoresis, the result as shown in Figure 4, the product band is single, clear, bright, the size about 1400bp.With the clone products order-checking, find the conserved sequence of glutamic acid decarboxylase gene.
With BamH I and EcoR I goal gene and the pET28a (+) that amplification obtains carried out double digestion, the enzyme tangent condition is as follows: DNA 20 μ L, 10 * K buffer, 4 μ L, BamH I 4 μ L, EcoR I 4 μ L, ddH
2O 8 μ L, enzyme Qie Wendu is 35 ℃.
Plasmid enzyme restriction 1h, PCR product enzyme is cut 4h.Respectively enzyme being cut product carries out Purified in electrophoresis and cuts the glue recovery, product is reclaimed in the electrophoresis checking, then connects, and condition of contact is: 10 * buffer, 2 μ L, gad 10 μ L, plasmid 1 μ L, PEG30006 μ L, T4 ligase enzyme 1 μ L, connecting temperature is 16 ℃, connects 16 hours.
Ligation finishes rear 60 ℃ of insulation 5min deactivation ligase enzymes, gets respectively to connect product Transformed E .coli DH5 α competent cell (Calcium Chloride Method) again.Converted product is coated and is contained the LB film solid media that final concentration is 30 μ g/mL kantlex, cultivate after 18 hours for 37 ℃ and choose positive colony, incubated overnight positive colony in the LB liquid nutrient medium that contains 30 μ g/mL kantlex respectively, carry out BamH I and EcoR I double digestion, PCR checking behind the extraction plasmid, transformant contains recombinant plasmid pET28a (+)-gad (as shown in Figure 1).Through order-checking, the GAD gene is shown in SEQ ID NO.3 among short lactobacillus (Lactobacillus brevis) the CGMCC NO.1306, and the GAD aminoacid sequence of coding is shown in SEQ ID NO.4.
Two, the expression of wild-type GAD and purifying
Get the plasmid pET28a (+) of the expression L-Glutamic decarboxylase of structure-gad Transformed E .coli BL21 (DE3) competent cell, obtain expressing the recombinant bacterial strain of short lactobacillus L-Glutamic decarboxylase.The picking thalline is containing on the LB solid medium flat board that final concentration is 30 μ g/mL kantlex in 37 ℃ of activation 10 hours, then picking list colony inoculation 20mL contains in the LB liquid nutrient medium of 30 μ g/mL kantlex, behind 37 ℃, 200rpm shaking table overnight incubation, the inoculum size by 2% is inoculated in the LB liquid nutrient medium that 100mL contains 30 μ g/mL kantlex at 37 ℃, 200rpm shaking table and cultivated 2～3 hours.Cell concentration (OD in fermented liquid
600) reach at 0.6～0.8 o'clock, adding final concentration in the nutrient solution is the isopropyl-β-D-thiogalactoside(IPTG) (IPTG) of 1.0mmol/L, changes culture temperature to 30 ℃, continues to cultivate 6～8 hours.After cultivate finishing, centrifugal 10min collects thalline under 4 ℃, the condition of 8000 * g.
Then adopt broken born of the same parents' damping fluid re-suspended cell, then use Ni-NTA (QIAGEN) to carry out metal chelate chromatography, obtain the pure enzyme of wild-type GAD, its operation steps and agents useful for same are such as " expression and purification of mutant enzyme ".
Three, the acquisition of rite-directed mutagenesis PCR product
The dNTPs that adds 200 μ mol in the Eppendorf pipe of 200 μ L, each 1 μ mol of upstream and downstream primer, about 10ngpET28a (+)-gad recombinant plasmid is as template DNA, 10 * Pfu Buffer of 5 μ L, 2.5U the Pfu archaeal dna polymerase, then adding sterile distilled water to cumulative volume is 50 μ L.Reaction parameter is behind 95 ℃ of denaturation 5min, 95 ℃ of sex change 1min, and 53 ℃ of annealing 2min, 72 ℃ are extended 14min, and through 18 circulations, last 72 ℃ are extended 10min.
Upstream primer: 5 '-CTTGGGT
CCCGAACTACCTACGATGGCC-3 ' (shown in SEQ ID NO.7);
Downstream primer: 5 '-GTAGTTC
GGGACCCAAGTAGCTGACCT-3 ' (shown in SEQ ID NO.8);
Wherein, the nucleotide sequence of underscore mark is combined as the codon of coding GAD the 311st amino acids residue.The codon of wild-type GAD gene in this position is the codon GGT of coding glycine; In designed primer, the codon of this position replaces with the codon CCC of coding proline(Pro).Adopt this to primer, according to the rite-directed mutagenesis principle, can in the increase GAD gene that obtains, introduce codon specifically and replace, make the 311st amino acids residue of the GAD of gained genes encoding become proline(Pro), and no longer be the glycine of wild-type.The principle of rite-directed mutagenesis as shown in Figure 3.
Four, the conversion of rite-directed mutagenesis PCR product and checking
The rite-directed mutagenesis PCR product of above-mentioned acquisition is digested with Dpn I enzyme, with the degraded wild plasmid, eliminate the individual possibility that occurs of wild-type in the transformant.The system of digestion is: PCR product 10 μ L, and 10 * buffer, 2 μ L, Dpn I 1 μ L adds ddH
2O to cumulative volume be 20 μ L.
The product CaCl that digestion is later
2Method transforms e. coli bl21 (DE3), and concrete operations are as follows:
(1) from-70 ℃ of refrigerators, gets the competent cell suspension of 100 μ L, melt on ice;
(2) add rite-directed mutagenesis PCR digestion product 5 μ L, mixing is placed 30min on ice gently;
(3) in 42 ℃ of water-bath heat shock 110s, then place immediately 2min on ice;
(4) Xiang Guanzhong adds 900 μ LSOC substratum, mixing, and 37 ℃ of shaking culture 1h make cell recovery;
(5) above-mentioned bacterium liquid is shaken up, gets and be coated in right amount on the LB flat board that contains 30 μ g/mL kantlex, face up and place half an hour, after bacterium liquid is absorbed by substratum, be inverted dull and stereotyped, 37 ℃ of overnight incubation.
After son to be transformed grew, several clones of random choose extracted plasmid, carry out full-automatic dna sequencing, and the confirmation mutational site is Gly
311→ Pro (as shown in Figure 2).
Five, the expression and purification of mutant enzyme
Transformant is inoculated in the LB liquid nutrient medium that 20mL contains 30 μ g/mL kantlex, 37 ℃, the 180rpm shaken overnight is cultivated, and then is inoculated in the 100mL LB liquid nutrient medium that contains 30 μ g/mL kantlex with 1% inoculum size, 37 ℃, 180rpm is cultured to OD
600Be 0.6～0.8 o'clock, add IPTG (final concentration is 0.5mM), 30 ℃, 150rpm continues to induce 8h.After inducing end, 4 ℃ of lower centrifugal 10min of 5000 * g collect bacterial sediment.With pH 8.0 phosphoric acid buffer washed twice, eliminate broken born of the same parents' damping fluid (50mM potassium phosphate buffer of using again original fermented solution volume 1/10 behind the substratum, 1mMPMSF, 300mM NaCl, 10mM imidazoles, pH 8.0) re-suspended cell, ultrasonic broken born of the same parents 90 times (300W, work 3s, gap 6s) in ice bath, the centrifugal 10min of 15000 * g collects supernatant, namely obtains containing the crude enzyme liquid of GAD.
Employing Ni-NTA affinity chromatography is carried out separation and purification to the crude enzyme liquid of gained of upper step.Through loading (loading), cleaning (washing) and wash-out (eluting), collect elutriant, dialysis is removed small molecules and is namely obtained pure enzyme.Suitably after the dilution, measure the concentration of pure enzyme with the Xylene Brilliant Cyanine G method.
Used damping fluid is formulated as follows:
Lysis buffer (disruption buffer): the 50mM SODIUM PHOSPHATE, MONOBASIC, 1mM phenylmethylsulfonyl fluoride (PMSF), 300mMNaCl, the 10mM imidazoles, pH 8.0.
Wash post damping fluid (wash buffer): the 50mM SODIUM PHOSPHATE, MONOBASIC, 1mM phenylmethylsulfonyl fluoride (PMSF), 300mMNaCl, the 20mM imidazoles, pH 8.0.
Elution buffer (elution buffer): the 50mM SODIUM PHOSPHATE, MONOBASIC, 1mM phenylmethylsulfonyl fluoride (PMSF), 300mMNaCl, the 250mM imidazoles, pH 8.0.
Six, the sudden change Thermostability is investigated
With the pure enzyme of G311P that obtains respectively after being incubated 0,10,30,60,90min in the situation of 60 ℃ and 70 ℃, ice bath 10min.Get the pure enzyme of 10 μ L, (pH 4.8 to be incorporated in 200 μ L substrate solutions of 48 ℃ of preheatings, 0.1M citric acid-Sodium phosphate dibasic damping fluid contains 0.01mM PLP, 50mM substrate L-MSG), react 10min at 48 ℃ behind the mixing rapidly, reaction is put into rapidly boiling water bath 10min with termination reaction after finishing, and is centrifugal, collects supernatant liquor, adopt the amount of the GABA of high effective liquid chromatography for measuring reaction generation, to measure the residual enzyme activity of mutant enzyme G311P.Under similarity condition, with the reaction of wild-type GAD in contrast.The HPLC operational condition is as follows: chromatography column is Hypersil ODS2C18 (250mm * 4.6mm) (Dalian is according to Lyntech Corporation (US) 10177 South 77th East Avenue Tulsa, Oklahoma 74133 U.S.), the ultraviolet detection wavelength is 254nm, sample size is 10 μ L, 25 ℃ of control column temperatures, mobile phase A is methyl alcohol, Mobile phase B is tetrahydrofuran (THF): methyl alcohol: and 0.05M sodium-acetate (pH 6.2) (5: 75: 420, V/V).The gradient elution program sees the following form:
Obtain result such as Fig. 5 and 6.As seen, mutant enzyme G311P compares with wild-type enzyme, can keep better the L-Glutamic decarboxylase vigor under higher temperature, and this characteristic will be conducive to the industry preparation that GAD is used for GABA better.
Above-described embodiment is described preferred implementation of the present invention; be not that scope of the present invention is limited; design under the prerequisite of spirit not breaking away from the present invention; various distortion and improvement that the common engineering technical personnel in this area make technical scheme of the present invention all should fall in the definite protection domain of claims of the present invention.
Claims (10)
1. the thermally-stabilised variant G311P of L-Glutamic decarboxylase gene, it is characterized in that: the nucleotides sequence of described gene is listed in the 311st rite-directed mutagenesis.
2. the thermally-stabilised variant G311P of L-Glutamic decarboxylase according to claim 1 gene, it is characterized in that: the codon GGT that the nucleotide sequence of described gene is the 311st sports CCC.
3. the thermally-stabilised variant G311P of L-Glutamic decarboxylase according to claim 2 gene, it is characterized in that: the nucleotide sequence of described gene is shown in SEQ ID NO.1.
4. the L-Glutamic decarboxylase of the thermally-stabilised variant G311P of each described L-Glutamic decarboxylase of claim 1-3 genes encoding.
5. L-Glutamic decarboxylase according to claim 4, it is characterized in that: the aminoacid sequence of described L-Glutamic decarboxylase is shown in SEQ ID NO.2.
6. expression of GAD unit that comprises the thermally-stabilised variant G311P of each described L-Glutamic decarboxylase of claim 1-3 gene.
7. recombinant plasmid that comprises the thermally-stabilised variant G311P of each described L-Glutamic decarboxylase of claim 1-3 gene.
8. transformant that comprises the thermally-stabilised variant G311P of each described L-Glutamic decarboxylase of claim 1-3 gene.
The thermally-stabilised variant G311P of each described L-Glutamic decarboxylase of claim 1-3 gene under condition more than 60 ℃, catalysis Pidolidone or its salt generates the application in the method for γ-aminobutyric acid.
Claim 4 or 5 described L-Glutamic decarboxylases under condition more than 60 ℃, catalysis Pidolidone or its salt generates the application in the method for γ-aminobutyric acid.
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| CN105462949A (en) * | 2015-12-17 | 2016-04-06 | 浙江大学宁波理工学院 | Glutamic acid decarboxylase mutant and preparation method thereof and application |
| CN108467860A (en) * | 2018-03-28 | 2018-08-31 | 江南大学 | A kind of method of highly producing gamma-aminobutyric acid |
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| CN108467860A (en) * | 2018-03-28 | 2018-08-31 | 江南大学 | A kind of method of highly producing gamma-aminobutyric acid |
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