CN102080090A - Cloning, expression and application of Lactobacillus brevis glutamate decarboxylase gene - Google Patents
Cloning, expression and application of Lactobacillus brevis glutamate decarboxylase gene Download PDFInfo
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Abstract
The invention discloses cloning, expression and application of a Lactobacillus brevis glutamate decarboxylase gene. The gene is derived from Lactobacillus brevis CGMCC No.1306, and is obtained by amplifying a Lactobacillus brevis genome DNA through PCR (polymerase chain reaction); and the full length of the gene is 1407bp. BamH I and EcoRI restriction enzyme recognition sequences are respectively added to both ends of the gene; and the gene is connected with pET-28a(+) which is digested by the same restriction enzymes, and is converted into colibacillus expression host bacteria BL21 (DE3), thereby realizing the recombinant expression in the colibacillus; and the molecular weight of the expression product glutamate decarboxylase is 53538.6Da. The recombinant glutamate decarboxylase, or the engineering bacteria for expressing the recombinant glutamate decarboxylase can convert L-glutamic acid or salt thereof into gamma-aminobutyric acid by decarboxylation, and has the advantages of high conversion efficiency, high product purity and high production efficiency.
Description
Technical field
The present invention relates to a kind of clone, expression and application of short lactobacillus glutamic acid decarboxylase gene, belong to technical field of bioengineering.More specifically, the present invention relates to the L-Glutamic decarboxylase of a kind of glutamic acid decarboxylase gene that from short lactobacillus, utilizes the PCR method screening and cloning to make new advances, its expression, reach this enzyme or express the application of engineering bacteria in the preparation γ-An Jidingsuan of this enzyme.
Background technology
γ-An Jidingsuan (being called for short GABA) claims aminobutyric acid again, is a kind of important nonprotein amino acid.It is not only a kind of important neurotransmitter of central nervous system, and human body is also had the physiological function of multiple beneficial.The lot of documents report, GABA has hypotensive and cholesterol, treatment epilepsy, the calmness of calming the nerves, alleviate depression symptom, cerebral function improvement and long-term memory, adjusting hormone secretion, prevent diabetes, the sharp kidney of strong liver, improves climacteric syndrome and improves the functions such as ability in vitro fertilization of sperm.Studies show that the aging of people's brain might cause because its brain can't obtain the chemicals γ-An Jidingsuan (GABA) of q.s.Take in the activity that GABA can improve the glucose phosphate esterase, thereby promote the energy metabolism of brain, the activation cerebral blood flow increases the oxygen supply amount, finally recovers function of brain cell, improves nervous function.
Along with the further investigation to GABA physiologically active and effect, GABA also receives publicity day by day.GABA becomes and had a kind ofly both had remarkable pharmacological action, can be used for disease treatment, physiologically active substance with health role again, as to can be used for daily " integration of drinking and medicinal herbs " taken.The pharmaceutical factory of State Food and Drug Administration's approval production gamma aminobutyric acid (γ-An Jidingsuan) sheet and gamma aminobutyric acid injection liquid has reached 24, is mainly used in the various types of hepatic coma of treatment.Japan PCI Company utilizes the fermentative Production γ-An Jidingsuan, as functional food ingredient, has been widely used in goods such as beverage, jam, cake, biscuit, food flavouring.Therefore in recent years the GABA demand is also grown with each passing day.
The method for preparing GABA mainly comprises chemical synthesis and biological synthesis process two big classes.
Modal chemical synthesis process is exactly (temperature of reaction is up to 180 ℃) reaction under strong condition with O-phthalic imide potassium and γ-neoprene cyanogen, and products therefrom and vitriol oil back hydrolysis get through crystallization and purification again.Other has with pyrrolidone as raw material, makes the patent report of GABA through calcium hydroxide, bicarbonate of ammonia hydrolysis.Though chemosynthesis GABA is swift in response, have severe reaction conditions, poor stability, a shortcoming such as energy consumption is big, cost is high, side reaction is many and environmental pollution is serious.
Biological synthesis process is to utilize biological intravital L-Glutamic decarboxylase (Glutamate decarboxylase GAD) as catalyzer, generates GABA with L-L-glutamic acid or its sodium salt α-decarboxylation.Biological synthesis process comprises two kinds of plants enriched method and microorganism (enzyme) catalysis methods.Plants enriched method wherein is to utilize GAD catalysis L-L-glutamic acid or its sodium salt generation GABA in the plant.But the GABA that plant can produce is pettiness extremely, so this method is difficult to use in the mass preparation of GABA, only is used for increasing the GABA content of farm crop such as rice, tealeaves at present, to improve the economic worth of these crops.Microorganism (enzyme) catalysis method is to utilize microorganism (or enzyme) catalysis L-L-glutamic acid or its sodium salt with GAD vigor to prepare GABA.Compare with additive method, the synthetic GABA of microorganism (enzyme) catalysis method has many advantages, and the most outstanding is, and mild condition, specificity are strong, by product is few (enzyme process), safe, is the desirable approach of mass preparation food or pharmaceutical grade GABA.
The key that microorganism (enzyme) catalysis method prepares GABA is the microorganism strains or the enzyme that will obtain high vigor (L-Glutamic decarboxylase vigor).But at present, utilizing microorganism (enzyme) catalysis method to prepare a problem that γ-An Jidingsuan exists is usually facultative anaerobic bacteria of used bacterial strain, so poor growth, culture cycle is long, causes the production efficiency of γ-An Jidingsuan low.Though also there is clone's glutamic acid decarboxylase gene in intestinal bacteria, to carry out the patent (application number 200710022222.3) of heterogenous expression, but the L-Glutamic decarboxylase vigor that obtains not high, and, therefore also to consume extra energy because the optimum temperuture of enzyme is higher than normal temperature.In addition, for microorganism (enzyme) catalysis method prepares γ-An Jidingsuan, because the complicacy of bacterial metabolism usually also has other because the impurity that self metabolism of thalline is produced in the product that is obtained except γ-An Jidingsuan.This need carry out the product that complicated separation and purification just can obtain conforming with specification of quality to product, has increased production cost.
Summary of the invention
The technical problem to be solved in the present invention is the above-mentioned deficiency at prior art, providing a kind of is the short lactobacillus (Lactobacillus brevis) of CGMCC NO.1306 from preserving number, utilizes the PCR method screening and cloning to go out the short lactobacillus glutamic acid decarboxylase gene.
Concrete, the invention provides a kind of short lactobacillus glutamic acid decarboxylase gene, the dna sequence dna of this gene is SEQID NO.1, total length 1407bp is the short lactobacillus (Lactobacillus brevis) of CGMCC NO.1306 from preserving number.This gene is a dna sequence dna of not reporting as yet, and its based composition feature is as shown in table 1 below:
Table 1 short lactobacillus (Lactobacillus brevis) CGMCC NO.1306
Glutamic acid decarboxylase gene based composition feature
The derivation aminoacid sequence of this genes encoding is SEQ ID NO.2.
Another technical problem that the present invention will solve provides a kind of expression of above-mentioned short lactobacillus glutamic acid decarboxylase gene, obtains expression product---the reorganization L-Glutamic decarboxylase.Concrete: this short lactobacillus glutamic acid decarboxylase gene is expressed in engineering bacterias such as intestinal bacteria, genus bacillus by general genetic engineering means; Can produce the reorganization L-Glutamic decarboxylase on a large scale by heterogenous expression, the molecular weight of this expression product reorganization L-Glutamic decarboxylase is 53538.6Da.
The 3rd technical problem that the present invention will solve is: a kind of above-mentioned reorganization L-Glutamic decarboxylase is provided or expresses the purposes of engineering bacteria in the preparation γ-An Jidingsuan of this recombinase, concrete: utilize reorganization L-Glutamic decarboxylase that is obtained or the engineering bacteria of expressing this recombinase to change L-glutamic acid or its salt into γ-An Jidingsuan efficiently, can be used for the production preparation of γ-An Jidingsuan.In the process of preparation γ-An Jidingsuan, optimal reaction temperature is 35～40 ℃, and optimum response pH is 4.4.
Advantage of the present invention and beneficial effect:
Inventor's (see that application number is: 200510049188.X, denomination of invention is " produce γ-An Jidingsuan short lactobacillus and uses thereof " patent) in previous work is separated to a strain and has short lactobacillus (Lactobacillus brevis) CGMCC NO.1306 than high gamma-aminobutyric acid output.Because therefore the output height of this bacterial strain in γ-An Jidingsuan is produced can infer that the L-Glutamic decarboxylase vigor of its expression is higher, the biology preparation of γ-An Jidingsuan had important value.By analyzing several lactobacillus glutamic acid decarboxylase gene sequences, we have designed different degenerated primers, by optimizing the PCR condition, the clone has obtained glutamic acid decarboxylase gene from short lactobacillus (Lactobacillus brevis) the CGMCC NO.1306 genomic dna that extracts.
Add BamH I and EcoRI restriction enzyme recognition sequence at these gene two ends respectively, be connected and transformed into escherichia coli expressive host bacterium BL21 (DE3), realized recombinant expressed in intestinal bacteria with plasmid pET-28a (+) through the same restrictions enzymic digestion.
Utilize short lactobacillus glutamic acid decarboxylase gene of the present invention, heterogenous expression by engineering bacterias such as intestinal bacteria, can overcome the shortcoming that growth velocity is slow, growth cycle is long that the amphimicrobian growth of short lactobacillus battalion is caused, at short notice the scale operation L-Glutamic decarboxylase.When the preparation γ-An Jidingsuan, both can directly utilize the recombinant bacterial strain of expressing L-Glutamic decarboxylase to carry out the cell transformation of γ-An Jidingsuan, also can carry out the enzymatic conversion method of γ-An Jidingsuan with the L-Glutamic decarboxylase of reorganization.When adopting enzymatic conversion method, compare with enzymatic conversion method of the prior art, because this enzyme catalysis vigor height, the catalysis optimum temperuture is near room temperature, preparation during γ-An Jidingsuan not only the product purity height, substrate conversion is thorough, production efficiency is high, and does not need extra heat energy, production cost low.
Description of drawings
Fig. 1. existing lactic acid bacteria glutamic acid decarboxylase gene 5 ' terminal sequence is relatively
Fig. 1 is that present sequence is derived from the aminoacid sequence (N end) of the L-Glutamic decarboxylase of milk-acid bacteria and corresponding base sequence (5 ' end) for known six kinds.As seen from the figure, their base sequence has similarity in various degree, and preceding four kinds of similarities are higher, back two kinds lower.Therefore, four kinds of 5 ' base sequences with back two kinds of glutamic acid decarboxylase genes are foundation in the past respectively, have designed the upstream degenerated primer of two correspondences respectively.
Fig. 2. existing lactic acid bacteria glutamic acid decarboxylase gene 3 ' terminal sequence is relatively
Fig. 2 is that present sequence is derived from the aminoacid sequence (C end) of the L-Glutamic decarboxylase of milk-acid bacteria and corresponding base sequence (3 ' end) for known six kinds.Different with Fig. 1 is wherein to have the base sequence of five kinds of L-Glutamic decarboxylases that very high similarity is arranged.Therefore, the base sequence of holding with these five kinds of glutamic acid decarboxylase genes 3 ' is a foundation, the downstream degenerated primer of a correspondence of design.
Embodiment
Bacterial strain and the substratum that the present invention relates to are as follows:
Bacterial strain: short lactobacillus (Lactobacillus brevis) CGMCC NO.1306 is this laboratory screening, and escherichia coli DH5a and e. coli bl21 (λ DE3) are this laboratory preservation.
Substratum:
(1) 1000mL GYP solid medium: glucose 10g, yeast extract paste 10g, peptone 5g, sodium acetate 2g, MgSO
47H
2O 20mg, MnSO
44H
2O 1mg, NaCl 1mg, FeSO
47H
2O 1mg, agar 15g, pH 6.8.
(2) 1000mL GYP liquid nutrient medium: glucose 10g, yeast extract paste 10g, peptone 5g, sodium acetate 2g, MgSO
47H
2O 20mg, MnSO
44H
2O 1mg, NaCl 1mg, FeSO
47H
2O 1mg, pH 6.6-7.0.
(3) 1000ml LB solid medium: Tryptones 10g, yeast extract 5g, NaCl 10g, agar 15g, pH 7.0.
(4) 1000ml LB liquid nutrient medium: Tryptones 10g, yeast extract 5g, NaCl 10g, pH 7.0.
The clone of embodiment 1 short lactobacillus glutamic acid decarboxylase gene
The picking preserving number is that the preservation strain short lactobacillus (Lactobacillus brevis) of CGMCC NO.1306 activates 24h at 30 ℃ on agar slant culture-medium, be inoculated in then on the 25ml GYP liquid nutrient medium, in volume is the Erlenmeyer flask of 250ml, under 30 ℃, leave standstill and be cultured to thalline and reach exponential growth mid-term (about 24h), get 4.5ml bacterium liquid abandoning supernatant after 10000rpm is centrifugal a minute then, adopt Shanghai to give birth to genome that the worker produces and extract test kit, by specification extracts genome.Through the electrophoresis checking, extracting product is single band, size and genome sizableness, and no RNA pollutes.
Existing bacterium GAD gene order in the search GenBank database is analyzed the GAD gene order design primer of having chosen 5 milk-acid bacterias that may be approaching with the aimed strain sibship after contrasting.Owing to still have the difference of 10%-30% between them, therefore constitute the preference of (respectively as depicted in figs. 1 and 2) and codon according to their two terminal sequences, adopt primer-design software DNAMAN 6.0 to design two upstream degenerated primers:
Upstream degenerated primer 1:5 '-cg
GgatccAtgaaYaaRaaYgaYcaRgaRc-3 ';
Upstream degenerated primer 2:5 '-cg
GgatccAtggcWatgttRtaYggWaaac-3 ';
With downstream degenerated primer: 5 '-gg
GaattcTtagtgHgtgaaYccgtattt-3 '.
Wherein, in the degenerated primer of upstream "
Ggatcc" be restriction endonuclease BamH I restriction enzyme site, in the degenerated primer of downstream "
Gaattc" be restriction endonuclease EcoR I restriction enzyme site.
Use upstream degenerated primer 1 (Up1) and upstream degenerated primer 2 (Up2) and downstream degenerated primer (Down1) pairing respectively, select and optimize different templates/Pfu/ primer ratio, annealing temperature, cycle index, with target gene group (short lactobacillus (Lactobacillus brevis) CGMCC NO.1306 genomic dna) is that template is carried out pcr amplification, the PCR result suitable with the clip size that is expanded, that abundance is appropriate and non-specific band is few.
Through more different upstream and downstream degenerated primer pairing and template/Pfu/ primer ratios, annealing temperature, cycle index is determined reaction conditions, has finally obtained a DNA band.
Determined PCR system consists of (unit: μ l): ultrapure water, 38.5; Pfu buffer, 5; Upstream degenerated primer 1,0.5; Downstream degenerated primer 2,0.5; DNTP, 4; Template, 1; Pfu, 0.5; The reaction cumulative volume is 50.
Used PCR condition is: enter amplification cycles behind 94 ℃ of sex change 5min, i.e. and 94 ℃ of sex change 1.5min, 57 ℃ of annealing 1min, 72 ℃ are extended 3.5min, circulate altogether 32 times, extend 8min at 72 ℃ at last.Gained PCR product is checked through gel electrophoresis, and the products therefrom band is single, clear, bright, and size is between 1000～1500bp and near 1500bp, and negative control does not have band.
Clone's product is served Hai Shenggong and is checked order, and finds that it has the conserved sequence of L-Glutamic decarboxylase, is confirmed that it is the goal gene glutamic acid decarboxylase gene.
Embodiment 2 goal gene construction of prokaryotic expression vector
Because when clone's goal gene, introduced the restriction enzyme site (BamH I and EcoR I) of restriction endonuclease at the two ends of clone's primer, so when construction of expression vector, as long as the plasmid that selection needs, adopt BamH I and EcoR I that goal gene and vector plasmid are carried out double digestion respectively, double digestion method of attachment according to routine connects goal gene and vector plasmid then, can obtain the carrier that can be used for expressing.Particularly,, adopted pET-28a (+), goal gene and pET-28a (+) have been carried out double digestion respectively according to the described double digestion condition of table 2 as vector plasmid at present embodiment.
Table 2 double digestion reaction conditions
Annotate: enzyme Qie Wendu is 35 ℃.
Plasmid enzyme restriction 1h, PCR product enzyme is cut 4h.Respectively enzyme is cut product and carry out the electrophoresis purifying and cut the glue recovery, the electrophoresis checking is reclaimed product and is also estimated carrier and the concentration that is inserted into, and carries out the preferred of ligation condition by condition shown in the table 3 then.
Table 3 ligation condition is investigated
Annotate: connecting temperature is 16 ℃, connects 16 hours.
Carry out the double digestion of the pcr amplification product of plasmid and purifying respectively by table 2 condition, products therefrom carries out gel electrophoresis to be separated, and the purpose fragment of cutting glue recovery, cmy vector and PCR product respectively, products therefrom sampling carrying out gel electrophoresis conclusive evidence.Connect according to the listed condition of table 3 then, ligation finishes back 60 ℃ of insulation 5min deactivation ligase enzymes, gets respectively to connect product transformed into escherichia coli DH5 α competent cell again.
Each condition of contact product transforms CaCl by the thermal transition method respectively
2The competent cell of method preparation, converted product is coated and is contained the LB solid medium 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 carries out BamH I and EcoR I double digestion, PCR checking behind the extraction plasmid respectively.
Found that, from the connection product transformant of condition of contact 3 correspondences, obtained containing the plasmid of purpose band, illustrate that condition of contact 3 successfully realized being connected of purpose fragment and vector plasmid, successfully made up plasmid pET-28a (+)-gad that expresses L-Glutamic decarboxylase.Get the plasmid of this transformant and serve the Hai Shenggong order-checking, obtained the GAD gene of Lactobacillus brevis CGMCC NO.1306, its sequence is seen SEQ ID NO.1, L-Glutamic decarboxylase aminoacid sequence that it is corresponding such as SEQ ID NO.2.
Get plasmid pET-28a (+)-gad transformed into escherichia coli BL21 (λ DE3) competent cell of the expression L-Glutamic decarboxylase of structure, obtain expressing the recombinant bacterial strain (BL21 (λ DE3)-pET-28a (+)-gad) 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, picking list colony inoculation 20ml contains the LB liquid nutrient medium of 30 μ g/ml kantlex then, placing the 50ml of 250ml Erlenmeyer flask to contain the LB liquid nutrient medium of 30 μ g/ml kantlex by 2% inoculum size inoculation behind 37 ℃, 200rpm shaking table overnight incubation, is that the constant temperature oscillator of 200rpm was cultivated 2～3 hours at 37 ℃, rotating speed.Cell concentration (OD in fermented liquid
600) reach at 0.6～0.8 o'clock, in nutrient solution, add the expression that inductor is induced L-Glutamic decarboxylase.The available inductor comprises isopropyl-(IPTG), lactose etc., and the concentration of inductor is between 0.1～1mmol/L.In the present embodiment, the inductor of employing is IPTG, and preferred inductor concentration is 0.75～1.0mmol/L.Induce the back to change about culture temperature to 30 ℃, be preferably 30 ℃, continue then to cultivate 5～8 hours.After cultivating end, at 4 ℃, the centrifugal 10min of 8000rpm collects thalline.Promptly obtain having the cell of L-Glutamic decarboxylase vigor.This somatic cells can be used for whole-cell catalytic L-glutamic acid or glutaminate prepares γ-An Jidingsuan.
The separation and purification of embodiment 4 short lactobacillus L-Glutamic decarboxylases
The cell with L-Glutamic decarboxylase vigor that embodiment 3 obtains can also obtain high purity, highly active L-Glutamic decarboxylase through proteic separation and purification, adopting pure enzyme to carry out enzyme process catalysis L-glutamic acid or glutaminate prepares γ-An Jidingsuan can avoid introducing other impurity, product purity height in product, can effectively reduce the separation and purification cost of finished product, enhance productivity.
The separation and purification of L-Glutamic decarboxylase has several different methods, has adopted metal chelate chromatography to carry out the purifying of enzyme in the present embodiment.Its process is that behind the escherichia coli expression that carries out L-Glutamic decarboxylase, the collection somatic cells adopts broken born of the same parents' damping fluid re-suspended cell then, uses nickel Ago-Gel medium Ni-NTA (QIAGEN) to carry out metal chelate chromatography then.
Broken born of the same parents' damping fluid consists of: 50mM NaH
2PO
4(M is mol/l, down together), 300mM NaCl, the 10mM imidazoles, regulating pH with NaOH is 8.0.With the resuspended centrifugal thalline of broken born of the same parents' damping fluid, carry out ultrasonic broken born of the same parents with ultrasonic cell pulverization instrument then, with abundant release glutamate decarboxylase protein.Used ultrasonic broken born of the same parents' condition is: ultrasonic power 250～300W, and working hour 3s, intermittent time 3s works 180 times, adopts the ice bath insulation.After ultrasonic the finishing at 4 ℃, centrifugal 30min under the condition of 12000rpm, supernatant liquor is crude enzyme liquid.
Metal chelate chromatography adopts non-sex change condition to carry out purifying according to QIAGEN company specification sheets.Particularly, at first Ni-NTA is carried out balance, add crude enzyme liquid then and adsorb with broken born of the same parents' damping fluid.Absorption can be adopted Static Adsorption, also can adopt the mode of post to carry out dynamic adsorption.On chromatography column, wash (wash) and wash-out (elution) after the adsorption equilibrium.Dcq buffer liquid consists of: 50mM NaH
2PO
4, 300mM NaCl, the 20mM imidazoles, regulating pH with NaOH is 8.0.Flushing does not come off the back with elution buffer wash-out target protein until there being albumen again from post, collects elution fraction and just obtains L-Glutamic decarboxylase.Elution buffer consists of: 50mM NaH
2PO
4, 300mM NaCl, the 200mM imidazoles, regulating pH with NaOH is 8.0.Through the check of SDS-PAGE electrophoresis, the gained L-Glutamic decarboxylase is single band, proves to have obtained the pure enzyme of L-Glutamic decarboxylase.
With the engineering bacteria cell of the expression L-Glutamic decarboxylase that obtains among the resuspended embodiment 3 of stroke-physiological saline solution, and then centrifugal collection thalline.Get the 0.5g wet thallus, be suspended in citric acid-Sodium phosphate dibasic buffer system of 25mL, L-Sodium Glutamate content is 60mM, and the control temperature of reaction system is at 35～40 ℃, and system pH is preferably 4～4.5 between 4～6.Reacted 3 hours, reaction solution is centrifugal to be got alpha-aminobutyric acid content through efficient liquid phase chromatographic analysis and is about 6g/L.Embodiment 6 transforms glutaminate with L-Glutamic decarboxylase and prepares γ-An Jidingsuan
With the pure enzyme preparation feedback of the L-Glutamic decarboxylase system of preparation among citric acid-Sodium phosphate dibasic buffer system dilution embodiment 5, system pH is preferably 4～4.5 between 4～6.Adding a certain amount of L-glutamic acid or its salt in system carries out catalysis and can obtain γ-An Jidingsuan.In the present embodiment, the catalytic substrate of selection is the L-Sodium Glutamate, and concentration of substrate is 60mM, and the control temperature of reaction system reacted 2 hours at 35～40 ℃, and reaction solution is centrifugal to get the about 6g/L of alpha-aminobutyric acid content through efficient liquid phase chromatographic analysis.
Claims (4)
1. short lactobacillus glutamic acid decarboxylase gene, it is characterized in that: the dna sequence dna of this gene is SEQ ID NO.1, and total length 1407bp from deposit number is: the short lactobacillus of CGMCC NO.1306 (Lactobacillus brevis).
2. short lactobacillus glutamic acid decarboxylase gene according to claim 1 is characterized in that: the derivation aminoacid sequence of this genes encoding is SEQ ID NO.2.
3. the expression product of the described short lactobacillus glutamic acid decarboxylase gene of claim 1 reorganization L-Glutamic decarboxylase, molecular weight is 53538.6Da.
4. the expression product of the described short lactobacillus glutamic acid decarboxylase gene of claim 3 reorganization L-Glutamic decarboxylase or express the application of engineering bacteria in the preparation γ-An Jidingsuan of this enzyme.
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| CN102911927A (en) * | 2012-11-02 | 2013-02-06 | 浙江大学宁波理工学院 | Glutamate decarboxylase as well as coding genes and application thereof |
| CN103031322A (en) * | 2011-09-30 | 2013-04-10 | 浙江大学宁波理工学院 | Glutamate decarboxylase (GAD) thermally-stable variant G311P gene and application thereof |
| CN103773731A (en) * | 2014-01-03 | 2014-05-07 | 浙江大学宁波理工学院 | Method for improving apparent catalytic activity of glutamic acid decarboxylase recombinant engineering bacteria |
| CN103789246A (en) * | 2014-01-03 | 2014-05-14 | 浙江大学宁波理工学院 | Permeable glutamate decarboxylase engineering bacterium and preparation method thereof |
| CN104694524A (en) * | 2015-03-05 | 2015-06-10 | 浙江大学宁波理工学院 | Method for preparing glutamic acid decarboxylase mutant by utilizing ramachandran map information and mutant thereof |
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| CN106566823A (en) * | 2015-10-10 | 2017-04-19 | 中国科学院天津工业生物技术研究所 | Cloning of novel glutamate decarboxylase gene and application thereof |
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| CN103031322A (en) * | 2011-09-30 | 2013-04-10 | 浙江大学宁波理工学院 | Glutamate decarboxylase (GAD) thermally-stable variant G311P gene and application thereof |
| CN103031322B (en) * | 2011-09-30 | 2014-10-22 | 浙江大学宁波理工学院 | Glutamate decarboxylase (GAD) thermally-stable variant G311P gene and application thereof |
| CN102911927A (en) * | 2012-11-02 | 2013-02-06 | 浙江大学宁波理工学院 | Glutamate decarboxylase as well as coding genes and application thereof |
| CN102911927B (en) * | 2012-11-02 | 2014-03-12 | 浙江大学宁波理工学院 | Glutamate decarboxylase as well as coding genes and application thereof |
| CN103773731A (en) * | 2014-01-03 | 2014-05-07 | 浙江大学宁波理工学院 | Method for improving apparent catalytic activity of glutamic acid decarboxylase recombinant engineering bacteria |
| CN103789246A (en) * | 2014-01-03 | 2014-05-14 | 浙江大学宁波理工学院 | Permeable glutamate decarboxylase engineering bacterium and preparation method thereof |
| CN103789246B (en) * | 2014-01-03 | 2016-08-17 | 浙江大学宁波理工学院 | A kind of permeability glutamic acid decarboxylase enzyme engineering bacteria and preparation method thereof |
| CN104694524A (en) * | 2015-03-05 | 2015-06-10 | 浙江大学宁波理工学院 | Method for preparing glutamic acid decarboxylase mutant by utilizing ramachandran map information and mutant thereof |
| CN106566823A (en) * | 2015-10-10 | 2017-04-19 | 中国科学院天津工业生物技术研究所 | Cloning of novel glutamate decarboxylase gene and application thereof |
| CN105907742A (en) * | 2016-06-21 | 2016-08-31 | 浙江科技学院 | Carboxyl magnetic bead immobilized glutamic acid decarboxylase and preparation method and application thereof |
| CN105907742B (en) * | 2016-06-21 | 2019-04-16 | 浙江科技学院 | A kind of carboxyl magnetic bead immobilized glutamate decarboxylase and its preparation method and application |
| CN109182285A (en) * | 2018-10-10 | 2019-01-11 | 浙江卓运生物科技有限公司 | The lactic dehydrogenase in Kan Deleshi lactobacillus source and its application and preparation method |
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