CN106566823A - Cloning of novel glutamate decarboxylase gene and application thereof - Google Patents
Cloning of novel glutamate decarboxylase gene and application thereof Download PDFInfo
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Abstract
The invention relates to the cloning of a novel glutamate decarboxylase gene and the application thereof. A glutamate decarboxylase is obtained from bacillus, and then the glutamate decarboxylase is induced to express in escherichia coli. The study on the properties of the glutamate decarboxylase shows that, the glutamate decarboxylase is higher in activity at pH 4.0 to pH 6.0, and the optimum temperature of the glutamate decarboxylase is 45-65 DEG C. The Vmax of the glutamate decarboxylase is 150-200 U/mg, and the Km of the glutamate decarboxylase is 7-9 mmol/L. The recombinant escherichia coli containing the glutamate decarboxylase is subjected to whole-cell transformation, and then 500 g/L L-glutamic acid is added in batches. At 37 DEG C, the recombinant escherichia coli is converted for 12 hours in a sodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution at the pH 7.0, and 347.8 g/L gamma-aminobutyric acid is finally generated. The molar conversion rate is 99.4%. Compared with an existing glutamate decarboxylase, the above novel glutamate decarboxylase is higher in catalytic efficiency and relatively wide in pH range, thus being more suitable for industrial application.
Description
Technical field
The present invention relates to a kind of new glutamate decarboxylase, and its application in γ-aminobutyric acid production, belong to biocatalysis field.
Background technology
Glutamate decarboxylase is a kind of P5P (PLP) dependent form enzyme, can be catalyzed L-Glutamic Acid decarboxylation and generate γ-aminobutyric acid（GABA）And carbon dioxide.Product γ-aminobutyric acid is a kind of important inhibitory neurotransmitter, has anxiety, adjusts the different physiological roles such as blood pressure, treatment epilepticss, has important using value in industries such as food, medicine.The main method of production γ-aminobutyric acid has chemical synthesiss, plants enriched method and microbe fermentation method at present.Microbe fermentation method refers to that the GAD living things catalysis L-Glutamic Acid being contained within using organism or L-Glutamic Acid salt are occurred decarboxylic reaction and generates GABA.Because its reaction condition is gentle, reaction time is short, environmentally safe the advantages of receive more and more attention.
The bacterium that GABA is produced using fermentation method has yeast, monascuses and lactic acid bacteria etc., and corresponding yield is generally 4.3 g/L[1]、9.18 g/L[2]With 10.78 g/L[3], the lactic acid bacteria maximum output of Jing mutation and fermentation optimization is up to 100 g/L[4].By the glutamate decarboxylase of Lactobacillus brevis in Corynebacterium glutamicum heterogenous expression, the GABA yield for obtaining be 30.18 g/L[5].The glutamate decarboxylase catalytic efficiency that these fermentation methods product GABA is used is relatively low, and optimum pH slant acidity, and the pH of culture medium need to be dropped to 4.5 or less than 4.5 can just make which play catalysiss.By the glutamic acid decarboxylase gene of escherichia coli or Lactobacillus brevis in expression in escherichia coli, GABA is produced using resting cell method, yield can reach 280-300 g/L respectively[6]With 278.3 g/L[7], although high catalytic efficiency, but still need to for conversion environment pH to drop to 4.5 or less than 4.5, it is larger to equipment loss.
Less than 5.0, when pH is more than 6.0, enzyme activity drastically can decline the optimum pH of the glutamate decarboxylase relatively low and enzyme of general activity having now been found that, this characteristic significantly limit glutamate decarboxylase application in the industry.
List of references：
1. Zheng swan goose, Zhao Wei are prosperous beautiful uncommon. and response phase method optimization candida mycoderma Y6 produces γ-aminobutyric acid fermentation technology [J]. Food Science, 2015,36 (09):130-135.
2. leaf inkstone. γ-aminobutyric acid process conditions [J] are produced in response phase method optimization monascuses X27 liquid fermentation. Chinese grain and oil journal, 2010,25 (9): 107-110.
3. MengHe Bi LiGe. the medium optimization [J] of γ-aminobutyric acid lactic acid bacteria is produced in traditional dairy products. food industry science and technology, 2009,7: 124-127.
4. summer river, prunus mume (sieb.) sieb.et zucc. are happy, Huang Jun etc. produce the screening and mutation [J] of the lactic acid bacteria of γ-aminobutyric acid. nuclear agricultural science report, and 2006,20 (5): 379-382.
5. F Shi. J Jiang. Y Li, et al. Enhancement of c-aminobutyric
acid production in recombinant Corynebacterium
glutamicum by co-expressing two glutamate decarboxylase genes from Lactobacillus brevis[J].
J Ind Microbiol Biotechnol, 2013, 40: 1285–1296.
6. A Plohov, M Gusytiner, T Yampolskaya, et al.Preparation of
γ-aminobutyric
acid using E. coli cells with high activity of
glutamate decarboxylase[J]. Appl
Biochem Biotech, 2000, 88: 257-265.
7. field Ganoderma, Xu Meijuan, Rao Xiaoming. one plant of recombination bacillus coli/pET-28a-lpadStructure and its Efficient Conversion γ-aminobutyric acid conversion condition optimization [J]. biological engineering journal, 2012,28 (1): 65-75.
The content of the invention
Present invention is primarily targeted at being found that a kind of new glutamate decarboxylase, and construct expression strain.By glutamate decarboxylase abduction delivering after purification, determine zymologic property.Zymologic property research shows that the optimum temperature of the enzyme is 45 DEG C ~ 60 DEG C, more stable at 30 DEG C；Optimum pH is 4.0 ~ 6.0, most stable in pH7.0；Vmax is 150 ~ 200 U/mg, and Km is 7 ~ 9 mmol/L.
Resting cell is tested, and the total amount of feeding of L-Glutamic Acid is 500 g/L, converts the γ-aminobutyric acid that 12h ultimately generates 347.92 g/L, and molar yield is 99.41%.
Description of the drawings
Figure of description 1 be BmGAD before purification after SDS-PAGE figure；
Figure of description 2 is BmGAD optimum temperature (A）And its temperature stability（B）；
Figure of description 3 is BmGAD optimum pH (A）And its pH stability（B）；
Figure of description 4 is to calculate Enzyme kinetic parameter using Lineweaer-Burk graphing methods；
Figure of description 5 is that HPLC methods determine GABA content in transformation system.
Specific implementation method
Specific implementation method of the present invention is illustrated below by way of specific embodiment, but these embodiments do not constitute the restriction to mode of the present invention, scope and effect.
Glutamic acid decarboxylase gene derives from Chinese microorganism strain preservation administrative center, bacterium numbering 10055.There are 1404 nucleotide sequences, encoding proteins size is 53 kDa.Find that the glutamic acid decarboxylase gene of bacillus megaterium and the glutamic acid decarboxylase gene similarity of escherichia coli and lactic acid bacteria respectively may be about 50% and 30% by sequence alignment.
Recombinant bacterial strain builds
With the glutamic acid decarboxylase gene sequential design PCR primer of bacillus megaterium type strain, as template, PCR obtains the genetic fragment of BmGAD to genome with bacillus megaterium, with pET21b as carrier, restriction enzyme site is Nde I and Hind III, construction recombination plasmid pET21b-Bmgad, by recombinant plasmid transformedE.coliDH5 α, Jing PCR, digestion verification correctly and after sequencing, conversionE.coliBL21, builds recombinant bacterial strainE.coliBL21（pET21b-Bmgad）.
Recombinant bacterium abduction delivering purification
By -80 DEG C of preservationsE.coliBL21（pET21b-Bmgad）Access containing amicillin resistance（100 μ g/ml of final concentration）LB culture medium, 37 DEG C of activated overnights, then by 2% inoculum concentration transfer it is new containing amicillin resistance（100 μ g/ml of final concentration）LB culture medium, when culture OD values are to 0.6 ~ 0.8, plus 0.2 ~ IPTG of 0.6 mmol/L, 25 DEG C of 6 ~ 8 h of induction.Jing Ni post affinitive layer purifications after the bacterium ultrasonication that will be collected, obtain pure enzyme BmGAD.
The measure of BmGAD zymologic properties
Catalysis per minute generates 1 μm of ol γ-aminobutyric acid and is defined as enzyme-activity unit U, specific enzyme activity U/mg.Reaction system is 3mL disodium hydrogen phosphates-citrate buffer solution（The PLP of the sodium glutamate containing 50 mmol/L, 0.1 mmol/L）.Enzyme activity is measured using colorimetry, and protein concentration is determined using Coomassie Brilliant Blue.
Optimum temperature and temperature stability：35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C, 70 DEG C are selected, the reaction buffer of pH5 is measured to optimum temperature.Temperature stability determines 30 DEG C of selection, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, surveys once remaining enzyme activity every 1h, and sampling total time is 6h.
The measure of optimum pH and pH stability：Reacted in pH4.0, pH4.5, pH5.0, pH5.5, pH6.0, pH6.5, pH7.0 under the conditions of 50 DEG C respectively, determine the optimum pH of enzyme.It is, by BmGAD respectively in the buffer of pH4.0, pH4.5, pH5.0, pH5.5, pH6.0, pH6.5, pH7.0 is dissolved in, to survey remaining enzyme activity after 30 DEG C of 3 h of insulation that temperature stability is determined.
The measure that metal ion is affected on enzymatic activity：Add the Na of final concentration of 2 mmol/L respectively in reaction system+、Ca2+、Co2+、Mn2+、Zn2+、Fe2+、Fe3+、Cu2+, determine impact of the different metal ions to enzymatic activity.
The measure of kinetic constant：It is 1 mmol/L, 3 mmol/L, 5 mmol/L, 9 mmol/L, 15 mmol/L, 20 mmol/L, 30 mmol/L, 45 mmol/L, 70 mmol/L, 90 mmol/L to prepare pH5.0 concentration of sodium glutamate
Reaction buffer reacted respectively, Vmax and Km is tried to achieve using Lineweaer-Burk graphing methods.
The transformation experiment of recombinant bacterium
Disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution of the conversion buffer for pH 7.0, it is 15 g/L that bacterium is dense, and PLP concentration is 0.2 mmol/L, and conversion temperature is 37 DEG C.The total input amount of substrate glutamic acid is that 500 g/L are added using batch feeding, initial concentration is 100 g/L, respectively add the glutamic acid of 50 g/L respectively in 0.75 h, 1.5 h, 2.5 h, 3.5 h, 5 h, 6.5 h, 8 h, 10 h, final conversion ratio during 12 h, is measured by sampling.Transformation experiment result is measured using liquid chromatograph.
Sequence table
<110>Tianjin Institute of Industrial Biotechnology, Chinese Accademy of Sciences
<120>A kind of new glutamate decarboxylase and its application
<160> 2
<170> PatentIn
version 3.3
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<211> 467
<212> PRT
<213> Bacillus megaterium
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atgcttccgg aaacggctta tcaaatcatt catgacgaaa ttgctttaga cggaaatgcc 180
cgcttgaatt tagctacgtt tgttactacg tggatggagc ctgatgcaaa gcgtttgtac 240
ggagaatctt ttgataaaaa tatgatcgat aaagatgagt atccgcagac agcggctatt 300
gaagagagat gtgtacgtat tttagcggat ttgtggaatt cacctaatcc tgataccacg 360
atgggcgttt ctactacagg ttcatctgaa gcatgtatgc ttggtggact agcgttaaaa 420
agacgatggc agaaactgcg taaaagtaaa gggctatcaa cggaccgccc caatattgta 480
tttagttcat cggttcaagt ggtatgggag aagttcgcaa actattggga cgtagagcct 540
cgttatgtga atattaatcc agatcatcct tatttagatg cagaaggcgt gattaatgcg 600
gttgacgaaa atacaattgg cgtcgtaccg attcttggag tcacgtatac agggggttac 660
gaaccaatag ctgctatcgc aaaagcatta gatgagttac aggaaaaaac agggttggat 720
attcctatcc atgtagatgc tgcttctgga ggttttatcg ctccatttct tcaaccagac 780
cttatctggg atttccgctt gccgcgagta aagtccatta acgtgtcagg acacaagtat 840
ggtttagttt accctggctt gggatgggtg atttggagag aaaaagagga cttgcctgaa 900
gatcttattt tccgcgtttc ttatttaggg ggcaacatgc caacttttgc gctcaacttc 960
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atgcgcattg ttgttagaaa tgggttttca agagaccttg ctcatttatt tatggttaat 1320
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Claims (9)
1. a kind of glutamate decarboxylase, which has the following properties that：
（1）Molecular weight of albumen is about 53 kDa；
（2）Optimum pH：4.5~5.5；
（3）Optimum temperature：45℃~60℃.
2. the glutamate decarboxylase as described in right 1, it is characterised in that from bacillus.
3. the glutamate decarboxylase as described in right 1, its aminoacid sequence are sequence 1.
4. there is the enzyme with glutamate decarboxylase activity for replacing and/or lack and/or add or several aminoacid to obtain in the aminoacid as described in right 3.
5. the glutamate decarboxylase as described in right 1, its nucleotides sequence are classified as sequence 2.
6. the nucleotide as described in right 5 has more than 90% homology and encodes the DNA molecular for having identical function with right 1.
7. expression test kit, recombinant vector containing the DNA molecular having the right described in 5 or 6, recombinant bacterium.
8. the glutamate decarboxylase described in claim 1 generates the application in γ-aminobutyric acid in conversion L-Glutamic Acid or glutamate, Glu.
9. expression test kit as claimed in claim 7, recombinant vector and recombinant bacterium generate the application in γ-aminobutyric acid in conversion L-Glutamic Acid or glutamate, Glu.
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Cited By (6)
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| CN109722402A (en) * | 2017-10-28 | 2019-05-07 | 中国科学院天津工业生物技术研究所 | A kind of method of resting cell production γ-aminobutyric acid |
| CN110760533A (en) * | 2019-12-05 | 2020-02-07 | 南阳师范学院 | Gene for coding glutamate decarboxylase, recombinant engineering bacterium and application thereof |
| CN111635898A (en) * | 2020-06-17 | 2020-09-08 | 中国科学院天津工业生物技术研究所 | Glutamic acid decarboxylase mutant and application thereof in preparation of gamma-aminobutyric acid |
| CN112251428A (en) * | 2020-12-21 | 2021-01-22 | 中国科学院天津工业生物技术研究所 | Glutamic acid decarboxylase mutant and application thereof in production of gamma-aminobutyric acid |
| CN114752544A (en) * | 2022-06-16 | 2022-07-15 | 森瑞斯生物科技(深圳)有限公司 | Method for producing gamma-aminobutyric acid by one-step method and strain construction thereof |
| CN116286760A (en) * | 2023-02-01 | 2023-06-23 | 西南大学 | Mulberry MnGAD gene cloning method, GAD prokaryotic expression method and GABA production method |
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