CN104212757A - Method for high-efficiently producing L-theanine through production of coli [gamma]-glutamylmethylamine synthetase with escherichia coli - Google Patents
Method for high-efficiently producing L-theanine through production of coli [gamma]-glutamylmethylamine synthetase with escherichia coli Download PDFInfo
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Abstract
The invention relates to a genetic engineered bacterium which contains a recombinant plasmid pET28a-GMAS, is 6585 bp in a plasmid size, is 1308 bp in a fragment size of a GMAS, is named as Escherichia coli and has a kanapenecilin resistance. Genes of the [gamma]-glutamylmethylamine synthetase are inducibly expressed by isopropylsulpho-[beta]-D-galactoside. The genetic engineered bacterium has a capability of biologically synthesizing theanine. By means of glutamic acid and ethylamine as substrates, a method in the invention is simple in operation method, is convenient to control in production, is high in conversion and production efficiency of the theanine and has an excellent industrial application prospect.
Description
Technical field
The invention belongs to technical field of bioengineering, be specifically related to a kind of gamma-glutamyl synthetic methylamine enzyme utilizing Recombinant organism to produce to catalyze and synthesize the method for L-thiamine.
Background technology
Theanine (theanine) is the distinctive seed amino acid of tealeaves, and be also one of taste compound of tealeaves, its content directly decides the quality of tealeaves.Meanwhile, theanine have loosen nerve, antidepressant, antitumor, hypotensive, improve the effect of the medicine such as immunizing power, also can apply with field of food, as the additive etc. as functional food.Therefore be more and more subject to people's attention, domestic and international market demand constantly increases.
At present, the production method of theanine, mainly contains three kinds.One extracts from tealeaves.Theanine in Tea content is low, and raw material sources are limited, and theanine output is very low, finally causes production cost too high.Second method is the method for chemosynthesis.The method can cause toxicant to remain, and can form D, L-type theanine isomer in resultant, splits process for refining complicated.The third utilizes microbial enzyme method to produce L-thiamine.At present, enzyme mainly L-Glutamine deaminase (CN1688705A, 2005 of production theanine reported of patent and document; JP2007185132A), gamma glutamyl transpeptidase (CN101343618A, 2008; Tea science, 2007,27(1), 61-66; Foodstuffs industry science and technology, 2008,29(2) 166-169) or glutamine synthetase etc., reaction substrate is glutamine and ethamine, compared with chemical synthesis, can not produce racemization phenomenon, and transformation efficiency is high, and reaction conditions is gentle.Receive extensive research, and enter industrial applications field.But to have no the gamma-glutamyl synthetic methylamine enzyme utilizing genetically engineered to originate be catalyzer, with the L-glutamic acid of more low cost and ethamine for substrate, efficient enzyme process prepares the bibliographical information of theanine.
Summary of the invention
The object of the present invention is to provide the strain Escherichia coli of a kind of Efficient Conversion L-glutamic acid and ethamine synthesis theanine, and this bacterial classification is preparing the application in L-thiamine.
technical scheme of the present invention is as follows:
Overall technological scheme is the extraction of physical environment soil bacteria genomic dna, the clone of gamma-glutamyl synthetic methylamine enzyme gene, adopt suitable expression vector and restriction enzyme, build recombinant expression vector, by the gene clone of gamma-glutamyl synthetic methylamine enzyme to suitable host cell, include but not limited to intestinal bacteria.
1. the extraction of physical environment soil bacteria genomic dna: in the sampling of many places Rhizosphere Soils in Tea Garden, the step be familiar with according to persons skilled in the art, kit method extract edatope microbial DNA.
2. the clone of gamma-glutamyl synthetic methylamine enzyme gene: according to the conserved sequence of gamma-glutamyl synthetic methylamine enzyme gene, design primer, according to step, preparation of reagents method that persons skilled in the art are familiar with, by pcr amplification gamma-glutamyl synthetic methylamine enzyme gene.
3. the structure of expression vector, conversion and transformant screening: the step be familiar with according to persons skilled in the art, reagent, completely can with the carrier obtained from commercial channels, restriction enzyme and method, target gene is cloned into carrier, transformed host cell, screening transformant.
4. the shake flask fermentation of recombinant bacterium: step, reagent, the method be familiar with according to persons skilled in the art, carries out shake flask fermentation, utilizes SDS-PAGE analyzing proteins expression.
5. theanine enzymatic Synthesis technique: according to the living features of enzyme itself, step, reagent, the method be familiar with according to persons skilled in the art, carry out enzymatic conversion method compound experiment, and reference is reported, utilizes the synthesis situation of Liquid Detection theanine.
invention effect
Beneficial effect of the present invention is embodied in, by building the colibacillus engineering of high expression gamma-glutamyl synthetic methylamine enzyme, while effectively obtaining high yield recombinase, effectively can utilize L-glutamic acid and ethamine synthesis L-thiamine, and transformation efficiency is about 70%, produces L-thiamine and provide for the L-glutamic acid industry making full use of low cost from now on changes into and effectively produce bacterial strain.
accompanying drawing explanation
Fig. 1 recombinant plasmid (pET-GMAS) collection of illustrative plates
Fig. 2 SDS-PAGE gel electrophoresis spectrum
Attached sequence gamma-glutamyl synthetic methylamine enzyme gene nucleotide series.
Embodiment
1
materials and methods
1.1 bacterial strains and plasmid
Intestinal bacteria (
escherichia coli) DH5 α, BL21(DE3) bacterial strain and plasmid vector pMD18-T simple vector, pET28a is this Laboratories Accession.
1.2 reagent and material
Experiment reagent: archaeal dna polymerase (Taq DNA Polymerase, PrimeSTAR
?hS DNA Polymerase), restriction enzyme (
ncoi and
bamHi), T4 DNA Ligase, dNTP, DNA marker DL10,000, Premixed Protein Marker (Low) is all purchased from Takara company; Power Soil DNA Isolation Kit is purchased from MOBIO Laboratories company, and plasmid Mini Kit, DNA purification kit are purchased from Beijing Ding Guo Bioisystech Co., Ltd; Sulfuric acid card receives mycin, penbritin, IPTG purchased from Genview company; All the other chemical reagent are analytical pure.
1.3
experimental technique
Bacterial genomes extraction, plasmid extraction and the recovery of DNA purifying etc. are all according to test kit operation instruction, preparation, the enzyme of E. coli competent are cut, enzyme connects, the conversion of carrier, the operation of the conventional molecular biological such as agarose gel electrophoresis and SDS-PAGE is with reference to " Molecular Cloning: A Laboratory guide " third edition (Science Press, 2002).
1.3.1
the extraction of environmental microorganism genomic dna
Collect tea place Rhizosphere Soils in Tea Garden soil, according to test kit Power Soil DNA Isolation Kit operation instruction, extract rhizosphere soil microorganism genomic dna.
1.3.2
the amplification of target gene gamma-glutamyl synthetic methylamine enzyme encoding sequence
Log according to Genbank
hyphomicrobiumsp. MC1(accession number NC_015717.1) encoding sequence design pair of primers (synthesis of Shanghai Ying Jun Bioisystech Co., Ltd), primer sequence and restriction enzyme site as follows:
PF:5 '-CATG
cCATGG (underscore is CTCAGGATCTTGCCCAGCTC-3 '
ncoi restriction enzyme site)
PR:5 '-CGC
gGATCC (underscore is TTAGCAGTCCAGCGTATGTGC-3 '
bamHi restriction enzyme site).
1.3.3
reaction system and condition:
PCR reaction system (50 μ l): each 1 μ l of 5 × PCR buffer 10 μ l, dNTP 4 μ l, primer PF and PR (10 μMs), template 0.5 μ l, PrimeSTAR
?hS DNA Polymerase 0.5 μ l, water to 50 μ l.
PCR reaction conditions (Touch down PCR): 95 DEG C of 5min; 98 DEG C of 10 sec, 60-51 DEG C 10 sec, 72 DEG C extend 1.5 min, 10 circulations; 98 DEG C of 10 sec, 55 DEG C of 10 sec, 72 DEG C extend 1.5 min, 20 circulations; 72 DEG C of 10 min reaction terminating.0.8 % agarose gel electrophoresis detects and purified pcr product.
PCR primer adds A reaction system (20 μ l): get 14.5 μ l PCR primer, adds 2 μ l 10 × Taq Buffer, 3 μ l dNTP, 2 U Taq DNA Polymerase, and after 72 DEG C of reaction 20 ~ 30 min, direct purification reclaims.
1.3.4
the Construction and identification of target gene cloning vector
According to pMD18-T simple vector test kit operation instructions, linking objective gene and carrier T, transform
e. colidH5 α, utilizes blue hickie to screen positive transformant, and selects part transformant to deliver to Sangon Biotech's order-checking.
1.3.5
target gene clone and qualification
In the sequencing result of related clones, select the transformant carrying interest genes, 37 DEG C of shake-flask culture that spend the night, extract recombinant plasmid, utilize
ncoi and
bamHi double digestion, reclaims target gene through agarose gel electrophoresis purifying.
Utilize simultaneously
ncoi and
bamHi double digestion pET28a carrier, and sepharose reclaims support products.
Then, T4 ligase enzyme 16 DEG C of enzymes that spend the night are utilized to connect pET28a carrier and target gene, product conversion
e. colidH5 α (CaCl
2method), select part transformant to check order through Sangon Biotech (Shanghai) Co., Ltd..
Select the transformant that sequencing result is correct, extract recombinant plasmid (pET-GMAS) and transform
e. colibL21 (CaCl
2method).
1.3.6
the expression of recombinant protein
Picking recombinant bacterium
e. colibL21 is inoculated in and receives in the LB substratum of mycin containing sulfuric acid card, and 37 DEG C of shaken overnight are cultivated.Culture being inoculated in the fresh sulfuric acid card that contains receives in the LB substratum of mycin, and inoculum size 2%, 500 mL shaking flask 37 DEG C shaking culture are to OD
600=0.6 ~ 0.8.30 DEG C of IPTG induction (final concentration 0.3mM), centrifugal collecting cell after 3h; 20 mMTris-Cl (pH8.0) re-suspended cell, ultrasonication, the upper cleer and peaceful precipitation of centrifugal collection respectively.Cleer and peaceful precipitation in SDS-PAGE analysis.With zero load
e. colibL21 is as negative control.
1.3.7
enzymatic conversion method produces theanine
As 1.3.6, ferment complete, 5000r/min, centrifugal 10min collects thalline.100mL fermented liquid concentration, to 10mL, is dissolved in the Tris-Cl damping fluid (pH8.0) of 20mM, ultrasonication 10min.Theanine building-up reactions system reference (Biosci. Biotechnol.Biochem., 71 (2), 545 – 552,2007), containing 50mM L-glutamic acid, 150mM ethylamine hydrochloride, 7.5mMATP, 30mM MgCl
2, 100mM imidazole buffer (pH7.75), enzyme liquid after appropriate cytoclasis, 30 DEG C of incubations.The detection method reference (Process Biochemistry, 45,1330 – 1333,2010) of theanine.
2
experimental result
2.1
the pcr amplification of the gamma-glutamyl synthetic methylamine enzyme gene being template with environmental microorganism genomic dna
With PF and PR for primer, with edatope genomic dna for template, pcr amplification is to the DNA fragmentation of about about 1300 bp.
2.2
the structure of target gene clone library
PCR primer and pMD18-T simple carrier enzyme are connected, transforms
e. colidH5 α, and utilize blue hickie screening positive clone, gather in the crops the transformant of about about 500.
2.3
the structure of the Cloning and Expression carrier of target gene
Select the interest genes transformant through order-checking, extract cloning vector, and double digestion results target gene, enzyme connects the pET28a carrier of two enzymic digestion, transforms
e. colidH5 α.Selection positive colony, extracts recombinant plasmid, and go forward side by side performing PCR and double digestion checking, result shows that enzyme is linked to be merit, and recombinant vectors collection of illustrative plates is as Fig. 1.
2.4
the expression of recombinant protein
Recombination bacillus coli BL21 (pET-GMAS), after IPTG induction, through SDS-PAGE gel electrophoresis analysis, there is protein band (Fig. 2) clearly at about about 44.3kDa, be gamma-glutamyl synthetic methylamine enzyme in expression product; After induction, thalline samples supernatant SDS-PAGE electrophoresis after ultrasonication, and this albumen exists in supernatant as seen, has good solubility.
2.5
enzymatic conversion method produces theanine
According to turning enzyme method described in 1.3.7, collected by centrifugation thalline, cytoclasis, carries out transformation experiment.Transformed continuously through 8 hours, 12000rpm is centrifugal.Carry out Liquid Detection according to described in 1.3.7, glutamic acid rotating rate is 69.8%, shows that recombinase has good enzymatic conversion active.
Sequence table
Claims (4)
1. produce a bacterial strain for gamma-glutamyl synthetic methylamine enzyme, this bacterial strain is Recombinant organism, it is characterized in that this bacterial strain is for get with recombination engineering plasmid pET28-GMAS transformation of E. coli.
2. engineering strain as claimed in claim 1, it is characterized in that, its Host Strains is selected from e. coli bl21, DH5 α.
3. engineering strain as claimed in claim 1, it is characterized in that, recombination engineering plasmid pET28-GMAS is containing, for example gamma-glutamyl synthetic methylamine enzyme gene nucleotide series wild in specification sheets.
4. as described in the arbitrary claim as described in claim 1-3, genetic engineering bacterium is mainly for the production of L-thiamine.
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Cited By (5)
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| CN105200075A (en) * | 2015-11-09 | 2015-12-30 | 四川同晟生物科技有限公司 | Plasmid for theanine production and construction and application method of corresponding engineering bacteria of plasmid |
| CN106893748A (en) * | 2015-12-21 | 2017-06-27 | 中国科学院天津工业生物技术研究所 | A kind of synthetic method of L-thiamine |
| CN106893699A (en) * | 2015-12-21 | 2017-06-27 | 中国科学院天津工业生物技术研究所 | A kind of crude enzyme preparation, its preparation method and application |
| CN110564789A (en) * | 2019-09-12 | 2019-12-13 | 河南巨龙生物工程股份有限公司 | Method for producing L-theanine by using escherichia coli fermentation |
| CN114134067A (en) * | 2021-10-19 | 2022-03-04 | 山东睿智医药科技有限公司 | Escherichia coli and application thereof |
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| CN101445788A (en) * | 2008-12-04 | 2009-06-03 | 中国农业科学院茶叶研究所 | Method for biosynthesizing theanine by gene-engineering strain |
| CN103409475A (en) * | 2013-07-18 | 2013-11-27 | 江南大学 | Method for synthesizing L-theanine through enzyme process |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105200075A (en) * | 2015-11-09 | 2015-12-30 | 四川同晟生物科技有限公司 | Plasmid for theanine production and construction and application method of corresponding engineering bacteria of plasmid |
| CN105200075B (en) * | 2015-11-09 | 2019-01-15 | 四川同晟生物医药有限公司 | The building and application method of plasmid and its corresponding engineering bacteria for theanine production |
| CN106893748A (en) * | 2015-12-21 | 2017-06-27 | 中国科学院天津工业生物技术研究所 | A kind of synthetic method of L-thiamine |
| CN106893699A (en) * | 2015-12-21 | 2017-06-27 | 中国科学院天津工业生物技术研究所 | A kind of crude enzyme preparation, its preparation method and application |
| CN106893699B (en) * | 2015-12-21 | 2020-11-03 | 中国科学院天津工业生物技术研究所 | Crude enzyme preparation, preparation method and application thereof |
| CN106893748B (en) * | 2015-12-21 | 2020-11-17 | 中国科学院天津工业生物技术研究所 | Method for synthesizing L-theanine |
| CN110564789A (en) * | 2019-09-12 | 2019-12-13 | 河南巨龙生物工程股份有限公司 | Method for producing L-theanine by using escherichia coli fermentation |
| CN110564789B (en) * | 2019-09-12 | 2021-05-04 | 河南巨龙生物工程股份有限公司 | Method for producing L-theanine by using escherichia coli fermentation |
| CN114134067A (en) * | 2021-10-19 | 2022-03-04 | 山东睿智医药科技有限公司 | Escherichia coli and application thereof |
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