CN101942424B - Coding gene of UDP (Uridine Diphosphate)-glucosyltransferase mutant and application thereof - Google Patents
Coding gene of UDP (Uridine Diphosphate)-glucosyltransferase mutant and application thereof Download PDFInfo
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- CN101942424B CN101942424B CN2010102619662A CN201010261966A CN101942424B CN 101942424 B CN101942424 B CN 101942424B CN 2010102619662 A CN2010102619662 A CN 2010102619662A CN 201010261966 A CN201010261966 A CN 201010261966A CN 101942424 B CN101942424 B CN 101942424B
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Abstract
The invention discloses a coding gene of UDP (Uridine Diphosphate)-glucosyltransferase mutant and application thereof, wherein the proteins are artificially synthesized, named as RsmUGT73B6 and shown in the following types: (1) the proteins formed by amino acid residue sequences of a sequence 3 in a sequence list; and (2) the proteins substituted and/or deleted and/or added by one or a plurality of amino acid residues of the amino acid residue sequences of a sequence 4 in the sequence list and synthesized with salisorosides rosavin corresponding to the proteins derived in (1). The experiment proves that the mutation research of UGT73B6 is carried out by adopting a directed mutagenesis (V361T/T373I/G376S) technology; the triplet mutant of UGT73B6 is named as RsmUGT73B6; and the result displays that the synthesis capacity of the mutated RsmUGT73B6 salisorosides rosavin is enhanced by 1.9 times of the synthesis capacity of UGT73B6.
Description
Technical field
The present invention relates to a kind of encoding sox and application thereof of UDP-glucanotransferase two mutants.
Background technology
Radix Rhodiolae (Rhodiola sachalinensis A.Bor) has another name called storehouse page or leaf Root of Kirilow Rhodiola; It is Crassulaceae (Crassulaceae) rhodiola (Rhodiola rosea L.) plant; Be perennial herb or undershrub, the normal tool meat root stock of crawling is one of rare medicinal plant in Changbai Mountain.The main active ingredient of Rhodida plant is salidroside (salidroside) and Jujubogenin tyrosol (tyrosol) etc.; The modern pharmacology result of study show salidroside have tangible anoxia, cold resistance, antifatigue, radioprotective, antiviral, delay effects such as body aging; For industries such as space flight, deep-sea, desert, plateau, physical culture is a kind of environmental adaptation medicine that has development prospect, uses frequent personnel, strong brain worker, mid-aged population to have positive nourishing health function to high radiation practitioner, microcomputer simultaneously.
Summary of the invention
An object of the present invention is to provide a kind of encoding sox and application thereof of UDP glucanotransferase two mutants.
Protein provided by the invention, synthetic, called after RsmUGT73B6 is following 1) or 2) protein:
1) protein of forming by the amino acid residue sequence of the sequence in the sequence table 4;
2) with 4 amino acid residue sequences of the sequence in the sequence table through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have UDP-glucanotransferase function and/or synthetic relevant with salidroside by 1) deutero-protein.
Above-mentioned sequence 4 is made up of 480 amino-acid residues, the replacement of above-mentioned one or several amino-acid residue and/or disappearance and/or be added to replacement and/or disappearance and/or the interpolation that is no more than 10 amino-acid residues.
Said proteinic encoding sox RsmUGT73B6 also is the scope that the present invention protects.
Said encoding sox is following 1)-5) in arbitrary described dna molecular:
1) dna molecular shown in the sequence 3 in the sequence table;
2) in the sequence table sequence 3 from the dna molecular shown in 5 ' terminal the 1st to the 1443rd Nucleotide;
3) in the sequence table sequence 3 from the dna molecular shown in 5 ' terminal the 4th to the 1443rd Nucleotide;
4) under stringent condition with 1) or 2) or 3) the dna sequence dna hybridization that limits and coding have the dna molecular shown in UDP-glucanotransferase function and/or the albumen synthetic relevant with salidroside;
5) with 1) or 2) or 3) dna sequence dna that limits has 70% at least, have 75% at least, have 80% at least, have 85% at least, have 90% at least, have 95% at least, have 96% at least, have 97% at least, have 98% or have 99% homology and coding at least and have the dna molecular shown in UDP-glucanotransferase function and/or the albumen synthetic relevant with salidroside at least.
Above-mentioned sequence 3 is made up of 1564 Nucleotide, the sequence of OFR be sequence 3 from 5 ' terminal 1-1443 position nucleotide sequence.
Said stringent condition can be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS,, and wash film with this solution 65 ℃ of hybridization down.
The recombinant vectors, reorganization bacterium, transgenic cell line, expression cassette or the recombinant virus that contain above-mentioned encoding sox also are the scopes that the present invention protects.
Said recombinant vectors is the recombinant vectors that obtains between the NdeI of above-mentioned encoding sox insertion carrier pET-30b (+) and SalI recognition site.
The above-mentioned encoding sox total length that increases or arbitrary segmental primer are to also being the scope that the present invention protects; Said primer is to as follows: a primer sequence is shown in sequence in the sequence table 5, and another primer sequence is shown in sequence in the sequence table 6.
The application of above-mentioned protein in the synthetic big glucoside of red scape, the application of above-mentioned encoding sox in synthetic salidroside all is the scope that the present invention will protect.Said synthetic salidroside is external synthetic salidroside.
Another object of the present invention provides a kind of method for preparing BENZALACETONE.
Method provided by the invention under the said proteinic katalysis of claim 1, obtains salidroside for being substrate with tyrosol and UDP-glucose.
Experiment of the present invention proves; Adopt rite-directed mutagenesis (V361T/T373I/G376S) technology that UGT73B6 has been carried out mutation research; The triplet two mutants called after RsmUGT73B6 of UGT73B6, the result shows that the RsmUGT73B6 salidroside synthesis capability after the sudden change has improved 1.9 times than UGT73B6.RsmUGT73B6 mutator gene and the biosynthesizing that is applied as salidroside thereof provide direct otherwise effective technique and application foundation.
Description of drawings
Fig. 1 is the RsmUGT73B6 construction of prokaryotic expression vector
Fig. 2 is that the NdeI and the BamHI double digestion of prokaryotic expression carrier identified
Fig. 3 is that UGT purifying SDS-PAGE analyzes
Fig. 4 is the HPLC detected result
Embodiment
Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.
Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.
The acquisition of the rite-directed mutagenesis of embodiment 1, UGT73B6
1.UGT73B6 rite-directed mutagenesis
Synthetic UGT73B6 (sequence 1 in the sequence table).
The rite-directed mutagenesis of UGT73B6 (V361T/T373I/G376S) adopts QuikChange site-directed mutagenesis kit (available from Stratagene; Catalog#200518) kit method, concrete mutant primer is (mutational site marks with underscore) as follows: UGT73B6 V361T/T373I/G376S (5 '-GGACCACCAGTCT
ACCGGGGGTTTTGTGTCACATTGTGGATGGAACTCA
ATTTTGGAA
AGTATCTCAGCAGGG-3 '), template is synthetic UGT73B6, carries out the PCR product that PCR obtains; This PCR product is sent to order-checking, and the result shows that this PCR product has the sequence of sequence 2 in the sequence table; The unnamed gene of this PCR product is RsmUGT73B6; OFR be in the sequence table sequence 3 from 5 ' terminal 1-1443 position Nucleotide, RsmUGT73B6 encoded protein called after RsmUGT73B6, this proteic aminoacid sequence are the sequence 4 in the sequence table.RsmUGT73B6 compares with UGT73B6, and the mutational site is: the 1081-1083 position sports ACC by GTG, and the 1117-1119 position sports ATT by ACA, and the 1126-1128 position sports AGT by GGT.RsmUGT73B6 compares with UGT73B6 (sequence 2), and the mutational site is the 361st and sports Thr (Threonine) by Val (Xie Ansuan), and the 373rd sports Ile (Isoleucine) by Thr (Threonine), and the 376th sports Ser (Serine) by glycocoll Gly.
2.RsmUGT73B6 heterogenous expression and protein purification
The ORF of RsmUGT73B6cDNA uses the N-end and the terminal primer of C-that contain specific limited property restriction enzyme site to carry out pcr amplification.Sense primer is: 5 '-GGAATTC
CATATGGGTTCTGAAACTCGGCC-3 ' (sequence 5), underscore is represented the NdeI restriction enzyme site; Antisense primer is: 5 '-CG
GGATCCCTAGACTTTCTTTAACTTGAGTTCC-3 ' (sequence 6), underscore is represented the BamHI restriction enzyme site.The PCR product (or sequence 3 of synthetic) that obtains with step 1) be a template, carries out PCR, after the PCR product that obtains (in the sequence 2 be nucleotide sequence from 5 ' terminal 4-1443) passes through the NdeI/BamHI double digestion; Be connected to the prokaryotic expression carrier pET-30b (+) that cuts through same enzyme (Novagen, Darmstadt, Germany) on; Obtain recombinant vectors (Fig. 1 is the carrier structure synoptic diagram); Recombinant vectors is carried out enzyme cut evaluation, result such as Fig. 2 (M, DL15000+2000Marker; 1-3, positive plasmid.) shown in, from figure, find out, obtain the dna fragmentation of about 1.5Kb, enzyme is cut the correct reorganization positive plasmid order-checking of qualification result, correct recombinant vectors called after pET-30b (+)-RsmUGT73B6 checks order.Through order-checking, pET-30b (+)-RsmUGT73B6 is that nucleotide sequence obtains between the NdeI of pET-30b (+) and BamHI recognition site, inserting in the sequence 2 from 5 ' terminal 4-1443, proves that further vector construction is correct.
Change pET-30b (+)-RsmUGT73B6 over to intestinal bacteria E.coli BL21-Rosetta (DE3) (TransGen; Beijing; China) in the bacterial strain; Obtain the reorganization bacterium, enzyme was cut evaluation after the bacterium of will recombinating was extracted plasmid, and obtaining size is reorganization bacterium called after BL21/pET-30b (+)-RsmUGT73B6 of the dna fragmentation of 1.5Kb.
Adopting uses the same method imports intestinal bacteria E.coli BL21-Rosetta (DE3) (TransGen with empty carrier pET-30b (+); Beijing; China) in the bacterial strain, obtain reorganization bacterium BL21/pET-30b (+), the bacterium of should recombinating again adopts aforesaid method fermentation, purifying to obtain control group albumen.
BL21/pET-30b (+)-RsmUGT73B6 is inserted 10ml contain kantlex (50 μ g ml
-1) and paraxin (34 μ gml
-1) the LB substratum, 37 ℃ of overnight cultures are diluted 100 times next day and are inserted in the 200ml LB substratum in 37 ℃, 200rpm and continue to be cultured to OD
600During for 0.6-0.8, adding final concentration is the IPTG of 1mM, is cooled to 30 ℃, behind the 6h, through centrifugal collection thalline and be resuspended in 3ml 0.1M potassium phosphate buffer (pH 7.5), the broken thalline of UW on ice.Broken on ice thalline homogenate is in 4 ℃ 10, and centrifugal 10min under the 000g collects supernatant.
Supernatant passes through Ni
2+-agarose column (Novagen). with the 0.1M potassium phosphate buffer washing Ni that contains 0.5M NaCl and 40mM imidazoles (imidazole)
2+Behind-the agarose column three times,, collect the washing elutriant with the 0.1M potassium phosphate buffer washing wash-out that contains 400mM imidazole; Active for the long-term recombinant protein that keeps, again elution buffer is passed through PD-10 post (Amersham Pharmacia Biotech, Uppsala; Sweden), with 0.1M Tris-HCl (pH7.5) the displacement damping fluid that contains 10% (v/v) glycerine wash-out again, collect elutriant; Obtain recombinant protein RsmUGT73B6, be stored in-80 ℃.
The purification efficiency of recombinant protein detects with SDS-PAGE.
Histidine six aggressiveness labels are constructed on the N-terminal of recombinant protein.The recombinant protein that above-mentioned purifying is obtained carries out the SDS-PAGE electrophoresis detection, result such as Fig. 3 (M, molecular mass standard; 1, the RsmUGT73B6 purification result; 2, the UGT73B6 purification result) shown in, the result shows that purified recombinant albumen forms a single band on the position of 50kDa.The target protein band is not arranged in the control group albumen.
The functional study of embodiment 2, two mutants
1, external enzymatic reaction and product analysis
The outer enzymatic reaction system of 250 μ l standard bodies comprises: 50mM Tris-HCl damping fluid (pH 7.5); 500 μ M tyrosols (Sigma-Aldrich Catalog #79058), the recombinant protein RsmUGT73B6 that 2mM UDP-glucose (Sigma-Aldrich Catalog#U4625) and 10 μ g are obtained by embodiment 1.37 ℃ of reaction 30min, reaction finishes the back and adds 250 μ l methyl alcohol, the centrifugal 10min of 12000g, reaction product is used for HPLC and analyzes.The recombinant protein UGT73B6 and the control group albumen that obtain with embodiment 1 are contrast.
1 enzyme activity unit (U) is defined as the enzyme amount of catalysis tyrosol generation 1nmol salidroside in 1 hour.Reaction times is 60min, and measuring temperature is 37 ℃.
The analysis of enzymatic prepn is used and is equipped with Kromosil C18 reversed-phase column (5 μ m, 250mm * 4.6mm; Macherey Nagel) Waters Alliance 2695 highly effective liquid phase chromatographic systems (HPLC) are accomplished.Moving phase is water (A) and methyl alcohol (B), and flow velocity is 0.6ml min
-1, used following gradient condition: 30%B3 minute, 30-70%B 27 minutes, 70-80% B 2 minutes, 80-95%B 3 minutes and 95%B 5 minutes.The detection wavelength is 275nm.(Shanghai Tongtian Biotechnology Co., Ltd., Catalog E-0069) carries out quantitatively with the salidroside standard substance.
For performance liquid chromatography-mass spectroscopy, liquid phase separation is accomplished by Shimadzu LC-10ADvp HPLC system.Mass spectroscopy by Shimadzu LCMS-2010A mass spectrometer system accomplish (Shimadazu, Kyoto, Japan).The ShimadzuLC/ESIMS-2010 software system are accomplished DAP.Liquid phase chromatogram condition is with aforementioned.The righttest mass spectrum condition is following: all m/z POP scopes are set in 120-350; The dry gas flow velocity, 1.5L min
-1The CDL temperature, 250 ℃; The block temperature, 200 ℃; Probe voltage+4.5kV.
(A is the UGT73B6 catalytic result for the qualitative and quantitative analysis results of enzymatic prepn such as Fig. 4; B is the RsmUGT73B6 catalytic result) shown in; Show that the RT of the salidroside that RsmUGT73B6 catalysis generates is 8.3 minutes, the RT of the salidroside that UGT73B6 catalysis generates is 8.3 minutes.
RsmUGT73B6 and tyrosol A and UDP-glucose are hatched in the standard enzymatic reaction system jointly and enzymatic reaction system pH value is 7.5 o'clock, and for synthesizing of salidroside, the enzyme of RsmUGT73B6 is 4263U mg than living
-1UGT73B6 enzyme be 1470U mg than living
-1Therefore, it is principal product that RsmUGT73B6 can generate salidroside efficiently, and active in UGT73B6.Control group albumen does not have enzymic activity.
Mass spectrometric detection result is 300.31 for salidroside standard substance molecular weight, and the molecular weight that RsmUGT73B6 catalysis generates salidroside is 300.31, and the molecular weight that UGT73B6 catalysis generates salidroside is 300.31, shows that the material that obtains is a salidroside.
Proof: RsmUGT73B6 is the uridine diphosphoglucose based transferase.
The mensuration of recombinant protein kinetic constant is under the situation of UDP-glucose concn saturated (concentration is 2mM/l), with tyrosol A between its K
mThe 0.2-6.0 scope in 5 concentration values calculate acquisition.
250 μ l 0.1M potassium phosphate buffer reaction systems of above-mentioned standard are used in experiment, each experiment repetition 3 times.The pH value of reaction system is 7.5, and above-mentioned reaction system places 37 ℃ after following 30 minutes, adds final concentration and be 5% methyl alcohol, uses the ETHYLE ACETATE extracting of 250 μ l afterwards and in 10, under the 000g centrifugal 10 minutes.After getting supernatant vacuum-drying, add the methanol aqueous solution of 50 μ l 50% (v/v).The HPLC condition determination is the same.K
mAnd V
MaxValue is drawn by the result of Lineweaver-Burke curve.
Two mutants RsmUGT73B6 enzymic activity (V
Max/ K
m) be 8.88, UGT73B6 enzymic activity (V
Max/ K
m) be 4.6; Two mutants RsmUGT73B6 improves 1.9 times than UGT73B6, and RsmUGT73B6 mutator gene and the biosynthesizing that is applied as salidroside thereof provide direct otherwise effective technique and application foundation.
Claims (8)
1. protein, the protein of forming by the amino acid residue sequence of the sequence in the sequence table 4.
2. the said proteinic encoding sox of claim 1.
3. encoding sox according to claim 2 is characterized in that: said encoding sox is following 1) or 2) described dna molecular:
1) dna molecular shown in the sequence 3 in the sequence table;
2) in the sequence table sequence 3 from the dna molecular shown in 5 ' terminal the 1st to the 1443rd Nucleotide.
4. the recombinant vectors, reorganization bacterium, transgenic cell line, expression cassette or the recombinant virus that contain claim 2 or 3 said encoding soxs.
5. recombinant vectors according to claim 4 is characterized in that: said recombinant vectors is for inserting the recombinant vectors that obtains between the MCS of carrier pET-30b (+) with claim 2 or 3 said encoding soxs.
6. the application of the said protein of claim 1 in synthetic salidroside, or claim 2 or the application of 3 said encoding soxs in synthetic salidroside.
7. application according to claim 6 is characterized in that: said synthetic salidroside is external synthetic salidroside.
8. method for preparing salidroside is characterized in that: said method under the said proteinic katalysis of claim 1, obtains salidroside for being substrate with tyrosol and UDP-glucose.
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| WO2020097922A1 (en) * | 2018-11-16 | 2020-05-22 | 邦泰生物工程(深圳)有限公司 | Udp-glucosyltransferase mutant, use thereof and method for preparing rebaudioside d |
| CN111909909A (en) * | 2019-05-09 | 2020-11-10 | 湖北工业大学 | Glycosyltransferase mutant and application thereof in synthesizing beta-D-glucoside |
| CN110592043B (en) * | 2019-11-01 | 2022-10-18 | 广西师范大学 | UDP-glucosyltransferase mutant and application thereof |
| CN112813084B (en) * | 2021-02-26 | 2022-07-19 | 云南农业大学 | Carbon glycosyltransferase DhCGT1 gene in pseudo-anethod plants and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1302895A (en) * | 1999-11-02 | 2001-07-11 | 上海生元基因开发有限公司 | Human UDP glucose-glycoprotein glycosyl transferase and itscoding sequence |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1302895A (en) * | 1999-11-02 | 2001-07-11 | 上海生元基因开发有限公司 | Human UDP glucose-glycoprotein glycosyl transferase and itscoding sequence |
Non-Patent Citations (2)
| Title |
|---|
| L Q Ma et al.Molecular cloning and overexpression of a novel UDP-glucosyltransferase elevating salidroside levels in Rhodiola sachalinensis.《Plant cell rep》.2007,第26卷摘要、991页倒数第2段、993页右栏倒数第1段. * |
| 周文灵等.植物糖基转移酶及其在代谢工程中的应用.《生物技术》.2009,第19卷(第6期),95-98. * |
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