CN109957555A - A kind of glycosyl transferase mutant and its application in catalysis Gastrodin biosynthesis - Google Patents
A kind of glycosyl transferase mutant and its application in catalysis Gastrodin biosynthesis Download PDFInfo
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Abstract
The present invention provides a kind of glycosyl transferase mutant and its applications in catalysis Gastrodin biosynthesis, belong to gene engineering technology field.The glycosyl transferase mutant has the amino acid sequence as shown in SEQ ID No.1 in sequence table.The present invention is using the mutant UGT73B6MK of the glycosyl transferase UGT73B6 in the page root of kirilow rhodiola of plant library as parent, it is alanine Ala (A) by the site mutation of 389 phenylalanine Phe (F), improves the enzyme activity for catalyzing and synthesizing Gastrodin.Glycosyl transferase mutant after engineered, external enzyme activity is parent UGT73B6MK4.9 times, internal conversion ratio is parent UGT73B6MK3.8 times, have better catalytic activity, be more suitable for the industrialized production of Gastrodin.
Description
Technical field
The invention belongs to gene engineering technology fields, and in particular to a kind of glycosyl transferase mutant and its in catalysis Rhizoma Gastrodiae
Application in plain biosynthesis.
Background technique
Glycosyl transferase (Glycosyltransferases, GT, EC 2.4.x.y) is capable of the sugar such as UDP- of catalytic activation
Glucose is connected to different acceptor molecules, in albumen, nucleic acid, oligosaccharides, rouge and small molecule.Glycosylation can change receptor point
The activity of son, the water solubility, secretory and the stability that increase acceptor molecule etc. especially determine that the biology of some compounds is living
Property.Having many important activity ingredients in plant source natural product is glycation product, such as plant- based medicine Gastrodin
(Gastrodin) be substrate hydroxy-benzyl alcohol is occurred under glycosyl transferase catalytic action phenolic hydroxyl group glucosyl reaction
And it synthesizes.
Gastrodin (Gastrodin, GAS) has the feature that chemical name is 4- (hydroxymethyl) phenyl
Beta-D-glucopyranoside, molecular formula C13H18O7, molecular weight 286.1053, No. CAS is 62499-27-8, structure
Formula are as follows:
Gastrodin aglycon has the feature that chemical name is to hydroxy-benzyl alcohol (4-Hydroxybenzyl alcohol)
P-Hydroxybenzyl alcohol, molecular formula C7H8O2, molecular weight 124.0524, No. CAS is 623-05-2, structural formula
Are as follows:
Gastrodin has calm, anticonvulsion, anti-inflammatory, analgesia, expansion blood vessel, anti-oxidant, enhancing body's immunity and resists
The effects of senile dementia, is clinically widely used in the auxiliary of neurasthenia and neurasthenia syndrome, dizziness, headache and insane carbuncle
Help treatment.The country is using Gastrodin as the Medicines and Health Product of effective component such as Injectio of gastrodine, and Rhizoma Gastrodiae cellulose capsule, dispersible tablet etc. are
Largely listed or obtained clinical certification.Currently, the production of Gastrodin mainly passes through chemical synthesis or carries out to Rhizoma Gastrodiae plant
Extraction, however chemical synthesis there are high by-product, it is seriously polluted the problems such as, there are resource constraints etc. for plant extraction.Pass through micro- life
Object cell directly converts and tissue cultures produce Gastrodin etc. and only exist in the laboratory research stage, and there are period length, low outputs etc.
Problem.Urgently exploitation green, efficient, large-scale production Gastrodin mode.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of glycosyl transferase mutant and its in catalysis Gastrodin biology
Application in synthesis, the glycosyl transferase mutant have the characteristics that high enzyme activity, are conducive to industrial biological method production day
Numb element.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides a kind of glycosyl transferase mutant, have the amino as shown in SEQ ID No.1 in sequence table
Acid sequence.
The present invention provides the glycosyl transferase mutant code gene, have such as SEQ ID No.2 in sequence table
Shown in nucleotide sequence.
It include the glycosyl transferase in the recombinant plasmid the present invention provides a kind of glycosyl transferase recombinant plasmid
Mutant code gene.
The present invention provides a kind of glycosyl transferase recombinant bacterial strain, the recombinant bacterial strain is that conversion has the recombinant plasmid
Expression vector.
The present invention provides the glycosyl transferase mutant or the glycosyl transferase recombinant bacterial strain to close in catalysis substrate
At the application in Gastrodin.
Preferably, catalysis substrate is to hydroxy-benzyl alcohol and/or UDP-glucose.
Preferably, the temperature of the catalysis is 28~35 DEG C.
Preferably, during the glycosyl transferase recombinant bacterial strain catalysis substrate synthetic gastrodin, the glycosyl transfer
Enzyme recombinant bacterial strain carried out under the isopropyl-beta D-thio galactopyranoside solution condition of 0.1~1mmol/L induction 15~
25h。
The present invention provides a kind of glycosyl transferase mutant, have such as SEQ ID No.1 amino acid sequence in sequence table.
The present invention is with the mutant UGT73B6 of the glycosyl transferase UGT73B6 in the page root of kirilow rhodiola of plant libraryMKIt, will as parent
The site mutation of 389 phenylalanine Phe (F) sports alanine Ala, and (glycosyl transferase shown in A, SEQ ID No.1 is prominent
Variant), improve the enzyme activity for catalyzing and synthesizing Gastrodin.The present invention provides the glycosyl transferase mutant or the glycosyls
Application of the transferase recombinant bacterial strain in catalysis Gastrodin biosynthesis.The transfer of glycosyl shown in SEQ ID No.1 after engineered
Enzyme mutant UGT73B6MK+FAIt is catalyzed the vigor for being converted into Gastrodin to hydroxy-benzyl alcohol to improve a lot, external enzyme activity is parent
4.9 times of UGT73B6MK, internal conversion ratio are 3.8 times of parent UGT73B6MK, have better catalytic activity, are more suitable for
The industrialized production of Gastrodin.
Detailed description of the invention
Fig. 1 is 2 mutant UGT73B6 of embodimentMK+FAAnd parent UGT73B6MKPurifying protein SDS-PAGE electrophoresis,
It is the albumen purified that middle arrow, which is marked,；Wherein Lane1 is albumen Marker, and Lane2 is the parent UGT73B6 of purifyingMK, Lane3
For mutant UGT73B6MK+FA；
Fig. 2 is mutant recombinant bacterial strain and parent's recombinant bacterial strain Yield comparison in embodiment 4；Wherein Fig. 2-a is parent's weight
Group bacterial strain BL21 (DE3) pET28a-ugt73b6MK, Fig. 2-b is mutant recombinant bacterium BL21 (DE3) pET28a-ugt73b6MK +FA, Fig. 2-c is mutant recombinant bacterium BL21 (DE3) pET28a-ugt73b6MK+FS；
Fig. 3 is HPLC and the LC-MS mirror of 3 mutant recombinant bacterial strain of embodiment and parent's recombinant bacterial strain Synthesis Gastrodin
Determine map；Wherein Fig. 3-a is mutant recombinant bacterium BL21 (DE3) pET28a-ugt73b6MK+FAFermentation liquid HPLC map；Fig. 3-b
For parent's recombinant bacterial strain BL21 (DE3) pET28a-ugt73b6MKFermentation liquid HPLC map；Fig. 3-c is Gastrodin mark product HPLC；Figure
3-d is the LC-MS map of peak I；Fig. 3-e is the LC-MS map of peak II.
Specific embodiment
The present invention provides a kind of glycosyl transferase mutant, have the amino as shown in SEQ ID No.1 in sequence table
Acid sequence.
The present invention provides the glycosyl transferase mutant code gene, have such as SEQ ID No.2 in sequence table
Shown in nucleotide sequence.
In the present invention, a kind of preparation method of glycosyl transferase mutant code gene preferably includes following steps: since
The mutant UGT73B6 of glycosyl transferase UGT73B6 in the page root of kirilow rhodiola of plant libraryMKAs parent, with rite-directed mutagenesis
Method obtains mutant code gene, is specifically alanine Ala (A), shape by the site mutation of 389 phenylalanine Phe (F)
At mutant be greatly improved in terms of the enzyme activity for catalyzing and synthesizing Gastrodin.In the present invention, the side of the rite-directed mutagenesis
Method, comprising the following steps:
1) rite-directed mutagenesis primer is designed；
2) with UGT73B6 containing parentMKThe expression plasmid pET28a-ugt73B6 of geneMKFor template, in the step 1)
Rite-directed mutagenesis primer carries out PCR amplification, obtains PCR product；
3) the obtained PCR product is digested into removal pET28a-ugt73B6MKTemplate obtains postdigestive PCR product；
4) postdigestive PCR product being converted into bacillus coli DH 5 alpha, picking transformant, culture, plasmid extract, DNA sequencing,
Sequence analysis obtains the mutant code gene for occurring correctly to be mutated.
The present invention is not particularly limited the method for the primer of design rite-directed mutagenesis, and use is well known to those skilled in the art
Primer design method.In the present invention, the site of rite-directed mutagenesis is the 389th amino acids residue of glycosyl transferase.This
In invention, the 389th Phe codon TTC sports the high frequency AC pulse Link GCG of Ala.
In the present invention, sporting rite-directed mutagenesis primer used in Ala is preferably F389A-PS, F389A-PA.The F389A-
PS has the nucleotide sequence as shown in SEQ ID No.3 in sequence table.The F389A-PA has such as SEQ ID in sequence table
Nucleotide sequence shown in No.4.
As shown in table 1, wherein underscore is the base after mutating for No.3~4 SEQ ID.
1 mutant primer of table
| Primer | Primer sequence |
| F389A-PS | 5’-GTTACTTGGCCGGTGGCGGCTGAACAGTTCTAC-3’(SEQ ID No.3) |
| F389A-PA | 5’-GTAGAACTGTTCAGCCGCCACCGGCCAAGTAAC-3’(SEQ ID No.4) |
After obtaining rite-directed mutagenesis primer, the present invention is with UGT73B6 containing parentMKThe expression plasmid pET28a- of gene
ugt73B6MKFor template, PCR amplification is carried out with the rite-directed mutagenesis primer, obtains PCR product.
In the present invention, the parent UGT73B6MKThe expression plasmid pET28a-ugt73B6 of geneMKIn Patent No.
201510160496.3 Chinese patent in disclose.
In the present invention, the rite-directed mutagenesis PCR reaction system is preferably as follows:
| Reactive component | Volume (μ L) |
| 5 × Q5 buffer | 10 |
| dNTPs(2mM) | 5 |
| Upstream primer (25 μM) | 1 |
| Downstream primer (25 μM) | 1 |
| Template (20ng/ μ L) | 4 |
| Q5DNA polymerase (2.5U/ μ L) | 0.5 |
| ddH2O | 50 |
In the present invention, the rite-directed mutagenesis PCR amplification condition is preferably as follows: 98 DEG C of 30s；98 DEG C of 10s, 60 DEG C of 15s, 72 DEG C
2min 30s, 30 circulations；72℃5min.
After obtaining the PCR product, the PCR product is digested removal pET28a-ugt73B6 by the present inventionMKTemplate obtains
Postdigestive PCR product.
In the present invention, the digestion is preferably DpnI enzyme with enzyme.The digestions condition of the DpnI enzyme is preferably as follows: taking
1 μ LDpnI enzyme is added in 20 μ L of PCR product after amplification, and 37 DEG C of digestion are overnight.
Postdigestive PCR product is obtained, postdigestive PCR product is converted bacillus coli DH 5 alpha, culture, picking by the present invention
Transformant, extracts plasmid, Song Jinwei intelligence company DNA sequencing, analyzes to obtain the 389th Phe codon TTC by sequence and sports
The mutant of the high frequency AC pulse Link GCG of Ala.
In the present invention, the plasmid pET28a is purchased from Novagen, article No. 69864.
The present invention is not particularly limited connection method, using connection type well-known to those skilled in the art.
The present invention is not particularly limited the method for the conversion, using method for transformation well-known to those skilled in the art
?.The high frequency AC pulse Link GCG that the 389th Phe codon TTC sports Ala will be obtained and be named as ugt73B6MK+FAAnd containing prominent
The plasmid of variant gene is pET28a-ugt73B6MK+FA。
In the present invention, the type of the expression vector is not particularly limited, and is carried using eukaryotic expression known in the art
Body and prokaryotic expression carrier.The prokaryotic expression carrier is preferably bacillus coli DH 5 alpha.The bacillus coli DH 5 alpha comes
Source is not particularly limited, using known commercial goods.The bacillus coli DH 5 alpha is for all genes in the present invention
Clone.The carrier for expression of eukaryon is preferably saccharomyces cerevisiae.The source of the saccharomyces cerevisiae is not particularly limited, using ability
Commercial goods known to field technique personnel.
The present invention is not particularly limited the sequencing, is using sequencing mode mode well-known to those skilled in the art
It can.In the embodiment of the present invention, the sequencing trust money Wei Zhi company is carried out.
It include that the glycosyl transferase is prominent the present invention provides a kind of glycosyl transferase recombinant plasmid, in the recombinant plasmid
Variant encoding gene.The plasmid is not particularly limited, using plasmid known in the art.
The present invention provides a kind of glycosyl transferase recombinant bacterial strain, the recombinant bacterial strain is that conversion has the recombinant plasmid
Escherichia coli.
In the present invention, the expression vector of the glycosyl transferase recombinant bacterial strain is Escherichia coli.The kind of the Escherichia coli
Class is preferably bacterial strain BL21 (DE3).The present invention is not particularly limited the source of the bacterial strain BL21 (DE3), using this field
The source bacterial strain BL21 (DE3) known to technical staff.
The present invention provides the glycosyl transferase mutant or the glycosyl transferase recombinant bacterial strain in catalysis Gastrodin
Application in biosynthesis.
In the present invention, the glycosyl transferase mutant UGT73B6MK+FAWith the full cellular forms of engineering bacteria using catalysis, or
Person is with glycosyl transferase mutant UGT73B6MK+FAThick enzyme is used through partially purified or Economical Purification form.The present invention
In, the enzyme is including the use of this field immobilization technology by glycosyl transferase mutant UGT73B6 of the inventionMK+FAFixation is made
Change the solidification enzyme of enzyme or immobilized cell form.
In the present invention, the thick enzyme or the separating and extracting process through partially purified or Economical Purification enzyme do not have special limit
System, using thick enzyme extraction scheme known in the art.
In the present invention, catalysis substrate is preferably to hydroxy-benzyl alcohol and/or UDP-glucose.
In the present invention, the concentration of the substrate of the catalysis is preferably 2~5mmol/L.
In the present invention, the temperature of catalysis is preferably 28~35 DEG C, and more preferably 30 DEG C.
In the present invention, the concentration of glycosyl transferase mutant is preferably 80~100 μ g/ μ L.In the present invention, glycosyl transferase
Mutant is catalyzed in Mg2+Participate in lower carry out；The Mg2+Concentration be 4~6mmol/L.In the present invention, the catalysis of recombinant bacterial strain
When, condition of culture is as follows: the temperature of culture is 37 DEG C of cultures；The time of culture be preferably culture solution OD value be 0.6~0.8 when
Add substrate.Isopropyl-beta D-thio galactopyranoside of the glycosyl transferase recombinant bacterial strain in 0.1~1mmol/L is molten
15~25h of induction is carried out under the conditions of liquid.
Below with reference to embodiment to a kind of glycosyl transferase mutant provided by the invention and its in catalysis Gastrodin biology
Application in synthesis is described in detail, but they cannot be interpreted as limiting the scope of the present invention.
Embodiment 1
Mutant UGT73B6 is constructed using site-directed mutagenesis techniqueMK+FA.Rite-directed mutagenesis primer is designed, to contain parent
UGT73B6MKThe expression plasmid pET28a-ugt73B6 of geneMK(patent No. 201510160496.3) is template, carries out PCR expansion
Increase.Rite-directed mutagenesis PCR reaction system:
| Reactive component | Volume (μ L) |
| 5 × Q5 buffer | 10 |
| dNTPs(2mM) | 5 |
| Upstream primer (25 μM) | 1 |
| Downstream primer (25 μM) | 1 |
| Template (20ng/ μ L) | 4 |
| Q5DNA polymerase (2.5U/ μ L) | 0.5 |
| ddH2O | to50 |
PCR amplification condition: 98 DEG C of 30s；98 DEG C of 10s, 60 DEG C of 15s, 72 DEG C of 2min 30s, 30 circulations；72℃5min.
It is F389A-PS, F389A-PA that 389 Phe, which sport mutant primer used in Ala, as shown in table 1, wherein lower stroke
Line is the base after mutating.
1 mutant primer of table
| Primer | Primer sequence |
| F389A-PS | 5’-GTTACTTGGCCGGTGGCGGCTGAACAGTTCTAC-3’ |
| F389A-PA | 5’-GTAGAACTGTTCAGCCGCCACCGGCCAAGTAAC-3’ |
The PCR product of amplification DpnI enzyme direct enzyme cutting digests removal pET28a-ugt73B6MKTemplate.DpnI enzyme digestion disappears
Change condition: 1 μ L DpnI enzyme is added in the 20 μ L of PCR product after taking amplification, and 37 DEG C of digestion are overnight.
Postdigestive PCR product, directly conversion bacillus coli DH 5 alpha, culture, picking transformant extract plasmid, send gold only
Intelligence carries out DNA sequencing, and sequence, which is analyzed, determines UGT73B6MKThe height that 389th Phe codon TTC of gene sports Ala is frequent
Numeral GCG, i.e. acquisition ugt73B6 containing mutant geneMK+FAOr the plasmid pET28a-ugt73B6 containing mutant geneMK+FA。
Embodiment 2
Mutant UGT73B6MK+FAAnd parent UGT73B6MKRecombinant expression and purifying
By plasmid pET28a-ugt73B6MK, pET28a-ugt73b6MK+FALarge intestine is converted respectively with the method for chemical conversion
Bacillus strain BL21 (DE3).Recombinant bacterium construction method is as follows: take 100 μ L competent E.coli bacterial strain BL21 (DE3) cells in
On ice, 1 μ L plasmid is added after ten minutes, mixes gently, on ice place 30 minutes after, 42 DEG C heat shock 90 seconds, take out immediately in ice
It is upper to place 2 minutes, 800 μ L nonreactive LB liquid mediums are added, 37 DEG C, the recovery of 200rpm shaking table is cultivated 40 minutes, then by bacterium
Liquid is coated on the LB plate containing kanamycins.The conversion bacterial strain of expression vector is carried using kanamycin resistance screening and is passed through
It extracts plasmid and carries out digestion verification, obtain recombinant bacterial strain BL21 (DE3) pET28a-ugt73b6 containing mutantMK+FAAnd parent's weight
Group bacterial strain BL21 (DE3) pET28a-ugt73b6MK。
Protein induced expression, purifying: picking mutant strain and parent strain clone, until added with 50mg/L kanamycins
Then the liquid of 50mg/L kanamycins is added in 37 DEG C of overnight incubations in LB liquid medium with the ratio access 1000mL of 1:100
In body LB culture medium, 37 DEG C of cultures to OD are 0.6~0.8, and IPTG (the isopropyl-beta D-thio galactopyranose of 0.1mM is added
Glycosides) carry out 16 DEG C Fiber differentiation 20 hours.4 DEG C, 3000rpm is centrifuged 10min and collects thallus, is resuspended in the lysis buffer of 50mL
(50mM Tris/HCl, pH=8.0,10mM imidazoles, 10% glycerol).Thallus is resuspended, it is broken through French high pressure cell cracker
Broken 3 times, 10,000g, 4 DEG C of centrifugation 30min, supernatant immediately with balanced with lysis buffer after 2mL nickel column agarose resin
(Ni-NTAresin, QIAGENValencia, CA) mixing washes nickel column with the lysis buffer of 100mL after 4 DEG C combine 50min.
Then nickel column is eluted with 3mL elution buffer (50mM Tris/HCl, pH=8.0,0.5M imidazoles, 10% glycerol), collection is washed
De- liquid balances removal imidazoles with 50mM Tris/HCl (pH=8.0) buffer containing 10%glycerol to bag filter.Purifying
Albumen is concentrated in Milipore super filter tube (3kDa).Bradford protein determination kit measures the protein concentration after concentration and divides
Dress, -80 DEG C of preservations.
Albumen after purification carries out SDS-PAGE, as shown in Figure 1, albumen size about 54kDa, purified mutant
UGT73B6MK+FAAnd parent UGT73B6MKProtein concentration be respectively about 20mg/mL, mutant protein expression quantity is without obvious poor
Not.Purified albumen carries out external enzyme activity determination.
Embodiment 3
Mutant UGT73B6MK+FAAnd UGT73B6MKActivity ratio compared with
(1) internal transformation experiment
Each bacterial strain clone is picked them separately, including recombinant bacterial strain BL21 (DE3) pET28a-ugt73b6 containing mutantMK+FA,
Parent's recombinant bacterial strain BL21 (DE3) pET28a-ugt73b6MKAnd the bacterial strain BL21 (DE3) of this laboratory patent applied for
pET28a-ugt73b6FS+MK(patent No. 201510160497.8), until in LB liquid medium added with 50mg/L kanamycins
Then 37 DEG C of overnight incubations are added in the LB liquid medium of 50mg/L kanamycins, 37 DEG C with the ratio access 50mL of 1:100
Culture is 0.6-0.8 to OD, and the IPTG (isopropyl-beta D-thio galactopyranoside) that 0.1mM is added carries out 16 DEG C of induction trainings
It supports 20 hours.By the bacterium solution after induction, 4000rpm is centrifuged 10 minutes, removes supernatant, collects thallus, it is fresh to be forwarded to 50mL
M9Y culture medium, and addition 2mM substrate is fed to hydroxy-benzyl alcohol.It is subsequently placed in 30 DEG C, 200rpm shaken cultivation 84 hours, from 24
Hour begins, and takes within every 12 hours fermentation liquid, and centrifuging and taking supernatant, carries out Gastrodin LC-MS identification and HPLC measures Gastrodin yield,
And compare the internal conversion ratio of mutant protein and Parent Protease.
LC-MS qualification result shows that peak I is Gastrodin, and II is added substrate to hydroxy-benzyl alcohol, such as Fig. 3-d, Fig. 3-
e.Internal transformation experiment the result shows that, 2mM substrate is fed in shaking flask incubation, the Gastrodin yield of each bacterial strain became at 72 hours
In stabilization.72 hours mutant UGT73B6MK+FAThe yield for converting Gastrodin is 574mg/L, compares parent UGT73B6MKConversion
The yield of Gastrodin is 161mg/L, compares the mutant UGT73B6 of patent applied forFS+MKConversion Gastrodin yield be
317mg/L such as Fig. 2.UGT73B6MK+FAConversion 2mM substrate is parent to hydroxy-benzyl alcohol synthetic gastrodin conversion ratio in vivo
UGT73B6MK3.8 times, be the mutant ugt73b6 of patent applied forFS+MK1.7 times.And it adds substrate 24 hours, dashes forward
Variant UGT73B6MK+FAInverted 70% substrate, has converted 2mM at culture 72 hours to hydroxy-benzyl alcohol synthetic gastrodin completely,
Such as Fig. 2.
(2) vitro conversion is tested
Enzymatic reaction temperature is 30 DEG C, and reaction system is 100 μ L, Tris/HCl containing 50mM (pH=8.0), 5mM
MgCl2, the 100 pure enzymes of μ g, the concentration to hydroxy-benzyl alcohol and UDP-glucose is respectively 5mM, and the reaction time is 10min (via preliminary
Test determines), 4 μ L 10%TFA are added and terminate enzyme reaction.Reaction product 10,000g are centrifuged 10min, and 50 μ L supernatants is taken to carry out
HPLC analysis, measures the yield of synthetic gastrodin, calculates enzymatic reaction initial velocity.
The result shows that mutant UGT73B6MK+FAThe initial velocity of reaction for converting Gastrodin is 2.68 μM/min, parent
UGT73B6MKThe initial velocity of reaction for converting Gastrodin is 0.5 μM/min, shows mutant UGT73B6MK+FAEnzyme activity is parent
UGT73B6MK4.9 times.
Embodiment 4
Detection to hydroxy-benzyl alcohol and Gastrodin
(1) HPLC is detected
Agilent liquid chromatograph, determination condition: C18 column (4.6 × 250mm)；Detection wavelength 224nm；Mobile phase A=water
(containing 0.1% volume formic acid), B=methanol；Flow velocity=1ml/min；Condition of gradient elution: 0-20min, 10% volume B；Sample volume
50μL。
(2) LC-MS is analyzed
The condition of LC-MS analysis: C18 column (4.6 × 250mm)；Detection wavelength 224nm；Mobile phase A=water (contains 0.1 body
Product % formic acid), B=methanol；Flow velocity=1ml/min；Elution requirement: 0~35min, 10% volume B；20 μ L of sample volume；ESI is just
Ion source, molecular weight scanning range 50~800.
Testing result is as shown in Figure 3.Testing result is as shown in Figure 3.Show mutant UGT73B6MK+FAConversion of substrate is to hydroxyl
Base benzylalcohol (peak II) synthetic gastrodin (peak I), and conversion 2mM substrate is parent to hydroxy-benzyl alcohol synthetic gastrodin conversion ratio in vivo
This UGT73B6MK3.8 times.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
<110>Tianjin Institute of Industrial Biotechnology, Chinese Accademy of Sciences
<120>a kind of glycosyl transferase mutant and its application in catalysis Gastrodin biosynthesis
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 480
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 1
Met Gly Ser Glu Thr Arg Pro Leu Ser Ile Phe Phe Phe Pro Phe Met
1 5 10 15
Ala His Gly His Met Ile Pro Met Val Asp Met Ala Arg Leu Phe Ala
20 25 30
Ser Gln Gly Val Arg Cys Thr Ile Val Thr Thr Pro Gly Asn Gln Pro
35 40 45
Leu Ile Ala Arg Ser Ile Gly Lys Val Gln Leu Leu Gly Phe Glu Ile
50 55 60
Gly Val Thr Thr Ile Pro Phe Arg Gly Thr Glu Phe Gly Leu Pro Asp
65 70 75 80
Gly Cys Glu Asn Leu Asp Ser Val Pro Ser Pro Gln His Val Phe His
85 90 95
Phe Phe Glu Ala Ala Gly Ser Leu Arg Glu Pro Phe Glu Gln Leu Leu
100 105 110
Glu Glu His Lys Pro Asp Cys Val Val Gly Asp Met Phe Phe Pro Trp
115 120 125
Ser Thr Asp Ser Ala Ala Lys Phe Gly Ile Pro Arg Leu Val Phe His
130 135 140
Gly Thr Ser Tyr Phe Ala Leu Cys Ala Gly Glu Ala Val Arg Ile His
145 150 155 160
Lys Pro Tyr Leu Ser Val Ser Ser Asp Asp Glu Pro Phe Val Ile Pro
165 170 175
Gly Leu Pro Asp Glu Ile Lys Leu Thr Lys Ser Gln Leu Pro Met His
180 185 190
Leu Leu Glu Gly Lys Lys Asp Ser Val Leu Ala Gln Leu Leu Asp Glu
195 200 205
Val Lys Glu Thr Glu Val Ser Ser Tyr Gly Val Ile Val Asn Ser Ile
210 215 220
Tyr Glu Leu Glu Pro Ala Tyr Ala Asp Tyr Phe Arg Asn Val Leu Lys
225 230 235 240
Arg Arg Ala Trp Glu Ile Gly Pro Leu Ser Leu Cys Asn Arg Asp Val
245 250 255
Glu Glu Lys Ala Met Arg Gly Lys Gln Ala Ala Ile Asp Gln His Glu
260 265 270
Cys Leu Lys Trp Leu Asp Ser Lys Glu Pro Asp Ser Val Val Tyr Val
275 280 285
Cys Phe Gly Ser Thr Cys Lys Phe Pro Asp Asp Gln Leu Ala Glu Ile
290 295 300
Ala Ser Gly Leu Glu Ala Ser Gly Gln Gln Phe Ile Trp Val Ile Arg
305 310 315 320
Arg Met Ser Asp Asp Ser Lys Glu Asp Tyr Leu Pro Lys Gly Phe Glu
325 330 335
Glu Arg Val Lys Asp Arg Ala Leu Leu Ile Arg Gly Trp Ala Pro Gln
340 345 350
Val Leu Ile Leu Asp His Gln Ser Val Gly Gly Phe Val Ser His Cys
355 360 365
Gly Trp Asn Ser Thr Leu Glu Gly Ile Ser Ala Gly Leu Pro Met Val
370 375 380
Thr Trp Pro Val Ala Ala Glu Gln Phe Tyr Asn Glu Lys Leu Leu Thr
385 390 395 400
Glu Val Leu Lys Ile Gly Val Ala Val Gly Ala Arg Lys Trp Arg Gln
405 410 415
Leu Val Gly Asp Phe Val His Lys Asp Ala Ile Gln Arg Ala Val Arg
420 425 430
Glu Ile Met Glu Gly Glu Glu Ala Glu Glu Arg Arg Ile Ile Ala Arg
435 440 445
Gln Met Gly Lys Met Ala Lys Arg Ala Val Glu Lys Asp Gly Ser Ser
450 455 460
Trp Thr Asn Leu Asn Asn Leu Leu Gln Glu Leu Lys Leu Lys Lys Val
465 470 475 480
<210> 2
<211> 1440
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
atgggctctg aaactcgccc gctgagcatc ttcttttttc cgtttatggc gcatggccac 60
atgattccga tggtggatat ggcacgtctg tttgcttctc agggtgtgcg ttgcaccatt 120
gttaccactc cgggtaacca gccgctgatt gctcgctcta tcggtaaggt tcagctgctg 180
ggttttgaaa ttggtgtgac tactatcccg ttccgcggta ctgagttcgg cctgccggat 240
ggctgtgaaa acctggatag cgtgccgagc ccgcagcatg tgtttcattt ctttgaggca 300
gcgggtagcc tgcgtgagcc gtttgaacag ctgctggaag agcacaaacc ggactgtgtt 360
gtgggcgata tgttctttcc gtggtctacc gactctgcgg ctaaattcgg tattccgcgc 420
ctggttttcc acggtacctc ctacttcgcg ctgtgcgctg gcgaagcagt gcgtattcat 480
aagccgtacc tgtctgtgtc ttctgatgat gaaccgttcg ttattccggg cctgccggac 540
gagatcaaac tgaccaagtc ccagctgccg atgcacctgc tggagggtaa gaaagactct 600
gttctggcac agctgctgga tgaggtgaaa gaaactgagg tttcctctta cggtgttatc 660
gttaactcta tctacgaact ggaaccggct tacgcagatt acttccgtaa cgttctgaag 720
cgccgtgcgt gggagatcgg tccgctgtct ctgtgtaacc gtgacgttga agagaaagcg 780
atgcgtggta agcaggctgc tatcgatcag catgaatgcc tgaaatggct ggattccaaa 840
gaaccggatt ccgttgttta cgtttgtttt ggtagcactt gcaaattccc ggatgatcag 900
ctggcggaaa tcgcgtctgg tctggaggca agcggccagc agttcatctg ggttattcgc 960
cgtatgtctg acgactctaa ggaagactac ctgccgaaag gtttcgaaga gcgtgttaag 1020
gaccgtgctc tgctgattcg cggttgggct ccgcaggttc tgatcctgga ccatcagtcc 1080
gttggcggtt ttgtttctca ctgtggttgg aactctaccc tggaaggcat cagcgcgggt 1140
ctgccgatgg ttacttggcc ggtggcggct gaacagttct acaacgaaaa actgctgacc 1200
gaggtgctga aaatcggtgt tgcagtgggt gctcgtaagt ggcgtcagct ggtgggtgac 1260
ttcgttcaca aagacgctat tcagcgtgcg gtgcgtgaaa ttatggaggg cgaagaggcg 1320
gaggaacgtc gtatcatcgc gcgtcagatg ggtaaaatgg cgaaacgcgc ggtggagaag 1380
gacggtagct cttggaccaa cctgaacaac ctgctgcagg aactgaagct gaagaaggtt 1440
<210> 3
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
gttacttggc cggtggcggc tgaacagttc tac 33
<210> 4
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
gtagaactgt tcagccgcca ccggccaagt aac 33
Claims (8)
1. a kind of glycosyl transferase mutant, which is characterized in that have the amino acid sequence as shown in SEQ ID No .1
Column.
2. glycosyl transferase mutant code gene described in claim 1, which is characterized in that have as in sequence table
Nucleotide sequence shown in SEQIDNo.2.
3. a kind of glycosyl transferase recombinant plasmid, which is characterized in that include glycosyl as claimed in claim 2 in the recombinant plasmid
Transferase mutant code gene.
4. a kind of glycosyl transferase recombinant bacterial strain, which is characterized in that the recombinant bacterial strain includes recombinant plasmid described in claim 3
Expression vector.
5. glycosyl transferase recombinant bacterial strain described in glycosyl transferase mutant or claim 4 described in claim 1 is at catalysis bottom
Application in object synthetic gastrodin.
6. application according to claim 5, which is characterized in that the substrate is to hydroxy-benzyl alcohol and/or UDP-glucose.
7. application according to claim 5 or 6, which is characterized in that the temperature of the catalysis is 28~35 DEG C.
8. application according to claim 7, which is characterized in that the glycosyl transferase recombinant bacterial strain catalysis substrate synthesizes day
During numb element, isopropyl-beta D-thio galactopyranosyl of the glycosyl transferase recombinant bacterial strain in 0.1~1.0mmol/L
15~25h of induction is carried out under the conditions of glycoside solution.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111019918A (en) * | 2019-11-20 | 2020-04-17 | 广东广业清怡食品科技有限公司 | Glycosyltransferase mutant and application thereof |
| CN113755354A (en) * | 2020-07-16 | 2021-12-07 | 中国科学院天津工业生物技术研究所 | Recombinant saccharomyces cerevisiae for producing gastrodin by using glucose and application thereof |
| CN114058602A (en) * | 2020-07-30 | 2022-02-18 | 中国中医科学院中药研究所 | Sinkiang lithospermum caffeic acid and rosmarinic acid glycosyl transferase as well as coding gene and application |
| CN115058400A (en) * | 2022-04-19 | 2022-09-16 | 湖北大学 | Application of glycosyl transferase RrUGT3 from rose in biosynthesis of gastrodin |
| CN117737029A (en) * | 2023-12-25 | 2024-03-22 | 皖西学院 | Glycosyltransferase mutant and application thereof in synthesis of collaterals plug |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104774816A (en) * | 2015-04-07 | 2015-07-15 | 中国科学院天津工业生物技术研究所 | Glycosyl transferase synthesized by catalytic gastrodine as well as gene of encoding enzyme and application |
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2017
- 2017-12-29 CN CN201711485080.4A patent/CN109957555B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104774816A (en) * | 2015-04-07 | 2015-07-15 | 中国科学院天津工业生物技术研究所 | Glycosyl transferase synthesized by catalytic gastrodine as well as gene of encoding enzyme and application |
Non-Patent Citations (1)
| Title |
|---|
| GAVIN J. WILLIAMS: "Optimizing glycosyltransferase specificity via ‘hot spot’saturation mutagenesis presents a new catalyst for novobiocin", 《CHEM BIOL.》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111019918A (en) * | 2019-11-20 | 2020-04-17 | 广东广业清怡食品科技有限公司 | Glycosyltransferase mutant and application thereof |
| CN113755354A (en) * | 2020-07-16 | 2021-12-07 | 中国科学院天津工业生物技术研究所 | Recombinant saccharomyces cerevisiae for producing gastrodin by using glucose and application thereof |
| CN113755354B (en) * | 2020-07-16 | 2024-01-16 | 中国科学院天津工业生物技术研究所 | Recombinant saccharomyces cerevisiae for producing gastrodin by utilizing glucose and application thereof |
| CN114058602A (en) * | 2020-07-30 | 2022-02-18 | 中国中医科学院中药研究所 | Sinkiang lithospermum caffeic acid and rosmarinic acid glycosyl transferase as well as coding gene and application |
| CN114058602B (en) * | 2020-07-30 | 2023-08-22 | 中国中医科学院中药研究所 | Sinkiang lithospermum caffeic acid and rosmarinic acid glycosyltransferase, and coding gene and application thereof |
| CN115058400A (en) * | 2022-04-19 | 2022-09-16 | 湖北大学 | Application of glycosyl transferase RrUGT3 from rose in biosynthesis of gastrodin |
| CN115058400B (en) * | 2022-04-19 | 2023-04-25 | 湖北大学 | Application of glycosyltransferase RrUGT3 from roses in biosynthesis of gastrodin |
| CN117737029A (en) * | 2023-12-25 | 2024-03-22 | 皖西学院 | Glycosyltransferase mutant and application thereof in synthesis of collaterals plug |
| CN117737029B (en) * | 2023-12-25 | 2024-06-07 | 皖西学院 | Glycosyltransferase mutant and application thereof in synthesis of collaterals plug |
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| CN109957555B (en) | 2021-04-02 |
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