CN109295027A

CN109295027A - A kind of glycosyl transferase mutant

Info

Publication number: CN109295027A
Application number: CN201811311058.2A
Authority: CN
Inventors: 范文超; 高书良; 王金刚; 梁岩; 袁圣伦; 任亮
Original assignee: Zhejiang Huari Biotechnology Co Ltd
Current assignee: Zhejiang Huari Biotechnology Co Ltd
Priority date: 2018-11-06
Filing date: 2018-11-06
Publication date: 2019-02-01
Anticipated expiration: 2038-11-06
Also published as: CN109295027B

Abstract

The present invention obtains the glycosyl transferase mutant SEQ ID NOs:3-6 of enzyme activity raising by the method for directed evolution, these mutant or its expression microorganism energy efficient catalytic protopanoxadiol prepare Ginsenoside compound K by glycosyl transfer reaction, have industrialized developing and application prospect.

Description

A kind of glycosyl transferase mutant

Technical field

The invention belongs to biocatalysis fields, specifically, being related to a kind of glycosyl transferase mutant and its in synthesis people Join the purposes in saponin(e CK.

Background technique

Ginsenoside is the general name for the saponin(e being separated to from ginseng and its congener (such as Radix Notoginseng, American Ginseng), is belonged to It is the main effective ingredient in ginseng in Triterpene saponins.Currently, at least 60 kinds of saponin(es have been isolated from ginseng, In some ginsenosides be proven to have extensive physiological function and medical value: including antitumor, immunological regulation, it is antifatigue, The functions such as shield heart, protect liver.According to the form of aglycon aglucon, ginsenoside is typically divided into three classes, oleanane type, protoplast's ginseng two Alcohol type (PPD type) and protopanaxatriol type (PPT type).Wherein, the ginsenoside compound K (CK) as shown in following formula I is The most biological ingredient with pharmaceutical active of PPD type ginsenoside.Ginsenoside compound K has good anti-tumor activity, can induce Apoptosis of tumor cells inhibits Nasopharyngeal neoplasms.It is tested in conjunction with radiation and chemotherapy, and the effect of radiation and chemotherapy can be enhanced Fruit.In addition to this, Ginsenoside compound K also has antiallergic activity, anti-inflammatory activity, and can play neuroprotection, resists Diabetes effect and function of anti-skin aging.The pharmacological activity of Ginsenoside compound K has the spy of multiple target point, high activity and hypotoxicity Point.

Ginsenosides compound main source is by directly extracting in plant ginseng, due to Ginseng Growth all the time Period is long, growing environment requirement is harsh, is unable to satisfy social demand.It can be fermented by enzymatic or microbial engineering bacteria at present Ginsenoside compound is synthesized, but yield is lower, needs more Optimization Works and improve fermentation yield.Ginsenoside compound K is in ginseng Middle content is extremely low, it is the main generation that protopanaxadiol-type's saponin(e (PPD type) is generated after microbial hydrolytic in human body intestinal canal Thank to product.Studies have shown that most protopanaxadiol-type's saponin(es are only metabolized as just be absorbed by the body after CK, so ginseng soap Glycosides CK is directly by body absorption and the real entity that plays a role, and other saponin(es are prodrug.

Effect of the protopanoxadiol (PPD) in UDP glycosyl transferase (uridine 5'-diphosphate glucosyltransferase, UGT) CK is produced down.Therefore it develops the glycosyl transferase that specificity is high, enzyme activity is high and produces the effective of Ginsenoside compound K as a kind of Approach.

Summary of the invention

In order to develop the UDP glycosyl transferase (UGT) of high catalytic efficiency, the present invention is with the source ginseng Panax ginseng Uridine 5'-diphosphate (UDP) glycosyl transferase UGT1 (GenBank:AIE12479.1, i.e. SEQ ID NO:1) based on, lead to The method for crossing fallibility PCR establishes glycosyltransferase gene mutation library, then obtains enzyme activity by screening-gene mutation library and obviously mention High glycosyl transferase mutant.

Therefore, the first purpose of this invention is to provide a kind of glycosyl transferase, amino acid sequence are as follows:

SEQ ID NO:3, for SEQ ID NO:1 the 82nd H replaces with A, the 175th S replaces with the mutation of A Body, it may be assumed that

MKSELIFLPAPAIGHLVGMVEMAKLFISRHENLSVTVLIAKFYMDTGVDNYNKSLLTNPTPRLTIVNL PETDPQNYMLKPRAAIFPSVIETQKTHVRDIISGMTQSESTQVVGLLADLLFINIMDIANEFNVPTYVYSPAGAGH LGLAFHLQTLNDKKQDVTEFRNSDTELLVPAFANPVPAEVLPSMYVDKEGGYDYLFSLFRRCRESKAIIINTFEEL EPYAINSLRMDSMIPPIYPVGPILNLNGDGQNSDEAAVILGWLDDQPPSSVVFLCFGSYGSFQENQVKEIAMGLER SGHRFLWSLRPSIPKGETKLQLKYSNLKEILPVGFLDRTSCVGKVIGWAPQVAVLGHESVGGFLSHCGWNSTLESV WCGVPVATWPMYGEQQLNAFEMVKELGIAVEIEVDYKKDYFNMKNDFIVRAEEIETKIKKLMMDENNSEIRKKVKE MKEKSRAAMSENGSSYNSLAKLFEEIM(SEQ ID NO:3)；

SEQ ID NO:4, for SEQ ID NO:1 the 82nd H replaces with A, the 88th V replaces with A, the 122nd N replace with K, the 129th E replaces with the mutant of T, it may be assumed that

MKSELIFLPAPAIGHLVGMVEMAKLFISRHENLSVTVLIAKFYMDTGVDNYNKSLLTNPTPRLTIVNL PETDPQNYMLKPRAAIFPSAIETQKTHVRDIISGMTQSESTQVVGLLADLLFIKIMDIANTFNVPTYVYSPAGAGH LGLAFHLQTLNDKKQDVTEFRNSDTELLVPSFANPVPAEVLPSMYVDKEGGYDYLFSLFRRCRESKAIIINTFEEL EPYAINSLRMDSMIPPIYPVGPILNLNGDGQNSDEAAVILGWLDDQPPSSVVFLCFGSYGSFQENQVKEIAMGLER SGHRFLWSLRPSIPKGETKLQLKYSNLKEILPVGFLDRTSCVGKVIGWAPQVAVLGHESVGGFLSHCGWNSTLESV WCGVPVATWPMYGEQQLNAFEMVKELGIAVEIEVDYKKDYFNMKNDFIVRAEEIETKIKKLMMDENNSEIRKKVKE MKEKSRAAMSENGSSYNSLAKLFEEIM(SEQ ID NO:4)；

SEQ ID NO:5, for SEQ ID NO:1 the 82nd H replaces with A, the 88th V replaces with A, the 122nd N replace with K, the 129th E replaces with T, the 315th K replaces with the mutant of D, it may be assumed that

MKSELIFLPAPAIGHLVGMVEMAKLFISRHENLSVTVLIAKFYMDTGVDNYNKSLLTNPTPRLTIVNL PETDPQNYMLKPRAAIFPSAIETQKTHVRDIISGMTQSESTQVVGLLADLLFIKIMDIANTFNVPTYVYSPAGAGH LGLAFHLQTLNDKKQDVTEFRNSDTELLVPSFANPVPAEVLPSMYVDKEGGYDYLFSLFRRCRESKAIIINTFEEL EPYAINSLRMDSMIPPIYPVGPILNLNGDGQNSDEAAVILGWLDDQPPSSVVFLCFGSYGSFQENQVKEIAMGLER SGHRFLWSLRPSIPKGETDLQLKYSNLKEILPVGFLDRTSCVGKVIGWAPQVAVLGHESVGGFLSHCGWNSTLESV WCGVPVATWPMYGEQQLNAFEMVKELGIAVEIEVDYKKDYFNMKNDFIVRAEEIETKIKKLMMDENNSEIRKKVKE MKEKSRAAMSENGSSYNSLAKLFEEIM(SEQ ID NO:5)；Or

SEQ ID NO:6, for SEQ ID NO:1 the 82nd H replaces with A, the 88th V replaces with A, the 122nd N replace with K, the 129th E replaces with T, the 175th S replaces with the mutant of A, it may be assumed that

MKSELIFLPAPAIGHLVGMVEMAKLFISRHENLSVTVLIAKFYMDTGVDNYNKSLLTNPTPRLTIVNL PETDPQNYMLKPRAAIFPSAIETQKTHVRDIISGMTQSESTQVVGLLADLLFIKIMDIANTFNVPTYVYSPAGAGH LGLAFHLQTLNDKKQDVTEFRNSDTELLVPAFANPVPAEVLPSMYVDKEGGYDYLFSLFRRCRESKAIIINTFEEL EPYAINSLRMDSMIPPIYPVGPILNLNGDGQNSDEAAVILGWLDDQPPSSVVFLCFGSYGSFQENQVKEIAMGLER SGHRFLWSLRPSIPKGETKLQLKYSNLKEILPVGFLDRTSCVGKVIGWAPQVAVLGHESVGGFLSHCGWNSTLESV WCGVPVATWPMYGEQQLNAFEMVKELGIAVEIEVDYKKDYFNMKNDFIVRAEEIETKIKKLMMDENNSEIRKKVKE MKEKSRAAMSENGSSYNSLAKLFEEIM(SEQ ID NO:6)。

It is preferred that the amino acid sequence of above-mentioned glycosyl transferase is SEQ ID NO:6.

Second object of the present invention is to provide the gene for encoding above-mentioned glycosyl transferase.

Preferably, the gene for encoding above-mentioned glycosyl transferase SEQ ID NO:6 has following base sequences:

ATGAAAAGCGAGCTCATCTTTCTGCCAGCGCCAGCGATTGGCCATCTGGTGGGCATGGTGGAAATGGC CAAGCTGTTCATTAGCCGTCACGAGAACCTCAGTGTGACGGTGCTGATCGCCAAGTTTTACATGGACACGGGCGTG GACAACTATAACAAGAGTCTGCTGACCAATCCGACCCCGCGCCTCACGATTGTGAACCTCCCGGAAACCGACCCGC AGAATTACATGCTGAAGCCACGCGCCGCGATTTTCCCGAGCGCGATTGAAACGCAAAAGACCCACGTGCGCGACAT CATCAGCGGCATGACCCAGAGCGAAAGCACCCAAGTGGTTGGTCTGCTGGCGGATCTGCTGTTCATCAAGATCATG GATATCGCCAACACGTTCAACGTGCCAACGTATGTGTACAGTCCAGCGGGTGCCGGTCATCTGGGTCTGGCCTTCC ATCTGCAAACCCTCAACGACAAGAAGCAAGATGTGACCGAGTTCCGCAATAGCGATACGGAACTGCTCGTGCCAGC CTTCGCGAATCCAGTTCCAGCGGAGGTTCTGCCAAGCATGTACGTGGACAAAGAGGGCGGCTATGACTATCTGTTT AGTCTGTTCCGCCGCTGTCGCGAAAGCAAGGCCATCATCATCAACACCTTTGAAGAACTCGAGCCGTACGCGATTA ACAGTCTGCGCATGGACAGCATGATTCCGCCGATCTATCCAGTTGGTCCGATCCTCAATCTGAACGGCGATGGCCA AAACAGTGATGAAGCCGCGGTGATCCTCGGCTGGCTGGATGATCAACCGCCAAGCAGCGTTGTGTTCCTCTGCTTC GGCAGCTATGGCAGCTTTCAAGAAAACCAAGTTAAAGAGATCGCGATGGGTCTGGAACGTAGCGGCCATCGCTTTC TCTGGAGTCTGCGTCCGAGCATCCCAAAGGGCGAAACGAAGCTGCAACTGAAGTACAGCAATCTGAAGGAAATCCT CCCAGTGGGTTTTCTGGATCGCACCAGCTGCGTTGGCAAAGTGATTGGTTGGGCCCCACAAGTTGCGGTTCTGGGT CACGAAAGCGTTGGTGGCTTTCTGAGCCATTGTGGCTGGAACAGTACGCTGGAAAGCGTGTGGTGCGGCGTTCCAG TTGCCACGTGGCCAATGTATGGCGAACAGCAGCTGAACGCCTTCGAGATGGTTAAGGAACTGGGCATCGCCGTGGA GATCGAAGTTGACTACAAGAAAGATTACTTTAACATGAAGAATGACTTCATCGTGCGCGCCGAGGAGATCGAGACC AAAATCAAAAAACTGATGATGGATGAAAATAATAGCGAAATCCGCAAGAAAGTGAAAGAGATGAAGGAAAAAAGCC GTGCGGCGATGAGCGAGAATGGCAGCAGCTACAATAGTCTGGCCAAGCTCTTCGAGGAAATCATGTAA(SEQ ID NO:7)。

Third object of the present invention is to provide the plasmid comprising said gene.

Fourth object of the present invention is to provide the microorganism for having converted above-mentioned plasmid.

Preferably, mentioned microorganism is selected from Escherichia coli, yeast, hay bacillus.More preferable e. coli bl21 (DE3).

Of the invention the 5th is designed to provide purposes of the above-mentioned glycosyl transferase in production Ginsenoside compound K.

In one embodiment, such use is using protopanoxadiol as substrate raw material, under glycosyl transferase catalysis Ginsenoside compound K is synthesized by glycosyl transfer reaction.

Preferably, above-mentioned enzymic catalytic reaction is in pH8.0,35 DEG C of temperature progress.

The mutant SEQ ID NOs:4-6 that the present invention constructs significantly improves glycosyl transfer reaction catalysis activity, can be efficient Rate catalysis substrate protopanoxadiol generate Ginsenoside compound K, the enzyme-catalyzed reaction condition is mild, has and industrialized developing and answers Use prospect.

Specific embodiment

The present invention is described in further details below in conjunction with specific embodiment.It should be understood that following embodiment is only used for The bright present invention is not for limiting the scope of the invention.

Additive amount, content and the concentration of many kinds of substance is referred to herein, wherein the percentage composition, except special instruction Outside, all refer to mass percentage.

It in the embodiments herein, is illustrated if do not made for reaction temperature or operation temperature, the temperature is logical Often refer to room temperature (15-30 DEG C).

For simplicity, amino acid abbreviations herein both can be used English trigram, can also use English-word Mother, this be it is well known to those skilled in the art, these abbreviations are listed in the following table:

1 amino acid bilingual of table and abbreviation

Alanine	Alanine	A or Ala	Aliphatic category
				Arginine	Arginine	R or Arg	Basic amine group acids
Asparagine	Asparagine	N or Asn	Amides
				Aspartic acid	Aspartic acid	D or Asp	Acidic amino acid class
Cysteine	Cysteine	C or Cys	Sulfur-bearing class
				Glutamine	Glutamine	Q or Gln	Amides
Glutamic acid	Glutamic acid	E or Glu	Acidic amino acid class
				Glycine	Glycine	G or Gly	Aliphatic category
Histidine	Histidine	H or His	Basic amine group acids
				Isoleucine	Isoleucine	I or Ile	Aliphatic category
Leucine	Leucine	L or Leu	Aliphatic category
				Lysine	Lysine	K or Lys	Basic amine group acids
Methionine	Methionine	M or Met	Sulfur-bearing class
				Phenylalanine	Phenylalanine	F or Phe	Aromatic
Proline	Proline	P or Pro	Imino acid
				Serine	Serine	S or Ser	Hydroxy kind
Threonine	Threonine	T or Thr	Hydroxy kind
				Tryptophan	Tryptophan	W or Trp	Aromatic
Tyrosine	Tyrosine	Y or Tyr	Aromatic
				Valine	Valine	V or Val	Aliphatic category

As the foundation forms of building glycosyl transferase mutant, the wild type from ginseng Panax ginseng is urinated The amino acid sequence of glycosides 5 '-diphosphonic acid (UDP) glycosyl transferase UGT1 (GenBank:AIE12479.1) is SEQ ID NO:1:

MKSELIFLPAPAIGHLVGMVEMAKLFISRHENLSVTVLIAKFYMDTGVDNYNKSLLTNPTPRLTIVNL PETDPQNYMLKPRHAIFPSVIETQKTHVRDIISGMTQSESTQVVGLLADLLFINIMDIANEFNVPTYVYSPAGAGH LGLAFHLQTLNDKKQDVTEFRNSDTELLVPSFANPVPAEVLPSMYVDKEGGYDYLFSLFRRCRESKAIIINTFEEL EPYAINSLRMDSMIPPIYPVGPILNLNGDGQNSDEAAVILGWLDDQPPSSVVFLCFGSYGSFQENQVKEIAMGLER SGHRFLWSLRPSIPKGETKLQLKYSNLKEILPVGFLDRTSCVGKVIGWAPQVAVLGHESVGGFLSHCGWNSTLESV WCGVPVATWPMYGEQQLNAFEMVKELGIAVEIEVDYKKDYFNMKNDFIVRAEEIETKIKKLMMDENNSEIRKKVKE MKEKSRAAMSENGSSYNSLAKLFEEIM(SEQ ID NO:1)。

When using Escherichia coli as host expresses wild type glycosyl transferase SEQ ID NO:1, it is excellent codon has been carried out Change, coding gene sequence is SEQ ID NO:2:

ATGAAATCTGAATTAATCTTTTTACCAGCACCAGCTATTGGTCACTTGGTCGGTATGGTCGAGATGGC TAAGTTGTTTATTTCAAGACATGAAAATTTGTCAGTCACTGTTTTGATTGCAAAATTCTATATGGACACTGGTGTT GATAACTATAATAAGTCTTTATTAACAAATCCAACTCCAAGATTAACTATTGTTAATTTGCCAGAAACTGACCCAC AAAACTACATGTTGAAACCAAGACATGCTATTTTTCCATCTGTTATTGAAACTCAGAAAACTCATGTTAGAGATAT TATTTCTGGTATGACACAATCTGAGTCTACACAAGTTGTCGGTTTGTTGGCAGACTTGTTATTTATAAATATTATG GATATTGCTAATGAGTTTAACGTTCCTACTTACGTTTACTCACCAGCTGGTGCTGGTCATTTGGGTTTGGCTTTCC ACTTGCAAACTTTAAATGATAAAAAACAAGATGTTACAGAATTTAGAAACTCTGATACTGAATTATTGGTTCCATC TTTCGCAAACCCCGTTCCCGCTGAGGTTTTGCCATCTATGTACGTTGATAAGGAGGGTGGTTACGATTACTTATTT TCTTTGTTCAGAAGATGTAGAGAATCTAAGGCAATTATTATTAATACTTTTGAGGAATTAGAGCCATACGCAATTA ATTCTTTAAGAATGGACTCTATGATTCCACCAATTTATCCAGTCGGTCCTATATTGAACTTGAACGGTGACGGTCA AAACTCTGACGAGGCTGCTGTCATTTTGGGTTGGTTGGACGACCAACCACCTTCTTCTGTCGTCTTTTTATGTTTT GGTTCTTACGGTTCATTCCAAGAAAATCAAGTTAAAGAGATTGCAATGGGTTTGGAGCGTTCTGGTCATAGGTTCT TGTGGTCTTTAAGACCATCTATTCCTAAGGGTGAGACTAAATTGCAATTAAAATACTCTAACTTAAAAGAAATTTT GCCAGTTGGTTTTTTGGATAGAACTTCATGCGTTGGTAAAGTCATTGGTTGGGCTCCACAAGTTGCTGTCTTGGGT CATGAATCTGTTGGTGGTTTTTTGTCACACTGTGGTTGGAACTCTACTTTGGAATCAGTCTGGTGCGGTGTTCCAG TTGCTACTTGGCCAATGTACGGTGAACAGCAATTGAATGCTTTTGAAATGGTTAAAGAATTAGGTATTGCTGTTGA AATAGAGGTTGATTACAAAAAAGATTATTTTAATATGAAAAATGATTTTATTGTCAGAGCTGAAGAAATTGAAACA AAGATTAAGAAATTAATGATGGATGAGAATAATTCTGAGATTAGAAAAAAGGTTAAAGAAATGAAGGAAAAATCCC GTGCTGCTATGTCTGAAAACGGTTCTTCTTATAACTCTTTGGCTAAATTATTCGAAGAAATTATGTAG(SEQ ID NO:2)。

In order to obtain the higher glycosyl transferase mutant of enzymatic activity, gene order SEQ of the present invention to glycosyl transferase ID NO:2 carries out point mutation.The variant amino acid sequence that one or more amino acid sites replace is obtained by fallibility round pcr Column, filter out several sites that enzyme activity can be improved, then in a manner of pinpointing combinatorial mutagenesis, obtain 3 plants of enzyme activities and obviously mention High mutant.

Herein, " glycosyl transferase " specifically refers to " uridine 5'-diphosphate (UDP) glycosyl transferase ", also referred to as It " UDP- glycosyl transferase " or is abbreviated as " UGT ".

Herein, term " wild (type) ", " wild enzyme ", " wild-type enzyme " and " WT " indicates identical meaning, is all Refer to the glycosyl transferase UGT1 (SEQ ID NO:1) not being genetically engineered.

Herein, term " protopanaxadiol-type's saponin(e " is sometimes referred to simply as " protopanoxadiol " or " PPD ".

4 glycosyl transferase mutant SEQ ID NOs:3-6 that the present invention filters out have the characteristics that it is common, i.e., relatively In SEQ ID NO:1, there is the forward mutation assay H82A in a site, implies that the rite-directed mutagenesis of at least the 82nd amino acids may It can cause the variation of enzymatic activity；SEQ ID NOs:4-6 have the characteristics that it is common, i.e., relative to SEQ ID NO:1, all there are four Forward mutation assay H82A, V88A, N122K and the E129T in site.

Glycosyl transferase mutant (SEQ ID NOs:3-6) of the invention is since amino acid sequence is clear, this field Technical staff is readily available its encoding gene, the expression cassette comprising these genes and plasmid and the conversion comprising the plasmid Body.These genes, expression cassette, plasmid, transformant can pass through genetic engineering building mode well-known to those skilled in the art It obtains.

Above-mentioned transformant host can be the microorganism of any suitable expression glycosyl transferase mutant, include bacterium and true Bacterium.Preferred microorganism is Escherichia coli, Pichia pastoris, saccharomyces cerevisiae or hay bacillus, preferably Escherichia coli, more preferably big Enterobacteria BL21 (DE3).

When as being that biocatalyst is used to produce Ginsenoside compound K, the shape of enzyme can be presented in glycosyl transferase of the invention The form of formula or thallus.The form of the enzyme includes resolvase, immobilised enzymes, including purifying enzyme, thick enzyme, fermentation liquid, carrier Fixed enzyme etc.；The form of the thallus includes survival thallus and dead thallus.

Glycosyl transferase of the invention isolate and purify including immobilised enzymes technology of preparing is also those skilled in the art institute It is well known.

Embodiment

Material and method

Full genome synthesis herein is completed by Suzhou Jin Weizhi Biotechnology Co., Ltd；Expression vector is by Zhejiang Hua Rui Bioisystech Co., Ltd's subclone preparation.Primer synthesis and sequencing are all complete by raw work bioengineering Shanghai limited liability company At.

Molecular biology experiment herein includes plasmid construction, digestion, connection, competent cell preparation, conversion, culture Basigamy etc., referring especially to " Molecular Cloning:A Laboratory guide " (third edition), J. Pehanorm Brooker, D.W. Russell (beauty) is compiled Write, Huang Peitang etc. is translated, Science Press, Beijing, 2002) it carries out.Specific experiment item can be determined by simple experiment when necessary Part.

The reaction condition or kit specification that PCR amplification experiment is provided according to plasmid or DNA profiling supplier carry out.It must It can be adjusted by simple experiment when wanting.

The high performance liquid chromatography (HPLC) of 1.PPD and CK content measures

Shimadzu LC-20A liquid chromatograph, Shodex C18-120-5 4E column (5 μm, 4.6mm × 250mm) chromatographic column, 35 DEG C of column temperature, mobile phase A water, Mobile phase B acetonitrile, gradient elution: 0min (35%B), 55min (90% ), B 50-55min (90%B) and 55-65min (35%B).Flow velocity: 0.8ml/min, wavelength 203nm.

PPD is purchased from Wuhan ChemFaces Bioisystech Co., Ltd.CK standard items are purchased from Sigma-Aldrich company.

2.UDP glycosyl transferase (UGT) enzyme activity determination

UGT enzyme activity determination is carried out in 100 μ L reaction systems (scalable).100 μ L reaction systems include: 100mM phosphate buffer (pH8.0), 1%Tween-20,5mM UDP-glucose (UDP-glucose), 5mM substrate PPD, 35 DEG C reaction overnight.Isometric n-butanol is added and terminates reaction, and carries out mixing extracting 1h.12000rpm is centrifuged 10min, draws Upper organic phase, evaporated in vacuo, product are dissolved in methanol solution, are quantified with high performance liquid chromatography to substrate and product CK Analysis.

Enzyme activity definition: under the conditions of pH8.0,35 DEG C of temperature, it is catalyzed PPD per minute and generates 1 micromole (μm ol) ginseng soap Enzyme amount required for glycosides CK is defined as 1 unit (U).

3. the high flux screening of glycosyl transferase mutant reacts:

Reference literature (Gantt, RW；Peltier-Pain,P；Cournoyer,WJ；Thorson,JS.Using simple donors to drive the equilibria of glycosyltransferase-catalyzed Reactions.Nature Chemical Biology.2011,7 (10): method 685-691.), in the 20mM of 100 μ l 2- chloro-4 nitrophenyl-β-D- pyrans the Portugal of substrate PPD, 0.5mM in Tris-HCl buffer (pH 8.0) comprising 0.25mM UDP-glucose (the UDP- of polyglycoside (2-chloro-4-nitrophenyl β-D-glucopyranoside), 1mM Glucose), 1% (v/v) Tween 80,11 μM of OleD mutant TDP-16 (include mutation P67T S132F A242L Q268V), 50 μ l glycosyl transferase solution.30 DEG C of microwell plate incubations, reaction process monitor 20min under the conditions of 410nm.

4. culture medium

Fermentation medium: peptone 20g/L, yeast extract 5g/L, glycerol 4.0mL/L, fumaric acid (fumaric acid) 2g/ L, potassium dihydrogen phosphate 1g/L, NH₄NO₃8g/L, ammonium hydroxide adjust pH to 7.5,121 DEG C of sterilizing 20min.

LB culture medium: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride, pH7.2,121 DEG C of high-temperature sterilizations 20min.(solid medium separately adds 20g/L agar powder).

TB culture medium: 24g/L yeast extract, 12g/L tryptone, 16.43g/L K₂HPO₄.3H₂O、2.31g/L KH₂PO₄, 5g/L glycerol, pH7.0-7.5,121 DEG C of high-temperature sterilization 20min.

20X electricity turns mother liquor: 80g/L glycine, 2% Tween 80.

The building of 1 wild type UGT1 of embodiment expression bacterial strain

The gene order SEQ ID NO:2 of composite coding UGT1, both ends are designed restriction enzyme site NdeI and BamHI, are cloned into Corresponding site on pSH plasmid obtains recombinant plasmid pSH-UGT1, is then transformed into Bacillus coli expression host with electrotransformation In BL21 (DE3) competent cell, the LB culture medium flat plate containing kanamycins, 37 DEG C of overnight incubations, picking individual colonies, inoculation are applied Into the test tube containing LB culture medium, overnight incubation, thalline were collected by centrifugation, extracts plasmid, and gene sequencing determines correctly, obtains table Up to recombination engineered strain BL21 (DE3)/pSH-UGT1 of wild type UGT1.

Determined amino acid sequence is UGT1.

Monoclonal is selected from the plate containing UGT1 engineering strain, is inoculated into 5mL LB culture medium, 37 DEG C were cultivated； 1%v/v is inoculated into the 1000mL shaking flask containing 100mL fermentation medium and cultivates 4-6 hours, OD₆₀₀Reach 1.2-1.5, is added The IPTG of 0.2mM is induced, and cools to 25 DEG C of cultures 10-16 hour, is centrifuged acquisition thallus, -80 DEG C freeze 24 hours it is spare.

2 fallibility PCR method of embodiment constructs UGT1 random mutation point library

Using pSH-UGT1 as template, random mutant library is constructed using fallibility round pcr.

Forward primer UGT1-Nde-F:5 '-CAT ATGAAATCTGAATTAATCTTTT-3 ',

Reverse primer UGT1-Bam-R:5 '-GGATCC CTACATAATTTCTTCGAATAAT-3 '.

50 μ L fallibility PCR reaction systems include: 50ng plasmid template pSH-UGT1,30pmol pair of primers UGT1-Nde-F And UGT1-Bam-R, 1X Taq buffer, 0.2mM dGTP, 0.2mM dATP, 1mM dCTP, 1mM dTTP, 7mM MgCl₂, (0mM、0.05mM、0.1mM、0.15mM、0.2mM)MnCl₂, the Taq enzyme (fermentas) of 2.5 units.PCR reaction condition Are as follows: 95 DEG C of 5min；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 2min/kbp；30 circulations；72℃10min.Glue recycling 2.0kb dashes forward at random Become segment as big primer, be MegaPrimer PCR:94 DEG C 5min with KOD-plus archaeal dna polymerase,；98 DEG C of 10s, 60 DEG C 30s, 68 DEG C of 2min/kbp, 25 circulations；68℃10min.DpnI digested plasmid template, electrotransformation e. coli E.coli BL21 (DE3), obtains more than 10⁴The random mutation library of a clone.

The high flux screening of embodiment 3UGT1 mutant library

Transformant in 3.1 selection UGT1 mutant libraries is inoculated into the 96 hole depth well culture plates containing 700 μ L LB culture mediums In, 100 μ g/mL kanamycins are contained in culture medium, after 37 DEG C of culture 6h, after addition final concentration 0.1mM IPTG, are cooled to 25 DEG C, Overnight incubation.5000rpm is centrifuged 10min, abandons supernatant, is placed in -70 DEG C of freezing 1h, and room temperature melts 30min.200 μ L cracking is added Thallus is resuspended in liquid (group is divided into 20mM Tris-HCl, pH8.0,2mM Isosorbide-5-Nitrae-dithiothreitol (DTT), 4000U lysozyme), and 37 DEG C incubate 1h is educated, is sufficiently centrifuged, clear enzyme solution in acquisition.

Using the enzyme solution of above-mentioned acquisition, 96 orifice plates of enzyme reaction are transferred to, as previously described " glycosyl transferase mutant High flux screening reaction " method implements screening, with wild type UGT1 (UGT1-WT) for negative control, is assessed according to light absorption value anti- Answer intensity.

In random mutation library, by being screened to about 2000 mutant clones, the more excellent mutant such as institute in table 1 of screening Show.Further verifying is done to this few plant mutant body clone.

Table 1, mutant enzyme activity

Strain number	Enzyme activity (%)
		UGT1-WT	100
UGT1-533	354
		UGT1-764	548
UGT1-988	452

The vigor of 3.2 high enzyme activity mutant is verified

Picking clone to be verified is inoculated into the LB culture medium containing 100 μ g/mL kanamycins, the bottled liquid of 250ml triangle 25ml culture medium, 37 DEG C of culture 6h are cooled to 25 DEG C, overnight incubation after final concentration 0.1mM IPTG is added.Thallus is collected, is abandoned Supernatant, by the thallus of every 5ml bacterium solution, adding 1ml lysate, (group is divided into 20mM Tris-HCl, pH8.0,2mM1, bis- sulphur threose of 4- Alcohol, 20000U lysozyme), thallus is resuspended, 37 DEG C of incubation 1h are sufficiently centrifuged, clear enzyme solution in acquisition.

Using upper clear enzyme solution, referring to " UDP glycosyl transferase enzyme activity determination " method above, using 1ml reaction system, into Row enzyme activity confirmatory reaction.

Plasmid is stripped to clone simultaneously, plasmid send gold dimension intelligence in Suzhou to be sequenced, confirms mutational site.

According to the quantitative detection to product CK and substrate PPD, mutant enzyme activity verification result is as shown in table 2.

Table 2, the verifying of mutant enzyme vigor

Strain number	Mutating acid site	Enzyme activity (%)
			UGT1-WT	-	100
UGT1-533	H82A,S175A	432
			UGT1-764	H82A,V88A,N122K,E129T	530
UGT1-988	H82A,V88A,N122K,E129T,K315D	478

The building of 4 combination mutant of embodiment

According to the amino acid mutation site identified in table 2, it is total to devise combination H82A, V88A, N122K, E129T, S175A The mutein sequence UGT1sm in 5 sites, and full genome is carried out by Suzhou gold dimension intelligence and is synthesized, carry out Bacillus coli expression Codon optimization, obtained coding gene sequence are SEQ ID NO:7.Coding gene sequence both ends design restriction enzyme site NdeI and BamHI is cloned into corresponding site on pSH plasmid, obtains recombinant plasmid pSH-UGT1sm, is then transformed into large intestine with electrotransformation In bacillus expressive host BL21 (DE3) competent cell, the LB culture medium flat plate containing kanamycins, 37 DEG C of overnight incubations are applied.It chooses Menu bacterium colony is inoculated into the test tube containing LB culture medium, overnight incubation.Thalline were collected by centrifugation, extracts plasmid, and gene sequencing is true It is fixed correct, obtain recombination engineered strain BL21 (DE3)/pSH-UGT1sm of expression wild type UGT1.

Referring to method same as Example 3, enzyme activity determination is carried out to mutant SEQ ID NO:6, as a result relatively Vigor is as shown in table 3.

Table 3, the verifying of mutant SEQ ID NO:6 enzyme activity

Strain number	Mutating acid site	Enzyme activity (%)
			UGT1-WT	-	100
UGT1sm	H82A,V88A,N122K,E129T,S175A	620

Wild type glycosyl transferase UGT1, mutant SEQ ID are compared it can be seen from table 2 and the experimental result of table 3 NOs:4-6 significantly improves enzyme activity, and wherein the enzyme activity of SEQ ID NOs:6 improves more than 5 times, can expeditiously be catalyzed Substrate protopanoxadiol PPD generates Ginsenoside compound K, has industrialized developing potentiality.

Sequence table

<110>Zhejiang Hua Rui Bioisystech Co., Ltd

<120>a kind of glycosyl transferase mutant

<130> SHPI1812099

<160> 7

<170> SIPOSequenceListing 1.0

<210> 1

<211> 475

<212> PRT

<213> GenBank: AIE12479.1

<400> 1

Met Lys Ser Glu Leu Ile Phe Leu Pro Ala Pro Ala Ile Gly His Leu

1 5 10 15

Val Gly Met Val Glu Met Ala Lys Leu Phe Ile Ser Arg His Glu Asn

20 25 30

Leu Ser Val Thr Val Leu Ile Ala Lys Phe Tyr Met Asp Thr Gly Val

35 40 45

Asp Asn Tyr Asn Lys Ser Leu Leu Thr Asn Pro Thr Pro Arg Leu Thr

50 55 60

Ile Val Asn Leu Pro Glu Thr Asp Pro Gln Asn Tyr Met Leu Lys Pro

65 70 75 80

Arg His Ala Ile Phe Pro Ser Val Ile Glu Thr Gln Lys Thr His Val

85 90 95

Arg Asp Ile Ile Ser Gly Met Thr Gln Ser Glu Ser Thr Gln Val Val

100 105 110

Gly Leu Leu Ala Asp Leu Leu Phe Ile Asn Ile Met Asp Ile Ala Asn

115 120 125

Glu Phe Asn Val Pro Thr Tyr Val Tyr Ser Pro Ala Gly Ala Gly His

130 135 140

Leu Gly Leu Ala Phe His Leu Gln Thr Leu Asn Asp Lys Lys Gln Asp

145 150 155 160

Val Thr Glu Phe Arg Asn Ser Asp Thr Glu Leu Leu Val Pro Ser Phe

165 170 175

Ala Asn Pro Val Pro Ala Glu Val Leu Pro Ser Met Tyr Val Asp Lys

180 185 190

Glu Gly Gly Tyr Asp Tyr Leu Phe Ser Leu Phe Arg Arg Cys Arg Glu

195 200 205

Ser Lys Ala Ile Ile Ile Asn Thr Phe Glu Glu Leu Glu Pro Tyr Ala

210 215 220

Ile Asn Ser Leu Arg Met Asp Ser Met Ile Pro Pro Ile Tyr Pro Val

225 230 235 240

Gly Pro Ile Leu Asn Leu Asn Gly Asp Gly Gln Asn Ser Asp Glu Ala

245 250 255

Ala Val Ile Leu Gly Trp Leu Asp Asp Gln Pro Pro Ser Ser Val Val

260 265 270

Phe Leu Cys Phe Gly Ser Tyr Gly Ser Phe Gln Glu Asn Gln Val Lys

275 280 285

Glu Ile Ala Met Gly Leu Glu Arg Ser Gly His Arg Phe Leu Trp Ser

290 295 300

Leu Arg Pro Ser Ile Pro Lys Gly Glu Thr Lys Leu Gln Leu Lys Tyr

305 310 315 320

Ser Asn Leu Lys Glu Ile Leu Pro Val Gly Phe Leu Asp Arg Thr Ser

325 330 335

Cys Val Gly Lys Val Ile Gly Trp Ala Pro Gln Val Ala Val Leu Gly

340 345 350

His Glu Ser Val Gly Gly Phe Leu Ser His Cys Gly Trp Asn Ser Thr

355 360 365

Leu Glu Ser Val Trp Cys Gly Val Pro Val Ala Thr Trp Pro Met Tyr

370 375 380

Gly Glu Gln Gln Leu Asn Ala Phe Glu Met Val Lys Glu Leu Gly Ile

385 390 395 400

Ala Val Glu Ile Glu Val Asp Tyr Lys Lys Asp Tyr Phe Asn Met Lys

405 410 415

Asn Asp Phe Ile Val Arg Ala Glu Glu Ile Glu Thr Lys Ile Lys Lys

420 425 430

Leu Met Met Asp Glu Asn Asn Ser Glu Ile Arg Lys Lys Val Lys Glu

435 440 445

Met Lys Glu Lys Ser Arg Ala Ala Met Ser Glu Asn Gly Ser Ser Tyr

450 455 460

Asn Ser Leu Ala Lys Leu Phe Glu Glu Ile Met

465 470 475

<210> 2

<211> 1428

<212> DNA

<213>artificial sequence ()

<400> 2

atgaaatctg aattaatctt tttaccagca ccagctattg gtcacttggt cggtatggtc 60

gagatggcta agttgtttat ttcaagacat gaaaatttgt cagtcactgt tttgattgca 120

aaattctata tggacactgg tgttgataac tataataagt ctttattaac aaatccaact 180

ccaagattaa ctattgttaa tttgccagaa actgacccac aaaactacat gttgaaacca 240

agacatgcta tttttccatc tgttattgaa actcagaaaa ctcatgttag agatattatt 300

tctggtatga cacaatctga gtctacacaa gttgtcggtt tgttggcaga cttgttattt 360

ataaatatta tggatattgc taatgagttt aacgttccta cttacgttta ctcaccagct 420

ggtgctggtc atttgggttt ggctttccac ttgcaaactt taaatgataa aaaacaagat 480

gttacagaat ttagaaactc tgatactgaa ttattggttc catctttcgc aaaccccgtt 540

cccgctgagg ttttgccatc tatgtacgtt gataaggagg gtggttacga ttacttattt 600

tctttgttca gaagatgtag agaatctaag gcaattatta ttaatacttt tgaggaatta 660

gagccatacg caattaattc tttaagaatg gactctatga ttccaccaat ttatccagtc 720

ggtcctatat tgaacttgaa cggtgacggt caaaactctg acgaggctgc tgtcattttg 780

ggttggttgg acgaccaacc accttcttct gtcgtctttt tatgttttgg ttcttacggt 840

tcattccaag aaaatcaagt taaagagatt gcaatgggtt tggagcgttc tggtcatagg 900

ttcttgtggt ctttaagacc atctattcct aagggtgaga ctaaattgca attaaaatac 960

tctaacttaa aagaaatttt gccagttggt tttttggata gaacttcatg cgttggtaaa 1020

gtcattggtt gggctccaca agttgctgtc ttgggtcatg aatctgttgg tggttttttg 1080

tcacactgtg gttggaactc tactttggaa tcagtctggt gcggtgttcc agttgctact 1140

tggccaatgt acggtgaaca gcaattgaat gcttttgaaa tggttaaaga attaggtatt 1200

gctgttgaaa tagaggttga ttacaaaaaa gattatttta atatgaaaaa tgattttatt 1260

gtcagagctg aagaaattga aacaaagatt aagaaattaa tgatggatga gaataattct 1320

gagattagaa aaaaggttaa agaaatgaag gaaaaatccc gtgctgctat gtctgaaaac 1380

ggttcttctt ataactcttt ggctaaatta ttcgaagaaa ttatgtag 1428

<210> 3

<211> 475

<212> PRT

<213>artificial sequence ()

<400> 3

Met Lys Ser Glu Leu Ile Phe Leu Pro Ala Pro Ala Ile Gly His Leu

1 5 10 15

Val Gly Met Val Glu Met Ala Lys Leu Phe Ile Ser Arg His Glu Asn

20 25 30

Leu Ser Val Thr Val Leu Ile Ala Lys Phe Tyr Met Asp Thr Gly Val

35 40 45

Asp Asn Tyr Asn Lys Ser Leu Leu Thr Asn Pro Thr Pro Arg Leu Thr

50 55 60

Ile Val Asn Leu Pro Glu Thr Asp Pro Gln Asn Tyr Met Leu Lys Pro

65 70 75 80

Arg Ala Ala Ile Phe Pro Ser Val Ile Glu Thr Gln Lys Thr His Val

85 90 95

Arg Asp Ile Ile Ser Gly Met Thr Gln Ser Glu Ser Thr Gln Val Val

100 105 110

Gly Leu Leu Ala Asp Leu Leu Phe Ile Asn Ile Met Asp Ile Ala Asn

115 120 125

Glu Phe Asn Val Pro Thr Tyr Val Tyr Ser Pro Ala Gly Ala Gly His

130 135 140

Leu Gly Leu Ala Phe His Leu Gln Thr Leu Asn Asp Lys Lys Gln Asp

145 150 155 160

Val Thr Glu Phe Arg Asn Ser Asp Thr Glu Leu Leu Val Pro Ala Phe

165 170 175

Ala Asn Pro Val Pro Ala Glu Val Leu Pro Ser Met Tyr Val Asp Lys

180 185 190

Glu Gly Gly Tyr Asp Tyr Leu Phe Ser Leu Phe Arg Arg Cys Arg Glu

195 200 205

Ser Lys Ala Ile Ile Ile Asn Thr Phe Glu Glu Leu Glu Pro Tyr Ala

210 215 220

Ile Asn Ser Leu Arg Met Asp Ser Met Ile Pro Pro Ile Tyr Pro Val

225 230 235 240

Gly Pro Ile Leu Asn Leu Asn Gly Asp Gly Gln Asn Ser Asp Glu Ala

245 250 255

Ala Val Ile Leu Gly Trp Leu Asp Asp Gln Pro Pro Ser Ser Val Val

260 265 270

Phe Leu Cys Phe Gly Ser Tyr Gly Ser Phe Gln Glu Asn Gln Val Lys

275 280 285

Glu Ile Ala Met Gly Leu Glu Arg Ser Gly His Arg Phe Leu Trp Ser

290 295 300

Leu Arg Pro Ser Ile Pro Lys Gly Glu Thr Lys Leu Gln Leu Lys Tyr

305 310 315 320

Ser Asn Leu Lys Glu Ile Leu Pro Val Gly Phe Leu Asp Arg Thr Ser

325 330 335

Cys Val Gly Lys Val Ile Gly Trp Ala Pro Gln Val Ala Val Leu Gly

340 345 350

His Glu Ser Val Gly Gly Phe Leu Ser His Cys Gly Trp Asn Ser Thr

355 360 365

Leu Glu Ser Val Trp Cys Gly Val Pro Val Ala Thr Trp Pro Met Tyr

370 375 380

Gly Glu Gln Gln Leu Asn Ala Phe Glu Met Val Lys Glu Leu Gly Ile

385 390 395 400

Ala Val Glu Ile Glu Val Asp Tyr Lys Lys Asp Tyr Phe Asn Met Lys

405 410 415

Asn Asp Phe Ile Val Arg Ala Glu Glu Ile Glu Thr Lys Ile Lys Lys

420 425 430

Leu Met Met Asp Glu Asn Asn Ser Glu Ile Arg Lys Lys Val Lys Glu

435 440 445

Met Lys Glu Lys Ser Arg Ala Ala Met Ser Glu Asn Gly Ser Ser Tyr

450 455 460

Asn Ser Leu Ala Lys Leu Phe Glu Glu Ile Met

465 470 475

<210> 4

<211> 475

<212> PRT

<213>artificial sequence ()

<400> 4

Met Lys Ser Glu Leu Ile Phe Leu Pro Ala Pro Ala Ile Gly His Leu

1 5 10 15

Val Gly Met Val Glu Met Ala Lys Leu Phe Ile Ser Arg His Glu Asn

20 25 30

Leu Ser Val Thr Val Leu Ile Ala Lys Phe Tyr Met Asp Thr Gly Val

35 40 45

Asp Asn Tyr Asn Lys Ser Leu Leu Thr Asn Pro Thr Pro Arg Leu Thr

50 55 60

Ile Val Asn Leu Pro Glu Thr Asp Pro Gln Asn Tyr Met Leu Lys Pro

65 70 75 80

Arg Ala Ala Ile Phe Pro Ser Ala Ile Glu Thr Gln Lys Thr His Val

85 90 95

Arg Asp Ile Ile Ser Gly Met Thr Gln Ser Glu Ser Thr Gln Val Val

100 105 110

Gly Leu Leu Ala Asp Leu Leu Phe Ile Lys Ile Met Asp Ile Ala Asn

115 120 125

Thr Phe Asn Val Pro Thr Tyr Val Tyr Ser Pro Ala Gly Ala Gly His

130 135 140

Leu Gly Leu Ala Phe His Leu Gln Thr Leu Asn Asp Lys Lys Gln Asp

145 150 155 160

Val Thr Glu Phe Arg Asn Ser Asp Thr Glu Leu Leu Val Pro Ser Phe

165 170 175

Ala Asn Pro Val Pro Ala Glu Val Leu Pro Ser Met Tyr Val Asp Lys

180 185 190

Glu Gly Gly Tyr Asp Tyr Leu Phe Ser Leu Phe Arg Arg Cys Arg Glu

195 200 205

Ser Lys Ala Ile Ile Ile Asn Thr Phe Glu Glu Leu Glu Pro Tyr Ala

210 215 220

Ile Asn Ser Leu Arg Met Asp Ser Met Ile Pro Pro Ile Tyr Pro Val

225 230 235 240

Gly Pro Ile Leu Asn Leu Asn Gly Asp Gly Gln Asn Ser Asp Glu Ala

245 250 255

Ala Val Ile Leu Gly Trp Leu Asp Asp Gln Pro Pro Ser Ser Val Val

260 265 270

Phe Leu Cys Phe Gly Ser Tyr Gly Ser Phe Gln Glu Asn Gln Val Lys

275 280 285

Glu Ile Ala Met Gly Leu Glu Arg Ser Gly His Arg Phe Leu Trp Ser

290 295 300

Leu Arg Pro Ser Ile Pro Lys Gly Glu Thr Lys Leu Gln Leu Lys Tyr

305 310 315 320

Ser Asn Leu Lys Glu Ile Leu Pro Val Gly Phe Leu Asp Arg Thr Ser

325 330 335

Cys Val Gly Lys Val Ile Gly Trp Ala Pro Gln Val Ala Val Leu Gly

340 345 350

His Glu Ser Val Gly Gly Phe Leu Ser His Cys Gly Trp Asn Ser Thr

355 360 365

Leu Glu Ser Val Trp Cys Gly Val Pro Val Ala Thr Trp Pro Met Tyr

370 375 380

Gly Glu Gln Gln Leu Asn Ala Phe Glu Met Val Lys Glu Leu Gly Ile

385 390 395 400

Ala Val Glu Ile Glu Val Asp Tyr Lys Lys Asp Tyr Phe Asn Met Lys

405 410 415

Asn Asp Phe Ile Val Arg Ala Glu Glu Ile Glu Thr Lys Ile Lys Lys

420 425 430

Leu Met Met Asp Glu Asn Asn Ser Glu Ile Arg Lys Lys Val Lys Glu

435 440 445

Met Lys Glu Lys Ser Arg Ala Ala Met Ser Glu Asn Gly Ser Ser Tyr

450 455 460

Asn Ser Leu Ala Lys Leu Phe Glu Glu Ile Met

465 470 475

<210> 5

<211> 475

<212> PRT

<213>artificial sequence ()

<400> 5

Met Lys Ser Glu Leu Ile Phe Leu Pro Ala Pro Ala Ile Gly His Leu

1 5 10 15

Val Gly Met Val Glu Met Ala Lys Leu Phe Ile Ser Arg His Glu Asn

20 25 30

Leu Ser Val Thr Val Leu Ile Ala Lys Phe Tyr Met Asp Thr Gly Val

35 40 45

Asp Asn Tyr Asn Lys Ser Leu Leu Thr Asn Pro Thr Pro Arg Leu Thr

50 55 60

Ile Val Asn Leu Pro Glu Thr Asp Pro Gln Asn Tyr Met Leu Lys Pro

65 70 75 80

Arg Ala Ala Ile Phe Pro Ser Ala Ile Glu Thr Gln Lys Thr His Val

85 90 95

Arg Asp Ile Ile Ser Gly Met Thr Gln Ser Glu Ser Thr Gln Val Val

100 105 110

Gly Leu Leu Ala Asp Leu Leu Phe Ile Lys Ile Met Asp Ile Ala Asn

115 120 125

Thr Phe Asn Val Pro Thr Tyr Val Tyr Ser Pro Ala Gly Ala Gly His

130 135 140

Leu Gly Leu Ala Phe His Leu Gln Thr Leu Asn Asp Lys Lys Gln Asp

145 150 155 160

Val Thr Glu Phe Arg Asn Ser Asp Thr Glu Leu Leu Val Pro Ser Phe

165 170 175

Ala Asn Pro Val Pro Ala Glu Val Leu Pro Ser Met Tyr Val Asp Lys

180 185 190

Glu Gly Gly Tyr Asp Tyr Leu Phe Ser Leu Phe Arg Arg Cys Arg Glu

195 200 205

Ser Lys Ala Ile Ile Ile Asn Thr Phe Glu Glu Leu Glu Pro Tyr Ala

210 215 220

Ile Asn Ser Leu Arg Met Asp Ser Met Ile Pro Pro Ile Tyr Pro Val

225 230 235 240

Gly Pro Ile Leu Asn Leu Asn Gly Asp Gly Gln Asn Ser Asp Glu Ala

245 250 255

Ala Val Ile Leu Gly Trp Leu Asp Asp Gln Pro Pro Ser Ser Val Val

260 265 270

Phe Leu Cys Phe Gly Ser Tyr Gly Ser Phe Gln Glu Asn Gln Val Lys

275 280 285

Glu Ile Ala Met Gly Leu Glu Arg Ser Gly His Arg Phe Leu Trp Ser

290 295 300

Leu Arg Pro Ser Ile Pro Lys Gly Glu Thr Asp Leu Gln Leu Lys Tyr

305 310 315 320

Ser Asn Leu Lys Glu Ile Leu Pro Val Gly Phe Leu Asp Arg Thr Ser

325 330 335

Cys Val Gly Lys Val Ile Gly Trp Ala Pro Gln Val Ala Val Leu Gly

340 345 350

His Glu Ser Val Gly Gly Phe Leu Ser His Cys Gly Trp Asn Ser Thr

355 360 365

Leu Glu Ser Val Trp Cys Gly Val Pro Val Ala Thr Trp Pro Met Tyr

370 375 380

Gly Glu Gln Gln Leu Asn Ala Phe Glu Met Val Lys Glu Leu Gly Ile

385 390 395 400

Ala Val Glu Ile Glu Val Asp Tyr Lys Lys Asp Tyr Phe Asn Met Lys

405 410 415

Asn Asp Phe Ile Val Arg Ala Glu Glu Ile Glu Thr Lys Ile Lys Lys

420 425 430

Leu Met Met Asp Glu Asn Asn Ser Glu Ile Arg Lys Lys Val Lys Glu

435 440 445

Met Lys Glu Lys Ser Arg Ala Ala Met Ser Glu Asn Gly Ser Ser Tyr

450 455 460

Asn Ser Leu Ala Lys Leu Phe Glu Glu Ile Met

465 470 475

<210> 6

<211> 475

<212> PRT

<213>artificial sequence ()

<400> 6

Met Lys Ser Glu Leu Ile Phe Leu Pro Ala Pro Ala Ile Gly His Leu

1 5 10 15

Val Gly Met Val Glu Met Ala Lys Leu Phe Ile Ser Arg His Glu Asn

20 25 30

Leu Ser Val Thr Val Leu Ile Ala Lys Phe Tyr Met Asp Thr Gly Val

35 40 45

Asp Asn Tyr Asn Lys Ser Leu Leu Thr Asn Pro Thr Pro Arg Leu Thr

50 55 60

Ile Val Asn Leu Pro Glu Thr Asp Pro Gln Asn Tyr Met Leu Lys Pro

65 70 75 80

Arg Ala Ala Ile Phe Pro Ser Ala Ile Glu Thr Gln Lys Thr His Val

85 90 95

Arg Asp Ile Ile Ser Gly Met Thr Gln Ser Glu Ser Thr Gln Val Val

100 105 110

Gly Leu Leu Ala Asp Leu Leu Phe Ile Lys Ile Met Asp Ile Ala Asn

115 120 125

Thr Phe Asn Val Pro Thr Tyr Val Tyr Ser Pro Ala Gly Ala Gly His

130 135 140

Leu Gly Leu Ala Phe His Leu Gln Thr Leu Asn Asp Lys Lys Gln Asp

145 150 155 160

Val Thr Glu Phe Arg Asn Ser Asp Thr Glu Leu Leu Val Pro Ala Phe

165 170 175

Ala Asn Pro Val Pro Ala Glu Val Leu Pro Ser Met Tyr Val Asp Lys

180 185 190

Glu Gly Gly Tyr Asp Tyr Leu Phe Ser Leu Phe Arg Arg Cys Arg Glu

195 200 205

Ser Lys Ala Ile Ile Ile Asn Thr Phe Glu Glu Leu Glu Pro Tyr Ala

210 215 220

Ile Asn Ser Leu Arg Met Asp Ser Met Ile Pro Pro Ile Tyr Pro Val

225 230 235 240

Gly Pro Ile Leu Asn Leu Asn Gly Asp Gly Gln Asn Ser Asp Glu Ala

245 250 255

Ala Val Ile Leu Gly Trp Leu Asp Asp Gln Pro Pro Ser Ser Val Val

260 265 270

Phe Leu Cys Phe Gly Ser Tyr Gly Ser Phe Gln Glu Asn Gln Val Lys

275 280 285

Glu Ile Ala Met Gly Leu Glu Arg Ser Gly His Arg Phe Leu Trp Ser

290 295 300

Leu Arg Pro Ser Ile Pro Lys Gly Glu Thr Lys Leu Gln Leu Lys Tyr

305 310 315 320

Ser Asn Leu Lys Glu Ile Leu Pro Val Gly Phe Leu Asp Arg Thr Ser

325 330 335

Cys Val Gly Lys Val Ile Gly Trp Ala Pro Gln Val Ala Val Leu Gly

340 345 350

His Glu Ser Val Gly Gly Phe Leu Ser His Cys Gly Trp Asn Ser Thr

355 360 365

Leu Glu Ser Val Trp Cys Gly Val Pro Val Ala Thr Trp Pro Met Tyr

370 375 380

Gly Glu Gln Gln Leu Asn Ala Phe Glu Met Val Lys Glu Leu Gly Ile

385 390 395 400

Ala Val Glu Ile Glu Val Asp Tyr Lys Lys Asp Tyr Phe Asn Met Lys

405 410 415

Asn Asp Phe Ile Val Arg Ala Glu Glu Ile Glu Thr Lys Ile Lys Lys

420 425 430

Leu Met Met Asp Glu Asn Asn Ser Glu Ile Arg Lys Lys Val Lys Glu

435 440 445

Met Lys Glu Lys Ser Arg Ala Ala Met Ser Glu Asn Gly Ser Ser Tyr

450 455 460

Asn Ser Leu Ala Lys Leu Phe Glu Glu Ile Met

465 470 475

<210> 7

<211> 1428

<212> DNA

<213>artificial sequence ()

<400> 7

atgaaaagcg agctcatctt tctgccagcg ccagcgattg gccatctggt gggcatggtg 60

gaaatggcca agctgttcat tagccgtcac gagaacctca gtgtgacggt gctgatcgcc 120

aagttttaca tggacacggg cgtggacaac tataacaaga gtctgctgac caatccgacc 180

ccgcgcctca cgattgtgaa cctcccggaa accgacccgc agaattacat gctgaagcca 240

cgcgccgcga ttttcccgag cgcgattgaa acgcaaaaga cccacgtgcg cgacatcatc 300

agcggcatga cccagagcga aagcacccaa gtggttggtc tgctggcgga tctgctgttc 360

atcaagatca tggatatcgc caacacgttc aacgtgccaa cgtatgtgta cagtccagcg 420

ggtgccggtc atctgggtct ggccttccat ctgcaaaccc tcaacgacaa gaagcaagat 480

gtgaccgagt tccgcaatag cgatacggaa ctgctcgtgc cagccttcgc gaatccagtt 540

ccagcggagg ttctgccaag catgtacgtg gacaaagagg gcggctatga ctatctgttt 600

agtctgttcc gccgctgtcg cgaaagcaag gccatcatca tcaacacctt tgaagaactc 660

gagccgtacg cgattaacag tctgcgcatg gacagcatga ttccgccgat ctatccagtt 720

ggtccgatcc tcaatctgaa cggcgatggc caaaacagtg atgaagccgc ggtgatcctc 780

ggctggctgg atgatcaacc gccaagcagc gttgtgttcc tctgcttcgg cagctatggc 840

agctttcaag aaaaccaagt taaagagatc gcgatgggtc tggaacgtag cggccatcgc 900

tttctctgga gtctgcgtcc gagcatccca aagggcgaaa cgaagctgca actgaagtac 960

agcaatctga aggaaatcct cccagtgggt tttctggatc gcaccagctg cgttggcaaa 1020

gtgattggtt gggccccaca agttgcggtt ctgggtcacg aaagcgttgg tggctttctg 1080

agccattgtg gctggaacag tacgctggaa agcgtgtggt gcggcgttcc agttgccacg 1140

tggccaatgt atggcgaaca gcagctgaac gccttcgaga tggttaagga actgggcatc 1200

gccgtggaga tcgaagttga ctacaagaaa gattacttta acatgaagaa tgacttcatc 1260

gtgcgcgccg aggagatcga gaccaaaatc aaaaaactga tgatggatga aaataatagc 1320

gaaatccgca agaaagtgaa agagatgaag gaaaaaagcc gtgcggcgat gagcgagaat 1380

ggcagcagct acaatagtct ggccaagctc ttcgaggaaa tcatgtaa 1428

Claims

1. a kind of glycosyl transferase, amino acid sequence are as follows:

SEQ ID NO:3, for SEQ ID NO:1 the 82nd H replaces with A, the 175th S replaces with the mutant of A；

SEQ ID NO:4, for SEQ ID NO:1 the 82nd H replaces with A, the 88th V replaces with A, the 122nd N is replaced It is changed to K, the 129th E replaces with the mutant of T；

SEQ ID NO:5, for SEQ ID NO:1 the 82nd H replaces with A, the 88th V replaces with A, the 122nd N is replaced It is changed to K, the 129th E replaces with T, the 315th K replaces with the mutant of D；Or

SEQ ID NO:6, for SEQ ID NO:1 the 82nd H replaces with A, the 88th V replaces with A, the 122nd N is replaced It is changed to K, the 129th E replaces with T, the 175th S replaces with the mutant of A.

2. glycosyl transferase as described in claim 1, which is characterized in that the amino acid sequence is SEQ ID NO:6.

3. encoding the gene of glycosyl transferase as described in claim 1.

4. encoding the gene of glycosyl transferase as claimed in claim 2, sequence is SEQ ID NO:7.

5. including the plasmid of gene as claimed in claim 2.

6. having converted the microorganism of plasmid as claimed in claim 5.

7. microorganism as claimed in claim 6, the microorganism is selected from Escherichia coli, yeast, hay bacillus.

8. microorganism as claimed in claim 6 is e. coli bl21 (DE3).

9. glycosyl transferase as described in claim 1 or microorganism as claimed in claim 6 are in synthesis Ginsenoside compound K Purposes.

10. purposes as claimed in claim 9, which is characterized in that using protopanoxadiol as substrate raw material, urged in glycosyl transferase Ginsenoside compound K is prepared by glycosyl transfer reaction under change.