CN108611344A - The preparation and application of AtAGM2 and AtAGM3 encoding genes and enzyme - Google Patents

Abstract

The present invention discloses two phosphoacetylglucosamine mutase (Arabidopsis thaliana N acetylpHospHoglucosamine mutase 2,3 for deriving from arabidopsis (Arabidopsis thaliana)；AtAGM2, AtAGM3) gene order and its application.The present invention provides a kind of methods preparing acetyl glucosamine transphosphorylase, it i.e. will be in the gene cloning to coli expression carrier of enzyme, obtain can heterogenous expression mutase E. coli recombinant stain, the mutase activity that AtAGM2 all has with AtAGM3. two kinds of AGM albumen catalysis pbosphohexose isomers is prepared with recombinant bacterial strain heterogenous expression, can be applied to enzyme law catalysis synthesis uridine 5'-diphosphate acetylglucosamine (UDP GlcNAc) or production pbosphohexose isomers field.

Description

The preparation and application of AtAGM2 and AtAGM3 encoding genes and enzyme

Technical field

The present invention relates to the gene order of two kinds of phosphoacetylglucosamine mutases AtAGM2 and AtAGM3 and its preparations The application of method more particularly to both enzymes in nucleotide sugar and the production of hexosephosphate isomers.The present invention also provides this The zymologic property of the recombinant plasmid and recombination engineered strain and two kinds of enzymes of two kinds of phosphoacetylglucosamine mutases.

Background technology

Acetylglucosamine (GlcNAc, N- acetyl-β-D- Glucosamines) is a kind of amino sugar derivative of glucose. In vivo, it is a kind of monosaccharide with important biochemical functions for being only second to glucose.Often in the form of UDP-GlcNAc Participate in a variety of glycoconjugates in main many bioprocesses including nervous system and immune system and organism Structure composition.In vivo, the main path for generating UDP-GlcNAc is aminohexose approach (Hexosamine biosynthesis pathway).This approach is the branch of glycolysis, all with the intermediate product fructose-of hexose metabolism approach 6- phosphoric acid (Fructose-6-P) is starting material, under the concerted catalysis effect of a variety of enzymes, finally synthesizes UDP-GlcNAc.Root It is different according to the source of enzyme involved by the sequence and route of synthesis for being catalyzed reaction in building-up process, it is divided into eukaryon, protokaryon and quasi- bacterium The UDP-GlcNAc route of synthesis of virus.Currently, aminohexose approach is widely studied, but the amino in plant oneself Third enzyme phosphoacetylglucosamine mutase in sugared approach is not yet studied and is applied so far, thus the amino in plant oneself Sugared approach is not got through yet.

In addition UDP-GlcNAc can be largely used to the production of oligosaccharides as active nucleoside sugar important in organism, into And it is developed to pharmaceuticals or functional material.But due to the limitation of its production method, its supply is still smaller at present, price It is very high.Had research at present using the multistep enzymic catalytic reaction system of acellular catalysis, be coupled glucokinase, AGM with Three enzymes of GlcNAc-1-P uridines transferase (GlmU) produce UDP-GlcNAc using GlcNAc as substrate.Either internal table Up to system or vivoexpression system, the soluble-expression level of the AGM l from yeast is very low, affects the effect of Product formation Rate and cost.So providing a kind of high activity, the AGM of high expression has a very important significance.

The price of hexosephosphate is very expensive, is presently mainly chemically synthesized, and production stage is cumbersome.Sigma with And the reserves of the companies such as Santa Cruz seldom even run out of goods.Due to the limitation of synthetic method, lead to different different of hexosephosphate Price variance is huge (dozens or even hundreds of times) between structure body；Such as the 29 yuan/mg of price of GlcN-6-P, and the price of GlcN-1-P It is then 460/mg.So exploring the preparation method of new hexosephosphate, the preparation method of especially expensive hexosephosphate It is imperative.

Present Research for above-mentioned phosphoacetylglucosamine mutase and its problem of exist in the application, the present invention Disclose two phosphoacetylglucosamine mutases for deriving from arabidopsis (Arabidopsis thaliana) The gene order of (Arabidopsis thaliana N-acetylpHospHoglucosamine mutase, AtAGM) and its Preparation method.Both enzymes all have the activity for being catalyzed different pbosphohexose isomers, with another come in arabidopsis Phosphoacetylglucosamine mutase AtAGM1 is compared, and the efficiency that three is catalyzed reaction is not much different, and has similar enzyme Property is learned, can be applied in the production that acellular enzyme process catalyzes and synthesizes UDP-GlcNAc or pbosphohexose isomers.

Invention content

The first purpose of the invention is to provide the phosphoacetylglucosamine mutase AtAGM2 of two plant origins with AtAGM3 and its encoding gene.

Second object of the present invention, which is to provide, a kind of preparing phosphoacetylglucosamine mutase AtAGM2 and AtAGM3 Method.

Third object of the present invention is to provide containing the phosphoacetylglucosamine mutase AtAGM2 with The recombinant expression plasmid and recombination engineered strain of AtAGM3.

Phosphoacetylglucosamine mutase AtAGM2 provided by the present invention derives from arabidopsis (Arabidopsis Thaliana), amino acid sequence has one kind in following feature or two kinds：

1) 1-625 amino acid residue sequences of the SEQ ID NO.2 since aminoterminal in sequence table is active The amino acid sequence of phosphoacetylglucosamine mutase AtAGM, 620-625 are the amino acid sequence of His-Tag.

2) by the SEQ ID NO.2 in sequence table 1-625 amino acids residues since aminoterminal carry out one or More than two amino acid substitutions, deletions, or additions and formed with the constant amino acid of phosphoacetylglucosamine mutase activity Sequence.

The present invention also provides the encoding genes of above-mentioned phosphoacetylglucosamine mutase AtAGM2, derive from arabidopsis (Arabidopsis thaliana), nucleotide sequence have the one or two or more kinds in following feature：

1) in sequence table SEQ ID NO.1 DNA (DNA) sequence；

2) in polynucleotide SEQ ID NO.2 amino acid sequences DNA (DNA) sequence；

3) one or more nucleosides is carried out to DNA (DNA) sequence of SEQ ID NO.1 in sequence table Coding has the active nucleotide sequence of phosphoacetylglucosamine mutase obtained from acid replaces, misses or adds.

The there is provided phosphoacetylglucosamine mutase AtAGM3 of invention, derives from arabidopsis (Arabidopsis Thaliana), amino acid sequence has one kind in following feature or two kinds：

1) 1-623 amino acid residue sequences of the SEQ ID NO.4 since aminoterminal in sequence table is active The amino acid sequence of phosphoacetylglucosamine mutase AtAGM, 518-623 are the amino acid sequence of His-Tag.

2) by the SEQ ID NO.4 in sequence table 1-623 amino acids residues since aminoterminal carry out one or More than two amino acid substitutions, deletions, or additions and formed with the constant amino acid of phosphoacetylglucosamine mutase activity Sequence.

The present invention also provides the encoding genes of above-mentioned phosphoacetylglucosamine mutase AtAGM3, derive from arabidopsis (Arabidopsis thaliana), nucleotide sequence have the one or two or more kinds in following feature：

1) in sequence table SEQ ID NO.3 DNA (DNA) sequence；

2) in polynucleotide SEQ ID NO.4 amino acid sequences DNA (DNA) sequence；

3) one or more nucleosides is carried out to DNA (DNA) sequence of SEQ ID NO.3 in sequence table Coding has the active nucleotide sequence of phosphoacetylglucosamine mutase obtained from acid replaces, misses or adds.

The amino acid sequence and its nucleotide of the phosphoacetylglucosamine mutase AtAGM2 and AtAGM3 of the present invention are compiled Code sequence can also be according to the amino acid sequence of the AtAGM2 and AtAGM3 of prediction and its nucleotide coding sequence is artificial synthesized obtains .

The method of Prepare restructuring enzyme AtAGM2 and AtAGM3, be by phosphoacetylglucosamine mutase AtAGM2 or The encoding gene of AtAGM3 is cloned into recombinant expression carrier, imports host cell, obtains the acetylglucosamine phosphorus of recombinant expression Sour mutase

The encoding gene of above-mentioned phosphoacetylglucosamine mutase AtAGM2 or AtAGM3, nucleotide sequence have such as One or two or more kinds in lower feature：

1) DNA (DNA) sequence with SEQ ID NO.1 or SEQ ID NO.3 in sequence table, 2) coding DNA (DNA) sequence of SEQ ID NO.2 or SEQ ID NO.4 amino acid sequences,

3) to DNA (DNA) sequence of SEQ ID NO.1 in sequence table or SEQ ID NO.3 carry out one or Coding has the active nucleosides of phosphoacetylglucosamine mutase obtained from more than two nucleotide replace, miss or add Acid sequence.

The expression vector of the recombinant expression phosphoacetylglucosamine mutase or AtAGM2 or AtAGM3 can be Coli expression carrier, Yeast expression carrier, hay bacillus expression vector, lactic acid bacteria expression vectors, streptomyces expression vector, Phage vector, filamentous fungi expression vector, plant expression vector, insect expression vector or mammalian cell expression vector Deng.

Recombinant bacterium or transgenic cell for recombinantly expressing phosphoacetylglucosamine mutase AtAGM2 or AtAGM3 System, can be e. coli host cell (such as Escherichia coli BL21, Escherichia coli JM109, Escherichia coli DH5 α etc.), yeast host cells (such as Saccharomyces cerevisiae, Pichia Pastoris, Kluyveromyces lactis etc.), hay bacillus host cell (such as Bacillus subtilis R25, Bacillus subtilis 9920 etc.), lactic acid bacteria host cell (such as Lactic acid bacteria COCC101), put Line bacterium host cell (such as Streptomyces spp.), filamentous fungal host cell (such as Trichoderma viride, Trichoderma reesei, Aspergillus niger, Aspergillus nidulans etc.), insect cell (such as Bombyx mori, Antharaea eucalypti etc.) or mammalian cell (such as Chinese hamster ovary cell CHO, immature storehouse Mouse kidney cell BHK, CHL cells CHL etc.).

Above-mentioned phosphoacetylglucosamine mutase can be applied in hexosephosphate is produced with nucleotide sugar, including with One in lower application or two kind or more：

1) in the transformation for realizing 1,6 position isomer of hexosephosphate, the application in corresponding isomer is produced；

2) in UDP-GlcNAc (UDP-GlcN；The application in the production of nucleotide sugars such as UDP-Glc)；

Description of the drawings

Fig. 1：N-Acetyl-D-glucosamine transphosphorylase Gene A tagm2 schemes with the detection of Atagm3 agarose gel electrophoresis.

Fig. 2：The SDS-PAGE figures of phosphoacetylglucosamine mutase AtAGM2 expression and purifying.The sample being respectively added point It is not：1-E.coli BL21 (DE3)/pET28a-AtAGM2 induces thalline；2-E.coli BL21(DE3)/pET28a- AtAGM2 does not induce thalline；M- pre-dyed Protein Markers；Remaining is the imidazole elution of various concentration.The item of arrow meaning Band is AtAGM2 protein bands.

Fig. 3：The SDS-PAGE figures of phosphoacetylglucosamine mutase AtAGM3 expression and purifying.The sample being respectively added point It is not：1-E.coli BL21 (DE3)/pET28a-AtAGM3 does not induce thalline；2,3-E.coli BL21 (DE3)/pET28a- Thalline after AtAGM3 inductions；M- pre-dyed Protein Markers；Remaining is the imidazole elution of various concentration.The item of arrow meaning Band is AtAGM3 protein bands.

Fig. 4：Relative activity figure of the AtAGM2 enzymes at differential responses system pH.

Fig. 5：Relative activity line chart of AtAGM2 enzymes at a temperature of differential responses.

Fig. 6：Relative activity figure of the AtAGM3 enzymes at differential responses system pH.

Fig. 7：Relative activity line chart of AtAGM3 enzymes at a temperature of differential responses.

Fig. 8：Relative activity figure of the AtAGM2 enzymes under different metal ions catalysis.

Fig. 9：Relative activity figure of the AtAGM3 enzymes under different metal ions catalysis.

Specific implementation mode

The information of SEQ ID NO.1

(a) sequence signature

Length：1878 nucleotide

Type：Nucleotide

Chain：It is single-stranded

(b) molecule type：DNA

Sequence description：SEQ ID NO.1

ATGGAAGGAAAGGTTTTCCAAAACTTTAATGTAGTACAGAGCTGCTACCGACAAAATAAGCAGTTTAAG ACACGATACCAAAGAGAACCTGACCTGTTCATGTCTACTTTACTTCCCTGTCCAAGAGAGAAGATGGCATTTAACCT CAACTCTTCCATGCGTGCCCACACTTTGTCTAAATACCAGTTTGTTCTTTCAAAGCAAAGAACTTTTTACTGCAATG CTACTTCGTCAAGTGCTACTGTGCCATCTCTTGACAAAAATGATTTTCTGAAGCTCCAAAACGGCAGTGATATTCGG GGTGTAGCGGTCACTGGGGTTGAGGGGGAACCTGTAAGCCTTCCTGAACCAGTGACTGAAGCCATAGCTGCTGCTTT TGGGCAATGGCTGTTACACAAGAAGAAGGCTGAATCCCGGCGTTTGAGAGTATCTGTTGGCCATGACTCTCGCATCT CTGCACAAACTTTGCTGGAGGCGGTTTCTCGAGGTCTTGGTGTTTCTGGATTAGATGTTGTTCAGTTTGGATTAGCA TCAACACCAGCAATGTTTAATAGCACATTGACTGAAGATGAGTCATTCTTGTGCCCAGCTGATGGGGCTATTATGAT AACAGCAAGCCATCTTCCTTACAACAGGAACGGTTTCAAGTTCTTTACCAGTGATGGAGGACTTGGGAAGGTTGATA TCAAGAACATTTTGGAGCGAGCTGCAGATATTTACAAGAAGCTTTCTGATGAAAATTTGAGGAAATCACAAAGAGAA AGTTCTTCTATTACAAAGGTTGACTACATGTCAGTATACACCTCTGGTCTTGTAAAGGCAGTCCGGAAAGCAGCAGG AGATTTGGAGAAGCCTCTAGAGGGATTTCATATAGTTGTTGATGCTGGAAATGGAGCTGGAGGATTTTTTGCTGCCA AGGTGCTTGAGCCTTTAGGAGCAATTACTTCTGGCAGTCAATTTCTGGAACCAGATGGTATGTTCCCAAATCATATC CCTAATCCGGAAGATAAGGCGGCAATGGAAGCTATAACCAAGGCTGTTCTTGATAATAAGGCTGATTTGGGTATCAT CTTTGATACTGATGTTGATAGGTCTGCTGCTGTGGATTCATCTGGCCGTGAATTCAACCGTAATCGTCTTATTGCCT TGCTATCAGCCATTGTTCTAGAGGAACACCCTGGCACAACTATAGTTACGGATAGTGTCACTTCGGACGGTCTGACC TCATTTATTGAGAAGAAGCTTGGCGGAAAGCATCACAGGTTCAAAAGAGGTTACAAGAATGTCATTGACGAAGCTAT TCGCTTGAACTCGGTTGGGGAAGAATCACATCTGGCTATAGAAACCAGTGGTCATGGAGCTCTAAAGGAAAACCATT GGCTCGACGATGGGGCCTATCTCATGGTAAAAATCCTGAACAAACTAGCTGCGGCCCGAGCTGCTGGTCAAGGGAGT GGCAGCAAAGTTTTAACAGATCTTGTTGAAGGTCTGGAAGAGCCCAAAGTGGCTTTAGAACTGAGGCTTAAAATCGA CAAGAATCACCCTGACCTTGAAGGAAGTGATTTCCGGGAGTATGGAGAGAAGGTCCTGCAACACGTGTCGAACTCAA TAGAAACAAATCCAAATCTTATAATAGCTCCAGTTAACTACGAAGGGATCCGCGTTTCGGGCTTTGGTGGATGGTTT CTTCTCAGACTTTCTCTCCATGATCCTGTTCTTCCCCTTAACATCGAGGCACAGAGTGAGGATGATGCTGTGAAATT AGGCCTTGTGGTTGCTACGACAGTGAAGGAGTTCAATGCTTTGGACACCTGTGCCTTGTCCAACCTCACTCACTCCT CCGCGGCCGCACTCGAGCACCACCACCACCACCACTGA

The information of SEQ ID NO.2

(a) sequence signature

Length：625 amino acid

Type：Amino acid

Chain：It is single-stranded

(b) molecule type：Albumen

Sequence description：SEQ ID NO.2

MEGKVFQNFNVVQSCYRQNKQFKTRYQREPDLFMSTLLPCPREKMAFNLNSSMRAHTLSKYQFVLSKQRTFYCNATS SSATVPSLDKNDFLKLQNGSDIRGVAVTGVEGEPVSLPEPVTEAIAAAFGQWLLHKKKAESRRLRVSVGHDSRISAQ TLLEAVSRGLGVSGLDVVQFGLASTPAMFNSTLTEDESFLCPADGAIMITASHLPYNRNGFKFFTSDGGLGKVDIKN ILERAADIYKKLSDENLRKSQRESSSITKVDYMSVYTSGLVKAVRKAAGDLEKPLEGFHIVVDAGNGAGGFFAAKVL EPLGAITSGSQFLEPDGMFPNHIPNPEDKAAMEAITKAVLDNKADLGIIFDTDVDRSAAVDSSGREFNRNRLIALLS AIVLEEHPGTTIVTDSVTSDGLTSFIEKKLGGKHHRFKRGYKNVIDEAIRLNSVGEESHLAIETSGHGALKENHWLD DGAYLMVKILNKLAAARAAGQGSGSKVLTDLVEGLEEPKVALELRLKIDKNHPDLEGSDFREYGEKVLQHVSNSIET NPNLIIAPVNYEGIRVSGFGGWFLLRLSLHDPVLPLNIEAQSEDDAVKLGLVVATTVKEFNALDTCALSNLTHSSAA ALEHHHHHH-

The information of SEQ ID NO.3

(a) sequence signature

Length：1872 nucleotide

Type：Nucleotide

Chain：It is single-stranded

(b) molecule type：DNA

Sequence description：SEQ ID NO.3

ATGGCGTCGACTTCAACATCATCTTTAATGGCTTCTAAAACTGTAATCTCCAAAACAGCTCTGTTTTCT TCCTTACCGGGAATAGTCAGCCGGAGTTTTTTAACATTCGCACCGGCTTCTCCTTCCGTTAAACCCCTTAGGATAAG ATCTTCAAATGTTACTAAGTTCGACGAAGTAACCAACAGTCTTGACGAAGACATGGACCAGATTCGACGGTTACAAA ACGGTTCTGACGTGAGAGGAGTCGCATTGGAAGGAGAGAAAGGTCGAACAGTTGACCTAACGCCTGCAGCTGTTGAA GCAATCGCAGAGAGCTTTGGAGAATGGGTTGCAGCAACGGAGAGTAACGGAAACGGCGTCATTAAGATTTCTCTCGG ACGGGATCCACGTGTTTCCGGTGGGAAGCTAAGCACGGCAGTGTTTGCCGGCTTAGCTCGTGCAGGCTGTTTAGCTT TTGACATGGGTTTAGCTACAGCGCCAGCTTGCTTCATGAGCACGTTACTCTCTCCATTCGAATACGACGCTTCAATT ATGATGACAGCTTCTCATTTACCGTATACAAGAAACGGACTCAAGTTCTTTACCAAGAGAGGAGGATTAACGTCTCC TGAAGTGGAGAAGATATGCGATTTAGCTGCGCGAAAGTACGCTACTAGGCAGACTAAAGTCTCTACATTGATCAGAA CGCGACCGCAGCAAGTTGATTTTATGAGCGCTTACTCTAAGCACCTTAGAGAAATCATTAAAGAGAGAATCAATCAC CCTGAACACTATGACACTCCTCTCAAAGGATTTCAGATAGTTGTGAATGCGGGTAATGGGTCAGGAGGCTTCTTTAC GTGGGACGTTCTAGACAAGTTAGGAGCCGATACATTCGGTTCGCTCTATCTAAACCCTGACGGGATGTTCCCTAATC ACATTCCTAATCCGGAAAACAAAATCGCAATGCAACACACCCGAGCCGCGGTTCTTGAGAACTCAGCAGATCTCGGG GTTGTGTTTGATACGGATGTTGACAGGAGTGGAGTGGTGGATAACAAAGGAAATCCTATCAACGGAGATAAGCTTAT TGCGCTTATGTCAGCTATAGTGCTTAAAGAACATCCAGGAAGTACAGTAGTGACTGACGCAAGAACGAGTATGGGGC TAACTAGGTTTATAACGGAGCGAGGAGGGAGGCATTGTTTGTATAGAGTAGGGTATAGAAACGTGATTGACAAGGGA GTAGAGCTGAACAAAGACGGCATCGAGACTCATCTCATGATGGAAACTTCAGGACATGGTGCGGTTAAGGAGAATCA CTTCTTGGATGATGGTGCATACATGGTGGTGAAGATCATAATTGAAATGGTGAGAATGAGACTCGCGGGATCAAATG AAGGTATCGGTAGTTTGATCGAAGATCTTGAGGAGCCGTTAGAAGCGGTTGAGCTTCGGTTGAATATTTTATCAGAG CCAAGAGATGCCAAAGCAAAAGGCATTGAAGCCATTGAGACTTTCAGGCAATACATTGAGGAAGGAAAACTGAAAGG GTGGGAATTGGGCACGTGTGGGGATTGTTGGGTTACTGAAGGTTGCTTGGTGGACTCAAATGATCATCCATCTGCTA TTGATGCTCACATGTACAGGGCAAGAGTGAGTGATGAAGAGAGTGGGGAAGAGTATGGTTGGGTGCATATGAGGCAG AGTATTCATAACCCTAACATCGCACTTAATATGCAATCAATGCTTCCTGGTGGATGTCTCTCCATGACAAGAATCTT CAGAGACCAGTTTCTTGAAGCTAGTGGGGTGGCTAGATTCCTGGATATAAGTGACTTCGACAATTACATCGGAGGTC AATCTCTCGAGCACCACCACCACCACCACTGA

The information of SEQ ID NO.4

(a) sequence signature

Length：623 amino acid

Type：Amino acid

Chain：It is single-stranded

(b) molecule type：Albumen

Sequence description：SEQ ID NO.4

MASTSTSSLMASKTVISKTALFSSLPGIVSRSFLTFAPASPSVKPLRIRSSNVTKFDEVTNSLDEDMDQ IRRLQNGSDVRGVALEGEKGRTVDLTPAAVEAIAESFGEWVAATESNGNGVIKISLGRDPRVSGGKLSTAVFAGLAR AGCLAFDMGLATAP

ACFMSTLLSPFEYDASIMMTASHLPYTRNGLKFFTKRGGLTSPEVEKICDLAARKYATRQTKVSTLIRTRPQQVDFM SAYSKHLREIIKERINHPEHYDTPLKGFQIVVNAGNGSGGFFTWDVLDKLGADTFGSLYLNPDGMFPNHIPNPENKI AMQHTRAAVLENSADLGVVFDTDVDRSGVVDNKGNPINGDKLIALMSAIVLKEHPGSTVVTDARTSMGLTRFITERG GRHCLYRVGYRNVIDKGVELNKDGIETHLMMETSGHGAVKENHFLDDGAYMVVKIIIEMVRMRLAGSNEGIGSLIED LEEPLEAVELRLNILSEPRDAKAKGIEAIETFRQYIEEGKLKGWELGTCGDCWVTEGCLVDSNDHPSAIDAHMYRAR VSDEESGEEYGWVHMRQSIHNPNIALNMQSMLPGGCLSMTRIFRDQFLEASGVARFLDISDFDNYIGGQSLEHHHHH H-

The clone of embodiment 1 phosphoacetylglucosamine mutase AtAGM2 and AtAGM3 full-length genes

To intending south in The National Center for Biotechnology Information (NCBI) database After the pbosphohexose mutase gene of mustard is analyzed, the base of two not yet clear functions in hexose displacement enzyme family is had selected Cause.After sequence analysis, design primer is as follows：

Agm2-F:5’-GCGTCCATGGAAGGAAAGGTTTTCCAAAAC-3’；Agm2-R:5’- ATATATGCGGCCGCGGAGGAGTGAGTG-3 ' expands the gene order of Atagm2.Agm3-F:5’- AACTCCATGGCGTCGACTTCAACATCATC-3’；Agm3-R:5 '-ACTGCTCGAGAGATTGACCTCCGATGTAA-3 ' come Expand the gene order of Atagm3.

With reference to the mRNA of RNA extracts kits (Bo Maide biologies, article No. RN0112) operating procedure extraction Arabidopsis leaf. Using the cDNA that the RNA of the arabidopsis of extraction is inverted PCR amplification is carried out as template.PCR reaction conditions are：94 DEG C of 2min, 1 is followed Ring；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 2min, 30 cycles；72 DEG C of 5min, 1 cycle.PCR product carries out Ago-Gel electricity After swimming analysis, gel extraction (as shown in Figure 1) is carried out to target fragment, prokaryotic expression carrier pET28a is connected to after double digestion It is sequenced after upper.

2 phosphoacetylglucosamine mutase AtAGM2 of embodiment and AtAGM3 gene sequencings

The result of sequencing uses the Basic Local Alignment Search Tool in GenBank databases (BLAST) it analyzes, 8.0 softwares of Vector NTI Suite carry out Multiple Sequence Alignment, analytical sequence information.

The phosphoacetylglucosamine mutase gene 2 (being named as AtAGM2) of acquisition encodes head of district 1878bp, nucleosides Acid sequence is as shown in SEQ ID NO 1.AtAGM2 encodes 625 amino acid and a terminator codon, and amino acid sequence is such as Shown in SEQ ID NO 3, protein theoretical molecular weight is 67.0kDa, and prediction isoelectric point is 6.1.The nucleotide sequence of AtAGM2 In the genome of arabidopsis on No. five chromosomes of arabidopsis (locus-tag=" AT5G17530 ").

The phosphoacetylglucosamine mutase gene 3 (being named as AtAGM3) of acquisition encodes head of district 1872bp, nucleosides Acid sequence is as shown in SEQ ID NO 2.AtAGM encodes 623 amino acid and a terminator codon, and amino acid sequence is such as Shown in SEQ ID NO 4, protein theoretical molecular weight is 67.3kDa, and prediction isoelectric point is 5.62.The nucleotide sequence of AtAGM3 In the genome of arabidopsis on the No.1 chromosome of arabidopsis (locus-tag=" AT1G70820 ").

The pbosphohexose mutase gene that AtAGM2 and AtAGM3 is assumed that, belongs to pbosphohexose mutase (α-D- Phosphohexomutases) the member of family.

There is also another phosphoacetylglucosamine mutase AtAGM1, the encoding genes of the enzyme for arabidopsis (AT5G18070) coding head of district 1710bp encodes 556 amino acid, the AtAGM1 that Recombinant protein expression obtains, albumen Molecular weight is located in 61.5KDa, the nucleotide sequence that prediction isoelectric point is 5.35.AtAGM1 on No. five chromosomes of arabidopsis (locus-tag=" AT5G18070 "), activity have been accredited.

The gene similitude of AtAGM2 and AtAGM3 is up to 36.39%, and they are relatively low with the sequence similarity of AtAGM1, Only 15.45% and 12.28%, but some sequences are highly conserved.And the molecular weight of albumen and isoelectric point phase of three It is poor little.

3 AtAGM2 of embodiment and recombinant expression and purifying of the AtAGM3 genes in Escherichia coli

Sequencing result shows to be inserted into AtAGM2 genes shown in SEQ ID NO 1 on pET28a, and direction of insertion is just Really, it was demonstrated that the recombinant plasmid of structure is correct, which is named as pET28a-AtAGM2.

Meanwhile the recombinant plasmid of AtAGM3 also builds success, and AtAGM3 shown in SEQ ID NO 3 is inserted on pET28a Gene, and direction of insertion is correct, which is named as pET28a-AtAGM3.

PET28a-AtAGM2 (or pET28a-AtAGM3) conversion coli strain BL21 (DE3) are subjected to induction table It reaches.The seed liquor being incubated overnight is added in fresh LB culture mediums with 1% inoculum concentration and is enlarged culture in 37 DEG C, works as bacterium IPTG is added when the OD600nm=0.6-0.8 of liquid, makes its final concentration of 0.5mM, 16 DEG C carry out staying overnight induction.After bacterial cell disruption High speed centrifugation takes supernatant to carry out ni-sepharose purification, and gradient imidazoles elutes (20-500mM imidazoles, 20mM Tris-HCl, PH7.6).With Polyacrylamide gel electrophoresis detects expression and the purifying situation of phosphoacetylglucosamine mutase AtAGM2 (or AtAGM3), After electrophoresis uses 12% separation gel, 80V voltages to flatten, changes 120V voltages and run through separation gel.As a result pure as shown in Fig. 2 (or 3) The molecular weight phase of phosphoacetylglucosamine mutase AtAGM2 (or AtAGM3) position on running gel and prediction after change It coincide.

The zymologic property of embodiment 4 phosphoacetylglucosamine mutase AtAGM2 and AtAGM3

(1) vitality test of phosphoacetylglucosamine mutase AtAGM

The general system for measuring AtAGM enzyme activity is as follows：Different hexosephosphate substrate (GlcNAc-1-P, GlcNAc-6-P, GlcN-1-P, GlcN-6-P, Glc-1-P, Glc-6-P) it is used as substrate, every group of substrate that reaction system (300 μ L) is added：20mM PBS,pH 7.6,5mM MgSO₄, 10 μM of Glc-1,6-2P are added suitable recombinase AtAGM, react 10min.By reactant After system boils immediately, 200 μ L 200mM NaOH are added in removing protein, every group of substrate.With HAPEC-PAD method detection architecture midsoles The consumption of object and the generation situation of product.Since the reaction of AtAGM catalysis is reversible reaction, and also intermediate product hexose- 1,6-2P generation, so the amount of substrate (nmol) of enzyme activity (nmol/min/mg) 1mg albumen consumption per minute indicates.Egg White concentration is measured using green skies BCA determination of protein concentration kits.

Ion-exchange chromatography system used in HAPEC-PAD method Enzyme activity assays includes wearing peace Bio-LC gradient mixings pump, GM- 3 (4mm) gradient vortex mixers, ion-exchange chromatography CarboPac PA-100column (4 × 250mm), AgCl reference electrodes with Electrochemical detector.Pulse potential for detection, which changes, is：T=0s, E=0.10v；T=0.20s, E=0.10v；T= 0.40s, E=0.10v；T=0.41s, E=-2.00v；T=0.42s, E=-2.00v；T=0.43s, E=0.60v；T= 0.44s, E=-0.10v；Mobile phase used in t=0.50s, E=-0.10v.：A, 100mM NaOH aqueous solutions；B, 800mM second Sour sodium and 100mM NaOH aqueous solutions.Mobile phase elution requirement is 0-5min, 90%A+10%B；6-15min, 10%-90%B； 16-18min, 10%A+90%B；19-20min, 90%A+10%B. overall flow rate are 0.5ml/min, and detection column temperature is 30 DEG C, into Sample volume is 20 μ L

(2) phosphoacetylglucosamine mutase AtAGM substrate specificities

Since AtAGM substrate selectives are wider, GlcNAc-6-P and GlcNAc-1-P can be catalyzed；GlcN-6-P with GlcN-1-P；Several conversions between isomers of Glc-6-P and Glc-1-P.And AtAGM catalysis is reversible reaction, so The substrate specificity of AtAGM is detected using above six kinds of substrates.Using the general system mentioned in embodiment 4 (1), with Hexosephosphate substrate (GlcNAc-1-P, GlcNAc-6-P, GlcN-1-P, GlcN-6-P, Glc-1-P, Glc- different 0.1mM 6-P) it is used as substrate, every group of substrate reactions that 0.5 μ g AtAGM2 (or AtAGM3 or AtAGM1) are added, are reacted in 30 DEG C of water-baths 10min.Albumen concentration is measured using green skies BCA determination of protein concentration kits.

The results are shown in Table 1, under reaction condition as above, identical concentration of substrate, and vigor of the AtAGM to different substrates It is different.AtAGM2 with AtAGM3 with identify before come AtAGM1 compared with, although three kinds of AGM enzymes pair, six kinds of substrates are shown not Same catalytic rate, but still have certain general character.Universal law is that the vigor of positive reaction is more than back reaction (hexose -6-P Activity be higher than corresponding hexose -1-P).But generally speaking the activity of AtAGM2 and AtAGM3 will be less than AtAGM1, especially On its main function substrate GlcNAc-1-P and GlcNAc-6-P.And AtAGM1 and AtAGM3 urges Glc-1-P Change rate and be higher than Glc-6-P, this is with AtAGM2 and differs.

Table 1：The substrate specificity of phosphoacetylglucosamine mutase AtAGM2 and AtAGM3.

(3) influences of the pH to recombinase AtAGM

Using the general system mentioned in embodiment 4 (1), under conditions of 30 DEG C, respectively with 30 μM of GlcNAc-6-P For substrate, in pH 3.6-10.6 (pH 3.6-5.6HAc-NaAc, Ph 6.6-7.6Na₂HPO₄-NaH₂PO₄, pH 8.6Tris- HCl, pH 9.6-10.6Gly-NaOH) in reaction system, 5 μ g enzymes measure its activity according to standard detecting method.According to enzyme not Block diagram is drawn with the relative activity under pH, and line chart determines the optimal reaction pH of enzyme.As a contrast with the enzyme of inactivation, with The highest value of activity is 100%, measures the relative activity of the enzyme at each reaction pH.

The results are shown in Figure 4, and optimal pH range when AtAGM2 is using GlcNAc-6-P as substrate is in neutral slant acidity range It is interior, optimal pH 5.6.As shown in fig. 6, optimal pH range when AtAGM3 is using GlcNAc-6-P as substrate is in neutrality, it is most suitable PH is 7.0.Compared with the optimal pH of AtAGM1 is 7.6, three's optimal pH is not much different, and is in neutral range.

(4) influence of the temperature to recombinase AtAGM

Using the general system mentioned in embodiment 4 (1), using 30 μM of GlcNAc-1-P or GlcNAc-6-P as substrate, Measure the activity of recombinase according to standard method at 4-80 DEG C respectively, the relative activity such as Fig. 5 of 5 μ g enzymes at different temperatures, 7 It is shown.It is control with the enzyme of inactivation, is the opposite enzyme activity of 100% calculating to react highest enzyme activity.AtAGM2 is with GlcNAc-6-P Optimal reactive temperature as substrate is 30 DEG C (Fig. 5), and AtAGM2 is with the optimal reactive temperature using GlcNAc-6-P as substrate It is 20 DEG C (Fig. 7).Compared with the optimum temperature range of AtAGM1 is 25-30 DEG C, requirement of the three to reaction temperature also substantially phase Together.

(5) EDTA, SDS and metal ion etc. are on the active influences of AtAGM

Using the general system mentioned in embodiment 4 (1), using 30 μM of GlcNAc-6-P as substrate, in reaction system Various concentration of metal ions are set in 10mM, enzyme activity is detected according to standard method.As a contrast with the enzyme of inactivation, most with activity High value is 100%.The results are shown in Figure 8, Mg²⁺Have the effect of being significantly improved, some ions such as Ca to the enzyme activity of AtAGM2^{2 +}, Zn²⁺There is apparent inhibiting effect Deng to reaction.As shown in figure 9, Mg²⁺Also has the work that is significantly improved to the enzyme activity of AtAGM3 With some ions such as Fe²⁺, wait has apparent inhibiting effect to reaction.For AtAGM1, Mg²⁺Presence can also promote The progress of reaction.Illustrate Mg²⁺The phenomenon that activating reaction is the general character of all AtAGM.

SEQUENCE LISTING

<110>Dalian Inst of Chemicophysics, Chinese Academy of Sciences

<120>The preparation and application of AtAGM2 and AtAGM3 encoding genes and enzyme

<130>

<160> 8

<170> PatentIn version 3.5

<210> 1

<211> 1878

<212> DNA

<213>Arabidopsis（Arabidopsis thaliana）

<220>

<221> DNA

<222> (1)..(1878)

<400> 1

atggaaggaa aggttttcca aaactttaat gtagtacaga gctgctaccg acaaaataag 60

cagtttaaga cacgatacca aagagaacct gacctgttca tgtctacttt acttccctgt 120

ccaagagaga agatggcatt taacctcaac tcttccatgc gtgcccacac tttgtctaaa 180

taccagtttg ttctttcaaa gcaaagaact ttttactgca atgctacttc gtcaagtgct 240

actgtgccat ctcttgacaa aaatgatttt ctgaagctcc aaaacggcag tgatattcgg 300

ggtgtagcgg tcactggggt tgagggggaa cctgtaagcc ttcctgaacc agtgactgaa 360

gccatagctg ctgcttttgg gcaatggctg ttacacaaga agaaggctga atcccggcgt 420

ttgagagtat ctgttggcca tgactctcgc atctctgcac aaactttgct ggaggcggtt 480

tctcgaggtc ttggtgtttc tggattagat gttgttcagt ttggattagc atcaacacca 540

gcaatgttta atagcacatt gactgaagat gagtcattct tgtgcccagc tgatggggct 600

attatgataa cagcaagcca tcttccttac aacaggaacg gtttcaagtt ctttaccagt 660

gatggaggac ttgggaaggt tgatatcaag aacattttgg agcgagctgc agatatttac 720

aagaagcttt ctgatgaaaa tttgaggaaa tcacaaagag aaagttcttc tattacaaag 780

gttgactaca tgtcagtata cacctctggt cttgtaaagg cagtccggaa agcagcagga 840

gatttggaga agcctctaga gggatttcat atagttgttg atgctggaaa tggagctgga 900

ggattttttg ctgccaaggt gcttgagcct ttaggagcaa ttacttctgg cagtcaattt 960

ctggaaccag atggtatgtt cccaaatcat atccctaatc cggaagataa ggcggcaatg 1020

gaagctataa ccaaggctgt tcttgataat aaggctgatt tgggtatcat ctttgatact 1080

gatgttgata ggtctgctgc tgtggattca tctggccgtg aattcaaccg taatcgtctt 1140

attgccttgc tatcagccat tgttctagag gaacaccctg gcacaactat agttacggat 1200

agtgtcactt cggacggtct gacctcattt attgagaaga agcttggcgg aaagcatcac 1260

aggttcaaaa gaggttacaa gaatgtcatt gacgaagcta ttcgcttgaa ctcggttggg 1320

gaagaatcac atctggctat agaaaccagt ggtcatggag ctctaaagga aaaccattgg 1380

ctcgacgatg gggcctatct catggtaaaa atcctgaaca aactagctgc ggcccgagct 1440

gctggtcaag ggagtggcag caaagtttta acagatcttg ttgaaggtct ggaagagccc 1500

aaagtggctt tagaactgag gcttaaaatc gacaagaatc accctgacct tgaaggaagt 1560

gatttccggg agtatggaga gaaggtcctg caacacgtgt cgaactcaat agaaacaaat 1620

ccaaatctta taatagctcc agttaactac gaagggatcc gcgtttcggg ctttggtgga 1680

tggtttcttc tcagactttc tctccatgat cctgttcttc cccttaacat cgaggcacag 1740

agtgaggatg atgctgtgaa attaggcctt gtggttgcta cgacagtgaa ggagttcaat 1800

gctttggaca cctgtgcctt gtccaacctc actcactcct ccgcggccgc actcgagcac 1860

caccaccacc accactga 1878

<210> 2

<211> 625

<212> PRT

<213>Arabidopsis（Arabidopsis thaliana）

<220>

<221> PRT

<222> (1)..(625)

<400> 2

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

1 5 10 15

Arg Gln Asn Lys Gln Phe Lys Thr Arg Tyr Gln Arg Glu Pro Asp Leu

20 25 30

Phe Met Ser Thr Leu Leu Pro Cys Pro Arg Glu Lys Met Ala Phe Asn

35 40 45

Leu Asn Ser Ser Met Arg Ala His Thr Leu Ser Lys Tyr Gln Phe Val

50 55 60

Leu Ser Lys Gln Arg Thr Phe Tyr Cys Asn Ala Thr Ser Ser Ser Ala

65 70 75 80

Thr Val Pro Ser Leu Asp Lys Asn Asp Phe Leu Lys Leu Gln Asn Gly

85 90 95

Ser Asp Ile Arg Gly Val Ala Val Thr Gly Val Glu Gly Glu Pro Val

100 105 110

Ser Leu Pro Glu Pro Val Thr Glu Ala Ile Ala Ala Ala Phe Gly Gln

115 120 125

Trp Leu Leu His Lys Lys Lys Ala Glu Ser Arg Arg Leu Arg Val Ser

130 135 140

Val Gly His Asp Ser Arg Ile Ser Ala Gln Thr Leu Leu Glu Ala Val

145 150 155 160

Ser Arg Gly Leu Gly Val Ser Gly Leu Asp Val Val Gln Phe Gly Leu

165 170 175

Ala Ser Thr Pro Ala Met Phe Asn Ser Thr Leu Thr Glu Asp Glu Ser

180 185 190

Phe Leu Cys Pro Ala Asp Gly Ala Ile Met Ile Thr Ala Ser His Leu

195 200 205

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

210 215 220

Gly Lys Val Asp Ile Lys Asn Ile Leu Glu Arg Ala Ala Asp Ile Tyr

225 230 235 240

Lys Lys Leu Ser Asp Glu Asn Leu Arg Lys Ser Gln Arg Glu Ser Ser

245 250 255

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

260 265 270

Lys Ala Val Arg Lys Ala Ala Gly Asp Leu Glu Lys Pro Leu Glu Gly

275 280 285

Phe His Ile Val Val Asp Ala Gly Asn Gly Ala Gly Gly Phe Phe Ala

290 295 300

Ala Lys Val Leu Glu Pro Leu Gly Ala Ile Thr Ser Gly Ser Gln Phe

305 310 315 320

Leu Glu Pro Asp Gly Met Phe Pro Asn His Ile Pro Asn Pro Glu Asp

325 330 335

Lys Ala Ala Met Glu Ala Ile Thr Lys Ala Val Leu Asp Asn Lys Ala

340 345 350

Asp Leu Gly Ile Ile Phe Asp Thr Asp Val Asp Arg Ser Ala Ala Val

355 360 365

Asp Ser Ser Gly Arg Glu Phe Asn Arg Asn Arg Leu Ile Ala Leu Leu

370 375 380

Ser Ala Ile Val Leu Glu Glu His Pro Gly Thr Thr Ile Val Thr Asp

385 390 395 400

Ser Val Thr Ser Asp Gly Leu Thr Ser Phe Ile Glu Lys Lys Leu Gly

405 410 415

Gly Lys His His Arg Phe Lys Arg Gly Tyr Lys Asn Val Ile Asp Glu

420 425 430

Ala Ile Arg Leu Asn Ser Val Gly Glu Glu Ser His Leu Ala Ile Glu

435 440 445

Thr Ser Gly His Gly Ala Leu Lys Glu Asn His Trp Leu Asp Asp Gly

450 455 460

Ala Tyr Leu Met Val Lys Ile Leu Asn Lys Leu Ala Ala Ala Arg Ala

465 470 475 480

Ala Gly Gln Gly Ser Gly Ser Lys Val Leu Thr Asp Leu Val Glu Gly

485 490 495

Leu Glu Glu Pro Lys Val Ala Leu Glu Leu Arg Leu Lys Ile Asp Lys

500 505 510

Asn His Pro Asp Leu Glu Gly Ser Asp Phe Arg Glu Tyr Gly Glu Lys

515 520 525

Val Leu Gln His Val Ser Asn Ser Ile Glu Thr Asn Pro Asn Leu Ile

530 535 540

Ile Ala Pro Val Asn Tyr Glu Gly Ile Arg Val Ser Gly Phe Gly Gly

545 550 555 560

Trp Phe Leu Leu Arg Leu Ser Leu His Asp Pro Val Leu Pro Leu Asn

565 570 575

Ile Glu Ala Gln Ser Glu Asp Asp Ala Val Lys Leu Gly Leu Val Val

580 585 590

Ala Thr Thr Val Lys Glu Phe Asn Ala Leu Asp Thr Cys Ala Leu Ser

595 600 605

Asn Leu Thr His Ser Ser Ala Ala Ala Leu Glu His His His His His

610 615 620

His

625

<210> 3

<211> 1872

<212> DNA

<213>Arabidopsis（Arabidopsis thaliana）

<220>

<221> DNA

<222> (1)..(1872)

<400> 3

atggcgtcga cttcaacatc atctttaatg gcttctaaaa ctgtaatctc caaaacagct 60

ctgttttctt ccttaccggg aatagtcagc cggagttttt taacattcgc accggcttct 120

ccttccgtta aaccccttag gataagatct tcaaatgtta ctaagttcga cgaagtaacc 180

aacagtcttg acgaagacat ggaccagatt cgacggttac aaaacggttc tgacgtgaga 240

ggagtcgcat tggaaggaga gaaaggtcga acagttgacc taacgcctgc agctgttgaa 300

gcaatcgcag agagctttgg agaatgggtt gcagcaacgg agagtaacgg aaacggcgtc 360

attaagattt ctctcggacg ggatccacgt gtttccggtg ggaagctaag cacggcagtg 420

tttgccggct tagctcgtgc aggctgttta gcttttgaca tgggtttagc tacagcgcca 480

gcttgcttca tgagcacgtt actctctcca ttcgaatacg acgcttcaat tatgatgaca 540

gcttctcatt taccgtatac aagaaacgga ctcaagttct ttaccaagag aggaggatta 600

acgtctcctg aagtggagaa gatatgcgat ttagctgcgc gaaagtacgc tactaggcag 660

actaaagtct ctacattgat cagaacgcga ccgcagcaag ttgattttat gagcgcttac 720

tctaagcacc ttagagaaat cattaaagag agaatcaatc accctgaaca ctatgacact 780

cctctcaaag gatttcagat agttgtgaat gcgggtaatg ggtcaggagg cttctttacg 840

tgggacgttc tagacaagtt aggagccgat acattcggtt cgctctatct aaaccctgac 900

gggatgttcc ctaatcacat tcctaatccg gaaaacaaaa tcgcaatgca acacacccga 960

gccgcggttc ttgagaactc agcagatctc ggggttgtgt ttgatacgga tgttgacagg 1020

agtggagtgg tggataacaa aggaaatcct atcaacggag ataagcttat tgcgcttatg 1080

tcagctatag tgcttaaaga acatccagga agtacagtag tgactgacgc aagaacgagt 1140

atggggctaa ctaggtttat aacggagcga ggagggaggc attgtttgta tagagtaggg 1200

tatagaaacg tgattgacaa gggagtagag ctgaacaaag acggcatcga gactcatctc 1260

atgatggaaa cttcaggaca tggtgcggtt aaggagaatc acttcttgga tgatggtgca 1320

tacatggtgg tgaagatcat aattgaaatg gtgagaatga gactcgcggg atcaaatgaa 1380

ggtatcggta gtttgatcga agatcttgag gagccgttag aagcggttga gcttcggttg 1440

aatattttat cagagccaag agatgccaaa gcaaaaggca ttgaagccat tgagactttc 1500

aggcaataca ttgaggaagg aaaactgaaa gggtgggaat tgggcacgtg tggggattgt 1560

tgggttactg aaggttgctt ggtggactca aatgatcatc catctgctat tgatgctcac 1620

atgtacaggg caagagtgag tgatgaagag agtggggaag agtatggttg ggtgcatatg 1680

aggcagagta ttcataaccc taacatcgca cttaatatgc aatcaatgct tcctggtgga 1740

tgtctctcca tgacaagaat cttcagagac cagtttcttg aagctagtgg ggtggctaga 1800

ttcctggata taagtgactt cgacaattac atcggaggtc aatctctcga gcaccaccac 1860

caccaccact ga 1872

<210> 4

<211> 623

<212> PRT

<213> MASTSTSSLMASKTVISKTALFSSLPGIVSRSFLTFAPASPSVKPLRIRSSNVTKFDEVTNSLDEDM DQIRRLQNGSDVRGVALEGEKGRTVDLTPAAVEAIAESFGEWVAATESNGNGVIKISLGRDPRVSGGKLSTAVFAGL ARAGCLAFDMGLATAP

<220>

<221> PRT

<222> (1)..(623)

<400> 4

Met Ala Ser Thr Ser Thr Ser Ser Leu Met Ala Ser Lys Thr Val Ile

1 5 10 15

Ser Lys Thr Ala Leu Phe Ser Ser Leu Pro Gly Ile Val Ser Arg Ser

20 25 30

Phe Leu Thr Phe Ala Pro Ala Ser Pro Ser Val Lys Pro Leu Arg Ile

35 40 45

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

50 55 60

Glu Asp Met Asp Gln Ile Arg Arg Leu Gln Asn Gly Ser Asp Val Arg

65 70 75 80

Gly Val Ala Leu Glu Gly Glu Lys Gly Arg Thr Val Asp Leu Thr Pro

85 90 95

Ala Ala Val Glu Ala Ile Ala Glu Ser Phe Gly Glu Trp Val Ala Ala

100 105 110

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

115 120 125

Pro Arg Val Ser Gly Gly Lys Leu Ser Thr Ala Val Phe Ala Gly Leu

130 135 140

Ala Arg Ala Gly Cys Leu Ala Phe Asp Met Gly Leu Ala Thr Ala Pro

145 150 155 160

Ala Cys Phe Met Ser Thr Leu Leu Ser Pro Phe Glu Tyr Asp Ala Ser

165 170 175

Ile Met Met Thr Ala Ser His Leu Pro Tyr Thr Arg Asn Gly Leu Lys

180 185 190

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

195 200 205

Cys Asp Leu Ala Ala Arg Lys Tyr Ala Thr Arg Gln Thr Lys Val Ser

210 215 220

Thr Leu Ile Arg Thr Arg Pro Gln Gln Val Asp Phe Met Ser Ala Tyr

225 230 235 240

Ser Lys His Leu Arg Glu Ile Ile Lys Glu Arg Ile Asn His Pro Glu

245 250 255

His Tyr Asp Thr Pro Leu Lys Gly Phe Gln Ile Val Val Asn Ala Gly

260 265 270

Asn Gly Ser Gly Gly Phe Phe Thr Trp Asp Val Leu Asp Lys Leu Gly

275 280 285

Ala Asp Thr Phe Gly Ser Leu Tyr Leu Asn Pro Asp Gly Met Phe Pro

290 295 300

Asn His Ile Pro Asn Pro Glu Asn Lys Ile Ala Met Gln His Thr Arg

305 310 315 320

Ala Ala Val Leu Glu Asn Ser Ala Asp Leu Gly Val Val Phe Asp Thr

325 330 335

Asp Val Asp Arg Ser Gly Val Val Asp Asn Lys Gly Asn Pro Ile Asn

340 345 350

Gly Asp Lys Leu Ile Ala Leu Met Ser Ala Ile Val Leu Lys Glu His

355 360 365

Pro Gly Ser Thr Val Val Thr Asp Ala Arg Thr Ser Met Gly Leu Thr

370 375 380

Arg Phe Ile Thr Glu Arg Gly Gly Arg His Cys Leu Tyr Arg Val Gly

385 390 395 400

Tyr Arg Asn Val Ile Asp Lys Gly Val Glu Leu Asn Lys Asp Gly Ile

405 410 415

Glu Thr His Leu Met Met Glu Thr Ser Gly His Gly Ala Val Lys Glu

420 425 430

Asn His Phe Leu Asp Asp Gly Ala Tyr Met Val Val Lys Ile Ile Ile

435 440 445

Glu Met Val Arg Met Arg Leu Ala Gly Ser Asn Glu Gly Ile Gly Ser

450 455 460

Leu Ile Glu Asp Leu Glu Glu Pro Leu Glu Ala Val Glu Leu Arg Leu

465 470 475 480

Asn Ile Leu Ser Glu Pro Arg Asp Ala Lys Ala Lys Gly Ile Glu Ala

485 490 495

Ile Glu Thr Phe Arg Gln Tyr Ile Glu Glu Gly Lys Leu Lys Gly Trp

500 505 510

Glu Leu Gly Thr Cys Gly Asp Cys Trp Val Thr Glu Gly Cys Leu Val

515 520 525

Asp Ser Asn Asp His Pro Ser Ala Ile Asp Ala His Met Tyr Arg Ala

530 535 540

Arg Val Ser Asp Glu Glu Ser Gly Glu Glu Tyr Gly Trp Val His Met

545 550 555 560

Arg Gln Ser Ile His Asn Pro Asn Ile Ala Leu Asn Met Gln Ser Met

565 570 575

Leu Pro Gly Gly Cys Leu Ser Met Thr Arg Ile Phe Arg Asp Gln Phe

580 585 590

Leu Glu Ala Ser Gly Val Ala Arg Phe Leu Asp Ile Ser Asp Phe Asp

595 600 605

Asn Tyr Ile Gly Gly Gln Ser Leu Glu His His His His His His

610 615 620

<210> 5

<211> 30

<212> DNA

<213>It is artificial synthesized

<220>

<221> DNA

<222> (1)..(30)

<400> 5

gcgtccatgg aaggaaaggt tttccaaaac 30

<210> 6

<211> 27

<212> DNA

<213>It is artificial synthesized

<220>

<221> DNA

<222> (1)..(27)

<400> 6

atatatgcgg ccgcggagga gtgagtg 27

<210> 7

<211> 29

<212> DNA

<213>It is artificial synthesized

<220>

<221> DNA

<222> (1)..(29)

<400> 7

aactccatgg cgtcgacttc aacatcatc 29

<210> 8

<211> 29

<212> DNA

<213>It is artificial synthesized

<220>

<221> DNA

<222> (1)..(29)

<400> 8

actgctcgag agattgacct ccgatgtaa 29

Claims (8)

1. a kind of encoding gene of phosphoacetylglucosamine mutase AtAGM2, nucleotide sequence has in following feature It is one or two or more kinds of：

1) DNA (DNA) sequence with SEQ ID NO.1 in sequence table；

2) DNA (DNA) sequence of SEQ ID NO.2 amino acid sequences is encoded；

3) one or more nucleotide is carried out to DNA (DNA) sequence of SEQ ID NO.1 in sequence table to take Coding has the active nucleotide sequence of phosphoacetylglucosamine mutase obtained from generation, missing or addition.

2. a kind of encoding gene of phosphoacetylglucosamine mutase AtAGM3, nucleotide sequence has in following feature It is one or two or more kinds of：

1) DNA (DNA) sequence with SEQ ID NO.3 in sequence table；

2) DNA (DNA) sequence of SEQ ID NO.4 amino acid sequences is encoded；

3) one or more nucleotide is carried out to DNA (DNA) sequence of SEQ ID NO.3 in sequence table to take Coding has the active nucleotide sequence of phosphoacetylglucosamine mutase obtained from generation, missing or addition.

3. a kind of acetyl grape of the encoding gene coding of phosphoacetylglucosamine mutase AtAGM2 described in claim 1 Osamine transphosphorylase, it is characterised in that：Its amino acid sequence has one kind or two kinds in following feature：

1) 1-625 amino acids residue sequences of the SEQ ID NO.2 since aminoterminal in sequence table；

2) one or more amino acid substitution, missing are carried out to amino acid sequence shown in SEQ ID NO.2 in sequence table Or addition and formed have the active amino acid sequence of phosphoacetylglucosamine mutase.

4. a kind of acetyl grape of the encoding gene coding of the phosphoacetylglucosamine mutase AtAGM3 described in claim 2 Osamine transphosphorylase, it is characterised in that：Its amino acid sequence has one kind or two kinds in following feature：

1) 1-623 amino acids residue sequences of the SEQ ID NO.4 since aminoterminal in sequence table；

2) one or more amino acid substitution, missing are carried out to amino acid sequence shown in SEQ ID NO.4 in sequence table Or addition and formed have the active amino acid sequence of phosphoacetylglucosamine mutase.

5. the application of the phosphoacetylglucosamine mutase described in a kind of claim 3 or 4, it is characterised in that：The enzyme can be with To acetylglucosamnie-6-phosphate (GlcNAc-6-P), acetylglucosamine -1- phosphoric acid (GlcNAc-1-P), gucosamine -6- Phosphoric acid (GlcN-6-P), gucosamine -1- phosphoric acid (GlcN-1-P) and G-6-P (Glc-6-P), glucose -1- phosphorus One or two or more kinds of pbosphohexoses in sour (Glc-1-P) etc. have displacement activity.

6. the preparation method of the phosphoacetylglucosamine mutase described in a kind of claim 3 and 4, it is characterised in that：Being will The encoding gene of phosphoacetylglucosamine mutase AtAGM2 or AtAGM3 are cloned into recombinant expression carrier, and it is thin to import host Born of the same parents obtain the phosphoacetylglucosamine mutase of recombinant expression；

The encoding gene of above-mentioned phosphoacetylglucosamine mutase AtAGM, nucleotide sequence have one in following feature Kind or two kinds or more：

1) DNA (DNA) sequence with SEQ ID NO.1 or SEQ ID NO.3 in sequence table,

2) DNA (DNA) sequence of SEQ ID NO.2 or SEQ ID NO.4 amino acid sequences is encoded,

3) one or two is carried out to DNA (DNA) sequence of SEQ ID NO.1 in sequence table or SEQ ID NO.3 Coding has the active nucleotides sequence of phosphoacetylglucosamine mutase obtained from the above nucleotide replaces, misses or adds Row；

The expression vector of the recombinant expression phosphoacetylglucosamine mutase, refers to coli expression carrier, yeast Expression vector, hay bacillus expression vector, lactic acid bacteria expression vectors, streptomyces expression vector, phage vector, filamentous fungi table One or two or more kinds up in carrier, plant expression vector, insect expression vector or mammalian cell expression vector.

7. according to claim 6 the method, it is characterised in that：Weight for recombinantly expressing phosphoacetylglucosamine mutase Group bacterium or transgenic cell line, refer to e. coli host cell Escherichia coli BL21, Escherichia coli JM109, Escherichia coli DH5 α, yeast host cells Saccharomyces cerevisiae, Pichia Pastoris, Kluyveromyces lactis, hay bacillus host cell Bacillus subtilis R25, Bacillus Subtilis 9920, lactic acid bacteria host cell Lactic acid bacteria COCC101, actinomyces host cell Streptomyces spp, filamentous fungal host cell Trichoderma viride, Trichoderma reesei, Aspergillus niger, Aspergillus nidulans, insect cell Bombyx mori, Antharaea Eucalypti, mammalian cell Chinese hamster ovary cell CHO, baby hamster kidney cell BHK, CHL cells One or two or more kinds in CHL.

8. the phosphoacetylglucosamine mutase described in a kind of claim 3 or 4 is in hexosephosphate and nucleotide sugar production Application, it is characterised in that：Including in applying below one or two kind or more：

1) in the transformation for realizing 1,6 position isomer of hexosephosphate, the application in corresponding isomer is produced；

2) in UDP-GlcNAc (UDP-GlcN；The application in the production of nucleotide sugars such as UDP-Glc).