CN110157693A - A kind of -6 phosphate synthase mutant of Glucosamine - Google Patents

A kind of -6 phosphate synthase mutant of Glucosamine Download PDF

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CN110157693A
CN110157693A CN201910439712.6A CN201910439712A CN110157693A CN 110157693 A CN110157693 A CN 110157693A CN 201910439712 A CN201910439712 A CN 201910439712A CN 110157693 A CN110157693 A CN 110157693A
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glucosamine
phosphate synthase
leu
acetylglucosamine
ala
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刘龙
陈坚
堵国成
宋阳
顾洋
李江华
赵雅雯
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Jiangnan University
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1096—Transferases (2.) transferring nitrogenous groups (2.6)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y206/00—Transferases transferring nitrogenous groups (2.6)
    • C12Y206/01—Transaminases (2.6.1)
    • C12Y206/01016—Glutamine-fructose-6-phosphate transaminase (isomerizing) (2.6.1.16), i.e. glucosamine-6-phosphate-synthase

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Abstract

The invention discloses a kind of -6 phosphate synthase mutant of Glucosamine, belong to genetic engineering and enzyme engineering field.The present invention provides the Glucosamine -6- phosphate synthase mutant from E.coli using the method for fixed point saturation mutation Glucosamine -6- phosphate synthase.Compared to wild type Glucosamine -6- phosphotransferase, mutant significantly improves the catalysis activity of fructose-1, 6-diphosphate, and the yield of acetylglucosamine is higher, is more suitable for industrialized production.

Description

A kind of -6 phosphate synthase mutant of Glucosamine
Technical field
The present invention relates to a kind of -6 phosphate synthase mutant of Glucosamine, belong to genetic engineering and enzyme engineering field.
Background technique
Acetylglucosamine is a kind of intracorporal monosaccharide of biology, be widely present in bacterium, yeast, mould, plant and In animal body.In human body, acetylglucosamine is the synthesis precursor of glycosaminoglycan disaccharide unit, to repairing and maintain soft Bone and joint tissue function play a significant role.Therefore, acetylglucosamine is widely used as drug and nutritious food addition To treat and repair joint injury.In addition, acetylglucosamine also has many applications in cosmetics and pharmaceutical field.Mesh Before, acetylglucosamine mainly uses chitin in acidolysis shrimp shell or crab shell to produce, and the waste liquid that the method generates is to environment dirt Contaminate more serious, and obtained product easily causes allergic reaction, and the crowd for being not suitable for seafood allergy takes.
Biosynthesis to GlcN is research shows that in Escherichia coli, by glutamine as amino group donor, 6- phosphoric acid fruit Sugar generates GlcN, which is in GlcN route of synthesis under the catalytic action of Glucosamine -6- phosphate synthase (GlmS) First rate-limiting reaction.And -6 phosphate synthase of Glucosamine is a kind of Double bottom object desmoenzyme, belongs to transamination enzyme, is urged Change vigor is lower.
Summary of the invention
The purpose of the present invention is to provide a kind of -6 phosphate synthase mutant of Glucosamine first, is by amino acid sequence The 38th alanine mutation of the wild GlmS as shown in SEQ ID NO.2 is that glutamic acid or the 249th arginine are prominent Become tryptophan or the 471st glycine mutation is serine.
The present invention also provides the methods for obtaining -6 phosphate synthase mutant of Glucosamine, with pET28a-glmS- Gna1 is template, by rite-directed mutagenesis by the gene of encoding amino acid sequence wild type GlmS as shown in SEQ ID NO.2 into Row mutation, the carrier for carrying the gene of encoding mutant body is transferred to E.coli BL21 (DE3), culture recombinant bacterium obtains amino - 6 phosphate synthase mutant of glucose.
The nucleotide sequence of the wild type GlmS is encoded as shown in SEQ ID NO.1, derives from Escherichia coli Escherichia coli BL21(DE3)。
The present invention also provides the methods for improving acetylglucosamine yield, are with Escherichia coli BL21 It (DE3) is host, recombinant expression encodes the gene of -6 phosphate synthase mutant of Glucosamine, obtains recombinant bacterium；It will Recombination bacillus coli with 5~8% inoculum concentration is transferred to fermentation medium after activating with seed culture medium, in 35~37 DEG C, 200~ It is cultivated under the conditions of 220rpm, OD600IPTG induction -6 phosphate synthase of Glucosamine of 0.4mM is added when reaching 0.6 or so Expression enables recombinant bacterium to produce acetylglucosamine by substrate of glucose.
Seed culture medium (g/L): tryptose arteries and veins 10, yeast powder 5, NaCl 10.
Fermentation medium (g/L): tryptone 12, yeast powder 24, dipotassium hydrogen phosphate 12.54, potassium dihydrogen phosphate 2.31 are sweet Oil 4, glucose 30.
Beneficial effects of the present invention: 3 significant mutant G471S, R249W, A38E are constructed, raising is realized The effect of GlmS catalysis activity, the synthesis of acetylglucosamine more advantageous than wild type GlmS.Compared to wild type GlmS enzyme, dash forward Variant G471S, R249W, A38T are after the 16h that ferments, using glucose as substrate, G471S acetylglucosamine yield 36.975mg/L improves 22.62%, R249W yield compared with bacterium germination out and reaches 148.23mg/L, strain yield of setting out 4.66 again.A38E yield reaches 127.56mg/L, is 4.01 times of starting strain yield.
Specific embodiment
The measuring method of acetylglucosamine:
High performance liquid chromatography (HPLC) detection method: Agilent 1260, RID detector, HPX-87H column (Bio-Rad Hercules, CA), mobile phase: 5mM H2SO4, flow velocity 0.6mL/min, 35 DEG C of column temperature, 10 μ L of sampling volume.
Seed culture medium (g/L): tryptose arteries and veins 10, yeast powder 5, NaCl 10.
Fermentation medium (g/L): tryptone 12, yeast powder 24, dipotassium hydrogen phosphate 12.54, potassium dihydrogen phosphate 2.31 are sweet Oil 4, glucose 30.
The determination in 1 mutational site of embodiment
The catalyst mechanism of GlmS defers to orderly reaction mechanism, and fructose-1, 6-diphosphate is first in conjunction with glutamine, with glutamy The isomerization of the hydrolysis of amine, transamination and amino sugar, GlmS successively glutamate release and GlcN-6-P.The GlmS of Escherichia coli For homodimer, the isomery enzyme domains in two different monomers are two paddy ammonia on the inside of dimer, on the outside of dimer The structural domain that amide combines.It is determined by the PDB crystal structure analysis (PDB is encoded to 1JXA) of the GlmS from E.coli G471, R249, A38 are possible raising enzyme activity site.
The preparation of embodiment 2 mutant G471S, R249W, A38E
(1) rite-directed mutagenesis
Using fast PCR technology, to express the plasmid pET28a-glmS-gna1 for the gene for having wild type GlmS (construction method of pET28a-glmS-gna1 is referring to CN103589696A) is template, carries out rite-directed mutagenesis.
Introduce the primer of G471S mutation:
Forward primer: 5 '-GCTGTTCCTGGCCGTGGCGATC-3 '
Reverse primer: 5 '-GATCGCCACGGCCAGGAACAGC-3 '
Introduce the primer of R249W mutation
Forward primer: 5 '-CGGGCGATAAAGGCATTTACTGGCACTACATGCAGA-3 '
Reverse primer: 5 '-TCTGCATGTAGTGCCAGTAAATGCCTTTATCGCCCG-3 '
Introduce the primer of A38E mutation
Forward primer: 5 '-CCGTTGTTGATGAAGAAGGTCATATGACCCGCC-3 '
Reverse primer: 5 '-GGCGGGTCATATGACCTTCTTCATCAACAACGG-3 '
PCR reaction system is equal are as follows: 5 ﹡ PrimerStar Buffer, 10 4 μ L of μ L, dNTPs (each 2.5mM), forward primer (10 μM) 1 μ L, 1 μ L of reverse primer (10 μM), 1 μ L, PrimerStar HS DNA Ploymerase (2.5U/ μ L) of template DNA 32.5 μ L of distilled water is added in 0.5 μ L.
It is equal that PCR reacts amplification condition are as follows: 98 DEG C of initial denaturation 4min；Subsequent 98 DEG C of 10s, 55 DEG C of 15s, 72 DEG C of 8min carry out 30 A circulation, last 72 DEG C of heat preservations 10min.
By PCR product after Dpn I digests 2h, the competence for being transferred to E. coli BL21 (DE3) respectively is thin In born of the same parents, it is applied to overnight incubation in the LB solid medium containing agar, picking single bacterium, which is fallen in LB liquid medium, cultivated Plasmid is extracted after night and carries out sequence verification.
(2) expression and determination of yield of mutant
Picking is sequenced in correct mutant and LB culture medium, and at 37 DEG C, overnight incubation under 200rpm is connect with 2% (v/v) Kind amount is inoculated into TB culture medium, in 37 DEG C, 200rpm culture to OD600It is 0.6,0.4mM IPTG induction is added, is sent out after induction Ferment 12h.
In the fermentation supernatant for expressing the recombinant bacterium of G471S, R249W, A38E, acetylglucosamine yield is respectively 36.975mg/L, 148.23mg/L, 127.56mg/L improve 22.6%, 366.4%, 301.7% compared to wild type.Recombination is big Enterobacteria seed culture and fermentation medium:
Seed culture medium (g/L): tryptose arteries and veins 10, yeast powder 5, NaCl 10.
Fermentation medium (g/L): tryptone 12, yeast powder 24, dipotassium hydrogen phosphate 12.54, potassium dihydrogen phosphate 2.31 are sweet Oil 4, glucose 30.
3 fermenting and producing acetylglucosamine of embodiment
With LB slant medium culture recombination bacillus coli, after cultivating 12h, take the intervention of 1 ring equipped with 20mL kind with oese Cultivated in the triangular flask of the 250mL of sub- culture medium, in seed culture medium according to 50 μ g/mL be added kanamycins, 37 DEG C, 12h is cultivated under 200rpm.Seed is transferred to fermentation medium with 5% inoculum concentration, cultivates under the conditions of 37 DEG C, 200rpm, OD600 The IPTG induction of 0.4mM is added when equal to 0.6, cultivates 16h after induction.Measure acetylglucosamine in final fermented supernatant fluid Yield, the results showed that express acetylglucosamine yield 36.975mg/L in the fermentation supernatant of the recombinant bacterium of G471S, with go out Bacterium germination is compared to improve 22.62%；It expresses yield in the fermentation supernatant of the recombinant bacterium of R249W and reaches 148.23mg/L, be bacterium germination out 4.66 times of kind yield；It expresses yield in the fermentation supernatant of the recombinant bacterium of A38E and reaches 127.56mg/L, be starting strain yield 4.01 times.By improving GlmS catalysis activity, the accumulation for improving acetylglucosamine in recombination bacillus coli is realized.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention Enclosing subject to the definition of the claims.
SEQUENCE LISTING
<110>Southern Yangtze University
<120>a kind of -6 phosphate synthase mutant of Glucosamine
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 1830
<212> DNA
<213>Escherichia coli
<400> 1
atgtgtggaa ttgttggcgc gatcgcgcaa cgtgatgtag cagaaatcct tcttgaaggt 60
ttacgtcgtc tggaataccg cggatatgac tctgccggtc tggccgttgt tgatgcggaa 120
ggtcatatga cccgcctgcg tcgcctcggt aaagtccaga tgctggctca ggcagcggaa 180
gaacatcctc tgcatggcgg caccggtatt gctcatactc gctgggcgac acacggtgaa 240
ccttcagaag tgaatgcgca tccgcatgtt tctgaacaca ttgtggtggt gcataacggc 300
atcatcgaaa accatgaacc gctgcgtgaa gagctaaaag cgcgtggcta taccttcgtt 360
tctgaaaccg acaccgaagt gattgcccat ctggtgaact gggagctgaa acaaggcggg 420
actctgcgtg aggccgttct gcgtgctatc ccgcagctgc gtggtgcgta cggtacagtg 480
atcatggact cccgtcaccc ggataccctg ctggcggcac gttctggtag tccgctggtg 540
attggcctgg ggatgggcga aaactttatc gcttctgacc agctggcgct gttgccggtg 600
acccgtcgct ttatcttcct tgaagagggc gatattgcgg aaatcactcg ccgttcggta 660
aacatcttcg ataaaactgg cgcggaagta aaacgtcagg atatcgaatc caatctgcaa 720
tatgacgcgg gcgataaagg catttaccgt cactacatgc agaaagagat ctacgaacag 780
ccgaacgcga tcaaaaacac ccttaccgga cgcatcagcc acggtcaggt tgatttaagc 840
gagctgggac cgaacgccga cgaactgctg tcgaaggttg agcatattca gatcctcgcc 900
tgtggtactt cttataactc cggtatggtt tcccgctact ggtttgaatc gctagcaggt 960
attccgtgcg acgtcgaaat cgcctctgaa ttccgctatc gcaaatctgc cgtgcgtcgt 1020
aacagcctga tgatcacctt gtcacagtct ggcgaaaccg cggataccct ggctggcctg 1080
cgtctgtcga aagagctggg ttaccttggt tcactggcaa tctgtaacgt tccgggttct 1140
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ttgccgagcc gtattgagca gatgctgtct caggacaaac gcattgaagc tctggcagaa 1380
gatttctctg acaaacatca cgcgctgttc ctgggccgtg gcgatcagta cccaatcgcg 1440
ctggaaggcg cattgaagct gaaagagatc tcttacattc acgctgaagc ctacgctgca 1500
ggtgaactga aacacggtcc gctggcgctg attgatgccg atatgccggt tatcgtcgtt 1560
gcaccgaaca acgaattgct ggaaaaacta aaatccaaca ttgaagaagt tcgcgcgcgt 1620
ggcggtcagt tgtatgtctt cgccgatcag gatgcgggtt ttgtaagtag cgataacatg 1680
cacatcatcg agatgccgca tgtggaagag gtgattgcac caatcttcta caccgttccg 1740
ctgcagctac tggcttatca cgtcgcgctg atcaaaggta ccgacgttga ccagccgcgt 1800
aacctggcaa aatcggttac ggttgagtaa 1830
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Met Cys Gly Ile Val Gly Ala Ile Ala Gln Arg Asp Val Ala Glu Ile
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His Gly Gly Thr Gly Ile Ala His Thr Arg Trp Ala Thr His Gly Glu
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Pro Ser Glu Val Asn Ala His Pro His Val Ser Glu His Ile Val Val
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Val His Asn Gly Ile Ile Glu Asn His Glu Pro Leu Arg Glu Glu Leu
100 105 110
Lys Ala Arg Gly Tyr Thr Phe Val Ser Glu Thr Asp Thr Glu Val Ile
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Ala His Leu Val Asn Trp Glu Leu Lys Gln Gly Gly Thr Leu Arg Glu
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Ala Val Leu Arg Ala Ile Pro Gln Leu Arg Gly Ala Tyr Gly Thr Val
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Ile Met Asp Ser Arg His Pro Asp Thr Leu Leu Ala Ala Arg Ser Gly
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Ser Pro Leu Val Ile Gly Leu Gly Met Gly Glu Asn Phe Ile Ala Ser
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Asp Gln Leu Ala Leu Leu Pro Val Thr Arg Arg Phe Ile Phe Leu Glu
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Lys Thr Gly Ala Glu Val Lys Arg Gln Asp Ile Glu Ser Asn Leu Gln
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Tyr Asp Ala Gly Asp Lys Gly Ile Tyr Arg His Tyr Met Gln Lys Glu
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Ser His Gly Gln Val Asp Leu Ser Glu Leu Gly Pro Asn Ala Asp Glu
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Leu Leu Ser Lys Val Glu His Ile Gln Ile Leu Ala Cys Gly Thr Ser
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Tyr Asn Ser Gly Met Val Ser Arg Tyr Trp Phe Glu Ser Leu Ala Gly
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Ile Pro Cys Asp Val Glu Ile Ala Ser Glu Phe Arg Tyr Arg Lys Ser
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Ala Val Arg Arg Asn Ser Leu Met Ile Thr Leu Ser Gln Ser Gly Glu
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Thr Ala Asp Thr Leu Ala Gly Leu Arg Leu Ser Lys Glu Leu Gly Tyr
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370 375 380
Glu Ser Asp Leu Ala Leu Met Thr Asn Ala Gly Thr Glu Ile Gly Val
385 390 395 400
Ala Ser Thr Lys Ala Phe Thr Thr Gln Leu Thr Val Leu Leu Met Leu
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Val Ala Lys Leu Ser Arg Leu Lys Gly Leu Asp Ala Ser Ile Glu His
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Asp Ile Val His Gly Leu Gln Ala Leu Pro Ser Arg Ile Glu Gln Met
435 440 445
Leu Ser Gln Asp Lys Arg Ile Glu Ala Leu Ala Glu Asp Phe Ser Asp
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Lys His His Ala Leu Phe Leu Gly Arg Gly Asp Gln Tyr Pro Ile Ala
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Ala Asp Met Pro Val Ile Val Val Ala Pro Asn Asn Glu Leu Leu Glu
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Lys Leu Lys Ser Asn Ile Glu Glu Val Arg Ala Arg Gly Gly Gln Leu
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Tyr Val Phe Ala Asp Gln Asp Ala Gly Phe Val Ser Ser Asp Asn Met
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His Ile Ile Glu Met Pro His Val Glu Glu Val Ile Ala Pro Ile Phe
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Glu
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cgggcgataa aggcatttac tggcactaca tgcaga 36
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ccgttgttga tgaagaaggt catatgaccc gcc 33
<210> 8
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ggcgggtcat atgaccttct tcatcaacaa cgg 33

Claims (9)

1. a kind of -6 phosphate synthase mutant of Glucosamine, which is characterized in that be by amino acid sequence such as SEQ ID NO.2 Shown in wild GlmS the 471st glycine mutation be serine.
2. encoding the gene of -6 phosphate synthase mutant of Glucosamine described in claim 1.
3. carrying the carrier or cell of gene described in claim 2.
4. a kind of method for obtaining -6 phosphate synthase mutant of Glucosamine described in claim 1, which is characterized in that with PET28a-glmS-gna1 is template, by rite-directed mutagenesis by encoding amino acid sequence wild type as shown in SEQ ID NO.2 What the gene of GlmS was mutated, the carrier for carrying the gene of encoding mutant body is transferred to E.coli BL21 (DE3), culture weight Group bacterium obtains -6 phosphate synthase mutant of Glucosamine.
5. a kind of method for producing acetylglucosamine, which is characterized in that be with Escherichia coli BL21 (DE3) For host, recombinant expression encodes the gene of -6 phosphate synthase mutant of Glucosamine described in claim 1, is recombinated Bacterium；Fermentation medium is transferred to 5~8% inoculum concentration after recombination bacillus coli is activated with seed culture medium, in 35~37 DEG C, It is cultivated under the conditions of 200~220rpm, OD600IPTG induction -6 phosphoric acid of Glucosamine that 0.4mM is added when reaching 0.6 or so closes At the expression of enzyme, recombinant bacterium is enable to produce acetylglucosamine by substrate of glucose.
6. a kind of method for producing acetylglucosamine according to claim 5, which is characterized in that seed culture medium is pressed G/L meter contains: tryptose arteries and veins 10, yeast powder 5, NaCl10.
7. a kind of method for producing acetylglucosamine according to claim 5, which is characterized in that fermentation medium is pressed G/L meter contains: tryptone 12, yeast powder 24, dipotassium hydrogen phosphate 12.54, potassium dihydrogen phosphate 2.31, glycerol 4, glucose 30.
8. the method for -6 phosphate synthase mutant of Glucosamine production acetylglucosamine described in application claim 1.
9. according to the method described in claim 8, it is characterized in that, being with -6 phosphate synthase mutant of Glucosamine Catalyst is catalyzed glucose production acetylglucosamine.
CN201910439712.6A 2016-09-29 2016-09-29 A kind of -6 phosphate synthase mutant of Glucosamine Pending CN110157693A (en)

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CN112899248A (en) * 2021-01-20 2021-06-04 江南大学 Glucosamine-6 phosphate synthetase mutant and application thereof
CN114891772A (en) * 2022-05-27 2022-08-12 湖南福来格生物技术有限公司 Deacetylase mutant and application thereof

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CN113046335B (en) * 2019-12-27 2023-05-26 中国科学院天津工业生物技术研究所 Bionic coenzyme-favored glucose 6-phosphate dehydrogenase mutant and application thereof
CN112048486B (en) * 2020-07-23 2021-11-09 中国水产科学研究院南海水产研究所 Penaeus monodon PmGFPT2 gene and application thereof
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