CN107236696A - A kind of sucrose phosphorylase recombined bacillus subtilis in expression L. mesenteroides sources - Google Patents
A kind of sucrose phosphorylase recombined bacillus subtilis in expression L. mesenteroides sources Download PDFInfo
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- CN107236696A CN107236696A CN201710637427.6A CN201710637427A CN107236696A CN 107236696 A CN107236696 A CN 107236696A CN 201710637427 A CN201710637427 A CN 201710637427A CN 107236696 A CN107236696 A CN 107236696A
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- C12P19/44—Preparation of O-glycosides, e.g. glucosides
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- C12Y—ENZYMES
- C12Y204/00—Glycosyltransferases (2.4)
- C12Y204/01—Hexosyltransferases (2.4.1)
- C12Y204/01007—Sucrose phosphorylase (2.4.1.7)
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Abstract
The invention discloses a kind of sucrose phosphorylase recombined bacillus subtilis in expression L.mesenteroides sources, belong to genetic engineering and technical field of enzyme engineering.The present invention obtains Leuconostoc mesenteroides sources sucrose phosphorylase target gene by artificial synthesized SPase genes, design primer, PCR, construction recombination plasmid pBSMuL3 SP, and convert to bacillus subtilis Bacillus subtilis (CCTCC M2016536), obtain recombined bacillus subtilis.Using this bacillus subtilis as strain, fermenting and producing SPase.Recombinant sucrose phosphorylase is applied to prepare α ursin there is relatively good effect.The present invention recombinantly expresses sucrose phosphorylase by expressive host of the bacillus subtilis of food security, and producing enzyme level is high, is easy to isolate and purify, and fermentation raw material wide material sources, and production cost is relatively low.
Description
Technical field
The present invention relates to a kind of sucrose phosphorylase recombined bacillus subtilis in expression L.mesenteroides sources,
Belong to genetic engineering and technical field of enzyme engineering.
Background technology
Sucrose phosphorylase (EC 2.4.1.7, Sucrose Phosphorylase, SPase), is one in GH13 families
Member, is the enzyme-specific of catalysis transfer glucoside bond.The reaction of the type of major catalytic 2：One is by Cori's eater Cori
Glucosyl group be transferred to acceptor；Two be that the glucosyl group in sucrose is transferred into acceptor, and acceptor includes inorganic phosphate, water, contained
The material of phenolic hydroxyl group, alcoholic extract hydroxyl group and carboxyl.Converted product is alpha-arbutin when using phenolic hydroxyl group as acceptor.Alpha-arbutin conduct
A kind of high effect medicine, cosmetic additive agent strongly promoted in the world, at home and abroad has huge demand, in cosmetics
Whitening spot-removing field occupies consequence.According to market value about 3800 yuan of the per kilogram of investigation alpha-arbutin, and its raw material pair
About 66 yuan of per kilograms of the market value of benzenediol, so alpha-arbutin is a kind of product of high added value, if it is possible to realize production
The production of industry metaplasia will bring very big economic well-being of workers and staff.Sucrose phosphorylase can also catalyze and synthesize the derivative of some unstable materials
Thing, improves its stability.Therefore, sucrose phosphorylase has a wide range of applications in terms of food, cosmetics and medicine.
Sucrose phosphorylase belongs to endocellular enzyme, and the gene of enzyme is mainly derived from Leuconostoc mesenteroides (Leuconostoc
Mesenteroides), Streptococcus mutans (Streptococcus mutans), Pseudomonas saccharophila (Pseudomonas
Saccharophila), bifidobacterium longum (Bidifobacterium longum), bacillus megaterium (Bacillus
Megaterium), bifidobacterium adolescentis (Bifidobacterium adolescentis).Current L.mesenteroides comes
The sucrose phosphorylase in source only obtains heterogenous expression in Escherichia coli, but Escherichia coli easily produce endotoxin, is not suitable for
Applied to industries such as medicine, cosmetics.
The content of the invention
To solve the above problems, the present invention is suitable for the bacillus subtilis that food uses first constructs weight for Host Strains
The sucrose phosphorylase bacterial strain in group expression L.mesenteroides sources, electrophoresis result shows that sucrose phosphorylase obtains height
Horizontal expression, is that the preparation of industrialization of alpha-arbutin is laid a good foundation.
First purpose of the present invention is to provide a kind of recombination expression L.mesenteroides sources sucrose phosphorylase
Recombined bacillus subtilis.
In one embodiment of the invention, the amino acid sequence of the sucrose phosphorylase such as SEQ ID NO.1 institutes
Show.
In one embodiment of the invention, the recombined bacillus subtilis is using deposit number as CCTCC M
2016536 bacillus subtilis is host.
In one embodiment of the invention, the recombined bacillus subtilis is with bacillus subtilis (Bacillus
Subtilis) 168 be host.
In one embodiment of the invention, the expression vector is pBSMuL3.
Second object of the present invention is to provide the construction method of the recombined bacillus subtilis.
In one embodiment of the invention, methods described be by encoding amino acid sequence as shown in SEQ ID NO.1
The gene of sucrose phosphorylase be connected with expression vector, be transferred in bacillus subtilis.
In one embodiment of the invention, sucrose phosphorus of the encoding amino acid sequence as shown in SEQ ID NO.1
The nucleotide sequence of the gene of phosphorylase is as shown in SEQ ID NO.2.
In one embodiment of the invention, it is CCTCC M that the bacillus subtilis, which includes deposit number,
2016536 bacillus subtilis or bacillus subtilis 168.
In one embodiment of the invention, the expression vector is pBSMuL3.
In one embodiment of the invention, the construction method of the recombined bacillus subtilis is specific as follows：
1) gene of sucrose phosphorylase of the amplification coding amino acid sequence as shown in SEQ ID NO.1；
2) target gene cloned is connected to pMD-18T, is configured to recombinant vector pMD-18T-sp, conversion is extremely
E.coliJM109 is further cloned, and target gene is connected into expression vector pBSMuL3 after digestion, pBSMuL3-sp is named as.
3) recombinant plasmid pBSMuL3-sp is entered into bacillus subtilis expressive host by electricity conversion, recombinantly expressed
The Bacillus subtilis genes engineering bacteria of sucrose phosphorylase.
Third object of the present invention is to provide application of the bacillus subtilis in production sucrose phosphorylase, institute
State using being that the bacillus subtilis is seeded in fermentation medium, in 30~35 DEG C, 200~250rpm cultures 24~
72h。
Fourth object of the present invention is to provide application of the bacillus subtilis in production alpha-arbutin.
In one embodiment of the invention, the application is to cultivate described bacillus subtilis after 24~48h
Zymotic fluid centrifugation obtain crude enzyme liquid；Using mol ratio as 1:1~8 hydroquinones and sucrose is substrate, by 5000~10000U/
The addition of g substrates adds enzyme liquid, and in 30~35 DEG C, pH 6.8~7.2 reacts 12~48h.
The 5th purpose of the present invention is to provide a kind of sucrose phosphorylase, its amino acid sequence such as SEQ ID NO.1 institutes
Show.
The present invention also provide the genetic engineering bacterium food, medicine, chemical field application.
Beneficial effect：The present invention constructs expression by host of the bacillus subtilis of food access
The recombined bacillus subtilis of the sucrose phosphorylase in L.mesenteroides sources, enzyme activity reaches 162.19U/mL, heavy using this
Group enzyme production alpha-arbutin, it react 24 hours at 30 DEG C, then with substrate conversion during saccharification ferment treatment 3 hours at 40 DEG C
Rate, up to 83.6%, is that the preparation of industrialization of alpha-arbutin is laid a good foundation.
Brief description of the drawings
Fig. 1 recombinant plasmids pBSMuL3-sp building process figure；
Fig. 2 pMD18T-sp double digestion proof diagrams；From left to right, swimming lane 1 is 5000Marker, and 2-3 is sucrose phosphorylase
Connect expression vector digestion verification；
The double digestion proof diagram of Fig. 3 pBSMuL3-sp recombinant plasmids；From left to right, swimming lane 1 is 10000bp Marker, swimming
Road 2~3 is that sucrose phosphorylase connects expression vector digestion verification；
Fig. 4 pBSMuL3-sp recombinant bacterium shake flask fermentation SDS-PAGEs, swimming lane 1~3 is the extracellular supernatants of 13h, 13h is broken
Wall supernatant, 13h broken walls precipitation；Swimming lane 4 is middle-molecular-weihydroxyethyl albumen marker, and swimming lane 5~7 is the extracellular supernatants of 24h, on 24h broken walls
Clearly, 24h broken walls are precipitated；Swimming lane 8~10 is the extracellular supernatants of 43h, 43h broken walls supernatant, 43h broken walls precipitation；
The enzymatic conversion HPLC chromatogram of Fig. 5 maximum conversions.
Embodiment
Bacillus subtilis Bacillus subtilis (deposit number is CCTCC M 2016536) are in Application No.
201611025858.9 patent application disclosed in.
The assay method of enzyme activity：By 1mL 5% sucrose solution and 0.9mL 50mmol/L, pH 6.7 phosphoric acid buffer
Liquid is fully mixed, and 10min is preheated at 30 DEG C, is added 3mLDNS after adding 100ul enzyme liquid, reaction 10min, is boiled 7min rapid
Cooling, plus distilled water are settled to survey absorbance under 15ml, 540nm and (are equally operated by catalyst of the enzyme liquid of inactivation and be used as blank
Control).
Enzyme activity is defined：Enzyme amount needed for sucrose hydrolysis per minute is generated into 1 μm of ol fructose is defined as sucrose phosphorylase
The enzyme activity (U) of one unit.
The HPLC detections of product：The amount of alpha-arbutin in end reaction system is determined using HPLC.Chromatographic condition is：
Carrying out the specific chromatographic condition of product analysis using HPLC (high performance liquid chromatography) is:The HPLC chromatogram instruments of Agilent 1200,
Agilent automatic samplers, 5 μm of Agilent SB-Aq (4.6mm × 250mm), Agilent LC-9A UV-detectors system
System；Mobile phase is according to the recipe configuration of the 10mM of 80% (volume fraction) methanol solution of phosphoric acid,diluted -20%, the flow velocity of mobile phase
For 0.6mLmin-1；Ultraviolet detection wavelength is 281nm, and alpha-arbutin and monose in determination sample are distinguished with C-18 posts and sugared post
With the content of polysaccharide, 35 DEG C of column temperature.
The calculation formula for turning glycosides efficiency D of alpha-arbutin is：
D (%)=M1/M2* 100%
Wherein：
D：Hydroquinones is converted into the molar yield (%) of alpha-arbutin；
M1：The molal quantity (mol) of the alpha-arbutin generated in enzymatic conversion reaction；
M2：The molal quantity (mol) for the hydroquinones put into reaction solution.
Embodiment 1：The clone of Leuconostoc mesenteroides saccharose phosphorylation enzyme coding gene and the structure of expression vector
The structure of expression vector is carried out by Fig. 1 flow, primers F, R are designed according to the saccharose phosphorylation enzyme gene of synthesis：
F：5’-GCGAAGCTTAAGGAGGATATTATGGAAATTCAGAACAAGGC-3’
R：5’-GCGGGATCCTTAATTCTGGGTCAGATTATCGC-3’
The letter expression that the restriction enzyme site is underlined.PCR system is：20 μM of primers Fs and each 0.5 μ L of R,
μ L, the 2.5U/ μ L of 4 μ L, 5 × PS Buffer of dNTPMix 10 PrimeStar polymerase 0.5uL, the μ L of template 0.5, plus double steamings
The μ L of water polishing 50.PCR conditions：94 DEG C of pre-degeneration 4min；98 DEG C of denaturation 10s, 55 DEG C of annealing 10s, 72 DEG C extend 1min50s, 30
Individual circulation.PCR primer is subjected to glue reclaim plus A is connected with cloning vector pMD-18T after purification, target gene is reclaimed in digestion will
It carries out double digestion with expression vector pBSMuL3, and is stayed overnight in 16 DEG C of connections, Transformed E .coli JM109, and coating contains ammonia benzyl
The LB flat boards of (100 μ g/mL) resistance, 37 DEG C of culture 10-12h, picking transformant extracts recombinant plasmid and double digestion checking.Such as
There are two bar segments shown in Fig. 3, after digestion, size is respectively 7000bp or so (expression vector pBSMuL3) and 1473bp (sucrose phosphorus
Phosphorylase) it is successful connection.Then to verifying that correct recombinant plasmid determines DNA sequence dna, positive clone molecule is pBSMuL3-sp.
Embodiment 2：Recombinant plasmid pBSMuL3-sp conversion
1) fresh LB flat boards (LB solid mediums：Peptone 10g/L, yeast extract 5g/L, NaCl 10g/L, 0.2g/L fine jade
Cosmetics) choose bacillus subtilis Bacillus subtilis (deposit number be CCTCC M 2016536) single bacterium colony and be inoculated in
In 5mlLB fluid nutrient mediums, 37 DEG C, 200rpm cultures 10h.
2) 2.5mL is taken to transfer into 40mL plus 0.5M sorbierite LB culture mediums, 37 DEG C, 200rpm concussion and cultivates are four small
When.
3) whole bacterium solution ice-water bath 10min are taken, then 5000rpm, 4 DEG C, centrifugation 5min collects thalline.
4) buffer solution (sorbierite 0.5M, mannitol 0.5M, glucose 10%) washing thalline is turned with the electricity of 40ml precoolings,
5000rpm, 4 DEG C, centrifugation 5min removes supernatant, so rinsing 4 times.
5) thalline after washing is resuspended in into 1mL electricity to turn in culture medium, be dispensed into 1.5mL EP pipes, often pipe fills 300 μ l
Competent cell.
6) 10 μ L recombinant plasmids will be added in 300 μ L competent cells, ice bath is incubated 15min, adds the electric revolving cup of precooling
In (2mm), electric shock is once.Electroporation is set：2.4kv, 25uF, 200 Ω, shock by electricity 1 time.
7) electric shock finish add immediately 1mL recovery mediums RM (sorbierite 0.5M, mannitol 0.38M, peptone 10g/L,
Yeast extract 5g/L, NaCl 10g/L) pressure-vaccum, 37 DEG C, 200rpm, after recovery 3h, coated plate.37 DEG C, incubated overnight, picking colony is extremely
In LB kanamycins culture mediums, verified, expression vector and target gene stripe size are corresponding i.e. checking just after digestion
Really, shake flask fermentation producing enzyme is then carried out.
Embodiment 3:Shake flask fermentation producing enzyme
By the recombined bacillus subtilis inoculation obtained in embodiment 2 in LB culture mediums, 8h is cultivated at 37 DEG C
It is forwarded to afterwards with 5% inoculum concentration in TB fermentation mediums, first puts to 37 DEG C, the incubated 2h of 200rpm, then move to 33 DEG C of constant temperature
Cultivate 48h producing enzymes.After fermentation ends, supernatant as crude enzyme liquid is collected by centrifugation.
LB culture mediums (g/L)：Peptone 10, yeast extract 5, NaCl 10.
TB culture mediums (g/L)：Peptone 10, dusty yeast 24, glycerine 5, K2HPO4·3H2O 16.43, KH2PO42.31。
Enzyme activity in crude enzyme liquid is measured, as a result shown, enzyme activity is 136U/mL.Protein electrophoresis result is shown in
There is a band (Fig. 4) consistent with theoretical molecular at 53kDa.
Embodiment 4：Enzyme transforming process prepares alpha-arbutin
10mL enzymatic conversion system is carried out in 50mL closed container.Using hydroquinones as acceptor molecule, sucrose is made
SPase Transglycosylations are carried out for donor.Reaction system includes：Added in 20mM sodium phosphate buffers (pH 7.0) and supply acceptor
Molar ratio be 4:1 sucrose and the SPase of HQ and 7500U/g hydroquinones, 24h is reacted by it at 30 DEG C.Boiling water
5min terminating reactions are heated in bath.Then centrifuged and passed through with saccharification ferment treatment 3h and further heat inactivation at 40 DEG C
HPLC analyzes product.Conversion ratio is up to 83.64% after reaction terminates, and chromatographic peak is shown in Fig. 5.
Embodiment 5
Recombined bacillus subtilis is built using the same policy of embodiment 1~3, difference is, host is replaced with into withered grass
Bacillus 168, after testing, enzyme activity reach 113U/mL.
Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with this skill
The people of art, without departing from the spirit and scope of the present invention, can do various changes and modification, therefore the protection model of the present invention
Enclose being defined of being defined by claims.
Sequence table
<110>Southern Yangtze University
<120>A kind of sucrose phosphorylase recombined bacillus subtilis in expression L. mesenteroides sources
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 490
<212> PRT
<213>Leuconostoc mesenteroides Leuconostoc mesenteroides
<400> 1
Met Glu Ile Gln Asn Lys Ala Met Leu Ile Thr Tyr Ala Asp Ser Leu
1 5 10 15
Gly Lys Asn Leu Lys Asp Val His Gln Val Leu Lys Glu Asp Ile Gly
20 25 30
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Asp Arg Gly Phe Ala Pro Ala Asp Tyr Thr Arg Val Asp Ala Ala Phe
50 55 60
Gly Asp Trp Ala Asp Val Glu Ala Leu Gly Glu Glu Tyr Tyr Leu Met
65 70 75 80
Phe Asp Phe Met Ile Asn His Ile Ser Arg Glu Ser Val Met Tyr Gln
85 90 95
Asp Phe Lys Lys Asn His Asp Asp Ser Lys Tyr Lys Asp Phe Phe Ile
100 105 110
Arg Trp Glu Lys Phe Trp Ala Lys Ala Gly Glu Asn Arg Pro Thr Gln
115 120 125
Ala Asp Val Asp Leu Ile Tyr Lys Arg Lys Asp Lys Ala Pro Thr Gln
130 135 140
Glu Ile Thr Phe Asp Asp Gly Thr Thr Glu Asn Leu Trp Asn Thr Phe
145 150 155 160
Gly Glu Glu Gln Ile Asp Ile Asp Val Asn Ser Ala Ile Ala Lys Glu
165 170 175
Phe Ile Lys Thr Thr Leu Glu Asp Met Val Lys His Gly Ala Asn Leu
180 185 190
Ile Arg Leu Asp Ala Phe Ala Tyr Ala Val Lys Lys Val Asp Thr Asn
195 200 205
Asp Phe Phe Val Glu Pro Glu Ile Trp Asp Thr Leu Asn Glu Val Arg
210 215 220
Glu Ile Leu Thr Pro Leu Lys Ala Glu Ile Leu Pro Glu Ile His Glu
225 230 235 240
His Tyr Ser Ile Pro Lys Lys Ile Asn Asp His Gly Tyr Phe Thr Tyr
245 250 255
Asp Phe Ala Leu Pro Met Thr Thr Leu Tyr Thr Leu Tyr Ser Gly Lys
260 265 270
Thr Asn Gln Leu Ala Lys Trp Leu Lys Met Ser Pro Met Lys Gln Phe
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Thr Thr Leu Asp Thr His Asp Gly Ile Gly Val Val Asp Ala Arg Asp
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Ile Leu Thr Asp Asp Glu Ile Asp Tyr Ala Ser Glu Gln Leu Tyr Lys
305 310 315 320
Val Gly Ala Asn Val Lys Lys Thr Tyr Ser Ser Ala Ser Tyr Asn Asn
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Leu Asp Ile Tyr Gln Ile Asn Ser Thr Tyr Tyr Ser Ala Leu Gly Asn
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Asp Asp Ala Ala Tyr Leu Leu Ser Arg Val Phe Gln Val Phe Ala Pro
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Gly Ile Pro Gln Ile Tyr Tyr Val Gly Leu Leu Ala Gly Glu Asn Asp
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Ile Ala Leu Leu Glu Ser Thr Lys Glu Gly Arg Asn Ile Asn Arg His
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Tyr Tyr Thr Arg Glu Glu Val Lys Ser Glu Val Lys Arg Pro Val Val
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Ala Asn Leu Leu Lys Leu Leu Ser Trp Arg Asn Glu Ser Pro Ala Phe
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Asp Leu Ala Gly Ser Ile Thr Val Asp Thr Pro Thr Asp Thr Thr Ile
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Val Val Thr Arg Gln Asp Glu Asn Gly Gln Asn Lys Ala Val Leu Thr
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Ala Asp Ala Ala Asn Lys Thr Phe Glu Ile Val Glu Asn Gly Gln Thr
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<213>Leuconostoc mesenteroides Leuconostoc mesenteroides
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aaagatgttc atcaggttct gaaagaagat attggagatg ccattggcgg tgttcatctg 120
ctgccgtttt tcccgtcaac cggcgatcgc ggctttgccc cagcagatta tacccgcgtg 180
gatgcagcct ttggcgattg ggccgatgtg gaagccttag gtgaagaata ttatctgatg 240
tttgatttta tgattaatca tatttcacgc gaatcagtta tgtatcagga ttttaagaag 300
aaccatgatg atagtaaata taaggacttc tttattcgct gggaaaaatt ttgggccaaa 360
gcaggcgaaa atcgtccgac ccaggccgat gtggatctga tctataagcg caaagataaa 420
gccccgaccc aggaaattac ctttgatgat gggaccaccg aaaatctgtg gaataccttt 480
ggcgaagaac agattgatat tgatgtgaat agcgccattg ccaaagagtt cattaaaacc 540
accttagaag atatggttaa acatggcgcc aaccttattc gcttagatgc ctttgcctat 600
gccgttaaaa aagtggatac caatgacttc ttcgttgaac cggaaatttg ggataccctg 660
aacgaagtgc gcgaaattct gaccccgctg aaagccgaaa ttctgccgga aattcatgaa 720
cattatagca ttccgaagaa aattaatgat catggctatt ttacctatga ttttgcactg 780
ccgatgacca ccctgtatac cctgtatagc ggcaaaacca atcagttagc caaatggctg 840
aaaatgtctc cgatgaaaca gtttaccacc ttagataccc atgatggcat tggtgttgtg 900
gatgcacgcg atattctgac cgatgatgaa attgattatg caagcgaaca gctgtataag 960
gtgggcgcca atgttaaaaa gacctatagc tcagcaagct ataacaatct ggacatctat 1020
cagattaata gtacctatta tagtgcactg ggtaatgatg atgcagcata tctgctgtct 1080
cgcgtgtttc aggtgtttgc accgggcatt ccgcaaatct actatgtggg cttactggcc 1140
ggtgaaaatg atattgccct gctggaatca accaaagaag gtcgtaatat taatcgtcat 1200
tattataccc gcgaagaagt taaatcagaa gttaaacgtc cggttgttgc caacctgctg 1260
aaactgctgt cttggcgtaa cgagtctccg gcctttgatc tggcaggctc tattaccgtg 1320
gataccccga ccgataccac cattgttgtg acccgccagg atgaaaatgg tcagaataag 1380
gcagtgctga ccgcagatgc agccaataag acctttgaaa ttgtggaaaa tggtcagacc 1440
gttatgtcta gcgataatct gacccagaat taa 1473
Claims (10)
1. a kind of genetic engineering bacterium, it is characterised in that using bacillus subtilis as host, express amino acid sequence such as SEQ ID
Sucrose phosphorylase shown in NO.1.
2. genetic engineering bacterium according to claim 1, it is characterised in that using deposit number as CCTCC M's 2016536
Bacillus subtilis, or bacillus subtilis 168 are host.
3. genetic engineering bacterium according to claim 1 or 2, it is characterised in that using pBSMuL3 as expression vector.
4. build the method for the genetic engineering bacterium described in claim 1, it is characterised in that by encoding amino acid sequence such as SEQ ID
The gene of sucrose phosphorylase shown in NO.1 is connected with expression vector, is transferred in bacillus subtilis.
5. method according to claim 4, it is characterised in that it is CCTCC that the bacillus subtilis, which includes deposit number,
M 2016536 bacillus subtilis or bacillus subtilis 168.
6. method according to claim 4, it is characterised in that the expression vector is pBSMuL3.
7. application of the genetic engineering bacterium described in claim 1 in production sucrose phosphorylase, it is characterised in that the application is
Genetic engineering bacterium described in claim 1 is seeded in fermentation medium, in 30~35 DEG C, 200~250rpm cultures 24~
72h。
8. application of the genetic engineering bacterium described in claim 1 in production alpha-arbutin.
9. apply according to claim 8, it is characterised in that the application is by the genetic engineering bacterium described in claim 1
Zymotic fluid centrifugation after 24~48h of culture obtains crude enzyme liquid；Using mol ratio as 1:1~8 hydroquinones and sucrose is substrate, is pressed
The addition of 5000~10000U/g substrates adds enzyme liquid, and in 30~35 DEG C, pH 6.8~7.2 reacts 12~48h.
10. genetic engineering bacterium described in claim 1 food, medicine, chemical field application.
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Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107236695A (en) * | 2017-07-20 | 2017-10-10 | 金韵 | A kind of genetic engineering bacterium for expressing sucrose phosphorylase and its application |
| CN108102996A (en) * | 2018-02-12 | 2018-06-01 | 江南大学 | A kind of method of the high efficient expression maltogenic amylase in bacillus subtilis |
| CN109234220A (en) * | 2018-11-02 | 2019-01-18 | 南京工业大学 | One bacillus subtilis gene recombination bacterium and its construction method and application |
| CN109306357A (en) * | 2018-11-09 | 2019-02-05 | 沈阳农业大学 | A kind of method that high efficient expression prepares sucrose phosphorylase |
| CN109371006A (en) * | 2018-12-12 | 2019-02-22 | 江南大学（如皋）食品生物技术研究所 | A kind of process for fixation of sucrose phosphorylase |
| CN109988799A (en) * | 2019-01-24 | 2019-07-09 | 浙江工业大学 | A kind of glycerol -2- alpha-glucosyl enzyme is preparing the application in 2- α-glycosylglycerol |
| CN110343654A (en) * | 2019-08-15 | 2019-10-18 | 江南大学 | A kind of genetic engineering bacterium producing sucrose phosphorylase |
| CN111172128A (en) * | 2020-01-21 | 2020-05-19 | 浙江工业大学 | application of sucrose phosphorylase in preparation of 2-O- α -D-glucosyl-L-ascorbic acid |
| CN111172127A (en) * | 2020-01-17 | 2020-05-19 | 浙江工业大学 | Application of sucrose phosphorylase in preparation of glycerol glucoside |
| CN112300977A (en) * | 2020-11-18 | 2021-02-02 | 江南大学 | Gene engineering strain for synthesizing alpha-arbutin and construction method and application thereof |
| CN112375724A (en) * | 2020-11-18 | 2021-02-19 | 江南大学 | Gene engineering bacterium for efficiently synthesizing alpha-arbutin and construction method and application thereof |
| CN114774334A (en) * | 2022-06-20 | 2022-07-22 | 中国水产科学研究院黄海水产研究所 | Microbacterium abyssinicus SUC7, sucrose phosphorylase produced by same and application of sucrose phosphorylase |
| JP2022535648A (en) * | 2020-04-27 | 2022-08-10 | 江南大学 | Use of thermostable β-glucosidase in the production of gentiooligosaccharides |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002097077A1 (en) * | 2001-05-28 | 2002-12-05 | Ezaki Glico Co., Ltd. | Method of preparing sucrose phosphorylase |
| CN1845990A (en) * | 2003-09-04 | 2006-10-11 | 江崎格力高株式会社 | Method of making sucrose phosphorylase(SP) heat-stable |
| CN102174454A (en) * | 2011-01-17 | 2011-09-07 | 南京工业大学 | Escherichia coli engineering bacteria for expressing recombinant sucrose phosphorylase |
| CN103347999A (en) * | 2010-12-13 | 2013-10-09 | 麦兰特公司 | Method of producing succinic acid and other chemicals using sucrose-containing feedstock |
| CN106754466A (en) * | 2016-11-22 | 2017-05-31 | 江南大学 | It is a kind of for efficient exogenous protein expression and the bacillus subtilis of High Density Cultivation |
-
2017
- 2017-07-31 CN CN201710637427.6A patent/CN107236696B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002097077A1 (en) * | 2001-05-28 | 2002-12-05 | Ezaki Glico Co., Ltd. | Method of preparing sucrose phosphorylase |
| CN1845990A (en) * | 2003-09-04 | 2006-10-11 | 江崎格力高株式会社 | Method of making sucrose phosphorylase(SP) heat-stable |
| CN103347999A (en) * | 2010-12-13 | 2013-10-09 | 麦兰特公司 | Method of producing succinic acid and other chemicals using sucrose-containing feedstock |
| CN102174454A (en) * | 2011-01-17 | 2011-09-07 | 南京工业大学 | Escherichia coli engineering bacteria for expressing recombinant sucrose phosphorylase |
| CN106754466A (en) * | 2016-11-22 | 2017-05-31 | 江南大学 | It is a kind of for efficient exogenous protein expression and the bacillus subtilis of High Density Cultivation |
Non-Patent Citations (2)
| Title |
|---|
| 万月佳等: "重组大肠杆菌产蔗糖磷酸化酶的酶学性质及其催化合成α-熊果苷", 《生物工程学报》 * |
| 侯顾伟等: "蔗糖磷酸化酶制备及应用的研究进展", 《中国酿造》 * |
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| CN114774334B (en) * | 2022-06-20 | 2022-09-02 | 中国水产科学研究院黄海水产研究所 | Microbacterium abyssinicus SUC7, sucrose phosphorylase produced by same and application of sucrose phosphorylase |
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