CN105779425A - L-ribose isomerase and application thereof in preparation of L-ribose by biological method - Google Patents
L-ribose isomerase and application thereof in preparation of L-ribose by biological method Download PDFInfo
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- CN105779425A CN105779425A CN201610218779.3A CN201610218779A CN105779425A CN 105779425 A CN105779425 A CN 105779425A CN 201610218779 A CN201610218779 A CN 201610218779A CN 105779425 A CN105779425 A CN 105779425A
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- ribose isomerase
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- 101000804575 Cohnella laeviribosi D-lyxose ketol-isomerase Proteins 0.000 title claims abstract description 54
- PYMYPHUHKUWMLA-MROZADKFSA-N aldehydo-L-ribose Chemical compound OC[C@H](O)[C@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-MROZADKFSA-N 0.000 title claims abstract description 27
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 title claims abstract description 27
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 title claims abstract description 27
- 238000002360 preparation method Methods 0.000 title description 4
- 238000010170 biological method Methods 0.000 title 1
- ZAQJHHRNXZUBTE-UCORVYFPSA-N L-ribulose Chemical compound OC[C@H](O)[C@H](O)C(=O)CO ZAQJHHRNXZUBTE-UCORVYFPSA-N 0.000 claims abstract description 28
- 238000010276 construction Methods 0.000 claims abstract description 9
- 239000002773 nucleotide Substances 0.000 claims abstract description 8
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 8
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 3
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- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
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- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
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- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
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- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/90—Isomerases (5.)
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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
- C12P19/02—Monosaccharides
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y503/00—Intramolecular oxidoreductases (5.3)
- C12Y503/01—Intramolecular oxidoreductases (5.3) interconverting aldoses and ketoses (5.3.1)
- C12Y503/0102—Ribose isomerase (5.3.1.20)
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Abstract
The invention discloses L-ribose isomerase, wherein the amino acid sequence of the L-ribose isomerase is shown as SEQ ID NO.1, and the nucleotide sequence of the L-ribose isomerase is shown as SEQ ID NO. 2. The L-ribose isomerase has good thermal stability and pH stability, and the catalytic efficiency of the L-ribose isomerase on L-ribulose can reach more than 85%. The invention also discloses a recombinant strain containing the L-ribose isomerase and a construction method of the recombinant strain, and the catalytic efficiency of the recombinant strain on L-ribulose can reach more than 81%. The L-ribose isomerase described by the invention has good application prospect and economic value for industrial production of L-ribose.
Description
Technical field
The invention belongs to a kind of technical field, be specifically related to from fermentation unwrapping wire fiber bacterium (Actinotalea
Fermentans NX-1) L-ribose isomerase and application.
Background technology
L-ribose belongs to the rarest monosaccharide, is a kind of important medicine intermediate, has important medical value, by
The various L-ribose derivates of L-ribose synthesis, are widely used in antiviral and antitumor field.
The preparation of L-ribose at present mainly uses chemical method, needs the complex reaction of experience at least 7 steps, and faces by-product
The problems such as thing is many, low yield.L-ribose isomerase (L-RI) can prepare L-ribose with L-ribulose for raw material, possesses reaction
Gentle, productivity high, and L-ribulose can be obtained from L-arabinose catalysis by bioanalysis.Document is only reported at present
Three kinds of L-ribose isomerases, are respectively derived from Acinetobacter sp.DL-28, Geodermatophilus obscurus
DSM43160 and Cellulomonas parahominis MB426.But the catalytic efficiency that the L-RI having been found that is to L-ribulose
The highest, therefore excavating the L-RI to L-ribulose with high catalytic capability for producing L-ribose has extremely important meaning
Justice.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of L-ribose isomery to L-ribulose with high catalytic capability
Enzyme.
The present invention also to solve the technical problem that be to provide genetic engineering bacterium that a strain comprises above-mentioned L-ribose isomerase and
Its construction method.
The present invention finally to solve the technical problem that and be to provide above-mentioned L-ribose isomerase and said gene engineering bacteria in system
Application in standby L-ribose.
For solving above-mentioned technical problem, the present invention adopts the following technical scheme that
A kind of L-ribose isomerase, its aminoacid sequence is as shown in SEQ ID NO.1.This L-ribose isomerase is from fermentation
Unwrapping wire fiber bacterium (Actinotalea fermentans NX-1).
The gene of L-ribose isomerase described in coding claim 1, its nucleotide sequence is as shown in SEQ ID NO.2.
A kind of recombiant plasmid, this recombiant plasmid contains above-mentioned L-ribose isomerase gene.
A kind of recombinant bacterium, this recombinant bacterium contains above-mentioned L-ribose isomerase gene.
The construction method of above-mentioned recombinant bacterium, the method comprises the steps:
(1) nucleotide sequence shown in SEQ ID NO.2 is cloned on plasmid, obtains recombiant plasmid；
(2) by recombinant plasmid transformed to Host Strains, recombinant bacterium is i.e. obtained.
Wherein, described plasmid be pET-28a (+), described Host Strains is e. coli bl21 (DE3).
Concrete plasmid construction method is as follows with recombinant bacterium construction method:
(1) construction expression plasmid: utilize following primer sequence expand the nucleotide sequence shown in SEQ ID NO.2:
Primer 1:5 '-CGGGATCC(BamH I)ATGACCCGTACGTATGTGACCCGTC-3’；
Primer 2: 5 '-CCCAAGCTT(Hind III)TTAGCGAATGTGCGTCACCAGACGG-3’；
PCR amplification system is: each 1 μ L of genomic DNA 2 μ L, primer 1 and primer 2, dNTP2 μ L, 10 × Tag buffer 2.5
μ L, ExTag polymerase 0.5 μ L, ddH2O14μL；
PCR response procedures is: 94 DEG C of denaturations 5min, 94 DEG C of degeneration 30s；Then 54 DEG C of annealing 2min, 72 DEG C of extensions
5min, circulates 30 times, 4 DEG C of preservations；
Reclaim pcr amplification product, through restricted enzyme BamH I and Hind III double digestion, with through as double digestion
Plasmid pET-28a, be attached under the effect of T4 ligase, obtain recombiant plasmid pET-28a-afri；
(2) recombinant bacterium is built: converted to competence e. coli bl21 (DE3) by recombiant plasmid pET-28a-afri,
Coating, containing on the LB solid medium of 25 μ g/mL kanamycin, is cultivated 18～24h for 37 DEG C and is obtained preliminary positive colony；Through anti-
Property Screening of Media obtains positive colony: the LB liquid that the preliminary positive colony of picking contains 25 μ g/mL kanamycin in 5mL respectively
In culture medium, 37 DEG C, 200rpm overnight incubation, extract plasmid, through restricted enzyme BamH I and Hind III digestion plasmid,
Judge that the plasmid of DNA fragmentation with sequence table SEQ ID NO:1 is recombiant plasmid pET-28a-afri according to electrophoresis result, tool
The bacterium colony having this plasmid is positive colony, is recombinant bacterium.
Checking order recombiant plasmid pET-28a-afri, result shows that Insert Fragment is one and contains 747bp, coding
The protein of 249 aminoacid compositions.
The application in preparing L-ribose of the above-mentioned L-ribose isomerase.
Wherein, the L-ribulose with 1～100g/L is as substrate, and L-ribulose concentration is preferably 100g/L, adds L-ribose
Isomerase carries out enzymatic conversion reaction, and the consumption of enzyme is 10～500U, and the addition of L-ribose isomerase is preferably 20U；Reaction temperature
Spending 30～70 DEG C, reaction temperature is preferably 37 DEG C；Transformation time 1～12h, transformation time is preferably 3h.
Wherein, the enzyme of L-ribose isomerase definition method alive is: with L-ribulose as substrate, in the unit interval, catalysis generates
The enzyme amount of the L-ribose isomerase needed for 1 μm ol L-ribose is defined as 1U.
The preparation method of above-mentioned L-ribose isomerase:
The recombinant bacterium of the nucleotide sequence shown in SEQ ID NO.2 is inoculated in the LB that with the addition of 25 μ g/mL kanamycin
In fluid medium, 37 DEG C of shaking table overnight incubation；It is transferred to the LB containing 25 μ g/mL kanamycin again with the inoculum concentration of 5% (v/v)
In culture medium, 37 DEG C of fermentation culture 2～3h, to OD600The isopropyl-beta D-thio gala of 0.2～1mmol/L is added when being 0.6
Glucosides or the lactose of 0.5～20g/L, after continuing abduction delivering 6～20h, centrifugal collection thalline.
The thalline obtained is suspended from pH7.0 kaliumphosphate buffer, utilizes ultrasonic disruption cell, centrifugal, collect supernatant
Liquid (crude enzyme liquid), crude enzyme liquid, after 0.22 μm membrane filtration, uses the affine resin of Ni-NTA to be purified, obtains L-ribose isomery
The pure enzyme of enzyme.The present invention can utilize enzyme after purification or crude enzyme liquid to add in reaction system.
The application in preparing L-ribose of the above-mentioned recombinant bacterium.
Wherein, the L-ribulose with 1～100g/L is as substrate, and the addition of L-ribulose is 100g/L；Add recombinant bacterium
Carrying out conversion reaction, the addition of recombinant bacterium is 10～100g/L (calculating with thalline weight in wet base), and the addition of recombinant bacterium is 50g/L
Wet thallus；Reaction temperature 30～70 DEG C, reaction temperature is preferably 37 DEG C；Transformation time 1～48h, transformation time is preferably 3h.
The preparation method of above-mentioned recombinant bacterium:
The recombinant bacterium of the nucleotide sequence shown in SEQ ID NO.2 is inoculated in the LB that with the addition of 25 μ g/mL kanamycin
In fluid medium, 37 DEG C of shaking table overnight incubation；It is transferred to the LB containing 25 μ g/mL kanamycin again with the inoculum concentration of 5% (v/v)
In culture medium, 37 DEG C of fermentation culture 2～3h, to OD600The isopropyl-beta D-thio gala of 0.2～1mmol/L is added when being 0.6
Glucosides or the lactose of 0.5～20g/L, after continuing abduction delivering 6～20h, centrifugal collection thalline.
Beneficial effect: the invention provides a kind of L-ribose isomerase, this enzyme has preferable stability, and reaction temperature is
30～70 DEG C, reaction pH is 5.5～10, and this L-ribose isomerase demonstrates the catalytic efficiency high to L-ribulose.This is to L-
Ribulose has the L-ribose isomerase of high catalytic capability under the suitableeest catalytic condition, to the conversion ratio of L-ribulose up to 75%
Above, the industrialized production for L-ribose is with a wide range of applications and economic worth.
Accompanying drawing explanation
Fig. 1 is the structure schematic diagram of recombiant plasmid pET-28-afri.
Fig. 2 L-ribose isomerase prepares the response curve of L-ribose.
Detailed description of the invention
According to following embodiment, the present invention be may be better understood.But, as it will be easily appreciated by one skilled in the art that reality
Execute the content described by example and be merely to illustrate the present invention, and should be also without limitation on basis described in detail in claims
Invention.
Embodiment 1: the genome deriving from fermentation unwrapping wire fiber bacterium (Actinotalea fermentans NX-1) carries
Take.
Fermentation unwrapping wire fiber bacterium (Actinotalea fermentans NX-1) is this Laboratories Accession, uses Genomic
DNA Purification Kit (Takara, Dalian) extracting is in the fermentation unwrapping wire fiber bacterium of exponential phase
The genomic DNA of (Actinotalea fermentans NX-1), and with agarose gel electrophoresis to obtain bacterial genomes
Detect.
The clone of embodiment 2:L-ribose isomerase encoding gene (afri) and recombinant bacterium build.
The PCR amplification of 2.1 afri genes.
According to Unknown Function sequence (Genbank accession number No.WP_034244055) existing on GeneBank, use
VectorNTI software design primer Primer1 and Primer2, primer sequence is:
Primer 1:5 '-CGGGATCC(BamH I)ATGACCCGTACGTATGTGACCCGTC-3’；
Primer 2: 5 '-CCCAAGCTT(Hind III)TTAGCGAATGTGCGTCACCAGACGG-3’；
With embodiment 1 obtain genome as template, amplification fermentation unwrapping wire fiber bacterium genetic fragment.
PCR amplification system is: each 1 μ L of genomic DNA 2 μ L, primer Primer1 and Primer2, dNTP2 μ L, 10 × Tag
Buffer 2.5 μ L, Ex-Tag polymerase 0.5 μ L, ddH2O 14μL。
PCR response procedures is: 94 DEG C of denaturations 5min, 94 DEG C of degeneration 30s；Then 54 DEG C of annealing 2min, 72 DEG C of extensions
5min, circulates 30 times, 4 DEG C of preservations.
Reclaim test kit with the pillar rubber tapping of Axygen company after amplified band is cut glue to reclaim, be connected to Takara company
On pMD18-T carrier and convert e. coli jm109.Test by combining plasmid list double digestion on ampicillin/LB plates
Card, identifies positive colony, and carries out sequencing in Nanjing Jin Sirui biotechnology company.Full length sequence will be recorded exist
GenBank data base is analyzed, and determines entire reading frame therein by Vector NTI software.
The expression of 2.2 afri genes
Utilize pET-28a (+) plasmid (Novagen), construction of expression vector, express genes of interest, further confirm that gene
The correctness of clone.
2.2.1 restriction endonuclease reaction, purification and coupled reaction
PCR primer after purified, carries out enzyme action with the enzyme being pre-designed in primer sequence corresponding to restriction enzyme site anti-
Should.In this experiment, enzyme used is BamH I and Hind III.Enzyme action system is: PCR primer or plasmid solution 12.5 μ L,
BamH I 1 μ L, Hind III 1 μ L, 10 × buffer 2.5 μ L, ddH2O 8 μ L, cumulative volume 25 μ L.
Due to two selected restriction enzyme sites pET-28a (+) close proximity (about 30bp) on empty plasmid, therefore, enzyme
PCR primer and plasmid vector after cutting have only to i.e. can reach the purpose of purification through PCR cleaning agents box.
Through enzyme action PCR primer after purification and plasmid vector, may be used for coupled reaction.Coupled reaction system is: enzyme action
The PCR primer 4 μ L of purification, the plasmid 4 μ L of enzyme action purification, T4 ligase 1 μ L, 10 × ligase buffer 1 μ L.Obtain after connection
Recombiant plasmid pET-28-afri, its primary structure is as shown in Figure 1.
2.2.2 plasmid preparation and conversion
Plasmid extraction uses plasmid extraction kit, and the description with reference to manufacturer operates.Plasmid-transformed cells makes
Use Calcium Chloride Method.
2.2.3 the conversion of recombiant plasmid pET-28-afri
(1) 0.1-1 μ g recombiant plasmid pET-afri DNA is taken in 200 μ L competent cells, ice bath 30min.
(2) 42 DEG C of water-bath heat shock 90s, are quickly placed into 1-3min on ice.
(3) fresh LB fluid medium 800 μ L is added, in 37 DEG C of shaken cultivation 45min.
(4) take 200 μ L thalline and coat selectivity LB solid culture primary surface.Cultivate 12-16h for 37 DEG C to go out to single bacterium colony
Existing.
2.2.4 the qualification of recon
Positive bacterium colony is inoculated in the LB fluid medium containing kanamycin (25 μ g/mL) and carries out cultivating and extracting matter
Grain, uses BamH I and Hind respectively according to the enzyme action system in " restriction endonuclease reaction, purification and coupled reaction " and condition
III carries out list-double digestion to recombiant plasmid to be identified, digestion products carries out agarose gel electrophoresis qualification.
Confirming through electrophoresis result, this positive colony bacterium colony contains DNA fragmentation and inserts plasmid pET-28-afri, recombinates containing this
The recombination bacillus coli of plasmid pET-28-afri, is the recombination bacillus coli BL21-AFRI of conversion.Sequencing result shows to be inserted
Enter the open reading frame that fragment contains a long 747bp.
The abduction delivering of embodiment 3:L-ribose isomerase.
Recombination bacillus coli BL21-AFRI is inoculated in 5mL and with the addition of the LB fluid medium of 25 μ g/mL kanamycin
In, 37 DEG C of shaking table overnight incubation；It is transferred to equipped with 100mL LB culture medium that (that is mould containing 25 μ g/mL cards again with the inoculum concentration of 5%
Element) 500mL shaking flask in, 37 DEG C of shaking tables cultivate 2～3h, to OD600Add IPTG when being about 0.6 to carry out inducing (IPTG final concentration
1mmol/L), or interpolation 1g/L lactose is induced, after then proceeding to abduction delivering 6h, and centrifugal collection thalline.
Embodiment 4: the purification of restructuring L-ribose isomerase.
The thalline of the recombinant bacterium E.coli BL21-AFRI obtained is suspended from kaliumphosphate buffer (pH 7.0), uses physiology salt
Water cleans twice, uses sonicator smudge cells (400W, 30min), and 12000rpm is centrifuged 10min, gained supernatant
For Soluble target albumen (crude enzyme liquid).Sample, after 0.2 μm membrane filtration, is added in the affine resin of Ni-NTA by crude enzyme liquid,
Coutroi velocity is at about 15mL/h, with 10 times of bed volume Wash-Buffer (300mM NaCl, 50mM NaH2PO4, 10mM miaow
Azoles) rinse, finally with 10 times of bed volume Elution-Buffer (300mM NaCl, 50mM NaH2PO4, 250mM imidazoles) wash
Take off and collect target protein, by destination protein solution 4 DEG C in kaliumphosphate buffer (pH 7.0) dialyzed overnight.After purification
Destination protein enzyme activity reaches 12.3U/mg, is detected as single band through SDS-PAGE, and shows restructuring L-ribose isomerase egg
White molecular weight is 35kDa.
The enzyme of enzyme definition method alive: with L-ribulose as substrate, needed for catalysis generates 1 μm olL-ribose in the unit interval
The enzyme amount of L-ribose isomerase is defined as 1U.
Embodiment 5: restructuring L-ribose isomerase heat stability experiment.
Take restructuring L-ribose isomerase 50 μ L (20mg/mL) after purification in 40 DEG C, 50 DEG C, 60 DEG C, 65 DEG C, 70 DEG C and 75
DEG C different temperatures water-bath processes 1～3h, is then added to 50 μ L and contains 50mM Tris-hydrochloric acid and rush the reaction of liquid (pH 7.5)
In system, interpolation L-ribulose is to final concentration 100mM, and under optimal reactive temperature, water-bath 30min, is surveyed by liquid chromatograph
Determine L-ribose growing amount.Record and the results are shown in Table 1.
The impact on L-ribose isomerase of table 1 temperature
Embodiment 6: restructuring L-ribose isomerase pH stability experiment.
Take restructuring L-ribose isomerase 50 μ L (20mg/mL) after purification be respectively 5.5 in 90 μ L pH, 6.0,6.5,
7.5, room temperature treatment 0～12h under the conditions of 8.0,9.0 and 10.0, is then added to 50 μ L and contains 50mM Tris-hydrochloric acid and rush liquid
In the reaction system of (pH 7.5), add L-ribulose to final concentration 100mM, water-bath 30min under optimum temperature, pass through liquid
Phase chromatographic determination L-ribose growing amount.Record and the results are shown in Table 2.
Table 2 pH impact on L-ribose isomerase stability
Embodiment 7: the application of restructuring L-ribose isomerase.
The cumulative volume of enzymatic conversion reaction is 10mL, using 10g/L L-ribulose as conversion of substrate, is dissolved in 50mM
Tris-hydrochloric acid rushes in the reaction system of liquid (pH 7.5), takes 2mg embodiment 4 restructuring L-ribose isomerase after purification in system
In, react 12h under optimum temperature, by liquid chromatogram measuring L-ribose growing amount.After measured, in conversional solution, L-ribose concentration is
8.5g/L, restructuring L-ribose isomerase reaches 85% to the conversion ratio of substrate L-ribulose, sees Fig. 2.
Embodiment 8: produce the application of the genetic engineering bacterium of restructuring L-ribose isomerase.
The cumulative volume of conversion reaction is 100mL, using 100g/L L-ribulose as conversion of substrate, is suspended in 50mM
Tris-hydrochloric acid rushes in the reaction system of liquid (pH 7.5), takes the product restructuring L-ribose isomerase that 5g (wet thallus) embodiment 3 obtains
Genetic engineering bacterium in reaction system, under optimum temperature react 36h, by liquid chromatogram measuring L-ribose growing amount.Through surveying
Fixed, in conversional solution, L-ribulose concentration is 81g/L, and the conversion ratio of substrate L-ribulose is reached by restructuring L-ribose isomerase
81%.
Embodiment 9: derive from Actinotalea fermentans NX-1L-ribose isomerase and albumen WP_
034244055 catalytic performance contrast.
The cumulative volume of enzymatic conversion reaction is 10mL, using 10g/L L-ribulose as conversion of substrate, is dissolved in 50mM
In the reaction system of Tris-hydrochloride buffer (pH 7.5), take 2mg embodiment 4 restructuring L-ribose isomerase after purification and egg
(this gene is by the full genome synthesis of Nanjing Jin Sirui biotechnology company and to carry out albumen by embodiment 4 pure for white WP_034244055
Change) in system, react 12h under optimum temperature, by liquid chromatogram measuring L-ribose growing amount.After measured, Actinotalea
In fermentans NX-1 L-ribose isomerase conversional solution, L-ribose concentration is 8.5g/L, and restructuring L-ribose isomerase is to substrate
The conversion ratio of L-ribulose reaches 85%；In albumen WP_034244055 conversional solution, L-ribose concentration is 7.0g/L, L-core of recombinating
Sugar isomerase reaches 70% to the conversion ratio of substrate L-ribulose.
Claims (10)
1. a L-ribose isomerase, its aminoacid sequence is as shown in SEQ ID NO.1.
2. the gene of L-ribose isomerase described in coding claim 1, its nucleotide sequence is as shown in SEQ ID NO.2.
3. a recombiant plasmid, it is characterised in that this recombiant plasmid contains L-ribose isomerase gene described in claim 2.
4. a recombinant bacterium, it is characterised in that this recombinant bacterium contains L-ribose isomerase gene described in claim 2.
5. the construction method of recombinant bacterium described in claim 4, it is characterised in that the method comprises the steps:
(1) nucleotide sequence shown in SEQ ID NO.2 is cloned on plasmid, obtains recombiant plasmid；
(2) by recombinant plasmid transformed to Host Strains, recombinant bacterium is i.e. obtained.
6. according to claim, state the construction method of recombinant bacterium, it is characterised in that described plasmid be pET-28a (+), institute
The Host Strains stated is e. coli bl21 (DE3).
7. L-ribose isomerase application in preparing L-ribose described in claim 1.
Application the most according to claim 7, it is characterised in that the L-ribulose with 1～100g/L, as substrate, adds L-core
Sugar isomerase carries out enzymatic conversion reaction, and the consumption of enzyme is 10～500U, reaction temperature 30～70 DEG C, transformation time 1～20h.
9. the application in preparing L-ribose of the recombinant bacterium described in claim 4.
Application the most according to claim 9, it is characterised in that the L-ribulose with 1～100g/L, as substrate, adds weight
Group bacterium carries out conversion reaction, and the addition of recombinant bacterium is 10～100g/L, reaction temperature 30～70 DEG C, transformation time 1～48h.
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| CN108866120A (en) * | 2017-05-10 | 2018-11-23 | 韩国科学技术院 | Method for preparing L-ribose based on L-arabinose |
| CN110452942A (en) * | 2019-08-23 | 2019-11-15 | 华南师范大学 | Immobilized enzyme catalysis method prepares D- ribulose |
| CN110684761A (en) * | 2019-10-18 | 2020-01-14 | 南京工业大学 | L-ribose isomerase and application thereof in preparation of L-ribose by biological method |
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| CN110452942A (en) * | 2019-08-23 | 2019-11-15 | 华南师范大学 | Immobilized enzyme catalysis method prepares D- ribulose |
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