CN109371079A - A kind of biological synthesis method of uridine diphosphoglucose and uridine diphosphate glucuronate - Google Patents
A kind of biological synthesis method of uridine diphosphoglucose and uridine diphosphate glucuronate Download PDFInfo
- Publication number
- CN109371079A CN109371079A CN201811338833.3A CN201811338833A CN109371079A CN 109371079 A CN109371079 A CN 109371079A CN 201811338833 A CN201811338833 A CN 201811338833A CN 109371079 A CN109371079 A CN 109371079A
- Authority
- CN
- China
- Prior art keywords
- high temperature
- bacillus coli
- recombination bacillus
- reaction
- uridine
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- 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
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a kind of uridine diphosphoglucose and the biological synthesis methods of uridine diphosphate glucuronate.The present invention, for main initial feed, high temperature alpha-glucans phosphorylase and high temperature sugar -1- phosphoric acid nucleoside phosphorylase is recombinantly expressed in Escherichia coli respectively with soluble starch, synthesizes uridine diphosphoglucose using the thallus high temperature whole-cell catalytic after expression.On this basis, high temperature UDP-glucose dehydrogenase is recombinantly expressed in Escherichia coli, it is coupled the synthetic system high temperature whole-cell catalytic synthesis uridine diphosphate glucuronate of above-mentioned uridine diphosphoglucose, the Whole cell catalytic system of high temperature nadh oxidase is introduced in the synthetic system of uridine diphosphate glucuronate simultaneously, constitute the high temperature NAD+/NADH circulatory system, with reduce coenzyme NAD+use.The present invention successfully avoids the interference of the various metabolic pathways of thallus in synthesis process in the way of high temperature whole-cell catalytic, reduces purifying difficulty.
Description
Technical field
The present invention relates to a kind of uridine diphosphoglucose and the biological synthesis method of uridine diphosphate glucuronate,
Belong to biosynthesis technology field.
Background technique
In the research of carbohydrate, the uniform oligosaccharides of structure and sugared compound of sewing are information important in organism point
Son plays an important role in vital movement.Nowadays, they are more and more used as the probe or medicine of biological study
Object and the lead compound of vaccine discovery, the research synthesized to it also increasingly cause the concern of people.Chemical-enzymatic is as close
Research hotspot over year, the specificity with high efficiency and height, sewing in compound in the uniform oligosaccharides of composite structure and sugar has
Huge application potential.And in this synthesis process, the nucleotide carbohydrate of high energy as the donor substrate of monosaccharide be can not or
Scarce.
Nucleotide carbohydrate as in nature monosaccharide activate high energy forms, the transfer of Leloir type glycosyl, biosynthesis,
The many aspects such as glycosylation modified have important application, wherein especially with the carbohydrate of uridine 5'-diphosphate using relatively broad.Urine
Glycosides diphosphate glucose (UDP-Glc) can be used as a variety of glycosyls and turn as a member the most basic in uridine 5'-diphosphate carbohydrate
The monosaccharide donor substrate for moving enzyme, can be used as other uridine 5'-diphosphate carbohydrates such as uridine diphosphate galactose, uridine 5'-diphosphate
The precursor of xylose, uridine 5'-diphosphate rhamnose etc. also plays important in various aspects such as biological signaling pathway, target spot researchs
Effect.Derivative products uridine diphosphate glucuronate (UDP-GlcA) obtained from its dehydrogenation can be used as glycosaminoglycan sugar
The monosaccharide donor of chain elongation, is of great significance to the research of the glycosaminoglycan of different molecular weight size.
Time-consuming for traditional nucleotide sugar acquisition methods, involves great expense, is cumbersome, wherein direct extraction method source has
Limit, yield are extremely low, and chemical synthesis easily causes environment dirty because the inhomogeneity limiting factor of carbohydrate is more in synthesis process
Dye, in contrast specificity is high for enzymatic clarification, and synthesis process is efficiently quick, but enzyme molecule need to be purified, and easy in inactivation, to a large amount of
Product is prepared with certain limitation.Chinese patent literature CN104561195A discloses a kind of uridine diphosphoglucose
Preparation method, this method are in external (extracellular) reaction system, with uridine triphosphate (UTP) or its salt (such as sodium
Salt), maltodextrin be substrate, inorganic ions, dTT and Tris is added, with the uridine 5'-diphosphate grape of Recombinant protein expression
Sugared pyrophosphorylase crude enzyme liquid and maltodextrin phosphorylase crude enzyme liquid produce two phosphorus of uridine by bioconversion as catalyst
Sour grapes sugar.For the preparation method using maltodextrin as one of raw material, cost of material is higher, and needs to extract uridine 5'-diphosphate Portugal
The crude enzyme liquid of grape sugar pyrophosphorylase and maltodextrin phosphorylase, preparation step are complex.
Summary of the invention
In view of the deficiencies of the prior art, the present invention provides a kind of uridine diphosphoglucose and uridine diphosphoglucoses
The biological synthesis method of aldehydic acid is to use starches as raw material on the basis of traditional enzymatic clarification, with enzyme in Escherichia coli weight
Thallus after group expression carries out the reaction of high temperature whole-cell catalytic as catalyst, is a kind of easy, efficient, environmental protection and economy uridine
The biosynthesis side of diphosphate glucose (UDP-Glc) and its dehydrogenation derivative uridine diphosphate glucuronate (UDP-GlcA)
Method.
Technical scheme is as follows:
A kind of biological synthesis method of uridine diphosphoglucose, steps are as follows:
(1) in reaction solution, using soluble starch and phosphate as substrate, to contain the good high temperature alpha-glucans of inducing expression
The recombination bacillus coli of phosphorylase (Tm α GP) is catalyst, and biosynthesis obtains 1- phosphate-dextrose (1-P-Glc)；
(2) inorganic ions, sodium dihydrogen phosphate buffering is added in the 1- phosphate-dextrose (1-P-Glc) for taking step (1) to obtain
Liquid, uridine triphosphate (UTP), to contain the recombination large intestine bar of the good high temperature sugar -1- phosphoric acid nucleoside phosphorylase (StUSP) of inducing expression
Bacterium is catalyst, and biosynthesis obtains uridine diphosphoglucose (UDP-Glc).
It is preferred according to the present invention, final concentration of 45~55g/ of the soluble starch described in step (1) in reaction solution
L。
Preferred according to the present invention, phosphate described in step (1) is KH2PO4And K2HPO4, it is further preferred that will
KH2PO4And K2HPO4It is dissolved in the water and is configured to KP solution, PO in the KP solution4 3-Total concentration be 1M, pH be 6~7, institute
State PO in KP solution4 3-Final concentration of 0.2~0.9M in reaction solution；Most preferably, the pH of the KP solution is 7, the KP
PO in solution4 3-Final concentration of 0.7M in reaction solution.
It is preferred according to the present invention, good high temperature alpha-glucans phosphorylase (the Tm α of inducing expression is contained described in step (1)
GP recombination bacillus coli dosage) is 6~7g dry cell weight/L reaction solution.
Preferred according to the present invention, the reaction temperature of biosynthesis described in step (1) is 70 DEG C, and the reaction time is greater than
180min；It is further preferred that the reaction time is 360min.
Preferred according to the present invention, 1- phosphate-dextrose (1-P-Glc) is final concentration of in the reaction system in step (2)
0.4~0.6g/L.
Preferred according to the present invention, inorganic ions described in step (2) is magnesium ion, it is further preferred that the magnesium from
Son is generated by hydrolysis of magnesium chloride, final concentration of 10~20mM of magnesium ion.
Preferred according to the present invention, the group of phosphate sodium dihydrogen buffer solution described in step (2) is divided into NaH2PO40.2M,
NaOH adjusts pH 6~11；It is further preferred that the pH of the phosphate sodium dihydrogen buffer solution is 9.
It is preferred according to the present invention, final concentration of 0.05~3.0mM of uridine triphosphate described in step (2)；Further
Preferably, final concentration of 2mM.
It is preferred according to the present invention, the good high temperature sugar -1- phosphoric acid nucleoside phosphorylase of inducing expression is contained described in step (2)
(StUSP) dosage of recombination bacillus coli is 3.0~5.5g dry cell weight/L reaction solution, it is further preferred that dosage is
4.17g dry cell weight/L reaction solution.
Preferred according to the present invention, the reaction temperature of biosynthesis described in step (2) is 70~90 DEG C, and the reaction time is big
In 60min；It is further preferred that the reaction temperature of the biosynthesis is 80 DEG C, reaction time 180min.
According to the present invention preferably, the step of inducing expression includes:
Recombination bacillus coli containing target gene Tm α GP or the recombination bacillus coli containing target gene StUSP are connect
For kind in LB culture medium of the 30mL containing 100 μ g/mL ampicillins, 37 DEG C of 225rpm activate 12~14h；By the recombination after activation
Escherichia coli are inoculated in LB culture medium of the 250mL containing 100 μ g/mL ampicillins and expand culture, initial OD600Value is
0.05；37 DEG C of 225rpm are cultivated to OD600When value is 0.6~0.8, the IPTG induction of final concentration of 0.2mM, inductive condition is added
For 22 DEG C of 225rpm, 18~20h, the OD of recombination bacillus coli bacterium solution is measured600It is worth to get the good high temperature α-Portugal of inducing expression is contained
The recombination bacillus coli of phosphorylase (Tm α GP) contains the good high temperature sugar -1- phosphoric acid nucleoside phosphorylase of inducing expression
(StUSP) recombination bacillus coli.
It is further preferred that the recombination bacillus coli containing target gene Tm α GP and containing target gene StUSP's
Recombination bacillus coli is synthesized by Nanjing Jin Sirui company, wherein plasmid used in the recombination bacillus coli containing target gene Tm α GP
Carrier is pET20b, and bacterial strain uses therefor is E.coliBL21 (DE3)；Matter used in recombination bacillus coli containing target gene StUSP
Grain carrier is pET21b, and bacterial strain uses therefor is E.coliBL21 (DE3).
It is further preferred that the OD after the recombination bacillus coli inducing expression containing target gene Tm α GP600Value is
2.1~2.3；OD after the recombination bacillus coli inducing expression containing target gene StUSP600Value is 2.4~2.6.
A kind of biological synthesis method of uridine diphosphate glucuronate (UDP-GlcA), including the above-mentioned uridine of the present invention
It is the step of biological synthesis method of diphosphate glucose, further comprising the steps of:
In reaction solution, the uridine diphosphoglucose (UDP-Glc) obtained with above-mentioned biological synthesis method is substrate, is added
Enter phosphate sodium dihydrogen buffer solution, it is big with the recombination containing the good high temperature UDP-glucose dehydrogenase (PiUdh) of inducing expression
Enterobacteria is catalyst, and biosynthesis obtains uridine diphosphate glucuronate (UDP-GlcA)；It is utilized simultaneously containing induction table
Recombination bacillus coli up to good high temperature nadh oxidase (TkNOX) constructs high temperature NAD+/ NADH the circulatory system, consumption oxygen push
Cyclic process, so that synthetic system be made to be not required to that coenzyme NAD additionally is added+。
It is preferred according to the present invention, the uridine diphosphoglucose (UDP-Glc) in reaction solution final concentration of 1.2
~1.4g/L.
Preferred according to the present invention, the group of the phosphate sodium dihydrogen buffer solution is divided into NaH2PO40.5M, NaOH adjust pH 8
~11, it is further preferred that the pH of the phosphate sodium dihydrogen buffer solution is 10.
It is preferred according to the present invention, it is described to contain the good high temperature UDP-glucose dehydrogenase (PiUdh) of inducing expression
Recombination bacillus coli dosage be 7~8g dry cell weight/L reaction solution, it is further preferred that dosage be 7.28g dry cell weight/
L reaction solution.
It is preferred according to the present invention, the recombination large intestine bar containing the good high temperature nadh oxidase (TkNOX) of inducing expression
The dosage of bacterium is 4~5g dry cell weight/L reaction solution.
Preferred according to the present invention, the reaction temperature of the biosynthesis is 65 DEG C, and the reaction time is greater than 10min；Into one
Step is preferred, and the reaction time is 30min.
According to the present invention preferably, the step of inducing expression includes:
Recombination bacillus coli containing target gene PiUdh or the recombination bacillus coli containing target gene TkNOX are connect
For kind in LB culture medium of the 30mL containing 100 μ g/mL ampicillins, 37 DEG C of 225rpm activate 12~14h；By the recombination after activation
Escherichia coli are inoculated in LB culture medium of the 250mL containing 100 μ g/mL ampicillins and expand culture, initial OD600Value is
0.05；37 DEG C of 225rpm are cultivated to OD600When value is 0.6~0.8, the IPTG induction of final concentration of 0.2mM, inductive condition is added
For 22 DEG C of 225rpm, 18~20h, the OD of recombination bacillus coli bacterium solution is measured600It is worth to get the good high temperature uridine of inducing expression is contained
The recombination bacillus coli of diphosphate glucose dehydrogenase (PiUdh) contains the good high temperature nadh oxidase (TkNOX) of inducing expression
Recombination bacillus coli.
It is further preferred that the recombination bacillus coli containing target gene PiUdh and containing target gene TkNOX's
Recombination bacillus coli is synthesized by Nanjing Jin Sirui company, wherein plasmid used in the recombination bacillus coli containing target gene PiUdh
Carrier is pET15b, and bacterial strain uses therefor is E.coliBL21 (DE3)；Matter used in recombination bacillus coli containing target gene TkNOX
Grain carrier is pET21b, and bacterial strain uses therefor is E.coliBL21 (DE3).
It is further preferred that the OD after the recombination bacillus coli inducing expression containing target gene Tm α GP600Value is
2.2~2.5；OD after the recombination bacillus coli inducing expression containing target gene StUSP600Value is 2.2~2.5.
Technical characterstic of the invention:
The high temperature whole-cell catalytic synthetic method process of uridine diphosphoglucose (UDP-Glc) of the present invention is as schemed
Shown in 1.Recombination large intestine bar in reaction solution, first to contain the good high temperature alpha-glucans phosphorylase of inducing expression (Tm α GP)
Bacterium is catalyst, is catalyzed soluble starch and high-concentration phosphoric acid reactant salt obtains Cori ester (Glc-1-P)；Again to contain
The recombination bacillus coli for having the good high temperature sugar -1- phosphoric acid nucleoside phosphorylase (StUSP) of inducing expression is catalyst, is catalyzed first step institute
Obtained Glc-1-P reaction solution and uridine triphosphate (UTP) reaction obtains UDP-Glc.
The high temperature whole-cell catalytic synthetic method stream of uridine diphosphate glucuronate (UDP-GlcA) of the present invention
Journey is as shown in Figure 2.In reaction solution, to contain the weight of the good high temperature UDP-glucose dehydrogenase (PiUdh) of inducing expression
Group coli somatic is catalyst, is catalyzed the UDP-Glc reaction solution synthesized in the above method and is changed into UDP-GlcA, adds simultaneously
Enter the recombination bacillus coli containing the good high temperature nadh oxidase (TkNOX) of inducing expression, constructs high temperature NAD+/ NADH cyclic system
System, consumption oxygen pushes cyclic process, so as to so that synthetic system is not required to that coenzyme NAD additionally is added+。
The utility model has the advantages that
The method that biological synthesis method in the present invention utilizes high temperature whole-cell catalytic, eliminates the step of enzyme purification extraction
Suddenly, meanwhile, high temperature NAD is constructed using the recombination bacillus coli containing the good high temperature nadh oxidase (TkNOX) of inducing expression+/
After the NADH circulatory system, the NAD of thallus own level+The yield of UDP-GlcA can be made to reach 95% or more, without additionally adding
Add coenzyme NAD+, time and cost is greatly saved, simplifies operating procedure, and its catalytic carrier Escherichia coli is easy to change
It makes, it is low in cost, it is easy a large amount of obtain.Simultaneously under conditions of pyroreaction, extra contamination precipitation is precipitated in thallus, and interference produces
Object synthesis, the enzyme for promoting product to decompose and signal path lose activity, and are conducive to the synthesis of purpose carbohydrate, efficiently synthesize purpose sugar
The step of purifying after being also allowed to while class becomes more easy.In addition, the present invention using soluble starch as reaction substrate, reduces
Production cost.
Detailed description of the invention
Fig. 1: the high temperature whole-cell catalytic synthetic method flow chart of uridine diphosphoglucose (UDP-Glc)；
Fig. 2: the high temperature whole-cell catalytic synthetic method flow chart of uridine diphosphate glucuronate (UDP-GlcA)；
Fig. 3: the SDS-PAGE inspection of high temperature alpha-glucans phosphorylase (Tm α GP) solubility expression in recombination bacillus coli
Survey result figure；
Fig. 4: the SDS- of high temperature sugar -1- phosphoric acid nucleoside phosphorylase (StUSP) solubility expression in recombination bacillus coli
PAGE testing result figure；
Fig. 5: the TLC testing result figure of high temperature alpha-glucans phosphorylase (Tm α GP) high temperature whole-cell catalytic reactivity；
Fig. 6: high temperature alpha-glucans phosphorylase (Tm α GP) high temperature whole-cell catalytic reaction temperature TLC testing result figure；
Fig. 7: high temperature alpha-glucans phosphorylase (Tm α GP) high temperature whole-cell catalytic reacts KP solution ph TLC detection knot
Fruit figure；
Fig. 8: high temperature alpha-glucans phosphorylase (Tm α GP) high temperature whole-cell catalytic reaction time TLC testing result figure；
Fig. 9: the HPLC chromatogram of high temperature sugar -1- phosphoric acid nucleoside phosphorylase (StUSP) high temperature whole-cell catalytic reactivity；
In figure, abscissa is retention time, and ordinate is electrical signal intensity；
Figure 10: high temperature sugar -1- phosphoric acid nucleoside phosphorylase (StUSP) high temperature whole-cell catalytic reaction temperature curve graph；
In figure, abscissa is reaction temperature, and ordinate is conversion ratio；
Figure 11: high temperature sugar -1- phosphoric acid nucleoside phosphorylase (StUSP) high temperature whole-cell catalytic reacts NaH2PO4PH of cushioning fluid
Curve graph；
In figure, abscissa NaH2PO4PH of cushioning fluid, ordinate are conversion ratio；
Figure 12: high temperature sugar -1- phosphoric acid nucleoside phosphorylase (StUSP) high temperature whole-cell catalytic reaction time curve graph；
In figure, abscissa is the reaction time, and ordinate is conversion ratio；
The HPLC chromatogram of Figure 13: Tm α GP and StUSP high temperature whole-cell catalytic reaction synthesis UDP-Glc；
In figure, abscissa is retention time, and ordinate is electrical signal intensity；
The MS map of the UDP-Glc of Figure 14: Tm α GP and StUSP high temperature whole-cell catalytic synthesis；
The curve graph of StUSP thallus additional amount in the reaction of Figure 15: Tm α GP and StUSP high temperature whole-cell catalytic；
In figure, abscissa is StUSP dry cell weight, and ordinate is conversion ratio；
The curve graph of UTP additional amount in the reaction of Figure 16: Tm α GP and StUSP high temperature whole-cell catalytic；
In figure, abscissa is UTP additional amount, and ordinate is UTP conversion ratio；
Figure 17: high temperature UDP-glucose dehydrogenase (PiUdh) solubility expression in recombination bacillus coli
SDS-PAGE testing result figure；
Figure 18: the HPLC chromatogram of high temperature UDP-glucose dehydrogenase (PiUdh) high temperature whole-cell catalytic reaction；
In figure, abscissa is retention time, and ordinate is electrical signal intensity；
Figure 19: high temperature UDP-glucose dehydrogenase (PiUdh) high temperature whole-cell catalytic reaction temperature curve graph；
In figure, abscissa is reaction temperature, and ordinate is conversion ratio；
Figure 20: high temperature UDP-glucose dehydrogenase (PiUdh) high temperature whole-cell catalytic reacts NaH2PO4Buffer
PH value curve graph；
In figure, abscissa NaH2PO4PH of cushioning fluid, ordinate are conversion ratio；
Figure 21: high temperature UDP-glucose dehydrogenase (PiUdh) high temperature whole-cell catalytic reaction time curve graph；
In figure, abscissa is the reaction time, and ordinate is conversion ratio；
The HPLC chromatogram of Figure 22: Tm α GP, StUSP and PiUdh high temperature whole-cell catalytic reaction synthesis UDP-GlcA；
In figure, abscissa is retention time, and ordinate is electrical signal intensity；
The curve graph of PiUdh thallus additional amount in Figure 23: Tm α GP, the reaction of StUSP and PiUdh high temperature whole-cell catalytic；
In figure, abscissa is PiUdh dry cell weight, and ordinate is conversion ratio；
NAD in Figure 24: Tm α GP, the reaction of StUSP and PiUdh high temperature whole-cell catalytic+The curve graph of additional amount；
In figure, abscissa NAD+Additional amount, ordinate are conversion ratio；
Figure 25: the SDS-PAGE of high temperature nadh oxidase (TkNOX) solubility expression in recombination bacillus coli detects knot
Fruit figure；
The testing result that the reaction of Figure 26: TkNOX and PiUdh high temperature whole-cell catalytic influences UDP-GlcA synthesis；
In figure, ordinate is UDP-GlcA conversion ratio；
The MS figure of the high temperature whole-cell catalytic reaction synthesis UDP-GlcA of Figure 27: Tm α GP, StUSP, PiUdh and TkNOX
Spectrum.
Specific embodiment
The present invention is described below by specific embodiment.Unless specifically indicated, the technology hand used in the present invention
Duan Junwei method known in those skilled in the art.Following embodiment is intended to further illustrate the content of present invention, rather than limits
Protection scope of the present invention.
Substrate carbohydrate reagent of the present invention is purchased from sigma company, recombination bacillus coli be Nanjing gold this
Auspicious company's synthesis, bacterial strain uses therefor and plasmid vector are shown in Table 1.
The bacterial strain uses therefor of the present invention of table 1 and plasmid vector
TLC detection method of the present invention is as follows:
Silica gel plate: TLC Silica gel 60F254；Solvent: n-butanol: glacial acetic acid: water=2:1:1；Coloring agent: fennel
Fragrant aldehyde.
HPLC detection method of the present invention uses YMC-Pack Polyamine II column, and liquid phase systems are day
Island proper body fluid phase, ultraviolet detection system are SPD-20A.Specific method program includes two kinds according to the difference of detection ingredient:
The HPLC mobile phase condition of detection uridine diphosphoglucose (UDP-Glc) is shown in Table 2:
HPLC used in 2 UDP-Glc of table analyzes program
The HPLC mobile phase condition of detection uridine diphosphate glucuronate (UDP-GlcA) is shown in Table 3:
The HPLC of 3 UDP-GlcA of table analyzes program
The NADH detection kit from Amy victory company is also used in the present invention.
1. recombination bacillus coli high temperature alpha-glucans phosphorylase (Tm α GP) of embodiment and high temperature sugar -1- phosphoric acid nucleoside acid
Change the inducing expression of enzyme (StUSP)
By the synthetic recombination bacillus coli BL21 (DE3) containing target gene Tm α GP or StUSP of Jin Sirui company points
It is not inoculated in the LB culture medium of 30mL (ampicillin containing 100 μ g/mL), 37 DEG C of 225rpm activate 12~14h；Then,
Recombination bacillus coli BL21 (DE3) after activation is inoculated in the LB culture medium of 250mL (the ammonia benzyl mould containing 100 μ g/mL
Element) it expands culture, initial OD600Value is that 0.05,37 DEG C of 225rpm are cultivated to OD600When value reaches 0.6~0.8, it is added eventually
The IPTG that concentration is 0.2mM is induced, and inductive condition is 22 DEG C of 225rpm, 18~20h, measures the OD of recombination bacillus coli bacterium solution600
Value, wherein the OD after the recombination bacillus coli inducing expression containing target gene Tm α GP600Value is 2.1~2.3；Containing purposeful
OD after the recombination bacillus coli inducing expression of gene StUSP600Value is 2.4~2.6.
Above two recombination bacillus coli bacterium solution is taken, 8000rpm is centrifuged 20min and collects bacterial sediment, the bacterium of every 2mL bacterium solution
Body precipitating is with the resuspension of 100 μ L tri-distilled waters, and after 80 DEG C are boiled 10min, 12000rpm is centrifuged 20min, and supernatant is detected with SDS-PAGE
The expression of Tm α GP and StUSP.The SDS-PAGE result of Tm α GP and StUSP are shown in Fig. 3 and Fig. 4, detect purpose egg
It is white, illustrate that the two has solubility expression in recombination bacillus coli.
The high temperature whole-cell catalytic of 2. high temperature alpha-glucans phosphorylase of embodiment (Tm α GP) reacts
1) the high temperature whole-cell catalytic reaction of Tm α GP
Firstly, thalline were collected by centrifugation for the recombination bacillus coli bacterium solution containing the good Tm α GP of inducing expression prepared by embodiment 1
Precipitating, obtained bacterial sediment is frozen about one day in -20 DEG C, is taken out when reaction and is melted on ice, every 60mL bacterium solution is centrifuged to obtain
Bacterial sediment with 2mL tri-distilled water resuspension.Reaction system is as shown in table 4, wherein KP solution is by KH2PO4And K2HPO4Dissolution
Yu Shuizhong is formulated, KH in KP solution2PO4And K2HPO4Concentration be respectively 0.38M and 0.62M, pH 7, PO4 3-It is total dense
Degree is 1M, and the dosage of Tm α GP thallus is 6.6g dry cell weight/L reaction solution；In addition control group, respectively nothing in reaction solution are set
In soluble starch, reaction solution without in Tm α GP thallus, reaction solution add 1- phosphate-dextrose (1-P-Glc), each reaction solution in
70 DEG C of reactions 16h, ice bath 5min terminate reaction.Each reaction solution 12000rpm is centrifuged 20min, excessive calcium ion is added in supernatant
After desalination, one times is diluted, is detected with TLC.
The high temperature whole-cell catalytic reaction system of 4 Tm α GP of table
Testing result illustrates the high temperature whole-cell catalytic reaction of Tm α GP as shown in figure 5, there is 1-P-Glc generation in reaction group
With the activity that soluble starch is changed into 1-P-Glc.
2) the high temperature whole-cell catalytic reaction temperature of Tm α GP
Reaction system such as table 4, reaction temperature are respectively set 50 DEG C, and 60 DEG C, 70 DEG C, 75 DEG C, 80 DEG C amount to 5 temperature gradients
Point, three groups of each gradient are parallel.Remaining treatment conditions is same as above.
TLC testing result is as shown in fig. 6, the high temperature whole-cell catalytic reaction optimum temperature for illustrating Tm α GP is 70 DEG C.
3) the KP solution ph of the high temperature whole-cell catalytic reaction of Tm α GP
KP solution, such as table 4, is changed into the KP solution of different pH value, is arranged 3,4,5,6,7 by reaction system in addition to KP solution,
8,9,10,11,12 amount to 10 pH gradient points, and three groups of each gradient is parallel.Remaining treatment conditions is same as above.
Testing result is shown in Fig. 7, illustrates that the pH value of KP solution in the high temperature whole-cell catalytic reaction of Tm α GP is 6~7, most suitable
PH value is 7.
4) the high temperature whole-cell catalytic reaction time of Tm α GP
Reaction system such as table 4,0min, 5min, 10min, 20min, 30min, 60min is respectively set in the reaction time,
120min, 180min, 360min, 660min, 960min amount to 11 gradient points, and three groups of each gradient is parallel.Remaining processing item
Part is same as above.
Testing result is shown in Fig. 8, illustrates that the high temperature whole-cell catalytic reaction time of Tm α GP is greater than 180min and has Glc-1-P
It generates, the optimal reaction time is 360min.
The high temperature whole-cell catalytic of 3. high temperature sugar -1- phosphoric acid nucleoside phosphorylase (StUSP) of embodiment reacts
1) the high temperature whole-cell catalytic reaction of StUSP
Firstly, thalline were collected by centrifugation for the recombination bacillus coli bacterium solution containing the good StUSP of inducing expression prepared by embodiment 1
Precipitating, obtained bacterial sediment is frozen about one day in -20 DEG C, is taken out when reaction and is melted on ice, every 60mL bacterium solution is centrifuged to obtain
Bacterial sediment with 2mL tri-distilled water resuspension.Reaction system is as shown in table 5, wherein Mg2+It is generated by hydrolysis of magnesium chloride, biphosphate
The group of sodium buffer is divided into NaH2PO40.2M, NaOH adjust pH 7.5；In addition control group, respectively nothing in reaction solution are set
Uridine diphosphoglucose (UDP-Glc) is added in StUSP thallus, reaction solution, each reaction solution is in 80 DEG C of reaction 0.5h, ice bath
5min terminates reaction.Each reaction solution 12000rpm is centrifuged 20min, after supernatant is with 0.22 μm of membrane filtration, is described according to table 2
HPLC condition detected.
The high temperature whole-cell catalytic reaction system of 5 StUSP of table
Testing result is shown in Fig. 9, there is UDP-Glc generation in reaction group, illustrates that the high temperature whole-cell catalytic reaction of StUSP has
1-P-Glc is changed into the activity of UDP-Glc.
2) the high temperature whole-cell catalytic reaction temperature of StUSP
Reaction system such as table 5, reaction temperature are respectively set 60 DEG C, and 70 DEG C, 75 DEG C, 80 DEG C, 85 DEG C, 90 DEG C amount to 6 temperature
Gradient point is spent, three groups of each gradient is parallel.Remaining treatment conditions is same as above.
Testing result is shown in Figure 10, and when reaction temperature is 70~90 DEG C, the conversion ratio of 1-P-Glc is more than or equal to 50%, when anti-
The conversion ratio highest of 1-P-Glc when to answer temperature be 80 DEG C, illustrating the high temperature whole-cell catalytic reaction of StUSP, suitable temperature is 70
~90 DEG C, optimum temperature is 80 DEG C.
3) NaH of the high temperature whole-cell catalytic reaction of StUSP2PO4PH of cushioning fluid
Reaction system removes NaH2PO4Such as table 5 outside buffer, by NaH2PO4Buffer changes the NaH of different pH value into2PO4It is slow
Fliud flushing is respectively set 2.5,6,7,7.5,8,8.5,9,9.5,10,11,12 and amounts to 11 pH gradient points, and three groups of each gradient flat
Row.Remaining treatment conditions is same as above.
Testing result is shown in Figure 11, works as NaH2PO4The conversion ratio of 1-P-Glc is more than or equal to when the pH value of buffer is 6~11
50%, work as NaH2PO4The conversion ratio of 1-P-Glc is maximum when the pH value of buffer is 9, illustrates that the high temperature whole-cell catalytic of StUSP is anti-
The NaH that should be suitable for2PO4PH of cushioning fluid is 6~11, optimum pH 9.
4) the high temperature whole-cell catalytic reaction time of StUSP
Reaction system is removed NaH2PO4Buffer is changed to the NaH of pH=92PO4Such as table 5, reaction time point outside buffer
Not She Zhi 0min, 5min, 10min, 30min, 40min, 60min, 80min, 150min, 180min, 240min amounts to 10 ladders
Point is spent, three groups of each gradient is parallel.Remaining treatment conditions is same as above.
Testing result is shown in Figure 12, illustrates 1-P-Glc when the high temperature whole-cell catalytic reaction time of StUSP is greater than 60min
Conversion ratio is greater than 50%, and the optimal reaction time is 180min.
Embodiment 4. is made from starch high temperature whole-cell catalytic reaction synthesis two phosphorus of uridine of Tm α GP and StUSP coupling
Sour grapes sugar (UDP-Glc)
1) the high temperature whole-cell catalytic reaction of Tm α GP and StUSP
Firstly, the high temperature whole-cell catalytic reaction of Tm α GP is carried out by system in table 4,70 DEG C of 6h biosynthesis 1-P-Glc,
The high temperature whole-cell catalytic reaction solution of Tm α GP participates in the reaction of StUSP whole-cell catalytic, reaction system such as table 6, wherein Tm α GP is anti-
The concentration for answering 1-P-Glc in liquid is 11g/L, and additive amount is 16 μ L, Mg2+It is generated by hydrolysis of magnesium chloride, NaH2PO4The group of buffer
It is divided into NaH2PO40.2M, NaOH adjust pH 9；In addition control group is set, respectively without StUSP thallus, reaction solution in reaction solution
Middle addition UDP-Glc, 80 DEG C of reactions 3h, ice bath 5min terminate reaction.After 12000rpm is centrifuged 20min, supernatant is with 0.22 μm
After membrane filtration, detected according to the HPLC analysis condition of table 2.
The high temperature whole-cell catalytic reaction system of table 6 Tm α GP and StUSP
Testing result is shown in Figure 13, illustrates that the high temperature whole-cell catalytic reaction for being made from starch Tm α GP and StUSP coupling can
To synthesize UDP-Glc.
By supernatant after P2 molecular sieve purification, MS detection is carried out, MS result figure is shown in Figure 14.Mass Spectrometry Conditions are anion,
It has obtained and UDP-Glc (Mw=566.30) peak (565.05) that molecular weight after a proton is consistent is sloughed, it was demonstrated that product
UDP-Glc synthesis.
2) the StUSP thallus additional amount of the high temperature whole-cell catalytic reaction of Tm α GP and StUSP
Reaction system such as table 6, the additional amount of the above-mentioned Tm α GP reaction solution containing 1-P-Glc are 16 μ L.StUSP thallus weight
0 μ L, 3.33 μ L, 8.33 μ L, 16.6 μ L, 33.3 μ L, 50 μ L, 66.7 μ L is arranged altogether and amounts to 7 gradients, change H for suspension2O's adds
Amount, makes total volume remain 320 μ L, three groups of each gradient is parallel.Remaining condition is constant.
Testing result is shown in Figure 15, and when the additional amount of StUSP thallus is greater than 16.6 μ L, the conversion ratio of 1-P-Glc is greater than
The most suitable additional amount of 50%, StUSP thallus is 50 μ L, and conversion obtains as 4.17g (dry weight thallus)/L reaction solution.
3) the UTP dosage of the high temperature whole-cell catalytic reaction of Tm α GP and StUSP
Reaction system such as table 6, the additional amount of the above-mentioned Tm α GP reaction solution containing 1-P-Glc are that 16 μ L, StUSP thallus add
Enter amount and changes into 50 μ L.Changing the additional amount of UTP, 0mM, 0.5mM, 1mM is arranged in final concentration of the UTP in reaction solution altogether,
1.5mM, 2mM, 2.5mM, 3mM, 4mM, 5mM, 6mM amount to 10 gradients, change H2The dosage of O makes total volume remain 320 μ
L, three groups of each gradient are parallel.Remaining condition is constant.
Testing result is shown in Figure 16, and when the additional amount of UTP is 0.5~2.5mM, the conversion ratio of UTP is in 50% or more, UTP
Most suitable additional amount is 2mM.
The inducing expression of 5. recombination bacillus coli high temperature UDP-glucose dehydrogenase (PiUdh) of embodiment
The synthetic recombination bacillus coli BL21 (DE3) containing target gene PiUdh of Jin Sirui company is inoculated in
In the LB culture medium of 30mL (ampicillin containing 100 μ g/mL), 37 DEG C of 225rpm activate 12~14h；It then, will be after activation
Recombination bacillus coli be inoculated in the LB culture medium of 250mL (the ampicillin Amp containing 100 μ g/mL) and expand culture,
Initial OD600Value is that 0.05,37 DEG C of 225rpm are cultivated to OD600When value reaches 0.6~0.8, the IPTG of final concentration of 0.2mM is added
Induction, inductive condition are 22 DEG C of 225rpm, 18~20h, measure the OD of recombination bacillus coli bacterium solution600Value, contains target gene
OD after the recombination bacillus coli inducing expression of PiUdh6002.2~2.5.
Above-mentioned recombination bacillus coli bacterium solution is taken, 8000rpm is centrifuged 20min and collects bacterial sediment, and the thallus of every 2mL bacterium solution is heavy
It forms sediment with the resuspension of 100 μ L tri-distilled waters, after 80 DEG C are boiled 10min, 12000rpm is centrifuged 20min, and supernatant is detected with SDS-PAGE and expressed
Situation.The SDS-PAGE the result is shown in Figure 17 of PiUdh detects destination protein high temperature UDP-glucose dehydrogenase
(PiUdh), illustrate that PiUdh has solubility expression in recombination bacillus coli.
The high temperature whole-cell catalytic of 6. high temperature UDP-glucose dehydrogenase (PiUdh) of embodiment reacts
1) the high temperature whole-cell catalytic reaction of PiUdh
Firstly, by the above-mentioned recombination bacillus coli bacterium solution containing the good PiUdh of inducing expression thalline were collected by centrifugation precipitating, will
To bacterial sediment frozen in -20 DEG C about one day, take out when reaction and melt on ice, the bacterial sediment that every 60mL bacterium solution is centrifuged
With the resuspension of 2mL tri-distilled water.Reaction system is as shown in table 7, NaH2PO4The group of buffer is divided into NaH2PO40.5M, NaOH adjust pH
10；In addition control group is set, without adding uridine diphosphate glucuronate in PiUdh thallus, reaction solution respectively in reaction solution
(UDP-GlcA), uridine diphosphoglucose (UDP-Glc) is added in reaction solution, each reaction solution is in 70 DEG C of reaction 0.5h, ice bath
5min terminates reaction.Then 12000rpm is centrifuged 20min, after supernatant is with 0.22 μm of membrane filtration, according to the HPLC item of the description of table 3
Part is detected.
The high temperature Whole cell catalytic system of 7 PiUdh of table
Testing result is shown in Figure 18, illustrates that the high temperature whole-cell catalytic reaction of PiUdh has and UDP-Glc is changed into UDP-
The activity of GlcA.
2) the high temperature whole-cell catalytic reaction temperature of PiUdh
Reaction system such as table 7, reaction temperature are respectively set 50 DEG C, 60 DEG C, 65 DEG C, 70 DEG C, 75 DEG C, 80 DEG C, 85 DEG C total 7
A temperature gradient point, three groups of each gradient are parallel.It is detected using NADH kit, measures absorbance value at 460nm.
Testing result is shown in Figure 19, illustrates that the optimum temperature of the high temperature whole-cell catalytic reaction of PiUdh is 65 DEG C.
3) NaH of the high temperature whole-cell catalytic reaction of PiUdh2PO4PH of cushioning fluid
Reaction system removes NaH2PO4Such as table 7 outside buffer, by NaH2PO4Buffer changes the NaH of different pH value into2PO4It is slow
Fliud flushing is respectively set 2.5,4,5,6,7,8,9,10,11,12,13 and amounts to 11 pH gradient points, 65 DEG C of reaction 0.5h.Each ladder
Spend three groups it is parallel.It is detected using NADH kit, measures absorbance value at 460nm.
Testing result is shown in Figure 20, illustrates the NaH for being suitable in the high temperature whole-cell catalytic reaction of PiUdh2PO4PH of cushioning fluid
It is 8~11, optimum pH 10.
4) the high temperature whole-cell catalytic reaction time of PiUdh
Reaction system such as table 7,0min, 10min, 20min, 30min, 40min, 60min is respectively set in the reaction time,
90min, 120min, 150min, 180min amount to 10 gradient points, 65 DEG C of reactions.Three groups of each gradient is parallel.According to 3 institute of table
The HPLC program stated detects result.
Testing result is shown in Figure 21, illustrates UDP-Glc when the high temperature whole-cell catalytic reaction time of PiUdh is greater than 10min
For conversion ratio 50% or more, the optimal reaction time is 30min.
The high temperature whole-cell catalytic of embodiment 7.Tm α GP, StUSP and PiUdh reaction synthesis uridine diphosphoglucose aldehyde
Sour (UDP-GlcA)
1) the high temperature whole-cell catalytic reaction of Tm α GP, StUSP and PiUdh
Firstly, UDP-Glc is synthesized according to embodiment 4, after 12000rpm 20min centrifugal reaction solution removes thallus, with supernatant
Liquid participates in the reaction of PiUdh whole-cell catalytic as the substitute of UDP-Glc standard items in table 7, the concentration of UDP-Glc in supernatant
For 2.4g/L, additional amount is 200 μ L, changes H2The dosage of O makes total volume remain 400 μ L, NaH2PO4PH of buffer=10,
65 DEG C of reactions 30min, ice bath 5min terminate reaction.After 12000rpm is centrifuged 20min, after supernatant is with 0.22 μm of membrane filtration,
It is detected according to the HPLC analysis condition of table 3.
Testing result is shown in Figure 22, illustrates that the high temperature whole-cell catalytic reaction of Tm α GP, StUSP and PiUdh can synthesize UDP-
GlcA。
2) PiUdh thallus additional amount in the high temperature whole-cell catalytic reaction of Tm α GP, StUSP and PiUdh
UDP-Glc in table 7 is replaced with to the high temperature whole-cell catalytic reaction synthesis of Tm α GP and StUSP in embodiment 4
The supernatant of UDP-Glc, the concentration of UDP-Glc is 2.4g/L in supernatant, and additional amount is 200 μ L.In addition, every 90mL embodiment
After the PiUdh recombination bacillus coli bacterium solution centrifugation of 5 preparations, bacterial sediment is with the resuspension of 2mL tri-distilled water.The additional amount of PiUdh thallus
0 μ L, 1.1 μ L, 2.2 μ L, 5.6 μ L, 11.1 μ L, 16.7 μ L, 22.2 μ L, 33.3 μ L, 44.4 μ L, 66.7 μ L, 88.9 μ L are set altogether,
100 μ L amount to 12 gradients, change H2The dosage of O, makes total volume remain 400 μ L, and three groups of each gradient is parallel.Remaining condition
It is constant.
Testing result is shown in Figure 23, illustrate the most suitable additional amount of PiUdh be 88.9 μ L, conversion obtain for 7.28g (dry weight thallus)/
L reaction solution.
3) NAD in the high temperature whole-cell catalytic reaction of Tm α GP, StUSP and PiUdh+Dosage
UDP-Glc in table 7 is replaced with to the high temperature whole-cell catalytic reaction synthesis of Tm α GP and StUSP in embodiment 4
The supernatant of UDP-Glc, the concentration of UDP-Glc is 2.4g/L in supernatant, and additional amount is 200 μ L.In addition, every 60mL embodiment
After the PiUdh recombination bacillus coli bacterium solution centrifugation of 5 preparations, bacterial sediment is with the resuspension of 1mL tri-distilled water.The additional amount of PiUdh thallus
Become 66.7 μ L.Change NAD+Dosage, NAD+0mM, 0.05mM, 0.1mM, 0.3mM is arranged in final concentration in reaction solution altogether,
0.6mM, 0.9mM, 1.2mM, 1.5mM amount to 8 gradients, change H2The dosage of O makes total volume remain 400 μ L, each gradient
Three groups parallel.Remaining condition is constant.
Testing result is shown in Figure 24, is added without NAD+When, NAD that thallus itself contains+It can also make reaction.
High temperature whole-cell catalytic reaction synthesis two phosphorus of uridine that embodiment 8.Tm α GP, StUSP, PiUdh and TkNOX are coupled
Sour grapes uronic acid (UDP-GlcA)
1) inducing expression of recombination bacillus coli high temperature nadh oxidase (TkNOX)
The synthetic recombination bacillus coli BL21 (DE3) containing target gene TkNOX of Jin Sirui company is inoculated in
In the LB culture medium of 30mL (ampicillin containing 100 μ g/mL), 37 DEG C of 225rpm activate 12~14h；It then, will be after activation
Recombination bacillus coli BL21 (DE3) be inoculated in the LB culture medium of 250mL (ampicillin containing 100 μ g/ml) and expanded
Big culture, initial OD600Value is 0.05；37 DEG C of 225rpm are cultivated to OD600When value reaches 0.6~0.8, it is added final concentration of
The IPTG of 0.2mM is induced, and inductive condition is 22 DEG C of 225rpm, 18~20h, measures the OD of recombination bacillus coli bacterium solution600Value, contains
OD after having the recombination bacillus coli inducing expression of purpose gene TkNOX600Value is 2.2~2.5.
Above-mentioned recombination bacillus coli bacterium solution is taken, 8000rpm is centrifuged 20min and collects bacterial sediment, and the thallus of every 2ml bacterium solution is heavy
It forms sediment with the resuspension of 100 μ L tri-distilled waters, after 80 DEG C are boiled 10min, 12000rpm is centrifuged 20min, and supernatant is detected with SDS-PAGE and expressed
Situation.The SDS-PAGE result of TkNOX is shown in Figure 25, detects destination protein high temperature nadh oxidase (TkNOX), it was demonstrated that
TkNOX has solubility expression in recombination bacillus coli.
2) the high temperature whole-cell catalytic reaction of Tm α GP, StUSP, PiUdh and TkNOX
Firstly, by the above-mentioned recombination bacillus coli bacterium solution containing the good TkNOX of inducing expression thalline were collected by centrifugation precipitating, will
To bacterial sediment frozen in -20 DEG C about one day, take out when reaction and melt on ice, the bacterial sediment that every 60mL bacterium solution is centrifuged
With the resuspension of 1mL tri-distilled water.It is reacted by the reaction system of table 8, wherein UDP-Glc is Tm α GP and StUSP in embodiment 4
The reaction of high temperature whole-cell catalytic synthesizes the supernatant of UDP-Glc, and the concentration of UDP-Glc is 2.4g/L, PiUdh thallus in supernatant
Dosage be 7.28g dry cell weight/L reaction solution, the dosage of TkNOX thallus is 4.4g dry cell weight/L reaction solution；It is arranged simultaneously
Control group replaces with unloaded e. coli bl21 without TkNOX thallus in TkNOX thallus, reaction solution respectively in reaction solution
(DE3).Product UDP-GlcA is detected according to HPLC program described in table 3.
Tm α GP of 8 UDP-GlcA of table synthesis, StUSP, PiUdh and TkNOX high temperature whole-cell catalytic reaction system
Testing result is shown in Figure 26, illustrates that the reaction of TkNOX high temperature whole-cell catalytic can construct high temperature NAD+/ NADH cyclic system
System promotes the synthesis of UDP-GlcA, reduces NAD+Consumption.
According to condition iodine described in table 8,12000rpm is centrifuged 20min after completion of the reaction, and supernatant is through P2 molecular sieve
After purification, MS detection is carried out.MS result is shown in Figure 27.Mass Spectrometry Conditions are anion, have been obtained and UDP-GlcA (Mw=580.28)
The peak (579.19) that molecular weight after a proton is consistent is sloughed, and secondly peak (1159.19) are detected simultaneously again, it was demonstrated that
Product UDP-GlcA synthesis.
Claims (10)
1. a kind of biological synthesis method of uridine diphosphoglucose, which is characterized in that steps are as follows:
(1) in reaction solution, using soluble starch and phosphate as substrate, to contain the good high temperature alpha-glucans phosphoric acid of inducing expression
The recombination bacillus coli for changing enzyme is catalyst, and biosynthesis obtains 1- phosphate-dextrose；
(2) inorganic ions, phosphate sodium dihydrogen buffer solution, uridine triphosphate is added in the 1- phosphate-dextrose for taking step (1) to obtain,
Using the recombination bacillus coli containing the good high temperature sugar -1- phosphoric acid nucleoside phosphorylase of inducing expression as catalyst, biosynthesis is urinated
Glycosides diphosphate glucose.
2. biological synthesis method as described in claim 1, which is characterized in that in step (1) include it is following in one the more
:
I. final concentration of 45~55g/L of the soluble starch in reaction solution；
Ii. the phosphate is KH2PO4And K2HPO4, it is further preferred that by KH2PO4And K2HPO4It is dissolved in the water and is configured to
KP solution, PO in the KP solution4 3-Total concentration be 1M, pH is 6~7, PO in the KP solution4 3-End in reaction solution is dense
Degree is 0.2~0.9M；
Iii. the recombination bacillus coli dosage containing the good high temperature alpha-glucans phosphorylase of inducing expression is dry for 6~7g thallus
Weight/L reaction solution；
Iv. the reaction temperature of the biosynthesis is 70 DEG C, and the reaction time is greater than 180min.
3. biological synthesis method as described in claim 1, which is characterized in that include one or more in following in step (2)
:
I. the 1- phosphate-dextrose final concentration of 0.4~0.6g/L in the reaction system；
Ii. the inorganic ions is magnesium ion, it is further preferred that the magnesium ion is generated by hydrolysis of magnesium chloride, magnesium ion
Final concentration of 10~20mM；
Iii. the group of the phosphate sodium dihydrogen buffer solution is divided into NaH2PO40.2M, NaOH adjust pH 6~11；
Iv. final concentration of 0.05~3.0mM of the uridine triphosphate；
V. the dosage of the recombination bacillus coli containing the good high temperature sugar -1- phosphoric acid nucleoside phosphorylase of inducing expression be 3.0~
5.5g dry cell weight/L reaction solution；
Vi. the reaction temperature of the biosynthesis is 70~90 DEG C, and the reaction time is greater than 60min.
4. biological synthesis method as described in claim 1, which is characterized in that the step of inducing expression includes:
Recombination bacillus coli containing target gene Tm α GP or the recombination bacillus coli containing target gene StUSP are inoculated in
In LB culture medium of the 30mL containing 100 μ g/mL ampicillins, 37 DEG C of 225rpm activate 12~14h；By the recombination large intestine after activation
Bacillus is inoculated in LB culture medium of the 250mL containing 100 μ g/mL ampicillins and expands culture, initial OD600Value is 0.05；
37 DEG C of 225rpm are cultivated to OD600When value is 0.6~0.8, the IPTG induction of final concentration of 0.2mM is added, inductive condition is 22 DEG C
225rpm, 18~20h measure the OD of recombination bacillus coli bacterium solution600It is worth to get the good high temperature alpha-glucans phosphorus of inducing expression is contained
The recombination bacillus coli of phosphorylase or recombination bacillus coli containing the good high temperature sugar -1- phosphoric acid nucleoside phosphorylase of inducing expression；
Wherein, plasmid vector used in the recombination bacillus coli containing target gene Tm α GP is pET20b, and bacterial strain uses therefor is
E.coliBL21(DE3)；Plasmid vector used in the recombination bacillus coli containing target gene StUSP is pET21b, used
Bacterial strain is E.coliBL21 (DE3).
5. biological synthesis method as claimed in claim 4, which is characterized in that the recombination containing target gene Tm α GP is big
OD after enterobacteria inducing expression600Value is 2.1~2.3；The recombination bacillus coli containing target gene StUSP induces table
OD after reaching600Value is 2.4~2.6.
6. a kind of biological synthesis method of uridine diphosphate glucuronate, which is characterized in that including the above-mentioned uridine of the present invention
It is the step of biological synthesis method of diphosphate glucose, further comprising the steps of:
In reaction solution, sodium dihydrogen phosphate is added as substrate in the uridine diphosphoglucose obtained using above-mentioned biological synthesis method
Buffer, using the recombination bacillus coli containing the good high temperature UDP-glucose dehydrogenase of inducing expression as catalyst, biology
Synthesis obtains uridine diphosphate glucuronate；The recombination large intestine bar containing the good high temperature nadh oxidase of inducing expression is utilized simultaneously
Bacterium constructs high temperature NAD+/ NADH the circulatory system, consumption oxygen push cyclic process, thus make synthetic system be not required to additionally be added it is auxiliary
Enzyme NAD+。
7. biological synthesis method as claimed in claim 6, which is characterized in that including one or more in following:
I. final concentration of 1.2~1.4g/L of the uridine diphosphoglucose in reaction solution；
Ii. the group of the phosphate sodium dihydrogen buffer solution is divided into NaH2PO40.5M, NaOH adjust pH 8~11；
Iii. the dosage of the recombination bacillus coli containing the good high temperature UDP-glucose dehydrogenase of inducing expression be 7~
8g dry cell weight/L reaction solution；
Iv. the dosage of the recombination bacillus coli containing the good high temperature nadh oxidase of inducing expression is 4~5g dry cell weight/L
Reaction solution.
8. biological synthesis method as claimed in claim 6, which is characterized in that the reaction temperature of the biosynthesis is 65 DEG C,
Reaction time is greater than 10min.
9. biological synthesis method as claimed in claim 6, which is characterized in that the step of inducing expression includes:
Recombination bacillus coli containing target gene PiUdh or the recombination bacillus coli containing target gene TkNOX are inoculated in
In LB culture medium of the 30mL containing 100 μ g/mL ampicillins, 37 DEG C of 225rpm activate 12~14h；By the recombination large intestine after activation
Bacillus is inoculated in LB culture medium of the 250mL containing 100 μ g/mL ampicillins and expands culture, initial OD600Value is 0.05；
37 DEG C of 225rpm are cultivated to OD600When value is 0.6~0.8, the IPTG induction of final concentration of 0.2mM is added, inductive condition is 22 DEG C
225rpm, 18~20h measure the OD of recombination bacillus coli bacterium solution600It is worth to get the good high temperature uridine 5'-diphosphate of inducing expression is contained
The recombination bacillus coli of glucose dehydrogenase or recombination bacillus coli containing the good high temperature nadh oxidase of inducing expression；
Wherein, plasmid vector used in the recombination bacillus coli containing target gene PiUdh is pET15b, and bacterial strain uses therefor is
E.coliBL21(DE3)；Plasmid vector used in the recombination bacillus coli containing target gene TkNOX is pET21b, used
Bacterial strain is E.coliBL21 (DE3).
10. biological synthesis method as claimed in claim 6, which is characterized in that the recombination containing target gene Tm α GP is big
OD after enterobacteria inducing expression600Value is 2.2~2.5；The recombination bacillus coli containing target gene StUSP induces table
OD after reaching600Value is 2.2~2.5.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811338833.3A CN109371079B (en) | 2018-11-12 | 2018-11-12 | A kind of biological synthesis method of uridine diphosphoglucose and uridine diphosphate glucuronate |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811338833.3A CN109371079B (en) | 2018-11-12 | 2018-11-12 | A kind of biological synthesis method of uridine diphosphoglucose and uridine diphosphate glucuronate |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109371079A true CN109371079A (en) | 2019-02-22 |
| CN109371079B CN109371079B (en) | 2019-09-06 |
Family
ID=65384631
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811338833.3A Active CN109371079B (en) | 2018-11-12 | 2018-11-12 | A kind of biological synthesis method of uridine diphosphoglucose and uridine diphosphate glucuronate |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109371079B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111676259A (en) * | 2020-07-10 | 2020-09-18 | 山东大学 | Preparation method of sugar nucleotide and derivatives thereof |
| CN112608960A (en) * | 2020-12-30 | 2021-04-06 | 江南大学 | Preparation method of uridine diphosphate glucuronic acid |
| CN114507649A (en) * | 2022-02-16 | 2022-05-17 | 吉林大学 | Thermophilic enzyme and method for efficiently synthesizing UDP-glucose and UDP-glucuronic acid by one-pot method |
| CN114774495A (en) * | 2022-04-21 | 2022-07-22 | 山东大学 | Double-enzyme co-immobilization synthesis method of uridine diphosphate-N-acetylglucosamine |
| CN115290774A (en) * | 2022-07-21 | 2022-11-04 | 重庆医科大学 | Application of uridine diphosphate glucuronic acid in preparation of reagent for detecting liver cancer |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104561195A (en) * | 2013-10-22 | 2015-04-29 | 上海兆维科技发展有限公司 | Preparation method of uridine diphosphate glucose |
-
2018
- 2018-11-12 CN CN201811338833.3A patent/CN109371079B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104561195A (en) * | 2013-10-22 | 2015-04-29 | 上海兆维科技发展有限公司 | Preparation method of uridine diphosphate glucose |
Non-Patent Citations (2)
| Title |
|---|
| DONG Q, OUYANG LM, YU HL, XU JH: "Efficient biosynthesis of uridine diphosphate glucose from maltodextrin by multiple enzymes immobilized on magnetic nanoparticles", 《CARBOHYDRATE RESEARCH》 * |
| 陈圣 等: "生物法合成尿苷二磷酸葡萄糖的研究进展", 《中国生物工程杂志》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111676259A (en) * | 2020-07-10 | 2020-09-18 | 山东大学 | Preparation method of sugar nucleotide and derivatives thereof |
| CN112608960A (en) * | 2020-12-30 | 2021-04-06 | 江南大学 | Preparation method of uridine diphosphate glucuronic acid |
| CN114507649A (en) * | 2022-02-16 | 2022-05-17 | 吉林大学 | Thermophilic enzyme and method for efficiently synthesizing UDP-glucose and UDP-glucuronic acid by one-pot method |
| CN114507649B (en) * | 2022-02-16 | 2023-11-21 | 吉林大学 | Thermophilic enzyme and method for efficiently synthesizing UDP-glucose and UDP-glucuronic acid by one-pot method |
| CN114774495A (en) * | 2022-04-21 | 2022-07-22 | 山东大学 | Double-enzyme co-immobilization synthesis method of uridine diphosphate-N-acetylglucosamine |
| CN114774495B (en) * | 2022-04-21 | 2024-05-14 | 华熙唐安生物科技(山东)有限公司 | Double-enzyme co-immobilization synthesis method of uridine diphosphate-N-acetamido glucose |
| CN115290774A (en) * | 2022-07-21 | 2022-11-04 | 重庆医科大学 | Application of uridine diphosphate glucuronic acid in preparation of reagent for detecting liver cancer |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109371079B (en) | 2019-09-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109371079B (en) | A kind of biological synthesis method of uridine diphosphoglucose and uridine diphosphate glucuronate | |
| Ghalambor et al. | The Biosynthesis of Cell Wall Lipopolysaccharide in Escherichia coli: III. THE ISOLATION AND CHARACTERIZATION OF 3-DEOXYOCTULOSONIC ACID | |
| ES2340888T3 (en) | ENZYMATIC METHOD FOR PROCESSING ALFA-D-GLUCOSILGLICEROL (2-0-GLICERIL-ALFA-D-GLUCOPIRANOSIDA). | |
| Elbein et al. | The synthesis of cellulose by an enzyme system from a higher plant | |
| CN102220400B (en) | Method for synthesis of glutathione in vitro | |
| JP5100987B2 (en) | Method for producing aldonic acid | |
| CN113265434B (en) | Method for synthesizing UDP-galactose and method for synthesizing galactosyl compound | |
| EP3859003A1 (en) | Enzymatic preparation of glucosamine | |
| CN105886571B (en) | The synthetic method of Antigen of human blood group P1 pentasaccharides | |
| CN104561195B (en) | A kind of preparation method of uridine diphosphoglucose | |
| CN108018252B (en) | Preparation method of intermediate 2' -deoxyguanosine | |
| CN113322219B (en) | Method for synthesizing curcumin glucoside compound by biological method catalysis | |
| CN101555509A (en) | Method for synthesizing uridine phosphoryl compound by directional catalysis | |
| EP0385486B1 (en) | Process for producing fructose-1,6-diphosphate | |
| Frohnmeyer et al. | Gram‐Scale Production of GDP‐β‐l‐fucose with Multi‐Enzyme Cascades in a Repetitive‐Batch Mode | |
| CN116790649A (en) | Enzymatic synthesis method of UDP-glucuronic acid and UDP-N-acetamido glucose | |
| US6040158A (en) | Process for preparing sugar nucleotide | |
| Campbell et al. | Uridine diphospho‐α‐d‐gluco‐hexodialdose: Synthesis and kinetic competence in the reaction catalyzed by UDP‐glucose dehydrogenase | |
| WO2014199948A1 (en) | Production method for β-mannoside | |
| CN115819479A (en) | Alpha-salidroside and preparation method and application thereof | |
| JP3080771B2 (en) | Method for producing dinucleoside polyphosphate or derivative thereof | |
| Tai et al. | Highly efficient and regioselective production of an erythorbic acid glucoside using cyclodextrin glucanotransferase from Thermoanaerobacter sp. and amyloglucosidase | |
| JPH07508413A (en) | Purified sucrose-synthase, its production method and its use | |
| PT827549E (en) | PROCESS FOR THE GALACTOSILACAO ENZIMATICA OF MONOSACARIDOS AND OLIGOSACARIDOS | |
| Kragl et al. | Aldolases for use in carbohydrate synthesis: enzymatic reaction engineering as a tool for process optimization |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: A biosynthesis method of uridine diphosphate glucose and uridine diphosphate glucuronic acid Effective date of registration: 20211209 Granted publication date: 20190906 Pledgee: Chuzhou Wandong Rural Commercial Bank Co.,Ltd. Dawang sub branch Pledgor: ANHUI HEGENG BIO-TECH Co.,Ltd. Registration number: Y2021980014449 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20230313 Granted publication date: 20190906 Pledgee: Chuzhou Wandong Rural Commercial Bank Co.,Ltd. Dawang sub branch Pledgor: ANHUI HEGENG BIO-TECH Co.,Ltd. Registration number: Y2021980014449 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right |