CN106399427A - Tagatose preparation method - Google Patents
Tagatose preparation method Download PDFInfo
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- CN106399427A CN106399427A CN201610937656.5A CN201610937656A CN106399427A CN 106399427 A CN106399427 A CN 106399427A CN 201610937656 A CN201610937656 A CN 201610937656A CN 106399427 A CN106399427 A CN 106399427A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12P19/24—Preparation of compounds containing saccharide radicals produced by the action of an isomerase, e.g. fructose
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- 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
- C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
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Abstract
The invention discloses a tagatose preparation method and belongs to the field of multi-enzyme catalytic preparation of tagatose. Starch, cellulose, or their derivatives, or sucrose is used as substrate, and the substrate is efficiently catalyzed in a multi-enzyme reaction system by an in-vitro multi-enzyme molecular machine into tagatose. By adding enzymes capable of accelerating hydrolysis of starch, cellulose or sucrose and enzymes making use of the byproduct glucose, material conversion rate and tagatose yield are significantly increased. The method of the invention is high in tagatose yield, the materials are cheap, the production cost is low, and the method is suitable for industrial production of tagatose.
Description
Technical field
The present invention relates to the many enzyme catalysiss in vitro of a kind of preparation method of Tagatose, more particularly, to one kind are by starch or cellulose
And its method that derivant is converted into Tagatose, belong to the enzyme catalysiss preparation field of Tagatose.
Background technology
A kind of rare monosaccharide that Tagatose (D-Tagatose) is naturally-occurring, is the ketose form of galactose, Fructose
Epimer.Sweet taste characteristic is similar to sucrose, and the heat producing is only 1/3rd of sucrose, so being referred to as low grade fever
Amount sweeting agent.Tagatose have lower calorific value, zero glycemic index, blood glucose passivation, no dental caries, prebiotic function and
The excellent nutritive peculiarity such as antioxidant activity.Natural Tagatose is primarily present in the milk product such as yogurt, milk powder.Tagatose has
Four big functions：Low-yield, blood sugar lowering, improves intestinal microbial population and dental caries (Oh D-K:Tagatose:properties,
applications,and biotechnological processes.App.Microbiol.Biotechnol.2007,76:
1-8).
The method producing Tagatose has chemical synthesiss and two kinds of biotransformation method.Typically all with galactose as raw material, lead to
Cross chemical method or bioconversion carries out isomerization reaction and forms.Galactose raw material can be obtained by lactose hydrolysis, also has research to make
It is raw material with galactitol, be Tagatose through biological oxidation.But galactitol price is higher, industrialization wouldn't be suitable for use as at present
Raw materials for production.Chemical synthesiss are to be catalyst using soluble alkali metal salts or alkali salt, promote D- galactose in alkali
Property under the conditions of generate Tagatose, and form metal hydroxide-tagatose complex, then with acid neutralization acquisition D-Tag.Raw
Thing conversion method includes for galactitol being oxidized to Tagatose, with using isomerase produced by microorganism, galactose isomery is become tower
Two methods of lattice sugar.Because of chemical method high energy consumption, product is complicated, purification difficult, and side reaction is many, produces chemical contamination, therefore biological turn
Change method has preferable application prospect.The method that the more bioconversion of research produces Tagatose at present is to utilize L-arabinose
Isomery enzyme catalysiss D- galactose is converted into Tagatose, but the higher price of galactose have impact on the final price of Tagatose, leads
Cause cannot widely use (Rhimi M, Aghajari N, Juy M, Chouayekh H, Maguin E, Haser R, Bejar
S:Rational design of Bacillus stearothermophilus US100l-arabinose isomerase:
Potential applications for d-tagatose production.Biochim.2009,91:650-653.Oh
H-J,Kim H-J,Oh D-K:Increase in d-tagatose Production Rate by Site-directed
Mutagenesis of l-arabinose Isomerase from Geobacillus
thermodenitrificans.Biotechnol.Lett.2006,28:145-149.Bosshart A,Hee CS,
Bechtold M,Schirmer T,Panke S:Directed Divergent Evolution of a Thermostable
D-Tagatose Epimerase towards Improved Activity for Two Hexose
Substrates.ChemBioChem 2015,16:592-601.Men Y,Zhu Y,Zhang L,Kang Z,Izumori K,
Sun Y,Ma Y:Enzymatic conversion of D-galactose to D-tagatose:Cloning,
overexpression and characterization of l-arabinose isomerase from Pediococcus
pentosaceus PC-5.Microbiol.Res.2014,169:171-178.).
Korean science man invents the how enzymatic method of one kind by fructose converting for Tagatose, including using 6- phosphoric acid tower lattice
Sugared epimerase, 6- phosphoric acid Tagatose phosphatase are by fructose converting for Tagatose (Oh DK, HONG SH, Lee SH:
Aldolase,aldolase mutants and tagatose using the same production methods and
compositions for production.WO 2015016544 A1.Google Patents；2015.), but from Fructose
Producing fructose-1, 6-diphosphate needs ATP to carry out substrate phosphorylation to Fructose, leads to Tagatose production cost high, is not suitable with and gives birth on a large scale
Produce.
It would therefore be highly desirable to a kind of low cost of exploitation, low stain, the new method of the suitable large-scale production Tagatose of high yield.
Content of the invention
The technical problem to be solved is to provide a kind of preparation method of Tagatose, and the method passes through external multienzyme
Catalytic starch or cellulose and their derivant, or sucrose prepares Tagatose, has yield and the conversion ratio of Tagatose
Height, low production cost, the advantages of pollution-free.
For solving above-mentioned technical problem, the technical solution used in the present invention is：
The present invention discloses a kind of preparation method of starch Tagatose first, comprises the following steps：With starch or starch derivatives
Biological is substrate, adds and contains alpha-glucanses phosphorylase (α-Glucan phosphorylase, EC 2.4.1.1), glucose
Transphosphorylase (Phosphoglucomutase, EC 5.4.2.2), glucosephosphate isomerase (Phosphoglucose
Isomerase, EC 5.3.1.9), 6- phosphoric acid Tagatose epimerase (Tagatose 6-phosphate 4-
Epimerase) and the multienzyme catalytic materials of 6- phosphoric acid Tagatose phosphatase (Tagatose 6-phosphatase) to set up multienzyme anti-
Answer system, carry out enzymic catalytic reaction, obtain final product.
In order to reach more preferable effect it is preferred that conventionally carrying out point obtained enzymic catalytic reaction product
From, purification.
In described multienzymatic reaction system, the concentration of described substrate is 1-500g/L, the use of described alpha-glucanses phosphorylase
Measure as 0.1-1000U/mL, the consumption of described phosphoglucomutase is 0.1-1000U/mL, described glucose phosphate isomery
The consumption of enzyme is 0.1-1000U/mL, and the consumption of described 6- phosphoric acid Tagatose epimerase is 0.1-1000U/mL, described 6-
The consumption of phosphoric acid Tagatose phosphatase is 0.1-1000U/mL；Preferably, the concentration of described substrate is 100g/L, and described α-Portugal gathers
The consumption of saccharophosphorylase is 10U/mL, and the consumption of described phosphoglucomutase is 10U/mL, and described glucose phosphate is different
The consumption of structure enzyme is 10U/mL, and the consumption of described 6- phosphoric acid Tagatose epimerase is 10U/mL, described 6- phosphoric acid Tagatose
The consumption of phosphatase is 10U/mL.The condition of described enzymic catalytic reaction is：10-90 DEG C of reaction 1-100 hour；It is preferably, 37 DEG C
Reaction 24-40 hour.
In the preparation method of starch Tagatose of the present invention, described starch is preferably soluble starch；Described starch derivatives
Including：Any one or more in boiling starch, amylodextrin, maltodextrin, Fructus Hordei Germinatus polysaccharide or maltose according to appoint
The mixture of meaning ratio composition.
As the preferred technical solution of the present invention, also include in described multienzyme catalytic materials any one in (1), (2) or (3)
Kind：(1) any in starch debranching enzyme or maltose phosphorylase (maltose phosphorylase, EC 2.4.1.8)
One or two；(2) starch debranching enzyme or glucanotransferase (4-a-glucanotransferase, EC.2.4.1.25)
Any one of or two kinds；(3) any one in starch debranching enzyme, maltose phosphorylase or glucanotransferase or
Three kinds；Preferably, in multienzymatic reaction system, the consumption of described starch debranching enzyme is 0.1-500U/mL, described maltose phosphorus
The consumption of acidifying enzyme or glucanotransferase is 0.1-500U/mL；It is furthermore preferred that the consumption of described starch debranching enzyme is 1U/
ML, the consumption of described maltose phosphorylase or glucanotransferase is 1U/mL；Wherein, described starch debranching enzyme is different shallow lake
In powder enzyme (isoamylase, EC 3.2.1.68) or pullulanase (pullulanase, EC 3.2.1.41) any one or
Two kinds.
It is further preferred that for the yield improving Tagatose, remaining glucose is converted into Tagatose, described multienzyme
Also include in catalytic materials：Polyphosphoric acid glucokinase (polyphosphate glucokinase, EC 2.7.1.63) and poly- phosphorus
Hydrochlorate；Preferably, in multienzymatic reaction system, the consumption of described polyphosphoric acid glucokinase is 0.1-500U/mL, described poly- phosphorus
The consumption of hydrochlorate is 1-100mM；It is furthermore preferred that the consumption of described polyphosphoric acid glucokinase is 1U/mL, described Quadrafos
Consumption is 10mM.Wherein, described Quadrafos are preferably polyphosphate sodium.
After reaction terminates, remaining starch residue will be pure amylose, now can add a small amount of alpha amylase (EC
3.2.1.1) promote the hydrolysis of starch residue, improve the yield of Tagatose further.Preferably, in multienzymatic reaction system, institute
The consumption stating alpha amylase is 0.01-100U/ml, more preferably 0.1U/ml.
Also include in multienzymatic reaction system of the present invention：Buffer, inorganic phosphate radical and bivalence magnesium ion；Preferably,
The consumption of each composition is：Buffer 10-500mM, inorganic phosphate radical 2-100mM, bivalence magnesium ion 1-50mM；Wherein, described slow
Rushing liquid is phosphate buffer, and pH is 5.0-9.0；Preferably, pH is 7.0；It is furthermore preferred that the consumption of each composition is：Phosphate
Buffer 30mM (inorganic phosphate radical is avoided the need for using phosphate buffer), bivalence magnesium ion 5mM.
The present invention with starch or starch derivatives as substrate, add alpha-glucanses phosphorylase, phosphoglucomutase,
Multienzymatic reaction system prepared by glucosephosphate isomerase, 6- phosphoric acid Tagatose epimerase and 6- phosphoric acid Tagatose phosphatase,
Many enzymatic pathways include：By alpha-glucanses phosphorylase, one of starch or starch derivatives glucose unit is converted into
Cori's eater Cori；Cori's eater Cori is converted into by G6P by phosphoglucomutase；By glucose phosphate
G6P is converted into fructose-1, 6-diphosphate by isomerase；By 6- phosphoric acid Tagatose epimerase, fructose-1, 6-diphosphate is converted
For Tagatose -6- phosphoric acid；Tagatose -6- phosphoric acid dephosphorization is converted into by Tagatose and phosphoric acid by 6- phosphoric acid Tagatose phosphatase.By
It is can not be converse in the enzymic catalytic reaction that Tagatose -6- phosphoric acid dephosphorization is converted into by Tagatose by 6- phosphoric acid Tagatose phosphatase
Should, so this enzyme catalysiss system can obtain very high conversion ratio, and high yield pulp1 and high conversion can substantially reduce Tagatose
Separation costs.
Because starch is amylose (20-30%) and the mixture of amylopectin (70-80%).Propping up in amylopectin
Chain is with α -1, and 6 glycosidic bonds are connected with main chain, and alpha-glucanses phosphorylase is unable to decomposing alpha -1,6 glycosidic bonds.In order to improve
The conversion ratio of Tagatose, the present invention adds in multienzymatic reaction system being capable of α -1, the debranching enzyme of 6 glycosidic bonds in starch-splitting
Enzyme isoamylase or pullulanase.Because the amylatic final product of alpha-glucanses phosphorylase is maltose, for profit
With maltose, present invention addition maltose phosphorylase in reaction system further, maltose is decomposed into 1- phosphoric acid Fructus Vitis viniferae
Sugar and glucose；It is furthermore preferred that the present invention further adds polyphosphoric acid and polyphosphoric acid glucokinase in multienzymatic reaction system
Enzyme, converts glucose into G6P, is turned by 6- phosphoric acid Tagatose epimerase and 6- phosphoric acid Tagatose phosphatase
Turn to Tagatose, in starch and its derivant, all of glucose unit is converted into Tagatose, thus improving Tagatose the most at last
Yield and conversion ratio.Here, maltose phosphorylase can be replaced by glucanotransferase, and this enzyme can be by the oligomerization of short chain
Sugar polymerization becomes the oligosaccharide of long-chain, and the oligosaccharide of this long-chain can be re-used by alpha-glucanses phosphorylase, so as to
Enough improve the utilization rate of starch.
The invention also discloses the preparation method of another kind of cellulose Tagatose, comprise the following steps：With cellulose or fibre
The plain derivant of dimension is substrate, adds and contains cellulase, fibrous polysaccharaide phosphorylase (cellodextrin
Phosphorylase, EC 2.4.1.49), cellobiose phosphorylase (cellobiose phosphorylase, EC
2.4.1.20), phosphoglucomutase (EC 5.4.2.2), glucosephosphate isomerase (Phosphoglucose
Isomerase, EC 5.3.1.9), 6- phosphoric acid Tagatose epimerase (Tagatose 6-phosphate 4-
Epimerase) and the multienzyme catalytic materials of 6- phosphoric acid Tagatose phosphatase (Tagatose 6-phosphatase) to set up multienzyme anti-
Answer system, carry out enzymic catalytic reaction, obtain final product.
In order to reach more preferable effect it is preferred that conventionally carrying out point obtained enzymic catalytic reaction product
From, purification.
It is preferably, first cellulose or cellulose derivative and cellulase are mixed on ice-water bath, 4 DEG C of centrifugations, go
Clearly, obtain the mixture of cellulase and cellulose；Then, in the mixture of cellulase and cellulose, add fiber many
Saccharophosphorylase, cellobiose phosphorylase, phosphoglucomutase, glucosephosphate isomerase, 6- phosphoric acid Tagatose are poor
Set up multienzymatic reaction system to isomerase and 6- phosphoric acid Tagatose phosphatase.Wherein, the consumption of cellulose or cellulose derivative
Consumption for 1-500g/L, cellulase is 1-500U/ml；Preferably, the consumption of cellulose or cellulose derivative is 100g/
L, the consumption of cellulase is 10U/ml.This process can remove almost all of glucosidase in commercial cellulose enzyme,
Glucosidase hydrolysis fiber disaccharide can be avoided to generate substantial amounts of glucose, so that main hydrolyzate is cellobiose
And fibrous polysaccharaide.
In described multienzymatic reaction system, the concentration of the mixture of described cellulase and cellulose is 1-500g/L；Institute
The consumption stating fibrous polysaccharaide phosphorylase is 0.1-1000U/mL, and the consumption of described cellobiose phosphorylase is 0.1-1000U/
ML, the consumption of described phosphoglucomutase is 0.1-1000U/mL, and the consumption of described glucosephosphate isomerase is 0.1-
1000U/mL, the consumption of described 6- phosphoric acid Tagatose epimerase is 0.1-1000U/mL, described 6- phosphoric acid Tagatose phosphoric acid
The consumption of enzyme is 0.1-1000U/mL；Preferably, the concentration of the mixture of described cellulase and cellulose is 100g/L；Described
The consumption of fibrous polysaccharaide phosphorylase is 10U/mL, and the consumption of described cellobiose phosphorylase is 10U/mL, described glucose
The consumption of transphosphorylase is 10U/mL, and the consumption of described glucosephosphate isomerase is 10U/mL, described 6- phosphoric acid Tagatose
The consumption of epimerase is 10U/mL, and the consumption of described 6- phosphoric acid Tagatose phosphatase is 10U/mL.Described enzymic catalytic reaction
Condition be：10-80 DEG C of reaction 1-120 hour；It is preferably, 37 DEG C of reaction 48-96 hours, most preferably 72 hours.
In order to improve the yield of cellulose Tagatose further, also include in described multienzyme catalytic materials：Polyphosphoric acid glucose
Kinases (EC 2.7.1.63) and Quadrafos；Preferably, in multienzymatic reaction system, the use of described polyphosphoric acid glucokinase
Measure as 0.1-500U/mL, the consumption of described Quadrafos is 1-100mM；It is furthermore preferred that the use of described polyphosphoric acid glucokinase
Measure as 5U/mL, the consumption of described Quadrafos is 10mM；Wherein, described Quadrafos are preferably polyphosphate sodium.
Also include in multienzymatic reaction system of the present invention：Buffer, inorganic phosphate radical and bivalence magnesium ion；Preferably,
The consumption of each composition is：Buffer 10-500mM, inorganic phosphate radical 2-100mM, bivalence magnesium ion 1-50mM；Wherein, described slow
Rushing liquid is phosphate buffer；It is furthermore preferred that the pH value of described phosphate buffer is 5.0-9.0, most preferably 7.2；Excellent further
Choosing, the consumption of each composition is：Phosphate buffer 30mM (inorganic phosphate radical is avoided the need for using phosphate buffer), divalent magnesium from
Sub- 5mM.
Cellulose derivative of the present invention includes：Cellulose through pretreated product, fibrous polysaccharaide or fiber two
Any one in sugar.Described preprocess method includes：Acid-hydrolysis method, enzyme hydrolysis method or Physical；Preferably, described cellulose
Through pretreated product be cellulose through strong phosphoric acid process after product (Zhang, Y.H.P., et al. (2006). " A
Transition from Cellulose Swelling to Cellulose Dissolution by o-Phosphoric
Acid:Evidence from Enzymatic Hydrolysis and Supramolecular Structure."
Biomacromolecules 7(2):644-648.).
The present invention, with cellulose or cellulose derivative as substrate, adds cellulase, fibrous polysaccharaide phosphorylase, fiber
Two saccharophosphorylases, phosphoglucomutase, glucosephosphate isomerase, 6- phosphoric acid Tagatose epimerase and 6- phosphoric acid
Multienzymatic reaction system prepared by Tagatose phosphatase, and many enzymatic pathways include：Fiber is generated by cellulase hydrolysiss cellulose many
Sugar and cellobiose；By fibrous polysaccharaide phosphorylase, cellobiose phosphorylase by a Portugal of fibrous polysaccharaide or cellobiose
Grape sugar unit is converted into Cori's eater Cori；Cori's eater Cori is converted into by 6- phosphoric acid Fructus Vitis viniferae by phosphoglucomutase
Sugar；G6P is converted into by fructose-1, 6-diphosphate by glucosephosphate isomerase；By 6- phosphoric acid Tagatose epimerase
Fructose-1, 6-diphosphate is converted into Tagatose -6- phosphoric acid；By 6- phosphoric acid Tagatose phosphatase, Tagatose -6- phosphoric acid dephosphorization is converted into
Tagatose.The present invention adds polyphosphoric acid glucokinase and polyphosphoric acid in above-mentioned multienzymatic reaction system further, by cellulose
Final product glucose after hydrolysis is converted into G6P, poorer by glucosephosphate isomerase, 6- phosphoric acid Tagatose
It is converted into Tagatose to isomerase and 6- phosphoric acid Tagatose phosphatase, all of glucose in cellulose and its derivates the most at last
Unit is converted into Tagatose.
The present invention further discloses a kind of preparation method of sucrose Tagatose, comprise the following steps：With sucrose as substrate,
Add and contain sucrose phosphorylase, phosphoglucomutase, glucosephosphate isomerase, 6- phosphoric acid Tagatose epimerism
Enzyme, the multienzyme catalytic materials of 6- phosphoric acid Tagatose phosphatase are set up multienzyme catalystic converter system and are carried out enzymic catalytic reaction, obtain final product.
In order to reach more preferable effect it is preferred that conventionally carrying out point obtained enzymic catalytic reaction product
From, purification.
In described multienzyme catalystic converter system, the concentration of described sucrose is 10-300g/L, described sucrose phosphorylase
Consumption is 0.1-1000U/mL, and the consumption of described phosphoglucomutase is 0.1-1000U/mL, and described glucose phosphate is different
The consumption of structure enzyme is 0.1-1000U/mL, and the consumption of described 6- phosphoric acid Tagatose epimerase is 0.1-1000U/mL, described
The consumption of 6- phosphoric acid Tagatose phosphatase is 0.1-1000U/mL；Preferably, the concentration of described sucrose is 300g/L, described sucrose
The consumption of phosphorylase is 30U/mL, and the consumption of described phosphoglucomutase is 30U/mL, described glucose phosphate isomery
The consumption of enzyme is 30U/mL, and the consumption of described 6- phosphoric acid Tagatose epimerase is 30U/mL, described 6- phosphoric acid Tagatose phosphorus
The consumption of sour enzyme is 30U/mL.The condition of described enzymic catalytic reaction is：20-70 DEG C of reaction 2-100h；It is preferably, 37 DEG C of reactions
10-72h.
Wherein, also include in described multienzymatic reaction system：Buffer, inorganic phosphate radical and bivalence magnesium ion；Preferably, respectively
The consumption of composition is：Buffer 10-500mM, inorganic phosphate radical 2-100mM, bivalence magnesium ion 1-50mM；Wherein, described buffering
Liquid is phosphate buffer, and pH is 5.0-9.0；Preferably, pH is 7.2；It is furthermore preferred that the consumption of each composition is：Phosphate buffer
30mM (inorganic phosphate radical is avoided the need for using phosphate buffer), bivalence magnesium ion 5mM.
In order to improve the yield of sucrose Tagatose further, also include in described multienzyme catalystic converter system：Glucose is different
Structure enzyme, polyphosphoric acid glucokinase and Quadrafos；Preferably, every 10g/L sucrose, the consumption of glucose isomerase is 1U/mL,
The consumption of polyphosphoric acid glucokinase is 5U/mL, and the consumption of Quadrafos is 10mM；Wherein, described Quadrafos are preferably and gather
Sodium phosphate.
The present invention, with sucrose as substrate, adds different containing sucrose phosphorylase, phosphoglucomutase, glucose phosphate
Structure enzyme, 6- phosphoric acid Tagatose epimerase, 6- phosphoric acid Tagatose phosphatase prepare multienzymatic reaction system, many enzymatic pathways bag
Include：One of sucrose glucose unit is converted into by Cori ester by sucrose phosphorylase；Conjugated by glucose phosphate
Cori ester is converted into G-6-P by enzyme；By glucosephosphate isomerase, G-6-P is converted into
D-fructose-6-phosphoric acid；D-fructose-6-phosphoric acid is converted into by Tagatose -6- phosphoric acid by 6- phosphoric acid Tagatose epimerase；By 6- phosphoric acid
Tagatose -6- phosphoric acid dephosphorization is converted into Tagatose and phosphoric acid by Tagatose phosphatase.The present invention is in above-mentioned multienzymatic reaction system
Add glucose isomerase, polyphosphoric acid glucokinase and Quadrafos further, by the end-product glucose after sucrose hydrolysis
It is converted into G-6-P, then by glucosephosphate isomerase, 6- phosphoric acid Tagatose epimerase and 6- phosphoric acid Tagatose
Phosphatase is converted into Tagatose.
The present invention external multienzyme catalytic starch or cellulose and their derivant, or sucrose prepares the side of Tagatose
Method, described enzymic catalytic reaction can be carried out in one or more reactor substeps, preferably carries out in a reactor.
The enzyme that any one of multienzymatic reaction system of the present invention enzyme can have equal function by any one is replaced,
It can also be the mutant enzyme with equal function being obtained by protein engineering house of correction.
During Starch Conversion is tested by the present invention many enzyme catalysiss in vitro for Tagatose, in a reaction system, with solubility
Starch is substrate, adds alpha-glucanses phosphorylase, phosphoglucomutase, glucosephosphate isomerase, 6- phosphoric acid tower lattice
Sugared epimerase and 6- phosphoric acid Tagatose phosphatase carry out catalytic reaction, and the conversion ratio of Tagatose is 37%.And above-mentioned anti-
Answer and in system, add isoamylase, maltose phosphorylase, polyphosphoric acid glucokinase, glucanotransferase and poly- phosphorus further
Sour sodium, the conversion ratio of final Tagatose reaches 72%, and conversion ratio significantly improves.
The present invention many enzyme catalysiss in vitro convert cellulose in Tagatose experiment, in a reaction system, to live again
Amorphous cellulose be substrate, add cellulase, fibrous polysaccharaide phosphorylase, cellobiose phosphorylase, glucose
Transphosphorylase, glucosephosphate isomerase, 6- phosphoric acid Tagatose epimerase and 6- phosphoric acid Tagatose phosphatase are urged
Change reaction, the conversion ratio of Tagatose is 35%.Polyphosphoric acid glucokinase and polyphosphoric acid is it is possible to additionally incorporate in above-mentioned reaction system
Sodium, the conversion ratio of final Tagatose reaches more than 68%, and conversion ratio significantly improves.
The present invention many enzyme catalysiss in vitro are by sucrose inversion for, in Tagatose experiment, in a reaction system, with sucrose being
Substrate, adds sucrose phosphorylase, phosphoglucomutase, glucosephosphate isomerase, 6- phosphoric acid Tagatose epimerism
Enzyme, 6- phosphoric acid Tagatose phosphatase, glucose isomerase, polyphosphoric acid glucokinase and polyphosphate sodium carry out catalytic reaction,
The conversion ratio of whole Tagatose reaches 63%.
Technical solution of the present invention compared with prior art, has the advantages that：
(1) low in raw material price.The present invention with starch or cellulose and their derivant as raw material, or with sucrose
For raw material, rather than using expensive galactose.Therefore, the low production cost of Tagatose, is suitable to large-scale production.
(2) yield of Tagatose is high.The final step of the present invention many enzymic catalytic reactions in vitro, that is, by 6- phosphoric acid Tagatose phosphorus
The enzymic catalytic reaction that Tagatose -6- phosphoric acid dephosphorization is converted into Tagatose by sour enzyme is irreversible process, therefore can obtain very high
Tagatose conversion ratio, thus reducing the separation costs of Tagatose.
(3) preparation method of the present invention makees raw material without Fructose it is not necessary to ATP carries out substrate phosphorylation produces 6- phosphoric acid fruit
Sugar, substantially reduces the cost producing Tagatose.
The term definition that the present invention relates to
Unless otherwise defined, otherwise all technology used herein and scientific terminology all have with of the art
Those of ordinary skill's generally understood identical implication.
Term " enzymic catalytic reaction " means the chemical reaction carrying out under biocatalyzer-enzyme effect.
Brief description
Fig. 1 generates the schematic diagram of many in vitro enzymatic pathways of Tagatose for converted starch；Wherein, IA, isoamylase；
PA, pullulanase；α GP, alpha-glucanses phosphorylase；PGM, phosphoglucomutase；PGI, glucosephosphate isomerase；
T6E, 6- phosphoric acid Tagatose epimerase；T6P, 6- phosphoric acid Tagatose phosphatase；MP, (this enzyme can quilt for maltose phosphorylase
Glucanotransferase is replaced)；PPGK, polyphosphoric acid glucokinase；
Fig. 2 is to be detected with soluble starch as substrate using HPLC, the product after enzymatic reaction, and arrow indication represents
The characteristic peak of Tagatose；
Fig. 3 is the schematic diagram of many in vitro enzymatic pathways that conversion cellulose generates Tagatose；Wherein, Cellulase, fine
The plain enzyme of dimension；CDP, fibrous polysaccharaide phosphorylase；CBP, cellobiose phosphorylase；PGM, phosphoglucomutase；PGI, Portugal
Grape sugar phosphoric acid isomerase；T6E, 6- phosphoric acid Tagatose epimerase；T6P, 6- phosphoric acid Tagatose phosphatase；PPGK, polyphosphoric acid
Glucokinase；
Fig. 4 is the schematic diagram of many in vitro enzymatic pathways that conversion sucrose generates Tagatose；Wherein, SP, saccharose phosphorylation
Enzyme；GI, glucose isomerase；PGM, phosphoglucomutase；PGI, glucosephosphate isomerase；T6E, 6- phosphoric acid Tagatose
Epimerase；T6P, 6- phosphoric acid Tagatose phosphatase；PPGK, polyphosphoric acid glucokinase；
Fig. 5 is to be detected with sucrose as substrate with HPLC, the product after enzymatic reaction, and arrow indication represents Tagatose
Characteristic peak.
Specific embodiment
To further describe the present invention with reference to specific embodiment, advantages of the present invention and feature will be with description and
Apparent.It should be understood that described embodiment is only exemplary, any restriction is not constituted to the scope of the present invention.This area
Technical staff should be understood that lower without departing from the spirit and scope of the present invention can to the details of technical solution of the present invention and
Form is modified or is replaced, but these modifications or replacement each fall within protection scope of the present invention.
1st, experiment material
Soluble starch, soluble starch, ACROS Products, production code member：424490020；
Maltodextrin, ALDRICH Products, production code member 419672；
PET20b carrier, Novagen, Madison, WI；
Escherichia coli expression bacterium BL21 (DE3), Invitrogen, Carlsbad, CA；
Most of enzyme in the present invention (except Tagatose -6- phosphate epimerase, 6- phosphoric acid Tagatose phosphatase, gathers
Phosphoglucokinase and glucanotransferase) can be commercially available in Sigma company；But can all be according to gene engineering method
Obtained by prokaryotic expression；
Cellulase is bought from Sigma company, and production code member is C2730；
Maltose phosphorylase is bought from Sigma company, and production code member is M8284；
Alpha amylase is bought from Sigma company, and production code member is 10065；
Avicel, micro- crystalline cellulose, buy from Sigma company, production code member is 11365.
Starch Conversion is Tagatose by experimental example 1 many enzyme catalysiss in vitro
By an external multienzyme catalyst system and catalyzing Starch Conversion is Tagatose (Fig. 1).These key enzymes include：(1)α-
Glucosan phosphorylase (α GP, EC 2.4.1.1), discharges 1- phosphoric acid Fructus Vitis viniferae from the non-reducing end of starch plus 1 phosphate
Sugar；(2) phosphoglucomutase (PGM, EC 5.4.2.2), catalysis Cori's eater Cori is to G6P；(3) Fructus Vitis viniferae
Sugared phosphoric acid isomerase, G6P is converted into fructose-1, 6-diphosphate；(4) 6- phosphoric acid Tagatose epimerase, by 6- phosphorus
Tart fruit sugar is converted into Tagatose -6- phosphoric acid；(5) Tagatose -6- phosphoric acid dephosphorization is converted into tower lattice by 6- phosphoric acid Tagatose phosphatase
Sugar and phosphoric acid.
In the present invention, alpha-glucanses phosphorylase derives from Thermotoga maritima, volume on KEGG for the gene
Number be TM1168；Phosphoglucomutase also derives from Thermotoga maritima, and numbering on KEGG for the gene is
TM0769；Glucosephosphate isomerase derives from Clostridium thermocellum, and numbering on KEGG for the gene is
Cthe0217；6- phosphoric acid Tagatose epimerase comes from Agrobacterium fabrum, numbering on KEGG for the gene
For Atu3167；6- phosphoric acid Tagatose phosphatase derives from Archaeoglobus fulgidus, and numbering on KEGG for the gene is
AF_0444, these genomic DNAs all can obtain from the official website (www.atcc.org) of ATCC.This five genes are respectively
Obtained by PCR from corresponding genomic DNA with different primers, and pass through Simple Cloning (You, C., et al.
(2012)."Simple Cloning via Direct Transformation of PCR Product(DNA Multimer)
to Escherichia coli and Bacillus subtilis."Appl.Environ.Microbiol.78(5):1593-
1595.) method is cloned into pET20b carrier and (in (Novagen, Madison, WI), obtains corresponding expression vector pET20b-
Tm α GP, pET20b-TmPGM, pET20b-CtPGI, pET20b-AtaT6E and pET20b-AfT6P.This five plasmids all convert
To escherichia coli expression bacterium BL21 (DE3) (Invitrogen, Carlsbad, CA), and carry out protein expression and purification.
The divalent magnesium of the phosphate buffer containing 30mM (pH 7.0) in one 0.75 milliliter of reaction system, 5mM from
Son, the consumption of described alpha-glucanses phosphorylase is 10U/mL, and the consumption of described phosphoglucomutase is 10U/mL, described
The consumption of glucosephosphate isomerase is 10U/mL, and the consumption of described 6- phosphoric acid Tagatose epimerase is 10U/mL, described
The consumption of 6- phosphoric acid Tagatose phosphatase is 10U/mL, and the soluble starch of 100g/L carries out catalytic reaction at 37 DEG C, reacts 24
Individual hour.
According to the difference of retention time, HPLC can be used to distinguish Tagatose in reactant liquor, glucose, 1- phosphoric acid Fructus Vitis viniferae
Sugar or G6P；And Tagatose can be carried out with quantitation, the concentration of Tagatose and Tagatose characteristic peak in HPLC
Intensity is directly proportional；The mobile phase of HPLC is the dilute sulfuric acid of 5mM.
After reaction terminates, the final concentration of final Tagatose (Fig. 2) is 37g/L, and conversion ratio is 37%.
Starch Conversion is Tagatose by experimental example 2 many enzyme catalysiss in vitro
Alpha-glucanses phosphorylase, phosphoglucomutase, glucosephosphate isomerase, 6- phosphoric acid Tagatose difference are to different
The preparation of structure enzyme and 6- phosphoric acid Tagatose phosphatase is with experimental example 1.
The divalent magnesium of the phosphate buffer containing 30mM (pH 7.0) in one 0.75 milliliter of reaction system, 5mM from
Son, the consumption of described alpha-glucanses phosphorylase is 10U/mL, and the consumption of described phosphoglucomutase is 10U/mL, described
The consumption of glucosephosphate isomerase is 10U/mL, and the consumption of described 6- phosphoric acid Tagatose epimerase is 10U/mL, described
The consumption of 6- phosphoric acid Tagatose phosphatase is 10U/mL, and the soluble starch of 100g/L carries out catalytic reaction at 20 DEG C, reacts 24
Individual hour.
After reaction terminates, the final concentration of final Tagatose is 16g/L, and conversion ratio is 16%.
Starch Conversion is Tagatose by experimental example 3 many enzyme catalysiss in vitro
Alpha-glucanses phosphorylase, phosphoglucomutase, glucosephosphate isomerase, 6- phosphoric acid Tagatose difference are to different
The preparation of structure enzyme and 6- phosphoric acid Tagatose phosphatase is with experimental example 1.
The divalent magnesium of the phosphate buffer containing 30mM (pH 7.0) in one 0.75 milliliter of reaction system, 5mM from
Son, the consumption of described alpha-glucanses phosphorylase is 10U/mL, and the consumption of described phosphoglucomutase is 10U/mL, described
The consumption of glucosephosphate isomerase is 10U/mL, and the consumption of described 6- phosphoric acid Tagatose epimerase is 10U/mL, described
The consumption of 6- phosphoric acid Tagatose phosphatase is 10U/mL, and the soluble starch of 100g/L carries out catalytic reaction at 50 DEG C, reacts 24
Individual hour.
After reaction terminates, the final concentration of final Tagatose is 8g/L, and conversion ratio is 8%.
Experimental example 4 passes through process optimization and adds and promote amylolytic enzyme, using many enzyme catalysiss in vitro by Starch Conversion
For Tagatose
Because starch is that have branched chain, adopt merely alpha-glucanses phosphorylase can not completely by Starch Hydrolysis because
Alpha-glucanses phosphorylase only can act on α-Isosorbide-5-Nitrae glycosidic bond, and branched chain is with α -1, and 6 glycosidic bonds are connected with main chain.This
Need to add isoamylase (isoamylase, EC 3.2.1.68) hydrolyzing alpha -1,6 glycosidic bonds.Finally, starch is by both enzyme water
The final product of solution is maltose and glucose, in order to these final products are converted into Tagatose in addition it is also necessary to add maltose
Phosphorylase (maltose phosphorylase, EC 2.4.1.8) and polyphosphoric acid glucokinase (polyphosphate
Glucokinase, EC 2.7.1.63).
In the present invention, isoamylase derives from Sulfolobus tokodaii, and numbering on KEGG for the gene is
ST0928, the genomic DNA of this bacterial strain is German Albert-Ludwigs-The Georg of Freiburg
Fuchs professor's friendship provides.Polyphosphoric acid glucokinase derives from Thermobifida fusca, numbering on KEGG for the gene
For Tfu1811, the genomic DNA of this bacterial strain is David Wilson professor's friendship offer of Cornell Univ USA.Glucosan
Transferring enzyme derives from Thermococcus litoralis, and numbering on KEGG for the gene is OCC_10078, the gene of this bacterial strain
Group DNA can obtain from the official website (www.atcc.org) of ATCC.These three genes respectively with different primers from corresponding
Genomic DNA in obtained by PCR, and by Simple Cloning (You, C., et al. (2012). " Simple
Cloning via Direct Transformation of PCR Product(DNA Multimer)to Escherichia
coli and Bacillus subtilis."Appl.Environ.Microbiol.78(5):1593-1595.) method gram
In the grand carrier to pET20b, obtain corresponding expression vector pET20b-StIA, pET20b-TfuPPGK and pET20b-Ti4GT,
These three plasmids all convert to escherichia coli expression bacterium BL21 (DE3), and carry out protein expression and purification.
Alpha-glucanses phosphorylase, phosphoglucomutase, glucosephosphate isomerase, 6- phosphoric acid Tagatose difference are to different
The preparation of structure enzyme and 6- phosphoric acid Tagatose phosphatase is with experimental example 1；Maltose phosphorylase is bought from Sigma company, and product is compiled
Number be M8284.
The divalent magnesium of the phosphate buffer containing 30mM (pH 7.0) in one 0.75 milliliter of reaction system, 5mM from
Son, the consumption of described alpha-glucanses phosphorylase is 10U/mL, and the consumption of described phosphoglucomutase is 10U/mL, described
The consumption of glucosephosphate isomerase is 10U/mL, and the consumption of described 6- phosphoric acid Tagatose epimerase is 10U/mL, described
The consumption of 6- phosphoric acid Tagatose phosphatase is 10U/mL, the isoamylase of 1U/mL, the maltose phosphorylase of 1U/mL, 1U/mL
Polyphosphoric acid glucokinase, the glucanotransferase of 1U/ml, 10mM polyphosphate sodium, the soluble starch of 100g/L, at 37 DEG C
Carry out catalytic reaction, react 40 hours.The detection method of Tagatose with experimental example 1, final Tagatose (Fig. 2) final concentration of
72g/L, conversion ratio has reached 72%.
Experimental example 5 passes through process optimization and adds and promote amylolytic enzyme, using many enzyme catalysiss in vitro by Starch Conversion
For Tagatose
Alpha-glucanses phosphorylase, phosphoglucomutase, glucosephosphate isomerase, 6- phosphoric acid Tagatose difference are to different
The preparation of structure enzyme and 6- phosphoric acid Tagatose phosphatase is with experimental example 1；The preparation of polyphosphoric acid glucokinase is with experimental example 4, Pu Lu
Blue enzyme (pullulanase, EC 3.2.1.41) is bought from Sigma company, and production code member is P1067；Maltose phosphorylase from
Sigma company buys, and production code member is M8284.
The divalent magnesium of the phosphate buffer containing 30mM (pH 7.0) in one 0.75 milliliter of reaction system, 5mM from
Son, the consumption of described alpha-glucanses phosphorylase is 10U/mL, and the consumption of described phosphoglucomutase is 10U/mL, described
The consumption of glucosephosphate isomerase is 10U/mL, and the consumption of described 6- phosphoric acid Tagatose epimerase is 10U/mL, described
The consumption of 6- phosphoric acid Tagatose phosphatase is 10U/mL, the maltose phosphorylase of 1U/mL, the pullulanase of 1U/mL, 1U/mL
Polyphosphoric acid glucokinase, the glucanotransferase of 1U/ml, 10mM polyphosphate sodium, the soluble starch of 100g/L, at 37 DEG C
Carry out catalytic reaction, react 40 hours.The detection method of Tagatose is with experimental example 1, the final concentration of 73g/ of final Tagatose
L, conversion ratio has reached 73%.
Subsequently add a small amount of alpha amylase to promote the hydrolysis of residue starch in reaction system, improve the yield of Tagatose,
The consumption of alpha amylase is 0.1U/ml, continues to react 24 hours at 37 DEG C, the final concentration of 88g/L of final Tagatose (Fig. 2), turns
Rate has reached 88%.
Maltodextrin is converted into Tagatose to experimental example 6 by many enzyme catalysiss in vitro
Alpha-glucanses phosphorylase, phosphoglucomutase, glucosephosphate isomerase, 6- phosphoric acid Tagatose difference are to different
The preparation of structure enzyme and 6- phosphoric acid Tagatose phosphatase is with experimental example 1；Isoamylase, the preparation of polyphosphoric acid glucokinase are with experiment
Example 4, maltose phosphorylase is bought from Sigma company, and production code member is M8284.
The divalent magnesium of the phosphate buffer containing 30mM (pH 7.0) in one 0.75 milliliter of reaction system, 5mM from
Son, the consumption of described alpha-glucanses phosphorylase is 10U/mL, and the consumption of described phosphoglucomutase is 10U/mL, described
The consumption of glucosephosphate isomerase is 10U/mL, and the consumption of described 6- phosphoric acid Tagatose epimerase is 10U/mL, described
The consumption of 6- phosphoric acid Tagatose phosphatase is 10U/mL, the isoamylase of 1U/mL, the maltose phosphorylase of 1U/mL, 1U/mL
Polyphosphoric acid glucokinase, the glucanotransferase of 1U/ml, 10mM polyphosphate sodium, the maltodextrin (ALDRICH of 100g/L
Products, production code member 419672), carry out catalytic reaction at 37 DEG C, react 40 hours.The detection method of Tagatose is with real
Test example 1, the final concentration of 78g/L of final Tagatose, conversion ratio has reached 78%.
Many enzyme catalysiss convert cellulose into Tagatose to experimental example 7 in vitro
Fig. 3 is shown in by the schematic diagram that an external multienzyme catalyst system and catalyzing converts cellulose into Tagatose.
Cellulase comes from the product of Sigma company, and production code member is C2730；Phosphoglucomutase, Fructus Vitis viniferae
The preparation of sugared phosphoric acid isomerase, 6- phosphoric acid Tagatose epimerase and 6- phosphoric acid Tagatose phosphatase is with experimental example 1.
Fibrous polysaccharaide phosphorylase (Cthe_2989) and cellobiose phosphorylase (Cthe_0275) are all derived from
Clostridium thermocellum.This two genes respectively with different primers from corresponding genomic DNA (genome
DNA can from the official website of ATCC (www.atcc.org/) upper obtain) and in obtain by PCR, and by Simple Cloning
(You,C.,et al.(2012)."Simple Cloning via Direct Transformation of PCR Product
(DNA Multimer)to Escherichia coli and Bacillus subtilis."
Appl.Environ.Microbiol.78(5):Method 1593-1595.) is cloned in pET20b carrier, obtains corresponding table
Reach carrier pET20b-CthCDP and pET20b-CthCBP.This two plasmids all convert to escherichia coli expression bacterium BL21 (DE3)
In, and carry out protein expression and purification.
It is substrate that this experiment adopts micro- crystalline cellulose (Avicel).First by business-like cellulase (10U/ml) and
Cellulose (100g/L) mixes on ice-water bath, is positioned over 5 minutes in ice-water bath, is centrifuged at 4 DEG C, removes supernatant.It is precipitated as fiber
Element and the mixture of the cellulase being combined with cellulose.This process can remove almost all of in commercial cellulose enzyme
Glucosidase, so can avoid glucosidase hydrolysis fiber disaccharide to generate substantial amounts of glucose, so that main water
Solution product is cellobiose and fibrous polysaccharaide.
The divalent magnesium of the phosphate buffer containing 30mM (pH 7.2) in one 0.75 milliliter of reaction system, 5mM from
Son, the fibrous polysaccharaide phosphorylase of 10U/mL, 10U/mL cellobiose phosphorylase, the phosphoglucomutase of 10U/mL,
The glucosephosphate isomerase of 10U/mL, the 6- phosphoric acid Tagatose epimerase of 10U/mL, the 6- phosphoric acid Tagatose of 10U/mL
Phosphatase, the cellulose as above of 100g/L and the mixture of cellulase, carry out catalytic reaction at 37 DEG C, react 72
Hour.With experimental example 1, the final concentration of final Tagatose is 14g/L to the detection method of Tagatose, and conversion ratio is 14%.
Many enzyme catalysiss convert cellulose into Tagatose to experimental example 8 in vitro
Cellulase comes from the product of Sigma company, and production code member is C2730；Phosphoglucomutase, Fructus Vitis viniferae
Sugared phosphoric acid isomerase, 6- phosphoric acid Tagatose epimerase, the preparation of 6- phosphoric acid Tagatose phosphatase is with experimental example 1；Fiber is many
The preparation of saccharophosphorylase and cellobiose phosphorylase is with experimental example 7.
This experiment using live again amorphous cellulose (Regenerated Amorphous cellulose (RAC),
This is product after strong phosphoric acid process for the Avicel) (Zhang, Y.H.P., et al. (2006). " A Transition
from Cellulose Swelling to Cellulose Dissolution by o-Phosphoric Acid:
Evidence from Enzymatic Hydrolysis and Supramolecular Structure."
Biomacromolecules 7(2):644-648.) it is substrate.First by business-like cellulase (10U/ml) and this fiber
Plain (100g/L) mixes on ice-water bath, is positioned over 5 minutes in ice-water bath, is centrifuged at 4 DEG C, removes supernatant.Be precipitated as cellulose and
The mixture of the cellulase being combined with cellulose.
The divalent magnesium of the phosphate buffer containing 30mM (pH 7.2) in one 0.75 milliliter of reaction system, 5mM from
Son, the fibrous polysaccharaide phosphorylase of 10U/mL, 10U/mL cellobiose phosphorylase, the phosphoglucomutase of 10U/mL,
The glucosephosphate isomerase of 10U/mL, the 6- phosphoric acid Tagatose epimerase of 10U/mL, the 6- phosphoric acid Tagatose of 10U/mL
Phosphatase, the cellulose as above of 100g/L and the mixture of cellulase, carry out catalytic reaction at 37 DEG C, react 72
Hour.With experimental example 1, the final concentration of final Tagatose is 35g/L to the detection method of Tagatose, and conversion ratio is 35%.
Many enzyme catalysiss convert cellulose into Tagatose to experimental example 9 in vitro
Cellulase comes from the product of Sigma company, and production code member is C2730；Phosphoglucomutase, Fructus Vitis viniferae
Sugared phosphoric acid isomerase, 6- phosphoric acid Tagatose epimerase, the preparation of 6- phosphoric acid Tagatose phosphatase are with experimental example 1；Fiber is many
The preparation of saccharophosphorylase and cellobiose phosphorylase is with experimental example 7.
The divalent magnesium of the phosphate buffer containing 30mM (pH 7.2) in one 0.75 milliliter of reaction system, 5mM from
Son, the fibrous polysaccharaide phosphorylase of 10U/mL, 10U/mL cellobiose phosphorylase, the phosphoglucomutase of 10U/mL,
The glucosephosphate isomerase of 10U/mL, the 6- phosphoric acid Tagatose epimerase of 10U/mL, the 6- phosphoric acid Tagatose of 10U/mL
Phosphatase, the cellulose of experimental example 8 of 100g/L and the mixture of cellulase, carry out catalytic reaction at 25 DEG C, react 24
Hour.The final concentration of final Tagatose is 29g/L, and conversion ratio is 29%.
Many enzyme catalysiss convert cellulose into Tagatose to experimental example 10 in vitro
Cellulase comes from the product of Sigma company, and production code member is C2730；Phosphoglucomutase, Fructus Vitis viniferae
Sugared phosphoric acid isomerase, 6- phosphoric acid Tagatose epimerase, the preparation of 6- phosphoric acid Tagatose phosphatase are with experimental example 1；Fiber is many
The preparation of saccharophosphorylase and cellobiose phosphorylase is with experimental example 7.
The divalent magnesium of the phosphate buffer containing 30mM (pH 7.2) in one 0.75 milliliter of reaction system, 5mM from
Son, the fibrous polysaccharaide phosphorylase of 10U/mL, 10U/mL cellobiose phosphorylase, the phosphoglucomutase of 10U/mL,
The glucosephosphate isomerase of 10U/mL, the 6- phosphoric acid Tagatose epimerase of 10U/mL, the 6- phosphoric acid Tagatose of 10U/mL
Phosphatase, the cellulose of experimental example 8 of 100g/L and the mixture of cellulase, carry out catalytic reaction at 50 DEG C, react 24
Hour.The final concentration of final Tagatose is 9g/L, and conversion ratio is 9%.
Many enzyme catalysiss convert cellulose into Tagatose to experimental example 11 in vitro
Because the final product after cellulose hydrolysis is glucose, in order to be translated into Tagatose in addition it is also necessary to add poly-
Phosphoglucokinase and polyphosphoric acid.
Cellulase comes from the product of Sigma company, and production code member is C2730；Phosphoglucomutase, Fructus Vitis viniferae
Sugared phosphoric acid isomerase, 6- phosphoric acid Tagatose epimerase, the preparation of 6- phosphoric acid Tagatose phosphatase are with experimental example 1；Polyphosphoric acid
The preparation of glucokinase is with experimental example 4；Fibrous polysaccharaide phosphorylase, the preparation of cellobiose phosphorylase are with experimental example 7.
The divalent magnesium of the phosphate buffer containing 30mM (pH 7.2) in one 0.75 milliliter of reaction system, 5mM from
Son, the fibrous polysaccharaide phosphorylase of 10U/mL, 10U/mL cellobiose phosphorylase, the phosphoglucomutase of 10U/mL,
The glucosephosphate isomerase of 10U/mL, the 6- phosphoric acid Tagatose epimerase of 10U/mL, the 6- phosphoric acid Tagatose of 10U/mL
Phosphatase, the cellulose of experimental example 8 of 100g/L and the mixture of cellulase, the polyphosphoric acid glucokinase of 5U/mL, 10mM
Polyphosphate sodium, carries out catalytic reaction at 37 DEG C, reacts 72 hours.The detection method of Tagatose is with experimental example 1, final Tagatose
Final concentration of 68g/L, conversion ratio reached 68%.
Sucrose inversion is Tagatose by experimental example 12 many enzyme catalysiss in vitro
Sucrose inversion is shown in for the schematic diagram of Tagatose by Fig. 4 by an external multienzyme catalyst system and catalyzing.
Sucrose phosphorylase is derived from Thermoanaerobacterium thermosaccharolyticum JW/
SL-YS485, the enzyme of this coded by said gene is WP_015312040.1 in the numbering of ncbi database.This gene primer from
Obtained by PCR in corresponding genomic DNA, and pass through Simple Cloning (You, C., et al. (2012), " Simple
Cloning via Direct Transformation of PCR Product(DNA Multimer)to Escherichia
coli and Bacillus subtilis."Appl.Environ.Microbiol.78(5):1593-1595.)) method gram
In the grand carrier to pET20b, obtain corresponding expression vector pET20b-SP.This plasmid all converts to escherichia coli expression bacterium
In BL21 (DE3), and carry out protein expression and purification (Qi P, You C, Zhang YHP:One-Pot Enzymatic
Conversion of Sucrose to Synthetic Amylose by using Enzyme Cascades.ACS
Catal.2014,4:1311-1317.)
Glucose isomerase comes from the product of Sigma company, and production code member is G4166；Phosphoglucomutase,
The preparation of glucosephosphate isomerase, 6- phosphoric acid Tagatose epimerase and 6- phosphoric acid Tagatose phosphatase is with experimental example 1.Poly-
Phosphoglucokinase derives from Thermobifida fusca YX, and numbering on KEGG for the gene is Tfu1811；This base
Because being obtained by PCR from corresponding genomic DNA with primer, this plasmid all converts to escherichia coli expression bacterium BL21
(DE3) in, and it is cloned in pET20b carrier by the method for Simple Cloning (You, C., et al. (2012)), obtain
Obtain corresponding expression vector.This plasmid all converts to escherichia coli expression bacterium BL21 (DE3), and carry out protein expression with
Purification (Liao HH, Myung S, Zhang Y-HP.2012.One-step purification and immobilization
of thermophilic polyphosphate glucokinase from Thermobifida fusca YX:glucose-
6-phosphate generation without ATP Appl.Microbiol.Biotechnol.93:1109-1117).Portugal
Grape sugar transphosphorylase, glucosephosphate isomerase, 6- phosphoric acid Tagatose epimerase, the system of 6- phosphoric acid Tagatose phosphatase
For with experimental example 1.
The phosphate buffer containing 30mM (pH 7.2), 300g/L sucrose, 5mM in one 0.75 milliliter of reaction system
Bivalence magnesium ion, the sucrose phosphorylase of 30U/mL, the phosphoglucomutase of 30U/mL, the glucose phosphate of 30U/mL
Isomerase, the 6- phosphoric acid Tagatose epimerase of 30U/mL, the 6- phosphoric acid Tagatose phosphatase of 30U/mL, the Fructus Vitis viniferae of 30U/mL
Sugared isomerase, the polyphosphoric acid glucokinase of 150U/mL, 300mM polyphosphate sodium, carry out catalytic reaction at 37 DEG C, react 72
Hour.The final concentration of 188g/L of final Tagatose, conversion ratio has reached 63%, HPLC schemes as figure 5 illustrates.
Claims (18)
1. a kind of preparation method of Tagatose is it is characterised in that with starch or starch derivatives as substrate, adding and gather containing α-Portugal
Saccharophosphorylase, phosphoglucomutase, glucosephosphate isomerase, 6- phosphoric acid Tagatose epimerase, 6- phosphoric acid tower
Lattice Sugar-phosphatase and inorganic phosphate radical ion, set up multienzyme molecule machine and carry out multienzyme catalytic reaction, obtain final product Tagatose.
2. according to the preparation method described in claim 1 it is characterised in that：In described multienzymatic reaction system, described substrate dense
Spend for 1-500g/L, the consumption of described alpha-glucanses phosphorylase is 0.1-1000U/mL, described phosphoglucomutase
Consumption is 0.1-1000U/mL, and the consumption of described glucosephosphate isomerase is 0.1-1000U/mL, described 6- phosphoric acid Tagatose
The consumption of epimerase is 0.1-1000U/mL, and the consumption of described 6- phosphoric acid Tagatose phosphatase is 0.1-1000U/mL, institute
The concentration stating inorganic phosphate radical is 2-100mM；
Preferably, the concentration of described substrate is 100g/L, and the consumption of described alpha-glucanses phosphorylase is 10U/mL, described Fructus Vitis viniferae
The consumption of sugared transphosphorylase is 10U/mL, and the consumption of described glucosephosphate isomerase is 10U/mL, described 6- phosphoric acid tower lattice
The consumption of sugared epimerase is 10U/mL, and the consumption of described 6- phosphoric acid Tagatose phosphatase is 10U/mL, described inorganic phosphate
The concentration of root is 30mM.
3. according to the preparation method described in claim 1 it is characterised in that：The condition of described enzymic catalytic reaction is：10-90 DEG C anti-
Answer 1-100 hour；It is preferably, 37 DEG C of reaction 24-40 hours.
4. according to the preparation method described in claim 1 it is characterised in that：Described starch derivatives include boiling starch,
The mixture that any one or more in amylodextrin, maltodextrin, Fructus Hordei Germinatus polysaccharide or maltose forms according to arbitrary proportion.
5. according to the preparation method described in claim 1 it is characterised in that：Also include in described multienzyme catalytic materials (1), (2) or
Any one of (3)：(1) any one of starch debranching enzyme or maltose phosphorylase or two kinds；(2) starch-debranching
Any one of enzyme or glucanotransferase or two kinds；(3) starch debranching enzyme, maltose phosphorylase or glucosan transfer
Any one in enzyme or three kinds；
Preferably, in multienzymatic reaction system, the consumption of described starch debranching enzyme is 0.1-500U/mL, described maltose phosphorus
The consumption of acidifying enzyme is 0.1-500U/mL, and the consumption of described glucanotransferase is 0.1-500U/mL；
It is furthermore preferred that the consumption of described starch debranching enzyme is 1U/mL, the consumption of described maltose phosphorylase is 1U/mL, institute
The consumption stating glucanotransferase is 1U/mL；
Wherein, described starch debranching enzyme is any one in isoamylase or pullulanase or two kinds.
6. according to the preparation method described in claim 1 or 5 it is characterised in that：Also include in described multienzyme catalytic materials：Polyphosphoric acid
Glucokinase and Quadrafos；
Preferably, in multienzymatic reaction system, the consumption of described polyphosphoric acid glucokinase is 0.1-500U/mL, described poly- phosphorus
The consumption of hydrochlorate is 1-100mM；It is furthermore preferred that the consumption of described polyphosphoric acid glucokinase is 1U/mL, described Quadrafos
Consumption is 10mM；
Wherein, described Quadrafos are preferably polyphosphate sodium.
7. according to the preparation method described in claim 1 or 5 it is characterised in that：α starch is also included in described multienzyme catalytic materials
Enzyme；Preferably, in multienzymatic reaction system, the consumption of described alpha amylase is 0.01-100U/ml, more preferably 0.1U/ml.
8. according to the preparation method described in claim 1 it is characterised in that：Also include in described multienzymatic reaction system：Buffer
With bivalence magnesium ion；
Preferably, the consumption of each composition is：Buffer 10-500mM, bivalence magnesium ion 1-50mM；
Wherein, described buffer is phosphate buffer, and pH is 5.0-9.0；Preferably, pH is 7.0；
It is furthermore preferred that the consumption of each composition is：Phosphate buffer 30mM, bivalence magnesium ion 5mM.
9. a kind of preparation method of Tagatose is it is characterised in that comprise the following steps：With cellulose or cellulose derivative as bottom
Thing, adds and contains cellulase, fibrous polysaccharaide phosphorylase, cellobiose phosphorylase, phosphoglucomutase, glucose
Phosphoric acid isomerase, 6- phosphoric acid Tagatose epimerase, the multienzyme catalytic materials of 6- phosphoric acid Tagatose phosphatase and inorganic phosphate radical
Set up multienzymatic reaction system, carry out enzymic catalytic reaction, obtain final product Tagatose.
10. according to the preparation method described in claim 9 it is characterised in that：First by cellulose or cellulose derivative and fiber
Plain enzyme mixing, centrifugation, remove supernatant, obtain the mixture of cellulase and cellulose；Add fibrous polysaccharaide phosphorylase, fiber
Two saccharophosphorylases, phosphoglucomutase, glucosephosphate isomerase, 6- phosphoric acid Tagatose epimerase, 6- phosphoric acid
Tagatose phosphatase and inorganic phosphate radical, set up multienzyme molecule machine reaction system；
Wherein, the consumption of cellulose or cellulose derivative be 1-500g/L, cellulase consumption be 1-500U/ml；Preferably
, the consumption of cellulose or cellulose derivative is 100g/L, and the consumption of cellulase is 10U/ml.
11. according to the preparation method described in claim 9 or 10 it is characterised in that：In described multienzymatic reaction system, described fibre
The concentration of the mixture of the plain enzyme of dimension and cellulose is 1-500g/L；The consumption of described fibrous polysaccharaide phosphorylase is 0.1-1000U/
ML, the consumption of described cellobiose phosphorylase is 0.1-1000U/mL, and the consumption of described phosphoglucomutase is 0.1-
1000U/mL, the consumption of described glucosephosphate isomerase is 0.1-1000U/mL, described 6- phosphoric acid Tagatose epimerase
Consumption be 0.1-1000U/mL, the consumption of described 6- phosphoric acid Tagatose phosphatase is 0.1-1000U/mL, described inorganic phosphate
The concentration of root is 2-100mM；
Preferably, the concentration of the mixture of described cellulase and cellulose is 100g/L；Described fibrous polysaccharaide phosphorylase
Consumption is 10U/mL, and the consumption of described cellobiose phosphorylase is 10U/mL, and the consumption of described phosphoglucomutase is
10U/mL, the consumption of described glucosephosphate isomerase is 10U/mL, and the consumption of described 6- phosphoric acid Tagatose epimerase is
10U/mL, the consumption of described 6- phosphoric acid Tagatose phosphatase is 10U/mL, and the concentration of described inorganic phosphate radical is 30mM.
12. according to the preparation method described in claim 9 it is characterised in that：The condition of described enzymic catalytic reaction is：10-80℃
Reaction 1-120 hour；It is preferably, 37 DEG C of reaction 48-96 hours；
Described cellulose derivative includes cellulose through pretreated product；Preferably, described cellulose derivative is fine
Any one in dimension polysaccharide or cellobiose；
Described preprocess method includes acid-hydrolysis method, enzyme hydrolysis method or Physical；Preferably, described cellulose is after pretreatment
Product be cellulose through strong phosphoric acid process after product.
13. according to the preparation method described in claim 9 it is characterised in that：Also include in described multienzyme catalytic materials：Polyphosphoric acid Portugal
Glucokinase and Quadrafos；
Preferably, in multienzymatic reaction system, the consumption of described polyphosphoric acid glucokinase is 0.1-500U/mL, described poly- phosphorus
The consumption of hydrochlorate is 1-100mM；It is furthermore preferred that the consumption of described polyphosphoric acid glucokinase is 5U/mL, described Quadrafos
Consumption is 10mM；
Wherein, described Quadrafos are preferably polyphosphate sodium.
14. according to the preparation method described in claim 9 it is characterised in that：Also include in described multienzymatic reaction system：Buffering
Liquid, bivalence magnesium ion；
Preferably, the consumption of each composition is：Buffer 10-500mM, bivalence magnesium ion 1-50mM；
Wherein, preferred buffer is phosphate buffer；The pH value of described phosphate buffer is 5.0-9.0, preferably 7.2；
It is furthermore preferred that the consumption of each composition is：Phosphate buffer 30mM, bivalence magnesium ion 5mM.
A kind of 15. preparation methoies of Tagatose are it is characterised in that comprise the following steps：With sucrose as substrate, add and contain sucrose
Phosphorylase, phosphoglucomutase, glucosephosphate isomerase, 6- phosphoric acid Tagatose epimerase, 6- phosphoric acid tower lattice
The multienzyme catalytic materials of Sugar-phosphatase and inorganic phosphate radical are set up multienzyme molecule machine reaction system and are carried out enzymic catalytic reaction, obtain final product tower
Lattice sugar.
16. according to the preparation method described in claim 15 it is characterised in that in described multienzyme catalystic converter system, described
The concentration of sucrose is 10-300g/L, and the consumption of described sucrose phosphorylase is 0.1-1000U/mL, described glucose phosphate displacement
The consumption of enzyme is 0.1-1000U/mL, and the consumption of described glucosephosphate isomerase is 0.1-1000U/mL, described 6- phosphoric acid tower
The consumption of lattice sugar epimerase is 0.1-1000U/mL, and the consumption of described 6- phosphoric acid Tagatose phosphatase is 0.1-1000U/
ML, the concentration of described inorganic phosphate radical is 2-100mM；
Preferably, the concentration of described sucrose is 300g/L, and the consumption of described sucrose phosphorylase is 30U/mL, described glucose phosphorus
The consumption of sour mutase is 30U/mL, and the consumption of described glucosephosphate isomerase is 30U/mL, and described 6- phosphoric acid Tagatose is poor
It is 30U/mL to the consumption of isomerase, the consumption of described 6- phosphoric acid Tagatose phosphatase is 30U/mL, described inorganic phosphate radical
Concentration is 30mM；
The condition of described enzymic catalytic reaction is：20-70 DEG C of reaction 2-100h；It is preferably, 37 DEG C of reaction 10-72h.
17. according to the preparation method described in claim 15 it is characterised in that also including in described multienzymatic reaction system：Buffering
Liquid and bivalence magnesium ion；
Preferably, the consumption of each composition is：Buffer 10-500mM, bivalence magnesium ion 1-50mM；
Wherein, preferred buffer is phosphate buffer, and pH is 5.0-9.0；Preferably, pH is 7.2；
It is furthermore preferred that the consumption of each composition is：Phosphate buffer 30mM, bivalence magnesium ion 5mM.
18. according to the preparation method described in claim 15 it is characterised in that also including in described multienzyme catalystic converter system：
Glucose isomerase, polyphosphoric acid glucokinase and Quadrafos；
Preferably, every 10g/L sucrose, the consumption of glucose isomerase is 1U/mL, and the consumption of polyphosphoric acid glucokinase is 5U/
ML, the consumption of Quadrafos is 10mM；
Wherein, described Quadrafos are preferably polyphosphate sodium.
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