CN106399427B - The preparation method of Tagatose - Google Patents
The preparation method of Tagatose Download PDFInfo
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- CN106399427B CN106399427B CN201610937656.5A CN201610937656A CN106399427B CN 106399427 B CN106399427 B CN 106399427B CN 201610937656 A CN201610937656 A CN 201610937656A CN 106399427 B CN106399427 B CN 106399427B
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- 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 kind of preparation methods of Tagatose, belong to the multienzyme catalysis preparation field of Tagatose.The present invention is using starch, cellulose or their derivative or using sucrose as substrate, in a multienzymatic reaction system, by external multienzyme molecule machine efficient catalytic by substrate Efficient Conversion be Tagatose.The present invention can promote the enzyme of starch, cellulose or sucrose hydrolysis by addition and using the enzyme of by-product glucose, be obviously improved the transformation efficiency of raw material and the yield of Tagatose.The method of the present invention prepares that Tagatose yield is high, raw material is inexpensive, production cost is low, can realize the large-scale production of Tagatose.
Description
Technical field
The present invention relates to a kind of preparation methods of Tagatose more particularly to a kind of external multienzyme to be catalyzed starch or cellulose
And its method that derivative is converted into Tagatose, belong to the enzymatic preparation field of Tagatose.
Background technology
Tagatose (D-Tagatose) is a kind of naturally occurring rare monosaccharide, is the ketose form of galactolipin, fructose
Epimer.Sweet taste characteristic is similar to sucrose, and the heat generated is only the one third of sucrose, so being referred to as low-heat
Measure sweetener.Tagatose have lower calorific value, zero glycemic index, blood glucose passivation, without saprodontia, prebiotic function and
The excellent nutritive peculiarity such as antioxidant activity.Natural Tagatose is primarily present in the dairy products such as yogurt, milk powder.Tagatose has
Four big functions：Low energy, it is hypoglycemic, improve intestinal flora and anti-caries tooth (Oh D-K:Tagatose:properties,
applications,and biotechnological processes.App.Microbiol.Biotechnol.2007,76:
1-8)。
The method of production Tagatose has two kinds of chemical synthesis and biotransformation method.Generally all using galactolipin as raw material, lead to
It crosses chemical method or bioconversion carries out isomerization reaction and forms.Galactolipin raw material can be obtained by lactose hydrolysis, also have research to make
It is raw material with galactitol, is Tagatose through biological oxidation.But galactitol higher price wouldn't be suitable for use as industrializing at present
Raw materials for production.It is catalyst that chemical synthesis, which is using soluble alkali metal salts or alkali salt, promotes D- galactolipins in alkali
Property under the conditions of generate Tagatose, and form metal hydroxide-tagatose compound, then neutralized with acid and obtain D-Tag.It is raw
Object conversion method includes galactitol being oxidized to Tagatose, and utilize isomerase caused by microorganism by galactolipin isomery at tower
Two methods of lattice sugar.Because of chemical method high energy consumption, product is complicated, and purification difficult, side reaction is more, generates chemical contamination, therefore biology turns
Change method has preferable application prospect.The method for studying more bioconversion production Tagatose at present is to utilize L-arabinose
Isomery enzymatic D- galactolipins are converted into Tagatose, however the higher price of galactolipin affects the final price of Tagatose, leads
Cause can not 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.)。
The method that Korean science man invents a kind of catalysis of multienzyme will be fructose converting for Tagatose, including the use of 6- phosphoric acid tower lattice
Sugared epimerase, 6- phosphoric acid Tagatose phosphatases 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
Production fructose-1, 6-diphosphate needs ATP to carry out substrate phosphorylation to fructose, causes Tagatose production cost high, is not suitable with extensive raw
Production.
It would therefore be highly desirable to a kind of low cost is developed, and low stain, the new method of the suitable large-scale production Tagatose of high yield.
Invention content
Technical problem to be solved by the invention is to provide a kind of preparation method of Tagatose, this method passes through external multienzyme
Catalytic starch or cellulose and their derivative or sucrose prepare Tagatose, yield and conversion ratio with Tagatose
Height, production cost is low, it is pollution-free the advantages that.
In order to solve the above technical problems, the technical solution used in the present invention is：
The present invention discloses a kind of preparation method of starch Tagatose first, includes the following steps：With starch or starch derivatives
Biology is substrate, is added containing alpha-glucans 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 Tagatoses epimerase (Tagatose 6-phosphate 4-
Epimerase) and the multienzyme catalytic materials of 6- phosphoric acid Tagatoses phosphatase (Tagatose 6-phosphatase) to establish multienzyme anti-
Answer system, carry out enzymic catalytic reaction to get.
In order to reach better effect, it is preferred that conventionally divided acquired enzymic catalytic reaction product
From, purifying.
In the multienzymatic reaction system, a concentration of 1-500g/L of the substrate, the use of the alpha-glucans phosphorylase
Amount is 0.1-1000U/mL, and the dosage of the phosphoglucomutase is 0.1-1000U/mL, the glucose phosphate isomery
The dosage of enzyme is 0.1-1000U/mL, and the dosage of the 6- phosphoric acid Tagatose epimerase is 0.1-1000U/mL, the 6-
The dosage of phosphoric acid Tagatose phosphatase is 0.1-1000U/mL；Preferably, a concentration of 100g/L of the substrate, the α-Portugal are poly-
The dosage of saccharophosphorylase is 10U/mL, and the dosage of the phosphoglucomutase is 10U/mL, and the glucose phosphate is different
The dosage of structure enzyme is 10U/mL, and the dosage of the 6- phosphoric acid Tagatose epimerase is 10U/mL, the 6- phosphoric acid Tagatose
The dosage of phosphatase is 10U/mL.The condition of the enzymic catalytic reaction is：10-90 DEG C is reacted 1-100 hours；Preferably, 37 DEG C
Reaction 24-40 hours.
In the preparation method of starch Tagatose of the present invention, the starch is preferably soluble starch；The starch derivatives
Including：In boiling starch, amylodextrin, maltodextrin, malt polysaccharide or maltose any one or more according to appoint
The mixture of meaning ratio composition.
Further include any one in (1), (2) or (3) as the preferred technical solution of the present invention, in the multienzyme catalytic materials
Kind：(1) any in starch debranching enzyme or maltose phosphorylase (maltose phosphorylase, EC 2.4.1.8)
It is one or two kinds of；(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 dosage of the starch debranching enzyme is 0.1-500U/mL, the maltose phosphorus
The dosage of phosphorylase or glucanotransferase is 0.1-500U/mL；It is furthermore preferred that the dosage of the starch debranching enzyme is 1U/
The dosage of mL, the maltose phosphorylase or glucanotransferase is 1U/mL；Wherein, the 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 in order to improve the yield of Tagatose, it converts remaining glucose to Tagatose, the multienzyme
Further include in catalytic materials：Polyphosphoric acid glucokinase (polyphosphate glucokinase, EC 2.7.1.63) and poly- phosphorus
Hydrochlorate；Preferably, in multienzymatic reaction system, the dosage of the polyphosphoric acid glucokinase is 0.1-500U/mL, the poly- phosphorus
The dosage of hydrochlorate is 1-100mM；It is furthermore preferred that the dosage of the polyphosphoric acid glucokinase is 1U/mL, the Quadrafos
Dosage is 10mM.Wherein, the Quadrafos is preferably polyphosphate sodium.
After reaction, remaining starch residue will be pure amylose, and a small amount of alpha amylase (EC can be added at this time
3.2.1.1 the hydrolysis for) promoting starch residue, further increases the yield of Tagatose.Preferably, in multienzymatic reaction system, institute
The dosage for stating alpha amylase is 0.01-100U/ml, more preferably 0.1U/ml.
Further include in multienzymatic reaction system of the present invention：Buffer solution, inorganic phosphate radical and divalent magnesium ion；Preferably,
The dosage of each ingredient is：Buffer solution 10-500mM, inorganic phosphate radical 2-100mM, divalent magnesium ion 1-50mM；Wherein, described slow
Fliud flushing is phosphate buffer, pH 5.0-9.0；Preferably, 7.0 pH；It is furthermore preferred that the dosage of each ingredient is：Phosphate
Buffer solution 30mM (there is no need to inorganic phosphate radicals using phosphate buffer), divalent magnesium ion 5mM.
The present invention using starch or starch derivatives as substrate, be added alpha-glucans phosphorylases, phosphoglucomutase,
Glucosephosphate isomerase, 6- phosphoric acid Tagatose epimerases and 6- phosphoric acid Tagatose phosphatases prepare multienzymatic reaction system,
Multienzyme catalytic route includes：A glucose unit in starch or starch derivatives is converted to by alpha-glucans phosphorylase
Cori's eater Cori；It converts Cori's eater Cori to glucose 6-phosphate by phosphoglucomutase；By glucose phosphate
Isomerase converts glucose 6-phosphate to fructose-1, 6-diphosphate；Fructose-1, 6-diphosphate is converted by 6- phosphoric acid Tagatose epimerases
For Tagatose -6- phosphoric acid；It converts Tagatose -6- phosphoric acid dephosphorizations to Tagatose and phosphoric acid by 6- phosphoric acid Tagatose phosphatases.By
Being in the enzymic catalytic reaction for converting Tagatose -6- phosphoric acid dephosphorizations to by 6- phosphoric acid Tagatose phosphatases Tagatose can not be converse
It answers, so the enzymatic system can obtain very high conversion ratio, and high yield pulp1 and high conversion can substantially reduce Tagatose
Separation costs.
Since starch is the mixture of amylose (20-30%) and amylopectin (70-80%).Branch in amylopectin
Chain be with α -1, what 6 glycosidic bonds were connected with main chain, and alpha-glucans phosphorylase is unable to decomposing alpha -1,6 glycosidic bond.In order to improve
The conversion ratio of Tagatose, the present invention is added in multienzymatic reaction system being capable of α -1, the debranching enzyme of 6 glycosidic bonds in starch-splitting
Enzyme-isoamylase or Pullulanase.Since the final product that alpha-glucans phosphorylase hydrolyzes starch is maltose, for profit
With maltose, maltose phosphorylase is further added in the present invention in the reaction system, and maltose is decomposed into 1- phosphoric acid grapes
Sugar and glucose；It is furthermore preferred that polyphosphoric acid and polyphosphoric acid glucokinase is further added in the present invention in multienzymatic reaction system
Enzyme converts glucose into glucose 6-phosphate, is turned by 6- phosphoric acid Tagatose epimerases and 6- phosphoric acid Tagatose phosphatases
Tagatose is turned to, finally converts glucose unit all in starch and its derivative to Tagatose, to improve Tagatose
Yield and conversion ratio.Herein, maltose phosphorylase can be replaced by glucanotransferase, which can be by the oligomerization of short chain
Sugar polymerization becomes the oligosaccharide of long-chain, and the oligosaccharide of the long-chain can be re-used by alpha-glucans phosphorylase, so as to
Enough improve the utilization rate of starch.
The invention also discloses the preparation methods of another cellulose Tagatose, include the following steps：With cellulose or fibre
The plain derivative of dimension is substrate, is added containing 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 Tagatoses epimerase (Tagatose 6-phosphate 4-
Epimerase) and the multienzyme catalytic materials of 6- phosphoric acid Tagatoses phosphatase (Tagatose 6-phosphatase) to establish multienzyme anti-
Answer system, carry out enzymic catalytic reaction to get.
In order to reach better effect, it is preferred that conventionally divided acquired enzymic catalytic reaction product
From, purifying.
Preferably, first cellulose or cellulose derivative and cellulase are mixed on ice-water bath, 4 DEG C of centrifugations are gone
Clearly, the mixture of cellulase and cellulose is obtained；Then in the mixture of cellulase and cellulose, it is more to add fiber
Saccharophosphorylase, cellobiose phosphorylase, phosphoglucomutase, glucosephosphate isomerase, 6- phosphoric acid Tagatoses are poor
Multienzymatic reaction system is established to isomerase and 6- phosphoric acid Tagatose phosphatases.Wherein, the dosage of cellulose or cellulose derivative
Dosage for 1-500g/L, cellulase is 1-500U/ml；Preferably, the dosage of cellulose or cellulose derivative is 100g/
The dosage of L, cellulase are 10U/ml.The processing can remove almost all of glucuroide in commercial cellulose enzyme,
A large amount of glucose can be generated to avoid glucuroide hydrolysis fiber disaccharides, to make main hydrolysate be cellobiose
And fibrous polysaccharaide.
In the multienzymatic reaction system, a concentration of 1-500g/L of the mixture of the cellulase and cellulose；Institute
The dosage for stating fibrous polysaccharaide phosphorylase is 0.1-1000U/mL, and the dosage of the cellobiose phosphorylase is 0.1-1000U/
The dosage of mL, the phosphoglucomutase are 0.1-1000U/mL, and the dosage of the glucosephosphate isomerase is 0.1-
The dosage of 1000U/mL, the 6- phosphoric acid Tagatose epimerase are 0.1-1000U/mL, the 6- phosphoric acid Tagatose phosphoric acid
The dosage of enzyme is 0.1-1000U/mL；Preferably, a concentration of 100g/L of the mixture of the cellulase and cellulose；It is described
The dosage of fibrous polysaccharaide phosphorylase is 10U/mL, and the dosage of the cellobiose phosphorylase is 10U/mL, the glucose
The dosage of transphosphorylase is 10U/mL, and the dosage of the glucosephosphate isomerase is 10U/mL, the 6- phosphoric acid Tagatose
The dosage of epimerase is 10U/mL, and the dosage of the 6- phosphoric acid Tagatose phosphatase is 10U/mL.The enzymic catalytic reaction
Condition be：10-80 DEG C is reacted 1-120 hours；Preferably, it reacts 48-96 hours, most preferably 72 hours for 37 DEG C.
In order to further increase the yield of cellulose Tagatose, further include in the multienzyme catalytic materials：Polyphosphoric acid glucose
Kinases (EC 2.7.1.63) and Quadrafos；Preferably, in multienzymatic reaction system, the use of the polyphosphoric acid glucokinase
Amount is 0.1-500U/mL, and the dosage of the Quadrafos is 1-100mM；It is furthermore preferred that the use of the polyphosphoric acid glucokinase
Amount is 5U/mL, and the dosage of the Quadrafos is 10mM；Wherein, the Quadrafos is preferably polyphosphate sodium.
Further include in multienzymatic reaction system of the present invention：Buffer solution, inorganic phosphate radical and divalent magnesium ion；Preferably,
The dosage of each ingredient is：Buffer solution 10-500mM, inorganic phosphate radical 2-100mM, divalent magnesium ion 1-50mM；Wherein, described slow
Fliud flushing is phosphate buffer；It is furthermore preferred that the pH value of the phosphate buffer is 5.0-9.0, most preferably 7.2；It is further excellent
Choosing, the dosage of each ingredient is：Phosphate buffer 30mM (there is no need to inorganic phosphate radicals using phosphate buffer), divalent magnesium from
Sub- 5mM.
Cellulose derivative of the present invention includes：Cellulose passes through pretreated product, fibrous polysaccharaide or fiber two
Any one in sugar.The preprocess method includes：Acid-hydrolysis method, enzyme hydrolysis method or Physical；Preferably, the cellulose
It is cellulose by concentrated phosphoric acid treated product (Zhang, Y.H.P., et al. (2006) " A by pretreated product
Transition from Cellulose Swelling to Cellulose Dissolution by o-Phosphoric
Acid:Evidence from Enzymatic Hydrolysis and Supramolecular Structure."
Biomacromolecules 7(2):644-648.)。
Cellulase, fibrous polysaccharaide phosphorylase, fiber is added using cellulose or cellulose derivative as substrate in the present invention
Two saccharophosphorylases, phosphoglucomutase, glucosephosphate isomerase, 6- phosphoric acid Tagatose epimerases and 6- phosphoric acid
Tagatose phosphatase prepares multienzymatic reaction system, and multienzyme catalytic route includes：It is more at fiber by cellulose hydrolyzation cellulose raw
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；It converts Cori's eater Cori to 6- phosphoric acid grapes by phosphoglucomutase
Sugar；It converts glucose 6-phosphate to fructose-1, 6-diphosphate by glucosephosphate isomerase；By 6- phosphoric acid Tagatose epimerases
Convert fructose-1, 6-diphosphate to Tagatose -6- phosphoric acid；Tagatose -6- phosphoric acid dephosphorizations are converted to by 6- phosphoric acid Tagatose phosphatases
Tagatose.Polyphosphoric acid glucokinase and polyphosphoric acid is further added in above-mentioned multienzymatic reaction system in the present invention, by cellulose
Final product glucose after hydrolysis is converted into glucose 6-phosphate, then poor by glucosephosphate isomerase, 6- phosphoric acid Tagatoses
It is converted into Tagatose to isomerase and 6- phosphoric acid Tagatose phosphatases, finally by glucose all in cellulose and its derivates
Unit is converted into Tagatose.
The present invention further discloses a kind of preparation methods of sucrose Tagatose, include the following steps：Using sucrose as substrate,
It is added containing sucrose phosphorylase, phosphoglucomutase, glucosephosphate isomerase, 6- phosphoric acid Tagatose epimerisms
Enzyme, 6- phosphoric acid Tagatose phosphatases multienzyme catalytic materials establish multienzyme catalystic converter system carry out enzymic catalytic reaction to get.
In order to reach better effect, it is preferred that conventionally divided acquired enzymic catalytic reaction product
From, purifying.
In the multienzyme catalystic converter system, a concentration of 10-300g/L of the sucrose, the sucrose phosphorylase
Dosage is 0.1-1000U/mL, and the dosage of the phosphoglucomutase is 0.1-1000U/mL, and the glucose phosphate is different
The dosage of structure enzyme is 0.1-1000U/mL, and the dosage of the 6- phosphoric acid Tagatose epimerase is 0.1-1000U/mL, described
The dosage of 6- phosphoric acid Tagatose phosphatases is 0.1-1000U/mL；Preferably, a concentration of 300g/L of the sucrose, the sucrose
The dosage of phosphorylase is 30U/mL, and the dosage of the phosphoglucomutase is 30U/mL, the glucose phosphate isomery
The dosage of enzyme is 30U/mL, and the dosage of the 6- phosphoric acid Tagatose epimerase is 30U/mL, the 6- phosphoric acid Tagatose phosphorus
The dosage of sour enzyme is 30U/mL.The condition of the enzymic catalytic reaction is：20-70 DEG C of reaction 2-100h；Preferably, 37 DEG C of reactions
10-72h。
Wherein, further include in the multienzymatic reaction system：Buffer solution, inorganic phosphate radical and divalent magnesium ion；Preferably, respectively
The dosage of ingredient is：Buffer solution 10-500mM, inorganic phosphate radical 2-100mM, divalent magnesium ion 1-50mM；Wherein, the buffering
Liquid is phosphate buffer, pH 5.0-9.0；Preferably, 7.2 pH；It is furthermore preferred that the dosage of each ingredient is：Phosphate buffer
30mM (there is no need to inorganic phosphate radicals using phosphate buffer), divalent magnesium ion 5mM.
In order to further increase the yield of sucrose Tagatose, further include in the multienzyme catalystic converter system：Glucose is different
Structure enzyme, polyphosphoric acid glucokinase and Quadrafos；Preferably, per 10g/L sucrose, the dosage of glucose isomerase is 1U/mL,
The dosage of polyphosphoric acid glucokinase is 5U/mL, and the dosage of Quadrafos is 10mM；Wherein, the Quadrafos is preferably poly-
Sodium phosphate.
The present invention is added different containing sucrose phosphorylase, phosphoglucomutase, glucose phosphate using sucrose as substrate
Structure enzyme, 6- phosphoric acid Tagatoses epimerase, 6- phosphoric acid Tagatose phosphatases prepare multienzymatic reaction system, multienzyme catalytic route packet
It includes：It converts a glucose unit in sucrose to Cori ester by sucrose phosphorylase；It is conjugated by glucose phosphate
Enzyme converts Cori ester to G-6-P；G-6-P is converted to by glucosephosphate isomerase
Fructose-6-phosphate；It converts fructose-6-phosphate to Tagatose -6- phosphoric acid by 6- phosphoric acid Tagatose epimerases；By 6- phosphoric acid
Tagatose phosphatase converts Tagatose -6- phosphoric acid dephosphorizations to Tagatose and phosphoric acid.The present invention is in above-mentioned multienzymatic reaction system
Glucose isomerase, polyphosphoric acid glucokinase and Quadrafos is further added, by the final product glucose after sucrose hydrolysis
It is converted into G-6-P, then by glucosephosphate isomerase, 6- phosphoric acid Tagatose epimerases and 6- phosphoric acid Tagatoses
Phosphatase is converted into Tagatose.
The external multienzyme catalytic starch of the present invention or cellulose and their derivative or sucrose prepare the side of Tagatose
Method, the enzymic catalytic reaction can be carried out in one or more reactors substep, preferably be carried out in a reactor.
Any one of multienzymatic reaction system of the present invention enzyme can have the enzyme of same function to replace by any type,
Or the mutant enzyme with same function obtained by protein engineering house of correction.
Starch Conversion is in Tagatose experiment, in a reaction system, with solubility by the external multienzyme catalysis of the present invention
Starch is substrate, and alpha-glucans phosphorylase, phosphoglucomutase, glucosephosphate isomerase, 6- phosphoric acid tower lattice are added
Sugared epimerase and 6- phosphoric acid Tagatose phosphatases carry out catalysis reaction, and the conversion ratio of Tagatose is 37%.And above-mentioned anti-
It answers and isoamylase, maltose phosphorylase, polyphosphoric acid glucokinase, glucanotransferase and poly- phosphorus is further added in system
The conversion ratio of sour sodium, final Tagatose reaches 72%, and conversion ratio significantly improves.
The external multienzyme catalysis of the present invention converts cellulose into Tagatose experiment, in a reaction system, to live again
Amorphous cellulose be substrate, be added cellulase, fibrous polysaccharaide phosphorylase, cellobiose phosphorylase, glucose
Transphosphorylase, glucosephosphate isomerase, 6- phosphoric acid Tagatose epimerases and 6- phosphoric acid Tagatose phosphatases are urged
Change reaction, the conversion ratio of Tagatose is 35%.Polyphosphoric acid glucokinase and polyphosphoric acid are additionally incorporated in above-mentioned reaction system
The conversion ratio of sodium, final Tagatose reaches 68% or more, and conversion ratio significantly improves.
Sucrose inversion is in a reaction system, to be with sucrose in Tagatose experiment by the external multienzyme catalysis of the present invention
Sucrose phosphorylase, phosphoglucomutase, glucosephosphate isomerase, 6- phosphoric acid Tagatose epimerisms is added in substrate
Enzyme, 6- phosphoric acid Tagatoses phosphatase, glucose isomerase, polyphosphoric acid glucokinase and polyphosphate sodium carry out catalysis reaction, most
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 is using starch or cellulose and their derivative as raw material, or with sucrose
For raw material, rather than use expensive galactolipin.Therefore, the production cost of Tagatose is low, is suitable for large-scale production.
(2) yield of Tagatose is high.The final step of the present invention more enzymic catalytic reactions in vitro, i.e., by 6- phosphoric acid Tagatose phosphorus
It is irreversible procedure that sour enzyme, which converts Tagatose -6- phosphoric acid dephosphorizations to the enzymic catalytic reaction of Tagatose, therefore can be obtained very high
Tagatose conversion ratio, to reduce the separation costs of Tagatose.
(3) preparation method of the present invention makees raw material without fructose, does not need ATP and carries out substrate phosphorylation production 6- phosphoric acid fruits
Sugar substantially reduces the cost of production Tagatose.
The term definition involved in the present invention arrived
Unless otherwise defined, otherwise all technologies used herein and scientific terminology all have with it is of the art
Those of ordinary skill usually understands identical meaning.
Term " enzymic catalytic reaction " means the chemical reaction carried out under biocatalyst-enzyme effect.
Description of the drawings
Fig. 1 is the schematic diagram for the external multienzyme catalytic route that converted starch generates Tagatose；Wherein, IA, isoamylase；
PA, Pullulanase；α GP, alpha-glucans phosphorylase；PGM, phosphoglucomutase；PGI, glucosephosphate isomerase；
T6E, 6- phosphoric acid Tagatose epimerase；T6P, 6- phosphoric acid Tagatose phosphatase；MP, (enzyme can quilt for maltose phosphorylase
Glucanotransferase is replaced)；PPGK, polyphosphoric acid glucokinase；
Fig. 2 is to be detected using soluble starch as substrate using HPLC, and the product after enzymatic reaction, arrow meaning indicates
The characteristic peak of Tagatose；
Fig. 3 is the schematic diagram for converting cellulose raw into the external multienzyme catalytic route of Tagatose；Wherein, Cellulase, it is 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 for converting the external multienzyme catalytic route that 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 using sucrose as substrate with HPLC, and the product after enzymatic reaction, arrow meaning indicates Tagatose
Characteristic peak.
Specific implementation mode
The invention will now be further described with reference to specific embodiments, the advantages and features of the present invention will be with description and
It is apparent.It should be understood that described, examples are merely exemplary, does not constitute any restrictions to the scope of the present invention.This field
Technical staff should be understood that without departing from the spirit and scope of the invention can to the details of technical solution of the present invention and
Form is modified or is replaced, but these modifications or substitutions each fall within protection scope of the present invention.
1, experiment material
Soluble starch, soluble starch, ACROS Products, product identification：424490020；
Maltodextrin, ALDRICH Products, product identification 419672；
PET20b carriers, Novagen, Madison, WI；
Bacillus coli expression bacterium BL21 (DE3), Invitrogen, Carlsbad, CA；
(in addition to Tagatose -6- phosphate epimerases, 6- phosphoric acid Tagatose phosphatases gather most of enzyme in the present invention
Phosphoglucokinase and glucanotransferase) it can be commercially available in Sigma companies；But it can all be according to gene engineering method
It is obtained by prokaryotic expression；
Cellulase is bought from Sigma companies, product identification C2730；
Maltose phosphorylase is bought from Sigma companies, product identification M8284；
Alpha amylase is bought from Sigma companies, product identification 10065；
Avicel, micro- crystalline cellulose are bought, product identification 11365 from Sigma companies.
Starch Conversion is Tagatose by the catalysis of 1 external multienzyme of experimental example
By an external multienzyme catalyst system and catalyzing by Starch Conversion be Tagatose (Fig. 1).These key enzymes include：(1)α-
Glucosan phosphorylase (α GP, EC 2.4.1.1) releases 1- phosphoric acid grapes from the non-reducing end of starch plus 1 phosphate
Sugar；(2) phosphoglucomutase (PGM, EC 5.4.2.2), catalysis Cori's eater Cori to glucose 6-phosphate；(3) grape
Sugared phosphoric acid isomerase converts glucose 6-phosphate to fructose-1, 6-diphosphate；(4) 6- phosphoric acid Tagatose epimerase, by 6- phosphorus
Tart fruit sugar is converted into Tagatose -6- phosphoric acid；(5) 6- phosphoric acid Tagatose phosphatase converts Tagatose -6- phosphoric acid dephosphorizations to tower lattice
Sugar and phosphoric acid.
In the present invention, alpha-glucans phosphorylase derives from Thermotoga maritima, volume of the gene on KEGG
Number be TM1168；Phosphoglucomutase also derives from Thermotoga maritima, and number of the gene on KEGG is
TM0769；Glucosephosphate isomerase derives from Clostridium thermocellum, and number of the gene on KEGG is
Cthe0217；6- phosphoric acid Tagatose epimerases come from Agrobacterium fabrum, number of the gene on KEGG
For Atu3167；6- phosphoric acid Tagatose phosphatases derive from Archaeoglobus fulgidus, and number of the gene on KEGG is
AF_0444, these genomic DNAs can all be obtained from the official website (www.atcc.org) of ATCC.This five gene difference
It is obtained by PCR from corresponding genomic DNA with different primers, and passes 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 carriers 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
Into Bacillus coli expression bacterium BL21 (DE3) (Invitrogen, Carlsbad, CA), and carry out protein expression and purifying.
The phosphate buffer (pH 7.0) containing 30mM in one 0.75 milliliter of reaction system, the divalent magnesium of 5mM from
The dosage of son, the alpha-glucans phosphorylase is 10U/mL, and the dosage of the phosphoglucomutase is 10U/mL, described
The dosage of glucosephosphate isomerase is 10U/mL, and the dosage of the 6- phosphoric acid Tagatose epimerase is 10U/mL, described
The dosage of 6- phosphoric acid Tagatose phosphatases is 10U/mL, and the soluble starch of 100g/L carries out catalysis reaction, reaction 24 at 37 DEG C
A hour.
According to the difference of retention time, HPLC can be used for distinguishing Tagatose, glucose, the 1- phosphoric acid grapes in reaction solution
Sugar or glucose 6-phosphate；And Tagatose can be quantified, Tagatose characteristic peak in the concentration and HPLC of Tagatose
Intensity is directly proportional；The mobile phase of HPLC is the dilute sulfuric acid of 5mM.
After reaction, the final concentration of final Tagatose (Fig. 2) is 37g/L, conversion ratio 37%.
Starch Conversion is Tagatose by the catalysis of 2 external multienzyme of experimental example
Alpha-glucans phosphorylase, phosphoglucomutase, glucosephosphate isomerase, 6- phosphoric acid Tagatose differences are to different
The preparation of structure enzyme and 6- phosphoric acid Tagatose phosphatases is the same as experimental example 1.
The phosphate buffer (pH 7.0) containing 30mM in one 0.75 milliliter of reaction system, the divalent magnesium of 5mM from
The dosage of son, the alpha-glucans phosphorylase is 10U/mL, and the dosage of the phosphoglucomutase is 10U/mL, described
The dosage of glucosephosphate isomerase is 10U/mL, and the dosage of the 6- phosphoric acid Tagatose epimerase is 10U/mL, described
The dosage of 6- phosphoric acid Tagatose phosphatases is 10U/mL, and the soluble starch of 100g/L carries out catalysis reaction, reaction 24 at 20 DEG C
A hour.
After reaction, the final concentration of final Tagatose is 16g/L, conversion ratio 16%.
Starch Conversion is Tagatose by the catalysis of 3 external multienzyme of experimental example
Alpha-glucans phosphorylase, phosphoglucomutase, glucosephosphate isomerase, 6- phosphoric acid Tagatose differences are to different
The preparation of structure enzyme and 6- phosphoric acid Tagatose phosphatases is the same as experimental example 1.
The phosphate buffer (pH 7.0) containing 30mM in one 0.75 milliliter of reaction system, the divalent magnesium of 5mM from
The dosage of son, the alpha-glucans phosphorylase is 10U/mL, and the dosage of the phosphoglucomutase is 10U/mL, described
The dosage of glucosephosphate isomerase is 10U/mL, and the dosage of the 6- phosphoric acid Tagatose epimerase is 10U/mL, described
The dosage of 6- phosphoric acid Tagatose phosphatases is 10U/mL, and the soluble starch of 100g/L carries out catalysis reaction, reaction 24 at 50 DEG C
A hour.
After reaction, the final concentration of final Tagatose is 8g/L, conversion ratio 8%.
Experimental example 4 promotes amylolytic enzyme by process optimization and addition, is catalyzed Starch Conversion using external multienzyme
For Tagatose
Since starch is that have branched chain, merely using alpha-glucans phosphorylase can not completely by Starch Hydrolysis because
Alpha-glucans phosphorylase can only act on α-Isosorbide-5-Nitrae glycosidic bond, and branched chain be with α -1, what 6 glycosidic bonds were connect with main chain.This
It needs that isoamylase (isoamylase, EC 3.2.1.68) hydrolyzing alpha -1,6 glycosidic bond is added.Finally, starch is by both enzyme water
The final product of solution is maltose and glucose, in order to convert these final products to Tagatose, it is also necessary to maltose be added
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 number of the gene on KEGG is
The genomic DNA of ST0928, the bacterial strain are German Albert-Ludwigs-The Georg Fuchs of Freiburg
Friendship is taught to provide.Polyphosphoric acid glucokinase derives from Thermobifida fusca, and number of the gene on KEGG is
The genomic DNA of Tfu1811, the bacterial strain are David Wilson professor's friendship offers of Cornell Univ USA.Glucan turns
Enzyme source is moved in Thermococcus litoralis, number of the gene on KEGG is OCC_10078, the genome of the bacterial strain
DNA can be obtained from the official website (www.atcc.org) of ATCC.These three genes are respectively with different primers from corresponding
It is obtained by PCR in genomic DNA, and passes 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, corresponding expression vector pET20b-StIA, pET20b-TfuPPGK and pET20b-Ti4GT are obtained,
These three plasmids are all converted into Bacillus coli expression bacterium BL21 (DE3), and carry out protein expression and purifying.
Alpha-glucans phosphorylase, phosphoglucomutase, glucosephosphate isomerase, 6- phosphoric acid Tagatose differences are to different
The preparation of structure enzyme and 6- phosphoric acid Tagatose phosphatases is the same as experimental example 1；Maltose phosphorylase is bought from Sigma companies, and product is compiled
Number be M8284.
The phosphate buffer (pH 7.0) containing 30mM in one 0.75 milliliter of reaction system, the divalent magnesium of 5mM from
The dosage of son, the alpha-glucans phosphorylase is 10U/mL, and the dosage of the phosphoglucomutase is 10U/mL, described
The dosage of glucosephosphate isomerase is 10U/mL, and the dosage of the 6- phosphoric acid Tagatose epimerase is 10U/mL, described
The dosage of 6- phosphoric acid Tagatose phosphatases 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 sodiums, the soluble starch of 100g/L, at 37 DEG C
Catalysis reaction is carried out, 40 hours are reacted.The detection method of Tagatose with experimental example 1, final Tagatose (Fig. 2) it is final concentration of
72g/L, conversion ratio have reached 72%.
Experimental example 5 promotes amylolytic enzyme by process optimization and addition, is catalyzed Starch Conversion using external multienzyme
For Tagatose
Alpha-glucans phosphorylase, phosphoglucomutase, glucosephosphate isomerase, 6- phosphoric acid Tagatose differences are to different
The preparation of structure enzyme and 6- phosphoric acid Tagatose phosphatases is the same as experimental example 1；The preparation of polyphosphoric acid glucokinase is the same as experimental example 4, Pu Lu
Blue enzyme (pullulanase, EC 3.2.1.41) is bought from Sigma companies, product identification P1067；Maltose phosphorylase from
Sigma companies buy, product identification M8284.
The phosphate buffer (pH 7.0) containing 30mM in one 0.75 milliliter of reaction system, the divalent magnesium of 5mM from
The dosage of son, the alpha-glucans phosphorylase is 10U/mL, and the dosage of the phosphoglucomutase is 10U/mL, described
The dosage of glucosephosphate isomerase is 10U/mL, and the dosage of the 6- phosphoric acid Tagatose epimerase is 10U/mL, described
The dosage of 6- phosphoric acid Tagatose phosphatases 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 sodiums, the soluble starch of 100g/L, at 37 DEG C
Catalysis reaction is carried out, 40 hours are reacted.The detection method of Tagatose is the same as experimental example 1, the final concentration of 73g/ of final Tagatose
L, conversion ratio have reached 73%.
The hydrolysis that a small amount of alpha amylase promotes residue starch is then added in the reaction system, improves the yield of Tagatose,
The dosage of alpha amylase is 0.1U/ml, and continuation is reacted 24 hours at 37 DEG C, and the final concentration of 88g/L of final Tagatose (Fig. 2) turns
Rate has reached 88%.
The catalysis of 6 external multienzyme of experimental example converts maltodextrin to Tagatose
Alpha-glucans phosphorylase, phosphoglucomutase, glucosephosphate isomerase, 6- phosphoric acid Tagatose differences are to different
The preparation of structure enzyme and 6- phosphoric acid Tagatose phosphatases is the same as experimental example 1；The preparation of isoamylase, polyphosphoric acid glucokinase is the same as experiment
Example 4, maltose phosphorylase are bought from Sigma companies, product identification M8284.
The phosphate buffer (pH 7.0) containing 30mM in one 0.75 milliliter of reaction system, the divalent magnesium of 5mM from
The dosage of son, the alpha-glucans phosphorylase is 10U/mL, and the dosage of the phosphoglucomutase is 10U/mL, described
The dosage of glucosephosphate isomerase is 10U/mL, and the dosage of the 6- phosphoric acid Tagatose epimerase is 10U/mL, described
The dosage of 6- phosphoric acid Tagatose phosphatases 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 sodiums, the maltodextrin (ALDRICH of 100g/L
Products, product identification 419672), catalysis reaction is carried out at 37 DEG C, reacts 40 hours.The detection method of Tagatose is the same as real
Example 1, the final concentration of 78g/L of final Tagatose are tested, conversion ratio has reached 78%.
The catalysis of 7 external multienzyme of experimental example converts cellulose into Tagatose
The schematic diagram for converting cellulose into Tagatose by an external multienzyme catalyst system and catalyzing is shown in Fig. 3.
Cellulase comes from the product of Sigma companies, product identification C2730；Phosphoglucomutase, grape
The preparation of sugared phosphoric acid isomerase, 6- phosphoric acid Tagatose epimerases and 6- phosphoric acid Tagatose phosphatases is the same as experimental example 1.
Fibrous polysaccharaide phosphorylase (Cthe_2989) and cellobiose phosphorylase (Cthe_0275) are all derived from
Clostridium thermocellum.The two genes are respectively with different primers from corresponding genomic DNA (genome
DNA can from the official website of ATCC (www.atcc.org/) on obtain) in obtained by PCR, 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):Method 1593-1595.) is cloned into pET20b carriers, obtains corresponding table
Up to carrier pET20b-CthCDP and pET20b-CthCBP.The two plasmids are all converted to Bacillus coli expression bacterium BL21 (DE3)
In, and carry out protein expression and purifying.
This experiment uses micro- crystalline cellulose (Avicel) for substrate.First by commercialized cellulase (10U/ml) and
Cellulose (100g/L) mixes on ice-water bath, is positioned in ice-water bath 5 minutes, is centrifuged at 4 DEG C, removes supernatant.It is precipitated as fiber
The mixture of element and the cellulase that can be combined with cellulose.The processing can remove almost all of in commercial cellulose enzyme
Glucuroide can generate a large amount of glucose, to make main water to avoid glucuroide hydrolysis fiber disaccharides in this way
It is cellobiose and fibrous polysaccharaide to solve product.
The phosphate buffer (pH 7.2) containing 30mM in one 0.75 milliliter of reaction system, the divalent magnesium of 5mM from
Son, the fibrous polysaccharaide phosphorylase of 10U/mL, 10U/mL cellobiose phosphorylases, the phosphoglucomutase of 10U/mL,
The glucosephosphate isomerase of 10U/mL, the 6- phosphoric acid Tagatose epimerases of 10U/mL, the 6- phosphoric acid Tagatoses of 10U/mL
Phosphatase, the mixture of the cellulose and cellulase as described above of 100g/L carry out catalysis reaction at 37 DEG C, react 72
Hour.For the detection method of Tagatose with experimental example 1, the final concentration of final Tagatose is 14g/L, conversion ratio 14%.
The catalysis of 8 external multienzyme of experimental example converts cellulose into Tagatose
Cellulase comes from the product of Sigma companies, product identification C2730；Phosphoglucomutase, grape
Sugared phosphoric acid isomerase, 6- phosphoric acid Tagatose epimerases, the preparation of 6- phosphoric acid Tagatose phosphatases is the same as experimental example 1；Fiber is more
The preparation of saccharophosphorylase and cellobiose phosphorylase is the same as experimental example 7.
This experiment using live again amorphous cellulose (Regenerated Amorphous cellulose (RAC),
This is Avicel by concentrated phosphoric acid treated 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.) it is substrate.First by commercialized cellulase (10U/ml) and the fiber
Plain (100g/L) is mixed on ice-water bath, is positioned in ice-water bath 5 minutes, is centrifuged at 4 DEG C, is removed supernatant.Be precipitated as cellulose and
The mixture for the cellulase that can be combined with cellulose.
The phosphate buffer (pH 7.2) containing 30mM in one 0.75 milliliter of reaction system, the divalent magnesium of 5mM from
Son, the fibrous polysaccharaide phosphorylase of 10U/mL, 10U/mL cellobiose phosphorylases, the phosphoglucomutase of 10U/mL,
The glucosephosphate isomerase of 10U/mL, the 6- phosphoric acid Tagatose epimerases of 10U/mL, the 6- phosphoric acid Tagatoses of 10U/mL
Phosphatase, the mixture of the cellulose and cellulase as described above of 100g/L carry out catalysis reaction at 37 DEG C, react 72
Hour.For the detection method of Tagatose with experimental example 1, the final concentration of final Tagatose is 35g/L, conversion ratio 35%.
The catalysis of 9 external multienzyme of experimental example converts cellulose into Tagatose
Cellulase comes from the product of Sigma companies, product identification C2730；Phosphoglucomutase, grape
The preparation of sugared phosphoric acid isomerase, 6- phosphoric acid Tagatoses epimerase, 6- phosphoric acid Tagatose phosphatases is the same as experimental example 1；Fiber is more
The preparation of saccharophosphorylase and cellobiose phosphorylase is the same as experimental example 7.
The phosphate buffer (pH 7.2) containing 30mM in one 0.75 milliliter of reaction system, the divalent magnesium of 5mM from
Son, the fibrous polysaccharaide phosphorylase of 10U/mL, 10U/mL cellobiose phosphorylases, the phosphoglucomutase of 10U/mL,
The glucosephosphate isomerase of 10U/mL, the 6- phosphoric acid Tagatose epimerases of 10U/mL, the 6- phosphoric acid Tagatoses of 10U/mL
Phosphatase, the mixture of the cellulose and cellulase of the experimental example 8 of 100g/L carry out catalysis reaction at 25 DEG C, react 24
Hour.The final concentration of final Tagatose is 29g/L, conversion ratio 29%.
The catalysis of 10 external multienzyme of experimental example converts cellulose into Tagatose
Cellulase comes from the product of Sigma companies, product identification C2730；Phosphoglucomutase, grape
The preparation of sugared phosphoric acid isomerase, 6- phosphoric acid Tagatoses epimerase, 6- phosphoric acid Tagatose phosphatases is the same as experimental example 1；Fiber is more
The preparation of saccharophosphorylase and cellobiose phosphorylase is the same as experimental example 7.
The phosphate buffer (pH 7.2) containing 30mM in one 0.75 milliliter of reaction system, the divalent magnesium of 5mM from
Son, the fibrous polysaccharaide phosphorylase of 10U/mL, 10U/mL cellobiose phosphorylases, the phosphoglucomutase of 10U/mL,
The glucosephosphate isomerase of 10U/mL, the 6- phosphoric acid Tagatose epimerases of 10U/mL, the 6- phosphoric acid Tagatoses of 10U/mL
Phosphatase, the mixture of the cellulose and cellulase of the experimental example 8 of 100g/L carry out catalysis reaction at 50 DEG C, react 24
Hour.The final concentration of final Tagatose is 9g/L, conversion ratio 9%.
The catalysis of 11 external multienzyme of experimental example converts cellulose into Tagatose
Final product after being hydrolyzed due to cellulose is glucose, in order to be translated into Tagatose, it is also necessary to is added poly-
Phosphoglucokinase and polyphosphoric acid.
Cellulase comes from the product of Sigma companies, product identification C2730；Phosphoglucomutase, grape
The preparation of sugared phosphoric acid isomerase, 6- phosphoric acid Tagatoses epimerase, 6- phosphoric acid Tagatose phosphatases is the same as experimental example 1；Polyphosphoric acid
The preparation of glucokinase is the same as experimental example 4；The preparation of fibrous polysaccharaide phosphorylase, cellobiose phosphorylase is the same as experimental example 7.
The phosphate buffer (pH 7.2) containing 30mM in one 0.75 milliliter of reaction system, the divalent magnesium of 5mM from
Son, the fibrous polysaccharaide phosphorylase of 10U/mL, 10U/mL cellobiose phosphorylases, the phosphoglucomutase of 10U/mL,
The glucosephosphate isomerase of 10U/mL, the 6- phosphoric acid Tagatose epimerases of 10U/mL, the 6- phosphoric acid Tagatoses of 10U/mL
Phosphatase, the mixture of the cellulose and cellulase of the experimental example 8 of 100g/L, the polyphosphoric acid glucokinase of 5U/mL, 10mM
Polyphosphate sodium carries out catalysis reaction at 37 DEG C, reacts 72 hours.The detection method of Tagatose is the same as experimental example 1, final Tagatose
Final concentration of 68g/L, conversion ratio reached 68%.
Sucrose inversion is Tagatose by the catalysis of 12 external multienzyme of experimental example
The schematic diagram that sucrose inversion is Tagatose is shown in into Fig. 4 by an external multienzyme catalyst system and catalyzing.
Sucrose phosphorylase is derived from Thermoanaerobacterium thermosaccharolyticum JW/
SL-YS485, the enzyme of the coded by said gene are WP_015312040.1 in the number of ncbi database.This gene primer from
It is obtained by PCR in corresponding genomic DNA, and passes 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, corresponding expression vector pET20b-SP is obtained.This plasmid is all converted to Bacillus coli expression bacterium
In BL21 (DE3), and carry out protein expression and purifying (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 companies, product identification G4166；Phosphoglucomutase,
The preparation of glucosephosphate isomerase, 6- phosphoric acid Tagatose epimerases and 6- phosphoric acid Tagatose phosphatases is the same as experimental example 1.It is poly-
Phosphoglucokinase derives from Thermobifida fusca YX, and number of the gene on KEGG is Tfu1811；This base
Because being obtained by PCR from corresponding genomic DNA with primer, this plasmid is all converted to Bacillus coli expression bacterium BL21
(DE3) it in, and is cloned into pET20b carriers, is obtained by the method for Simple Cloning (You, C., et al. (2012))
Obtain corresponding expression vector.This plasmid is all converted into Bacillus coli expression bacterium BL21 (DE3), and carry out protein expression with
Purify (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
The system of grape sugar transphosphorylase, glucosephosphate isomerase, 6- phosphoric acid Tagatoses epimerase, 6- phosphoric acid Tagatose phosphatases
For with experimental example 1.
The phosphate buffer (pH 7.2) containing 30mM, 300g/L sucrose, 5mM in one 0.75 milliliter of reaction system
Divalent 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 epimerases of 30U/mL, the 6- phosphoric acid Tagatose phosphatases of 30U/mL, the grape of 30U/mL
Sugared isomerase, the polyphosphoric acid glucokinase of 150U/mL, 300mM polyphosphate sodiums carry out catalysis reaction at 37 DEG C, react 72
Hour.The final concentration of 188g/L of final Tagatose, conversion ratio have reached 63%, HPLC figures as figure 5 illustrates.
Claims (34)
1. a kind of preparation method of Tagatose, which is characterized in that using starch as substrate, alpha-glucans phosphorylase, grape is added
Sugared transphosphorylase, glucosephosphate isomerase, 6- phosphoric acid Tagatoses epimerase, 6- phosphoric acid Tagatose phosphatases and inorganic
Phosphate anion establishes multienzyme molecule machine and carries out multienzyme catalysis reaction to get Tagatose；Wherein, 6- phosphoric acid Tagatose is poor
Come to the enzyme gene of isomeraseAgrobacterium fabrum, number of the gene on KEGG is Atu3167.
2. a kind of preparation method of Tagatose, which is characterized in that using starch derivatives as substrate, alpha-glucans phosphorylation is added
Enzyme, phosphoglucomutase, glucosephosphate isomerase, 6- phosphoric acid Tagatoses epimerase, 6- phosphoric acid Tagatose phosphoric acid
Enzyme and inorganic phosphate radical ion establish multienzyme molecule machine and carry out multienzyme catalysis reaction to get Tagatose；Wherein, the shallow lake
Any one or more of powder derivative in boiling starch, amylodextrin, maltodextrin, malt polysaccharide or maltose
The mixture formed according to arbitrary proportion；
The enzyme gene of the 6- phosphoric acid Tagatose epimerase comes fromAgrobacterium fabrum, gene is in KEGG
On number be Atu3167.
3. according to preparation method as claimed in claim 1 or 2, it is characterised in that：A concentration of 100 g/L of the substrate, it is described
The dosage of alpha-glucans phosphorylase is 10U/mL, and the dosage of the phosphoglucomutase is 10 U/mL, the glucose
The dosage of phosphoric acid isomerase is 10 U/mL, and the dosage of the 6- phosphoric acid Tagatose epimerase is 10U/mL, the 6- phosphoric acid
The dosage of Tagatose phosphatase is 10U/mL, a concentration of 2-100 mM of the inorganic phosphate radical.
4. preparation method described in accordance with the claim 3, it is characterised in that：A concentration of 30 mM of the inorganic phosphate radical.
5. according to preparation method as claimed in claim 1 or 2, it is characterised in that：The condition of the enzymic catalytic reaction is：20℃,
37 DEG C or 50 DEG C are reacted 1-100 hours.
6. preparation method according to claim 5, it is characterised in that：The condition of the enzymic catalytic reaction is：37 DEG C of reactions
24-40 hours.
7. according to preparation method as claimed in claim 1 or 2, it is characterised in that：It is also wrapped in the system of more enzymic catalytic reactions
It includes（1）,（2）Or（3）Middle any type：（1）Any one of starch debranching enzyme or maltose phosphorylase or two kinds；
（2）Any one of starch debranching enzyme or glucanotransferase or two kinds；（3）Starch debranching enzyme, malt Sugar phosphorylation
Any one in enzyme or glucanotransferase or three kinds；Wherein, the starch debranching enzyme is isoamylase or Pullulanase
In any one.
8. preparation method according to claim 7, it is characterised in that：The dosage of the starch debranching enzyme is 1 U/mL,
The dosage of the maltose phosphorylase is 1U/mL, and the dosage of the glucanotransferase is 1U/mL.
9. according to preparation method as claimed in claim 1 or 2, it is characterised in that：It is also wrapped in the system of more enzymic catalytic reactions
It includes：Polyphosphoric acid glucokinase and Quadrafos；Wherein, the Quadrafos is polyphosphate sodium.
10. preparation method according to claim 9, it is characterised in that：The dosage of the polyphosphoric acid glucokinase is 1U/
The dosage of mL, the Quadrafos are 10mM.
11. according to preparation method as claimed in claim 1 or 2, it is characterised in that：It is also wrapped in the system of more enzymic catalytic reactions
Include alpha amylase.
12. preparation method according to claim 11, it is characterised in that：The dosage of the alpha amylase is 0. 1 U/ml.
13. according to preparation method as claimed in claim 1 or 2, it is characterised in that：It is also wrapped in the system of more enzymic catalytic reactions
It includes：Buffer solution and divalent magnesium ion；Wherein, the buffer solution is phosphate buffer, pH 5.0-9.0.
14. preparation method according to claim 13, it is characterised in that：The dosage of each ingredient is：Phosphate buffer 10-
500 mM, divalent magnesium ion 1-50 mM；Phosphate buffer pH is 7.0.
15. preparation method according to claim 14, it is characterised in that：The dosage of each ingredient is：Phosphate buffer 30
MM, 5 mM of divalent magnesium ion.
16. a kind of preparation method of Tagatose, which is characterized in that include the following steps：It is with cellulose or cellulose derivative
Substrate is added containing cellulase, fibrous polysaccharaide phosphorylase, cellobiose phosphorylase, phosphoglucomutase, grape
The multienzyme of sugared phosphoric acid isomerase, 6- phosphoric acid Tagatose epimerases, 6- phosphoric acid Tagatose phosphatases and inorganic phosphate radical is catalyzed
Object establishes multienzymatic reaction system, carries out enzymic catalytic reaction to get Tagatose；Wherein, the enzyme of 6- phosphoric acid Tagatose epimerase
Gene comes fromAgrobacterium fabrum, number of the gene on KEGG is Atu3167.
17. preparation method according to claim 16, 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 epimerases, 6- phosphoric acid
Tagatose phosphatase and inorganic phosphate radical establish multienzyme molecule machine reaction system；
Wherein, the dosage of cellulose or cellulose derivative is 100 g/L, the dosage of cellulase is 10U/ml.
18. preparation method according to claim 16, it is characterised in that：In the reaction system, the cellulase
With a concentration of 100 g/L of the mixture of cellulose；The dosage of the fibrous polysaccharaide phosphorylase is 10 U/mL, the fiber
The dosage of two saccharophosphorylases is 10 U/mL, and the dosage of the phosphoglucomutase is 10 U/mL, the glucose phosphorus
The dosage of acid isomer enzyme is 10U/mL, and the dosage of the 6- phosphoric acid Tagatose epimerase is 10 U/mL, the 6- phosphoric acid tower
The dosage of lattice Sugar-phosphatase is 10U/mL, a concentration of 2-100 mM of the inorganic phosphate radical.
19. preparation method according to claim 18, it is characterised in that：A concentration of 30 mM of the inorganic phosphate radical.
20. preparation method according to claim 16, it is characterised in that：The condition of the enzymic catalytic reaction is：25℃,37
DEG C or 50 DEG C react 1-120 hours；The cellulose derivative includes that cellulose passes through pretreated product；The pre- place
Reason method includes acid-hydrolysis method, enzyme hydrolysis method or Physical.
21. preparation method according to claim 20, it is characterised in that：The condition of the enzymic catalytic reaction is：37 DEG C anti-
It answers 48-96 hours；The cellulose derivative is any one in fibrous polysaccharaide or cellobiose；The cellulose is by pre-
Treated product is cellulose by concentrated phosphoric acid treated product.
22. preparation method according to claim 16, it is characterised in that：Further include in the multienzyme catalytic materials：Polyphosphoric acid
Glucokinase and Quadrafos；Wherein, the Quadrafos is polyphosphate sodium.
23. according to the preparation method described in claim 22, it is characterised in that：In multienzymatic reaction system, the polyphosphoric acid Portugal
The dosage of glucokinase is 5U/mL, and the dosage of the Quadrafos is 1-100 mM.
24. according to the preparation method described in claim 23, it is characterised in that：The dosage of the Quadrafos is 10 mM.
25. preparation method according to claim 16, it is characterised in that：Further include in the multienzymatic reaction system：Buffering
Liquid, divalent magnesium ion；The buffer solution is phosphate buffer；The pH value of the phosphate buffer is 5.0-9.0.
26. according to the preparation method described in claim 25, it is characterised in that：Phosphate buffer dosage is 10-500 mM, divalent
Magnesium ion dosage is 1-50 mM；
Wherein, the pH value of phosphate buffer is 7.2.
27. according to the preparation method described in claim 26, it is characterised in that：Phosphate buffer dosage be 30 mM, divalent magnesium from
Sub- dosage is 5 mM.
28. a kind of preparation method of Tagatose, which is characterized in that include the following steps：Using sucrose as substrate, addition contains sucrose
Phosphorylase, phosphoglucomutase, glucosephosphate isomerase, 6- phosphoric acid Tagatoses epimerase, 6- phosphoric acid tower lattice
The multienzyme catalytic materials of Sugar-phosphatase and inorganic phosphate radical establish multienzyme molecule machine reaction system and carry out enzymic catalytic reaction to get tower
Lattice sugar；Wherein, the enzyme gene of the 6- phosphoric acid Tagatose epimerase comes fromAgrobacterium fabrum, gene
Number on KEGG is Atu3167.
29. according to the preparation method described in claim 28, which is characterized in that in the multienzyme catalytic materials, the sucrose
The dosage of a concentration of 300 g/L, the sucrose phosphorylase are 30 U/mL, and the dosage of the phosphoglucomutase is 30
The dosage of U/mL, the glucosephosphate isomerase are 30 U/mL, and the dosage of the 6- phosphoric acid Tagatose epimerase is 30
The dosage of U/mL, the 6- phosphoric acid Tagatose phosphatase are 30 U/mL, a concentration of 2-100 mM of the inorganic phosphate radical；
The condition of the enzymic catalytic reaction is：37 DEG C of 72 h of reaction.
30. according to the preparation method described in claim 29, which is characterized in that a concentration of 30 mM of the inorganic phosphate radical.
31. according to the preparation method described in claim 28, which is characterized in that further include in the multienzyme catalytic materials：Buffer solution
With divalent magnesium ion；
Buffer solution dosage is 10-500 mM, and divalent magnesium ion dosage is 1-50 mM；The buffer solution is phosphate buffer, and pH is
5.0-9.0。
32. according to the preparation method described in claim 31, which is characterized in that phosphate buffer dosage be 30 mM, divalent magnesium from
Sub- dosage is 5 mM；Phosphate buffer pH is 7.2.
33. according to the preparation method described in claim 28, which is characterized in that further include in the multienzyme catalytic materials：Glucose
Isomerase, polyphosphoric acid glucokinase and Quadrafos.
34. according to the preparation method described in claim 33, which is characterized in that every 10 g/L sucrose, the use of glucose isomerase
Amount is 1U/mL, and the dosage of polyphosphoric acid glucokinase is 5 U/mL, and the dosage of Quadrafos is 10 mM；Wherein, the poly- phosphorus
Hydrochlorate is polyphosphate sodium.
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| US10138506B2 (en) | 2015-10-02 | 2018-11-27 | Bonumose Llc | Enzymatic production of D-tagatose |
| MA49847A (en) * | 2017-03-13 | 2021-06-02 | Bonumose Llc | ENZYMATIC PRODUCTION OF HEXOSES |
| MX2019011758A (en) * | 2017-03-31 | 2020-01-09 | Cj Cheiljedang Corp | Composition for tagatose production and method for tagatose production using same. |
| JP6961713B2 (en) * | 2017-03-31 | 2021-11-05 | シージェイ チェイルジェダン コーポレーションCj Cheiljedang Corporation | Composition for tagatose production and method for producing tagatose using it |
| CN109750024B (en) * | 2017-11-02 | 2021-12-14 | 中国科学院天津工业生物技术研究所 | 6-tagatose phosphate 4-position epimerase and application thereof |
| CN107974474B (en) * | 2018-01-09 | 2020-11-20 | 山东大学 | Method for producing D-tagatose |
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| KR102399441B1 (en) * | 2020-01-20 | 2022-05-18 | 씨제이제일제당 주식회사 | Tagatose production composition and tagatose manufacturing method using the same |
| CN112342178B (en) | 2020-11-05 | 2022-02-25 | 中国科学院天津工业生物技术研究所 | Recombinant microorganism, preparation method thereof and application thereof in producing tagatose |
| CN112342179B (en) * | 2021-01-05 | 2021-04-06 | 中国科学院天津工业生物技术研究所 | Bacillus subtilis genetic engineering bacteria for producing tagatose and method for preparing tagatose |
| CN112592378B (en) * | 2021-03-01 | 2021-06-01 | 中国科学院天津工业生物技术研究所 | Method for preparing high-purity crystalline tagatose |
| CN112708616B (en) * | 2021-03-29 | 2021-08-20 | 中国科学院天津工业生物技术研究所 | Method for producing tagatose by immobilized multienzyme system |
| CN112760316B (en) * | 2021-04-07 | 2021-08-24 | 中国科学院天津工业生物技术研究所 | Method for producing tagatose by artificial oil body immobilized multienzyme |
| CN112760317B (en) | 2021-04-07 | 2021-08-24 | 中国科学院天津工业生物技术研究所 | Method for producing tagatose by biomimetic silicified microcapsule immobilized multienzyme |
| CN113249371B (en) | 2021-07-05 | 2021-10-12 | 中国科学院天津工业生物技术研究所 | Preparation method and application of immobilized cells for tagatose production |
| CN117327684A (en) * | 2022-06-24 | 2024-01-02 | 天津怡和生物科技有限责任公司 | Enzyme for preparing tagatose, composition and application thereof |
| CN118530980A (en) * | 2024-04-30 | 2024-08-23 | 山东三元生物科技股份有限公司 | Method for producing tagatose by starch with extremely low side reaction product through one-step method |
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| US10138506B2 (en) * | 2015-10-02 | 2018-11-27 | Bonumose Llc | Enzymatic production of D-tagatose |
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