CN103789377A - Technique for developing allulose-containing functional jujube juice through biotransformation of jujube monosaccharide - Google Patents
Technique for developing allulose-containing functional jujube juice through biotransformation of jujube monosaccharide Download PDFInfo
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Abstract
The invention relates to a two-enzyme co-expression technique and a two-enzyme coupling and immobilizing technique, and specifically relates to a co-expression preparation method of a site-specific mutant glucose isomerase and a D-allulose 3-epimerase, a coupling and immobilizing technique of the two enzymes and application of the coupled and immobilized two enzymes in producing allulose from jujube condensed juice. In order to solve health problems of people due to high-calorific value sugar, the invention provides a method for promoting the production of low-calorific value D-allulose from the recombinase glucose isomerase and the D-allulose 3-epimerase through co-immobilization and two-enzyme coupling by virtue of a multiple-enzyme coupling and immobilizing technique with D-glucose and D-fructose in the jujube condensed juice as the substrates, and also provides a method for site-specific mutagenesis of an amino acid at the 146th site of the glucose isomerase and co-expression thereof with the D-allulose 3-epimerase.
Description
Technical field
The present invention relates to two enzyme coexpression technology and two enzyme coupling and immobilization technology, be specifically related to coupling and immobilization technology and the application of the fixing two enzymes of coupling in red date inspissated juice production psicose of a kind of rite-directed mutagenesis glucose isomerase and D-Psicose 3-epimerase coexpression preparation method and this pair of enzyme.
Background technology
Red date integrates nutrition, health care, medicinal function, lists first dietotherapeutic fruit of the Ministry of Health in.Red date is rich in glucide, and sugar degree occupies first of all kinds of fruits, also higher than sugaring raw material sugarcane, beet.Fresh jujube sugar degree reaches 20%-36%, dry jujube sugar degree is up to 50-80%, carbohydrate in jujube can be divided into following three classes: monose (being mainly glucose and fructose), disaccharide and polysaccharide, wherein fructose content accounts for 43.95%, glucose accounts for 42.32%, sucrose accounts for 3.85%, oligose and polysaccharide account for 9.86%.
China's red date saccharic aboundresources, is rich in the raw material such as glucose and fructose in red date inspissated juice.But fructose and glucose are all high energy capacity materials, absorption meeting brings huge physical stress to body, and the crowd's ratio that causes fat constantly increases, and diabetes and cardiovascular disorder crowd increase year by year, and present the trend of becoming younger.Although there do not have remarkable evidence to prove to be fat directly related with diabetes and cardiovascular disorder, to control energy and take in promotion health, reduce Fat Accumulation, the effect that reduces the generation of diabetes and cardiovascular disorder chronic diseases gains public acceptance.The high sugar food that the contains higher-energy shelf that fade out gradually, functional food low in calories, that have greater functionality nutritive property receives an acclaim in market.
D-Psicose is the epimer of D-Fructose, and closely similar with D-Fructose aspect sweetness intensities and kind.But, different from D-Fructose, in D-Psicose assimilation process in vivo, be difficult to metabolism, and there is the heat supply compared with low degree.Therefore, D-Psicose can be as the effective constituent of meals.The current sugar alcohol that is widely used as non-sugar sweetener can cause side effect in the time absorbing more than prescribed dose, as diarrhoea, and D-Psicose does not have such side effect, and D-Psicose has the function that the activity by suppressing steatogenesis enzyme slims the abdomen.Therefore, D-Psicose can be used as sweeting agent, and this sweeting agent is also to contributing to weight saving.In this connection, D-Psicose has attracted a large amount of interest as the sweeting agent of diet, and the needs of the method increase to exploitation High-efficient Production D-Psicose existing in foodstuffs industry.This is because D-Psicose, only as the intermediate in theriae processing or glucose isomerization reaction process, is very marginally present at occurring in nature, and can not chemosynthesis.
Xylose isomerase (xylose isomerase, Xiase, EC 5.3.1.5), claim again glucose isomerase (glucose isomerase, GIase), can catalysis D-Glucose outside born of the same parents to the isomerization reaction of D-Fructose, be that upper one of the key enzyme of high fructose syrup of preparing take starch as raw material is produced in foodstuffs industry.
D-Psicose 3-epimerase (D-psicose 3-epimerase, is abbreviated as DPE) is to realize at present a kind of Major Enzymes of D-Fructose to bio-transformation between D-Psicose, belongs to ketose 3-epimerization enzyme family, and the suitableeest substrate is D-Psicose.Therefore, using gene engineering technique is prepared the recombinase of glucose isomerase and D-Psicose 3-epimerase, can effectively D-Glucose be converted into D-Fructose, be converted into D-Psicose by D-Fructose again.
In single enzyme application, exist to environment sensitive, the unstable easy inactivation of character the problem of the difficult separating-purifying of product.Enzyme immobilization technology can not only be saved the cost of enzyme, improve the stability in use of enzyme, simplify later separation step, more can be by several enzymes being carried out to coimmobilization to realize the concerted reaction of multi-enzyme system, in reaction process, reduce the sphere of action of enzyme-to-substrate, increase enzyme reactant concn around, improve the efficiency of reaction.Along with the research application of multi-enzyme system, multi-enzyme system altogether fixing and coreaction has become an active research field, is one of the forward position in enzyme engineering field.
The immobilization of multi-enzyme system can be divided into the multienzyme co-immobilization technology that the mixed immobilization zymotechnic that is based upon on single enzyme immobilization basis and new development are got up.Mixed immobilization enzyme is that single enzyme is respectively fixed on different carriers and is mixed in same reaction system, but because this kind of enzyme is fixed on different carriers, in reaction, material transfer will overcome double diffusion barrier, and enzymatic reaction is restricted, and need to further improve carrier mass-transfer performance.
Multienzyme co-immobilization refers to plurality of enzymes is fixed in identical carrier, the feature of different enzymes can be given full play to and is mutually combined, and gets rid of the interfering factors of measuring in thing, thereby utilizes the synergistic effect of enzyme to make catalytic efficiency higher than single immobilized enzyme.The application of multienzyme co-immobilization technology has also solved the mass transfer problem between multienzyme in mixed immobilization, by having reduced mass transfer Distance Shortened the transhipment time of substrate, efficiency and the life cycle of multi-enzyme system have been improved simultaneously, more be conducive to the separation and Extraction of product, reduced from every side production cost.Along with the further investigation of immobilization technology, a large amount of novel comprehensive fixation supports and easy process for fixation are used widely, and this will make multienzyme linked reaction system in suitability for industrialized production, bring into play larger effect.
Using gene engineering technique builds reconstitution cell and by recombinant cell culture, makes that the synthetic other biological in-vivo content of its excess is atomic but to have very high economic worth recombinant protein be one of most active research direction in modern biotechnology.China Science & Technology University's Life Science College is exclusively born State Scientific and Technological Commission's 863 projects " protein engineering of glucose isomerase ", complete at present the protein engineering transformation of glucose isomerase through tackling of key scientific and technical problems in 15 years, obtained the gene recombination glucose isomerase of character improvement; And utilize the high gene recombination streptomycete industrial producing strain of gene engineering method construction expression amount, possess the ripe scientific and technological condition of gene recombination glucose isomerase industrialization.Some investigators from jerusalem artichoke pseudomonas (
pseudomonas cichorii) in separation and purification obtain D-tagatose 3-epimerase (DTE), the bio-reactor made from recombinant expressed enzyme immobilization is take the fructose of 60% (w/v) as substrate, transformation efficiency reaches 25%.Investigator also by agrobacterium tumefaciens (
agrobacterium tumefaciens) in the DNA recombinant expression of D-tagatose 3-epimerase albuminoid obtain D-Psicose 3-epimerase (D-psicose 3-epimerase); In addition, some investigators from soil, screen obtain root nodule bacterium (
sinorhizobium sp.), the D-Fructose of its infiltrationization cell take 70% is as substrate, and after 15h reaction, D-Psicose concentration reaches 37mg/mL.The at present not open document that glucose isomerase and D-Psicose 3-epimerase coexpression is obtained simultaneously to two kinds of enzymes, the also disclosure to 146 amino acids rite-directed mutagenesises to glucose isomerase not.
Summary of the invention
The health problem causing to people for solving high calorie sugar, the invention provides and a kind of as substrate, adopt multienzyme coupling and immobilization technology by the method that after recombinase glucose isomerase, D-Psicose 3-epimerase co-immobilization, two enzyme couplings promote to produce D-Psicose low in calories using D-Glucose, D-Fructose in red date inspissated juice; Provide glucose isomerase 146 amino acids rite-directed mutagenesises simultaneously, and with the method for D-Psicose 3-epimerase coexpression.
For solving the problems of the technologies described above, the technical solution adopted in the present invention is: a kind of bio-transformation red date monose exploitation, containing the technology of the functional jujube juice of psicose, is carried out according to following steps:
The first step, obtains goal gene
Be proline(Pro) by the arginine rite-directed mutagenesis of 146 of glucose isomerase enzyme amino acid sequence, then nucleotide sequence and the D-Psicose 3-epimerase nucleotide sequence of the glucose isomerase after sudden change carried out respectively to pcr amplification; By after the nucleotide sequence of the glucose isomerase of the described rite-directed mutagenesis after amplification and D-Psicose 3-epimerase nucleotide sequence difference purifying, clone respectively glucose isomerase nucleotide sequence and the D-Psicose 3-epimerase nucleotide sequence of described rite-directed mutagenesis;
Second step, the structure of recombinant expression plasmid
The glucose isomerase nucleotide sequence of rite-directed mutagenesis obtained above and pCDFDuet-1 carrier are used respectively
bam HIwith
kpn Icarry out double digestion, D-Psicose 3-epimerase nucleotide sequence and pCDFDuet-1 carrier are used respectively
bgl IIwith
hind IIIcarry out double digestion, enzyme is cut after the purified recovery of product, uses T
4ligase enzyme spends the night in 16 ℃ of connections, transforms
e.colidH5 α competent cell, streptomycin resistance plate screening transformant, identify respectively, and picking positive transformant checks order by PCR and double digestion method; Product proceeds to abduction delivering in Host Strains by the plasmid of positive transformant again and obtains recombinant bacterium;
The 3rd step, by recombinant bacterium through centrifugal collection, and ultrasonication, recentrifuge obtains the glucose isomerase of rite-directed mutagenesis and the crude enzyme liquid of D-Psicose 3-epimerase coexpression;
The 4th step, immobilization
The crude enzyme liquid obtaining is added in resin, every 1.0g resin enzyme concentration: crude enzyme liquid prepared by 0.5 ~ 2ml gene recombination, adsorption temp is 25 ℃ ~ 35 ℃, absorption pH=6.5 ~ 7.5, adsorption time 100 ~ 140min, then add linking agent, crosslinked being fixed of 25 ~ 35min enzyme, described linking agent volume parts is 0.03% ~ 0.06%;
The 5th step, catalysis jujube juice is produced psicose
Jujube juice is added in described immobilized enzyme, control temperature: 60 ~ 70 ℃; PH value: 5.0 ~ 7.0; The weight part ratio of described jujube juice and immobilized enzyme is 1 ~ 5:1; 5 ~ 10 as a child completed catalyzed reaction, obtained D-Psicose mixed solution.
In the described the first step, the step of rite-directed mutagenesis is:
Take pET-28a-GI as template, the upstream and downstream mutant primer of design complete complementary pairing; Carry out PCR reaction by following program: 95 ℃ of 3 min; 94 ℃ of 30s, 60 ℃ of 30 s, 68 ℃ of 6min, totally 16 circulations; 68 ℃ of 10min; PCR product, after DpnI digestion, transforms
e.colidH5 α competent cell, coats the LB flat board containing 10% kantlex; Picking transformant is cultivated and is checked order, if not correctly sudden change of order-checking discovery, picking list bacterium colony checks order or again after sudden change, checks order again, until suddenly change successfully;
Described upstream and downstream mutant primer is:
F-AACTTATTTACCCATCCTCGCTTTGTCCATGGC
R-GCCATGGACAAAGCGAGGATGGGTAAATAAGTT。
The condition that glucose isomerase nucleotide sequence after described the first step rite-directed mutagenesis and D-Psicose 3-epimerase nucleotide sequence carry out pcr amplification is:
Sudden change glucose isomerase nucleotide sequence primer:
Upstream primer: 5 '-GA
aGATCTcATGCCTTATTTTGACAACATCAGCA-3 '
Downstream primer: 5 '-GG
gGTACCtTAACGGGCTGCGCAAACTT-3 '
D-Psicose 3-epimerase nucleotide sequence primer:
Upstream primer: 5 '-CG
gGATCCgATGAAATATGGTATTTATTACGCT-3 '
Downstream primer: 5 '-CCC
aAGCTTgACTTCAAATACATGTTTTACA-3 '
PCR reaction system (50 μ L) is 10 × Taq buffered soln, 5 μ L, template DNA 1 μ L, and concentration is 10 μ molL
-1primer 2 μ L, concentration is 2.5mmolL
-1dNTPs 4 μ L, TaqDNA polysaccharase 0.3 μ L, ultrapure water 35.7 μ L, mix; Reaction conditions: 95 ℃ of 5min; 94 ℃ of 45s, 53 ℃ of 45s, 72 ℃ of 2min, 32 circulations; 72 ℃ of 10min.
In described second step, the glucose isomerase nucleotide sequence of described rite-directed mutagenesis and the double digestion enzyme of pCDFDuet-1 carrier are
bamHIwith
kpn I; The double digestion enzyme of described D-Psicose 3-epimerase nucleotide sequence and pCDFDuet-1 carrier is
bgl IIwith
hind III.
In described second step, the condition of abduction delivering is: the recombinant bacterium building is cultivated in substratum, spent the night in 37 ℃ of water-bath shaking culture, transfer in the 250mL triangular flask of 30mL substratum is housed by 1% inoculum size, continue to be cultured to OD
600be 0.6 ~ 0.8 o'clock, adding IPTG final concentration is 1mmolL
-1, 20 ℃ of abduction deliverings.
In described the 4th step, be 4:1 as the glucose isomerase of the rite-directed mutagenesis in preferred adjusting crude enzyme liquid and the weight part ratio of D-Psicose 3-epimerase.
Described resin is bisphenol-A epoxy vinyl ester.
Described linking agent is the CaCl of glutaraldehyde, saturated boric acid and 1wt%
2the NaNO of the mixing solutions of solution or 50 wt %
3the CaCl of solution and 1 wt %
2the mixing solutions of solution, preferably glutaraldehyde.
Compared with prior art the present invention has following beneficial effect.
1, glucose isomerase 146 amino acids in genus bacillus source are arginine (Arg), and at high temperature, deamination reaction easily occurs arginine.The α nook of Arg in the middle of 2 β-pleated sheet structures, Pro possesses certain rigid structure with respect to Arg, and the corner mobility of formation is little, may affect the thermostability of GI.After rite-directed mutagenesis, 146 amino acids arginine (Arg) become proline(Pro) (Pro), and Pro belongs to imino-acid, in the 2nd amino acid of genus bacillus glucose isomerase β-bend, have the effect of the thermostability of Enhancin.
2, ionic radius is larger, and its hydrated ionic radius is less, is more conducive to it and is drilled into the region of depression.For Mg
2+, Co
2+and Mn
2+plasma radius is less than the active ions of 0.08 nm, when after ion and enzyme combination, reactive site (hydrophobic region) is exposed, so that and Binding Capacity.Ca
2+plasma radius is greater than the inhibitor of 0.08 nm, and hydration radius is less, more easily pierces active pocket and enzyme is combined closely, and hydrophobic region is reduced, the effect that has on the contrary inhibitory enzyme to live.After rite-directed mutagenesis, glucose isomerase 146 amino acids arginine (Arg) become proline(Pro).The α nook of this amino acid in the middle of 2 β-pleated sheet structures, Pro possesses certain rigid structure with respect to Arg, and the corner mobility of formation is little.146 amino acids approach the active centre of GI, GI space conformation generation subtle change, Ca
2+be difficult for piercing active pocket, and enzyme active center is in conjunction with loose, has reduced interference, has improved fructose productive rate.
3, genetic engineering technique is prepared recombinase: glucose isomerase, D-Psicose 3-epimerase content that nature exists are few, vigor is low, wild strain is cultivated and is difficult for (anaerobism) and enzyme preparation more difficult (producing enzyme level low), scale operation purifying process is comparatively complicated, has limited its broadened application.Therefore, build engineering bacteria by gene recombination technology, it is significant that secreting, expressing high reactivity glucuroide is realized industrialization to it.
4, the selection of bacterial classification: through screening after, derive from bacillus (
bacillus sp.) glucose isomerase enzyme activity vigor compared with other bacterial classification source the highest; Derive from
ruminococcus sp.d-Psicose 3-epimerase compared with deriving from the D-Psicose 3-epimerase, D-tagatose 3-epimerase of other bacterial classification, thermostability is best, D-Fructose transforms and generates the most effective of D-Psicose, is more suitable for the industrial production of D-Fructose generation D-Psicose.
5, the selection of plasmid: the present invention need express glucose isomerase and two kinds of enzymes of D-Psicose 3-epimerase simultaneously, the pCDFDuet-1 selecting is protokaryon co-expression plasmid, containing two T7 promotors, comprise two polygene binding sites, can two target proteins of coexpression, overcome the uncompatibility of two plasmids in same host.
6, the zymologic property of recombinase: the character of heterogenous expression product (recombinase) usually changes because of the physiological characteristic of molecule manipulation and the expression host cell adopting, this result of study shows, recombinase glucose isomerase (GI) compared with natural enzyme, does not have change in essence with the main zymologic property of D-Psicose 3-epimerase (DPE).Wherein, optimum temperature, the suitableeest action pH, thermostability are similar to natural enzyme; Aspect metal ion dependency, when two enzyme coexpressions, remove Mg
2+outside dependency changes to some extent, without significantly changing.
7, in single enzymatic reaction, the synthetic precursor of enzyme process generally obtains by multistep chemosynthesis reaction, and the synthetic cost that it is too high and loaded down with trivial details step have often affected the competitive power of target product; And in substrate coupling multienzymatic reaction system, can be from wide material sources or compound with low cost, the synthetic intermediate obtaining with a kind of enzyme catalysis, obtain target product through another kind of enzyme catalysis again, reduce the separation and Extraction step of intermediate, shorten reaction process, can greatly reduce separation costs and reduce environmental pollution.
8, the present invention is fixed on two kinds of enzymes in identical carrier, the characteristic of two kinds of different enzymes is given full play to and is mutually combined, get rid of the interfering factors of measuring in thing, thereby utilize the synergistic effect of enzyme, the transfer time of substrate that reduced mass transfer Distance Shortened, has improved efficiency and the life cycle of dual-enzyme system, is conducive to the separation and Extraction of product, reduce production cost, made catalytic efficiency higher than single immobilized enzyme and mixed immobilization enzyme.
9, the mode that being fixed of spent ion exchange resin can be adsorbed by ion-exchange, but absorption method is fixed on the firm not of zymoprotein combination on resin, in use easily come off, thus the method that the present invention has selected absorption to be combined with cross-linked phase, to increase immobilization effect; Select 6 factors such as enzyme concentration, adsorption time, absorption pH, adsorption temp, dosage of crosslinking agent, crosslinking time to carry out immobilization optimization experiment.
10, the explanation of the present invention to coupling and immobilization technology scheme: the preparation of GI and DPE immobilized enzyme: take pretreated resin as carrier, add the enzyme liquid of appropriate amount, and add a certain amount of glutaraldehyde to be cross-linked.Select enzyme concentration, adsorption time, absorption pH, adsorption temp, dosage of crosslinking agent, 6 factors of crosslinking time to carry out immobilization optimization experiment.By measuring the rate of recovery of two enzymes, show that optimum fixing condition is: every 1.0g resin enzyme concentration 0.5 ~ 2ml crude enzyme liquid is (when enzyme concentration exceedes 2ml, enzyme molecular density on carrier increases, steric effect aggravation, enzyme molecular inactivation, can cause the immobilized enzyme rate of recovery to decline), adsorption time 100 ~ 140min, adsorption temp is 25 ℃ ~ 35 ℃, absorption pH=6.5 ~ 7.5, crosslinking time 25 ~ 35min, glutaraldehyde volume fraction is 0.03% ~ 0.06%, with this understanding, the two enzyme rate of recovery reach respectively 20%, 25%.
By after two kinds of enzyme co-immobilizations of above-mentioned preparation so that in jujube juice, glucose, fructose are as substrate, linked reaction is produced D-Psicose low in calories.
(1) for guaranteeing the original flavor of jujube juice, pH value is carried out to adjusting among a small circle, find pH5.0 ~ 7.0 o'clock, can generate psicose more than 70g/L.HPLC detects red date sugar conversion results as shown in Figure 1.
The primitive reaction condition of (2) two enzymes in jujube juice: temperature: 60 ~ 70 ℃; PH value: 5.0 ~ 7.0; The ratio of jujube juice and immobilized enzyme is 3:1 ~ 1:1; 6 ~ 10 as a child reached balance, sampling per hour.Fig. 2 is glucose and the fructose content of simulation jujube juice, and two enzyme couplings promote the reaction process that psicose generates.
When reaction reaches balance, glucose, fructose, the ratio of psicose is approximately 45:40:15.Take 1L jujube juice as raw material, can generate psicose and be approximately 85g.In red date concentrated solution, rare sugared content reaches 7.0%.
(3) simulation jujube juice glucose, fructose content, get 10mL solution, and Fig. 3 is for adding 7.35mg Ca
2+rear two enzyme coupling generates the balanced reaction of psicose.
Result shows: jujube juice complicated component, Ca
2+content is too much understood the activity of severe inhibition GI, and when fructose generates psicose, when balance is carried out to the right, glucose cannot regeneration fructose.GI in the present invention is the enzyme producing after rite-directed mutagenesis, and when thermostability improves, the variation of space conformation has reduced Ca
2+to the restraining effect of reaction process.
Accompanying drawing explanation
Fig. 1 is the red date sugar conversion figure that HPLC detects.
Fig. 2 is not for adding Ca with sugared content in simulation jujube juice
2+for two enzyme linked reaction fate maps of reaction solution.
Fig. 3 adds Ca with sugared content in simulation jujube juice
2+for two enzyme linked reaction fate maps of reaction solution.
Fig. 4 is that the crude enzyme liquid of the enzymatic glucose isomerase that contains rite-directed mutagenesis is thermally-stabilised.
Fig. 5 is the crude enzyme liquid of the enzymatic glucose isomerase that contains rite-directed mutagenesis and the thermostability comparison diagram of mutant enzyme glucose isomerase crude enzyme liquid in the time of 80 ℃ not.
Fig. 6 is the enzymatic glucose isomerase (Line1) of the rite-directed mutagenesis after codon optimized and the SDS-PAGE comparison diagram of the enzymatic glucose isomerase (Line2) of sudden change.
Fig. 7 is pCDFDuet-1 plasmid vector collection of illustrative plates.
Fig. 8 is protein expression SDS-PAGE analysis chart.
Fig. 9 is temperature, the affect broken line graph of pH value on psicose growing amount.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described.
The present invention adopts the enzyme source of genus bacillus as glucose isomerase, and intestinal bacteria are as recombinase nucleotide sequence Host Strains.
The idiographic flow that first three step of the present invention adopts genetic engineering technique to prepare coexpression recombinase is:
Bacterial screening
gI rite-directed mutagenesis
carrier is selected
obtain goal gene
construction of recombinant expression plasmid
the abduction delivering of target protein
sDS-PAGE
analysis and Identification
zymologic property is analyzed.
one, enzyme gene source:
Filter out through primary dcreening operation and multiple sieve, Molecular Identification and zymologic property research the bacterial strain that glucose isomerase activity is the highest, utilize the BLAST software in NCBI to carry out 16SrDNA sequential analysis discovery to the highest bacterial strain of activity, this bacterial strain and bacillus (
bacillus sp.) homology is nearest.Again according to homologous sequence search, and download the 16SrDNA sequence of some relevant bacterial classifications, utilize MEGA4.0 software to carry out constructing system evolutionary tree, further explore this bacterial strain and genus bacillus (
bacillus sp.). sibship, find this bacterial strain with
bacillus sp.evolutionary distance is the shortest, and this result of comparing with BLAST is consistent.
D-Psicose 3-epimerase (D-psicose 3-epimerase), according to the sequence of the DPE of agrobacterium tumefaciens, BLAST on NCBI finds, provides " conservative putative protein " sequence (ZP_04858451) synthetic according to Takara Bio Inc company
ruminococcus sp.DPEthe complete nucleotide sequence of gene.
to the selection in glucose isomerase rite-directed mutagenesis site
The glucose isomerase of different sources is carried out to sequence alignment of protein, and by DS software, different thermophilic bacterium glucose isomerase three-dimensional structures are analyzed, 146 amino acids that discovery derives from thermophilic bacterium glucose isomerase are proline(Pro) (Pro), and the glucose isomerase in this genus bacillus source is arginine (Arg).At high temperature, easily there is deamination reaction in arginine, the α nook of this amino acid in the middle of 2 β-pleated sheet structures, and Pro possesses certain rigid structure with respect to Arg, and the corner mobility of formation is little, may affect the thermostability of GI.Therefore select site R146P to carry out site-directed mutagenesis.
to the rite-directed mutagenesis of glucose isomerase
Primer: F-AACTTATTTACCCATCCTCGCTTTGTCCATGGC
R-GCCATGGACAAAGCGAGGATGGGTAAATAAGTT
Carrier: pET28a-GI
Adopt the method for amplification plasmid, take pET-28a-GI as template, the upstream and downstream mutant primer of design complete complementary pairing.Carry out PCR reaction by following program: 95 ℃ of 3min; 94 ℃ of 30s, 60 ℃ of 30s, 68 ℃ of 6min, totally 16 circulations; 68 ℃ of 10min.PCR product, after DpnI digestion, transforms
e.colidH5 α competence, coats the LB(LB-Kana containing 10% kantlex) flat board.The order-checking of Jin Wei intelligence bio tech ltd is cultivated and delivered to picking transformant.If not correctly sudden change of order-checking discovery, picking list bacterium colony checks order or again after sudden change, checks order again, until suddenly change successfully.
The glucose isomerase of sudden change has been carried out to thermal stability determination simultaneously:
Saltant type crude enzyme liquid is incubated processing in 60,70,80,90 ℃ of water-baths in buffer system, sampling is at regular intervals measured glucose isomerase enzyme and lived, and residual enzyme result of variations alive is shown in Fig. 4.Fig. 4 result shows, 60 ℃ time, is incubated 24h, can also retain 90% enzyme work; 70 ℃ and 80 ℃ can retain respectively 78% and 50% enzyme work, under 90 ℃ of ultrahigh-temperature, also can keep the stability of 4h after insulation 24h.And can find out in Fig. 5, wild-type crude enzyme liquid only has 35% enzyme work at 80 ℃ of insulation 24h, compare saltant, has reduced by 15%.Illustrate and be mutated into after Pro, strengthened the thermostability of this glucose isomerase.The Pro that this result hint glucose isomerase is 146 plays vital effect to maintaining its thermostability impact.Pro due to its structure entropy than other amino acid little and foldable more, once and folding, need the very high energy could unfolding, do not affecting under the prerequisite of its higher structure like this, Pro substitutes the thermostability that can improve whole protein molecule.Pro belongs to imino-acid, it is generally acknowledged, when Pro is at the N-terminal of α spiral or in the time that 2 β-pleated sheet structures are middle, has the effect of the thermostability of Enhancin.146 amino acids of genus bacillus glucose isomerase are sported to Pro, in the 2nd amino acid of β-bend, think and have very large effect to maintaining its thermostability by the Pro of 146.
Also glucose isomerase codon is optimized to be increased in the high efficient expression in intestinal bacteria: wild-type glucose isomerase is sent to Jin Weizhi company and carry out codon optimized, the mensuration of this glucose isomerase protein content is take wild-type glucose isomerase as contrast, by gel imaging instrument detection computations, restructuring wild type strain with and codon optimize recombinant bacterial strain, its glucose isomerase protein content accounts for respectively 15% and 25% of soluble proteins, as shown in Figure 6.
two, the selection of genophore:
PCDFDuet-1 plasmid (as shown in Figure 7), containing two open reading frame, comprises two polygene binding sites, has a T before each site
7lacpromotor and a ribosome bind site, can two target proteins of coexpression; Also can with pACYCDuet-1 (Cat.No.71147-3), pRSFDuet-1 (Cat. No. 71341-3), pETDuet-1 (Cat. No. 71146-3) 4 ~ 8 target proteins of coexpression in a suitable host strain that combine.The present invention selects pCDFDuet-1 plasmid to glucose isomerase and two kinds of enzyme coexpressions of D-Psicose 3-epimerase.
three, obtain goal gene:
(1) amplification of design of primers and object fragment:
According to the upper hot genus bacillus of GenBank (
bacillus sp.) the Nucleotide over design primer of glucose isomerase.Upstream primer: 5 '-GA
aGATCT(underscore part is CATGCCTTATTTTGACAACATCAGCA-3 '
bgl IIrestriction enzyme site), downstream primer: 5 '-GG
gGTACC(underscore part is TTAACGGGCTGCGCAAACTT-3 '
kpn Irestriction enzyme site).On NCBI
ruminococcus sp.the Nucleotide over design upstream primer of D-Psicose 3-epimerase (DPE): 5 '-CG
gGATCC(underscore part is GATGAAATATGGTATTTATTACGCT-3 '
bamH Irestriction enzyme site), downstream primer: 5 '-CCC
aAGCTT(underscore part is GACTTCAAATACATGTTTTACA-3 '
hind IIIrestriction enzyme site).
PCR reaction system (50 μ L) is: 10 × Taq buffered soln, 5 μ L template DNA 1 μ L, concentration is 10 μ molL
-1primer 2 μ L, dNTPs(2.5mmolL
-1) 4 μ L, TaqDNA polysaccharase 0.3 μ L, ultrapure water 35.7 μ L, mix.Reaction conditions: 95 ℃ of 5min; 94 ℃ of 45s, 53 ℃ of 45s, 72 ℃ of 2min, 32 circulations; 72 ℃ of 10min.
(2) clone of gene and sequential analysis:
To after pcr amplification product purifying, be connected on pCDFDuet-1 plasmid vector, transform
e
.coli
dH5 α competent cell.Screening obtains and contains
dpewith
xylAgene masculine clone compares at NCBI and existing glucose isomerase enzyme sequence, D-Psicose 3-epimerization enzyme sequence after order-checking.
Four
, recombinant expression plasmid structure
Glucose isomerase enzyme mutant gene obtained above and pCDFDuet-1 are used respectively
bamHIwith
kpn Icarry out double digestion, D-Psicose 3-epimerase gene and pCDFDuet-1 are used respectively
bgl IIwith
hind IIIcarry out double digestion, enzyme is cut after the purified recovery of product, uses T
4ligase enzyme spends the night in 16 ℃ of connections, Transformed E.
colidH5 α competent cell, streptomycin resistance plate screening transformant, identify respectively, and picking positive transformant checks order by PCR and double digestion method.Product proceeds to abduction delivering in expressive host bacterium by the plasmid of positive transformant again.
five, the abduction delivering of target protein
The recombinant bacterial strain building is cultivated in substratum, spent the night in 37 ℃ of water-bath shaking culture, transfer in the 250mL triangular flask of 30mL substratum is housed by 1% inoculum size, continue to be cultured to OD
600be 0.6 ~ 0.8 o'clock, adding IPTG final concentration is (1mmolL
-1), 20 ℃ of abduction deliverings.After 16h, 4000rmin
-1frozen centrifugation is collected thalline, thalline is suspended from the phosphate buffer solution of pH value 7.0 to ice-bath ultrasonic.Ultrasonic power is 200W, every ultrasonic 5s interval 8s, totally 70 times.
six, SDS-PAGE Analysis and Identification
By centrifugal after the cell pyroprocessing with after ultrasonic disruption, collect supernatant liquor, be crude enzyme liquid.Get respectively cleer and peaceful whole cell and carry out SDS-PAGE, resolving gel concentration is 10%, and concentrated gum concentration is 5%, coomassie brilliant blue R_250 dyeing, and Fig. 8 is the protein expression situation detecting.
Through SDS-PAGE electrophoretic analysis, be that an obvious protein band is expressed at 43kD place at relative molecular mass as can be seen from Figure 8, the same with the glucose isomerase size of report.There is an obvious protein band at relative molecular mass 33kD place, show that RDPE expresses successfully.Recombinant protein all exists with soluble form with this understanding.
seven, the zymologic property analysis of glucose isomerase and D-Psicose 3-epimerase
Above-mentioned recombinant protein glucose isomerase (GI) and D-Psicose 3-epimerase (DPE) are carried out to zymologic property analysis.
(1) temperature, pH value and metal ion impact and the thermostability on psicose growing amount
The speed of the D-Psicose 3-epimerization enzymic activity of the glucose isomerase of BGI and RDPE take its isomerization fructose as D-Psicose is determined.Reaction system is 1mL(1mol/L glucose; 100mmol/L PIPES damping fluid, pH7.0; 1mmol/L MgCl
2).30min is carried out in isomerization reaction at 65 ℃.Spectrophotometric determination, the psicose HPLC for fructose that form measure.Isomerase vigor definition: the needed enzyme amount of per minute generation 1mg fructose is 1 glucose isomerase enzyme activity unit under testing conditions; It is a D-Psicose 3-epimerization enzyme activity unit that per minute generates the required enzyme amount of 1mg psicose.
1. study BGI and the optimum temperature of RDPE coexpression, the suitableeest action pH and metal ion dependency.Fig. 9 is temperature, the affect broken line graph of pH value on psicose growing amount.
Under differing temps, measure the enzyme of BGI and RDPE and live, live as reference (being designated as 100%) using the enzyme recording at 65 ℃, result is as shown in Fig. 9: the optimum temperature of BGI and RDPE coexpression is 60 ℃ ~ 70 ℃.Under different pH conditions, measure the enzyme of BGI and RDPE and live, live as reference (being designated as 100 %) using the enzyme recording under pH7.0, result as shown in Figure 9: the suitableeest action pH of BTGI and RDPE coexpression is 7.0.Under different ions, measure the enzyme of BGI and RDPE and live, with 10 mmol/L CoCl
2the enzyme recording under existence is lived as reference (being designated as 100 %), and result shows that the activity of BTGI and RDPE coexpression relies on Mg
2+.
In sum, 65 ° of C of optimum temperuture after two enzyme coexpressions, optimum pH is 7.0, adds Mg
2+there is the effect of very strong promotion to generating psicose.
2. thermal stability determination is at 20mmol/L PBS damping fluid (pH7.0) 1mmol/L MgCl
2in carry out; PH Stability Determination carries out in the damping fluid (pH5.0 ~ 11.0) adapting to.
BGI and RDPE are placed in to pH7.0 damping fluid, and under differing temps, temperature is bathed different time, then measures remaining enzyme and lives, and thermostability experimental result shows, BTGI and RDPE be at 70 ℃, and 65 ℃, the transformation period of living of the enzyme at 55 ℃ is respectively 4h, 8h and 24h.When 70 ° of C, after insulation 4h, almost there is no psicose conversion capability, when 65 ° of C, after insulation 8h, lose psicose conversion capability, 55 ° of C, after insulation 24h, can also keep 80% activity.
By BGI and RDPE in different pH damping fluids at 65 ℃ temperature bathe 10 min, cooling in ice bath after, measure remaining enzyme and live, result shows that BTGI and RDPE have good stability between pH7.0 ~ 8.0.
The crude enzyme liquid obtaining take aforesaid method is enzyme source, in conjunction with following specific embodiment, the two enzyme couplings of the present invention and immobilized technology catalysis jujube juice is prepared to psicose and is described further.
A kind of method that adopts two enzyme coupling immobilization catalysis jujube juice to produce psicose is carried out according to following steps:
The first step, immobilization
Crude enzyme liquid is added in bisphenol-A epoxide vinylester resin, every 1.0g resin enzyme concentration 2ml crude enzyme liquid, adsorption temp is 35 ℃, absorption pH=7.5, adsorption time 140min, then adds linking agent glutaraldehyde, crosslinking time 35min, described linking agent volume parts is 0.06%, being fixed enzyme;
Second step, two enzyme coupling catalysis jujube juice is produced psicose
Jujube juice is added in described immobilized enzyme, control temperature: 70 ℃; PH value: 7.0; The weight part ratio of described jujube juice and immobilized enzyme is 2:1; 10 as a child completed catalyzed reaction, obtained psicose mixed solution.
A kind of method that adopts two enzyme coupling immobilization catalysis jujube juice to produce psicose is carried out according to following steps:
The first step, immobilization
Crude enzyme liquid is added in bisphenol-A epoxide vinylester resin, every 1.0g resin enzyme concentration 0.5ml crude enzyme liquid, in crude enzyme liquid, the ratio of two kinds of enzymes is 4:1, adsorption temp is 25 ℃, absorption pH6.5, adsorption time 100min, then add linking agent glutaraldehyde, crosslinking time 25min, described linking agent volume parts is 0.03%, being fixed enzyme;
Second step, two enzyme coupling catalysis jujube juice is produced psicose
Jujube juice is added in described immobilized enzyme, control temperature: 60 ℃; PH value: 5.0; The weight part ratio of described jujube juice and immobilized enzyme is 1:1; 6 as a child completed catalyzed reaction, obtained psicose mixed solution.
Embodiment 3
A kind of method that adopts two enzyme coupling immobilization catalysis jujube juice to produce psicose is carried out according to following steps:
The first step, immobilization
Crude enzyme liquid is added in bisphenol-A epoxide vinylester resin, every 1.0g resin enzyme concentration 1ml crude enzyme liquid, adsorption temp is 30 ℃, absorption pH=7, adsorption time 120min, then adds linking agent glutaraldehyde, crosslinking time 30min, described linking agent volume parts is 0.05%, being fixed enzyme;
Second step, catalysis jujube juice is produced psicose
Jujube juice is added in described immobilized enzyme, control temperature: 65 ℃; PH value: 6.0; The weight part ratio of described jujube juice and immobilized enzyme is 3:1; 8 as a child completed catalyzed reaction, obtained psicose mixed solution.
The present invention can summarize with other the specific form without prejudice to spirit of the present invention or principal character.Therefore, no matter from that, above-mentioned embodiment of the present invention all can only think explanation of the present invention can not limit invention, claims have been pointed out scope of the present invention, and scope of the present invention is not pointed out in above-mentioned explanation, therefore, any variation in implication and the scope suitable with claims of the present invention, all should think to be included in the scope of claims.
Claims (8)
1. the exploitation of bio-transformation red date monose, containing a technology for the functional jujube juice of psicose, is characterized in that carrying out according to following steps:
The first step, obtains goal gene
Be proline(Pro) by the arginine rite-directed mutagenesis of 146 of glucose isomerase enzyme amino acid sequence, then nucleotide sequence and the D-Psicose 3-epimerase nucleotide sequence of the glucose isomerase after sudden change carried out respectively to pcr amplification; By after the nucleotide sequence of the glucose isomerase of the described rite-directed mutagenesis after amplification and D-Psicose 3-epimerase nucleotide sequence difference purifying, clone respectively glucose isomerase nucleotide sequence and the D-Psicose 3-epimerase nucleotide sequence of described rite-directed mutagenesis;
Second step, the structure of recombinant expression plasmid
The glucose isomerase nucleotide sequence of rite-directed mutagenesis obtained above and pCDFDuet-1 carrier are used respectively
bam HIwith
kpn Icarry out double digestion, D-Psicose 3-epimerase nucleotide sequence and pCDFDuet-1 carrier are used respectively
bgl IIwith
hind IIIcarry out double digestion, enzyme is cut after the purified recovery of product, uses T
4ligase enzyme spends the night in 16 ℃ of connections, transforms
e.colidH5 α competent cell, streptomycin resistance plate screening transformant, identify respectively, and picking positive transformant checks order by PCR and double digestion method; Product proceeds to abduction delivering in Host Strains by the plasmid of positive transformant again and obtains recombinant bacterium;
The 3rd step, by recombinant bacterium through centrifugal collection, and ultrasonication, recentrifuge obtains the glucose isomerase of rite-directed mutagenesis and the crude enzyme liquid of D-Psicose 3-epimerase coexpression;
The 4th step, immobilization
The crude enzyme liquid obtaining is added in resin, every 1.0g resin enzyme concentration: crude enzyme liquid prepared by 0.5 ~ 2ml gene recombination, adsorption temp is 25 ℃ ~ 35 ℃, absorption pH=6.5 ~ 7.5, adsorption time 100 ~ 140min, then add linking agent, crosslinked being fixed of 25 ~ 35min enzyme, described linking agent volume parts is 0.03% ~ 0.06%;
The 5th step, catalysis jujube juice is produced psicose
Jujube juice is added in described immobilized enzyme, control temperature: 60 ~ 70 ℃; PH value: 5.0 ~ 7.0; The weight part ratio of described jujube juice and immobilized enzyme is 1 ~ 5:1; 5 ~ 10 as a child completed catalyzed reaction, obtained D-Psicose mixed solution.
2. a kind of bio-transformation red date monose exploitation according to claim 1, containing the technology of the functional jujube juice of psicose, is characterized in that the step of rite-directed mutagenesis in the described the first step is:
Take pET-28a-GI as template, the upstream and downstream mutant primer of design complete complementary pairing; Carry out PCR reaction by following program: 95 ℃ of 3 min; 94 ℃ of 30s, 60 ℃ of 30 s, 68 ℃ of 6min, totally 16 circulations; 68 ℃ of 10min; PCR product, after DpnI digestion, transforms
e.colidH5 α competent cell, coats the LB flat board containing 10% kantlex; Picking transformant is cultivated and is checked order, if not correctly sudden change of order-checking discovery, picking list bacterium colony checks order or again after sudden change, checks order again, until suddenly change successfully;
Described upstream and downstream mutant primer is:
F-AACTTATTTACCCATCCTCGCTTTGTCCATGGC
R-GCCATGGACAAAGCGAGGATGGGTAAATAAGTT。
3. a kind of bio-transformation red date monose exploitation according to claim 1 is containing the technology of the functional jujube juice of psicose, it is characterized in that the condition that glucose isomerase nucleotide sequence after described the first step rite-directed mutagenesis and D-Psicose 3-epimerase nucleotide sequence carry out pcr amplification is:
Sudden change glucose isomerase nucleotide sequence primer:
Upstream primer: 5 '-GA
aGATCTcATGCCTTATTTTGACAACATCAGCA-3 '
Downstream primer: 5 '-GG
gGTACCtTAACGGGCTGCGCAAACTT-3 '
D-Psicose 3-epimerase nucleotide sequence primer:
Upstream primer: 5 '-CG
gGATCCgATGAAATATGGTATTTATTACGCT-3 '
Downstream primer: 5 '-CCC
aAGCTTgACTTCAAATACATGTTTTACA-3 '
PCR reaction system (50 μ L) is 10 × Taq buffered soln, 5 μ L, template DNA 1 μ L, and concentration is 10 μ molL
-1primer 2 μ L, concentration is 2.5mmolL
-1dNTPs 4 μ L, TaqDNA polysaccharase 0.3 μ L, ultrapure water 35.7 μ L, mix; Reaction conditions: 95 ℃ of 5min; 94 ℃ of 45s, 53 ℃ of 45s, 72 ℃ of 2min, 32 circulations; 72 ℃ of 10min.
4. a kind of bio-transformation red date monose exploitation according to claim 1 is containing the technology of the functional jujube juice of psicose, it is characterized in that: in described second step, the glucose isomerase nucleotide sequence of described rite-directed mutagenesis and the double digestion enzyme of pCDFDuet-1 carrier are
bamHIwith
kpn I; The double digestion enzyme of described D-Psicose 3-epimerase nucleotide sequence and pCDFDuet-1 carrier is
bgl IIwith
hind III.
5. a kind of bio-transformation red date monose exploitation according to claim 1 is containing the technology of the functional jujube juice of psicose, it is characterized in that: in described second step, the condition of abduction delivering is: the recombinant bacterium building is cultivated in substratum, spend the night in 37 ℃ of water-bath shaking culture, transfer in the 250mL triangular flask of 30mL substratum is housed by 1% inoculum size, continue to be cultured to OD
600be 0.6 ~ 0.8 o'clock, adding IPTG final concentration is 1mmolL
-1, 20 ℃ of abduction deliverings.
6. a kind of bio-transformation red date monose exploitation according to claim 1 is containing the technology of the functional jujube juice of psicose, it is characterized in that: in described the 4th step, the weight part ratio of the glucose isomerase of the rite-directed mutagenesis in crude enzyme liquid and D-Psicose 3-epimerase is 4:1.
7. a kind of bio-transformation red date monose exploitation according to claim 1, containing the technology of the functional jujube juice of psicose, is characterized in that: described resin is bisphenol-A epoxy vinyl ester.
8. a kind of bio-transformation red date monose exploitation according to claim 1, containing the technology of the functional jujube juice of psicose, is characterized in that: described linking agent is the CaCl of glutaraldehyde, saturated boric acid and 1wt%
2the NaNO of the mixing solutions of solution or 50 wt %
3the CaCl of solution and 1 wt %
2the mixing solutions of solution.
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| CN108013306A (en) * | 2017-10-28 | 2018-05-11 | 中国科学院天津工业生物技术研究所 | The preparation method of juice drinks rich in psicose |
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| CN114592015A (en) * | 2022-04-18 | 2022-06-07 | 河北农业大学 | Method for converting fructose in jujube fruits into psicose by using psicose epimerase and application of method |
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| CN106879991A (en) * | 2017-02-19 | 2017-06-23 | 沧州恩际生物制品有限公司 | A kind of hypoglycemic processing method for jujube class extract solution |
| CN106879991B (en) * | 2017-02-19 | 2021-03-26 | 沧州恩际生物制品有限公司 | Blood sugar reducing treatment method for jujube extract |
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| WO2021244005A1 (en) * | 2020-06-03 | 2021-12-09 | 中国科学院天津工业生物技术研究所 | Allulose 3-epimerase mutant, engineered bacterium expressing same, and immobilized enzyme and immobilization method thereof |
| CN114592015A (en) * | 2022-04-18 | 2022-06-07 | 河北农业大学 | Method for converting fructose in jujube fruits into psicose by using psicose epimerase and application of method |
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