CN1786170A - High efficiency transglyco beta galactoside gene - Google Patents
High efficiency transglyco beta galactoside gene Download PDFInfo
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Abstract
The invention discloses a high efficiency turning glycosyl beta-galactosidase gene. The genes are gained by enterobacter cloacae B5 CGMCC No.1401 gene set DNA under PCR amplification. The whole length of gene nucleotide sequence is 3090 basic groups, and coding 1029 amino acid. The recombinase features are that enzyme is quart-subunit albumen; polymer molecular weight are 391kD; mono subunit are 97.8kD; the enzyme to lactose Km value is 0.32mmol/L, Vmax is 210.70umol/(L.min); and it to onitrophenol-beta-D-galactoside Km value is close to 0; its optimum reaction temperature is 35 centigrade degree; the pH value is from 6.5-9.5; it can be stored at least two months at room temperature; oligomerization galactose yield is 55%; receptor substrate specificity of the enzyme turning glycosyl is very wild; it can used to compose multi galactoside compounds. The gained enzyme gene can be used to modify gene to gain new beta-galactoside synthetase, and improve its combined efficiency fundamentally.
Description
Invention field
The present invention relates to a kind of beta-galactosidase gene, relate in particular to the application of a kind of high efficiency transglyco beta galactoside gene and described high efficiency transglyco beta galactoside gene thereof.
Background of invention
Oligose is one of key element of mammalian cell surface glycoprotein and glycolipid and microbe-derived physiologically active substance, has powerful functions such as bioinformation transmission.Thereby oligose is used for the structural analysis of drug modification, oligose and the development of oligose synthetic technology has just caused people's very big concern.Though oligose is generally acknowledged to have sizable potential as a kind of therapy by scientist, its important effect is not so far still embodied fully.One of reason of its slower development is a very difficulty of the synthetic oligose that is sufficient to clinical usage quantity.It is loaded down with trivial details that a plurality of hydroxyls that react make the chemical method synthesis step of specificity glycosidic link on the saccharide compound, is not suitable for scale operation.Enzyme process catalysis oligosaccharide synthesis relies on its simple steps, special regioselectivity and stereoselectivity, is becoming oligose and glycoconjugate synthetic main flow.
There are two fermentoids to be used for the synthetic of catalysis glycosidic link: glycosyltransferase and Glycosylase.Glycosyltransferase has been used to produce some oligose, but enzyme difficult obtains and nucleosides substrate expensive limited its widespread use.And the catalytic oligomeric glucide building-up reactions approach of Glycosylase is simple, does not need other cofactor, and substrate is generally low-cost monose and disaccharide.Enzyme more easily obtains, stable in properties.But, in the building-up reactions that Glycosylase carried out, change the newly-generated oligomeric glucide of glycosyl also can be again by enzyme as substrate hydrolysis, reaction is in hydrolysis and synthetic dynamic balance state, the output that causes changeing the glycosyl product is generally lower.In order to break this balance, scientist has carried out unremitting effort.Utilize the thermodynamics and the principle of dynamics of enzyme reaction to strengthen the output (10-40%) that concentration of substrate, raising temperature of reaction etc. can improve the glucosides product to a certain extent; (80-90% v/v) or two-phase reaction system, reduces the concentration and the activity of water, impels molecular balance to carry out to glucosides synthetic direction, has further improved the output (40-60%) of glucosides product to adopt high levels of organic solvents.But in the reaction system effective concentration of water keep and low water surrounding under the high reactivity of water all limited the final application of output and these methods of product to a certain extent.
In recent years, the development and progress of Protocols in Molecular Biology has promoted the research in Glycosylase oligosaccharide synthesis field greatly.1998, scientist carries out molecular modification to Glycosylase, replaces the nucleophilic catalysis amino-acid residue at the enzyme catalysis center with non-nucleophilic amino-acid residue, has produced a class novel active enzyme, catalytic synthesis, so called after glucosides synthetic enzyme (Glycosynthases).The appearance of glucosides synthetic enzyme has thoroughly solved the problem that product is hydrolyzed, and has fundamentally improved the commentaries on classics glycosyl efficient (changeing glycosyl efficient can reach more than 90%) of Glycosylase.Up to the present, Fa Zhan glucosides synthetic enzyme has 13 kinds in the world, derive from 11 kinds of different microorganisms and plant, obtain by beta-glucosidase, beta-glucan glycosides enzyme, beta-galactosidase enzymes, beta-Mannosidase, beta-mannase glycosides enzyme, alpha-glucosidase etc. transformations respectively.The focus of the synthetic area research of oligose is exactly an efficient commentaries on classics glycosyl Glycosylase of seeking different sources at present, and obtains the enzyme gene, obtains the glucosides synthetic enzyme so that carry out genetic modification on this basis.
Beta-galactosidase enzymes (EC3.2.1.23) is the important glycoside hydrolase of a class, and its hydrolytic activity is known for people already and utilized, and its commentaries on classics glycosyl activity just drew attention up to the eighties, was used to synthesis of oligonucleotides semi-lactosi and galactoside compound.Oligomeric galactose is one of natural constituents in the breast milk, and its most basic most important physiological function comes from its proliferation function to bifidus bacillus, has good thermostability and acid resistance, is widely used in foodstuff manufacturing and pharmaceutical industries.At present, the industrial production of oligomeric galactose is mainly synthetic by microbial enzyme method, is that substrate commentaries on classics glycosyl is synthetic with the lactose by beta-galactosidase enzymes promptly.The development research of China's oligomeric galactose also is in initial period, and autonomous microbial enzyme method industrial production still belongs to blank, the whole dependence on import of the Sumylact L that uses in the dairy industry.Therefore, obtaining high efficiency transglyco beta galactoside spontaneous development synthetic to oligose and dairy industry has very important significance.Yet glycosyl transferred beta-galactosidase is followed the mechanism of action of retention of configuration Glycosylase, catalyzed reversible reaction, and the product productive rate is subjected to certain limitation, so there is limitation when synthetic galactoside compound.This just needs further research enzyme gene, for molecular modification provides the basis, to obtain beta galactose glycosides synthetic enzyme, fundamentally improves combined coefficient.
At present, the research of external beta galactose glycosides synthetic enzyme also is confined to intestinal bacteria, the special synthetic β-1,6 of this enzyme key.Domestic beta galactose glycosides synthetic enzyme research is still blank, and the research of bacterium transglyco beta galactoside gene does not appear in the newspapers.Because the Glycosylase regioselectivity difference of different sources, so seek the high efficiency transglyco beta galactoside gene of different sources, new enzyme source can be provided for the molecular modification of Glycosylase, the efficient galactoside compound that synthesizes different of bondings is had significance.
By retrieval, the report that does not have enterobacter cloacae (Enterobacter cloacae) transglyco beta galactoside gene at present in the world.
Summary of the invention
The purpose of this invention is to provide a kind of new high efficiency transglyco beta galactoside gene,, and fill up the blank of domestic bacterium transglyco beta galactoside gene research for the acquisition of beta galactose glycosides synthetic enzyme provides new enzyme source.
Transglyco beta galactoside gene involved in the present invention derives from enterobacter cloacae (Enterobacter cloacae) B5 CGMCC No.1401, and feature is as follows:
(1) has nucleotide sequence shown in SEQ ID No.1, total length 3090 bases;
(2), have and be different from nucleotide sequence shown in the SEQ ID No.1 but the aminoacid sequence identical nucleotide sequence coded with SEQID No.1 because the degeneracy of genetic code;
(3) 1029 amino acid of this genes encoding, sequence is shown in SEQ ID No.1;
The application of above-mentioned high efficiency transglyco beta galactoside gene, its method be, with this gene clone to the pET-15b plasmid expression vector, transformed into escherichia coli BL21 (DE3), abduction delivering reorganization beta-galactosidase enzymes, feature is as follows:
(1) said gene is four protein subunits at the recombinase of expression in escherichia coli, and the polymer molecule amount is 391kD, and single molecular weight subunit is 97.8kD; Enzyme is to the K of lactose
mValue is 0.32mmol/L, V
MaxBe 210.70 μ mol/ (Lmin); Enzyme is to o-NP-β-D-galactoside (o-nitrophenyl-β-D-galactopyranoside, K ONPG)
mValue approaches 0; Enzyme is 30 ℃～42 ℃ to the temperature of the suitable reaction of ONPG, and the pH of suitable reaction is 6.5～10.6; Enzyme solution with the dipotassium hydrogen phosphate of pH7.0-potassium phosphate buffer preparation can stablize preservation at least 2 hours under 35 ℃ of conditions of temperature, can stablize preservation at least 2 months at 25 ℃; Enzyme is when preserving for 4 ℃, and stable at least preservation is 24 hours in pH6.5～10.6 scopes; Hg
2+, Cu
2+, Ag
+The activity of strongly inhibited enzyme, Co
2+, Mg
2+, Mn
2+, DTT, EDTA, imidazoles do not have restraining effect to enzymic activity;
(2) said gene has the glycosyl of commentaries on classics activity in the reaction system of recombinase at hydrolyzes lactose of expression in escherichia coli, has oligomeric galactose to generate content and Japanese goods Oligomate 50 in the reaction
Quite, up to about 55%, and the product kind has certain difference; Enzyme can be transferred to galactosyl on multiple sugar, the alkylol cpd, as semi-lactosi, wood sugar, fructose, seminose, glucose, sorbose, sucrose, cellobiose, sorbyl alcohol, inositol, N.F,USP MANNITOL etc. when being glycosyl donor with ONPG; It is subjected to sugared body substrate specificity widely, can be in order to synthetic multiple galactoside compound.
Wherein, above-mentioned recombinase is 35 ℃ to the optimal reactive temperature of ONPG.
Wherein, above-mentioned recombinase is 6.5～9.5 to the optimal reaction pH of ONPG.
In the buffering range of above-mentioned pH6.5～10.6, pH5.5～8.5 scopes are used dipotassium hydrogen phosphate-potassium phosphate buffer, and pH8.7～10.6 scopes are used glycine-sodium hydrate buffer solution.
In the application of above-mentioned high efficiency transglyco beta galactoside gene, with this gene clone to the pET-15b plasmid expression vector, can be used for genetic modification, promptly the enzyme gene is carried out rite-directed mutagenesis, replace the nucleophilic catalysis amino-acid residue at the enzyme catalysis center with non-nucleophilic amino-acid residue, to obtain new beta galactose glycosides synthetic enzyme, thoroughly remove hydrolysis reaction, fundamentally improve and change glucosides efficient.
Description of drawings
The recombinase that Fig. 1 expresses for beta-galactosidase gene of the present invention is the commentaries on classics glycosyl reaction of substrate with the lactose.
Wherein: Lane 1, lactose and the reaction of inactivator liquid; Lane 2,3, lactose and pure enzyme reaction; Lane 4, Oligomate50
The recombinase that Fig. 2 expresses for beta-galactosidase gene of the present invention is glycosyl donor with ONPG, and multiple sugar, alkylol cpd are the commentaries on classics glycosyl reaction of glycosyl acceptor.
The glycosyl acceptor that shows from left to right among the figure is followed successively by semi-lactosi, cellobiose, inositol, N.F,USP MANNITOL, wood sugar, fructose, glucose, sucrose, sorbose, sorbyl alcohol, seminose.4 samples of the reaction concurrent of every kind of acceptor molecule, preceding 2 is that control reaction was taken a sample at 0 hour, 4 hours, back 2 is to change glycosyl to be reflected at 4 hours, to take a sample in 12 hours.
Sugared spot among the figure shown in the arrow is for changeing the galactoside compound that glycosyl generates.
Embodiment
The extraction of embodiment 1 enterobacter cloacae (Enterobacter cloacae) B5 genomic dna
Enterobacter cloacae (Enterobacter cloacae) B5 CGMCC No.1401 is inoculated in the 5mLLB substratum, and 37 ℃ of shaking tables were cultivated 16 hours, got the 1.5mL culture, 12,000 rev/mins centrifugal 2 minutes, the gained precipitation is suspended from the TE damping fluid of 565 μ L; Add the sodium lauryl sulphate (SDS) of 30 μ L10% (mass volume ratio) and the Proteinase K of 5 μ L20mg/mL, mixing was in 37 ℃ of incubations 1 hour; Add the abundant mixing of 100 μ L5mol/L NaCl, add 80 μ L CTAB/NaCl solution again, mixing was in 65 ℃ of incubations 20 minutes; Ice bath is 30 minutes then; Add isopyknic phenol/chloroform/primary isoamyl alcohol (25: 24: 1, v/v/v), mixing; 12,000 rev/mins centrifugal 5 minutes, supernatant liquor is changed in the new pipe, add isopyknic phenol/chloroform/primary isoamyl alcohol again, mixing; 12,000 rev/mins centrifugal 5 minutes, supernatant is changed in the new pipe, add 2 times of volume dehydrated alcohols, mixing precipitates up to DNA gently; 12,000 rev/mins centrifugal 5 minutes, DNA precipitation is with 70% washing with alcohol 2 times; Vacuum-drying 10 minutes heavily is dissolved in the TE damping fluid of 50 μ L.
Above-mentioned LB culture medium prescription is: peptone 10g/L, yeast powder 5g/L, NaCl 7g/L, pH7.0～7.5,121 ℃ sterilization 20 minutes;
The TE buffer formulation is as follows: the Tutofusin tris of 10mmol/L (Tris), and the disodium ethylene diamine tetraacetate of 1mmol/L (EDTA), transferring pH is 8.0.
The CTAB/NaCl solution formula is as follows: the cetyltriethylammonium bromide of 10% (w/v) (CTAB) is dissolved among the NaCl of 0.7mol/L.
The pcr amplification of embodiment 2 E.cloacae B5 beta-galactosidase genes
Design primer, introducing can insert the BamHI restriction enzyme site (the pET-15b plasmid vector has N end 6His mark, is convenient to the purifying of expressing protein) of pET-15b plasmid (Novagen), and sequence is as follows:
F-B5：5’-GATCGGATCCGATGCCCAACACTCTATCG-3’
R-B5：5’-GAAGGGATCCTTAAGGGTTCTGCTGCCA-3’
Enterobacter cloacae genomic dna with extraction is a template, adopts above-mentioned primer to carry out pcr amplification.Pcr amplification system cumulative volume is 50 μ L, contains ultrapure water 35 μ L, 10 * PCR buffer, 5 μ L, dNTP2.4mmol/L, each 1 μ mol/L of primer, MgCl
21.5mmol/L, template DNA 100ng, TaKaRa LA Taq 2.5U.Pcr amplification condition: 95 ℃ of pre-sex change 5 minutes; React 28 circulations (72 ℃ were extended 3 minutes for 95 ℃ of sex change 1 minute, 52 ℃ of annealing 1 minute); 72 ℃ were extended 10 minutes after 28 loop ends.The PCR product is about about 3.1kb, through agarose gel electrophoresis, and ethidium bromide staining, uv analyzer detects, and reclaims then, and products therefrom is the full gene fragment of E.cloacae B5 beta-galactosidase enzymes.
The clone and the determined dna sequence of embodiment 3 PCR products
The PCR recovery product of embodiment 2 is connected to (TA clone) on the pMD18-T plasmid vector, Transformed E scherichia coli Top10 competent cell, coating penbritin flat board, with colony polymerase chain reaction (PCR) method screening reorganization bacterium, order-checking then, order-checking is finished by Shanghai Bo Ya Bioisystech Co., Ltd, the sequence that records is analyzed, show an open reading frame, by 3090 based compositions, 1029 amino acid of encoding are for E.cloacae B5 beta-galactosidase enzymes complete genome sequence, shown in SEQ ID No.1.
The correct reorganization bacterium that embodiment 3 is screened is inoculated in 30mL LB ammonia benzyl (100 μ g/mL) nutrient solution, 37 ℃ of shaking tables are cultivated, extract recombinant plasmid, add BamHI (TaKaRa) and cut 3 hours, carry out agarose gel electrophoresis, ethidium bromide staining in 30 ℃ of enzymes, uv analyzer detects, reclaim the purpose fragment, adopt same enzyme blanking method to handle the pET-15b plasmid vector simultaneously, and dephosphorylation.The enzyme gene fragment for preparing is mixed by a certain percentage with the pET-15b carrier, adopt to connect test kit (TaKaRa DNA Ligation Kit Ver.2.0) in 16 ℃ of connections 8 hours.Adopt CaCl then
2Conversion method transforms host bacterium E.coli BL21 (DE3), transformed bacteria liquid coating penbritin flat board, 37 ℃ of incubated overnight.Extract positive bacterium colony plasmid, the BamHI enzyme is cut checking, the reorganization bacterium that plasmid enzyme restriction is correct is expressed experiment, promptly recombinate bacterium behind the IPTG inducing culture, centrifugal acquisition mycetocyte, measure mycetocyte beta-galactosidase enzymes hydrolytic activity and change the glycosyl activity, complete satisfactory reorganization bacterium is preserved as bacterial classification.Simultaneously correct recombinant plasmid is checked order, order-checking is finished by Shanghai Bo Ya Bioisystech Co., Ltd, and the enzyme gene order that records is shown in SEQ ID No.1.
Above-mentioned beta-galactosidase enzymes hydrolytic activity is measured:
Get the 50mg mycetocyte, add the ONPG solution 450 μ L of 2mmol/L, 40 ℃ were reacted 10 minutes, added the Na of 1mL0.5mol/L
2CO
3The solution termination reaction, 12,000 rev/mins centrifugal 2 minutes, supernatant liquor is surveyed OD
400The unit of activity of enzyme regulation: the enzyme amount that discharges 1 μ mol o-NP with 1 minute hydrolysis ONPG is an enzyme activity unit (U).
Above-mentioned beta-galactosidase enzymes changes the glycosyl activity determination method:
Get the 50mg mycetocyte, be suspended in the potassium phosphate buffer of 50 μ LpH7.0,50mmol/L, after-20 ℃ of following temperature are freezing fully, put room temperature and thaw, repeat freeze thawing again 2 times, the gained suspension is the beta-galactosidase enzymes crude enzyme liquid.Get 100 μ L crude enzyme liquids, add 30% (w/v) lactose solution of 300 μ LpH7.0 potassium phosphate buffers preparations, in 50 ℃ of reactions 4 hours, 12,000 rev/mins centrifugal 5 minutes, supernatant liquor promptly contains reaction product---oligomeric galactose.
Reaction product is carried out thin-layer chromatography, and (Thin-Layer Chromatography TLC) analyzes, and determines to change the glycosyl activity.TLC thin plate (Silica gel60, No.553, Merck) behind the point sample, at developing agent (propyl carbinol: ethanol: water=5: 3: 2, v/v/v) middle exhibition layer, spray painting developer (3 of 20% sulphuric acid soln+0.5%, the 5-orcin), in 120 ℃ of bakings 10 minutes, sugared spot colour developing, if generated new oligosaccharides spot near the lactose spot except that the monose spot, then beta-galactosidase enzymes has the glycosyl of commentaries on classics activity.
The expression and purification of embodiment 5 E.cloacae B5 beta-galactosidase genes in intestinal bacteria
E.coli BL21 (DE3) the reorganization bacterium that embodiment 4 is preserved is inoculated in 200mL LB ammonia benzyl nutrient solution, cultivates 12 hours for 37 ℃, transfers in 2L LB ammonia benzyl nutrient solution, and 37 ℃ of cultivations are measured the absorbancy of nutrient solution under 600nm and changed.When light absorption value reaches 0.5～0.8, add IPTG to final concentration 1mmol/L, continue again to cultivate 3～5 hours, in 12000 rev/mins centrifugal 3 minutes, obtain cell precipitation, be suspended in the potassium phosphate buffer of 80mLpH7.0,50mmol/L.Ultrasonic disruption cell in ice-water bath, broken liquid in 4 ℃ with 12000 rev/mins centrifugal 20 minutes, the gained supernatant is crude enzyme liquid.
Crude enzyme liquid is removed the foreign protein of not being with the 6His mark with Ni-NTAAgarose (QIAGEN) affinity column chromatography, has obtained the pure beta-galactosidase enzymes of electrophoresis with DEAE Sepharose Fast Flow column chromatography then.
The basic enzymatic property of embodiment 6 E.cloacae B5 reorganization beta-galactosidase enzymes
(1) molecular weight of enzyme
The mensuration employing polyacrylamide gel electrophoresis of zymoprotein molecular weight (polyacrylamide gel electrophoresis, PAGE).PAGE with standard protein schemes the relative migration value R of working sample and standard protein per sample
f, with the R of standard protein
fValue is made typical curve to the logarithm of molecular weight.Again according to the R of pure enzyme sample
fValue just can be tried to achieve its molecular weight.SDS-PAGE adopts vertical board-like discontinuous system electrophoresis mode, and the electrophoretic separation gum concentration is 10%, and concentrated glue is 4%; Native gradient PAGE (the non-gradient polyacrylamide gel electrophoresis of dissociating) adopts the vertical tabular polyacrylamide gel of 5～10% linear gradient, 4 ℃ of electrophoresis.
Native gradient PAGE shows that the molecular weight of zymoprotein is about 391kD, and SDS-PAGE shows that zymoprotein list molecular weight subunit is about 97.8kD, shows that this enzyme is one four protein subunit.
(2) kinetic parameter of enzyme
The mensuration of the kinetic parameter of enzyme adopts two counting backward techniques.
(lactose solution of concentration range 0.043～0.34mol/L) mixes with 32.5 μ L different concns with the pure enzymes of 2.5 μ L (4.07U/mL), in 50 ℃ of reactions 30 minutes, measure the glucose content of hydrolysis with Reagent kit of glucose (Fenghui Medical Science and Technology Co., Ltd., Shanghai), calculate hydrolysis rate, record the K of enzyme lactose with the double-reciprocal plot method
mValue is 0.32mol/L, V
MaxBe 210.70 μ mol/ (Lmin).
(concentration range 0.14～1.4mmol/L) is mixed, and in 40 ℃ of reactions 3 minutes, adds the Na of 1mL0.5mol/L with the ONPG of 450 μ L different concns with the pure enzyme of one times of 50 μ L dilution
2CO
3The solution termination reaction is measured OD
400, calculate hydrolysis rate, record the K of enzyme with the double-reciprocal plot method to ONPG
mValue approaches 0, illustrates that this enzyme is very high to the avidity of ONPG.
(3) temperature and pH are to the influence of enzymic activity
In suitable temperature range (25 ℃～60 ℃), per 5 ℃ is that the relative enzyme of a gradient mensuration enzyme under this temperature lived.The result shows: the temperature of the suitable reaction of enzyme is 30 ℃～42 ℃, and optimal reactive temperature is 35 ℃.Enzyme is measured relative enzyme in insulation under the said temperature gradient after 2 hours live.The result shows: enzyme is more stable below 35 ℃, and inactivation is very fast more than 45 ℃, and 60 ℃ lose all enzymes and live.
With the damping fluid preparation enzyme reaction system of different pH, measure relative enzyme and live, the result shows: the pH scope of the suitable reaction of enzyme is wider, is pH6.5～10.6, and optimal reaction pH is 6.5～9.5; Enzyme in the damping fluid of different pH 4 ℃ preserved 24 hours, measure relative enzyme and live, the result shows: enzyme is stable in pH6.5～10.6 scopes.
The enzyme solution of pH7.0 potassium phosphate buffer preparation is put room temperature preservation, measure the stability of enzyme at room temperature (25 ℃), the result shows: enzyme can be stablized preservation at least 2 months at 25 ℃.
Above-mentioned enzyme activity determination method is: the pure enzyme that 50 μ L dilution is a times mixes with the ONPG solution of 450 μ L2mmol/L, in 40 ℃ of reactions 10 minutes, adds the Na of 1mL0.5mol/L
2CO
3The solution termination reaction is measured OD
400
Above-mentioned different pH damping fluid is respectively: acetic acid-sodium-acetate buffer pH3.6～5.0; Dipotassium hydrogen phosphate-potassium phosphate buffer pH5.5～8.5; Glycine-sodium hydrate buffer solution pH8.7～10.6; Sodium bicarbonate-sodium hydrate buffer solution pH10.0～11.0.
(4) part metals ion and compound are to the influence of enzymic activity
With ONPG is substrate, and adding final concentration in the enzyme activity determination reaction system respectively is each metal ion species of 1mmol/L, and the SDS of 10mmol/L, DTT, EDTA, imidazoles are measured relative enzyme and lived.The result shows, Hg
2+, Cu
2+, Ag
+The activity of strongly inhibited enzyme, Zn
2+With SDS enzyme also there are in various degree deactivation, Co
2+, Mg
2+, Mn
2+, DTT, EDTA, imidazoles do not have restraining effect to enzymic activity.
Embodiment 7 E.cloacae B5 recombinases are the commentaries on classics glycosyl reaction of substrate with the lactose
With 30% (w/v) lactose solution of 20 μ L pH7.0 potassium phosphate buffers preparations, with the pure enzyme of 5 μ L in 50 ℃ of reactions 4 hours, 100 ℃ are boiled 5 minutes inactivator liquid, (Thin-Layer Chromatography TLC) analyzes to carry out thin-layer chromatography then.The TLC thin plate (Silica gel60, No.553 is Merck) behind the point sample, at developing agent (propyl carbinol: ethanol: water=5: 3: 2, v/v/v) middle exhibition layer, spray painting developer (3 of 20% sulphuric acid soln+0.5%, the 5-orcin), in 120 ℃ of bakings 10 minutes, sugared spot colour developing.As Fig. 1, except that the monose spot, generated new oligosaccharides spot near the lactose spot, be to change the oligose semi-lactosi that glycosyl generates, by efficient liquid phase chromatographic analysis (High Performance LiquidChromatography, HPLC) and the thin layer scanning analysis, its content and Japanese goods Oligomate 50
Quite, up to about 55%, and kind has certain difference.
Above-mentioned HPLC analyzes equipment used and condition: Tianjin, island (SHIMADZU) high performance liquid chromatograph; Tianjin, island RID-10A differential detector; BIO-RAD Aminex HPX-42C post (300mm * 7.8mm); Moving phase is tri-distilled water, and flow velocity is 0.2mL/min, 80 ℃ of column temperatures; Interpretation of result software is Class-VP6.0.Sample with 0.2 μ m membrane filtration after, be diluted to the sugar soln of 5% (w/v), sample introduction analysis.
Above-mentioned thin layer scanning is analyzed equipment used and condition: Tianjin, island (SHIMADZU) CS-9301 dual wavelength flying spot thin layer chromatography scanner, the detection wavelength is 550nm.Above-mentioned thin layer chromatography board is carried out scanning analysis.
Embodiment 8 E.cloacae B5 recombinases are subjected to sugared body substrate specificity widely
Recombinase has and is subjected to sugared body substrate specificity widely, can with ONPG glycosyl donor, galactosyl is transferred on multiple sugar, the alkylol cpd, as semi-lactosi, wood sugar, fructose, seminose, glucose, sucrose, cellobiose, sorbose, sorbyl alcohol, inositol, N.F,USP MANNITOL etc.
100mmol/L sugar or pure receptor solution, 5 μ L100mmol/L ONPG and the pure enzyme of 5 μ L of 20 μ L 50mmol/L pH7.0 potassium phosphate buffers preparations react in 50 ℃, respectively sampling in 4 hours, 12 hours.Do control experiment simultaneously, do not add glycosyl donor ONPG, sampling in 0 hour, 4 hours.Carry out TLC then and analyze, as shown in Figure 2, when ONPG is glycosyl donor, new sugared spot has appearred below the semi-lactosi spot of original saccharide acceptor (alcohol because of can not develop the color except) and hydrolysis generation, be to change the galactoside compound that glycosyl generates, the reaction times is long more, and the product growing amount is big more.In control experiment, do not generate new sugared spot, illustrate that above-mentioned acceptor molecule can not issue in the effect of enzyme and be conigenous commentaries on classics.Recombinase is subjected to sugared body substrate characteristic widely, can be in order to synthetic multiple galactoside compound.
Sequence table
SEQ?ID?No.1
＜110〉Shandong University
＜120〉a kind of high efficiency transglyco beta galactoside gene
<141>2005-9-26
<211>3090
<212>DNA
＜213〉enterobacter cloacae (Enterobacter cloacae)
＜221〉enterobacter cloacae (Enterobacter cloacae) B5 CGMCC No.1401 glycosyl transferred beta-galactoside
The enzyme gene
<222>(1)...(3090)
<400>
atg?ccc?aac?act?cta?tcg?ctg?act?ctc?agc?gcc?att?ctg?gcc?cga?cgg?gac?tgg?gaa?aac 60
Met?Pro?Asn?Thr?Leu?Ser?Leu?Thr?Leu?Ser?Ala?Ile?Leu?Ala?Arg?Arg?Asp?Trp?Glu?Asn
1 5 10 15 20
ccg?ggt?gtc?acc?cag?tgg?aac?cgt?ctg?gag?gca?cat?gcg?ccg?tta?cac?agc?tgg?cgt?ctt 120
Pro?Gly?Val?Thr?Gln?Trp?Asn?Arg?Leu?Glu?Ala?His?Ala?Pro?Leu?His?Ser?Trp?Arg?Leu
25 30 35 40
gaa?cag?cct?gcc?ctg?gat?gat?gct?gca?tcc?gcc?agc?aga?cgc?tcc?ctc?aat?ggg?gtg?tgg 180
Glu?Gln?Pro?Ala?Leu?Asp?Asp?Ala?Ala?Ser?Ala?Ser?Arg?Arg?Ser?Leu?Asn?Gly?Val?Trp
45 50 55 60
cga?ttt?aac?tac?ttt?cct?gca?cct?gag?caa?atc?ccg?gag?gcg?tgg?gta?acc?gaa?gat?tgt 240
Arg?Phe?Asn?Tyr?Phe?Pro?Ala?Pro?Glu?Gln?Ile?Pro?Glu?Ala?Trp?Val?Thr?Glu?Asp?Cys
65 70 75 80
gca?gac?gcc?gtt?ccg?atg?ccc?gtg?ccg?tcg?aat?tgg?cag?atg?cag?ggg?ttt?gac?acg?ccc 300
Ala?Asp?Ala?Val?Pro?Met?Pro?Val?Pro?Ser?Asn?Trp?Gln?Met?Gln?Gly?Phe?Asp?Thr?Pro
85 90 95 100
atc?tac?acc?aac?gtc?acc?tat?cct?atc?ccc?gtt?aat?ccg?cct?ttt?gtg?ccg?cag?gaa?aac 360
Ile?Tyr?Thr?Asn?Val?Thr?Tyr?Pro?Ile?Pro?Val?Asn?Pro?Pro?Phe?Val?Pro?Gln?Glu?Asn
105 110 115 120
cca?acc?ggc?tgt?tac?tcg?ctc?aca?ttt?gat?gtg?gat?gac?gcg?tgg?ctt?cag?agc?ggg?cag 420
Pro?Thr?Gly?Cys?Tyr?Ser?Leu?Thr?Phe?Asp?Val?Asp?Asp?Ala?Trp?Leu?Gln?Ser?Gly?Gln
125 130 135 140
acc?cgc?atc?atc?ttt?gac?ggc?gtg?aac?tct?gcg?ttt?cat?ctg?tgg?tgc?aac?ggg?cgg?tgg 480
Thr?Arg?Ile?Ile?Phe?Asp?Gly?Val?Asn?Ser?Ala?Phe?His?Leu?Trp?Cys?Asn?Gly?Arg?Trp
145 150 155 160
atg?ggc?tat?tcc?cag?gac?agc?agg?ctg?cct?gcc?gaa?ttc?aat?ctc?tcg?acg?gtt?ctg?cgg 540
Met?Gly?Tyr?Ser?Gln?Asp?Ser?Arg?Leu?Pro?Ala?Glu?Phe?Asn?Leu?Ser?Thr?Val?Leu?Arg
165 170 175 180
ccc?ggt?gag?aac?cgc?ctg?gcg?gtc?atg?gtg?ctg?cgc?tgg?tgc?gac?ggc?agc?tat?ctg?gaa 600
Pro?Gly?Glu?Asn?Arg?Leu?Ala?Val?Met?Val?Leu?Arg?Trp?Cys?Asp?Gly?Ser?Tyr?Leu?Glu
185 190 195 200
gat?cag?gac?atg?tgg?cgc?atg?agc?gga?att?ttc?cgc?gat?gtg?acg?ctg?ctg?cat?aaa?ccc 660
Asp?Gln?Asp?Met?Trp?Arg?Met?Ser?Gly?Ile?Phe?Arg?Asp?Val?Thr?Leu?Leu?His?Lys?Pro
205 210 215 220
gaa?acg?cag?att?gcc?gat?tat?cgc?gtg?gtg?acg?gac?ctg?aat?gcg?gag?ctg?gac?cgc?gcg 720
Glu?Thr?Gln?Ile?Ala?Asp?Tyr?Arg?Val?Val?Thr?Asp?Leu?Asn?Ala?Glu?Leu?Asp?Arg?Ala
225 230 235 240
gtg?ctt?aag?gca?gat?gtc?gcg?ctg?gca?ggg?gcg?ggc?ttt?gca?gac?tgc?gag?gtg?gtc?ttt 780
Val?Leu?Lys?Ala?Asp?Val?Ala?Leu?Ala?Gly?Ala?Gly?Phe?Ala?Asp?Cys?Glu?Val?Val?Phe
245 250 255 260
acc?ctg?tgg?cgc?aag?ggt?gaa?aaa?tgc?gcc?agc?gtt?tcc?cgg?cgt?ccg?ggg?tct?gcc?gtt 840
Thr?Leu?Trp?Arg?Lys?Gly?Glu?Lys?Cys?Ala?Ser?Val?Ser?Arg?Arg?Pro?Gly?Ser?Ala?Val
265 270 275 280
gtg?gac?gag?cgt?ggc?agc?tgg?gac?gaa?cgc?tta?acg?gtg?gcg?atc?ccc?att?gac?cgc?ccc 900
Val?Asp?Glu?Arg?Gly?Ser?Trp?Asp?Glu?Arg?Leu?Thr?Val?Ala?Ile?Pro?Ile?Asp?Arg?Pro
285 290 295 300
gcg?ctc?tgg?agt?gct?gaa?acg?ccg?gaa?ctg?tat?cgc?ctg?acg?atg?gcg?ctt?ctc?ggc?ccg 960
Ala?Leu?Trp?Ser?Ala?Glu?Thr?Pro?Glu?Leu?Tyr?Arg?Leu?Thr?Met?Ala?Leu?Leu?Gly Pro
305 310 315 320
cag?ggt?gag?gtg?ctg?gag?gtt?gag?gcg?tgc?gat?gtg?ggc?ttc?cgc?cgc?gtt?gac?atc?agc 1020
Gln?Gly?Glu?Val?Leu?Glu?Val?Glu?Ala?Cys?Asp?Val?Gly?Phe?Arg?Arg?Val?Asp?Ile?Ser
325 330 335 340
aac?ggc?ctg?ctg?aag?ctt?aac?ggt?aag?ccg?ctg?ctg?atc?cgc?ggg?gtt?aac?cga?ctc?gag 1080
Asn?Gly?Leu?Leu?Lys?Leu?Asn?Gly?Lys?Pro?Leu?Leu?Ile?Arg?Gly?Val?Asn?Arg?Leu?Glu
345 350 355 360
cat?cac?ccg?gaa?aac?ggt?cag?gtg?atg?gac?gag?gcg?acc?atg?cgt?cgc?gac?atc?gaa?atc 1140
His?His?Pro?Glu?Asn?Gly?Gln?Val?Met?Asp?Glu?Ala?Thr?Met?Arg?Arg?Asp?Ile?Glu?Ile
365 370 375 380
atg?aag?cag?cat?aac?ttc?aac?gcc?gtt?cgt?tgc?tcg?cac?tac?ccg?aac?cat?ccg?ctg?tgg 1200
Met?Lys?Gln?His?Asn?Phe?Asn?Ala?Val?Arg?Cys?Ser?His?Tyr?Pro?Asn?His?Pro?Leu?Trp
385 390 395 400
tac?cgg?ctt?tgc?gat?cgc?tac?ggg?ctg?tac?gtc?gtt?gac?gaa?gcc?aat?att?gaa?acc?cac 1260
Tyr?Arg?Leu?Cys?Asp?Arg?Tyr?Gly?Leu?Tyr?Val?Val?Asp?Glu?Ala?Asn?Ile?Glu?Thr?His
405 410 415 420
ggc?atg?gtg?ccg?atg?agc?cgc?ctc?gct?gac?gat?cca?cgc?tgg?ctg?ccc?gcc?atg?agt?gag 1320
Gly?Met?Val?Pro?Met?Ser?Arg?Leu?Ala?Asp?Asp?Pro?Arg?Trp?Leu?Pro?Ala?Met?Ser?Glu
425 430 435 440
cgc?gta?acc?cgc?atg?gtg?cag?cgc?gat?cgc?aat?cat?ccc?tca?att?atc?atc?tgg?tcg?ctg 1380
Arg?Val?Thr?Arg?Met?Val?Gln?Arg?Asp?Arg?Asn?His?Pro?Ser?Ile?Ile?Ile?Trp?Ser?Leu
445 450 455 460
ggt?aac?gag?tcc?ggc?cac?ggc?gcg?aat?cat?gat?gcg?ctg?tac?cgc?tgg?ctg?aaa?acc?acc 1440
Gly?Asn?Glu?Ser?Gly?His?Gly?Ala?Asn?His?Asp?Ala?Leu?Tyr?Arg?Trp?Leu?Lys?Thr?Thr
465 470 475 480
gat?ccc?acg?cgc?ccg?gta?cag?tac?gaa?ggc?ggc?ggg?gcg?aat?acg?gcg?gcg?acc?gat?att 1500
Asp?Pro?Thr?Arg?Pro?Val?Gln?Tyr?Glu?Gly?Gly?Gly?Ala?Asn?Thr?Ala?Ala?Thr?Asp?Ile
485 490 495 500
gtt?tgc?ccg?atg?tac?gcc?cgg?gtc?gat?tgg?gat?cag?cct?ttc?ccg?gcg?gtg?cct?aaa?tgg 1560
Val?Cys?Pro?Met?Tyr?Ala?Arg?Val?Asp?Trp?Asp?Gln?Pro?Phe?Pro?Ala?Val?Pro?Lys?Trp
505 510 515 520
tca?atc?aag?aaa?tgg?atc?ggc?atg?ccc?gac?gaa?acg?cgt?ccg?ctg?atc?ctc?tgt?gaa?tac 1620
Ser?Ile?Lys?Lys?Trp?Ile?Gly?Met?Pro?Asp?Glu?Thr?Arg?Pro?Leu?Ile?Leu?Cys?Glu?Tyr
525 530 535 540
gcc?cac?gcg?atg?gga?aac?agc?ttc?ggc?ggg?ttt?gcg?aaa?tac?tgg?cag?gcg?ttt?cgc?agc 1680
Ala?His?Ala?Met?Gly?Asn?Ser?Phe?Gly?Gly?Phe?Ala?Lys?Tyr?Trp?Gln?Ala?Phe?Arg?Ser
545 550 555 560
cat?cct?cgc?ctg?cag?ggt?ggg?ttt?gtc?tgg?gac?tgg?gtc?gat?cag?gcg?ttg?acg?aag?aaa 1740
His?Pro?Arg?Leu?Gln?Gly?Gly?Phe?Val?Trp?Asp?Trp?Val?Asp?Gln?Ala?Leu?Thr?Lys?Lys
565 570 575 580
gac?gaa?aag?ggc?aac?gcg?ttc?tgg?gcc?tac?ggc?ggg?gac?ttt?ggc?gat?acg?ccg?aac?gat 1800
Asp?Glu?Lys?Gly?Asn?Ala?Phe?Trp?Ala?Tyr?Gly?Gly?Asp?Phe?Gly?Asp?Thr?Pro?Asn?Asp
585 590 595 600
cgg?cag?ttc?tgc?ctc?aat?ggc?ctg?gtc?ttc?ccc?gat?cgc?acg?ccg?cac?ccg?gcg?ctg?tac 1860
Arg?Gln?Phe?Cys?Leu?Asn?Gly?Leu?Val?Phe?Pro?Asp?Arg?Thr?Pro?His?Pro?Ala?Leu?Tyr
605 610 615 620
gag?gcg?cag?cgc?gcc?cag?cag?ttc?ttt?acc?ttt?acg?ctg?gtc?agc?acc?tct?ccg?ctg?atg 1920
Glu?Ala?Gln?Arg?Ala?Gln?Gln?Phe?Phe?Thr?Phe?Thr?Leu?Val?Ser?Thr?Ser?Pro?Leu?Met
625 630 635 640
att?gag?gtg?caa?agc?ggc?tac?ctg?ttc?cgc?cct?acc?gat?aac?gag?gtg?ctg?agc?tgg?acg 1980
Ile?Glu?Val?Gln?Ser?Gly?Tyr?Leu?Phe?Arg?Pro?Thr?Asp?Asn?Glu?Val?Leu?Ser?Trp?Thr
645 650 655 660
gtt?gcc?cgg?gat?ggg?aag?gta?ctg?gct?tcg?ggc?gag?gtc?acg?ctg?gcg?ata?gcc?cct?gaa 2040
Val?Ala?Arg?Asp?Gly?Lys?Val?Leu?Ala?Ser?Gly?Glu?Val?Thr?Leu?Ala?Ile?Ala?Pro?Glu
665 670 675 680
ggc?gtt?cag?cgt?ctg?gag?atc?gct?ctg?ccg?gaa?ttg?aaa?gcc?ggg?ccg?ggt?gaa?atc?tgg 2100
Gly?Val?Gln?Arg?Leu?Glu?Ile?Ala?Leu?Pro?Glu?Leu?Lys?Ala?Gly?Pro?Gly?Glu?Ile?Trp
685 690 695 700
ctg?aac?gtt?gag?gtt?cgc?cag?cct?cgg?gca?acg?ccg?tgg?tca?ccg?gca?ggc?cat?cgc?tgc 2160
Leu?Asn?Val?Glu?Val?Arg?Gln?Pro?Arg?Ala?Thr?Pro?Trp?Ser?Pro?Ala?Gly?His?Arg?Cys
705 710 715 720
gcg?tgg?gag?cag?tgg?ccg?ctt?ccg?gcc?cca?ctc?ttt?att?gca?ccg?cca?gcc?tct?acg?ggc 2220
Ala?Trp?Glu?Gln?Trp?Pro?Leu?Pro?Ala?Pro?Leu?Phe?Ile?Ala?Pro?Pro?Ala?Ser?Thr?Gly
725 730 735 740
gag?ccg?ccg?gtt?tta?acg?caa?aac?gat?cgc?atc?ctg?gag?gtc?aca?cat?cgt?caa?cag?cgc 2280
Glu?Pro?Pro?Val?Leu?Thr?Gln?Asn?Asp?Arg?Ile?Leu?Glu?Val?Thr?His?Arg?Gln?Gln?Arg
745 750 755 760
tgg?cag?ttc?gat?cgc?gca?tcc?gga?tac?ctg?act?caa?tgg?tgg?cga?aat?ggc?gtt?gaa?acg 2340
Trp?Gln?Phe?Asp?Arg?Ala?Ser?Gly?Tyr?Leu?Thr?Gln?Trp?Trp?Arg?Asn?Gly?Val?Glu?Thr
765 770 775 780
ctg?ctt?tca?ccc?gtg?acg?gat?aac?gtc?agc?cgc?gcg?ccg?ctg?gac?aac?gac?att?ggg?gtg 2400
Leu?Leu?Ser?Pro?Val?Thr?Asp?Asn?Val?Ser?Arg?Ala?Pro?Leu?Asp?Asn?Asp?Ile?Gly?Val
785 790 795 800
agc?gaa?gcg?acg?cgc?atc?gat?cca?aac?gcg?tgg?gtt?gaa?cgc?tgg?aaa?gcg?gcg?ggc?atg 2460
Ser?Glu?Ala?Thr?Arg?Ile?Asp?Pro?Asn?Ala?Trp?Val?Glu?Arg?Trp?Lys?Ala?Ala?Gly?Met
805 810 815 820
tac?gat?ctc?acc?tcg?cgc?atg?ctg?cac?tgt?gag?gca?gag?cag?cat?gcg?cgt?gag?gtg?gtg 2520
Tyr?Asp?Leu?Thr?Ser?Arg?Met?Leu?His?Cys?Glu?Ala?Glu?Gln?His?Ala?Arg?Glu?Val?Val
825 830 835 840
gtt?acc?acg?ctt?aat?gtc?ctg?gag?cat?cgc?ggc?agg?gcg?ctg?ttc?ctg?agc?cgt?aaa?atc 2580
Val?Thr?Thr?Leu?Asn?Val?Leu?Glu?His?Arg?Gly?Arg?Ala?Leu?Phe?Leu?Ser?Arg?Lys?Ile
845 850 855 860
tgg?cgg?ctg?gac?gag?cag?ggg?gtt?ctg?cat?ggc?gac?att?cag?gtc?gat?att?gcg?tct?gat 2640
Trp?Arg?Leu?Asp?Glu?Gln?Gly?Val?Leu?His?Gly?Asp?Ile?Gln?Val?Asp?Ile?Ala?Ser?Asp
865 870 875 880
atc?ccg?aag?ccc?gcg?cgc?att?ggc?ctg?agc?gtt?cat?ctc?gct?gaa?acg?cca?gaa?aag?gtt 2700
Ile?Pro?Lys?Pro?Ala?Arg?Ile?Gly?Leu?Ser?Val?His?Leu?Ala?Glu?Thr?Pro?Glu?Lys?Val
885 890 895 900
gac?tgg?ctg?ggg?ctg?ggg?ccg?cat?gaa?aac?tac?ccg?gac?aga?aag?ctg?gcg?gcg?cag?cag 2760
Asp?Trp?Leu?Gly?Leu?Gly?Pro?His?Glu?Asn?Tyr?Pro?Asp?Arg?Lys?Leu?Ala?Ala?Gln?Gln
905 910 915 920
ggg?cgc?tgg?aca?ctg?ccc?ctg?gca?gac?atg?cac?acg?ccg?tat?atc?ttc?ccg?acg?gaa?aat 2820
Gly?Arg?Trp?Thr?Leu?Pro?Leu?Ala?Asp?Met?His?Thr?Pro?Tyr?Ile?Phe?Pro?Thr?Glu?Asn
925 930 935 940
ggt?ttg?cgc?tgc?gat?acc?cgc?aaa?ctt?gtg?ctg?ggg?gcg?cat?cag?ctg?aat?ggc?gcg?ttc 2880
Gly?Leu?Arg?Cys?Asp?Thr?Arg?Lys?Leu?Val?Leu?Gly?Ala?His?Gln?Leu?Asn?Gly?Ala?Phe
945 950 955 960
cat?ttc?tca?gtg?ggc?cgc?tat?agc?cag?cag?caa?ctg?cgt?gag?aca?acc?cat?cat?cat?ctg 2940
His?Phe?Ser?Val?Gly?Arg?Tyr?Ser?Gln?Gln?Gln?Leu?Arg?Glu?Thr?Thr?His?His?His?Leu
965 970 975 980
ctg?cgg?gaa?gag?ccg?ggg?ggc?tgg?ctc?aac?ctc?gat?gcg?ttt?cat?atg?ggg?gtg?ggc?ggc 3000
Leu?Arg?Glu?Glu?Pro?Gly?Gly?Trp?Leu?Asn?Leu?Asp?Ala?Phe?His?Met?Gly?Val?Gly?Gly
985 990 995 1000
gat?gac?tcc?tgg?agc?ccc?agc?gtt?tcg?ccg?gaa?ttt?atc?ctg?cag?acg?cgc?cag?ctt?cgc 3060
Asp?Asp?Ser?Trp?Ser?Pro?Ser?Val?Ser?Pro?Glu?Phe?Ile?Leu?Gln?Thr?Arg?Gln?Leu?Arg
1005 1010 1015 1020
tat?acc?ttt?agc?tgg?cag?cag?aac?cct?taa 3120
Tyr?Thr?Phe?Ser?Trp?Gln?Gln?Asn?Pro
1025
Claims (8)
1. high efficiency transglyco beta galactoside gene has following feature:
(1) has nucleotide sequence shown in SEQ ID No.1, total length 3090 bases;
(2), have and be different from nucleotide sequence shown in the SEQ ID No.1 but the aminoacid sequence identical nucleotide sequence coded with SEQID No.1 because the degeneracy of genetic code;
(3) 1029 amino acid of this genes encoding, sequence is shown in SEQ ID No.1.
2. the application of high efficiency transglyco beta galactoside gene according to claim 1 is characterized in that, with described gene clone to the pET-15b plasmid expression vector, at expression in escherichia coli reorganization beta-galactosidase enzymes;
Wherein: said gene is four protein subunits at the recombinase of expression in escherichia coli, and the polymer molecule amount is 391kD, and single molecular weight subunit is 97.8kD; Enzyme is to the K of lactose
mValue is 0.32mmol/L, V
MaxBe 210.70 μ mol/ (Lmin); Enzyme is to o-NP-β-D-galactoside (o-nitrophenyl-β-D-galactopyranoside, K ONPG)
mValue approaches 0; Enzyme is 30～42 ℃ to the temperature of the suitable reaction of ONPG, and the pH of suitable reaction is 6.5～10.6; Enzyme solution with the dipotassium hydrogen phosphate of pH7.0-potassium phosphate buffer preparation can stablize preservation at least 2 hours under 35 ℃ of conditions of temperature, can stablize preservation at least 2 months at 25 ℃; Enzyme is when preserving for 4 ℃, and stable at least preservation is 24 hours in pH 6.5～10.6 scopes; Hg
2+, Cu
2+, Ag
+The activity of strongly inhibited enzyme, Co
2+, Mg
2+, Mn
2+, DTT, EDTA, imidazoles do not have restraining effect to enzymic activity; Enzyme has the glycosyl of commentaries on classics activity in the reaction system of hydrolyzes lactose, have oligomeric galactose to generate in the reaction, and productive rate reaches 55%; Enzyme can be transferred to galactosyl on multiple sugar, the alkylol cpd when being glycosyl donor with ONPG.
3. as the application of high efficiency transglyco beta galactoside gene as described in the claim 2, it is characterized in that described enzyme is 35 ℃ to the optimal reactive temperature of ONPG.
4. as the application of high efficiency transglyco beta galactoside gene as described in the claim 2, it is characterized in that described enzyme is 6.5～9.5 to the optimal reaction pH of ONPG.
5. as the application of high efficiency transglyco beta galactoside gene as described in claim 2 or 4, it is characterized in that, in the buffering range of described pH 6.5～10.6, pH5.5～8.5 scopes are used dipotassium hydrogen phosphate-potassium phosphate buffer, and pH8.7～10.6 scopes are used glycine-sodium hydrate buffer solution.
6. as the application of high efficiency transglyco beta galactoside gene as described in the claim 2, it is characterized in that described sugar, alkylol cpd are meant semi-lactosi, wood sugar, fructose, seminose, glucose, sorbose, sucrose, cellobiose, sorbyl alcohol, inositol, N.F,USP MANNITOL.
7. as the application of high efficiency transglyco beta galactoside gene as described in claim 2 or 6, it is characterized in that, described enzyme has and is subjected to sugared body substrate specificity widely, galactosyl is transferred on described sugar, the alkylol cpd through enzyme, can be in order to synthetic multiple galactoside compound.
8. the application of the described high efficiency transglyco beta galactoside gene of claim 1, it is characterized in that, with described gene clone to the pET-15b plasmid expression vector, can be used for genetic modification, promptly the enzyme gene is carried out rite-directed mutagenesis, replace the nucleophilic catalysis amino-acid residue at the enzyme catalysis center with non-nucleophilic amino-acid residue, to obtain new beta galactose glycosides synthetic enzyme.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100448979A CN100370029C (en) | 2005-10-11 | 2005-10-11 | High efficiency transglyco beta galactoside gene |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100448979A CN100370029C (en) | 2005-10-11 | 2005-10-11 | High efficiency transglyco beta galactoside gene |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1786170A true CN1786170A (en) | 2006-06-14 |
| CN100370029C CN100370029C (en) | 2008-02-20 |
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|---|---|---|---|
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103276030A (en) * | 2013-06-25 | 2013-09-04 | 山东大学 | Preparation method of resveratrol galactoside |
| CN105765067A (en) * | 2013-09-30 | 2016-07-13 | 天野酶株式会社 | Modified beta-galactosidase |
| CN106543244A (en) * | 2016-11-08 | 2017-03-29 | 山东大学 | The preparation method of galactose type rhodioside and its derivant |
| CN112725313A (en) * | 2021-01-27 | 2021-04-30 | 中国海洋大学 | Preparation and application of beta-galactosidase |
| CN113265434A (en) * | 2021-05-19 | 2021-08-17 | 吉林大学 | Method for synthesizing UDP-galactose and galactosyl compound |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3212798B2 (en) * | 1994-06-14 | 2001-09-25 | 寳酒造株式会社 | Hyperthermostable β-galactosidase gene |
| DK38893D0 (en) * | 1993-03-31 | 1993-03-31 | Novo Nordisk As | DNA |
| HUP0102605A2 (en) * | 1998-06-09 | 2001-11-28 | U.S. Department Of Agriculture | Genes coding for tomato betha-galactosidase polypeptides |
-
2005
- 2005-10-11 CN CNB2005100448979A patent/CN100370029C/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103276030A (en) * | 2013-06-25 | 2013-09-04 | 山东大学 | Preparation method of resveratrol galactoside |
| CN105765067A (en) * | 2013-09-30 | 2016-07-13 | 天野酶株式会社 | Modified beta-galactosidase |
| CN106543244A (en) * | 2016-11-08 | 2017-03-29 | 山东大学 | The preparation method of galactose type rhodioside and its derivant |
| CN106543244B (en) * | 2016-11-08 | 2019-07-26 | 山东大学 | The preparation method of galactose type rhodioside and its derivative |
| CN112725313A (en) * | 2021-01-27 | 2021-04-30 | 中国海洋大学 | Preparation and application of beta-galactosidase |
| CN113265434A (en) * | 2021-05-19 | 2021-08-17 | 吉林大学 | Method for synthesizing UDP-galactose and galactosyl compound |
| CN113265434B (en) * | 2021-05-19 | 2023-05-02 | 吉林大学 | Method for synthesizing UDP-galactose and method for synthesizing galactosyl compound |
Also Published As
| Publication number | Publication date |
|---|---|
| CN100370029C (en) | 2008-02-20 |
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