CN102911923A - Alpha-glycosidase, coding gene, vector, engineering bacterium and application of alpha-glycosidase, coding gene, vector and engineering bacterium - Google Patents
Alpha-glycosidase, coding gene, vector, engineering bacterium and application of alpha-glycosidase, coding gene, vector and engineering bacterium Download PDFInfo
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Abstract
The invention provides alpha-glycosidase, a coding gene, a vector, an engineering bacterium and application of the alpha-glycosidase, the coding gene, the vector and the engineering bacterium. An amino acid sequence of the alpha-glycosidase is shown as a sequence of SEQ ID NO.1. As compared with Xanthomonas campestris, the novel recombination gomphosis alpha-glycosidase is glycosidase, and has certain obvious functional improvement including that the enzymatic activity is improved by about 6 times, the activity is high under a neutral pH (potential of hydrogen) condition, and the activity under the condition that the pH is 6.0 is about 30 times of the activity of glycosidase of the Xanthomonas campestris. Besides, the novel functional improvement of the actual sequence is realized as compared with the actual sequence, and the alpha-glycosidase can be widely applied to galactosylated modification economical chemical compounds and has wide application prospect. The alpha-glycosidase XcG-A is easy to realize prokaryotic expression, and can be efficiently produced by the recombinant vector, a strain and a preparation method, and the preparation method for the alpha-glycosidase is simple, convenient and efficient, easy to realize abundant expression and suitable for industrial production.
Description
(1) technical field
The present invention relates to a kind of alpha-glycosidase (being alpha-glucosidase), encoding gene, carrier, engineering bacteria and application of grand gene source.
(2) background technology
(α-glycosidase) is a kind of in the Starch Hydrolysis enzyme to alpha-glucosidase, it can be from the α-glucose glycosidic bond of the non-reduced terminal hydrolysis substrate of polysaccharide, produce alpha-D-glucose, this enzyme also possesses the transglycosylase activity, can be with glycosyl donors such as maltose, the catalysis glycosylation is modified the acceptor compound of multiple hydroxyl.At present, alpha-glucosidase has been widely used in comprising the technical fields such as Starch Hydrolysis, zymamsis and chemosynthesis.But the industrial application class alpha-glucosidase of developing at present especially possesses the enzyme of higher transglycosylation, and also very limited, people's exigence is developed more, possesses the new enzyme of alpha-glucosidase of industrial required zymologic property.
In the comparatively thorough alpha-glucosidase of at present known research, wherein the most attractive is the Glycosylase that derives from Xanthomonas campestris, this enzyme can be widely used in and turn the glucosides reaction, the a series of multifarious glucosides acceptor that turns of catalysis comprises quinhydrones, catechin, menthol, elemenol, glycerine and simple alcohol compound.What deserves to be explained is, the alpha-glucosaccharase enzyme catalysis turn the feature that glucosides reaction possesses does not need expensive activation glycosyl donor, traditional glycosyltransferase of comparing, this is enzymatic to turn the glucosides reaction and has obvious technical superiority.Simultaneously, the new glucosides class substrate that generates because turning the glucosides catalytic reaction, can give economic compounds acceptor new beneficial property, comprise improve thermostability, improve water-soluble, strengthen the biological activity of resistance of oxidation and raising etc., so this enzyme possesses wide application space.
Technique of metagenome is a kind of new microbial gene excavating resource technology of the eighties of last century appearance nineties, but be different from traditional culturing micro-organisms technology, using this technology directly to carry out genome to " all " of environmental microorganism extracts, can totally study the microorganism in the environmental sample, the appearance of this technology and utilization, so that originally surpass 99% can not the culturing micro-organisms genetic resources research and development become reality, this has expanded the exploration space of microbial gene resource greatly.
(3) summary of the invention
The object of the invention provides a kind of alpha-glycosidase (being alpha-glucosidase), encoding gene, carrier, engineering bacteria and application of grand gene source.
A kind of alpha-glycosidase XcG-A of grand gene source, its aminoacid sequence is shown in SEQ ID NO.1:
MSQTPWWRGAVIYQIYPRSFLDSNGDGVGDLPGIIAKLDYIAGLGVDAIWISPFFKSPMADFGYDISDFRDVDPMFGTLADFDRLLAKAHALGLRVMIDQVLGHSSVEHEWFKESRESRDNPKADWYVWADARPDGTPPNNWLSIFGGVAWKWEPRRGQYYLHNFLSSQPDLNFHCPDVRAAMLDSVKFWLDRGVDGFRLDSINFPFHDAQLRDNPPKPVEARSGRGFSADNPYAFQYHYYNNTQPENLALLEELRALMDRYPGVATLGEISSEDSLATMAEYVTEQRLHMGYSFELLVDDFSAAFIRGTVEQLEAKMSDGWPCWAISNHDVRRAVTRWGGATPSPALASQLVALVCSLRGSVCLYQGEELGLSEAEVAFEDLQDPYGITFWPTFKGRDGCRTPMPWTDAPSAGFTSGKPWLPLAASHRAAAVSVQQDDAHSVLSAVRDFLAWRKEMPALREGSIAFYDTAEPVLMFRREHLGQVMLLAFNLSADPADLALPAGEWEQIDVPGVELGAMEGGHLRLAGHGVVAAVGRG。
The XcG-A of alpha-glycosidase described in the present invention compares with the alpha-glycosidase that derives from Xanthomonas campestris, possesses the features such as active height and pH tolerance range be wide.
Because the singularity of aminoacid sequence; any fragment or its variant that contains the peptide protein of aminoacid sequence shown in the SEQ ID NO.1; such as its examples of conservative variations, bioactive fragment or derivative; as long as the fragment of this peptide protein or peptide protein variant and aforementioned amino acid sequence homology all belong to the row of protection domain of the present invention more than 95%.Concrete described change can comprise amino acid whose disappearance, insertion or replacement in the aminoacid sequence; Wherein, for the conservative property change of variant, the amino acid of replacing has the structure similar to original acid or chemical property, and as replacing Isoleucine with leucine, variant also can have non-conservation and change, as replacing glycine with tryptophane.The fragment of peptide protein of the present invention, derivative or analogue refer to basically keep the identical biological function of alpha-glycosidase of the present invention or active peptide protein, can be under conditions: (I) one or more amino-acid residues are guarded or non-conservative amino-acid residue (preferably conservative amino acid residues) replaces, and the amino acid that replaces can be also can not encoded by genetic codon; Certain group on (II) one or more amino-acid residues is replaced by other group; (III) mature peptide albumen and another kind of compound (such as the compound that prolongs the peptide protein transformation period, for example polyoxyethylene glycol) merge; (IV) additional aminoacid sequence is integrated into ripe peptide protein and the peptide protein sequence that forms sequence or the proteinogen sequence of this peptide protein of purifying (as be used for).
Described peptide protein can be recombinant protein, native protein or synthetic proteins, it can be the product of pure natural purifying, or the product of chemosynthesis, or use recombinant technology from protokaryon or eucaryon host (for example: bacterium, yeast, higher plant, insect and mammalian cell), to produce.The host used according to the recombinant production scheme, peptide protein of the present invention can be glycosylated.Peptide protein of the present invention can also comprise or not comprise initial methionine residues.
The invention still further relates to the gene of the described alpha-glycosidase XcG-A of coding.
Concrete, described gene nucleotide series can be shown in SEQ ID NO.2:
GAATTCATGT?CGCAGACACC?ATGGTGGCGC?GGGGCCGTCA?TCTATCAGAT?TTATCCGCGT?AGTTTTCTGG?ATTCCAATGG?GGATGGCGTA?GGCGATCTGC?CGGGCATCAT?TGCCAAGCTC?GACTACATCG?CCGGGCTGGG?CGTGGATGCG?ATCTGGATTT?CGCCGTTTTT?CAAGTCGCCG?ATGGCCGACT?TCGGGTATGA?CATCTCGGAC?TTCCGCGACG?TGGACCCGAT?GTTCGGCACG?TTGGCCGACT?TCGACCGGCT?GCTGGCGAAG?GCGCATGCAC?TCGGGCTGCG?GGTGATGATC?GACCAGGTGC?TGGGCCACTC?GTCGGTGGAG?CACGAGTGGT?TCAAGGAAAG?CCGCGAAAGC?CGCGACAACC?CCAAGGCCGA?CTGGTACGTG?TGGGCCGACG?CGCGCCCGGA?CGGCACGCCG?CCGAACAACT?GGCTGTCGAT?CTTCGGTGGC?GTGGCCTGGA?AGTGGGAGCC?TCGGCGCGGC?CAGTACTACC?TGCACAACTT?CCTGTCCTCC?CAGCCCGACC?TCAACTTCCA?TTGCCCGGAC?GTGCGCGCGG?CGATGCTGGA?CAGCGTGAAG?TTCTGGCTGG?ACCGGGGCGT?GGATGGCTTC?CGCCTGGATT?CGATCAACTT?CCCGTTCCAC?GACGCACAGC?TGCGTGACAA?CCCGCCCAAG?CCGGTGGAAG?CGCGCAGCGG?GCGCGGCTTC?AGCGCGGACA?ACCCGTACGC?GTTCCAGTAC?CACTACTACA?ACAACACCCA?GCCCGAGAAC?CTGGCGCTGC?TGGAAGAACT?GCGCGCGCTG?ATGGACCGGT?ACCCCGGGGT?GGCGACGCTG?GGCGAGATTT?CCTCCGAGGA?CTCGCTGGCG?ACGATGGCCG?AATATGTCAC?CGAACAGCGC?CTGCACATGG?GCTACAGCTT?CGAGCTGCTG?GTCGATGACT?TCAGCGCGGC?CTTCATCCGC?GGCACGGTGG?AGCAACTGGA?AGCAAAGATG?AGTGATGGCT?GGCCGTGCTG?GGCCATCTCC?AACCACGACG?TGCGGCGCGC?GGTGACCCGT?TGGGGTGGCG?CGACGCCCTC?GCCGGCGCTT?GCGTCGCAGC?TGGTGGCGCT?GGTGTGCTCG?CTGCGTGGCT?CCGTCTGCCT?GTACCAGGGT?GAGGAGCTGG?GCCTGAGTGA?GGCAGAGGTG?GCGTTCGAGG?ACCTGCAGGA?TCCGTATGGG?ATTACCTTCT?GGCCGACCTT?CAAGGGCCGG?GATGGCTGCC?GTACGCCGAT?GCCGTGGACC?GACGCGCCAT?CTGCCGGATT?CACCAGCGGC?AAGCCTTGGC?TGCCGTTAGC?TGCGTCGCAT?CGTGCCGCTG?CTGTGAGCGT?GCAACAAGAC?GATGCGCATT?CCGTGTTGAG?TGCAGTACGG?GATTTTCTAG?CTTGGCGCAA?GGAGATGCCG?GCGCTGCGTG?AGGGATCCAT?CGCTTTCTAC?GATACGGCCG?AACCGGTGCT?GATGTTCCGG?CGCGAGCATT?TGGGTCAGGT?CATGCTGTTG?GCGTTCAATC?TGTCCGCCGA?TCCTGCCGAC?CTGGCCTTGC?CTGCCGGCGA?GTGGGAGCAG?ATCGATGTAC?CTGGTGTCGA?GCTTGGGGCG?ATGGAGGGCG?GACACCTACG?GCTGGCCGGG?CATGGGGTCG?TTGCTGCTGT?CGGTCGTGGC?TGAAAGCTT。
Because the singularity of nucleotide sequence, the variant of polynucleotide shown in any SEQ ID NO:2 as long as itself and this polynucleotide have 70% above homology, all belongs to the row of protection domain of the present invention.The variant of described polynucleotide refers to a kind of polynucleotide sequence that one or more Nucleotide change that has.The variant of these polynucleotide can make living displacement varient or the varient of non-life, comprises replacing varient, deletion mutation body and inserting varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be replacement, disappearance or the insertion of polynucleotide, but can be from the function of the peptide protein that changes in fact its coding.
Main points of the present invention have been to provide the nucleotide sequence shown in the aminoacid sequence shown in the SEQ ID NO:1 and the SEQ ID NO:2, in the situation of known this aminoacid sequence and nucleotide sequence, the acquisition of this aminoacid sequence and nucleotide sequence, and the acquisition of related vector, host cell, all be apparent to those skilled in the art.
The invention still further relates to the recombinant vectors that contains described gene, and transform the genetic engineering bacterium that obtains by described recombinant vectors.
The invention still further relates to the application of described gene in preparation restructuring alpha-glycosidase XcG-A.Concrete, described being applied as: make up the recombinant vectors that contains described encoding gene, described recombinant vectors is changed in the intestinal bacteria, the recombination engineering bacteria that obtains carries out inducing culture, nutrient solution separates and to obtain containing the somatic cells of alpha-glycosidase of recombinating, and somatic cells is broken through cell, separation and purification obtains described restructuring alpha-glycosidase XcG-A.
The experiment proved that alpha-glycosidase XcG-A of the present invention expresses output in Host Strains high, for example in dust Xi Shi intestinal bacteria, cross and express that expression amount accounts for 40% ~ 50% of crude enzyme liquid supernatant total protein concentration.Compare with conventional alpha-glycosidase, have very high activity, at pH8.0, under 25 ℃ of conditions, XcG-A is 1.35U/mg to the enzyme activity of pNPG.At K
+And NH
4+Vigor can improve 2-3 doubly in the solvent.
Alpha-glycosidase XcG-A of the present invention has the high expression level amount, high reactivity, substrate specificity, simultaneously alpha-glycosidase XcG-A of the present invention can turn glucosides with corresponding substrate for catalysis maltose, generation menthyl-glycoside, Resorcinol glucosides, elemenol glucosides, puerarin glucosides, Eugenol glucosides etc.
The preparation method of high expression level of the present invention, highly active alpha-glycosidase XcG-A and zymetology characterize:
(1), alpha-glycosidase XcG-A full length gene of the present invention obtains.Design alpha-glycosidase degenerated primer, take all macro genome DNAs of this laboratory as template, angle and get 3 genes of homology about 70%, carry out external chimeric construct with the genomic dna that extracts from Xanthomonas campestris afterwards, obtain total length alpha-glycosidase gene, XcG-A, the alpha-glycosidase Nucleotide full length sequence shown in the SEQ ID NO.2.
(2), the structure that contains the expression vector system of goal gene.With the alpha-glycosidase gene clone described in the step (1) to expression vector, such as pET28a.
(3), the recombinant vectors that contains alpha-glycosidase XcG-A gene in the step (2) is changed in the heterogenous expression host cell, such as (Escherichiacoli) BL21, under conditions suitable for the expression, cultivate recombinant host cell.
(4), separation and purification goes out the high expression level described in the present invention, highly active alpha-glycosidase XcG-A from the culture of step (3).
(5), pass through as above preparation method, the alpha-glycosidase that obtains is carried out further zymetology characteristic present, comprise the tolerance, substrate hydrolysis specificity of enzymic activity, optimum temperuture and thermostability, optimal pH, pH stability and heavy metal ion solvent etc.
The invention still further relates to described alpha-glycosidase XcG-A in the application in the glucosides reaction of turning of catalysis maltose and hydroxyl substrate.
Described hydroxyl substrate is the common hydroxyl substrate that can turn with maltose glucosides reaction in this area, is preferably one of following: menthol, Resorcinol, elemenol, puerarin, Eugenol.
The present invention is according to the Glycosylase sequence (XcG) of the Xanthomonas campestris in the database, designed conservative site primer, and then in grand genome, amplified homologous fragment, adopt external chimeric mode, made up the new enzyme of mosaic type (XcG-A) that comprises the new homologous fragment of grand genome, it is carried out the function sign, and applied it to the turning in the glucosides reaction of hydroxyl substrate of series.The Glycosylase of the relative Xanthomonas campestris of this restructuring new enzyme of mosaic type, have some obvious revising of the functions, comprise that enzyme activity has improved about 6 times, vigor under the centering pH condition is higher, 30 times of 6.0 times vigor of pH Glycosylases that is Xanthomonas campestris approximately, realize the newer functional promotion of sequence in function, can be widely used in simultaneously glycosylation modified economic compounds, possessed wide application prospect.
Beneficial effect of the present invention is mainly reflected in: alpha-glycosidase XcG-A of the present invention is easy to prokaryotic expression, can adopt the recombinant vectors described in this patent, bacterial strain and preparation method's High-efficient Production alpha-glycosidase of the present invention; And preparation method's simple and effective of enzyme of the present invention is easy to great expression, is suitable for suitability for industrialized production.
(4) description of drawings
Fig. 1 is that alpha-glycosidase XcG-A and maltose (maltose), menthol (menthol) substrate turn glucosides reaction thin-layer chromatogram; M ': menthol; M '-G:menthol-glucose; M '-G-G:menthol-glucose-glucose; M:maltose.
Fig. 2 is that alpha-glycosidase XcG-A and maltose, elemenol (elemenol) substrate turn glucosides reaction thin-layer chromatogram; E:elemenol; E-G:elemenol-glucose; E-G-G:elemenol-glucose-glucose; M:maltose.
Fig. 3 is that alpha-glycosidase XcG-A and maltose, Resorcinol (hydroquinone) substrate turn glucosides reaction thin-layer chromatogram; H:hydroquinone; α-A: α-arbutin; α-A-G: α-arbutin-glucose; G:glucose; M:maltose.
Fig. 4 is that alpha-glycosidase XcG-A and maltose, puerarin (puerarin) substrate turn glucosides reaction thin-layer chromatogram; P:puerarin; P-G:puerarin-glucose; M:maltose.
Fig. 5 is that alpha-glycosidase XcG-A and maltose, Eugenol (eugenol) substrate turn glucosides reaction thin-layer chromatogram; E:eugenol; E-G:eugenol-glucose; E-G-G:eugenol-glucose-glucose; M:maltose.
(5) embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Experimental technique among the present invention is ordinary method if no special instructions, specifically can be referring to " Molecular Cloning:A LaboratoryManual " (Sambrook and Russell, ed.2001).
Restriction enzyme EcoR I, the Hind III that uses in the embodiments of the invention is all available from the precious biotechnology in Dalian company limited; Bacillus coli DH 5 alpha, BL21(DE3) available from Novagen company; Primer is synthetic to be finished by the living worker's biotechnology in Shanghai company limited with the sequence examining order.
Embodiment 1: the acquisition of alpha-glycosidase XcG-A
Gather respectively pedotheque from 46 places all over China, adopt Mo Bio Power Soil DNA Isolation Kit(M) (Carlsbad, CA) test kit extracts the total macro genome DNA of sample, use Copy Control Fosmid Library production kit(Epicentre, USA) build the grand genomic library of storehouse test kit constructing environment.Take the alpha-glucosaccharase enzyme sequence (Genebank:AEL07660.1 etc.) that derives from Xanthomonas campestris as homing sequence, from ncbi database, download the aminoacid sequence of GHF13-23 subfamily, the over-designed degenerated primer, take the soil metagenome DNA of above-mentioned structure as template, use the method for degenerated primer pcr amplification, angle and get goal gene.
Extract Xanthomonas campestris genomic dna, alpha-glycosidase sequence (Genebank:CP002789.1) the design two ends primer according to its report obtains full-length gene by pcr amplification, called after XcG.At primer 5 ' end design EcoR I, Hind III restriction enzyme site, be connected to the pET28a carrier, be converted into intestinal bacteria (Escherichia coli DH5 α), extract recombinant plasmid, as the template of chimeric restructuring.
The goal gene that to from soil metagenome, transfer, in the overlap section primer of the two ends design of obtaining the homologous gene fragment and the XcG full-length gene method by external chimeric construct, homologous gene is fitted on the XcG gene, obtains mosaic type total length alpha-glycosidase gene, called after XcG-A.As template, carry out secondary PCR with chimeric PCR product, with EcoR I, Hind III double digestion PCR product, be connected on the pET28a carrier, change among the cloning host bacterium E.coli DH5 α, identify order-checking, namely obtain the full length sequence of alpha-glycosidase recombinant bacterium.
Embodiment 2: the structure that contains the recombinant bacterium of alpha-glycosidase XcG-A
According to two alpha-glycosidase full-length gene orders, design amplifies the upstream primer XcG-A-F(5 ' of complete coding reading frame-CCGGAATTCATGTCGCAGACACCATG-3 ') downstream primer XcG-A-R(5 '-CCCAAGCTTTCAGCCACGACCGACA-3 '), and (expression vector described in the present invention is pET28a to introduce respectively restriction endonuclease sites in the upstream and downstream primer, choosing the upstream restriction enzyme site according to this carrier is the EcoR I, and the downstream is the Hind III).Take the plasmid that contains XcG-A as template, behind pcr amplification, the alpha-glycosidase nucleotide fragments (SEQ ID N0.1) that amplification is obtained carries out double digestion (EcoR I, Hind III), be connected on the expression vector pET28a with corresponding restriction enzyme site, change among the expressive host E.coli BL21 with the CaCl chemical transformation, construct E.coli BL21/pET28a/XcG-A alpha-glycosidase expression system.
Embodiment 3: the purifying of the abduction delivering of recombinant bacterium and alpha-glycosidase XcG-A
The alpha-glycosidase expression system E.coli BL21/pET28a/ XcG-A that obtains is seeded in Luria-Bertani (LB) liquid nutrient medium that contains 100ug/ml kantlex (sigma, USA), and 37 ℃ are cultured to logarithmic phase (OD
600Be about 0.6) after to add final concentration be the inductor IPTG(genview of 1 mM, USA), 25 ℃, behind the 180rpm inducing culture 20h, centrifugal collection thalline, resuspended with the damping fluid of 60ml PBS damping fluid (pH 7.0), the ultrasonication bacteria suspension, centrifugal removal precipitation obtains the crude enzyme liquid that supernatant is alpha-glycosidase albumen.Cellulose acetate membrane filtration crude enzyme liquid with 0.45um carries out the affinity chromatography purifying subsequently, obtains the pure enzyme of alpha-glycosidase XcG-A described in the present invention.
Embodiment 4: the vitality test of alpha-glycosidase XcG-A
According to the relevant measuring method of alpha-glycosidase, adopt colorimetric method to carry out the vitality test of alpha-glycosidase XcG-A.With pNPG(p-nitrophenyl-α-D-glucopyranoside) as substrate, within the unit time (per minute), generate the rate calculations enzyme activity of product with the enzyme liquid of unit mass.Specific implementation method is as follows:
Alpha-glycosidase XcG-A is dissolved in the 50mM PBS damping fluid (pH 7.0) makes enzyme liquid, getting 50ul enzyme liquid is added to 450 μ l and contains substrate pNPG(reaction solution final concentration 1mM) PBS buffered soln (pH7.0, reaction solution final concentration 50mM) in, use Shimadzu ultra-violet and visible spectrophotometer (shimadzu UV-2550) to measure 37 ℃ of lower 405nm wavelength place light absorption value kinetic curves.The kinetic curve that the setting deionized water replaces enzyme liquid to record is made as blank, measures and obtains enzymolysis kinetics parameter K value, calculates enzymic catalytic reaction speed.
Formula is calculated in enzyme work:
U=[1000?×（△A?V?/tε）×?10
-3]/M
U is specific activity of enzyme U/mg, and △ A is light absorption value poor of the light absorption value of sample catalyzed reaction and corresponding blank, and V is the volume L of reaction system, and t is reaction times min, and ε is molar absorptivity 1.6 * 10
3L/(molcm), M is the enzyme amount mg that adds in the reaction system.
As calculated, the specific activity of enzyme of alpha-glycosidase XcG-A of the present invention is 1.35U/mg.
Embodiment 5: alpha-glycosidase XcG-A and maltose, menthol substrate turn the glucosides reaction:
For measuring alpha-glycosidase XcG-A to the glucosides ability that turns of menthol, get 0.9mL 1mg/mL enzyme liquid, add 25mg menthol, 0.36g maltose, 0.1mL 100mM Na
2HPO
4-NaH
2PO
4(pH7.0) damping fluid, 30 ℃ of concussion reaction 12h, afterwards in 100 ℃ of water-bath 5min termination reactions, centrifugal removal precipitation, with standard substance such as glucose, maltose in contrast, carry out thin-layer chromatographic analysis, choose the GF254 silica-gel plate and place developping agent (propyl carbinol: acetone: water=4:1:1), 2-3cm place, upper end takes out chromatography to developping agent from silica-gel plate, dries up.Evenly spray 20%(v/v) ethanol solution of sulfuric acid, place 110 ℃ of baking 10min, observe the colour developing phenomenon.(Fig. 1) can see from thin-layer chromatogram, and reaction has generated menthyl-glycoside menthol-glucose; With menthyl-glycoside glucosides menthol-glucose-glucose.
Embodiment 6: alpha-glycosidase XcG-A and maltose, elemenol substrate turn the glucosides reaction:
Be to measure alpha-glycosidase XcG-A to the glucosides ability that turns of elemenol, get 0.9mL 1mg/mL enzyme liquid, 0.36g maltose, 1 μ l concentration and be 99% elemenol, add 0.1mL 100mM Na
2HPO
4-NaH
2PO
4(pH7.0) in the damping fluid, 30 ℃ of concussion reaction 12h, afterwards in 100 ℃ of water-bath 5min termination reactions, centrifugal removal precipitation, with standard substance such as glucose, maltose in contrast, carry out thin-layer chromatographic analysis, choose the GF254 silica-gel plate and place developping agent (propyl carbinol: acetone: water=4:1:1), 2 ~ 3cm place, upper end takes out chromatography to developping agent from silica-gel plate, dries up.Evenly spray 20%(v/v) ethanol solution of sulfuric acid, place 110 ℃ of baking 10min, observe the colour developing phenomenon.(Fig. 2) can see from thin-layer chromatogram, and reaction has generated elemene indican elemene-glucose and elemene indican glucosides elemene-glucose-glucose;
Embodiment 7: alpha-glycosidase XcG-A and maltose, Resorcinol substrate turn the glucosides reaction:
For measuring alpha-glycosidase XcG-A to the glucosides ability that turns of Resorcinol, get 0.9mL 1mg/mL enzyme liquid, 55mg Resorcinol, 0.216g maltose, add 0.1mL 100mM Na
2HPO
4-NaH
2PO
4(pH7.0) in the damping fluid, 30 ℃ of concussion reaction 12h, afterwards in 100 ℃ of water-bath 5min termination reactions, centrifugal removal precipitation, with standard substance such as glucose, maltose in contrast, carry out thin-layer chromatographic analysis, choose the GF254 silica-gel plate and place developping agent (propyl carbinol: acetone: water=4:1:1), 2-3cm place, upper end takes out chromatography to developping agent from silica-gel plate, dries up.Evenly spray 20%(v/v) ethanol solution of sulfuric acid, place 110 ℃ of baking 10min, observe the colour developing phenomenon.(Fig. 3) can see from thin-layer chromatogram, and the glucosides reaction that turns take Resorcinol as substrate has generated arbutin α-arbutin and arbutin glucosides α-arbutin-glucose.
Embodiment 8: alpha-glycosidase XcG-A and maltose, puerarin substrate turn the glucosides reaction:
For measuring alpha-glycosidase XcG-A to the glucosides ability that turns of puerarin, get 0.9mL 1mg/mL enzyme liquid, 25mg puerarin, 0.36g maltose, 0.1mL 100mM Na
2HPO
4-NaH
2PO
4(pH7.0) in the damping fluid, 30 ℃ of concussion reaction 12h, afterwards in 100 ℃ of water-bath 5min termination reactions, centrifugal removal precipitation, with standard substance such as glucose, maltose in contrast, carry out thin-layer chromatographic analysis, choose the GF254 silica-gel plate and place developping agent (propyl carbinol: acetone: water=4:1:1), 2-3cm place, upper end takes out chromatography to developping agent from silica-gel plate, dries up.Evenly spray 20%(v/v) ethanol solution of sulfuric acid, place 110 ℃ of baking 10min, observe the colour developing phenomenon.(Fig. 4) can see from thin-layer chromatogram, and reaction has generated puerarin glucosides puerarin-glucose.
Embodiment 9: alpha-glycosidase XcG-A and maltose, Eugenol substrate turn the glucosides reaction:
Be to measure alpha-glycosidase XcG-A to the glucosides ability that turns of Eugenol, get 0.9mL 1mg/mL enzyme liquid, 1 μ l concentration and be 99% Eugenol, 0.36g maltose, 0.1mL 100mM Na
2HPO
4-NaH
2PO
4(pH7.0) in the damping fluid, 30 ℃ of concussion reaction 12h, afterwards in 100 ℃ of water-bath 5min termination reactions, centrifugal removal precipitation, with standard substance such as glucose, maltose in contrast, carry out thin-layer chromatographic analysis, choose the GF254 silica-gel plate and place developping agent (propyl carbinol: acetone: water=4:1:1), 2 ~ 3cm place, upper end takes out chromatography to developping agent from silica-gel plate, dries up.Evenly spray 20%(v/v) ethanol solution of sulfuric acid, place 110 ℃ of baking 10min, observe the colour developing phenomenon.(Fig. 5) can see from thin-layer chromatogram, and reaction has generated Eugenol glucosides eugenol-glucose and Eugenol glucosides glucosides eugenol-glucose-glucose.
The above embodiment of the present invention only is for example of the present invention clearly is described, and is not to be restriction to embodiments of the present invention.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here can't give all embodiments exhaustive.Everyly belong to the row that apparent variation that technical scheme of the present invention extends out or change still are in protection scope of the present invention.
Claims (8)
1. the alpha-glycosidase XcG-A of a grand gene source, its aminoacid sequence is shown in SEQ ID NO.1.
2. the gene of coding claim 1 described alpha-glycosidase XcG-A.
3. gene as claimed in claim 2 is characterized in that described gene nucleotide series is shown in SEQ ID NO.2.
4. the recombinant vectors that contains claim 2 or 3 described genes.
5. transform the genetic engineering bacterium that obtains by the described recombinant vectors of claim 4.
6. claim 2 or the 3 described genes application in preparation restructuring alpha-glycosidase XcG-A.
7. alpha-glycosidase XcG-A claimed in claim 1 is in the application in the glucosides reaction of turning of catalysis maltose and hydroxyl substrate.
8. application as claimed in claim 7 is characterized in that described hydroxyl substrate is one of following: menthol, Resorcinol, elemenol, puerarin, Eugenol.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107267484A (en) * | 2017-07-18 | 2017-10-20 | 杭州师范大学 | A kind of α glycosidases, encoding gene, carrier, engineering bacteria and the application of grand gene source |
| CN107446844A (en) * | 2017-08-03 | 2017-12-08 | 浙江工业大学 | A kind of xanthomonas campestris and its application |
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| CN101363026A (en) * | 2007-08-09 | 2009-02-11 | 广西大学 | Gene encoding beta-glucosidase |
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| CN101363026A (en) * | 2007-08-09 | 2009-02-11 | 广西大学 | Gene encoding beta-glucosidase |
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| KIM SJ ET AL.: "Screening and characterization of an enzyme with beta-glucosidase activity from environmental DNA", 《J MICROBIOL BIOTECHNOL》, vol. 17, no. 6, 30 June 2007 (2007-06-30) * |
| VORHOELTER F.-J. ET AL.: "CAP51047.1", 《EMBL》, 8 April 2008 (2008-04-08) * |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107267484A (en) * | 2017-07-18 | 2017-10-20 | 杭州师范大学 | A kind of α glycosidases, encoding gene, carrier, engineering bacteria and the application of grand gene source |
| CN107446844A (en) * | 2017-08-03 | 2017-12-08 | 浙江工业大学 | A kind of xanthomonas campestris and its application |
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