CN100393873C - Starch mycose base synthetase, its coding gene, expression and engineering strain - Google Patents
Starch mycose base synthetase, its coding gene, expression and engineering strain Download PDFInfo
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Abstract
The present invention discloses a starch trehalose base synthetase, a coding gene, an expression method and an engineering bacterium thereof. The starch trehalose base synthetase provided by the present invention is a protein having the SEQ ID No. 1 amino acid residue sequence in the sequence table or formed by replacing, deleting or adding the SEQ ID No. 1 amino acid residue sequence by one or a plurality of amino acid residues, having the same activity with the SEQ ID No. 1 and derived from the SEQ ID No. 1. The starch trehalose base synthetase, the coding gene and the engineering bacterium-escherichia coli DE3 (pM75) CGMCC No. 1133 of the present invention can perform important functions in the production of trehalose.
Description
Technical field
The present invention relates to a kind of starch mycose-base synthetase and encoding gene and expression method and engineering bacteria in biotechnology and the enzyme engineering field.
Background technology
Trehalose is a kind of non-reducible disaccharide, is white crystal under the normality, tasty and refreshing sweetness and do not have aftertaste, and its sugariness is 45% of a granulated sugar; Because this sugar has the unique biological function; multiple biomacromolecule and cytolemma there is significant provide protection; its purposes and latent effect are very extensive, can be used as the protective material and the stablizer of biological products and active bacteria formulation, also can be used as natural additive in food and makeup.Traditional trehalose preparation method has yeast extraction process and phosphorylation enzyme process etc.In recent years, in bacterium, found to utilize the new enzyme of starch trehalose synthesis, i.e. the starch mycose-base synthetase.This starch enzyme process trehalose synthesis is the most rising a kind of in the various so far trehalose production methods.Japanese in 1993 finds that isolating root nodule bacterium (Rhizobium sp.) M-11 (CCTCC M94031) and Arthrobacter (Artobacter sp.) Q36 (CCTCC M94030) can produce the starch mycose-base synthetase from soil, this enzyme uses with mycose base hydrolase, hydrolyzable starch direct production trehalose (Fig. 1), mycose base hydrolase can be in the irreducibility Dian Fentang that the above method of hydrolysis under the normal atmosphere temperature neutral produces key between trehalose part and all the other glycosyl parts, generate trehalose.
Along with the develop rapidly of global biotechnology, the research of microbial genome is in the ascendant, and annual all have a large amount of new microbial enzyme genes to be found report.But according to statistics, the microbial enzyme gene of having reported at present mainly still comes from educable microorganism in the laboratory.Yet these microbial numbers are no more than 1% of all microorganism total amounts of estimating.Because the restriction of factors such as culture condition, separation means, microbial gene resources a large amount of in the environment are left unused in vain; And, need not separation, cultivation and evaluation through microorganism by culturing micro-organisms technology not, can directly obtain the DNA in the environmental sample and make up the gene library of associated sample; And, promptly obtained required genetic resources by suitable screening method.
One of key of culturing micro-organisms technology is not: obtain efficiently can carry out the mixing genomic dna of molecule manipulation and carry out purifying effectively from various samples.From environmental sample, extract the direct cracking process of the general employing of DNA.Directly cracking process is direct lysing cell released dna from environmental sample, reclaims DNA with the alkaline process extracting then, at last the method by ethanol sedimentation, CsCl density gradient centrifugation and hydroxyapatite column chromatography purification DNA.It can be divided into physics method and chemical method again according to the splitting action degree of pair cell.The former is general to adopt violent physical treatment to come lysing cell, and these physical treatments comprise methods such as repeatedly freeze thawing, ultrasonic wave, granulated glass sphere homogenate, microwave heating and grinding; These dna fragmentation length of handling gained are generally at 5kb to 10kb or littler.Chemical method is meant with chemical means handles lysing cell, and it comprises N,O-Diacetylmuramidase, Proteinase K, SDS, hot phenol, guanidine thiocyanate, proteolytic ferment, acetone and EDTA etc.; These dna fragmentations of handling gained are big (tens kb) generally.
The innovation and creation content
The purpose of this invention is to provide a kind of starch mycose-base synthetase and encoding gene thereof.
A kind of starch mycose-base synthetase, name is called G135, and it is to have SEQ ID № in the sequence table: 1 amino acid residue sequence or with SEQ ID №: 1 amino acid residue sequence is through replacement, disappearance or the interpolation of one or several amino-acid residue and have the № with SEQ ID: 1 is identical active by SEQ ID №: 1 deutero-protein.
SEQ ID № in the sequence table: 1 protein is made up of 732 amino-acid residues.
For the ease of purifying, can be at the terminal polypeptide label that increases of described starch mycose-base synthetase C-, as the aminoacid sequence of 6 Histidine polypeptide tails.
Starch mycose-base synthetase encoding gene has one of following nucleotide sequences:
1) the SEQ ID № in the sequence table: 2;
2) SEQ ID № in the code sequence tabulation: the polynucleotide of 1 protein sequence;
3) with sequence table in SEQ ID №: 2 dna sequence dnas that limit have 90% above homology, and the identical function protein DNA sequence of encoding.
SEQ ID № in the sequence table: 2 by 2243 based compositions, and its open reading frame is from the 1st-2199 bit base of 5 ' end.For the ease of purifying, can be at SEQ ID №: increase the encoding gene of polypeptide label behind 2 the 2196th bit base, as the nucleotide sequence of 6 the Histidine polypeptide tails of encoding.
The carrier, clone and the host bacterium that contain starch mycose-base synthetase encoding gene of the present invention; be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (be called for short CGMCC) as pM75 plasmid (Fig. 6) with on 04 20th, 2004, preserving number is engineering bacteria-colon bacillus (Escherichia coli) DE3 (pM75) of CGMCC № 1133, all belongs to protection scope of the present invention.
The pM75 size is 6.3kb, is made up of the 4.1kb fragment of starch mycose-base synthetase gene and carrier pET3a.Colon bacillus (Escherichia coli) DE3 (pM75) comprises this recombinant DNA molecules.
Another object of the present invention provides a kind of method of expressing the starch mycose-base synthetase.
The method of expression starch mycose-base synthetase provided by the present invention is that the recombinant expression vector that will contain above-mentioned starch mycose-base synthetase encoding gene imports the expressive host bacterium, expresses obtaining the starch mycose-base synthetase.
In the method, a principal host bacterium is expressed starch mycose-base synthetase gene of the present invention, and the gene that recombinant DNA molecules can be replicated and integrate in the host bacterium can keep with being stabilized, then available host/vector system be there is no particular restriction.For example, the host to be the member of EK system of e. coli k-12 and host can use for the member of the BS system of subtilis.Utilization comprises the EK system of in depth being studied on many kinds of carriers and the genetics can obtain good result, thereby is preferred.The specific example of host bacteria comprises DH5 α, HB101, BE3, TOP010 and the JM109 of EK system, and the BD170 of BS system, MI112 and ISW1214.
Wherein, the recombinant expression vector of described starch mycose-base synthetase encoding gene can obtain by starch mycose-base synthetase encoding gene of the present invention is cloned in the carrier that sets out according to ordinary method, the described carrier that sets out comprises pBR322, pET, pBV220, pBAD and the pUC18 that is used for the EK system, and the pUB110 and the pHY300PLK that are used for the BS system.
The method that the recombinant expression vector of described starch mycose-base synthetase encoding gene is transformed host bacteria there is no special restriction.Available Calcium Chloride Method (Mandel under the host's of EK system situation for example, M. and Higa, A., " molecular biology magazine ", 53,159 (1970)) available protoplasm body (Chang, C. and Cohen, and under the host's of BS system situation, S.N., Mol.Gen.Genet.), 168,111 (1978)).Can carry out the preliminary screening of recon microorganism (engineering bacteria) by the selection markers of carrier itself, utilize the character of starch mycose-base synthetase finally to determine engineering bacteria again, for example, when the pBR322 that utilizes the EK system makes carrier and utilizes the HindIII enzyme to cut starch mycose-base synthetase encoding gene of the present invention to be inserted into the HindIII restriction enzyme site of pBR322, the tetracycline resistance gene inactivation can come the preliminary screening transformant by AumprTets.Subsequently, selected transformant utilization such as photomechanical printing method are transferred on the agar plate that contains maltopentaose, cultivate then so that form bacterium colony.Therefore target recon microorganism is decomposed maltopentaose around colony, so by utilizing a kind of Tong Shiji solution can screen it maltopentaose dyeing contained in containing the agar plate of maltopentaose.
Described recombinant expression vector is preferably pM75, can utilize colon bacillus (Escherichia coli) DE3 (pM75) the CGMCC № 1133 scale operation starch mycose-base synthetases that contain this carrier, can use ordinary method from the culture that is obtained, to collect the starch mycose-base synthetase.
The DNA of starch mycose-base synthetase encoding gene of the present invention further can be used to prepare the probe that separates other homology starch mycose-base synthetase gene from other microorganism.
The DNA that does not cultivate the mud sample of gathering in the Tengchong hot spring of biotechnology extracting Yunnan that the present invention adopts the physics method to combine with chemical method, promptly before SDS/ Proteinase K heat treating process extracting DNA, be aided with the processing of some physical method, as repeatedly freeze thawing, liquid nitrogen grinding, granulated glass sphere homogenate etc., at last the DNA sample is carried out the purification process of CTAB and PVPP again, obtained to have the high purity DNA sample of higher yields.These DNA samples are again by several inscribe restriction enzyme digestion digestion, and fragment carries out electrophoretic analysis and screening is reclaimed as inserting fragment, and building with pUC18 is that carrier, bacillus coli DH 5 alpha are the not culturing micro-organisms genomic library of recipient cell.After the subclone that obtains in the not culturing micro-organisms genomic library to these structures carried out the liquid inducing culture, the method for utilizing detection starch mycose-base synthetase enzyme to live was screened; And detect the expression of foreign protein by protein electrophoresis; Obtained starch mycose-base synthetase enzyme male bacterial strain alive, by this recon extraction plasmid being carried out electrophoretic analysis and checking order, find to contain a kind of complete Glycosylase G135 gene order information of coding at the dna fragmentation of this about 4.1kb.This gene has the nucleotide sequence of sequence 2 in the sequence table, and its coding has the protein of the amino acid residue sequence of sequence 1 in the sequence table.The starch mycose-base synthetase encoding gene homology of starch mycose-base synthetase encoding gene of the present invention and other microorganisms is respectively 93% (sesame field sulfolobus solfataricus B12), 63% (sulfolobus acidocaldarius ATCC33909), 48% (Arthrobacter Q36), 48% (root nodule bacterium M11), 50% (tyrothricin).
Starch mycose-base synthetase of the present invention can (be up to about 75 ℃) under hot environment specificity is utilized starch partial hydrolysate, the efficient synthetic irreducibility Dian Fentang of trehalose structure as terminal units that have.Any material just can be used as the substrate of enzyme of the present invention so long as the reductibility Dian Fentang that the starch hydrolysis produces, as trisaccharide maltose, and maltotetrose, maltopentaose, MALTOHAXAOASE and Fructus Hordei Germinatus seven sugar etc.Except that these substrates, other by the reductibility starch partial hydrolysate for preparing with amylase or acid moieties hydrolyzed starch class material such as starch, amylopectin and amylose starch also as the substrate of enzyme of the present invention.
Can be used for the diastatic of partially hydrolysed starch can be (by Pergamon Press at Handbook of Amyloses and RelatedEnzymes; Tokyo, Japan (1988) publishes) in disclosed α-Dian Fenmei, maltopentaose forms amylase and MALTOHAXAOASE forms amylase etc.Also these amylase and debranching factor such as Starch debranching enzyme and isoamylase can be used in combination.
Starch mycose-base synthetase of the present invention has no particular limits the concentration as the starch partial hydrolysate of substrate, even can carry out enzyme reaction in the solution that contains 0.1% or 50% substrate, forms the irreducibility Dian Fentang.Also can use the soluble suspended substance that contains excessive substrate.The available temperature of reaction can be the temperature of enzyme non-inactivation of the present invention in starch mycose-base synthetase reaction of the present invention, promptly is up to about 75 ℃, preferably at 55-70 ℃.Available reaction pH is 4.5-8.0 in enzyme reaction of the present invention, preferably at pH 5.0-6.0.In actual applications, select the used time of enzyme reaction of the present invention, usually when with 0.1-100 unit/when the g substrate uses enzyme of the present invention, approximately need 0.1-100 hour according to the enzyme reaction condition.Starch mycose-base synthetase of the present invention and encoding gene thereof and engineering bacteria-colon bacillus (Escherichia coli) DE3 (pM75) CGMCC № 1133 will play a significant role in the production of trehalose.
Description of drawings
Fig. 1 is an enzyme process trehalose synthesis synoptic diagram
Fig. 2 is the optimal reactive temperature curve of starch mycose-base synthetase of the present invention
Fig. 3 is the optimal reaction pH curve of starch mycose-base synthetase of the present invention
Fig. 4 is the temperature-stable linearity curve of starch mycose-base synthetase of the present invention
Fig. 5 is the pH beta stability line of starch mycose-base synthetase of the present invention
Fig. 6 is the building process synoptic diagram of plasmid pM75
Embodiment
Will describe the present invention in more detail by embodiment below, these embodiment should not be considered to the present invention is defined in this.Concentration in an embodiment all is the % based on (W/V).
The expression of embodiment 1, starch mycose-base synthetase of the present invention
1, the clone of starch mycose-base synthetase encoding gene of the present invention
(1) extraction of the processing of environmental sample and DNA
From the Tengchong hot spring of Yunnan, gather mud sample, after 60 ℃ of oven dry, take by weighing a little sample, be positioned in the mortar, add liquid nitrogen then, fully be ground to sample and be the homogeneous powder powder.Sample after the grinding is positioned in the centrifuge tube of 50mL, and adding DNA extraction buffer (100mM Tris-HCl, pH 8.0,100mM EDTA, the 100mM sodium phosphate buffer, pH 8.0,1.5M NaCl, 1%CTAB), abundant mixing.Carry out three times the liquid nitrogen freeze thawing treatment then repeatedly.In mixed solution, add Proteinase K (20mg/mL) and SDS (10%) subsequently, behind the mixing, 60 ℃ water bath heat preservation 2-3 hour, turned upside down mixing several times every 15-20 minute; Centrifugal 10 minutes of 7000g room temperature is collected supernatant, adds entry in the precipitation, washes twice for 60 ℃, each 10 minutes; Merge three times supernatant, add twice of isopyknic phenol/chloroform/primary isoamyl alcohol (25: 24: 1) extracting; Aqueous phase adds pickling PVPP, 37 ℃ of water bath heat preservations 30 minutes, and via hole diameter is that the membrane filtration of 0.45nm is removed PVPP, adds the Virahol of 0.6 times of volume subsequently, room temperature is after 30 minutes, centrifugal 15 minutes of 4 ℃ of 12000g, 70% ethanol is washed; Precipitation is dissolved with TE damping fluid (10mMTris-HCl, pH 8.0,1mM EDTA pH 8.0).
(2) preparation of target DNA fragment
Respectively with the DNA sample for preparing in EcoRI or the two kinds of partially digested steps of restriction enzyme of PstI (1).Through 0.8% low melting-point agarose gel (containing 0.5 μ g/mL ethidium bromide) electrophoresis, under long wavelength (365nm) ultraviolet lamp, in molecular weight is that a kerf is cut at the 3kb place, insert the DEAE-cellulose membrane, continue electrophoresis, until molecular weight is on the DNA endonuclease bamhi arrival film of 8kb, stops electrophoresis, reclaims DNA from film.The DNA endonuclease bamhi that finally reclaims is dissolved in the TE damping fluid of 20 μ l.
(3) structure in environmental sample DNA library
The plasmid pUC18 that to cut through same EcoRI or PstI enzyme with calf intestinal alkaline phosphatase (CIAP) dephosphorylation after and the EcoRI of preparation in the step (2) or the dna fragmentation T that the PstI enzyme cuts back to close
4Dna ligase is at 16 ℃, after carrying out ligation 2-14 hour, carry out chemical conversion with competence E.coli DH5 α immediately, and ice was put 30 minutes subsequently, add SOC substratum (1% Tryptones, 0.5% yeast powder, 1%NaCl, 1% glucose, pH value 7.0) at 37 ℃, 225rpm shakes and recovers to cultivate 45-60 minute, be applied to (1% Tryptones on penbritin (Amp)-LB flat board, 0.5% yeast powder, 1%NaCl, 1.5% agar, 60 μ g/ml penbritins, 8 μ g/mL isopropylthio-s (IPTG) and 9 μ g/mL X-gal, pH value 7.0) cultivated the picking hickie 16-18 hour for 37 ℃.Positive bacterium colony is chosen to the LB solid medium that contains Amp, cultivate after 16-18 hour for 37 ℃, (1% Tryptones, 0.5% yeast powder, 1%NaCl) washes the bacterium colony on the plate with the LB liquid nutrient medium that contains Amp, add the LB liquid nutrient medium mixing that contains 10% glycerine, room temperature was placed 2 hours, liquid nitrogen flash freezer ,-70 ℃ of preservations.
(4) screening
Carry out the enzyme screening of living in the Tengchong hot spring microbial genome library that has made up from step (3), induce by cultivation, the enzyme that detects mycose-base synthetase is lived and is detected the expression of foreign protein by protein electrophoresis; Extract plasmid and carry out electrophoretic analysis and carry out the part order-checking having positive recombinant that enzymic activity expresses, find to contain the open reading frame of a part at the dna fragmentation of this plasmid.Utilize the information of the microorganism associated sugars glycoside enzyme gene sequence of having delivered in the gene pool, acquired part sequencing result is carried out internet retrieval and biosoftware analysis, obtain a series of Glycosylase gene order offset informations, determine to contain in this gene fragment the total conservative bioactive sequence of other several mycose-base synthetases.In turn all the other fragments are carried out full gene sequencing, splice complete novel enzyme gene G135 (starch mycose-base synthetase encoding gene of the present invention) sequence information.This gene has the nucleotide sequence of sequence 2 in the sequence table, and its coding has the protein of the amino acid residue sequence of sequence 1 in the sequence table.Through homology relatively, the result shows that the starch mycose-base synthetase encoding gene homology of starch mycose-base synthetase encoding gene of the present invention and other microorganisms is respectively 93% (sesame field sulfolobus solfataricus B12), 63% (sulfolobus acidocaldarius ATCC33909), 48% (Arthrobacter Q36), 48% (root nodule bacterium M11), 50% (tyrothricin).
Wherein, detect the activity of mycose-base synthetase of the present invention as follows: the 0.1ml enzyme solution is added in the 50mm acetate buffer solution (pH 5.5) of 0.1ml 2% (w/v) maltopentaose, 60 ℃ of reactions after 10 minutes boiling water bath 10 minutes with deactivation, 20 times of reaction mixture dilute with waters are got diluent 400ul then and are determined reducing power with the Somogyi-Nelson method.In contrast, 100 ℃ with enzyme solution preheating 15 minutes so that enzyme deactivation, detect by method same as described above.The enzymic activity of 1 unit is defined as: under this condition, per minute is removed the required enzyme amount of 1 μ mol maltopentaose reducing power.
Table 1.G135 Gene Sequence Analysis
2, the expression of starch mycose-base synthetase of the present invention
(1) structure of expression vector
With reference to known array, the PCR primer of design G135 gene (upstream: 5 '-GCGCCATATGATAATAGGTACGTA-3 ' downstream: 5 '-CGCGGATCCCTGAGTAATGCATATCTG-3 ') carry out pcr amplification.The pcr amplification condition is: 94 ℃ of pre-sex change 1 minute, and 94 ℃ of sex change 1 minute, 48 ℃ of annealing 1 minute, 71 ℃ were extended 2 minutes, 30 circulations, insulation is 10 minutes after 71 ℃, the about 2.2kb of electrophoresis detection amplified production.The amplified production size conforms to expected results.With fragment purification, cut through NdeI and BamHI enzyme, with through NdeI with after carrier pET-3a that the BamHI enzyme is cut links to each other, change among the recipient bacterium intestinal bacteria DE3, with NdeI and BamHI enzyme cut detect filter out contain recombinant plasmid bacterial strain colon bacillus (Escherichia coli) DE3 (pM75) (Fig. 6).
(2) inducing culture engineering bacteria colon bacillus (Escherichia coli) DE3 (pM75) expresses starch mycose-base synthetase of the present invention
Colon bacillus (Escherichia coli) DE3 (pM75) that obtains was contained in the 50ug/ml penbritin LB liquid nutrient medium shaking culture 12 hours in 2ml.The 2ml culture is inoculated in the 50ml LB substratum (containing the 50ug/ml penbritin), then 37 ℃ of shaking culture 2 hours, the cell density OD of nutrient solution in triangular flask
600During for the 0.6-0.8 left and right sides, the isopropylthio-solution that adds 5ul 20% carries out abduction delivering, under 37 ℃ of conditions, continue to cultivate 4 hours, in 6000 rev/mins centrifugal 10 minutes, collect thalline and nutrient solution supernatant respectively.To be suspended from by the cell that centrifugation is collected in the acetate buffer solution (pH5.5), and destroy cell by supersound process.Cell by supersound process after, by the centrifugation clear cell debris, and the cell conditioned medium liquid that is obtained made a kind of cell-free extract.With the cell-free extract of intestinal bacteria DE3 (pET) bacterial strain that changes pET-3a in contrast.Detect the activity of starch mycose-base synthetase in culture supernatant, the cell conditioned medium liquid respectively, the result is as shown in table 2.
Wherein, detect the activity of mycose-base synthetase of the present invention as follows: the 0.1ml enzyme solution is added in the 50mm acetate buffer solution (pH 5.5) of 0.1ml 2%w/v maltopentaose, 60 ℃ of reactions after 10 minutes boiling water bath 10 minutes with deactivation, 20 times of reaction mixture dilute with waters are got diluent 400ul then and are determined reducing power with the Somogyi-Nelson method.In contrast, 100 ℃ with enzyme solution preheating 15 minutes so that enzyme deactivation, detect by method same as described above.The enzymic activity of 1 unit is defined as: under this condition, per minute is removed the required enzyme amount of 1 μ mol maltopentaose reducing power.
Table 2
The purifying of embodiment 2, mycose-base synthetase of the present invention
Merge intestinal bacteria DE3 (pM75) culture supernatant and cell conditioned medium liquid crude enzyme liquid as mycose-base synthetase G135, and in 70 ℃ of water-baths the heating 30 minutes after recentrifuge, discard the heat denatured protein precipitation, collect supernatant, carry out ultrafiltration with VIVAFLOW50 ultra-filtration membrane bag, concentrated solution is gone up DEAE-Sepharose FAST FLOW ion exchange column and Phenyl-Sepharose hydrophobic chromatography and Sephacryl S-200 molecular gel Filter column continuously and is carried out separation and purification, its total enzyme activity, specific activity and yield are as shown in table 3, show to obtain electrophoretically pure zymoprotein.
Total enzyme activity, specific activity and the yield of new enzyme G135 in table 3. purification step.
The character of embodiment 3, mycose-base synthetase G135 of the present invention
The molecular weight that records mycose-base synthetase G135 of the present invention with SDS-PAGE is about 84kD, and is similar to The sequencing results.According to the analysis of enzymic activity being studied temperature and pH influence to this enzymic activity and stability.During pH 5.5, under condition of different temperatures, measure the vigor of G135 enzyme, obtain temperature-vigor curve (Fig. 2); In the time of 60 ℃, under condition of different pH, measure the vigor of G135 enzyme, obtain pH-vigor curve (Fig. 3); Enzyme liquid is incubated 60 minutes respectively in differing temps after, measuring residual enzyme activity separately, is 100% with uninsulated enzyme liquid vigor, obtains temperature-beta stability line (Fig. 4); Enzyme liquid being incubated 30 minutes at 30 ℃ respectively under condition of different pH, measuring residual enzyme activity separately, is 100% with untreated original enzyme liquid vigor, obtains pH-beta stability line (Fig. 5).The result shows that the optimal temperature of this enzyme is 75 ℃ when when pH5.5 preserves 30 minutes, and when preserving 30 minutes for 30 ℃, the appropriate pH of this enzyme is about 5.5; This enzyme is stable in temperature under up to about 70 ℃ and the about 4-11 of pH.
Sequence table
<160>2
<210>1
<211>732
<212>PRT
＜213〉the unknown
<400>1
Met?Ile?Ile?Gly?Thr?Tyr?Arg?Leu?Gln?Leu?Asn?Lys?Lys?Phe?Thr?Phe
1 5 10 15
Tyr?Asp?Val?Ile?Glu?Asn?Leu?Asp?Tyr?Phe?Lys?Glu?Leu?Gly?Val?Ser
20 25 30
His?Leu?Tyr?Leu?Ser?Pro?Ile?Leu?Asn?Gly?Arg?Pro?Gly?Ser?Ala?His
35 40 45
Gly?Tyr?Asp?Val?Val?Asp?His?Ser?Glu?Ile?Asn?Glu?Glu?Leu?Gly?Gly
50 55 60
Lys?Glu?Gly?Tyr?Phe?Thr?Leu?Val?Lys?Glu?Ala?Lys?Ser?Arg?Gly?Leu
65 70 75 80
Gly?Ile?Ile?Gln?Asp?Ile?Val?Pro?Asn?His?Met?Ala?Ile?His?His?Thr
85 90 95
Asn?Trp?Arg?Leu?Met?Asp?Leu?Leu?Lys?Asn?Trp?Lys?Asn?Ser?Lys?Tyr
100 105 110
Tyr?Asn?Tyr?Phe?Asp?His?Tyr?Asp?Asp?Asn?Lys?Ile?Ile?Leu?Pro?Ile
115 120 125
Leu?Glu?Asp?Glu?Leu?Asp?Thr?Val?Ile?Asp?Lys?Gly?Leu?Ile?Lys?Val
130 135 140
Gln?Lys?Asp?Lys?Ile?Glu?Tyr?Arg?Gly?Phe?Ile?Leu?Pro?Ile?Asn?Asp
145 150 155 160
Glu?Gly?Val?Glu?Phe?Leu?Lys?Lys?Ile?Asn?Cys?Phe?Asp?Asn?Ser?Cys
165 170 175
Leu?Lys?Lys?Glu?Asp?Ile?Lys?Lys?Leu?Leu?Leu?Met?Gln?Tyr?Tyr?Arg
180 185 190
Leu?Thr?Tyr?Trp?Lys?Lys?Asp?Tyr?Pro?Asn?Tyr?Arg?Arg?Phe?Phe?Ala
195 200 205
Val?Asn?Asp?Leu?Ile?Ala?Val?Arg?Val?Glu?Leu?Asp?Glu?Val?Phe?Arg
210 215 220
Glu?Ser?His?Glu?Ile?Ile?Gly?Lys?Leu?Leu?Val?Asp?Gly?Leu?Arg?Ile
225 230 235 240
Asp?His?Ile?Asp?Gly?Leu?Tyr?Asn?Pro?Lys?Glu?Tyr?Leu?Asp?Lys?Leu
245 250 255
Arg?Gln?Leu?Val?Gly?Asn?Asp?Lys?Ile?Ile?Tyr?Val?Glu?Lys?Ile?Leu
260 265 270
Ser?Ile?Asn?Glu?Lys?Leu?Arg?Asp?Asp?Trp?Lys?Val?Asp?Gly?Thr?Thr
275 280 285
Gly?Tyr?Asp?Phe?Val?Asn?Tyr?Val?Asn?Met?Leu?Leu?Val?Asp?Gly?Ser
290 295 300
Gly?Glu?Glu?Glu?Leu?Thr?Lys?Phe?Tyr?Glu?Asn?Phe?Ile?Gly?Arg?Lys
305 310 315 320
Ile?Asn?Ile?Asp?Glu?Leu?Ile?Ile?Gln?Ser?Lys?Lys?Leu?Val?Ala?Asn
325 330 335
Gln?Leu?Phe?Lys?Ala?Asp?Ile?Glu?Thr?Leu?Ser?Asn?Leu?Leu?Asn?Val
340 345 350
Asn?Tyr?Asp?Tyr?Leu?Val?Asp?Phe?Leu?Ala?Cys?Met?Lys?Lys?Tyr?Arg
355 360 365
Thr?Tyr?Leu?Pro?Tyr?Glu?Asp?Ile?Asn?Gly?Ile?Arg?Glu?Cys?Asp?Lys
370 375 380
Glu?Gly?Lys?Leu?Lys?Asp?Glu?Lys?Gly?Ile?Met?Arg?Leu?Gln?Gln?Tyr
385 390 395 400
Met?Pro?Ala?Ile?Phe?Pro?Lys?Gly?Tyr?Glu?Asp?Thr?Thr?Leu?Phe?Ile
405 410 415
Tyr?Asn?Arg?Leu?Ile?Ser?Leu?Asn?Glu?Val?Gly?Ser?Asp?Leu?Arg?Arg
420 425 430
Phe?Ser?Leu?Ser?Ile?Asp?Asp?Phe?His?Asn?Phe?Asn?Gln?Ser?Arg?Val
435 440 445
Asn?Thr?Ile?Ser?Met?Asn?Thr?Leu?Ser?Thr?His?Asp?Thr?Lys?Phe?Ser
450 455 460
Glu?Glu?Leu?Arg?Ala?Arg?Ile?Ser?Val?Leu?Ser?Glu?Ile?Pro?Lys?Glu
465 470 475 480
Trp?Glu?Glu?Arg?Val?Ile?Tyr?Trp?His?Asp?Leu?Leu?Arg?Pro?Asn?Ile
485 490 495
Asp?Lys?Asn?Asp?Glu?Tyr?Arg?Phe?Tyr?Gln?Thr?Leu?Val?Gly?Ser?Tyr
500 505 510
Glu?Gly?Phe?Asp?Asn?Lys?Glu?Arg?Ile?Lys?Asn?His?Met?Ile?Lys?Val
515 520 525
Ile?Arg?Glu?Ala?Lys?Val?His?Thr?Thr?Trp?Glu?Asn?Pro?Asn?Ile?Glu
530 535 540
Tyr?Glu?Asn?Lys?Val?Leu?Asp?Phe?Ile?Asp?Glu?Ala?Phe?Glu?Asn?Ser
545 550 555 560
Asn?Phe?Thr?Asn?Asp?Phe?Glu?Thr?Phe?Glu?Lys?Arg?Ile?Val?Tyr?Phe
565 570 575
Ala?Tyr?Met?Lys?Ser?Leu?Val?Ala?Thr?Thr?Leu?Lys?Phe?Leu?Ser?Pro
580 585 590
Gly?Val?Pro?Asp?Ile?Tyr?Gln?Gly?Thr?Glu?Val?Trp?Arg?Phe?Leu?Leu
595 600 605
Thr?Asp?Pro?Asp?Asn?Arg?Met?Pro?Val?Asp?Phe?Lys?Lys?Leu?Arg?Glu
610 615 620
Leu?Leu?Asn?Asn?Leu?Thr?Gln?Lys?Asn?Leu?Glu?Leu?Ser?Asp?Pro?Arg
625 630 635 640
Val?Lys?Met?Leu?Tyr?Val?Lys?Lys?Leu?Leu?Gln?Leu?Arg?Arg?Glu?Tyr
645 650 655
Ser?Leu?Asn?Asp?Tyr?Lys?Pro?Leu?Pro?Phe?Gly?Phe?Gln?Arg?Gly?Lys
660 665 670
Val?Thr?Val?Leu?Phe?Ser?Pro?Ile?Val?Thr?Arg?Glu?Val?Lys?Glu?Lys
675 680 685
Ile?Ser?Ile?Arg?Gln?Lys?Ser?Val?Asp?Trp?Ile?Arg?Asn?Glu?Glu?Ile
690 695 700
Ser?Ser?Gly?Val?Tyr?Asn?Leu?Ser?Glu?Leu?Ile?Gly?Glu?His?Arg?Val
705 710 715 720
Val?Ile?Leu?Thr?Glu?Lys?Val?Gly?Glu?Leu?Pro?Ile
725 730
<210>2
<211>2243
<212>DNA
＜213〉the unknown
<220>
<221>
<222>
<223>
<400>2
atgataatag?gtacgtatag?gctacagctc?aataagaaat?tcacttttta?tgatgtaata 60
gaaaatttgg?attattttaa?agaattagga?gtatcacatc?tatacctatc?tccaatactt 120
aacggtaggc?cagggagtgc?tcacggttac?gatgtagtag?accatagtga?aattaatgag 180
gaattaggag?ggaaagaggg?atattttaca?ctagtcaagg?aagctaagag?tagaggttta 240
ggaatcatac?aagatatagt?gccaaatcac?atggcaatac?atcatactaa?ttggaggctt 300
atggatctac?taaagaattg?gaaaaatagt?aaatattaca?actattttga?tcattatgat 360
gataacaaaa?taatccttcc?aattcttgag?gacgagttgg?ataccgttat?agataaggga 420
ttgataaaag?tacaaaagga?taaaatagag?tataggggat?tcatattacc?aataaatgat 480
gaaggagtcg?agttcttgaa?aaaaattaat?tgctttgata?attcatgttt?aaagaaagag 540
gatataaaga?aattactatt?aatgcaatac?tataggttaa?cttactggaa?aaaagattac 600
ccaaattata?ggagattttt?cgcggtaaat?gatttgatag?ctgttagagt?agagttggat 660
gaagtattta?gagagtccca?tgagataatt?ggcaagctac?ttgttgacgg?tttaagaatt 720
gaccacatag?atggactata?taaccctaag?gagtatttag?ataagctaag?acagttagta 780
ggaaatgata?agataatata?cgtagagaag?atattatcaa?tcaacgagaa?attaagagat 840
gattggaaag?tagatggtac?tactggatat?gatttcctga?actacgttaa?tatgctatta 900
gtagatggaa?gtggtgagga?ggagttaact?aagttttatg?agaatttcat?tggaagaaaa 960
atcaatatag?acgagttaat?aatacaaagt?aaaaagttag?ttgcaaatca?gttgtttaaa 1020
gctgatattg?aaacattaag?caacttactg?aacgttaatt?acgattattt?agtagatttt 1080
ctagcatgta?tgaaaaaata?caggacgtat?ttaccatatg?aggatattaa?cggaataagg 1140
gaatgcgata?aggagggaaa?gttaaaagat?garaagggaa?tcatgagact?ccaacaatac 1200
atgccagcaa?tcttccctaa?gggctatgag?gatactaccc?tcttcatcta?caatagatta 1260
atttccctta?acgaggttgg?gagcgaccta?agaagattca?gtttaagcat?agacgatttt 1320
cataacttta?accaaagcag?agtaaatacc?atttcaatga?acactctctc?tacgcatgat 1380
actaagttca?gtgaagagct?tagagctaga?atatcagtac?tatctgagat?accaaaggag 1440
tgggaggaga?gggtaatata?ctggcatgat?ttgttaaggc?caaatataga?taaaaatgac 1500
gagtatagat?tttatcaaac?acttgtagga?agttacgagg?gatttgataa?taaggagaga 1560
attaagaacc?acatgattaa?ggtcataaga?gaagctaagg?tacatacaac?gtgggaaaat 1620
cctaatatag?agtatgaaaa?taaagttttg?gatttcatag?atgaagcgtt?cgagaacagt 1680
aattttacaa?atgattttga?aacttttgaa?aagagaatag?tttatttcgc?ttatatgaaa 1740
tcattagttg?caacgacact?taaattcctt?tcgcctggtg?taccagatat?ttatcaagga 1800
actgaagttt?ggagattctt?acttacagac?ccagataaca?gaatgccggt?ggatttcaag 1860
aaactaaggg?aattattaaa?taatttgact?caaaagaact?tagaactctc?agatccaaga 1920
gtcaaaatgt?tatatgttaa?gaaattgcta?caacttagaa?gagagtactc?actaaacgat 1980
tataaaccat?taccctttgg?cttccaaagg?ggaaaagtaa?ctgtcctttt?ctcaccaata 2040
gtgactaggg?aggttaaaga?gaagattagt?ataaggcaaa?aaagcgttga?ttggatcaga 2100
aatgaggaaa?ttagtagtgg?agtatacaat?ttaagtgagt?tgattgggga?gcatagagtc 2160
gttatattaa?ctgaaaaagt?gggtgaacta?cctatataga?tttattcctg?aactactctt 2220
gtcagatatg?cattactcag?atc 2243
Claims (12)
1. starch mycose-base synthetase, it is to have SEQ ID № in the sequence table: 1 amino acid residue sequence or with SEQ ID №: 1 amino acid residue sequence is through replacement, disappearance or the interpolation of one or several amino-acid residue and have the № with SEQ ID: 1 is identical active by SEQ ID №: 1 deutero-protein.
2. enzyme according to claim 1 is characterized in that: described starch mycose-base synthetase has SEQ ID № in the sequence table: 1 amino acid residue sequence.
3. starch mycose-base synthetase encoding gene has one of following nucleotide sequences:
1) the SEQ ID № in the sequence table: 2;
2) SEQ ID № in the code sequence tabulation: the polynucleotide of 1 protein sequence;
3) with sequence table in SEQ ID №: 2 dna sequence dnas that limit have 90% above homology, and the identical function protein DNA sequence of encoding.
4. gene according to claim 3 is characterized in that: described starch mycose-base synthetase encoding gene has SEQ ID № in the sequence table: 2 nucleotide sequence.
5. the carrier that contains the described gene of claim 3.
6. the transgenic cell line that contains the described gene of claim 3.
7. the host bacterium that contains the described gene of claim 3.
8. colon bacillus (Escherichia coli) DE3 (pM75) CGMCC № 1133.
9. a method of expressing the starch mycose-base synthetase is that the recombinant expression vector that will contain starch mycose-base synthetase encoding gene imports the expressive host bacterium, expresses obtaining the starch mycose-base synthetase; Described starch mycose-base synthetase encoding gene has one of following nucleotide sequences:
1) the SEQ ID № in the sequence table: 2;
2) SEQ ID № in the code sequence tabulation: the polynucleotide of 1 protein sequence;
3) with sequence table in SEQ ID №: 2 dna sequence dnas that limit have 90% above homology, and the identical function protein DNA sequence of encoding.
10. method according to claim 9 is characterized in that: member of EK system that described host bacterium is an e. coli k-12 or the member of BS system of subtilis.
11. method according to claim 10 is characterized in that: the described EK member of system comprises DH5 α, HB101, BE3, TOPO10 and JM109; The described BS member of system comprises BD170, MI112 and ISW1214.
12. according to claim 9,10 or 11 described methods, it is characterized in that: the carrier that sets out that is used to make up described recombinant expression vector comprises pBR322, pET, pBV220, pBAD and the pUC18 that is used for the EK system, and the pUB110 and the pHY300PLK that are used for the BS system; Described recombinant expression vector is pM75.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1364899A (en) * | 2001-01-10 | 2002-08-21 | 中国科学院微生物研究所 | Mycose base hydrolase and its preparation and use |
| CN1398966A (en) * | 2001-07-27 | 2003-02-26 | 吴襟 | Acidophilic thermostable mycose-base synthetase and its prepn and use |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1364899A (en) * | 2001-01-10 | 2002-08-21 | 中国科学院微生物研究所 | Mycose base hydrolase and its preparation and use |
| CN1398966A (en) * | 2001-07-27 | 2003-02-26 | 吴襟 | Acidophilic thermostable mycose-base synthetase and its prepn and use |
Non-Patent Citations (1)
| Title |
|---|
| 耐热古菌芝田硫化叶菌海藻糖生成相关酶的基因克隆、表达和序列分析. 吴襟，于炜婷，王辉，刘莉，王绍校，张树政.生物化学与生物物理进展，第30卷第5期. 2003 * |
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