CN101481696B - Cold adapted endo beta-xylanase gene XynA and use - Google Patents
Cold adapted endo beta-xylanase gene XynA and use Download PDFInfo
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Abstract
The invention relates to a gene XynA of cold adapted endo-beta-xylanase and an application thereof. The genome DNA fragment sequence is shown as SEQ ID N o. 1. The cold adapted beta-xylanase gene XynA is applicable for preparing beta-xylanase XynA. The obtained beta-xylanase XynA is used as neutral cold adapted hydrolase for hydrolyzing oat xylan, beech xylan and birch xylan to produce xylobiose and xylotriose. Optimum enzyme activity temperature is 30 DEG C and optimum pH is 7.0.
Description
Technical field
The present invention relates to a kind of gene of novel suitable cold endo-xylanase, belong to biological technical field.
Background technology
Beta-xylanase be by bacterium and fungus secretion can degradation of xylan glycoside hydrolase, the overwhelming majority belongs to the 10th family and the 11st family, finds in recent years that the minority beta-xylanase belonged to the 8th family.The beta-xylanase nature difference of the 10th family of different sources is little; And the enzyme substrates specificity of the 11st family is stronger, and molecular weight is lower, and the nature difference of different ramose enzymes is bigger.The beta-xylanase of the 10th family can hydrolysis closes on the glycosidic link of tapping point, and the non-reducing end glycosidic link.For the beta-xylanase of the 10th family, between two tapping points, have two non-replacement xylose residues of successive just can satisfy the requirement of its effect, and 3 such residues of the enzyme require of the 11st family could act on.The beta-xylanase of the 10th family not only has effect for xylan, and low-molecular-weight cellulosic substrate is also had effect, particularly aromatic base-cellobiose and cell-oligosaccharide.The enzyme of the 10th family also discharges xylose residues or the fragrance-xylose residues adjacent with substituted xylose residues.In addition, the enzyme of this family has higher enzymolysis activity to wood oligose.The crystal structure analysis of enzyme, and kinetics and end product analysis revealed during the wood oligose of the different sizes of enzymolysis, the enzyme of the 10th family has 4 substrate binding sites usually.The beta-xylanase of the 11st family is proper beta-xylanase, only for the substrate that contains wood sugar effect is arranged.Catalysis is more single than the 10th family, and its enzymolysis product needs the further enzymolysis of the 10th family.Identical with the 10th family, the enzyme of the 11st family can act on the aromatic base β-glycosidic link of xylo-bioses and xylotriose, but to the not effect of aromatic base cellobiose.In addition, work has serious obstruction to substituting group and β-1,3 key to enzyme, makes its enzymolysis product molecular weight than the 10th family's height.There is the three-dimensional structure of several beta-xylanases determined in the 10th family and the 11st family respectively.The enzymolysis nature difference of the 10th family and the beta-xylanase of the 11st family mainly is to be caused by the different of tertiary structure.The beta-xylanase molecule of the 11st family is little and tight, and mainly is made up of βZhe Die (β-pleated sheets).Catalyst structure domain is slit-like, can be in conjunction with 5-7 xylose residues.The beta-xylanase of the 10th family presents (beta/alpha)
8Tubbiness is folding, and its substrate binding site is in the crack darker in the tertiary structure unlike the enzyme of the 11st family, and molecule is big, snappiness is better, so show relatively poor Substratspezifitaet with respect to the 11st family.
Beta-xylanase has a wide range of applications in industries such as papermaking, food and feed.In the pulping and paper-making industry, the use of beta-xylanase can reduce the consumption of acid bleaching agent in the pulp bleaching process (as chlorine, dioxide peroxide), reduces and pollutes.In food service industry, beta-xylanase can decompose from natural hemicellulose such as cotton seed hulls, bagasse and corn cob and obtains xylo-oligosaccharide, and this sugar is the current remarkable a kind of functional oligose that is subjected to most.Its sugariness is 40% of a sucrose, can not cause that glucose level rises significantly in the blood plasma after edible.Can be made into the food of different sugarinesses with xylo-oligosaccharide, can replace glucose, as diabetics's dietetic food.The use of beta-xylanase can be converted into nutritional substance with the non-starch polysaccharide in the feed in feedstuff industry, eliminates its anti-oxidant action, thereby improves the transformation efficiency of feed.Beta-xylanase is applied to China grass degumming, can reduces environmental pollution, reduce the damage of degumming technology fiber.
Because the extensive use of beta-xylanase is all paid attention to by the countries in the world scientist the research and development of beta-xylanase always.The novel beta-xylanase that exploitation has new purposes has very important theory significance and using value.Since the seventies in 20th century, along with gene recombination technology and development of molecular biology, the encoding gene of many beta-xylanases and primary structure are studied in great detail, and the minority enzyme are arranged by crystallization.
In beta-xylanase, fit cold zytase owing to have very high activity at low temperatures, therefore at association with pulp bleaching, wood oligose production, warm zytase has more superiority in industries such as flour modification and the feed ratio, has been subjected to investigator's extensive concern.Studies show that in recent years, exist a large amount of microorganisms in the sea ice of polar region, these microorganism long term growth have formed cryogenic special construction of adaptation and special mechanism under cryogenic extreme environment, therefore, sea ice microorganism in polar region is a good material of finding novel suitable cold enzyme.But because difficulties such as sampling and cultivation are at present relatively less to the research of polar region sea ice microorganism.
Summary of the invention
At the deficiencies in the prior art, the invention provides gene xynA of a kind of suitable cold beta-xylanase XynA newly and preparation method thereof.The present invention also provides a kind of polar region sea ice bacterium-cold-adaptive microbe bacterium strain Glaciecolamesophila SM0901 that is used to prepare gene xynA.
The genomic DNA fragment sequence that contains suitable cold xylanase gene xynA of the present invention is shown in SEQ ID NO.1:
SEQ?ID?NO.1：
atgttaagaa?agttcatgct?aagagggcgt?ttgaggaaag?aattgatact?caagttaaac 60
ctgcttaaac?ccaacatgct?aacgataaaa?ccttgcttgt?tggcactcgc?gttagcggcg 120
acatccactg?taagccaggc?agctacagcc?gtgagctcaa?atgactcagc?cttgagcctt 180
aaaaatgagg?cagtgaatcc?taaaaacgag?gctgtgagcc?aaacaaaaag?tctgaaagcc 240
catttcagta?agcagttttt?agtgggcagt?gctattaacg?cacagcaagc?taaacgcacc 300
gaacaagaca?ctgatgcctt?aatcatcacc?caatttaata?cgattacccc?tgaaaacgaa 360
ttgaagtggg?agcgcattca?tccaaagcct?gatgcttatg?acttctctct?gtccgatgag 420
tacgtacatt?acggcttagc?caataatatg?ttcatcatag?ggcacacgtt?ggtttggcac 480
agccaaacac?cggattgggt?atttgaaaac?gcacaaggcg?agttgttaac?gagagaggcg 540
ttgttagctc?gcatgaaaga?gcacattcac?accgttgtga?gtcgctacaa?aggcaaaatc 600
aaaggctggg?atgtggtcaa?tgaagcgctc?aatgaagatg?gtagcctgcg?ggattctaag 660
tggcggcaga?ttattggcga?tgacttcatt?gaaaaagcct?ttacctatgc?ccacgctgca 720
gatccagatg?ctaaattgta?ttacaacgat?tacaacttat?ataaaccaga?aaaaagtgcc 780
ggtgcagcaa?aactaatcaa?gtcattacaa?gacaaaggga?tccctgttca?cggtgtcggc 840
ttacaaggcc?actactcgtt?aacgcaccct?gccttaaatg?aactagacga?tgcattaacg 900
ctgtttgctt?ccttgggaat?tgaatctatg?atcaccgagt?tagatgtatc?cgtattaccc 960
tttcccagtg?aagctataca?aggggcagat?atcagccaag?atttagcctt?gaacaaagcc 1020
ttaaacccct?accctgatgg?gttaccagaa?gcgcagcagg?acgcattaac?tgctcgatac 1080
aaggagatat?tttcggtatt?tctaacgcac?caagacacgc?taacacgcgt?tacgttttgg 1140
ggcgtgaatg?atgccaacag?ttggcgtaac?aattggccaa?tgcgcgggcg?caccgattac 1200
ccgttactgt?ttgatcgaaa?cagtgaaatt?aagcccgcct?atcgtgctgt?catgacacta 1260
actattgact?ga 1272
The gene xynA of the suitable cold beta-xylanase of the present invention is the genomic dna that extracts from marine bacteria Glaciecola mesophila DSM 15026T.Described marine bacteria Glaciecola mesophila DSM 15026T separates in the marine invertebrate body to obtain, and this bacterial strain is preserved in the German DSMZ of DSMZ, preserving number: Glaciecola mesophila DSM15026T, but market is buied.Beta-xylanase gene order design primer according to existing Pseudoalteromonas (Pseudoalteromonas) in the database.Then, utilize round pcr from the genomic dna of Glaciecola mesophila SM0901, to clone the gene of coding beta-xylanase.Nucleotide sequence to this gene is measured.Sequencing result shows in the dna fragmentation of acquisition and contains an open reading frame with 1272 Nucleotide that this open reading frame promptly is the gene xynA of coding beta-xylanase, 423 amino acid of encoding altogether.Therefore, the precursor of beta-xylanase XynA is one and contains 423 amino acid whose polypeptide.
The structure of the precursor of beta-xylanase XynA comprises signal peptide and catalyst structure domain two portions.The gene xynA of beta-xylanase is carried out heterogenous expression and purifying in intestinal bacteria, obtain ripe activated beta-xylanase XynA.Wherein, maturing enzyme contains 373 amino acid, is a zytase of not reporting as yet with new sequence in lytic enzyme the 10th family.
Nucleotide and coded amino acid corresponding relation are as follows among the gene xynA of above-mentioned suitable cold beta-xylanase, and signal peptide cutting site indicates with arrow, and terminator represents that with asterisk underscore partly is the maturing enzyme sequence, and first amino acid label of maturing enzyme is+1:
The present invention is with a kind of beta-xylanase called after of bacterial strain Glaciecola mesophila DSM 15026T excretory XynA.The unnamed gene of this beta-xylanase XynA is xynA.The expression of this beta-xylanase XynA is extracted among the embodiment 2 and will elaborates.
The technical term that the present invention uses:
1.CTAB/NaCl method: be meant at containing the more bacterium of polysaccharose substance, extracting on the bacterial genomes DNA ordinary method basis, add CTAB (bromination n-Hexadecane trimethyl ammonium) and sodium-chlor and remove polysaccharose substance, thereby reach a kind of method of extraction effect preferably.The CTAB/NaCl method is the genome DNA extracting method of this area routine.
2.PCR technology: the abbreviation of chain polymerization enzyme reaction technology is to utilize archaeal dna polymerase, template, and primer and dNTP carry out the technology of amplification in vitro specific DNA fragments.
3.CDD analysis software: English full name is Conserved Domain Database, and Chinese is the conserved structure regional data base, and it is a kind of software that is used for inferring and analyzing the structural domain information of specific amino acids sequence formation that provides on the NCBI website.
The cloning process of the suitable cold beta-xylanase gene XynA of the present invention, step is as follows:
1, utilize the CTAB/NaCl method to extract the genomic dna of Glaciecola mesophila SM0901.
2, according in the database existing from Pseudoalteromonas atlantica T6c bacterium genome sequence known beta-xylanase gene order design primer.
3, then, utilize the gene of round pcr clones coding zytase XynA from the genomic dna of Glaciecola mesophila DSM 15026T.Obtaining the dna fragmentation of a 1272bp at last, is the open reading frame of a coding beta-xylanase XynA, and initiator codon is positioned at 1bp, and terminator codon is positioned at 1270bp, 423 amino acid of encoding altogether.
4, utilize the CDD analysis software to carrying out analysis revealed according to the gene order deduced amino acid, the structure of the precursor of beta-xylanase XynA comprises signal peptide and catalyst structure domain, and the precursor of beta-xylanase XynA is one and contains 423 amino acid whose polypeptide.
The preparation of beta-xylanase XynA:
Beta-xylanase gene xynA is carried out heterogenous expression and purifying in intestinal bacteria, obtain the ripe beta-xylanase XynA of a molecular weight 42.97kDa.Beta-xylanase XynA to purifying carries out property testing.
Analyze ordinary methods such as hydrolysate, the work of the suitableeest enzyme, optimal pH and measure by substrate specificity, HPLC suitable cold beta-xylanase XynA, the result shows this endonuclease capable hydrolysis oat xylan, the beech wood glycan, birch xylan, product is xylo-bioses and xylotriose, the suitableeest enzyme temperature alive is 30 ℃, and optimal pH is 7.0.Shown in Fig. 5-8.Therefore, beta-xylanase XynA is the suitable cold endo-xylanase of a neutrality.
The application of suitable cold beta-xylanase gene XynA:
The beta-xylanase XynA that gene XynA expresses and purifying obtains by suitable cold beta-xylanase fits cold lytic enzyme as neutrality, is used for hydrolysis oat xylan, beech wood glycan, birch xylan.Can be widely used in the industry production such as association with pulp bleaching, wood oligose production, flour modification or feed.
Beneficial effect of the present invention is as follows:
Encode a kind of suitable cold inscribe beta-xylanase XynA of new sequence of cloned genes xynA of the present invention.Compare with other beta-xylanase, beta-xylanase XynA has very high catalytic efficiency at 0-30 ℃, is applied to association with pulp bleaching, wood oligose production, and flour modification etc. can at room temperature be carried out, thus save energy.In addition, XynA is the restriction endonuclease of a strictness, and hydrolyzed xylan only produces oligosaccharides such as xylo-bioses and xylotriose, is suitable for very much wood oligose production, and this enzyme is suitable cold enzyme, thereby can at room temperature carry out wood oligose production with this enzyme.
Description of drawings
Fig. 1. the electrophorogram of the genomic dna of the Glaciecola mesophila DSM 15026T of extraction.The genomic dna of 1:Glaciecolamesophila DSM 15026T; 2:DNA molecular weight marker (marker).
Fig. 2. the electrophorogram of the gene fragment of the coding XynA by pcr amplification clone.1: the dna fragmentation of amplification; 2:DNA molecular weight marker (marker).
Fig. 3. in intestinal bacteria, carry out the suitable cold beta-xylanase XynA electrophorogram of heterogenous expression and purifying.1: molecular weight protein marker (marker); 2: the supernatant liquor electrophorogram of e. coli bl21 behind IPTG abduction delivering medium centrifugal that contains expression plasmid; 3: supernatant liquor dissolves and dialysis rear electrophoresis figure through ammonium sulfate precipitation and with 50mmol Tris-HCl; 4: the pure beta-xylanase XynA electrophorogram behind the sample process nickel post affinitive layer purification.
The pre-enzyme of Fig. 4 .XynA and maturing enzyme structural representation.
The substrate specificity of the suitable cold beta-xylanase XynA of Fig. 5.Wherein, B.W.X is a birch xylan, and Be.W.X is the beech wood glycan, and O.S.X is the oat xylan, CMC is a cellulose acetate, and Laminarin is a Sargassum polysaccharides, and Mannan is a mannosans, Chitosan is a chitin, and Starch is a starch, and N.A represents can not this kind of hydrolysis substrate.
Fig. 6 .HPLC analyzes beta-xylanase XynA hydrolysis beech wood glycan and wood oligose product curve.What curve was represented respectively from bottom to top successively among the figure is: standard substance, and the xylotriose enzymolysis, the Xylotetrose enzymolysis, wooden pentasaccharides enzymolysis, wooden six carbohydrases are separated, beech wood glycan enzymolysis.
Fig. 7 is the enzyme of a suitable cold beta-xylanase XynA temperature curve alive.
Fig. 8 is the enzyme of a suitable cold beta-xylanase XynA pH curve alive.
Embodiment
The present invention will be further described below in conjunction with drawings and Examples, but be not limited thereto.
Embodiment 1:
Suitable cold endo-xylanase XynA encoding gene fragment sequence such as SEQ ID NO.1.Nucleotide and coded amino acid corresponding relation are as previously mentioned in the gene.This genomic DNA fragment is 1272bp altogether, wherein contains the suitable cold endo-xylanase XynA of open reading frame coding of a 1272bp, and initiator codon is positioned at 1bp, and terminator codon is positioned at 1270bp, 423 amino acid of encoding altogether.
Embodiment 2: gene fragment clone method of the present invention
The bacterial classification source: Glaciecola mesophila DSM 15026T separates to obtain in the marine invertebrate body.Be stored in the German DSMZ of DSMZ, preserving number: DSM 15026T.Market is buied.
Concrete grammar is as follows:
1. fit the clone of cold endo-xylanase XynA encoding gene
1.1 the extraction of Glaciecola mesophila SM0901 genomic dna
With reference to " fine works molecular biology experiment guide ".
(1) cultivates the 50ml bacterial cultures to state of saturation, get the centrifugal 5min of culture 12000g of 15ml;
(2) throw out adds 5670 μ l TE, blows and beats repeatedly with suction pipe and makes it resuspended.Add 300 μ l 10%SDS and 30 μ l20mg/mL Proteinase Ks, mixing, 37 ℃ of incubation 2.5-3h;
(3) add 1000 μ l 5M NaCl, fully mixing adds 800 μ l CTAB/NaCl solution again, and mixing is in 65 ℃ of incubation 30-60min (beginning to remove the macromole of polysaccharide and other pollution) from this step;
(4) add isopyknic chloroform/primary isoamyl alcohol (24: 1), mixing, 4 ℃, the centrifugal 20min of 12000g changes supernatant liquor in the new pipe over to, shifts out supernatant as difficulty, removes boundary material with toothpick earlier;
(5) add isopyknic chloroform/primary isoamyl alcohol (24: 1), mixing, 4 ℃, the centrifugal 30min of 14000g forwards supernatant in the new pipe to;
(6) add isopyknic chloroform/primary isoamyl alcohol (24: 1), mixing, 4 ℃, the centrifugal 30min of 16000g forwards supernatant in the new pipe to;
(7) add 0.6 times of volume Virahol, mix (slowly turning upside down 3 minutes) gently, place 4 ℃ of refrigerators to keep 10min, the flocks ditch is gone out, change in the centrifuge tube of a 5ml with a thin curved glass rod;
(8) with washing in 70% ethanol of 3ml 2 times, use absolute ethanol washing again 2 times, the centrifugal 2min of each 12000g abandons supernatant, air-dry on the Bechtop (do not make the precipitation complete drying, otherwise extremely indissoluble);
(9) add 100-200 μ l TE damping fluid, 4 ℃ of dissolving DNAs that spend the night;
(10) adding 10mg/ml RNase is 50ug/mL to final concentration, 37 ℃ of incubation 1h;
(11) add equal-volume phenol/chloroform/primary isoamyl alcohol extracting 1 time, supernatant liquor is used chloroform/primary isoamyl alcohol extracting 1 time again;
(12) dehydrated alcohol of adding 1/10 volume 3M NaAc (pH5.2) and 2 times of volume precoolings in supernatant is inverted and is mixed;
(13) supernatant discarded gently after centrifugal, the DNA precipitation is respectively washed 1 time with 70% ethanol and dehydrated alcohol, is dissolved among an amount of TE buffer after dried slightly, measures DNA concentration and purity with ultraviolet spectrophotometer and is placed on 4 ℃ of refrigerators preservations (can in-20 ℃ of prolonged preservation).
1.2 design of primers is with synthetic
Two Auele Specific Primer XYL1:ATGTTAAGAAAGTTCATGC of beta-xylanase gene order design according to existing Pseudoalteromonas atlantica T6c bacterium in the database; XYL2:TCAGTCAATAGTTAGTGTC, synthetic by Bo Ya biotech company.
1.3 utilize PCR to carry out the gene order amplification
(1) being primer with XYL1 and XYL2, is template with the genomic dna, does pcr amplification.The PCR reaction conditions is: 95 ℃, and 5 minutes; 95 ℃ then, 1 minute; 50 ℃, 1 minute; 72 ℃, 1.5 minutes, after 32 circulations, 72 ℃, extended 10 minutes.
(2) pcr amplification product is carried out 1% agarose gel electrophoresis, the result shows the dna fragmentation that obtains a treaty 1300bp.DNA with Omega company reclaims test kit according to its explanation recovery amplification of DNA fragments then.
2. gene sequencing
(1) dna fragmentation is connected with cloning vector
Be connected on the pGEMT carrier of Promega company reclaiming dna fragmentation.The ligation system:
Exogenous dna fragment 3 μ l
T
4Ligase enzyme 1 μ l
Add ddH
2O to 10 μ l
Cover tight lid, finger flicks centrifuge tube, and the mixing sample changeed 2 seconds on whizzer, and sample is concentrated on the pipe end, and 16 ℃ of connections are spent the night.
(2) go up the competent method of preparation intestinal bacteria by " molecular cloning experiment guide " and prepare the bacillus coli DH 5 alpha competence.
(3) the reorganization pGEMT carrier that will connect by the heat shock method for transformation on " molecular cloning experiment guide " goes to the bacillus coli DH 5 alpha competence.
(4) bacillus coli DH 5 alpha of Zhuan Huaing is applied to the Mai Kangkai substratum that contains 100 μ g/L penbritins, 37 ℃ of incubated overnight, select white transformant as template, as primer, verify by bacterium colony PCR whether plasmid in the white transformant contains the dna fragmentation of pcr amplification with XYL1 and XYL2.
(5) transformant that plasmid is contained the dna fragmentation of pcr amplification is served the order-checking of sea living worker Bioisystech Co., Ltd.Therefore, obtain the sequence 1272bp of zytase XynA encoding gene xynA.Sequence is shown in SEQ ID NO.1.The open reading frame that wherein contains the coding zytase XynA of a 1272bp by the blastX software analysis discovery of NCBI website.Initiator codon is positioned at 1bp, and terminator codon is positioned at 1270bp, 423 amino acid of encoding altogether.The sequence of the complete genome xynA of zytase XynA and amino acid sequence coded thereof are as shown in Figure 2.
3. the expression of gene xynA in intestinal bacteria
3.1 the structure of expression vector pet-22b-xynA
(1) utilize low dose of plasmid extraction kit, step is to specifications extracted recombinant plasmid pGEMT-xynA. from the transformant that contains reorganization pGEMT plasmid
(3) according to two Auele Specific Primers of sequencing result design
XYA1:GCTTGGAC
CATATGWhat TTAAGAAAGTTCATGC marked with underscore is the NdeI restriction enzyme site.
XYA2:CGACG
CTCGAGGWhat TCAATAGTTAGTGTC marked with underscore is the XhoI restriction enzyme site.Synthetic by Bo Ya biotech company.
(3) utilizing PCR to carry out the gene order amplification, is primer with XYA1 and XYA2, is template with reorganization pGEMT plasmid, does pcr amplification.The PCR reaction conditions is: 95 ℃, and 5 minutes; 95 ℃ then, 1 minute; 50 ℃, 1 minute; 72 ℃, 1.5 minutes, after 32 circulations, 72 ℃, extended 10 minutes.
(4) pcr amplification product is carried out 1% agarose gel electrophoresis, the result shows the dna fragmentation that obtains a treaty 1300bp.
DNA with Omega company reclaims test kit according to its explanation recovery amplification of DNA fragments then.
(5) carry out double digestion with restriction enzyme NdeI and XhoI to reclaiming fragment and plasmid pet-22b.The endonuclease reaction system is as follows:
Enzyme cutting buffering liquid 2 μ l enzyme cutting buffering liquids 2 μ l
Reclaim dna fragmentation 10 μ l plasmid pet-22b 6 μ l
Add ddH
2O to 20 μ l adds ddH
2O to 20 μ l
Be placed in 37 ℃ of water-baths and reacted 3 hours.Enzyme is cut product carry out 1% agarose gel electrophoresis, the DNA with Omega company reclaims test kit according to its explanation recovery amplification of DNA fragments then.
(6) will be connected on the pet-22b carrier through the xynA gene fragment of double digestion.The ligation system:
Carrier pet-22b 1 μ l
XynA gene fragment 3 μ l
T
4Ligase enzyme 1 μ l
Add ddH
2O to 10 μ l
Cover tight lid, finger flicks centrifuge tube, and the mixing sample changeed 2 seconds on whizzer, and sample is concentrated on the pipe end, connects in 16 ℃ of shaking tables and spends the night.
(7) go up the competent method of preparation intestinal bacteria by " molecular cloning experiment guide " and prepare the bacillus coli DH 5 alpha competence.
(8) the reorganization pet-22b carrier that will connect by the heat shock method for transformation on " molecular cloning experiment guide " goes to the bacillus coli DH 5 alpha competence.
(9) bacillus coli DH 5 alpha of Zhuan Huaing is applied to the LB substratum that contains 100 μ g/mL penbritins, 37 ℃ of incubated overnight.
(10) picking list bacterium colony on flat board is connected to the LB liquid nutrient medium that 5mL contains 100 μ g/mL penbritins, 37 ℃ of incubated overnight.
(11) collect 3mL bacterium liquid, centrifugal 2 minutes of 12000g obtains thalline, extracts recombinant plasmid pet-22b-xynA with test kit.
Place-20 ℃ of preservations.
3.2 expression vector pet-22b-xynA is transformed in the e. coli bl21 (DE3).
(1) goes up the competent method of preparation intestinal bacteria by " molecular cloning experiment guide " and prepare the e. coli bl21 competence.
(2) the reorganization pet-22b carrier that will connect by the heat shock method for transformation on " molecular cloning experiment guide " goes to the e. coli bl21 competence.
(3) e. coli bl21 of Zhuan Huaing is applied to the LB substratum that contains 100 μ g/mL penbritins, 37 ℃ of incubated overnight.
3.3 gene xynA abduction delivering in intestinal bacteria.
(1) picking list bacterium colony on flat board is connected to the LB liquid nutrient medium that 20mL contains 100 μ g/mL penbritins, 37 ℃ of incubated overnight.
(2) 1% inoculum sizes are transferred to the fresh LB substratum that contains 100 μ g/mL penbritins.37 ℃ are cultured to bacteria concentration OD0.8, add IPTG to final concentration be 0.5mmol/L.Continuation was cultivated 20 hours at 15 ℃ of shaking tables.
4. the purifying of the zytase XynA of heterogenous expression
(1) collection is through the 1L LB nutrient solution of IPTG abduction delivering, and centrifugal 10 minutes of 12000g collects supernatant liquor.
(2) ammonium sulfate of adding 60% saturation ratio in the supernatant liquor placed 0 ℃ of refrigerator precipitation 8-12 hour.
(3) with above-mentioned precipitation 13000g, centrifugal 15 minutes, abandon supernatant liquor, keep precipitation.
(4) with Tris-HCl (pH7.5) the damping fluid dissolution precipitation of 50mmol/L, and with same buffer dialysis 3-4 time.
(5) nickel post affinity chromatography is carried out in the solution requirement to specifications after the dialysis.
(6) sample of collecting behind the chromatography detects purity with SDS-PAGE, proves the pure enzyme of the electrophoresis that obtains zytase XynA (Fig. 3).Tris-HCl (pH7.5) damping fluid with 20mmol/L is dialysed 3-4 time.Place-20 ℃ of preservations standby at last.
5. the property testing of zytase XynA
5.1 substrate specificity
(1) with 50mmol/L sodium phosphate buffer (pH7.0) compound concentration is 1% different substrate solutions, comprises the oat xylan, beech wood glycan, birch xylan, cellulose acetate, Sargassum polysaccharides, mannosans, chitin, starch.
(2) in the quantity tube of 20mL, add the different substrate solution of 0.9mL, place 30 ℃ of water-bath preheatings 5 minutes.Add the pure enzyme liquid of 0.1mL, reacted 10 minutes, add 1.5mLDNS solution termination reaction then.Quantity tube is placed boiling water bath heating 5 minutes, add the distilled water constant volume at last to 15mL.
(3) characterize hydrolysis degree by measuring the amount that 550nm place light absorption value calculates the reducing sugar of generation.
This enzyme to the hydrolytic activity of different substrates as shown in Figure 5.
5.2 HPLC analyzes hydrolysate
(1) with 50mmol/L sodium phosphate buffer (pH7.0) preparation 5mg/mL beech xylan solution, with the wood oligose solution (a wooden sugar-wooden six sugar) of tri-distilled water preparation 1mg/mL.
(2) reaction that in the eppendorf pipe, is hydrolyzed.Reaction system is as follows:
100 μ L 5mg/mL beech xylan solutions add the pure enzyme of 2 μ g, 15 ℃ of hydrolysis 10 hours.
The wood oligose solution of 50 μ L 1mg/mL adds the pure enzyme of 1 μ g, 15 ℃ of hydrolysis 10 hours.
Reaction soln is boiled 5 minutes termination reactions and makes protein denaturation, and centrifugal 5 minutes of 13000g removes denatured protein.Draw supernatant liquor with liquid-transfering gun and be placed on-20 ℃ of preservations by the filtration of 0.22 μ m cellulosefilm.
(3) will filter good sample in the previous step by high performance liquid chromatography technology (High performance liquidchromatography, HPLC) monose in the analysis enzymolysis solution and the amount of oligosaccharides, analytical column adopts Aminex HPX-42C (Bio-rad, 300mm * 7.8mm), detector is a RID-10A type differential refraction detector, with distilled water is moving phase, and flow velocity is 0.4ml/min, and column temperature is 75 ℃.
(4) the HPLC analytical results shows that zytase XynA enzymolysis beech wood glycan and the above wood oligose of tetrose only produce xylo-bioses and xylotriose (Fig. 6), can not the hydrolysis xylotriose, show that zytase XynA is the restriction endonuclease of a strictness.
The temperature 5.3 the suitableeest enzyme is lived
Under 0,5,10,15,20,25,30,35,40,45,50,55,60 ℃ of condition, measure the enzyme of beta-xylanase XynA respectively and live, to determine the optimal reactive temperature of enzyme.Measuring enzyme concrete grammar alive is:
The beech xylan solution of 90 μ L 1% adds the pure enzyme liquid of 10 μ L, and reaction is 10 minutes under the differing temps, adds 150 μ L DNS liquid termination reactions, boils 5 minutes.Add 1.25mL distilled water mixing, measure light absorption value in the 550nm place.Calculate the amount of the reducing sugar of generation according to the wood sugar typical curve.The result shows the suitableeest enzyme of this enzyme 30 ℃ of temperature (as Fig. 7) alive.
5.4 optimal pH
The enzyme of zytase is lived under pH3.0,4.0,5.0,5.5,6.0,7.0,8.0,9.0,10.0,11.0,12.0 conditions respectively, to determine the optimal pH of this enzyme.Buffer system (50mmol/L): pH3.0-6.0, citrate buffer solution; PH6.0-8.0, phosphoric acid buffer; PH8.0-9.0, the Tris-hydrochloride buffer; PH9.0-12.0, glycine-NaOH damping fluid.
Concrete grammar is 1% a beech xylan solution of preparing different pH with above-mentioned damping fluid, measures the enzyme of beta-xylanase XynA under condition of different pH according to the method in 5.3 then and lives.The result shows that the optimal pH of this enzyme is 7.0 (as Fig. 8).
5. result
Utilize the CTAB/NaCl method to extract the genomic dna (Fig. 1) of Glaciecola sp.GM.Obtain the nucleotide sequence of the gene xynA of coding beta-xylanase XynA through order-checking by pcr amplification obtain the encoding gene DNA fragment (Fig. 2) of beta-xylanase XynA.Gene xynA contains one 1272 the suitable cold endo-xylanase XynA of open reading frame coding, and initiator codon is positioned at 1bp, and terminator codon is positioned at 1270bp, 423 amino acid of encoding altogether.Utilize the structure of the CDD software analysis beta-xylanase XynA of NCBI website, the structure of the precursor of beta-xylanase XynA comprises signal peptide and catalyst structure domain two portions (Fig. 4).Gene xynA is carried out heterogenous expression and purifying in intestinal bacteria, obtain ripe activated beta-xylanase XynA (Fig. 3).The structure of maturing enzyme beta-xylanase XynA contains 373 amino acid whose catalyst structure domains as shown in Figure 4, is an enzyme of not reporting as yet with new sequence in lytic enzyme the 10th family.The wood oligose (Fig. 5) that this endonuclease capable hydrolyzed xylan and tetrose are above, the product of hydrolysis is xylo-bioses and xylotriose (Fig. 6), does not produce monose, and the suitableeest enzyme temperature alive is 30 ℃ (Fig. 7), and optimal pH is 7.0 (Fig. 8).Therefore, the beta-xylanase XynA of gene xynA coding is the suitable cold endo-xylanase of a neutrality.
Sequence table
＜110〉Shandong University
＜120〉a kind of gene xynA of suitable cold inscribe beta-xylanase
<160>1
<170>Patent?In 3.1
<210>1
<211>1272
<212>DNA
atgttaagaa?agttcatgct?aagagggcgt?ttgaggaaag?aattgatact?caagttaaac 60
ctgcttaaac?ccaacatgct?aacgataaaa?ccttgcttgt?tggcactcgc?gttagcggcg 120
acatccactg?taagccaggc?agctacagcc?gtgagctcaa?atgactcagc?cttgagcctt 180
aaaaatgagg?cagtgaatcc?taaaaacgag?gctgtgagcc?aaacaaaaag?tctgaaagcc 240
catttcagta?agcagttttt?agtgggcagt?gctattaacg?cacagcaagc?taaacgcacc 300
gaacaagaca?ctgatgcctt?aatcatcacc?caatttaata?cgattacccc?tgaaaacgaa 360
ttgaagtggg?agcgcattca?tccaaagcct?gatgcttatg?acttctctct?gtccgatgag 420
tacgtacatt?acggcttagc?caataatatg?ttcatcatag?ggcacacgtt?ggtttggcac 480
agccaaacac?cggattgggt?atttgaaaac?gcacaaggcg?agttgttaac?gagagaggcg 540
ttgttagctc?gcatgaaaga?gcacattcac?accgttgtga?gtcgctacaa?aggcaaaatc 600
aaaggctggg?atgtggtcaa?tgaagcgctc?aatgaagatg?gtagcctgcg?ggattctaag 660
tggcggcaga?ttattggcga?tgacttcatt?gaaaaagcct?ttacctatgc?ccacgctgca 720
gatccagatg?ctaaattgta?ttacaacgat?tacaacttat?ataaaccaga?aaaaagtgcc 780
ggtgcagcaa?aactaatcaa?gtcattacaa?gacaaaggga?tccctgttca?cggtgtcggc 840
ttacaaggcc?actactcgtt?aacgcaccct?gccttaaatg?aactagacga?tgcattaacg 900
ctgtttgctt?ccttgggaat?tgaatctatg?atcaccgagt?tagatgtatc?cgtattaccc 960
tttcccagtg?aagctataca?aggggcagat?atcagccaag?atttagcctt?gaacaaagcc 1020
ttaaacccct?accctgatgg?gttaccagaa?gcgcagcagg?acgcattaac?tgctcgatac 1080
aaggagatat?tttcggtatt?tctaacgcac?caagacacgc?taacacgcgt?tacgttttgg 1140
ggcgtgaatg?atgccaacag?ttggcgtaac?aattggccaa?tgcgcgggcg?caccgattac 1200
ccgttactgt?ttgatcgaaa?cagtgaaatt?aagcccgcct?atcgtgctgt?catgacacta 1260
actattgact?ga 1272
Claims (3)
1. a suitable cold beta-xylanase gene XynA is characterized in that this gene order is shown in SEQ ID NO.1.
2. the application of the described suitable cold beta-xylanase gene XynA of claim 1 is used for the preparation of beta-xylanase XynA, and step is as follows:
Beta-xylanase gene XynA is carried out heterogenous expression and purifying in intestinal bacteria, obtain the ripe beta-xylanase XynA of a molecular weight 42.97kDa.
3. as the application of beta-xylanase XynA as described in the claim 2, it is characterized in that gained beta-xylanase XynA is as the suitable cold lytic enzyme of neutrality, be used for hydrolysis oat xylan, the beech wood glycan, birch xylan, product is xylo-bioses and xylotriose, and the suitableeest enzyme temperature alive is 30 ℃, and optimal pH is 7.0.
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| CN1415016A (en) * | 1999-12-30 | 2003-04-30 | 金克克国际有限公司 | Trichoderma reesei xylanase |
| CN1185336C (en) * | 2002-04-19 | 2005-01-19 | 浙江大学 | Li's Trichoderma strains and use thereof |
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| CN1415016A (en) * | 1999-12-30 | 2003-04-30 | 金克克国际有限公司 | Trichoderma reesei xylanase |
| CN1185336C (en) * | 2002-04-19 | 2005-01-19 | 浙江大学 | Li's Trichoderma strains and use thereof |
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| Title |
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| 杜宗军等.青岛近海琼胶降解细菌的筛选和多样性分析.《中国海洋大学学报》.2007,第37卷(第2期),277-282. * |
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