CN105779491A - Thermophilic beta-1, 4 xylanase-His fusion protein, preparation method and application thereof, and construction of genetic engineering bacteria thereof - Google Patents
Thermophilic beta-1, 4 xylanase-His fusion protein, preparation method and application thereof, and construction of genetic engineering bacteria thereof Download PDFInfo
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Abstract
The invention provides a construction method of thermophilic beta-1, 4 xylanase-His fusion protein gene engineering bacteria, wherein the nucleotide sequence of the thermophilic beta-1, 4 xylanase-His fusion protein gene is shown as SEQ ID NO.1, and the construction method comprises the following steps: step A: culturing high-temperature alkane bacillus; and B: extracting genome DNA and constructing a fusion protein gene sequence; and C: designing a primer and fishing a thermophilic beta-1, 4 xylanase-His fusion protein target gene by a Polymerase Chain Reaction (PCR) method; step D: constructing a recombinant expression vector; step E: the recombinant expression vector is transformed into a host cell. The thermophilic beta-1, 4 xylanase has extremely high thermal stability, alkali resistance and resistance to metal ions, organic solvents and surfactants, and can be used for biomass energy production, functional sugar and functional sugar alcohol production.
Description
Technical field
The present invention relates to a kind of thermophilic β-construction method of Isosorbide-5-Nitrae xylanase-His antigen-4 fusion protein gene engineering bacteria, prepared thermophilic β-Isosorbide-5-Nitrae xylanase-His fusion protein and its preparation method and application.
Background technology
Xylan is the important component part of plant cell wall hemicellulose, it is the second abundant polysaccharide that is only second in cellulose, nature content, almost take up an area 1/3rd (Collinset.al.FEMSMicrobiologyReviews.2005,29:3-23) of ball renewable carbon source.Xylan wide material sources, can be obtained by the side-product of the industry such as forestry, agricultural, timber processing and papermaking, and its main chain is mainly made up of with β-Isosorbide-5-Nitrae-glycosidic bond xylan residue.And the difference according to source; the side chain of xylan can be replaced by residues such as arabinose, glucosan aldehydic acid, acetyl group; due to complicated, xylan needs can thoroughly to be degraded under the combined effect including xylanase, D-xylosidase, glycuronidase and α-L-arabinofuranosidase etc..
β-1,4 xylanase (Endo-β-1,4-xylanase, EC3.2.1.8) main from main chain internal action in xylose glycosidic bond, can single-minded degradation of xylan be oligomeric xylose and xylose, be one of most important enzyme (KulkamiN, ShendyeA in xylan degrading process, RaoM.Molecularandbiotechnologicalaspectsofxylanases [J] .FEMSMicrobiol.Rev.1999,23 (4): 411-456).The microorganism that can produce xylanase has a lot, including filamentous fungi, antibacterial, actinomycetes etc..But relatively the produced xylanase of fungus, bacterial xylanase has better heat stability, there is prospects for commercial application widely, and it is subject to common concern (BadalC.Hemicellulosebioconversion [J] .JIndMicrobiolBiotechnol of academia, 2003,30:279-291).
Xylanase Producing plant height temperature alkane ground bacillus (GeobacillusthermoleovoransDSM5366) is issued to optimum growh environment at the high temperature of 75 DEG C, there is good heat-resisting and acid resistance, be highly suitable for biomass economy and the functional sugar such as xylose, xylitol field.Being found by sequence alignment, it has relatively low homology with other xylanase sequence studied, it is intended that excavated the Novel xylanase source of good properties by this research.
Summary of the invention
It is an object of the invention to provide the construction method of a kind of thermophilic β-Isosorbide-5-Nitrae xylanase-His antigen-4 fusion protein gene engineering bacteria, to overcome the defect that the produced xylanase in existing xylanase source is low with the xylanase sequence homology studied；
It is a further object of the present invention to provide a kind of thermophilic β-Isosorbide-5-Nitrae xylanase-His fusion protein；
The preparation method that it is a further object of the present invention to provide a kind of thermophilic β-Isosorbide-5-Nitrae xylanase-His fusion protein；
It is a further object of the present invention to provide the application of a kind of thermophilic β-Isosorbide-5-Nitrae xylanase-His fusion protein.
The present invention is achieved through the following technical solutions: a kind of thermophilic β-1, the construction method of 4 xylanase-His antigen-4 fusion protein gene engineering bacterias, described thermophilic β-1, the nucleotide sequence of 4 xylanase-His antigen-4 fusion protein genes is such as shown in SEQIDNO.1, and described construction method comprises the following steps:
Step A: the cultivation of high temperature alkane ground bacillus
Configuration anaerobic culture medium, injects the ratio of 1 volume high temperature alkane ground bacillus, is inoculated in anaerobic culture medium by high temperature alkane ground bacillus and cultivates according to the anaerobic culture medium of 100 parts by volume；
Step B: the extraction of genomic DNA
The step A high temperature alkane ground bacillus cultivated is extracted by bacterial genomes DNA extraction kit the genome of high temperature alkane ground bacillus, obtains Genomic DNA solution；
Step C: design primer and take thermophilic β-Isosorbide-5-Nitrae xylanase-His fusion protein genes of interest with the fishing of polymerase chain reaction method
By adding 6 His labels before termination codon in downstream primer, synchronization gain fusion protein xyl-his in the process of polymerase chain reaction, wherein,
Forward primer: 5 ' CCAGTCCCATGGATGCGGAACGTCGTGCGTAA3 ', dashed part is the restriction enzyme site of NcoI；
Downstream primer: 5 ' CGACGACTCGAGCTAATGATGATGATGATGATGTTTGTGGTCGATAATAGCCCA3 ', dashed part is the restriction enzyme site of XhoI；
With step B gained Genomic DNA solution for template, polymerase chain reaction is carried out in the presence of described forward primer and downstream primer, obtain polymerase chain reaction (PCR) amplification product, again amplified production is purified, obtain the polymerase chain reaction product of purification, then carry out double digestion with restricted enzyme NcoI and XhoI, obtain thermophilic β-Isosorbide-5-Nitrae xylanase-His fusion protein genes of interest；PET-28a that it is carried out double digestion with same (+) plasmid adopts DNA ligase to be attached, the mode clicked is adopted to be transformed in bacillus coli DH 5 alpha, screen 5 clones to extract plasmids and carry out cloned sequence order-checking and identify, be successfully obtained plasmid pET-28a containing fusion protein (+)-xyl-His；
Step D: build recombinant expression carrier
By pET-28a (+)-xyl-His and pRSII418 adopts restricted enzyme NcoI and XhoI to carry out double digestion, the xyl-His fragment of acquisition and linearizing pRSII418 plasmid are attached, obtain recombinant expression carrier pRSII418-xyl-His；
Step E: recombinant expression carrier is transformed in host cell
Step D gained recombinant expression carrier is transformed in Pichia sp. and cultivates, Adenine nucleotides culture medium is adopted to screen, and plasmid in the clone filtered out is carried out sequence verification, it is finally obtained the Adenine nucleotides Pichia yeast engineering with thermophilic β-Isosorbide-5-Nitrae xylanase-His antigen-4 fusion protein gene.
Wherein, according to the anaerobic culture medium in the recipe configuration step A of Germany's Culture Collection.
Wherein, the reaction system of described polymerase chain reaction is prepared by the following method: archaeal dna polymerase 1 μ l, DNA polymerase buffer liquid 5 μ l, Genomic DNA solution 2 μ l as template, 2.5mM deoxynucleotide mixture 8 μ l, forward primer and downstream primer respectively add 40pmol, add ultra-pure water to cumulative volume 50 μ l.
Wherein, the response procedures of described polymerase chain reaction includes following two step:
Step one, 94 DEG C of denaturation 3min；
Step 2, then 98 DEG C degeneration 30s, annealing temperature is from 70-56 DEG C, and each circulation reduces by 1 DEG C, annealing time 30s, and 72 DEG C extend 60s, totally 15 circulations；98 DEG C of degeneration 30s, annealing temperature 55 DEG C, annealing time 30s, 72 DEG C of extensions 60s, totally 20 circulations more afterwards；Last 72 DEG C of extension 20min again.
Wherein, carrying out the enzyme action system of double digestion with restricted enzyme NcoI and XhoI and include described in described step C: the plasmid that 500ng is purified, forward primer and downstream primer each 2 μ l, restricted enzyme buffer 4.9 μ l.
Wherein, described deoxynucleotide mixture is the mixture of deoxyadenine thuja acid, deoxy-guanine thuja acid, deoxycytidylic acid and deoxythymidine acid, wherein the concentration of deoxyadenine thuja acid and deoxythymidine acid is 30nmol/L, and the concentration of deoxy-guanine thuja acid and deoxycytidylic acid is 20nmol/L.
Wherein, carrying out the enzyme action system of double digestion with restricted enzyme NcoI and XhoI and include described in step D: pRSII418 empty carrier 45 μ l, forward primer and downstream primer each 2 μ l, restricted enzyme buffer 6 μ l, add ultra-pure water to cumulative volume 50 μ l.
A kind of thermophilic β-Isosorbide-5-Nitrae xylanase-His fusion protein prepared with described thermophilic β-Isosorbide-5-Nitrae xylanase-His antigen-4 fusion protein gene engineering bacteria, the aminoacid sequence of described thermophilic β-Isosorbide-5-Nitrae xylanase-His fusion protein is such as shown in SEQIDNo.2.
Described thermophilic β-1, the preparation method of 4 xylanase-His fusion protein, by what build, there is thermophilic β-1, the Adenine nucleotides Pichia yeast engineering of 4 xylanase-His antigen-4 fusion protein genes is amplified cultivating, then pass through cell ultrasonication, cross the collection of nickel post, imidazole buffer eluting, repeatability separation purification of saltouing obtains thermophilic β-Isosorbide-5-Nitrae xylanase-His fusion protein.
The application in biomass energy production, xylose and xylan produce of the described thermophilic β-Isosorbide-5-Nitrae xylanase-His fusion protein.
The invention has the beneficial effects as follows: adopt Touch-downPCR method to effectively prevent owing to the 3 long Tm of causing of end primer are too high and there is the mismatch problems that hairpin structure causes；Select plasmid pET-28a (+) there is high copy number, it is possible to effectively realize the amplification of gene；Thermophilic β-Isosorbide-5-Nitrae the xylanase of the present invention has high heat stability, alkali resistance environment and the resistance to metal ion, organic solvent and surfactant, can be used in biomass energy production, functional sugar, functional sugar alcohol production.
Accompanying drawing explanation
Fig. 1 is the structure schematic diagram of recombinant expression carrier pRSII418-xyl-His；
Fig. 2 is thermophilic β-Isosorbide-5-Nitrae xylanase-His fusion protein S DS-PAGE electrophoretogram；
Fig. 3 is thermophilic β-Isosorbide-5-Nitrae xylanase-His fusion protein relative activity schematic diagram under different pH；
Fig. 4 is thermophilic β-Isosorbide-5-Nitrae xylanase-His fusion protein relative activity schematic diagram at different temperatures.
Detailed description of the invention
A kind of construction method of thermophilic β-Isosorbide-5-Nitrae xylanase-His antigen-4 fusion protein gene engineering bacteria, the nucleotide sequence of described thermophilic β-Isosorbide-5-Nitrae xylanase-His antigen-4 fusion protein gene is such as shown in SEQIDNO.1, and described construction method comprises the following steps:
Step A: the cultivation of high temperature alkane ground bacillus (GeobacillusthermoleovoransDSM5366)
According to the recipe configuration culture medium of DSMZ, inject the ratio of 1 volume high temperature alkane ground bacillus according to the anaerobic culture medium of 100 parts by volume, high temperature alkane ground bacillus is inoculated in culture medium and cultivates；
Step B, genomic DNA extraction
The step A high temperature alkane ground bacillus cultivated is extracted by bacterial genomes DNA extraction kit the genome of high temperature alkane ground bacillus, obtains Genomic DNA solution；
Step C: design of primers and with polymerase chain reaction (PCR) method fish take thermophilic β-1,4 xylanase-His fusion protein genes of interest, by adding 6 His labels, synchronization gain fusion protein xyl-his in PCR process before termination codon in downstream primer, wherein
Forward primer: 5 ' CCAGTCCCATGGATGCGGAACGTCGTGCGTAA3 ', dashed part is the restriction enzyme site of NcoI；
Downstream primer: 5 ' CGACGACTCGAGCTAATGATGATGATGATGATGTTTGTGGTCGATAATAGCCCA3 ', dashed part is the restriction enzyme site of XhoI；
With step B gained Genomic DNA solution for template, PCR reaction is carried out in the presence of above-mentioned forward primer and downstream primer, obtain pcr amplification product, again amplified production is purified, obtain the PCR primer of purification, then double digestion is carried out with restricted enzyme NcoI and XhoI, obtain thermophilic β-1, 4 xylanase-His fusion protein genes of interest, pET-28a that it is carried out double digestion with same (+) plasmid adopts the DNA ligase of Takara to be attached, the mode clicked is adopted to be transformed in bacillus coli DH 5 alpha, screen 5 clones extraction plasmids and carry out cloned sequence order-checking qualification, be successfully obtained plasmid pET-28a containing fusion protein (+)-xyl-His；
Step D: build recombinant expression carrier
By pET-28a (+)-xyl-His and pRSII418 adopts restricted enzyme NcoI and XhoI to carry out double digestion, the xyl-His fragment of acquisition and linearizing pRSII418 plasmid are attached, obtain recombinant expression carrier pRSII418-xyl-His；
Step E: recombinant expression carrier is transformed in host cell
Step D gained recombinant expression carrier is transformed into Pichia sp. (PMAD16 (Ade), buying from Invitrogen company) in cultivate, Adenine nucleotides culture medium is adopted to screen, and plasmid in the clone filtered out is carried out sequence verification, it is finally obtained the Adenine nucleotides Pichia yeast engineering with thermophilic β-Isosorbide-5-Nitrae xylanase-His antigen-4 fusion protein gene.
As preferred technical scheme, the reaction system of described PCR reaction is prepared by the following method:
Step one, archaeal dna polymerase (PfU enzyme, Takara company) 1 μ l, DNA polymerase buffer liquid (Primerstarbuffer) 5 μ l, Genomic DNA solution 2 μ l as template, 2.5mM deoxynucleotide mixture (dNTP) 8 μ l, upstream and downstream primer respectively adds 40pmol, adds ultra-pure water to cumulative volume 50 μ l；
The response procedures of step 2, described PCR reaction is: 94 DEG C of denaturation 3min；
Step 3, then 98 DEG C degeneration 30s, annealing temperature is from 70-56 DEG C, and each circulation reduces by 1 DEG C, annealing time 30s, and 72 DEG C extend 60s, totally 15 circulations；98 DEG C of degeneration 30s, annealing temperature 55 DEG C, annealing time 30s, 72 DEG C of extensions 60s, totally 20 circulations more afterwards；Last 72 DEG C of extension 20min again,
As preferred technical scheme, carrying out the enzyme action system of double digestion with restricted enzyme NcoI and XhoI and include described in described step C: the plasmid that 500ng is purified, forward primer and each 2 μ l of downstream primer, restricted enzyme buffer (FDbuffer) 4.9 μ l.
As preferred technical scheme, described deoxynucleotide mixture is the mixture of deoxyadenine thuja acid, deoxy-guanine thuja acid, deoxycytidylic acid and deoxythymidine acid, wherein deoxyadenine thuja acid and deoxythymidine every kind of concentration of acid are 30nmol/L, and deoxy-guanine thuja acid, deoxycytidylic acid every kind concentration are 20nmol/L.
As preferred technical scheme, carrying out the enzyme action system of double digestion with restricted enzyme NcoI and XhoI and include described in step D: pRSII418 empty carrier 100ng, forward primer and each 2 μ l of downstream primer, restricted enzyme buffer (FDbuffer) 6 μ l, adds ultra-pure water to cumulative volume 50 μ l.
A kind of thermophilic β-Isosorbide-5-Nitrae xylanase-His fusion protein prepared with above-mentioned thermophilic β-Isosorbide-5-Nitrae xylanase-His antigen-4 fusion protein gene engineering bacteria, its aminoacid sequence is such as shown in SEQIDNo.2.
This is thermophilic, and β-Isosorbide-5-Nitrae xylanase-His fusion protein has characteristics that
(1) optimal reactive temperature
Showing catalysis activity at 45-85 DEG C, optimal reactive temperature is 75 DEG C；
(2) optimal reaction pH
Showing catalysis activity within the scope of pH4.2-12.0, optimum pH is 8.0；
(3) substrate specificity
Can catalysis birch xylan (Birchwood) effectively, beech wood polysaccharide (Beechhood), Herba bromi japonici xylan (OatSpelts), high viscosity hydroxymethyl cellulose (high viscosity CMC), the hydrolysis of low viscosity hydroxymethyl cellulose (low viscosity CMC), the suitableeest substrate being wherein hydrolyzed is beech wood polysaccharide；
(4) heat stability
Being hatched in different temperatures by thermophilic β-Isosorbide-5-Nitrae xylanase-His fusion protein, show good heat stability, wherein insulation 6h remains in that the vigor of 70% at 75 DEG C, is incubated 3h and remains in that the vigor of 40% at 80 DEG C；
(5) monovalent metallic ion is had good resistance.
Above-mentioned thermophilic β-1, the preparation method of 4 xylanase-His fusion protein is, is amplified the Pichia sp. of structure cultivating, and then passes through cell ultrasonication, crosses the collection of nickel post, imidazole buffer eluting, repeatability separation purification of saltouing obtains thermophilic β-Isosorbide-5-Nitrae xylanase-His fusion protein.
During described thermophilic β-Isosorbide-5-Nitrae xylanase fusion protein produces for functional sugars such as biomass energy production, xylose and xylan.
High temperature alkane ground bacillus (GeobacillusthermoleovoransDSM5366) is the antibacterial that a strain is extremely thermophilic, it is possible to degraded cellulose, hemicellulose, its optimum growth temperature is 75 DEG C.Strain is bought in Germany Culture Collection DSMZ (DeutscheSammlungvonMikroorganismenundZellkulturen), it is analyzed finding by the arbitrarily primed PCR cellulose to high temperature alkane ground bacillus (GeobacillusthermoleovoransDSM5366) and hemicellulose metabolic enzyme system, this bacterium genome can encode a kind of thermophilic β-1, 4 xylanase-His fusion protein, its DNA molecular can will be expressed thermophilic β-1, the gene order of 4 xylanase-His fusion protein activity is called thermophilic β-1, 4 xylanase-His antigen-4 fusion protein gene xyl.Owing to thermophilic high temperature alkane ground bacillus (GeobacillusthermoleovoransDSM5366) is a strain extreme environment antibacterial, condition of culture is complicated, artificial culture is relatively difficult, cell density is too low, it is difficult to this enzyme of large batch of production, therefore directly produce with this bacterium of cultivation that thermophilic β-Isosorbide-5-Nitrae xylanase-His fusion protein cost is higher and the cycle is long.Thermophilic β-Isosorbide-5-Nitrae xylanase-His antigen-4 fusion protein gene is proceeded to easy cultivation, can in the room temperature host such as Pichia sp. of Fast-propagation, it is possible to efficiently solve the problems referred to above.
High temperature alkane ground bacillus (GeobacillusthermoleovoransDSM5366) is passed through the extraction of cultivation, genes of interest, obtain thermophilic β-1 of the present invention, 4 xylanase-His antigen-4 fusion protein genes, we entrust Beijing Sheng Gong Bioisystech Co., Ltd to carry out gene sequencing, and its nucleotide sequence is such as shown in sequence table SEQ IDNo.1.
Thermophilic β-Isosorbide-5-Nitrae xylanase-His antigen-4 fusion protein gene is loaded on pRSII418 systemic vectors, is then transferred in Pichia sp. (ADE deficiency) host, obtains the strain Pichia yeast engineering containing genes of interest.Express product be cell soluble protein, by cell ultrasonication, cross nickel post collect, imidazole buffer eluting, repeatability separation purification of saltouing obtains thermophilic β-Isosorbide-5-Nitrae xylanase-His fusion protein.
The thermophilic protein of engineering bacterium expression is thermophilic β-Isosorbide-5-Nitrae xylanase-His fusion protein, and its aminoacid sequence is such as shown in sequence table SEQ IDNo.2.The suitableeest catalytic temperature of this is thermophilic β-Isosorbide-5-Nitrae xylanase-His fusion protein is 70 DEG C, and optimum pH is 7.2, it is possible to catalysis xylan hydrolysis, has wide practical use.As in field of biological energy source, when producing bio-fuel with oxygenation pretreatment lignocellulose for substrate, adopt this enzyme can effectively tolerate alkali and process material pH, reduce after alkali processes and adjust pH step, simultaneously can well with high temperature fiber element enzyme effect, effectively reduce production cost；A large amount of hemicellulosic material is there is in remaining lignocellulose after extracting cellulose, after oxygenation pretreatment, waste liquid contains substantial amounts of oligomerization hemicellulose too, owing to this enzyme has good activity at alkaline range, and the metal ion in waste liquid is had fabulous resistance, may be used for producing the functional sugar such as xylose, arabinose, obtain the products such as xylitol further.
Embodiment 1: thermophilic β-1, the structure of 4 xylanase-His fusion protein engineering bacterias and the expression of enzyme thereof: 1, the extraction of the cultivation of high temperature alkane ground bacillus (GeobacillusthermoleovoransDSM5366) and genomic DNA thereof, formula according to DSMZ, configuration anaerobic culture medium (anaerocellummedium), it is sub-packed in anaerobism test tube, often pipe 5ml, air in test tube is drained after closing by culture medium with vacuum pump, and is filled with the mixing gas (80%N of a certain amount of nitrogen and carbon dioxide2And 20%CO2), anaerobic box is dissolved with culture medium 1ml and buys high temperature alkane ground bacillus (GeobacillusthermoleovoransDSM5366) in DSMZ, be inoculated in 5ml test tube according to the bacterium amount that connects of 1%, mixing, 70 DEG C of quiescent culture a couple of days, until antibacterial starts growth.Being then transferred in the conical flask equipped with 100ml culture medium 70 DEG C of cultivation a couple of days ,-80 DEG C of preservation thalline are standby.
Take high temperature alkane ground bacillus (GeobacillusthermoleovoransDSM5366) that 5ml small test tube is cultivated, extract the genome of antibacterial with the bacterial genomes DNA Mini Kit (BacteriaGenomicMiniPreparationKit) of Beijing Pu Boxin biotechnology Co., Ltd, it is standby that gained chromosomal DNA solution puts 4 DEG C of refrigerators.
2, design of primers and extract and obtain the preparation of thermophilic β-Isosorbide-5-Nitrae xylanase-His antigen-4 fusion protein gene and recombinant vector by PCR method；
Containing several sections of possible thermophilic β-Isosorbide-5-Nitrae xylanase-His antigen-4 fusion protein genes predicted in thermophilic bacteria high temperature alkane ground bacillus (GeobacillusthermoleovoransDSM5366) genome.The selected nucleotide sequence such as gene shown in table SEQIDNo.1 is as object of study, called after xyl.This enzyme gene is expanded from the genomic DNA of step 1 gained by PCR method and obtains.Two primers are the restriction endonuclease sites of the sequence according to gene and carrier and design, and entrust the synthesis of Shanghai Sheng Gong bio-engineering corporation.Forward primer 5 ' CCAGTCCCATGGATGCGGAACGTCGTGCGTAA3 ', dashed part；Restriction enzyme site for NcoI；Downstream primer: 5 ' CGACGACTCGAGCTAATGATGATGATGATGATGTTTGTGGTCGATAATAGCCCA3 ', dashed part is the restriction enzyme site of XhoI；Restriction enzyme site set by two primers and expression vector pET-28a (+) NcoI and XhoI match, be suitable at E. coli.
PCR reacts: containing 1 μ lPrimerstarDNA polymerase in 50 μ l reaction systems, Primerstarbuffer5 μ l, template DNA (genomic DNA) 2 μ l, (wherein deoxyadenine thuja acid and deoxythymidine every kind of concentration of acid are 30nmol/L to 2.5mMdNTP mixture, deoxy-guanine thuja acid, deoxycytidylic acid every kind concentration are 20nmol/L) 8 μ l, upstream and downstream primer respectively adds 40pmol, add aseptic ultra-pure water to cumulative volume 50 μ l.PCR response procedures is: 94 DEG C of denaturation 3min；Cyclic program is 98 DEG C of degeneration 30s, and annealing temperature is from 70-56 DEG C, and each circulation reduces by 1 DEG C, annealing time 30s, and 72 DEG C extend 60s, totally 15 circulations；98 DEG C of degeneration 30s, annealing temperature 55 DEG C, annealing time 30s, 72 DEG C of extensions 60s, totally 20 circulations more afterwards；Last 72 DEG C of extension 20min again.Detecting PCR primer with 1% agarose gel electrophoresis, molecular size range is 1224bp, consistent with the result of prediction.
PCR primer restricted enzyme NcoI and the XhoI of purification is carried out double digestion, and enzyme action system includes:
Adopting Tian Gen company plasmid extraction kit to extract pET-28 (a)-xyl-His plasmid, containing of 500ng adds each 2 μ l, the FD-buffer4.9 μ l of NcoI and XhoI in genes of interest plasmid, be incubated 2 hours at 37 DEG C.Carry out electrophoresis with the agarose gel of 0.8% after enzyme action, then reclaim the DNA fragmentation after enzyme action by DNA gel detection kit.
Enzyme action pRCII418 carrier is carried out with same restricted enzyme NcoI and XhoI, reaction system is 50 μ l, and system includes: each 2 μ l of pRCII418 empty carrier 45 μ l, forward primer and downstream primer, restricted enzyme buffer 6 μ l, adds ultra-pure water to cumulative volume 50 μ l.Being incubated 2 hours at 37 DEG C, then process dephosphorylation with phosphatase (FastAP) again, react about 20 minutes, the agarose gel with 0.8% carries out electrophoresis detection, then reclaims test kit recovery with glue.
Connected wood xylanase gene and carrier is come with connection test kit at 16 DEG C.Connection carrier is proceeded in DH5 α, carry out screening and the qualification of monoclonal bacterial strain with the agar plate containing kanamycin.One positive monoclonal of picking, join in the test tube containing kanamycin, 37 DEG C of 180rpm/min overnight incubation, take bacterium solution and carry out PCR checking, obtain a band being sized to 1224bp and be genes of interest, it was demonstrated that genes of interest has been connected with carrier and has been transformed in host.After order-checking is correct, extracts recombinant plasmid transformed and carry out expressing pRCII418-xyl-His in Pichia sp..Examining order is completed by Shanghai Hua Da gene.
3, recombinant vector expression in host bacteria:
The DNA plasmid of restructuring, pRCII418-xyl-His are transformed in Pichia sp..Pichia sp. preparation and vector method thereof are with reference to " Molecular Cloning: A Laboratory guide ".Picking positive transformants bacterium, puts 37 DEG C of shaken cultivation in the 5ml culture fluid without gland fat purine and overnight, is inoculated into next day in the fresh 2YT culture medium without gland fat purine of 100ml, and 37 DEG C are continued to cultivate 4h.The seed liquor tentatively amplified is accessed with the ratio of 1% in the 2YT culture medium of 1L and cultivate (37 DEG C, 120rpm/min), wherein without gland fat purine, the isopropyl-beta D-thio galactopyranoside (IPTG) of 200mg/ml is added when OD600 reaches about 0.8, reducing cultivation temperature to 22 DEG C, induction thalline expresses destination protein.4 DEG C of overnight abduction deliverings, obtain engineering bacteria of the present invention, 10000rpm, and 10min is centrifugal collects thalline.Then with the resuspended thalline of Tris-HCl buffer of 50mM, pH8.0, ultrasonication (1s × 1s, 30min) 14,000 obtains crude enzyme liquid in centrifugal 30 minutes afterwards.
The imidazole buffer eluting of debita spissitudo is adopted after the further nickel post excessively of crude enzyme liquid, obtain purpose enzyme, then under pH7.2,56 DEG C of conditions of temperature, the enzyme activity after different disposal and corresponding protein concentration is measured with beech wood polysaccharide for substrate, obtain the protein purification table after different disposal, as shown in table 1.
The protein purification table of table 1 is thermophilic β-Isosorbide-5-Nitrae xylanase-His fusion protein:
The purity of application SDS-PAGE (12%) electrophoresis detection recombiant protein, it is seen that a visible electrophoretic band near 46.5KDa, as shown in Figure 1.
Embodiment 2: the activity analysis of the thermophilic β that recombinates-Isosorbide-5-Nitrae xylanase-His fusion protein
Recombinate thermophilic β-1,4 xylanase-His fusion protein activity use DNS method to be analyzed: at pH7.2, under 70 DEG C of conditions, the reaction system of 300 μ l includes the 5 μ l enzyme liquid suitably diluted, the substrate (g/100mL) of 150 μ l2%, 60 μ l buffer (20mM) and 85 μ lddH2O, react 5min, add 600 μ lDNS and terminate reaction, boiling water boiling 5min, after cooling, 540nm measures OD value, with glucose as a standard curve, calculates enzyme activity.1 enzyme unit (U) alive is defined as the enzyme amount required for the xylan solution release 1umol reducing sugar of specified criteria degraded 1% per minute.
Embodiment 3: the property testing of the thermophilic β that recombinates-Isosorbide-5-Nitrae xylanase-His fusion protein.
The assay method of the optimum pH of thermophilic β-Isosorbide-5-Nitrae xylanase-His fusion protein of 1, recombinating and pH stability is as follows: the restructuring thermophilic β-Isosorbide-5-Nitrae xylanase of embodiment 1 purification is carried out enzymatic reaction under different pH to measure its optimum pH.Using beech wood polysaccharide as substrate, utilize the reaction system in embodiment 2, at 60 DEG C, carry out Xylanase activity mensuration with the buffer of 20mmol difference pH.Result is as shown in Figure 2, it was shown that the optimum pH of thermophilic β-Isosorbide-5-Nitrae xylanase-His fusion protein is 8.0, and in the scope of pH6.5-9.5, enzymatic activity can maintain more than the 60% of maximum enzyme activity.Thermophilic β-Isosorbide-5-Nitrae xylanase-His fusion protein room temperature condition in above-mentioned different pH buffer processes 240min, then measures enzymatic activity at 60 DEG C, with the pH patience of studying enzyme.Result shows that thermophilic β-Isosorbide-5-Nitrae xylanase-His fusion protein is all very stable between pH5-12, and after processing 240min within the scope of this pH, residual enzyme activity is more than 90%, and this illustrates that this enzyme has extraordinary pH stability.The buffer used is wide scope pHbuffer: by acetic acid, N-2-hydroxyethyl piperazine-N-2-ethyl sulfonic acid (HEPS), N-tri-(methylol) methyl-3-aminopropanesulfonicacid acid (TAPS), 3-cyclohexylamino propane sulfonic acid (CAPS) and 2-code quinoline ethyl sulfonic acid (MES)) based on, the buffer of accurate formulation 100mM difference pH at 70 DEG C respectively.
2, recombinate the optimal reactive temperature of thermophilic β-Isosorbide-5-Nitrae xylanase-His fusion protein and the assay method of heat stability；
Thermophilic β-1, the optimum temperature of 4 xylanase-His fusion protein be determined as using beech wood polysaccharide as substrate, the vigor of thermophilic β-Isosorbide-5-Nitrae xylanase-His fusion protein is measured in wide range buffer liquid (pH7.2) system and 40-85 DEG C of temperature range.Thermal stability determination is that thermophilic β-Isosorbide-5-Nitrae xylanase-His fusion protein processes different time at different temperatures, then carries out enzyme activity determination at 75 DEG C.Enzyme reaction optimum temperature measurement result is as shown in Figure 3, it was shown that optimum temperature is 70 DEG C.The heat stabilization test result of enzyme is as shown in Figure 4, show thermophilic β-1,4 xylanase-His fusion protein are very good at 60 DEG C of stability inferiors, are incubated the 4h enzyme activity that still can keep 70% at 75 DEG C, are incubated the 3h enzyme activity that still can keep 40% at 80 DEG C.
Certainly; the present invention also can have other various embodiments; when without departing substantially from present invention spirit and essence thereof, those of ordinary skill in the art can make various corresponding change and deformation according to the present invention, but these change accordingly and deform the protection domain that all should belong to the claims in the present invention.
Claims (10)
1. a thermophilic β-1, the construction method of 4 xylanase-His antigen-4 fusion protein gene engineering bacterias, it is characterised in that described thermophilic β-1, the nucleotide sequence of 4 xylanase-His antigen-4 fusion protein genes is such as shown in SEQIDNO.1, and described construction method comprises the following steps:
Step A: the cultivation of high temperature alkane ground bacillus
Configuration anaerobic culture medium, injects the ratio of 1 volume high temperature alkane ground bacillus, is inoculated in anaerobic culture medium by high temperature alkane ground bacillus and cultivates according to the anaerobic culture medium of 100 parts by volume；
Step B: the extraction of genomic DNA
The step A high temperature alkane ground bacillus cultivated is extracted by bacterial genomes DNA extraction kit the genome of high temperature alkane ground bacillus, obtains Genomic DNA solution；
Step C: design primer and take thermophilic β-Isosorbide-5-Nitrae xylanase-His fusion protein genes of interest with the fishing of polymerase chain reaction method
By adding 6 His labels before termination codon in downstream primer, synchronization gain fusion protein xyl-his in the process of polymerase chain reaction, wherein,
Forward primer: 5 ' CCAGTCCCATGGATGCGGAACGTCGTGCGTAA3 ', dashed part is the restriction enzyme site of NcoI；
Downstream primer: 5 ' CGACGACTCGAGCTAATGATGATGATGATGATGTTTGTGGTCGATAATAGCCCA3 ', dashed part is the restriction enzyme site of XhoI；
With step B gained Genomic DNA solution for template, polymerase chain reaction is carried out in the presence of described forward primer and downstream primer, obtain polymerase chain reaction (PCR) amplification product, again amplified production is purified, obtain the polymerase chain reaction product of purification, then carry out double digestion with restricted enzyme NcoI and XhoI, obtain thermophilic β-Isosorbide-5-Nitrae xylanase-His fusion protein genes of interest；PET-28a that it is carried out double digestion with same (+) plasmid adopts DNA ligase to be attached, the mode clicked is adopted to be transformed in bacillus coli DH 5 alpha, screen 5 clones to extract plasmids and carry out cloned sequence order-checking and identify, be successfully obtained plasmid pET-28a containing fusion protein (+)-xyl-His；
Step D: build recombinant expression carrier
By pET-28a (+)-xyl-His and pRSII418 adopts restricted enzyme NcoI and XhoI to carry out double digestion, the xyl-His fragment of acquisition and linearizing pRSII418 plasmid are attached, obtain recombinant expression carrier pRSII418-xyl-His；
Step E: recombinant expression carrier is transformed in host cell
Step D gained recombinant expression carrier is transformed in Pichia sp. and cultivates, Adenine nucleotides culture medium is adopted to screen, and plasmid in the clone filtered out is carried out sequence verification, it is finally obtained the Adenine nucleotides Pichia yeast engineering with thermophilic β-Isosorbide-5-Nitrae xylanase-His antigen-4 fusion protein gene.
2. the construction method of thermophilic β according to claim 1-Isosorbide-5-Nitrae xylanase-His antigen-4 fusion protein gene engineering bacteria, it is characterised in that according to the anaerobic culture medium in the recipe configuration step A of Germany's Culture Collection.
3. thermophilic β-1 according to claim 1, the construction method of 4 xylanase-His antigen-4 fusion protein gene engineering bacterias, it is characterized in that, the reaction system of described polymerase chain reaction is prepared by the following method: archaeal dna polymerase 1 μ l, DNA polymerase buffer liquid 5 μ l, as Genomic DNA solution 2 μ l, the 2.5mM deoxynucleotide mixture 8 μ l of template, forward primer and downstream primer respectively add 40pmol, add ultra-pure water to cumulative volume 50 μ l.
4. the construction method of thermophilic β according to claim 1-Isosorbide-5-Nitrae xylanase-His antigen-4 fusion protein gene engineering bacteria, it is characterised in that the response procedures of described polymerase chain reaction includes following two step:
Step one, 94 DEG C of denaturation 3min；
Step 2, then 98 DEG C degeneration 30s, annealing temperature is from 70-56 DEG C, and each circulation reduces by 1 DEG C, annealing time 30s, and 72 DEG C extend 60s, totally 15 circulations；98 DEG C of degeneration 30s, annealing temperature 55 DEG C, annealing time 30s, 72 DEG C of extensions 60s, totally 20 circulations more afterwards；Last 72 DEG C of extension 20min again.
5. thermophilic β-1 according to claim 1, the construction method of 4 xylanase-His antigen-4 fusion protein gene engineering bacterias, it is characterized in that, carrying out the enzyme action system of double digestion with restricted enzyme NcoI and XhoI and include described in described step C: the plasmid that 500ng is purified, forward primer and downstream primer each 2 μ l, restricted enzyme buffer 4.9 μ l.
6. thermophilic β-1 according to claim 3, the construction method of 4 xylanase-His antigen-4 fusion protein gene engineering bacterias, it is characterized in that, described deoxynucleotide mixture is the mixture of deoxyadenine thuja acid, deoxy-guanine thuja acid, deoxycytidylic acid and deoxythymidine acid, wherein the concentration of deoxyadenine thuja acid and deoxythymidine acid is 30nmol/L, and the concentration of deoxy-guanine thuja acid and deoxycytidylic acid is 20nmol/L.
7. thermophilic β-1 according to claim 1, the construction method of 4 xylanase-His antigen-4 fusion protein gene engineering bacterias, it is characterized in that, carrying out the enzyme action system of double digestion with restricted enzyme NcoI and XhoI and include described in step D: pRSII418 empty carrier 45 μ l, forward primer and each 2 μ l of downstream primer, restricted enzyme buffer 6 μ l, adds ultra-pure water to cumulative volume 50 μ l.
8. one kind with described thermophilic β-1, thermophilic β prepared by 4 xylanase-His antigen-4 fusion protein gene engineering bacterias-Isosorbide-5-Nitrae xylanase-His fusion protein, it is characterised in that, the aminoacid sequence of described thermophilic β-Isosorbide-5-Nitrae xylanase-His fusion protein is such as shown in SEQIDNo.2.
9. the thermophilic β-1 described in claim 8, the preparation method of 4 xylanase-His fusion protein, it is characterized in that, by what build, there is thermophilic β-1, the Adenine nucleotides Pichia yeast engineering of 4 xylanase-His antigen-4 fusion protein genes is amplified cultivating, then passing through cell ultrasonication, cross the collection of nickel post, imidazole buffer eluting, repeatability separation purification of saltouing obtains thermophilic β-Isosorbide-5-Nitrae xylanase-His fusion protein.
10. the thermophilic β described in claim 8-Isosorbide-5-Nitrae xylanase-His fusion protein application in biomass energy production, xylose and xylan produce.
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