CN110106159A

CN110106159A - A kind of high temperature-resisting cellulase, encoding gene and preparation method thereof

Info

Publication number: CN110106159A
Application number: CN201910408345.3A
Authority: CN
Inventors: 李荷; 王晓萌; 张梦乐
Original assignee: Guangdong Pharmaceutical University
Current assignee: Guangdong Pharmaceutical University
Priority date: 2019-05-16
Filing date: 2019-05-16
Publication date: 2019-08-09
Anticipated expiration: 2039-05-16
Also published as: CN110106159B

Abstract

The invention discloses a kind of high temperature-resisting cellulases, encoding gene and preparation method thereof, and the amino acid sequence of high temperature-resisting cellulase is as shown in SEQ ID NO.1；Its encoding gene is named as cel1029, and nucleotide sequence is as shown in SEQ ID NO.2.The invention also discloses the recombinant plasmid pET32a-cel1029 and recombination engineering that contain the high temperature-resisting cellulase gene, the invention also discloses the preparation method of the high temperature-resisting cellulase gene and the preparation methods of high temperature-resisting cellulase, by high temperature-resisting cellulase gene cel1029 overexpression in escherichia coli prokaryotic expression system, the high temperature-resisting cellulase and recombination high temperature-resistant cellulase solution expression with high efficiency in coli expression system.The high temperature-resisting cellulase obtained using the method, catalytic activity with higher and thermal stability have very big industrialized production and application prospect.

Description

A kind of high temperature-resisting cellulase, encoding gene and preparation method thereof

Technical field

The invention belongs to genetic engineering field, it is related to a kind of novel fire resistant cellulose enzyme gene and its coded product, especially It utilizes wild asafoetide distributed area of the method for Metagenomic library screening from the Shihezi South Mountain of South Border of Junggar Basin, Xinjiang, china Edaphon in obtain a kind of novel cellulose enzyme gene and its coded product.

Background technique

Cellulase is acted synergistically by three kinds of enzymes, effectively can handle and utilize cellulose using cellulase, this Sample, which allows for cellulase, becomes a kind of cheap carbon source, and application prospect is extensive.Cellulase has good in multiple fields Using can be improved the crushing juice rate of fruits and vegetables in garden stuff processing, increase mouthfeel, reduce the production time；For soy sauce, white wine Production link increases yield；It can be used as feed addictive, increase coarse-fibred utilization rate in livestock and poultry animal feed, improve livestock and poultry Growth rate；It can be used for biochemical method degraded cellulose and prepare bio-ethanol, produce the energy, pollution is few, and high conversion rate, this has become One of key issues studied both at home and abroad；In addition, using cellulase can to active material in tealeaves, Chinese medicine or other plants into Row extracts, and greatly improves its recovery rate；Cellulase has application in textile industry, paper-making industry and detergent.

Although having discovered that many cellulases from educable fungi and bacterium, due to certain limitations, such as Stability, hydrolysis efficiency, inhibiting effect cause them that can't preferably apply in industry side the sensibility etc. of by-product Face.Therefore, researcher is still striving to find new cellulase, such as thermophilic enzyme from multiple resources, high active enzyme and more Functional enzyme etc..In order to explore the cellulase that Anticipated transient without scram generates in soil, from special environmental sample collection soil sample, use The cellulase of the method screening high activity of macro genome is necessary.

Microorganism present in nature 99% is not educable, therefore micro- from being separated to traditional culture technique The method that new biocatalyst is screened in biology is very limited.Utilize technique of metagenome (Metagenomics), DNA is directly extracted from environment, is cloned into different carriers, and macro genomic library is constructed, then Therefrom screened.The technology is showed in excavation and using Anticipated transient without scram resource and in terms of screening novel bioactive substance Huge potentiality out, it has also become the Disciplinary Frontiers and hot spot of current microbiological research in the world.So far domestic and foreign scholars have been Be cloned into such as amylase, zytase, cellulase new gene by the technology, these enzymes have novel enzymatic property and Commercial application potentiality.Therefore this method provides new approaches for discovery cellulase new gene.

Summary of the invention

The first purpose of this invention is to solve cellulase obtained in the prior art due to certain limitations, such as stablizes Property, hydrolysis efficiency, inhibiting effect causes them that can't preferably apply in industrial aspect the reasons such as the sensibility of by-product The problem of, a kind of high temperature-resisting cellulase is provided.

Second object of the present invention is to provide the gene of high temperature-resisting cellulase.

Third object of the present invention is to provide a kind of recombinant plasmid pET32a- of high temperature-resisting cellulase gene cel1029。

Fourth object of the present invention is to provide the recombination engineering containing above-mentioned high temperature-resisting cellulase gene.

Of the invention the 5th is designed to provide a kind of preparation method of high temperature resistant recombinant fiber element enzyme gene.

Of the invention the 6th is designed to provide a kind of preparation method of high temperature resistant recombinant fiber element enzyme.

The first purpose of this invention is achieved by the following technical solution: the high temperature-resisting cellulase, it Amino acid sequence is as shown in SEQ ID NO.1.

SEQ ID NO.1:

MGSEHHHHHHEMKNSNHENGRSRFRWLGIFTSCLLVLSAASTYAMEPLTVNGNRILANGEVRSLAGPS FFWSNTGWGAERFYNQDAVSWVKDDWNATLVRASLGVDGEGGYLEDPAGNKSRVVALVEAAIANDLYVIIDWHSHH AEDHTALAVDFFEEMAQTYGHHDNVIYEIYNEPLQISWSGTIKPYAETVISAIRAIDPDNLIVVGTPSWSQDVDAA SWDPIQGYANIAYALHFYAGTHTQYLRDKAQTALNNGIALFVTEWGTVNANGDGGVAYDETQRWMDFLEANHISHA HWALNDKEKGA。

Second object of the present invention is achieved by the following technical solution: a kind of high temperature-resisting cellulase gene, It is named as cel1029, the nucleotide sequence of the gene is as shown in SEQ ID NO.2.

SEQ ID NO.2:

AGGGGGTATTATACTGTAGGGGGACGATCAAAAGCGTTGGCGTTCGTTAAATATTTACGAGTGCTGCC TCATGTCAAAGTCAGAAAAAATAGTATAGGAGGTAACATATGGGATCCGAACATCATCATCATCATCATGAAATGA AAAACTCAAACCATGAAAATGGCCGGAGCCGCTTTCGCTGGCTCGGCATTTTCACAAGTTGTTTGCTGGTCCTGTC TGCGGCCAGCACCTATGCGATGGAACCGCTGACCGTCAACGGCAACCGCATTCTCGCCAACGGCGAAGTGCGCAGC CTGGCCGGGCCCAGCTTTTTCTGGAGCAATACCGGCTGGGGCGCCGAGCGCTTCTATAACCAGGACGCGGTCAGCT GGGTGAAGGACGACTGGAATGCCACCCTGGTGCGTGCCTCCCTGGGCGTGGACGGGGAGGGCGGCTACCTGGAGGA TCCGGCCGGCAACAAGAGCCGCGTTGTGGCACTGGTGGAAGCCGCCATCGCCAATGACCTGTATGTGATTATCGAC TGGCATTCCCACCACGCCGAGGACCACACCGCCCTGGCGGTAGACTTCTTCGAGGAAATGGCGCAGACCTATGGCC ACCATGACAACGTCATCTATGAAATCTACAACGAGCCGCTGCAGATTTCCTGGAGCGGCACCATCAAACCCTACGC AGAGACAGTGATCTCTGCCATCCGCGCCATCGACCCAGACAACCTGATCGTGGTCGGCACGCCGAGCTGGTCGCAG GATGTGGACGCGGCCTCCTGGGATCCGATTCAGGGCTACGCCAATATTGCCTACGCGCTGCATTTTTACGCGGGCA CCCATACACAATATCTGCGGGACAAGGCGCAGACCGCGTTGAATAACGGTATTGCCCTGTTCGTCACCGAATGGGG GACCGTCAATGCCAATGGCGACGGTGGCGTGGCCTATGACGAAACCCAGCGTTGGATGGATTTTCTCGAGGCCAAT CACATCAGCCACGCCCACTGGGCATTGAACGACAAGGAAAAGGGCGCAT。

Third object of the present invention, the 4th purpose, the 5th purpose, the 6th purpose are come by following technical solution It realizes:

(1) soil metagenome library is established；

(2) soil metagenome library screening positive clone molecule and identification, in detail operation are as follows:

A. white colony is screened from soil metagenome library, cultivates, has on point to cellulase screening and culturing plate The bacterium colony of hydrolysis is cellulase-positive clone；

B. positive clone molecule is lined into LB plate culture, picking has the monoclonal of hydrolysis to extract plasmid PUC118-cel1029, and digestion verification；

C. plasmid pUC118-cel1029 Transformed E .coli DH5 α, bacterium solution are coated on cellulase screening and culturing medium, are seen It examines with the presence or absence of hydrolysis, has then for positive clone molecule.

(3) design primer sequence is as follows:

Cel1029-F:5 '-CGGGATCCATGGGATCCGAACATCATCATCATCATC-3’

(underscore part is BamH I restriction enzyme site)；

Cel1029-R:5 '-CCAAGCTTTGCGCCCTTTTCCTTGTCGTTCA-3’

(underscore part is III restriction enzyme site of Hind).

Using plasmid pUC118-cel1029 as template, PCR reaction is carried out, clones target gene fragment；

Primer system is as follows:

The reaction condition of PCR cycle is as follows:

First stage: 98 DEG C of denaturation 2min；

Second stage: 98 DEG C of denaturation 10sec, 64 DEG C of annealing 5sec, 72 DEG C of extension 5sec, totally 30 recycle；

Phase III: 72 DEG C of extension 8min are finally stored in 4 DEG C；

High temperature-resisting cellulase gene is obtained after purification.

High temperature-resisting cellulase gene after double digestion and pET32a (+) expression vector after double digestion are connected, obtained Connection product, i.e. recombinant plasmid pET32a-cel1029；

Recombinant plasmid pET32a-cel1029 is transformed into e. coli bl21 competent cell, by the suspension of thallus Renewal cultivation simultaneously cultivate on kalamycin resistance culture plate by dilution spread, and picking positive transformant obtains recombination engineering, i.e., Recombination engineering escherichia coli BL21-pET32a-cel1029 containing high temperature-resisting cellulase gene；

Recombination engineering is inoculated on the plate of that penicillin resistance containing card culture activation, selects recombination engineering again in containing Block in the fluid nutrient medium of that penicillin resistance and cultivate, then is forwarded to the fluid nutrient medium of that penicillin resistance containing card by inoculum concentration Middle culture, when bacterium colony grows to OD₆₀₀IPTG to final concentration 0.9mM is added when=0.8, centrifugation abandons supernatant, is crushed to obtain crude enzyme liquid, High temperature-resisting cellulase is obtained after purification.

Using metagenomics cloning process, the not educable limitation of traditional microbiological is avoided, is greatly improved in environment The utilization of resources of microorganism.Extract the wild asafoetide distributed area pedotheque in the Shihezi South Mountain of South Border of Junggar Basin, Xinjiang, china Total DNA simultaneously uses OMEGA company Gel Extraction Kit (D2500-01) kits, and total DNA after purification is passed through 30 DEG C of digestion 100min of BamH I are connected to carrier pUC118/BamH I (BAP), electroporated super to bacillus coli DH 5 alpha Competence establishes macro genomic library, is being the LB plate for screening substrate with CMCase by putting, by congo red staining, NaCl After decoloration, screening has the single colonie of hydrolysis to obtain positive clone molecule, analyzes through sequencing, BLAST and ORF Finder And design primer, to be cloned into target fragment.

Beneficial effects of the present invention:

1. the present invention is constructed from the wild asafoetide distributed area pedotheque in the Shihezi South Mountain of South Border of Junggar Basin, Xinjiang, china Macro genomic library in obtain a new cellulose enzyme gene cel1029, cel1029 is the complete of a 921bp size Cellulose enzyme gene is compared its nucleotide sequence progress homology searching analysis online using BLAST and shows cellulose enzyme gene Similitude is not present in cel1029 and known.

2. doing functional study to the novel cellulose enzyme gene by technique for gene engineering, find the sequence in Escherichia coli Efficient soluble-expression in BL21, the Ni-NTA through Novagen companyResins protein purification and SDS-PAGE electricity Swimming, obtains a single protein band, primarily determines that molecular weight is about 34kDa.Due to pET32a (+) expression vector codes sulphur Oxygen also albumen and six histidine tags etc. and destination protein amalgamation and expression, therefore the opposite of the novel cellulose enzyme is divided in electrophoresis Son amount is about 52kDa.

3. DNA nucleotide sequence shown in SEQ ID NO.2 is cloned into prokaryotic expression carrier pET32a (+) by the present invention On, it is transformed into e. coli bl21 competent cell, high temperature-resisting cellulase is obtained by the inducing expression to positive clone molecule, Its zymologic property is studied, as a result as follows:

(1) in coli expression system, which has solution expression with high efficiency.

It (2) is substrate with CMCase, the high temperature-resisting cellulase hydrolysing activity is 143.3U, measures high-temperature fibre element The optimal reactive temperature of enzyme is 55 DEG C, is between 4~60 DEG C in temperature, remaining enzyme activity can achieve 50% or more, show this Cellulase has advantage in terms of high temperature resistant and thermal stability；The optimal reaction pH of the high temperature-resisting cellulase is 6.0；1mM is dense The K of degree⁺、Na⁺、Fe²⁺、Mg²⁺And Ca²⁺Effect, and the Na of 10mM concentration are improved to the enzyme activity of the cellulase⁺、Mg²⁺With Mn²⁺, have certain facilitation to enzyme, illustrate that the high temperature-resisting cellulase has the resistance to metal ion characteristic of higher degree.

As it can be seen that high temperature-resisting cellulase gene provided by the invention, gene constructed into plasmid pET32a by this, then should Recombinant plasmid, which is transferred in escherichia coli prokaryotic expression system, to be expressed and is purified, and the product of the gene expression is with higher Catalytic activity has good high temperature resistant and thermal stability, has biggish industrialized production and application potential.

Detailed description of the invention

Fig. 1 is high temperature-resisting cellulase gene cel1029PCR amplified production agarose gel electrophoresis figure；

Wherein, M is 15000kb DNAmaker, and 1 is cel1029PCR amplified production；

Fig. 2 is the SDS-PAGE electrophoresis in embodiment 1；

Wherein, M is standard protein molecular weight maker, and 1 is the thick enzyme of high-temperature fibre element, and 2 be the high-temperature fibre element of purifying Enzyme；

Fig. 3 is to glucose standard curve；

Fig. 4 is the influence of inducing temperature and time to high temperature-resisting cellulase expression；

Fig. 5 is influence of the IPTG concentration to high temperature-resisting cellulase expression；

Wherein M is standard protein molecular weight maker；

Fig. 6 is the optimal pH of high temperature-resisting cellulase degradation substrate CMCase；

Fig. 7 is the optimum temperature of high temperature-resisting cellulase degradation substrate CMCase；

Fig. 8 is the result histogram that different metal ions influence high temperature-resisting cellulase degradation substrate active.

Specific embodiment

Below in conjunction with attached drawing and specific embodiment, the present invention will be described in detail, herein with schematic implementation of the invention Example and explanation are used to explain the present invention, but not as a limitation of the invention.

The present invention will be further described with reference to the accompanying drawings and examples, but the invention is not limited in any way.

The foundation of the macro genomic library of embodiment 1 and acquisition, the gene cloning and expression of positive clone molecule

1, the extraction of total DNA:

(1) the 5ml centrifuge tube after taking 12 high pressure sterilizations weighs the open country in the Shihezi South Mountain of South Border of Junggar Basin, Xinjiang, china The pedotheque in raw asafoetide distributed area, every pipe 1.25g are separately added into DNA Extraction buffer 1.5ml, the 130rpm/ on shaking table Under min, 37 DEG C of activation 30min.

(2) SDS that centrifuge tube is separately added into 300 μ l 10% is taken out.

(3) the water-bath 2h in 65 DEG C of water-baths, is gently mixed by inversion every 15mim or more.

(4) sample is taken out, after cooling, 4 DEG C, 10000rpm/min, is centrifuged 10min.

(5) it takes supernatant to be transferred to new 5ml centrifuge tube, 4 DEG C, 10000rpm/min, is centrifuged 20min.

(6) after taking supernatant, be added 0.7 times of volume isopropanol mix gently after, be stored at room temperature 1h.

(7) 4 DEG C, 10000rpm/min, it is centrifuged 30min.

(8) supernatant is abandoned, 75% ethanol washing being pre-chilled with 4 DEG C precipitates 1 time.4 DEG C, 10000rpm/min, it is centrifuged 10min. It dries at room temperature.

(9) every pipe adds the TE solution of 65 DEG C of 20 μ l preheatings, and in 65 DEG C of warm bath 5min, dissolves genomic DNA.

2, RNA isolation kit purifies DNA: recycling according to OMEGA company, U.S. Gel Extraction Kit (D2500-01) glue Kit specification carries out, and steps are as follows:

(1) electrophoresis: the soil genomic DNA of extraction electrophoresis (voltage 10V/cm) in 1% Ago-Gel, electrophoresis knot Shu Hou dyes 5min in EB solution；

(2) cut glue: the Ago-Gel after EB is dyed is observed in gel imager, and cuts genome containing soil The Ago-Gel band of DNA, is then rinsed with deionized water, is placed in 2ml centrifuge tube；

(3) it purifies: usingExtraction Kit kit recycles soil genomic DNA, specifically Steps are as follows:

1. weighing the quality of the Ago-Gel of the genomic DNA containing soil；

2. corresponding to the ratio of 1ml BingBuffer according to every 1g Ago-Gel, solution is added into centrifuge tube BingBuffer。

3. the centrifuge tube of the gel of Buffer containing Bing is placed in 60 DEG C of water-baths, vibrates and mix every 2-3min, until fine jade Sepharose melts completely；

4. HiBindDNA column is placed in 2ml collecting pipe, and step 3 solution obtained is transferred to HiBind DNA column In, 14,000rpm is centrifuged 1min after standing 2min, abandons filtrate；

5. the SPWWashBuffer of 700 μ l is added, 10,000rpm centrifugation 1min abandon filtrate, and repeat this step；

6. 14,000rpm is centrifuged 2min to dry column matrix；

7. HiBind DNA column is placed in new 1.5ml centrifuge tube, and appropriate ultrapure water is added dropwise to column center, stands After 2min, 14,000rpm centrifugation 2min.Tube bottom liquid is soil genomic DNA fragment after purification, and -20 DEG C save backup.

3, macro genome electrophoresis detection: with the purity and quality of 1% agarose gel electrophoresis detection total DNA, guarantee purifying The A of DNA afterwards₂₆₀/A₂₈₀Ratio is higher than 1.8.

4, the partially digested total DNA of restriction enzyme BamH I, enzyme of the recycling clip size in 2-8kb digestion total DNA: are used It is sliced section, specific method purifies DNA with RNA isolation kit in above-mentioned 2.Endonuclease reaction system is as follows:

30 DEG C, digestion 6h；

5, the electrophoresis detection of endonuclease bamhi: method is the same as macro genome electrophoresis detection.

6, the connection of endonuclease bamhi: the endonuclease bamhi and pUC118/BamHI (BAP) carrier that recycling is obtained are using T4DNA Ligase connects 12h at 16 DEG C overnight, and -20 DEG C of dehydrated alcohol 62.5 μ l and 2.5 μ being pre-chilled in advance are added after taking out connection product l CH₃COONa (3M) is mixed gently, and precipitates 1.5h at -20 DEG C.4 DEG C, 12000rpm/min, it is centrifuged 20min.Supernatant is abandoned, with- 70% ethyl alcohol of 20 DEG C of pre-coolings, gently washing precipitating, 4 DEG C, 12000rpm/min, is centrifuged 20min, supernatant is abandoned, in collecting pipe It precipitates, is dried in 60 DEG C of baking ovens, the dd H of 65 DEG C of 10 μ l preheatings are added₂The carrier DNA of connection is resuspended in O.

7, the preparation of the super competent cell of Escherichia coli electrotransformation, steps are as follows:

(1) E. coli DH5 α bacterium solution is lined into LB solid plate, 37 DEG C of inversion overnight incubations；

(2) the picking Escherichia coli single colonie from streak plate, is inoculated in the triangular flask containing 20ml LB, 37 DEG C, Shaken cultivation is stayed overnight under the conditions of 180rpm；

(3) bacterium solution that transfer 3ml is incubated overnight is into the fresh LB liquid medium of 300ml, under the conditions of 37 DEG C, 220rpm Shaken cultivation is to thallus OD₆₀₀For 0.35-0.4,15ml, 0.9M sucrose solution is added, training is vibrated under the conditions of 18 DEG C, 220rpm Support 4h；

(5) bacterium solution is placed in 30min on ice, be then transferred in the 50ml sterile centrifugation tube of pre-cooling, at 4 DEG C, 5,000g centrifugation 10min, abandon supernatant；

(6) ultrapure water of 40ml pre-cooling is added, cell is resuspended, whole process keeps sterile and operates on ice；Then 4 At DEG C, 5,000 × g is centrifuged 10min, abandons supernatant, and repeat this step three times；

(7) 10% glycerol of 20ml pre-cooling is added, cell is resuspended, at 4 DEG C, 5,000 × g is centrifuged 10min, supernatant is abandoned, And repeat this step twice；

(8) cell is resuspended in 10% glycerol that 1ml pre-cooling is added, and is dispensed with every 100 μ l bacterium solution of centrifuge tube.- 80 DEG C super Low temperature refrigerator saves backup.

8, the conversion of connection product:

(1) 100 μ lE.coli DH5 α electricity is taken to turn to be added after competent cell is placed on ice to melt 4min in centrifuge tube The connection product of endonuclease bamhi and pUC118/BamHI (BAP) carrier, after mixing gently, stands 25min on ice in step 6；

(2) 42 DEG C of water-bath heat shock 40s, go to stand 2min in ice bath rapidly；

(3) be added into centrifuge tube 46 DEG C of 500 μ l preheating SOC culture medium gently blow and beat mix after, 37 DEG C, 180rpm Shaken cultivation 50min；

(4) appropriate culture is taken to be coated on LAXI culture medium, 37 DEG C of overnight incubations are to get soil metagenome library.

9, the identification of library screening and positive clone molecule

White colony is selected, blue colonies are rejected, point screens LB culture to the cellulase that CMCase is screening substrate On (50 μ g/ml Kan) plate, for 24 hours, the congo red staining 15min of 1mg/mL is added in 37 DEG C of cultures, de- with the NaCl of 1mol/L Whether after color 15min, looking around has hydrolysis, and the bacterium colony for having hydrolysis is cellulase-positive clone.It will be fine After the plain enzyme positive clone of dimension lines LB plate (containing 50 μ g/ml Kan), the monoclonal upgrading grain of picking hydrolysis uses BamH Can I single endonuclease digestion, verifying cut out carrier and purpose band, if can be to illustrate that the plasmid is pUC118-cel1029, take 5 μ l should Plasmid Transformed E .coli DH5 α, will be coated on cellulase screening and culturing medium after bacterium liquid activation, 37 DEG C culture for 24 hours afterwards by Observation periphery of bacterial colonies whether there is hydrolysis after dyeing and decoloration.

10, the clone of genetic fragment

It is analyzed through sequencing, BLAST and ORF Finder, designs pair of primers: cel1029-F and cel1029-R, The one end cel1029-F introduces the BamHI restriction enzyme site that can be inserted into pET-32a (+) carrier, and the one end Cel1029-R introduces and can be inserted into III restriction enzyme site of Hind of pET-32a (+) carrier, primer sequence are as follows:

Cel1029-F:5 '-CGGGATCCATGGGATCCGAACATCATCATCATCATC-3’

(underscore part is BamH I restriction enzyme site)

Cel1029-R:5 '-CCAAGCTTTGCGCCCTTTTCCTTGTCGTTCA-3’

(underscore part is III restriction enzyme site of Hind)

Using plasmid pUC118-cel1029 as template, PCR reaction is carried out, system is as follows:

The reaction condition of PCR cycle is as follows:

First stage: 98 DEG C of denaturation 2min；

Phase III: 72 DEG C of extension 8min are finally stored in 4 DEG C.

PCR product is purified with above-mentioned OMEGA plastic recovery kit, as shown in Fig. 1, and by PCR product after purification With restricted quick restriction endonuclease BamH I, Hind III in 37 DEG C of double digestion 20min, pET-32a (+) expression vector is used BamH I, Hind III double digestion, with the T4DNA Ligase of TaKaRa by after double digestion PCR product and double digestion after PET-32a (+) expression vector obtains recombinant plasmid pET32a-cel1029 in 16 DEG C of connection 14h, takes the 3 μ l recombinant plasmids, adopts Calcium is transformed into heat shock method to turn in competence E.coliBL21 (DE3)；Recombination engineering is formed, conversion fluid coating, which contains, blocks that The LB solid medium of penicillin (100 μ g/ml), 37 DEG C of overnight incubations extract matter after 5 plants of single colonie inoculated and cultureds of random picking Grain DNA delivers sequencing after double digestion verifying.Above-mentioned digestion and coupled reaction system difference are as follows:

11, the inducing expression and purifying of destination protein high temperature-resisting cellulase

Recombination engineering is crossed into containing the LB solid medium for blocking that penicillin (100 μ g/ml), 37 DEG C of overnight incubations Activation, 1 recombination engineering of random picking are seeded in the LB liquid medium of that penicillin (100 μ g/ml) containing card, 37 DEG C, 180~220rpm/min shaking table culture is stayed overnight, and that penicillin (100 μ g/ml) containing card of 40mL is forwarded to by the inoculum concentration of 1:100 LB liquid medium in, when growing to OD₆₀₀IPTG to final concentration 0.9mM, 30 DEG C, 200rpm/min shaking table are added when=0.8 Culture 14 hours, 8,000rpm, it is centrifuged 10min, supernatant is abandoned, with sterile dd H₂O washing precipitating uses 2ml dd H afterwards twice₂O is heavy It forms sediment, Ultrasonic Cell Disruptor is crushed bacterium solution 15min, and solution becomes clarifying, as crude enzyme liquid.By thick enzyme Ni-NTA Agerose (Novagen) nickel column affinity column obtains high temperature-resisting cellulase after purification, and affinity column concrete operation step is produced by Novagen company Product specification carries out.

High temperature-resisting cellulase by the thick enzyme of acquisition and after purification carries out PAGE gel electrophoresis (10%) for thick enzyme Each component of albumen separates in liquid, is dyed with coomassie brilliant blue R_250, and albumen maker estimates the size of zymoprotein.Pass through egg White Purification Kit zymoprotein, SDS-PAGE electrophoresis obtain a single protein band.SDS-PAGE electrophoresis result table Bright, the encoded polypeptide of nucleotide sequence described in SEQ ID NO.2 obtains high efficient expression in e. coli bl21 (DE3), should The amino acid sequence of polypeptide as shown in SEQ ID NO.1, and all high temperature-resisting cellulases be it is soluble, without forgiving the bodily form At the molecular weight of recombinant protein cel1029 is about 34kDa (wherein fusion tag containing 17kDa) (such as 2 institute of attached drawing according to a preliminary estimate Show).

The optimization of 2 high temperature-resisting cellulase inducing expression condition of embodiment

(1) determination of best inducing temperature and time

The recombination engineering of hydrolysis circle is selected in 5ml LB liquid medium, 37 DEG C, 200rpm culture 12h are drawn 500 μ l culture solutions are transferred in 50ml LB liquid medium, 37 DEG C, 200rpm overnight incubation, and addition IPTG keeps its final concentration of Inducing temperature is set to 25 DEG C, 30 DEG C and 37 DEG C, induction time is set to 8h, 11h, 14h, 17h, 20h by 0.8mM For 24 hours, after preparing crude enzyme liquid, the zymologic property of thick enzyme activity is surveyed.Highest enzyme activity is set as 100%, selects best inducing expression Temperature and time, as a result as shown in Figure 4.

(2) the most preferably determination of induction IPTG concentration

The recombination engineering of hydrolysis circle is selected in 5ml LB liquid medium, 37 DEG C, 200rpm culture 12h inhale 500 μ l culture solutions are taken to be transferred in 50ml LB liquid medium, 37 DEG C, 200rpm overnight incubation, addition IPTG make its final concentration Respectively 0.1mM, 0.3mM, 0.5mM, 0.7mM, 0.9mM, 1.1mM, 1.3mM and 1.5mM, inducing temperature and induction time point It is not set as 30 DEG C and 14h, after preparing crude enzyme liquid, surveys the zymologic property of thick enzyme activity.Highest enzyme activity is set as 100%, and selection is most Good IPTG induced concentration, as a result as shown in Figure 5.

3 high temperature-resisting cellulase enzyme activity determination of embodiment

1, the measurement of enzyme activity

The enzymatic activity that the present invention detects high temperature-resisting cellulase using p-nitrophenol myristinate is substrate.Reaction System is 100 μ l, consisting of 90 μ l contain the Britton-Robinson (pH of the CMC (sodium carboxymethylcellulose) of 1mg/ml =6.0) three acid buffers and 10 μ l enzyme solutions.The reaction system reacts 20min at 55 DEG C, and 150 μ l are added after reaction DNS solution, 10min is reacted in boiling water, the extinction of the reduced sugar discharged during this is then measured at 540nm wavelength Degree, while the blank control of enzyme solution is not added.Its concentration is determined according to the standard curve of glucose.Enzyme-activity unit is defined as: Under reaction condition, it is catalyzed enzyme amount required for 1 μm of ol glucose per minute and is defined as 1 enzyme-activity unit (U).

2, the drafting of glucose standard curve

0.04g glucose is accurately weighed, is dissolved with tri- acid buffer of B-R of the pH 6.0 of 10ml, is made into the mark of 2mg/ml Quasi- glucose solution is added 3ml DNS reagent and boils 5min, 10ml distilled water is added after cooling and mixes, 200 μ l is taken to measure OD₅₄₀, it is 0.00mg sample as negative control using glucose content.With concentration of glucose to OD₅₄₀Value mapping is to get glucose mark Directrix curve, as a result as shown in Figure 3.

The drafting of 1 glucose standard curve of table

The zymologic property research of 4 high temperature-resisting cellulase of embodiment

1, high temperature-resisting cellulase optimal pH

When temperature is 50 DEG C, pH gradient is successively set as to 1.98,2.87,4.10,5.02,6.09,6.59,7.0, 7.54,7.96,8.95,9.91, measure the enzyme activity under condition of different pH.Highest enzyme activity is set to 100%, from result figure 6 In it can be seen that enzyme activity highest illustrates the optimal reaction pH=6 of high temperature-resisting cellulase when pH=6.Furthermore by 10 μ l enzyme solutions It is respectively placed in 80 μ l difference pH buffer liquid, after 4 DEG C of placement 12h, remaining enzyme activity is measured, with relative surplus enzyme activity pair PH mapping, pH at 6, enzyme activity be it is highest reached 130%, the pH that enzyme is placed on buffer is between 4~8, enzyme activity according to 40% or more can be so kept, it is relatively good to the tolerance of pH, illustrate that high temperature-resisting cellulase is a kind of partial neutral enzyme.

2, high temperature-resisting cellulase optimal reactive temperature and thermal stability

In pH=6, temperature gradient is successively set as to 4 DEG C, 20 DEG C, 30 DEG C, 40 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C, 70 DEG C and 80 DEG C, measure different temperatures under enzyme activity.Highest enzyme activity is set as 100%.As a result as shown in fig. 7, obtaining enzyme Optimal reactive temperature is 55 DEG C.Separately take identical diluted enzyme solution 40ul be respectively put into different temperatures (4,20,30,40,50,55, 60,70,80,90 DEG C) under 2h, then from 40ul take out 3 duplicate 10ul measure under optimum temperature and pH residue enzyme activity Power is between 4~60 DEG C in temperature, and remaining enzyme activity can achieve 50% or more, illustrate that high temperature-resisting cellulase has temperature Preferable tolerance.

3, influence of the metal ion to high temperature resistant DNA polymerase activity

Different metal ions is added in enzymatic reaction, its influence to high temperature resistant DNA polymerase activity is studied, with not Add the dilution enzyme solution of ion as control.As a result see Fig. 8:

Different metal ions are detected in the influence of enzymatic activity, enzyme activity in the enzymatic reaction for not adding metal ion is defined It is 100.It is mapped with enzyme activity to different metal ions, detects influence of the different ions to enzyme activity.As a result such as Fig. 8 institute Show, the K of 1mM concentration⁺、Na⁺、Fe²⁺、Mg²⁺And Ca²⁺There is facilitation to enzyme activity, wherein the K of 1mM⁺To the facilitation of enzyme Maximum, opposite enzyme activity can reach 150%；The Na of 10mM concentration⁺、Mg²⁺And Mn²⁺, there is certain facilitation to enzyme；1mM and 10mM concentration C o²⁺And Ni²⁺The vigor of enzyme is set to be reduced to 50% or so；In addition, 1mM and 10mM concentration C u²⁺And Zn²⁺Suppression to enzyme It is very big to make use.

It is provided for the embodiments of the invention technical solution above to be described in detail, specific case used herein The principle and embodiment of the embodiment of the present invention are expounded, the explanation of above embodiments is only applicable to help to understand this The principle of inventive embodiments；At the same time, for those skilled in the art, according to an embodiment of the present invention, in specific embodiment party There will be changes in formula and application range, in conclusion the contents of this specification are not to be construed as limiting the invention.

Sequence table

<110>Guangdong pharmaceutical university

<120>a kind of high temperature-resisting cellulase, encoding gene and preparation method thereof

<130> 2019

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His Glu Asn Gly Arg Ser Arg Phe Arg Trp Leu Gly Ile Phe Thr Ser

20 25 30

Cys Leu Leu Val Leu Ser Ala Ala Ser Thr Tyr Ala Met Glu Pro Leu

35 40 45

Thr Val Asn Gly Asn Arg Ile Leu Ala Asn Gly Glu Val Arg Ser Leu

50 55 60

Ala Gly Pro Ser Phe Phe Trp Ser Asn Thr Gly Trp Gly Ala Glu Arg

65 70 75 80

Phe Tyr Asn Gln Asp Ala Val Ser Trp Val Lys Asp Asp Trp Asn Ala

85 90 95

Thr Leu Val Arg Ala Ser Leu Gly Val Asp Gly Glu Gly Gly Tyr Leu

100 105 110

Glu Asp Pro Ala Gly Asn Lys Ser Arg Val Val Ala Leu Val Glu Ala

115 120 125

Ala Ile Ala Asn Asp Leu Tyr Val Ile Ile Asp Trp His Ser His His

130 135 140

Ala Glu Asp His Thr Ala Leu Ala Val Asp Phe Phe Glu Glu Met Ala

145 150 155 160

Gln Thr Tyr Gly His His Asp Asn Val Ile Tyr Glu Ile Tyr Asn Glu

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Pro Leu Gln Ile Ser Trp Ser Gly Thr Ile Lys Pro Tyr Ala Glu Thr

180 185 190

Val Ile Ser Ala Ile Arg Ala Ile Asp Pro Asp Asn Leu Ile Val Val

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Gly Thr Pro Ser Trp Ser Gln Asp Val Asp Ala Ala Ser Trp Asp Pro

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Ile Gln Gly Tyr Ala Asn Ile Ala Tyr Ala Leu His Phe Tyr Ala Gly

225 230 235 240

Thr His Thr Gln Tyr Leu Arg Asp Lys Ala Gln Thr Ala Leu Asn Asn

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Gly Ile Ala Leu Phe Val Thr Glu Trp Gly Thr Val Asn Ala Asn Gly

260 265 270

Asp Gly Gly Val Ala Tyr Asp Glu Thr Gln Arg Trp Met Asp Phe Leu

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Glu Ala Asn His Ile Ser His Ala His Trp Ala Leu Asn Asp Lys Glu

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agggggtatt atactgtagg gggacgatca aaagcgttgg cgttcgttaa atatttacga 60

gtgctgcctc atgtcaaagt cagaaaaaat agtataggag gtaacatatg ggatccgaac 120

atcatcatca tcatcatgaa atgaaaaact caaaccatga aaatggccgg agccgctttc 180

gctggctcgg cattttcaca agttgtttgc tggtcctgtc tgcggccagc acctatgcga 240

tggaaccgct gaccgtcaac ggcaaccgca ttctcgccaa cggcgaagtg cgcagcctgg 300

ccgggcccag ctttttctgg agcaataccg gctggggcgc cgagcgcttc tataaccagg 360

acgcggtcag ctgggtgaag gacgactgga atgccaccct ggtgcgtgcc tccctgggcg 420

tggacgggga gggcggctac ctggaggatc cggccggcaa caagagccgc gttgtggcac 480

tggtggaagc cgccatcgcc aatgacctgt atgtgattat cgactggcat tcccaccacg 540

ccgaggacca caccgccctg gcggtagact tcttcgagga aatggcgcag acctatggcc 600

accatgacaa cgtcatctat gaaatctaca acgagccgct gcagatttcc tggagcggca 660

ccatcaaacc ctacgcagag acagtgatct ctgccatccg cgccatcgac ccagacaacc 720

tgatcgtggt cggcacgccg agctggtcgc aggatgtgga cgcggcctcc tgggatccga 780

ttcagggcta cgccaatatt gcctacgcgc tgcattttta cgcgggcacc catacacaat 840

atctgcggga caaggcgcag accgcgttga ataacggtat tgccctgttc gtcaccgaat 900

gggggaccgt caatgccaat ggcgacggtg gcgtggccta tgacgaaacc cagcgttgga 960

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Claims

1. a kind of high temperature-resisting cellulase, which is characterized in that its amino acid sequence is as shown in SEQ ID NO.1.

2. a kind of high temperature-resisting cellulase gene, which is characterized in that high temperature-resisting cellulase as described in claim 1 is encoded, The unnamed gene is cel1029, and nucleotide sequence is as shown in SEQ ID NO.2.

3. the recombinant plasmid comprising high temperature-resisting cellulase gene described in claim 2.

4. the recombinant plasmid pET32a-cel1029 comprising high temperature-resisting cellulase gene described in claim 2.

5. the recombination engineering comprising high temperature-resisting cellulase gene described in claim 2.

6. a kind of preparation method of high temperature-resisting cellulase gene as claimed in claim 2, it is characterised in that: including following step It is rapid:

(1) soil metagenome library is established；

(2) soil metagenome library screening positive clone molecule and identification；

(3) design primer clones target gene fragment, obtains high temperature-resisting cellulase gene；

Primer sequence is as follows:

Cel1029-F:5 '-CGGGATCC(underscore part is BamH I enzyme to ATGGGATCCGAACATCATCATCATCATC-3 ' Enzyme site)；

Cel1029-R:5 '-CCAAGCTT(underscore part is the III digestion position Hind to TGCGCCCTTTTCCTTGTCGTTCA-3 ' Point).

7. a kind of preparation method of high temperature-resisting cellulase gene as claimed in claim 6, it is characterised in that: the step (2) detailed process is as follows:

A. white colony is screened from soil metagenome library, cultivates, has transparent on point to cellulase screening and culturing plate The bacterium colony of hydrolysis circle is cellulase-positive clone；

B. positive clone molecule is lined into LB plate culture, picking has the monoclonal of hydrolysis to extract plasmid pUC118- Cel1029, and digestion verification；

C. plasmid pUC118-cel1029 Transformed E .coli DH5 α, bacterium solution are coated on cellulase screening and culturing medium, and observation is It is no there are hydrolysis, have then for positive clone molecule.

8. a kind of preparation method of high temperature-resisting cellulase gene as claimed in claim 5, it is characterised in that: in step (3) The detailed operation of clone's target gene fragment is:

The reaction condition of PCR cycle is as follows:

First stage: 98 DEG C of denaturation 2min；

Phase III: 72 DEG C of extension 8min are finally stored in 4 DEG C；

High temperature-resisting cellulase gene is obtained after purification.

9. a kind of preparation method of high temperature-resisting cellulase as claimed in claim 2, which is characterized in that claim 1 High temperature-resisting cellulase gene is connect with pET-32a (+) expression vector, obtains connection product, that is, recombinant plasmid pET32a- Recombinant plasmid transformed is formed recombination engineering into e. coli bl21 competent cell, cultivates recombined engineering by cel1029 Bacterium is crushed, obtains crude enzyme liquid, purifies, obtains high temperature-resisting cellulase.

10. a kind of preparation method of high temperature-resisting cellulase according to claim 8, which is characterized in that detailed operation step Suddenly it is to connect the high temperature-resisting cellulase gene after double digestion and pET-32a (+) expression vector after double digestion, is connected It practices midwifery object, i.e. recombinant plasmid pET32a-cel1029；

Recombinant plasmid pET32a-cel1029 is transformed into e. coli bl21 competent cell, the suspension of thallus is restored It cultivates and dilution spread is cultivated on kalamycin resistance culture plate, picking positive transformant obtains recombination engineering, that is, contains The escherichia coli BL21-pET32a-cel1029 of high temperature-resisting cellulase gene；

Recombination engineering is inoculated on the plate of that penicillin resistance containing card culture activation, selects recombination engineering again and is inoculated in and contains Block in the fluid nutrient medium of that penicillin resistance and cultivate, then is forwarded to the fluid nutrient medium of that penicillin resistance containing card by inoculum concentration Middle culture, when growing to OD₆₀₀IPTG to final concentration 0.9mM is added when=0.8, continues to cultivate, centrifugation abandons supernatant, is crushed slightly Enzyme solution obtains high temperature-resisting cellulase after purification.