CN106191093B - The method of cellulose excision enzyme expression quantity under the conditions of enhancing B. amyloliquefaciens alkaline - Google Patents
The method of cellulose excision enzyme expression quantity under the conditions of enhancing B. amyloliquefaciens alkaline Download PDFInfo
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- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
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- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
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Abstract
The invention discloses a kind of methods of cellulose excision enzyme expression quantity under the conditions of enhancing B. amyloliquefaciens alkaline, belong to genetic engineering field.The method of the present invention includes the following steps: for the expression unit being made of basic promoter, SD sequence, signal coding sequence and the circumscribed enzyme coding gene of cellulose to be integrated into bacillus gene group by homologous recombination.The method of the present invention is concretely: sequence shown in SEQ ID NO.5 being connected on pHT01 carrier by restriction enzyme SacI, XbaI and DNA ligase and constructs homologous recombination vector；Bacillus licheniformis ATCC 9945a is converted with constructed homologous recombination vector, screens the successful bacterial strain of homologous recombination.The bacillus of the available cellulose excision enzyme of high efficient expression under alkaline condition by the method for the invention.The present invention realizes alkali control expression of the cellulose excision enzyme in bacillus.
Description
Technical field
The invention belongs to genetic engineering field, it is related to the circumscribed expression of enzymes of cellulose under the conditions of a kind of enhancing B. amyloliquefaciens alkaline
The method of amount.
Background technique
Cellulase has huge value in the use aspects to living resources.Cellulase can degrade natural fiber
Element is converted to glucose, and then produces a variety of industrial chemicals or biological products.If the bacterial strain of production cellulase is resistant to
Higher basicity, then may be implemented to produce cellulose under alkaline condition with microorganism on one side with alkaline degradation cellulose on one side
Enzyme degraded cellulose.
Bacillus can carry out normal growth and breeding in alkaline condition, and bacillus is a kind of the micro- of food safety
Biology, if with bacillus produce cellulase, such bacillus not only can with continuous expression cellulase under alkaline system,
And it does not need to be isolated and purified and bacillus is got rid of.
Summary of the invention
The purpose of the present invention is to provide a kind of sides of cellulose excision enzyme expression quantity under the conditions of enhancing B. amyloliquefaciens alkaline
Method.The object of the invention is also to provide a kind of bacillus for expressing cellulose excision enzyme under alkaline condition.
The purpose of the invention is achieved by the following technical solution:
A kind of method of cellulose excision enzyme expression quantity under the conditions of enhancing B. amyloliquefaciens alkaline, include the following steps: by by
The expression unit that basic promoter, SD sequence, signal coding sequence and the circumscribed enzyme coding gene of cellulose form, by homologous
Recombination and integration realizes bacillus high efficient expression cellulose excision enzyme under alkaline condition into bacillus gene group.It is described
Bacillus be preferably bacillus licheniformis；The alkaline condition is preferably that pH is 8 to 9；The cellulose excision enzyme
Encoding gene is preferably derived from aspergillus niger.
The sequence of the basic promoter is preferably as shown in SEQ ID NO.1；The SD sequence is preferably such as SEQ ID
Shown in NO.2；The signal coding sequence is preferably as shown in SEQ ID NO.3；The circumscribed enzyme coding gene of the cellulose
Sequence preferably as shown in SEQ ID NO.4.
Preferably, under the conditions of the described enhancing B. amyloliquefaciens alkaline cellulose excision enzyme expression quantity method, including it is as follows
Step: sequence shown in SEQ ID NO.5 is connected to pHT01 carrier by restriction enzyme SacI, XbaI and DNA ligase
Upper building homologous recombination vector；Bacillus licheniformis ATCC 9945a is converted with constructed homologous recombination vector, is screened homologous
Recombinate successful bacterial strain.
A kind of bacillus for expressing cellulose excision enzyme under alkaline condition, is obtained by the above method.
The present invention, will be by basic promoter, SD sequence, signal peptide by homologous recombination using bacillus as expressive host
The expression unit of coded sequence and the circumscribed enzyme coding gene composition of cellulose is integrated on its genome, so that bacillus be allowed to exist
High efficient expression cellulose excision enzyme under alkaline condition.The present invention realizes alkali control table of the cellulose excision enzyme in bacillus
It reaches.
Specific embodiment
Below with reference to embodiment, the present invention is described in further detail, and embodiments of the present invention are not limited thereto.
Embodiment 1
1, material
Bacillus licheniformis (Bacillus paralicheniformis) ATCC 9945a, bacillus coli DH 5 alpha, plasmid
DNA sequence dna 1(shown in pHT01, SEQ ID NO.5 includes upstream and downstream homology arm, basic promoter, SD sequence, signal peptide volume
Code sequence and the circumscribed enzyme coding gene of cellulose, are synthesized by biotech firm).
Plasmid extraction kit (B518188) is purchased from Shanghai bioengineering limited liability company.Plastic recovery kit (Gel
DNA Extraction Kit Ver.4.0), restriction enzyme and T4 DNA Ligase be purchased from Takara company.
LB culture medium: tryptone (Tryptone) 10g/L；Yeast extract (Yeast extract) 5g/L, sodium chloride
(NaCl) 10g/L.
2, the extraction of plasmid pHT01
Bacillus coli DH 5 alpha containing plasmid pHT01 cultivates 12h, culture temperature in the LB culture medium of the 5mg/L containing chloramphenicol
Degree is 30 DEG C, extracts plasmid with plasmid extraction kit (B518188).Extraction step is as follows:
(1) 0.5mL bacterium solution is taken, 10000rpm is centrifuged 3min and collects thallus, most or Aspirate medium.
(2) 700 μ L Lysis Buffer UF are added in bacterial sediment, suction is beaten or vibrated to thorough suspension thalline, room
Temperature places 3min.
(3) lysate is all carefully moved into adsorption column, is placed at room temperature for 1min, 8000rpm is centrifuged 2min.Outwell collecting pipe
In liquid, adsorption column is put into the same collecting pipe.
(4) 500 μ L Prewash Solution, 8000rpm are added into adsorption column and are centrifuged 2min.It outwells in collecting pipe
Liquid, adsorption column is put into the same collecting pipe.
(5) 500 μ L Wash Solution, 8000rpm are added into adsorption column and are centrifuged 1min.Outwell the liquid in collecting pipe
Adsorption column is put into the same collecting pipe by body.
(6) it repeats that 500 μ L Wash Solution, 8000rpm centrifugation 1min are added into adsorption column.Outwell collecting pipe
In liquid, adsorption column is put into the same collecting pipe.
(7) adsorption column and collecting pipe are put into centrifuge, 8000rpm is centrifuged 2min.
(8) adsorption column is put into clean 1.5mL centrifuge tube, 50 μ L Elution is added in adsorbed film center
Buffer, is stored at room temperature 2min, and 8000rpm is centrifuged 2min.Obtained plasmid DNA solution is placed in -20 DEG C of preservations.
3, the digestion of DNA sequence dna 1 shown in plasmid pHT01 and SEQ ID NO.5
Each 0.2 μ L of SacI and XbaI enzyme is added into the plasmid pHT01 that 25 μ L are extracted, enzyme cutting buffering liquid and ddH is added2O
24.6 μ L are added 5 μ L Loading Buffer, 60 DEG C of heat preservation 10min and obtain plasmid pHT01 digestion in 30 DEG C of heat preservation 60min
Liquid.
DNA sequence dna 1 dilutes 10 times with distilled water, takes 25 μ L, and each 0.2 μ L of SacI and XbaI enzyme is added, and digestion buffering is added
Liquid and ddH224.6 μ L of O is added 5 μ L Loading Buffer, 60 DEG C of heat preservation 10min and obtains DNA in 30 DEG C of heat preservation 60min
1 digested liquid of sequence.
4, digested liquid electrophoresis
(1) electrophoresis reagents are prepared
1) 5 × TBE(tris- boric acid and EDTA) buffer preparation (1000mL): Tris 54g, boric acid 27.5g,
0.5mol/L EDTA 20mL, is transferred to 8.0 for pH, is settled to 1000mL.4 DEG C of refrigerators save, and the used time dilutes 5 times.
2) preparation of sample loading buffer: 0.25% bromophenol blue, 40%(W/V) aqueous sucrose solution, 4 DEG C of refrigerators preservations.
3) preparation of ethidium bromide: weighing 0.1g ethidium bromide, is dissolved in 10mL water, is made into the mother of final concentration of 10mg/mL
Liquid, 4 DEG C of refrigerators save.When dyeing, the mother liquor of 12.5 μ L is drawn, is added in the water of 250mL, makes its final concentration of 0.5 μ g/mL,
It is uniformly mixed.
4) preparation of 100 × TE buffer: 1mol/L Tris-HCl(pH8.0), 100mmol/L EDTA.Weigh Tris
121.1g, EDTA-Na 237.23g first use 800mL distilled water, after heating stirring dissolution, then about with hydrochloric acid tune pH to 8.0(
Hydrochloric acid 20mL is added), then it is settled to 1000mL.
5) preparation of 100 × electrophoretic buffer: 4mol/L Tris-HCl(pH8.0), 2mol/L sodium acetate, 200mmol/L
EDTA.Tris 242.2g, anhydrous sodium acetate 82.03g, EDTA-Na 237.23g are weighed, 400mL distilled water heating stirring is first used
After dissolution, then with glacial acetic acid tune pH to 8.0(glacial acetic acid 50mL is about added), then it is settled to 500mL.Used time dilutes 100 times.
(2) electrophoresis method
1) electrophoresis tank is installed: the running gel bed of organic glass being cleaned, is dried, is sealed the opening at both ends with adhesive tape,
It is placed on horizontal workbench, plugs sample comb.
2) preparation of 1% Ago-Gel: weighing agarose and be dissolved in electrophoretic buffer, sets in micro-wave oven or boiling water bath
It is heated to dissolving completely, taking-up shakes up.
3) encapsulating: the agarose solution for being cooled to 60 DEG C is gently poured on electrophoresis tank level board.
4) after agarose gel solidification, electrophoretic buffer is added in electrophoresis tank, then extracts comb.
5) it is loaded: after the 4:1 mixing by volume of DNA sample and sample loading buffer, being added mixed liquor with micropipettor
Into sample cell, every slot adds 20 μ L, records the point sample order and sample-adding amount of sample.
6) electrophoresis: installing electrode cable, and loading wells one terminates cathode, and another termination anode opens power supply, adjusts voltage extremely
3-5V/cm, electrophoresis 1-3hr stop electrophoresis when bromophenol blue is moved on to away from gel front 1-2cm.
7) it dyes and observes: taking out gel, be placed in the dyeing liquor containing ethidium bromide and dye 30min, it can be in 254nm
Ultraviolet lamp under observe, have the position of fluorescent red-orange band, as DNA band.
After plasmid pHT01 digested liquid electrophoresis, there are two bands through dyeing observation, longer band is recycled in two bands.DNA sequence
After 1 digested liquid electrophoresis of column, there is a band through dyeing observation, recycle the electrophoretic band.
5, the recycling and purifying of endonuclease bamhi
Use the DNA fragmentation of the plastic recovery kit recovery purifying digestion of Takara.It is cut out in the UV lamp containing purposeful
The Ago-Gel of DNA exhausts the liquid of gel surface with paper handkerchief.
Blob of viscose weight is weighed, blob of viscose volume is calculated.It is added blob of viscose lysate Buffer GM, Buffer GM's into blob of viscose
3 gel volume of dosage.25 DEG C of dissolution blob of viscose 10min of room temperature after evenly mixing.After gel is completely dissolved, it is molten that 3M sodium acetate is added
10 μ L of liquid (pH5.2) is uniformly mixed to solution and is restored yellow.Spin Column in kit is placed in Collection
On Tube.0.2mL glue lysate is transferred in Spin Column, and 12000rpm is centrifuged 1 minute, abandons filtrate.By 700 μ L's
Buffer WB is added in Spin Column, and room temperature 12000rpm is centrifuged 30 seconds, abandons filtrate.Again by the Buffer of 700 μ L
WB is added in Spin Column, and room temperature 12000rpm is centrifuged 30 seconds, abandons filtrate.Spin Column is placed in
On Collection Tube, room temperature 12000rpm is centrifuged 1min.Spin Column is placed in the centrifuge tube of new 1.5mL
On, 30 μ L sterile purified waters or Elution Buffer is added in the centre of Spin Column film, is stored at room temperature 1 minute.Room
Warm 12000rpm is centrifuged 1min eluted dna.
6, the connection of endonuclease bamhi
Linked system are as follows: the 17 μ L of DNA sequence dna of digestion recycling, 1 μ L, the T4 DNA of pHT01 plasmid of digestion recycling
11 μ L of μ L, Buffer of ligase.4 DEG C connect 24 hours.
7, it converts
The 50 μ L of bacillus coli DH 5 alpha (without plasmid) competent cell for taking -80 DEG C of pipe preservations, sets and melts on ice, be added
The 5 above-mentioned connection products of μ L, gently rotating centrifugal pipe is to mix content, ice bath 30min；Centrifuge tube is placed in 42 DEG C of thermal shock 60-
It 90 seconds, then sets rapidly ice bath 3 minutes.500 μ L LB liquid mediums (without chloramphenicol) is added into centrifuge tube, mixes postposition
In 37 DEG C shaking table shaken cultivation 45 minutes (150 revs/min)；Purpose is to make relevant resistant maker gene expression on plasmid, is made
Thallus recovery.It draws 100 μ L culture solutions to be added on the LB solid agar medium of the 5mg/L containing chloramphenicol, with sterile elbow glass
Stick is gently uniformly spreadable by cell.By plate be placed in room temperature until liquid be absorbed, be inverted plate, 37 DEG C culture 12-16 hours.
Picking individual colonies, the transformant that preservation obtains.
Transformant is in the LB culture medium of the 5mg/L containing chloramphenicol, 30 DEG C of culture 12h.Use plasmid extraction kit
(B518188) plasmid is extracted.Extracted plasmid carries out digestion, sequencing identification, identifies that correct recombinant plasmid is named as pHT01-
1e。
8, plasmid pHT01-1e converts bacillus licheniformis
(1) preparation of bacillus licheniformis competent cell
Culture medium A (100mL): peptone 1g, yeast powder 0.5g, NaCl 1g, sorbierite 9g, pH value 7.0.
Solution B (100mL): sorbierite 9g, mannitol 9.25g, glucose 10g.
Solution C (10mL): 9mL solution B+1mL glycerol.
1) bacillus licheniformis ATCC 9945a connects strain in 2mL culture medium A, 37 DEG C, 200rpm be incubated overnight, about
10h。
2) bacterium solution for being incubated overnight 2mL is added in 98mL culture medium A, 37 DEG C, 3~4h of 200rpm culture.
3) by bacterium solution 10~15min of ice-water bath, then 4 DEG C, 5000rpm centrifugation 10min collection thallus.
4) it is resuspended with the solution B of 20mL pre-cooling, so rinsing 3 times.
5) thallus after washing is resuspended in 600 μ L solution Cs, is managed after mixing by the EP that the 60 every pipes of μ L are sub-packed in pre-cooling
In, obtain bacillus licheniformis competent cell.
(2) electrotransformation
Plasmid pHT01-1e is added in bacillus licheniformis competent cell and is added to after pre-cooling 10min on ice pre-
In cold electrotransformation cup.It is 1.5kV(P.LENGTH=100 μ s, PULSES=04, INTERVAL=555ms in voltage) under the conditions of
After electric shock, be rapidly added 500 μ L culture medium As, then go in EP pipe, 37 DEG C, 200rpm culture 3h after be coated on containing chloramphenicol
On the LB plate of 5mg/L.37 DEG C of culture 12h screen the successful transformant of homologous recombination, together from the single colonie grown on plate
It is the bacillus licheniformis engineering bacteria to be constructed that source, which recombinates successful transformant,.
The bacillus licheniformis engineering bacteria screened is inoculated in the 250mL triangular flask equipped with 100mL LB culture medium,
For 24 hours, survey culture solution cellulose excision enzyme enzyme activity is 0 to 37 DEG C of shaking table cultures；Adjusting culture solution pH with 0.1M NaOH is 8.5,37 DEG C
Shaking table continues to cultivate 4h, surveys culture solution cellulose excision enzyme enzyme activity 0.0026U/mL.It should be the result shows that bacillus licheniformis engineering
Bacterium constructs successfully.
Cellulose excision enzyme enzyme activity determination method are as follows: take 2g microcrystalline cellulose powder, be dissolved in 10mL, 0.2M acetic acid-acetic acid
In sodium buffer (pH4.8), culture solution 0.5mL is added, 50 DEG C of water-bath 30min, DNS methods [are shown in that document, Zhen Jing, Wang Jiwen are thanked
Precious grace waits screening, identification and characterization analysis [J] the microbiology of mono- plant of cellulose degradation fungi of to be notified to, and 2011,
38 (5): 709 714.] measurement generates the amount of reduced sugar.Enzyme activity definition: hydrolyzing microcrystalline cellulose per minute generates 1 μM of reduced sugar
The amount of required enzyme is 1U.
9, the control of bacillus licheniformis engineering bacteria alkali is expressed
(1) bacillus licheniformis engineering bacteria is inoculated in the 250mL equipped with LB culture medium with the oese of diameter about 0.2cm
In triangular flask, it is 6.5,37 DEG C of shaking table cultures with 0.1M lemon acid for adjusting pH that inoculum concentration, which is 1 ring/50mL, liquid amount 100mL,
Culture solution is obtained for 24 hours, and measuring culture solution cellulose excision enzyme enzyme activity is 0.
(2) bacillus licheniformis engineering bacteria is inoculated into LB culture medium according to above-mentioned inoculation condition, is adjusted with 1M NaOH
PH is that 8.5,37 DEG C of shaking table cultures obtain culture solution for 24 hours, measures culture solution cellulose excision enzyme enzyme activity 0.003U/mL.
From different culture pH to find out, in acid condition, without cellulose excision enzyme enzyme activity, illustrate without expressing fiber
Plain enzyme；Under alkaline condition, there is cellulose excision enzyme enzyme activity, illustrate that the expression of cellulose excision enzyme is activated.The above results table
The bright present invention realizes alkali control expression of the cellulose excision enzyme in bacillus licheniformis.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (3)
1. a kind of method of cellulose excision enzyme expression quantity under the conditions of enhancing B. amyloliquefaciens alkaline, it is characterised in that: including as follows
Step: sequence shown in SEQ ID NO.5 is connected to pHT01 carrier by restriction enzyme SacI, XbaI and DNA ligase
Upper building homologous recombination vector；Bacillus licheniformis ATCC 9945a is converted with constructed homologous recombination vector, is screened homologous
Recombinate successful bacterial strain.
2. according to the method described in claim 1, it is characterized by: the alkaline condition is pH8-9.
3. a kind of bacillus for expressing cellulose excision enzyme under alkaline condition, it is characterised in that: by described in claim 1
Method obtain.
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| CN1437656A (en) * | 2000-06-23 | 2003-08-20 | 诺维信公司 | Method for stable chromosomal multi-copy integration of genes |
| CN101298604A (en) * | 2008-06-25 | 2008-11-05 | 天津科技大学 | High-temperature acid-resistant alpha-amylase mutant strain and construction method thereof |
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| CN1437656A (en) * | 2000-06-23 | 2003-08-20 | 诺维信公司 | Method for stable chromosomal multi-copy integration of genes |
| CN101298604A (en) * | 2008-06-25 | 2008-11-05 | 天津科技大学 | High-temperature acid-resistant alpha-amylase mutant strain and construction method thereof |
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Effective date of registration: 20191212 Address after: 435400 Hubei Huanggang Wuxue Shi fo Si town Wu Shi Avenue Railway Station Industrial Park Patentee after: Hubei Zhongnonghuawei Biological Engineering Co. Ltd. Address before: 430068 Wuhan Province, Wuchang District, South Lake, Lee Ka pier village, No. 1, No. 1, No. Patentee before: Hubei University of Technology |