CN108823224A - The highly expressed heat-resisting acidic xylanase gene of one kind and its expression vector and application - Google Patents
The highly expressed heat-resisting acidic xylanase gene of one kind and its expression vector and application Download PDFInfo
- Publication number
- CN108823224A CN108823224A CN201810611547.3A CN201810611547A CN108823224A CN 108823224 A CN108823224 A CN 108823224A CN 201810611547 A CN201810611547 A CN 201810611547A CN 108823224 A CN108823224 A CN 108823224A
- Authority
- CN
- China
- Prior art keywords
- heat
- gene
- resisting
- recombinant
- acidic xylanase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2477—Hemicellulases not provided in a preceding group
- C12N9/248—Xylanases
- C12N9/2482—Endo-1,4-beta-xylanase (3.2.1.8)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Mycology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Medicinal Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of highly expressed heat-resisting acidic xylanase gene and its expression vector and applications.It obtains being suitable for the heat-resisting acidic xylanase gene XynZNOp of optimization expressed in Pichia pastoris after being transformed the heat-resisting acidic xylanase gene XynZN from aspergillus niger (Aspergillus niger).The present invention, which further constructs recombinant yeast pichia pastoris composing type and inducible vector and is transformed into screening in Pichia pastoris, obtains the recombinant bacterial strain of the heat-resisting acidic xylanase of high efficient expression, XynZNOp gene is significantly improved relative to the XynZN gene expression amount of wild type in recombinant bacterial strain as the result is shown, and increase rate is respectively 52.0% and 46.3% in composing type and induction type recombinant bacterial strain.Use the Pichia pastoris recombinant bacterial strain of the heat-resisting acidic xylanase XynZNOp optimization gene sequence construct of the present invention, destination protein expression quantity significantly improves, the production cost of heat-resisting acidic xylanase is greatly reduced, expanding production application is industrialized for it and lays a good foundation.
Description
Technical field
The invention belongs to genetic engineering fields, and in particular to heat-resisting acidic xylanase gene optimization sequence and contain
There are the recombinant expression carrier and recombinant host cell of optimization, it is poly- in the heat-resisting acid wood of production that the invention further relates to them
Application in carbohydrase.
Background technique
Xylan (xylan) is a kind of poly pentose, is the main component of hemicellulose in plant cell wall, accounts for plant
The 30% of dry cell weight is a kind of biological polyoses resource abundant, and content is only second to cellulose in nature.Zytase is
One of xylan hydrolysis enzyme system key enzyme, its β-Isosorbide-5-Nitrae-xylose glycosidic bond in a manner of inscribe in hydrolyzed xylan molecule,
Its hydrolysate has wood oligose, also there is the monosaccharide such as a small amount of xylose and arabinose.
Zytase has been successfully applied in the industry such as feed, food, medicine, papermaking now.Such as：Zytase is more early
Applied in feedstuff industry, because xylan is the typical anti-nutritional factors in feed, since the retentiveness of xylan, small molecule are inhaled
Surface-active is echoed, if anti-oxidant action can be formed to simple stomach or stomachless animals containing more xylan in feed, especially
It is that the very high feed of the xylans content such as barley, wheat, wheat bran and rice bran is more prominent.In this kind of high xylan content feed
Addition zytase can effectively release the anti-oxidant action of xylan.It is main to the anti-nutritional releasing mechanism of xylan
Have：Plant cell wall construction is destroyed, the release of feed nutrient is promoted；Gastrointestinal tract chyme viscosity is reduced, the suction of nutrient is improved
Receipts and transformation efficiency；The secretion for promoting animal endogenous digestive ferment, makes the Apparent metabolizable energy of feed be increased；Improve gastrointestinal tract
Micro-ecological environment.
It needs to undergo of short duration high-temperature process during feed granulating, in addition domestic animals and fowls class animal gastrointestinal tract is meta-acid
Property environment, zytase of the present invention can undergo high-temperature process and keep compared with high residue enzyme activity and in slant acidity
Higher catalysis activity is embodied in animal alimentary canal, therefore is particularly suitable for using in feed industry, is had a good application prospect.
The problem of zytase industry development must overcome at this stage is how to reduce it increased production cost its production
Amount.To solve this problem, people attempt to construct recombination work by mutagenesis wild mushroom breeding high-yield bacterium, using genetic engineering means
Journey bacterium, condition of culture some column means of optimization etc. improve the yield of zytase.
Currently, the excellent zytase production bacterial strain that each enterprise of China uses still depends on foreign technology, it is difficult to realize
The production domesticization of core technology, making enterprise, the production cost increases.As the modernization level of China's animal husbandry is higher and higher, animal
Nutrition is increasingly taken seriously, as important feed addictive, zytase demand it is also increasing, especially for production
The demand for the xylanase preparation that amount is high, thermal stability is good, good properties and enzymatic activity are high is also increasing.With biological skill
The continuous development of art has carried out clone to a variety of xylanase genes from microorganism using technique for gene engineering and has surveyed
Sequence has more deep understanding to the structure, function and regulating and expressing etc. of xylanase gene.With molecular biosciences
The further investigation of technology is produced the zytase of advantageous property using some high-effective microorganism bacterial strains and has become one kind newly
Effective way.It is poly- that various countries researcher has filtered out a large amount of wood with advantageous property from various different microorganisms
Carbohydrase gene, these zytases possess different property, such as height than living, heat-resisting, low temperature resistant, acidproof.In order to improve these
The production level of zytase, have researcher by these xylanase genes in the host strains such as Escherichia coli, Pichia pastoris into
The expression of row heterologous recombination.Studies have shown that is for the zytase of originated from fungus, and Pichia pastoris is as eukaryocyte host to expression
Albumen is able to carry out glycosylation modified, therefore is the ideal heterogenous expression host of originated from fungus zytase.From aspergillus niger
Xylanase heat resistance it is preferable, and have higher vigor under acidic environment, heat-resisting acidic xylanase optimized and is changed
Make, obtain can secreting, expressing amount is significantly increased in Pichia pastoris zytase optimization gene, have and huge apply valence
Value.
Summary of the invention
One of the object of the invention is to provide a kind of highly expressed heat-resisting acidic xylanase gene sequence by Optimizing Reconstruction
Column, the secreting, expressing of the heat-resisting acidic xylanase optimization gene in host cell, especially in Pichia pastoris host cell
Amount is high.
The second object of the present invention is to providing the recombination containing the highly expressed heat-resisting acidic xylanase optimization gene sequence
Expression vector.
The recombinant expression that the three of the object of the invention are to provide the highly expressed heat-resisting acidic xylanase optimization gene carries
The recombinant host cell of body.
The four of the object of the invention are to provide said gene or recombinant vector or recombinant host cell is applied to thermostable acid
The production of property zytase.
To achieve the above object, the present invention provides following technical solutions：
The gene order of heat-resisting acidic xylanase XynZN is optimized in the present invention, encodes albumen not changing it
In the case where matter amino acid sequence, codon preference is comprehensively considered, host's codon frequency, the deletion of problem codon,
The factors such as mRNA secondary structure optimize XynZN gene order, such as SEQ ID NO.1 institute of the gene XynZNOp after optimization
Show.
Another aspect of the present invention provides the recombinant expression carrier of the optimization gene containing the heat-resisting acidic xylanase
And the recombinant host cell containing the recombinant expression carrier；Preferably, the recombinant expression carrier is that recombinant eukaryon expression carries
Body, more preferably recombinant yeast expression vector, most preferably restructured Pichia pastoris in expression carrier.
Empty vectors are from commercial pGAPZ α A, pGAPZ α B, pGAPZ α C tri- in the recombinant eukaryon expression vector
Any one of kind of carrier, perhaps any one of commercial pPICZZ α A, pPICZZ α B and pPICZZ α tri- kinds of carriers of C or
PPIC9K or pPIC3.5K.
In the recombinant eukaryon expression vector empty vectors be preferably derived from commercial composing type carrier pGAPZ α A,
Any one of tri- kinds of pGAPZ α B, pGAPZ α C carriers, or commercial inducible vector pPICZZ α A, pPICZZ α B and
Any one of pPICZZ α tri- kinds of carriers of C.
It can by being connected with the expression regulation sequence of yeast for heat-resisting acidic xylanase optimization gene operability
Obtain the recombinant yeast expression vector of the heat-resisting acidic xylanase of the secreting, expressing in yeast cells；In order to reach better secretion
Expression effect, the present invention is preferably by the expression tune with Pichia pastoris of the heat-resisting acidic xylanase optimization gene operability
Control sequence be connected obtain can in Pichia pastoris secreting, expressing recombinant heat-proof acidic xylanase recombinant yeast pichia pastoris
Expression vector.
When selecting empty vectors is composing type carrier, the highly expressed heat-resisting acidic xylanase gene is being recombinated
It is located at α secretion guidance peptide gene sequence downstream in carrier, preferably by highly expressed heat-resisting acid xylanase sequence insertion
To between XhoI the and XbaI restriction enzyme site in composing type carrier, the nucleotide sequence is made to be located at GAP promoter downstream
And regulated and controled by it, obtain expression of recombinant yeast plasmid pGAPZ α-XynZNOp；
When selecting empty vectors is inducible vector, the highly expressed heat-resisting acidic xylanase gene is being recombinated
It is located at α secretion guidance peptide gene sequence downstream in carrier, preferably by highly expressed heat-resisting acid xylanase sequence insertion
Between XhoI and XbaI restriction enzyme site into inducible vector, the nucleotide sequence is made to be located at AOX1 promoter downstream
And regulated and controled by it, obtain expression of recombinant yeast plasmid pPICZ α-XynZNOp.
The present invention also provides a kind of application methods of heat-resisting acidic xylanase optimization gene, i.e. high efficient expression thermostable acid
Property zytase.Specifically include being connected with expression vector by the heat-resisting acidic xylanase optimization gene operability
It obtains recombinant expression carrier and the recombinant expression carrier is converted into host cell, obtain recombinant bacterial strain culture recombinant bacterial strain, recombinate
Heat-resisting acidic xylanase is expressed in a manner of composing type or induction type, after filtration sterilization, obtains heat-resisting acidic xylanase.On
It states in method, the recombinant expression carrier is preferably recombinant eukaryon expression vector, and more preferably restructured Pichia pastoris in expression carries
Body, most preferably pGAPZ α A and pPICZ α A recombinant expression carrier, the host cell is preferably yeast, is more preferably finished red
Yeast cells.
Effect of the invention is that：The present invention optimizes the codon of heat-resisting acidic xylanase gene.The present invention
The effect of codon optimization is demonstrated, it, will by taking pGAPZ α A (composing type) and pPICZ α A (induction type) carrier for expression of eukaryon as an example
XynZNOp gene after the XynZN gene being not optimised and optimization is respectively charged into pGAPZ α A (composing type) and pPICZ α A (induction
Type) in carrier for expression of eukaryon, and it is transferred to pichia pastoris X-33, through height copy screening, it is highest to obtain every kind of combination enzymatic productivity
Bacterial strain.Fermenting experiment the result shows that, under the regulation of the constitutive promoter GAP of carrier pGAPZ α A, heat-resisting acidic xylanase
Expression after gene optimization is relatively not optimised gene and improves 52.0%, and the heat-resisting acid Xylanase activity of fermented liquid supernatant reaches
2883.86U/mL, under the regulation of the inducible promoter AOX1 of carrier pPICZ α A, heat-resisting acidic xylanase gene optimization
Expression afterwards is relatively not optimised gene and improves 46.3%, and the heat-resisting acid Xylanase activity of fermented liquid supernatant reaches
4066.24U/mL.And the high temperature resistant and acid resistance of zytase are not affected.
Detailed description of the invention
Fig. 1：Codon adaptation indexI (the Codon of the present invention heat-resisting acidic xylanase gene optimization front and back
Adaptation Index：CAI) compare；
Fig. 2：The codon frequency of the present invention heat-resisting acidic xylanase gene optimization front and back is distributed (Codon
Frequency Distribution：CFD) compare；
Fig. 3：1% agarose gel electrophoresis of heat-resisting acidic xylanase gene PCR product of the present invention detects figure
Piece, swimming lane M：DL2000Plus DNA Marker (Vazyme Biotech Co., Ltd), swimming lane 1：The XynZN base being not optimised
Cause, swimming lane 2：XynZNOp gene after optimization；
Fig. 4：Composing type yeast expression vector pGAPZ α-XynZNOp signal comprising the XynZNOp gene after optimization
Figure；
Fig. 5：Methanol evoked pichia pastoris expression vector pPICZ α-XynZNOp comprising the XynZN gene after optimization shows
It is intended to；
Fig. 6：XynZN gene and XynZNOp base after optimization before restructured Pichia pastoris in expression comprising composing type carrier optimizes
Because institute's inulinase-producing activity compares figure；
Fig. 7：After restructured Pichia pastoris in expression comprising methanol evoked carrier optimizes preceding XynZN gene and optimization
XynZNOp gene institute inulinase-producing activity compares figure；
Fig. 8：React influence of the pH to recombination xylanase activity, (A) XynZN；(B)XynZNOp；
Fig. 9：The pH stability (corresponding pH is incubated for 2h) of recombined xylanase, (A) XynZN；(B)XynZNOp；
Figure 10：Influence of the reaction temperature to recombination xylanase activity, (A) XynZN；(B)XynZNOp；
Figure 11：The thermal stability of recombined xylanase (relevant temperature is incubated for 5min).
Specific embodiment
The invention will now be further described with reference to specific embodiments, the advantages and features of the present invention will be with description and
It is apparent.But examples are merely exemplary for these, and it is not intended to limit the scope of the present invention in any way.Those skilled in the art
Member it should be understood that without departing from the spirit and scope of the invention can details to technical solution of the present invention and form into
Row modifications or substitutions, but these modifications and replacement are fallen within the protection scope of the present invention.
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Embodiment one, the optimization design and synthesis of heat-resisting acidic xylanase gene
1, experimental material and source
Aspergillus niger is saved by this laboratory of inventor；
Optimization gene segment is synthesized by Nanjing Jin Sirui biotechnology company.
2, the optimization design of heat-resisting acidic xylanase gene
The present invention is first to the thermostable acid that clone obtains from aspergillus niger (Aspergillus niger, this experiment preservation)
The sequence of property zytase wild type gene XynZN is analyzed, right under the premise of not changing gene coding amino acid sequence
Heat-resisting acidity xylanase sequence is transformed, and restriction enzyme site XhoI and XbaI are shielded in sequence,
Restriction enzyme site XhoI and XbaI are added in sequence both ends, and the heat-resisting acidic xylanase gene after obtained optimization is named as
XynZNOp。
As a result：
Sequence after optimization is compared with original series, as shown in table 1, share 24% base it is optimised.Such as (Fig. 1) institute
Show, codon adaptation indexI (Codon Adaptation Index：CAI it) is improved by 0.66 before being not optimised to after optimizing
The range that 0.97, CAI value defines shows the codon preference of the gene in corresponding host more between 0~1 if higher
By force.As shown in (Fig. 2), gene optimization changes codon frequency distribution also, all Pichia pastoris low frequencies in sequence
Codon is replaced by high frequency or secondary high frequency AC pulse Link, and high frequency AC pulse Link of the frequency of use between 91% to 100% is dominant
The 87% of total sequence after change, and these high frequency AC pulse Links only account for the 37% of total sequence before optimizing.
XynZNOp optimization gene sequence table 1
(wherein vertical line indicates that corresponding base is identical up and down, and space indicates that corresponding base is different)
The high efficient expression of embodiment two, the heat-resisting acidic xylanase gene optimized
1, experimental material and source
Expression vector pGAPZ α A and pPICZ α A and Pichi strain X-33 are invitrogen Products；
YPD fluid nutrient medium：1% yeast powder, 2% peptone, 2% glucose；
YPS fluid nutrient medium：1% yeast powder, 2% peptone, 2% glucose；
YPDS agar medium：1% yeast powder, 2% peptone, 2% glucose, 0.1M sorbierite, 2% agar powder.
2, the building of expression vector pGAPZ α-XynZNOp and pPICZ α-XynZNOp
(1) will implement that the correctly XhoI and XbaI digestion with restriction enzyme of the plasmid containing XynZNOp is sequenced in one,
Obtained smaller fragment is the XynZNOp sequence for including XhoI and XbaI cohesive end, utilizes Ago-Gel reclaim reagent
Box recycles respective segments.
(2) by expression vector pGAPZ α A and pPICZ α A XhoI and XbaI digestion with restriction enzyme, agarose is utilized
Gel reclaims kit recycles respective segments, the XynZNOp sequence comprising XhoI and XbaI cohesive end that will be handled well in (1)
T4DNA ligase (Thermo Fisher Scientific, Catalog are used respectively with carrier pGAPZ α A and pPICZ α A
number：EL0011) correlation technique to specifications is attached reaction.
(3) connection product is converted to bacillus coli DH 5 alpha competent cell, and appropriate cell is taken to be coated on containing bleomycin
The plate of (zeocin, Thermo Fisher Scientific), 37 DEG C are incubated overnight, and screen positive transformants with bacterium colony PCR method
Son simultaneously carries out sequence verification, and correct recombinant plasmid will be sequenced and be respectively designated as pGAPZ α-XynZNOp and pPICZ α-
XynZNOp。
(4) the heat-resisting acidic xylanase XynZN gene recombination plasmid pGAPZ being not optimised is constructed by above-mentioned same method
α-XynZN and pPICZ α-XynZN.
3, the preparation of heat-resisting acidic xylanase expression bacterial strain, the screening of fermented and cultured and high vigor transformant
(1) recombinant plasmid pGAPZ α-XynZNOp and pPICZ α-XynZNOp is largely extracted, the plasmid of 20 μ g or more is obtained
Gene, after BglII restriction enzyme linearization process, after purifying using chloroform/phenol extraction method and linearisation is concentrated
Plasmid, so that the plasmid concentration of product is reached 1 μ g/ μ L or more.
(2) pichia pastoris X-33 competent cell is prepared, method is referring to invitrogen Pichia pastoris operation manual.
(3) pGAPZ α-XynZNOp and pPICZ α-XynZNOp linearisation product electric shock after purification is transferred to Pichia pastoris
X-33, shock voltage 1500V, electric shock time are 5ms.Cell is coated on containing 400 μ g/ml bleomycin after electric shock
YPDS agar plate is cultivated 48 hours in 30 DEG C, obtains the transformant containing pGAPZ α-XynZNOp and pPICZ α-XynZNOp.
(4) screening of high vigor transformant, method are as follows：
The screening of pGAPZ α-XynZNOp constitutive expression transformant microplate method：Picked clones are connected to the YPD liquid training of 120 μ L
It supports and (is placed in 96 porocyte culture plates) in base, be incubated overnight in 28 DEG C, 300rpm and take the switching of 20 μ L culture solutions in 100 μ afterwards for 24 hours
Continue 36h in the YPD fluid nutrient medium of L, culture solution is centrifuged 5min in 5000g, and supernatant is heat-resisting acidic xylanase fermentation
Crude liquid is chosen high vigor transformant after measuring enzyme activity with three above-mentioned incubations of Duplicate Samples repetition, is surveyed again after culture
Enzyme activity is measured to complete secondary screening.
The screening of pPICZ α-XynZNOp inducible expression transformant microplate method：Picked clones are connected to the YPD liquid training of 120 μ L
It supports and (is placed in 96 porocyte culture plates) in base, be incubated overnight in 28 DEG C, 300rpm and take the switching of 20 μ L culture solutions in 80 μ L afterwards for 24 hours
YPS fluid nutrient medium in；Continue culture and the YPS fluid nutrient medium that 20 μ L contain 3% methanol (w/w), inducing expression is added afterwards for 24 hours
YPS fluid nutrient medium of the 20 μ L containing 3% methanol (w/w) is added after 12h again and continues 12h；Culture solution is centrifuged 5min in 5000g,
Supernatant is heat-resisting acidic xylanase fermentation crude liquid, chooses high vigor transformant in three Duplicate Samples repetitions after measuring enzyme activity
Incubation is stated, enzyme activity is measured after culture again to complete secondary screening.
(5) the heat-resisting acidic xylanase XynZN gene recombination plasmid pGAPZ being not optimised is constructed by above-mentioned same method
α-XynZN and pPICZ α-XynZN is simultaneously transferred to pichia pastoris X-33 screening high dynamic strain.
(6) the horizontal fermented and cultured recombinant bacterial strain of shaking flask prepares heat-resisting acidic xylanase
The screening of composing type carrier pGAPZ α A recombinant bacterial strain：By screened after secondary screening be transferred to pGAPZ α-XynZN,
The recombinant bacterial strain of the Xylanase activity of pGAPZ α-XynZNOp carrier is incubated at respectively in 10mL YPD culture medium, in 30 DEG C with
After 250rpm revolving speed culture for 24 hours, 1mL culture solution is transferred into 50mL YPD culture medium, continues to train with 250rpm revolving speed in 28 DEG C
Support 48h.Take culture solution that must ferment crude enzyme liquid in 5000g centrifugation 5min, three groups of parallel laboratory tests are arranged in each sample.
The screening of methanol evoked carrier pPICZ α A recombinant bacterial strain：By screened after secondary screening be transferred to pPICZ α-XynZN,
The recombinant bacterial strain of the Xylanase activity of pPICZ α-XynZNOp carrier is incubated at respectively in 10mL YPD culture medium, in 30 DEG C with
After 250rpm revolving speed culture for 24 hours, switching 1mL culture solution continues to cultivate 36h into 50mL YPS culture medium, so far starts every 12h
Proper amount of methanol, which is added, makes final concentration of 0.5%, and continuous addition 4 times, 12h terminates to ferment after the 4th addition.Take culture solution in
5000g centrifugation 5min must ferment crude enzyme liquid, and three groups of parallel laboratory tests are arranged in each sample.
(7) the enzyme activity measurement method of shaking flask level is referring to national standard《GB/T 23874-2009 feed addictive thermostable acid
The measurement of property Xylanase activity》Described in method, the method for 96 orifice plate primary dcreening operations and secondary screening slightly improves, and specific method is such as
Under：It takes the heat-resisting acidic xylanase substrate (10g/L) of 50 μ L to be added in 96 hole PCR plates, is placed in PCR instrument and is preheated in certain temperature
5 minutes, 50 μ L are added through the appropriate diluted enzyme solution after identical preheating temperature, in identical thermotonus after mixing well
125 μ L DNS solution are added in 30min, mix well rear and 95 DEG C of heating 5min.The 170 above-mentioned reaction solutions of μ L are taken to be transferred to 96 holes
ELISA Plate measures absorbance in 540nm using microplate reader, the enzyme activity of each sample is calculated according to reading result.
Xylose standard curve, meter are made by the xylose solution that 1.000g/L xylose solution prepares 50-500mg/L various concentration
Calculate calibration curve equation.The definition of enzyme activity unit (U)：Under the determination condition of optimum temperature sum, it is catalyzed final concentration of wood
Enzyme amount needed for glycan substrate solution generates reduced sugar (indicating with xylose) is defined as an enzyme activity unit.
(8) the change of properties measurement of the heat-resisting acidic xylanase of sequence optimisation
The measurement of optimal pH：Under optimum temperature, 0.1M citrate-phosphate of the measurement recombined xylanase in pH 2.2-8.0
Enzymatic activity in disodium hydrogen buffer.3 Duplicate Samples of every group of measuring.
The measurement of pH stability：At room temperature in advance the recombined xylanase XynZN that respectively obtains step (6) and
XynZNOp enzyme solution is incubated in 2h in the buffer of pH 1.0-13.0, then measures the vigor of remaining zytase.Every group of experiment
Measure 3 Duplicate Samples.
The measurement of optimum temperature：In the buffer of optimal pH, (20~95 DEG C) are taken under different temperatures, respectively measurement recombination
The enzyme activity of zytase XynZN and XynZNOp.3 Duplicate Samples of every group of measuring.
The measurement of temperature stability：Recombined xylanase XynZN and XynZNOp is incubated for 0- at 60-90 DEG C in advance
30min, then measure the vigor of remaining zytase.3 Duplicate Samples of every group of measuring.
As a result：
One, composition sequence PCR and its recombinant strains construct result
XynZNOp sequence size after removing signal peptide optimization is 621bp, and PCR product is through 1.0% agarose gel electrophoresis
Detection is consistent as shown in figure 3, the band of specificity occurs in the position between 500bp and 750bp with theoretical value size.It will close
It is connected into pGAPZ α A and pPICZ α A carrier respectively at good genetic fragment, obtained Vector map is as shown in Figure 4, Figure 5, in XhoI
XynZNOp sequence is inserted between XbaI restriction enzyme site, obtained recombinant vector is named as pGAPZ α-XynZNOp
With pPICZ α-XynZNOp, length is respectively 4136bp and 3690bp.It is screened after above-mentioned carrier conversion pichia pastoris X-33
High Xylanase activity bacterial strain is respectively designated as X-33/pGAPZ α-XynZNOp and X-33/pPICZ α-XynZNOp.
Two, the comparison of recombinant bacterial strain enzymatic productivity
It is tested by shake flask fermentation, is not optimised XynZN gene under the regulation of GAP promoter, maximum fermentative activity is
1897.28 ± 186.13U/mL, under the regulation of GAP promoter, maximum fermentative activity is the XynZNOp gene after optimization
2883.86 ± 230.43U/mL improves 52.0% compared with being not optimised gene；XynZN gene is not optimised in AOX1 promoter
Regulation under, maximum fermentative activity is 2779.38 ± 220.05U/mL, the tune of XynZNOp gene after optimization in GAP promoter
Under control, maximum fermentative activity is 4066.24 ± 248.92U/mL, and 46.3% is improved compared with being not optimised gene.
Three, the change of properties measurement of the heat-resisting acidic xylanase of sequence optimisation
By to recombined xylanase XynZNOp temperature and pH property after zytase XynZN before sequence optimisation and optimization
Measurement, obtain following result：(see Fig. 8-11)
Recombined xylanase optimal reaction pH be under conditions of 5.0, pH is 2.0-12.0 handle 2h after still have 78% with
On enzyme activity, there is very wide in range pH adaptability, recombined xylanase XynZNOp after optimization and zytase before optimizing
PH property no significant difference.
Recombined xylanase optimal reactive temperature is 50 DEG C, and the relative activity between 45~65 DEG C reaches 90% or more,
Residual enzyme activity after 80 DEG C of high-temperature process 5min still can reach 50% or more, the recombined xylanase XynZNOp after optimization
With the zytase temperature property no significant difference before optimization.
Sequence table
<110>Central South University
<120>The highly expressed heat-resisting acidic xylanase gene of one kind and its expression vector and application
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 621
<212> DNA
<213>Aspergillus niger (Aspergillus niger)
<400> 1
gttccccacg actctgtcgc ccagcgttcg gatgccttgc acatgctctc tgagcgctcg 60
accccgagct cgaccggcga gaacaacggc ttctactact ccttctggac cgacggcggt 120
ggcgacgtga cctacaccaa cggagatgct ggtgcctaca ctgttgagtg gtccaacgtg 180
ggcaactttg tcggtggaaa gggctggaac cccggaagtg cgcaggacat cacctacagc 240
ggcaccttca cccctagcgg caacggctat ctctccgtct atggctggac cactgacccc 300
ctgatcgagt actacatcgt cgagtcctac ggcgactaca accccggcag tggaggcaca 360
tacaagggca ccgtcacctc ggacggatcc gtttacgata tctacacggc tacccgtacc 420
aatgctgctt ccattcaggg aaccgctacc ttcactcagt actggtccgt ccgccagaac 480
aagagagttg gcggaactgt taccacctcc aaccacttca atgcttgggc taagctggga 540
atgaacctgg gtactcacaa ctaccagatc gtggctaccg agggttacca gagcagtgga 600
tcttcgtcca tcactgttca g 621
<210> 2
<211> 621
<212> DNA
<213>Unknown (Unknown)
<400> 2
gtgccgcacg acagcgttgc gcagcgtagc gatgcgctgc acatgctgag cgagcgttcc 60
accccttctt ctactggtga aaacaacgga ttctattact cattctggac tgatggtggt 120
ggagatgtta cctacacaaa cggtgatgct ggtgcttaca ctgttgaatg gtctaacgtt 180
ggtaattttg ttggtggtaa aggttggaac ccaggttctg ctcaagatat cacttactct 240
ggtactttta ctccttctgg taatggttac ttgtctgttt atggttggac tactgatcca 300
ttgatcgaat actacatcgt tgaatcttac ggagattata accctggttc tggtggtact 360
tacaaaggta ctgttacttc tgatggttct gtttacgata tctacactgc tactagaact 420
aacgctgctt ctattcaagg tactgctact tttactcaat actggtctgt tagacaaaat 480
aagagagttg gtggtactgt tactacttct aaccatttta atgcttgggc taaattgggt 540
atgaacttgg gtactcacaa ttatcaaatc gtcgctacag agggttatca atcatcaggt 600
tcatcatcca tcacagttca a 621
Claims (10)
1. a kind of highly expressed heat-resisting acidic xylanase gene, it is characterised in that：Its nucleotides sequence is classified as SEQ ID NO.1 institute
Show.
2. containing the recombinant expression carrier of highly expressed heat-resisting acidic xylanase gene described in claim 1.
3. the recombinant expression carrier of highly expressed heat-resisting acidic xylanase gene as claimed in claim 2, which is characterized in that institute
The recombinant expression carrier stated is recombinant eukaryon expression vector, preferred yeast expression vector；Further preferred Pichia anomala expression carries
Body.
4. the recombinant expression carrier of highly expressed heat-resisting acidic xylanase gene according to claim 3, feature exist
In empty vectors are from commercial pGAPZ α A, pGAPZ α B, tri- kinds of pGAPZ α C loads in the recombinant eukaryon expression vector
Any one of body, perhaps any one of commercial pPICZ α A, pPICZ α B and pPICZ α tri- kinds of carriers of C or pPIC9K,
Or pPIC3.5K.
5. the recombinant expression carrier of highly expressed heat-resisting acidic xylanase gene according to claim 4, feature exist
In, in the recombinant eukaryon expression vector empty vectors from commercial composing type carrier pGAPZ α A, pGAPZ α B,
Any one of pGAPZ α tri- kinds of carriers of C, or commercial inducible vector pPICZ α A, tri- kinds of C of pPICZ α B and pPICZ α loads
Any one of body.
6. the recombinant expression carrier of highly expressed heat-resisting acidic xylanase gene according to claim 5, feature exist
In,
When selecting empty vectors is composing type carrier, the highly expressed heat-resisting acidic xylanase gene is in recombinant vector
In be located at α secretion guidance peptide gene sequence downstream, highly expressed heat-resisting acid xylanase sequence is preferably inserted into group
Between XhoI and XbaI restriction enzyme site in shaping carrier, make the nucleotide sequence be located at GAP promoter downstream and by
It regulates and controls, and obtains expression of recombinant yeast plasmid pGAPZ α-XynZNOp；
When selecting empty vectors is inducible vector, the highly expressed heat-resisting acidic xylanase gene is in recombinant vector
In be located at α secretion guidance peptide gene sequence downstream, preferably highly expressed heat-resisting acid xylanase sequence is inserted into and is lured
Between XhoI and XbaI restriction enzyme site in conductivity type carrier, make the nucleotide sequence be located at AOX1 promoter downstream and by
It regulates and controls, and obtains expression of recombinant yeast plasmid pPICZ α-XynZNOp.
7. the gene work that the height comprising the described in any item recombinant expression carriers of claim 2-6 expresses heat-resisting acidic xylanase
Journey bacterium.
8. genetic engineering bacterium according to claim 7, it is characterised in that：The host cell of the genetic engineering bacterium is red to finish
Yeast cells.
9. genetic engineering bacterium according to claim 7, it is characterised in that：Be the described recombinant expression carrier importing is finished it is red
The recombinant yeast pichia pastoris cell obtained in yeast X-33 or GS115.
10. a kind of application of highly expressed heat-resisting acidic xylanase gene, it is characterised in that：It is poly- for expressing heat-resisting acid wood
Carbohydrase.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810611547.3A CN108823224A (en) | 2018-06-14 | 2018-06-14 | The highly expressed heat-resisting acidic xylanase gene of one kind and its expression vector and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810611547.3A CN108823224A (en) | 2018-06-14 | 2018-06-14 | The highly expressed heat-resisting acidic xylanase gene of one kind and its expression vector and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108823224A true CN108823224A (en) | 2018-11-16 |
Family
ID=64141660
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810611547.3A Withdrawn CN108823224A (en) | 2018-06-14 | 2018-06-14 | The highly expressed heat-resisting acidic xylanase gene of one kind and its expression vector and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108823224A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101955958A (en) * | 2010-08-17 | 2011-01-26 | 华南理工大学 | Genes, plasmid, bacterial strain and application of xylanase |
| CN102816749A (en) * | 2011-06-09 | 2012-12-12 | 中国科学院上海生命科学研究院 | Novel xylanase, and coding gene and application thereof |
-
2018
- 2018-06-14 CN CN201810611547.3A patent/CN108823224A/en not_active Withdrawn
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101955958A (en) * | 2010-08-17 | 2011-01-26 | 华南理工大学 | Genes, plasmid, bacterial strain and application of xylanase |
| CN102816749A (en) * | 2011-06-09 | 2012-12-12 | 中国科学院上海生命科学研究院 | Novel xylanase, and coding gene and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| BASSAM AL BALAA,ET AL: "Identification,Cloning, and Expression of the Scytalidium acidophilum XYL1 Gene Encoding for an Acidophilic Xylanase", 《BIOSCI.BIOTECHNOL.BIOCHEM》 * |
| GENBANK: "Aspergillus niger CBS 513.88 endo-1,4-beta-xylanase A,mRNA; XM_001388485.2", 《NCBI》 * |
| GUO,N.,ET AL: "Synthetic construct endo-1,4-beta-D-xylanase(xyl11Opt)gene,complete cds;Genbank:JQ710728.1", 《NCBI》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Hooker et al. | Leveraging anaerobic fungi for biotechnology | |
| CN114686386B (en) | Pichia pastoris for producing methanol mycoprotein and co-producing cellulase and application thereof | |
| CN110042092A (en) | The xylanase mutant and its encoding gene and application that first stability improves | |
| CN107164398A (en) | A kind of recombinant alpha galactosidase gene, carrier, engineering bacteria and its application | |
| CN105505969A (en) | Method for improving conversion rate of L-threonine and application of method | |
| CN104818260B (en) | A kind of resistant to elevated temperatures cupreum polygalacturonase mutant and its encoding gene and application | |
| CN112513249B (en) | Mutant strain of trichoderma filamentous fungus and method for producing protein | |
| CN103981112A (en) | Double-promoter multi-copy recombinant pichia pastoris strain for highly producing endo-inulinase | |
| CN101899458B (en) | High-yield and temperature-resistant beta-dextranase pichia pastoris and construction thereof | |
| CN103321074A (en) | Method for degrading plant lignin by using Bacillus subtilis engineering bacterial | |
| CN104789586A (en) | Escherichia coli genome integration vector, genetically engineered bacterium and application of genetically engineered bacterium to xylitol production | |
| CN109852650B (en) | Artificial aptamer enzyme regulated and controlled by theophylline and application | |
| CN108118006A (en) | A kind of heatproof zytase kluyveromyces marxianus engineered strain and its application | |
| CN105039276B (en) | Propiram synzyme, encoding gene, carrier and application | |
| CN108823224A (en) | The highly expressed heat-resisting acidic xylanase gene of one kind and its expression vector and application | |
| CN114381448B (en) | Glucanase mutant and application thereof | |
| CN103131718A (en) | Cloning of hypertonicity-resistant functional gene CgHog1 from Candida glycerinogenes and application thereof | |
| CN109929791A (en) | It is a kind of accumulate Glucosamine recombination bacillus coli and its application | |
| CN104611284A (en) | Strain for production of cyclodextrin glucosyltransferase and application of strain | |
| CN105316383B (en) | A method of improving the oxytetracycline yield of streptomycete by gene disruption | |
| CN107083375B (en) | Medium-temperature alpha-amylase and gene and application thereof | |
| CN103562216A (en) | Enhanced fermentation of cellodextrins and beta-D-glucose | |
| CN107488645A (en) | The lipase TTL mutant TTL-Ser61Asn and its gene and application that a kind of heat endurance improves | |
| JPWO2020075787A1 (en) | Trichoderma Risei mutant strain and protein production method | |
| Su et al. | Development and application of a novel screening method and experimental use of the mutant bacterial strain Clostridium beijerinckii NCIMB 8052 for production of butanol via fermentation of fresh cassava |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20181116 |
|
| WW01 | Invention patent application withdrawn after publication |