CN107502602A - The preparation and its application for the hot rod bacterium xylanase mutant that two heat resistances improve - Google Patents
The preparation and its application for the hot rod bacterium xylanase mutant that two heat resistances improve Download PDFInfo
- Publication number
- CN107502602A CN107502602A CN201710777945.8A CN201710777945A CN107502602A CN 107502602 A CN107502602 A CN 107502602A CN 201710777945 A CN201710777945 A CN 201710777945A CN 107502602 A CN107502602 A CN 107502602A
- Authority
- CN
- China
- Prior art keywords
- mutant
- mutational site
- replaced
- hot rod
- amino acid
- 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.)
- Pending
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)
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
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
Reset the invention discloses one kind using DNA molecular（DNA Shuffling）Technology obtains and derives from hot rod bacterium zytase multisite mutant and its encoding gene and application.The mutant one contains 6 amino acid mutation sites, and its amino acid sequence is as shown in SEQ ID No.1, and the nucleotide sequence that it is encoded is as shown in SEQ ID No.2.The mutant two contains 7 amino acid mutation sites, and its amino acid sequence is as shown in SEQ ID No.3, its nucleotide sequence such as SEQ ID No.4 encoded.Enzymatic assay shows that of the invention being compared from the zytase multisite mutant of hot rod bacterium and its gene of coding with wild type has higher heat resistance, and this characteristic will be that the mutant of the present invention is used to provide possibility in industrial production.
Description
Technical field
The invention belongs to bioengineering field, is related to two and derives from hot rod bacterium（Thermobacillus composti）'s
Zytase multisite mutant and its encoding gene and application, and in particular to one kind is reset using DNA molecular（DNA
Shuffling）Two multisite mutants are obtained to carrying out transformation from hot rod bacterium xylanase gene, and mutant is entered
Heat resistance and the analysis of other physiological biochemical characters are gone.
Background technology
Zytase（Xylanase）It is a kind of hydrolase being widely present in bacterium, fungi and animals and plants, mainly
Effect is hydrolyzed xylan.In recent years, with the weight that people develop to biomass resource in nature and agricultural by product
Go deep into depending on and to the gradual of xylo-oligosaccharide physiological function research, the research of properties of xylanase and characteristic becomes a heat
Point.Zytase can be applied not only to papermaking, food, feed, weaving and wine brewing etc., and be also a kind of regenerative resource.
At present, at home and abroad, existing substantial amounts of xylanase gene is cloned and expressed, but most of zytases are usually because be difficult to
Meet the extreme condition in commercial Application, such as in the industrial production, often require that zytase used can be in high temperature, extremely acid
Or higher enzyme activity is still kept in the environment of the alkali of pole, thus these requirements limit the broad field of application of zytase.
Therefore this just needs to transform xylan by genetic engineering means, new can be kept in the industrial production so as to obtain
The zytase that high enzyme is lived.
The content of the invention
The technical problems to be solved by the invention are that providing one kind derives from hot rod bacterium（Thermobacillus composti）Zytase multisite mutant and its encoding gene and application.Reset by using DNA molecular（DNA
Shuffling）Hot rod bacterium xylanase mutant library is obtained, then by dithering to hot rod bacterium xylanase mutant
Library is screened, and obtains two multisite mutants, its amino acid sequence such as SEQ ID No.1 and SEQ ID No.3 institutes
Show.Experiment proves that the mutant that the present invention obtains is compared with wild type has good heat resistance.
In order to reach object above, the present invention is realized using following technical scheme：
First, the present invention is according to the xylanase gene for deriving from hot rod bacterium（GenBank Accession No ：CP003255）
Take a successive PCR method【Nucleic Acids Research, 2004, 32, e98】According to pichia yeast codon
Preference synthesis comes from the xylanase gene of hot rod bacterium（XynB）.14 pairs of primers of design are used for the artificial conjunction of the xylanase gene
Into.
XynB genetic fragments are reclaimed using Ago-Gel QIAquick Gel Extraction Kit, with DNase I buffer solutions（50mmol/L
Tris-Cl pH7.4 + 1mmol/L MgCl2）100 μ l dissolve；0.1U DNase I are added, 25 DEG C are handled 15 minutes, 70 DEG C
Processing 10 minutes.10% acryl amide electrophoresis, saturating suction bag method reclaim 10 ~ 50bp DNA small fragments.Carry out Primerless PCR
Amplification, reaction system：The μ l 25mmol/L MgCl of+4 μ l 2.5mmol/L dNTPs of 5 μ l small fragments DNA+4.52 +
Taq2U + ddH2O to 50 μ l；Response procedures are：94 DEG C of 30s, 40 DEG C of 30s, 72 DEG C of 30s, totally 45 circulations.
Using primer free pcr amplification product as template, enter performing PCR amplification by primer of P1 and P14.Reaction system：5μl
μ l+the 2.5mmol/L of Primerless PCR primer+P1 0.2ng+P14+10 × PCR of 0.2ng Buffer 5
dNTPs 4μl + Taq2U + ddH2O to 50 μ l.Response procedures are：94 DEG C of 30s, 70 DEG C of 30s, 72 DEG C of 1.0 min, totally 35
Circulation, reclaim the XynB genetic fragments of rearrangement.
By the rearrangement XynB genetic fragments of above-mentioned recovery, after BamH I and Sac I double digestions, prokaryotic expression load is built into
Body pG251（CN1338515）Between promoter and t1t2 terminators, the carrier carries ampicillin resistance gene.Electric shocking method
Convert coli strain DH5 α and obtain mutant expression library, then with a large amount of extracts kits of plasmid（U.S. Omega is public
Department）Carry out plasmid extraction.Take the plasmid that 1 μ l are largely extracted after BamH I and Sac I double digestions with carrier for expression of eukaryon
pYM7909（Molecular Biology Report, 2012 Apr;39(4):3807-14）It is connected, takes that about 1 μ l's is above-mentioned heavy
Group plasmid linearization DNA carries out electroporated after being mixed with 80 μ l Pichia pastoris competent cells, is then coated on solid SD mountains
Pears alcohol culture medium（20g/L agar, 20g/L glucose, 146g/L sorbierites, SD）On, 28 DEG C culture 3 days after obtain white bacterium
Fall.White colony is inoculated in 48 orifice plates high temperature resistant colour developing screening is carried out at 70 DEG C.It is yellow that picking surveys presentation living under 70 DEG C of high temperature
The Positive mutants bacterium colony of color depth carries out the outer secreting, expressing purifying of Pichia pastoris, then carries out enzyme kinetic properties analysis to it
And the mutant obtained for screening is used in industrial production that provide can by Analysis of The Physiological And Biochemical Properties, these high-temperature stabilities
Energy.
The high temperature resistant plasmid obtained using progressively sequencing method to above-mentioned screening carries out complete sequence DNA sequencing.Sequencing knot
Fruit shows that mutant one contains 6 amino acid mutation sites on the same molecule, and mutant two contains 7 on the same molecule
Amino acid mutation site.Its amino acid sequence total length of the mutant one obtained is as shown in SEQ ID No.1, the nucleotides of coding
Sequence is as shown in SEQ ID No.2.Its amino acid sequence total length of mutant two is as shown in SEQ ID No.3, the nucleotides of coding
Sequence is as shown in SEQ ID No.4.
Term of the present invention is identical with its universal.
Described " nucleotides " and " primer " sequence is 5 ' ends to 3 ' ends.
Described " biological cell " refers to microorganism, plant cell or tissue.
Described " microorganism " refers to prokaryotic micro-organisms or eukaryotic microorganisms, and prokaryotic micro-organisms is mainly bacterium.
Brief description of the drawings
The optimal reaction pH of Fig. 1 enzymes.XynB-WT：Wild-type enzyme；XynB-C：The mutant one of the present invention；XynB-CE：This
The mutant two of invention.
The optimal reactive temperature of Fig. 2 enzymes.XynB-WT：Wild-type enzyme；XynB-C：The mutant one of the present invention；XynB-CE：
The mutant two of the present invention.
The stability of the temperature of Fig. 3 enzymes.XynB-WT：Wild-type enzyme；XynB-C：The mutant one of the present invention；XynB-CE：
The mutant two of the present invention.
Fig. 4 enzymes are in 65 DEG C and 70 DEG C of half-life period.XynB-WT：Wild-type enzyme；XynB-C：The mutant one of the present invention；
XynB-CE：The mutant two of the present invention.
Embodiment
The DNA molecular of the xylanase gene of embodiment 1 is reset（DNA Shuffling）
1.1 derive from the xylanase gene of hot rod bacteriumXynBSynthesis
According to the xylanase gene from hot rod bacterium（GenBank Accession No ：CP003255）, take and continuously prolong
Stretch PCR method【Nucleic Acids Research, 2004, 32, e98】According to pichia yeast codon-bias synthetic source
In the xylanase gene of hot rod bacterium（XynB）, design 14 pairs of primers and be used for the artificial synthesized of the xylanase gene.It is designed
Primer it is as follows：
1. P1: Tm=54, 60mer
GGA,TCC,AAC,ACC,TAC,TGG,CAG,TAC,TGG,ACT,GAT,GGT,ATC,GGG,TAT,GTC,AAC,GCA,
ACC,AAC
2. P2: Tm=54, 60mer
CAG,AGT,TGG,ACC,AAG,AGA,CAG,AGT,AGT,TGC,CAC,CTT,GAC,CGT,TGG,TTG,CGT,TGA,
CAT,ACC
3. P3: Tm=54, 60mer
TGT,CTC,TTG,GTC,CAA,CTC,TGG,TAA,CTT,CGT,CAT,TGG,TAA,GGG,TTG,GCA,GTA,TGG,
TGC,ACA
4. P4: Tm=54, 60mer
TGG,CTG,CCA,TGC,ACC,AGC,ATT,GTA,GTT,GAC,AAC,ACG,ATT,GTG,TGC,ACC,ATA,CTG,
CCA,ACC
5. P5: Tm=54, 60mer
ATG,CTG,GTG,CAT,GGC,AGC,CAA,ACG,GTA,ACG,CAT,ATC,TGA,CTC,TGT,ATG,GTT,GGA,
CTC,GTA
6. P6: Tm=54, 60mer
GAA,CCC,CAA,GAG,TCA,ACA,ACG,TAG,TAC,TCG,ATG,AGT,GGG,TTA,CGA,GTC,CAA,CCA,
TAC,AGA
7. P7: Tm=54, 60mer
GTT,GTT,GAC,TCT,TGG,GGT,TCC,TAC,CGT,CCT,ACT,GGT,GAC,TAC,CGT,GGT,TCT,GTC,
TAC,TCT
8. P8: Tm=54, 60mer
TGT,AAC,GCC,AAG,AGT,GAT,AGA,GGT,CAT,ACC,ATG,CAC,CAT,CAG,AGT,AGA,CAG,AAC,
CAC,GGT
9. P9: Tm=54, 60mer
CTA,TCA,CTC,TTG,GCG,TTA,CAA,CGC,ACC,ATC,CAT,CGA,TGG,TAC,TCA,GAC,CTT,CCA,
ACA,GTA
10. P10: Tm=54, 60mer
GTT,GGA,ACC,AGT,TGG,ACG,CTT,CTG,CTG,ACG,AAC,AGA,CCA,GTA,CTG,TTG,GAA,GGT,
CTG,AGT
11. P11: Tm=54, 60mer
AGC,GTC,CAA,CTG,GTT,CCA,ACG,TCT,CTA,TCA,CCT,TCG,AGA,ACC,ATG,TCA,ATG,CAT,
GGG,GTG
12. P12: Tm=54, 60mer
ACC,TGA,TAG,GAC,CAA,GAA,GAA,CCC,ATA,GGC,ATA,CCA,GCA,GCA,CCC,CAT,GCA,TTG,
ACA,TGG
13. P13: Tm=54, 60mer
TCT,TCT,TGG,TCC,TAT,CAG,GTT,CTT,GCA,ACT,GAA,GGT,TAC,TAC,TCT,TCT,GGT,TAC,
TCC,AAC
14. P14: Tm=54, 41mer
GAG,CTC,TTA,CCA,GAC,GGT,GAC,GTT,GGA,GTA,ACC,AGA,AGA,GTA,GTA,ACC,TTC,AGT,
TGC,AAG
Expanded using PCR, in 100 μ l reaction systems, the addition of P2 ~ P13 totally 12 primers is 2ng, and outside is drawn
Thing P1 and P14 addition are 30ng, and amplification condition is：94 DEG C of preheating 1min；94 DEG C of 30s, 52 DEG C of 30s, 72 DEG C of 1min, 72 DEG C
10min, the Taq archaeal dna polymerases used are KOD FX taq enzymes（Toyobo companies, Japan）, totally 30 circulations.
After PCR terminates, the recovery of 1% agarose gel, 10 μ l are taken directly to be connected with T/A cloning vectors（Dalian treasured biotech firm）.
4 DEG C of connections are stayed overnight, in Efficient Conversion DH5 α competence.Positive colony is obtained, extracts plasmid, sequencing.Obtain wild type hot rod
Bacterium xylanase gene（XynB）.
1.2 PCR expand xylanase gene and DNA molecular is reset
Positive plasmid containing XynB genes obtained as described above is template, and P1 and P14 are that primer expands xylanase gene,
Reaction condition is：94 DEG C of 10min pre-degenerations, 94 DEG C of denaturation 30s, 54 DEG C of annealing and 72 DEG C of extension 1min, totally 30 circulations, 1%
Agrose electrophoresis, saturating suction bag method recovery 525bp xylanase gene fragment.
1）DNase I degradation of dna and recovery small fragment
The xylanase gene fragment of above-mentioned recovery is with DNase I buffer solutions（50mmol/L Tris-Cl pH7.4 + 1mmol/
L MgCl2）100 μ l dissolve；0.1U DNase I are added, 25 DEG C are handled 15 minutes, and 70 DEG C are handled 10 minutes.10% acrylamide
Electrophoresis, saturating suction bag method reclaim 10 ~ 50bp small fragment.With 10 μ l 10 × primer free PCR buffer solutions（Primerless PCR
Buffer）（50mmol/L KCl + 10mmol/L Tris-Cl pH9.0 + 1% Triton）Dissolving precipitation.
2）Primer free PCR（Primerless PCR）
Carry out Primerless PCR amplifications.Reaction system：The μ of+4 μ l 2.5mmol/L dNTPs of 5 μ l small fragments DNA+4.5
l 25mmol/L MgCl2 + Taq2U + ddH2O to 50 μ l；Response procedures are：94 DEG C of 30s, 40 DEG C of 30s, 72 DEG C of 30s, totally 45
Individual circulation）, 2% Agrose electrophoresis detection PCR amplifications.
3）There is primer PCR（Primer PCR）
Using above-mentioned primer free pcr amplification product as template, PrimerPCR amplified reactions are carried out by primer of P1 and P14.Reactant
System：The μ l of 5 μ l Primerless PCR primer+P1 0.2ng+P14+10 × PCR of 0.2ng Buffer 5+
2.5mmol/L dNTPs 4μl + Taq2U + ddH2O to 50 μ l.Response procedures are：94 DEG C of 30s, 70 DEG C of 30s, 72 DEG C
1.0min, totally 35 circulation, 1%Agrose electrophoresis detections, reclaim 525bp reset xylanase gene fragment.
The fire resistant xylanase screening mutant of embodiment 2 and sequence analysis
The screening of 2.1 fire resistant xylanase mutant
By the xylanase gene fragment that above-mentioned recovery is reset after BamH I and Sac I double digestions, prokaryotic expression load is built into
Body pG251（CN1338515）Promoter and t1t2 terminators between, the carrier carries ampicillin resistance gene.Electric shock
Method conversion coli strain DH5 α obtain mutant expression library, then with a large amount of extracts kits of plasmid（U.S. Omega is public
Department）Carry out plasmid extraction.Take plasmid that 1 μ l largely extract again after BamH I and Sac I double digestions with carrier for expression of eukaryon
pYM7909（Molecular Biology Report, 2012 Apr;39(4):3807-14）It is connected, Efficient Conversion DH5 α impressions
In state, positive colony is obtained, extracts plasmid.Then take linearisation DNA of the about 1 μ l above-mentioned recombinant plasmid after BglII digestions
Carry out electroporated after being mixed with 80 μ l Pichia pastoris competent cells, be then coated on solid SD sorbierite culture mediums（20g/L
Agar, 20g/L glucose, 146g/L sorbierites, SD）On, 28 DEG C culture 3 days after obtain white colony.White colony is inoculated with
In 48 orifice plates containing 50 μ L BMMY fluid nutrient mediums (0.5% methanol), 28 DEG C, shaking table culture 1d；Then in 48 orifice plates
Add 100 μ L 10mM xylan solution in each hole, 70 DEG C of isothermal reactions 30 minutes；100 μ L DNS are added in every hole again
Colour developing, yellow color it is deep be filter out there are resistant to elevated temperatures mutant colonies.
The sequence analysis of 2.2 fire resistant xylanase mutant
DNA sequencing is carried out using the progressively sequencing methods fire resistant xylanase mutant obtained to 2.1 screenings.Analysis
As a result show, mutant one（XynB-C）Contain following 6 amino acid mutation sites on the same molecule, be specifically：It is mutated position
Point 1：Valine in V13A, i.e. xylanase amino acid sequence on the 13rd is replaced by alanine；Mutational site 2：S33T,
Serine on i.e. the 33rd is replaced by threonine；Mutational site 3：L80P, i.e., the leucine of the 117th replace with dried meat ammonia
Acid；Mutational site 4：S97C, i.e., the serine on the 97th are replaced by cysteine；Mutational site 5：F120S, i.e., the 120th
Phenylalanine on position is replaced by serine；Mutational site 6：N145C, i.e., the asparagine on the 145th are replaced by half
Cystine.Above-mentioned obtained mutational site is critical sites, and its amino acid sequence total length is compiled as shown in SEQ ID No.1
The nucleotide sequence of code is as shown in SEQ ID No.2.
Mutant two（XynB-CE）Contain following 7 amino acid mutation sites on the same molecule, be specifically：It is mutated position
Point 1：Valine in V13A, i.e. xylanase amino acid sequence on the 13rd is replaced by alanine；Mutational site 2：S33T,
Serine on i.e. the 33rd is replaced by threonine；Mutational site 3：L80P, i.e., the leucine of the 117th replace with dried meat ammonia
Acid；Mutational site 4：S97C, i.e., the serine on the 97th are replaced by cysteine；Mutational site 5：F120S, i.e., the 120th
Phenylalanine on position is replaced by serine；Mutational site 6：N145C, i.e., the asparagine on the 145th are replaced by half
Cystine；Mutational site 7：A153E, i.e., the alanine on the 153rd are replaced by glutamic acid.Above-mentioned obtained mutational site
It is critical sites, its amino acid sequence total length is as shown in SEQ ID No.3, the nucleotide sequence such as SEQ ID No.4 of coding
It is shown.
Expressed in the Pichia pastoris of the fire resistant xylanase mutant of embodiment 3
Expression of the 3.1 fire resistant xylanase mutant genes in Pichia pastoris
The xylanase mutant gene obtained using above-mentioned screening is entered performing PCR as masterplate, with primer P1 and P14 and expanded, with KOD
Plus（Toyobo Japan）For Taq archaeal dna polymerases, amplification condition is followed successively by：94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 60s, amplification 30
Individual circulation.After circulation terminates, 2U rTaq enzymes are added（Dalian Bao Bio-Engineering Company）, 72 DEG C of extension 90s, amplified fragments length
525bp.PCR primer after BamH I and Sac I double digestions with carrier for expression of eukaryon pYM7909（Molecular Biology
Report, 2012 Apr;39(4):3807-14）It is connected, in Efficient Conversion DH5 α competence, obtains positive colony, extract matter
Grain.Linearisation DNA of the about 1 μ l above-mentioned recombinant plasmid after BglII digestions is taken to be mixed with 80 μ l Pichia pastoris competent cells
After carry out it is electroporated（Novagen companies）, then it is coated on solid SD sorbierite culture mediums（20g/L agar, 20g/L grapes
Sugar, 146g/L sorbierites, SD）On, 28 DEG C culture 3 days after obtain white colony.White colony is inoculated in 100mL BMMY
In the shaking flask of fluid nutrient medium (0.5% methanol), 28 DEG C of shaking table cultures carry out classification to the bacterium solution of culture after 1 day, with ammonium sulfate heavy
Form sediment, then with gel-protein purification kit HisTrap HP（Amersham Biosciences companies）To being sunk through ammonium sulfate
The about 1 mL xylanase protein solution to form sediment carries out protein expression and purification, SDS-PAGE electrophoresis detections.
The fire resistant xylanase mutant enzyme activity determination of embodiment 4 and Physiological And Biochemical Parameters measure
4.1 fire resistant xylanase mutant enzyme activity determinations
The measure of xylanase activity is carried out with the albumen being purified into, determination of activity is using the fixed sugared methods of DNS.Reactant
It is to be：50 μ L zytase enzyme liquids, 100 μ L xylose solutions.10min is reacted at 45 DEG C, 150 μ L DNS is added immediately and terminates instead
Should, take 200 μ L mixed liquor ultraviolet scanners（TECAN Tecan i-control spectrophotometer）In wavelength
Light absorption value is surveyed under 545nm.With inactive enzyme（100 DEG C of 30 min of heating）As blank control.1 enzyme-activity unit U is defined as,
The enzyme amount required for 1 micro- mol xyloses is produced per min.Using Michaelis-Menten equation, double-reciprocal plot method, zytase is drawn out
Kinetic curve, so as to calculate the Km of zytase and enzymatic activity.
Fire resistant xylanase mutant enzyme activity parameter is as shown in table 1 below.As it can be seen from table 1 mutant XynB-C
Enzymatic activity is more or less the same with wild type, but mutant XynB-CE enzymatic activity is compared with wild type and is declined slightly.K m And one
Sample, mutant XynB-C'sK m It is more or less the same with wild type, but mutant XynB-CEK m Compare and be declined slightly with wild type.
The half-rate phase and enzyme kinetics parameter that 1 65 DEG C and 70 DEG C of table
4.2 fire resistant xylanase mutant physio-biochemical characteristics determine
The Analysis of The Physiological And Biochemical Properties of the present invention mainly has：The optimal reaction pH of enzyme, the optimal reactive temperature of enzyme, the temperature of enzyme
Stability and enzyme are in 65 DEG C and 70 DEG C of half-life period.
In the optimal reaction pH of studying enzyme, citrate-phosphate sodium buffer solution is selected（0.1M, pH 1.0-11.0）37
The activity of enzyme is determined at DEG C；It is under the conditions of optimal pH in the optimal reactive temperature of studying enzyme, measurement temperature is from 20 DEG C to 90
DEG C enzyme activity；In the temperature stability of studying enzyme, be under the conditions of optimal pH determine enzyme between 20 DEG C to 90 DEG C not
The residual activity of enzyme under synthermal processing.Enzyme is also determined under the conditions of optimal pH in 65 DEG C and 70 DEG C of half-life period simultaneously.With
The measure of upper each index is done 3 repetitions and tested.
As a result the optimum temperature for showing two mutant of the invention is 60 DEG C（Fig. 2）, optimum pH 6（Fig. 1）；It is wild
The enzymatic activity that the heat endurance of type enzyme remains between 20-60 DEG C is about 30-40 %, and the heat of two mutant of the present invention is steady
The qualitative enzymatic activity that still can retain average out to 50% between 20-60 DEG C（Fig. 3）；Half-life period measurement result is shown in 65 DEG C
Half-life period with wild-type enzyme at 70 DEG C is respectively 10 and 4 minutes, and the mutant one of the present invention（XynB-C）At 65 DEG C and 70
DEG C when half-life period be respectively 50 and 12 minutes, mutant two of the invention（XynB-CE）Half-life period in 65 DEG C and 70 DEG C
Respectively 60 and 18 minutes（Fig. 4 and table 1）.As can be seen here, two mutant of the invention are compared with very bright with wild type
Aobvious heat resistance, thus the mutant of the present invention is possibly used in industrial production.
Sequence table
<110>Academy of Agricultural Sciences, Shanghai City
<120>The preparation and its application for the hot rod bacterium xylanase mutant that two heat resistances improve
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 174
<212> PRT
<213>Hot rod bacterium (Thermobacillus composti)
<400> 1
Met Val Ser Gly Met Ser Thr Gln Pro Thr Val Lys Ala Ala Thr Thr
1 5 10 15
Leu Ser Leu Gly Pro Thr Leu Val Thr Ser Ser Leu Val Arg Val Gly
20 25 30
Thr Met Val His Thr Ile Val Leu Ser Thr Thr Met Leu Val His Gly
35 40 45
Ser Gln Thr Val Thr His Ile Trp Leu Cys Met Val Gly Leu Val Thr
50 55 60
His Ser Ser Ser Thr Thr Leu Leu Thr Leu Gly Val Pro Thr Val Pro
65 70 75 80
Leu Val Thr Thr Val Val Leu Ser Thr Leu Met Val His Gly Met Thr
85 90 95
Cys Ile Thr Leu Gly Val Thr Thr His His Pro Ser Met Val Leu Arg
100 105 110
Pro Ser Asn Ser Thr Gly Leu Ser Val Ser Arg Ser Val Gln Leu Val
115 120 125
Pro Thr Ser Leu Ser Pro Ser Arg Thr Met Ser Met His Gly Val Leu
130 135 140
Cys Val Cys Leu Trp Val Leu Leu Ala Pro Ile Arg Phe Leu Gln Leu
145 150 155 160
Lys Val Thr Thr Leu Leu Val Thr Pro Thr Ser Pro Ser Gly
165 170
<210> 2
<211> 525
<212> DNA
<213>Hot rod bacterium (Thermobacillus composti)
<400> 2
atggtatcgg gtatgtcaac gcaaccaacg gtcaaggcgg caactactct gtctcttggt 60
ccaactctgg taacttcgtc attggtaagg gttggcacta tggtgcacac aatcgtgttg 120
tcaactacaa tgctggtgca tggcagccaa acggtaacgc atatctggct ctgtatggtt 180
ggactcgtaa cccactcatc gagtactacg ttgttgactc ttggggttcc taccgtccca 240
ctggtgacta ccgtggttct gtctactctg atggtgcatg gtatgacctg tatcactctt 300
ggcgttacaa cgcaccatcc atcgatggta ctcagacctt ccaacagtac tggtctgtcc 360
gtcagcagaa gcgtccaact ggttccaacg tctctatcac cttcgagaac catgtcaatg 420
catggggtgc tgtgcgtatg cctatgggtt cttcttgctc ctatcaggtt cttgcaactg 480
aaggttacta ctcttctggt tactccaacg tcaccgtctg gttaa 525
<210> 3
<211> 174
<212> PRT
<213>Hot rod bacterium (Thermobacillus composti)
<400> 3
Met Val Ser Gly Met Ser Thr Gln Pro Thr Val Lys Ala Ala Thr Thr
1 5 10 15
Leu Ser Leu Gly Pro Thr Leu Val Thr Ser Ser Leu Val Arg Val Gly
20 25 30
Thr Met Val His Thr Ile Val Leu Ser Thr Thr Met Leu Val His Gly
35 40 45
Ser Gln Thr Val Thr His Ile Trp Leu Cys Met Val Gly Leu Val Thr
50 55 60
His Ser Ser Ser Thr Thr Leu Leu Thr Leu Gly Val Pro Thr Val Pro
65 70 75 80
Leu Val Thr Thr Val Val Leu Ser Thr Leu Met Val His Gly Met Thr
85 90 95
Cys Ile Thr Leu Gly Val Thr Thr His His Pro Ser Met Val Leu Arg
100 105 110
Pro Ser Asn Ser Thr Gly Leu Ser Val Ser Arg Ser Val Gln Leu Val
115 120 125
Pro Thr Ser Leu Ser Pro Ser Arg Thr Met Ser Met His Gly Val Leu
130 135 140
Cys Val Cys Leu Trp Val Leu Leu Glu Pro Ile Arg Phe Leu Gln Leu
145 150 155 160
Lys Val Thr Thr Leu Leu Val Thr Pro Thr Ser Pro Ser Gly
165 170
<210> 4
<211> 525
<212> DNA
<213>Hot rod bacterium (Thermobacillus composti)
<400> 4
atggtatcgg gtatgtcaac gcaaccaacg gtcaaggcgg caactactct gtctcttggt 60
ccaactctgg taacttcgtc attggtaagg gttggcacta tggtgcacac aatcgtgttg 120
tcaactacaa tgctggtgca tggcagccaa acggtaacgc atatctggct ctgtatggtt 180
ggactcgtaa cccactcatc gagtactacg ttgttgactc ttggggttcc taccgtccca 240
ctggtgacta ccgtggttct gtctactctg atggtgcatg gtatgacctg tatcactctt 300
ggcgttacaa cgcaccatcc atcgatggta ctcagacctt ccaacagtac tggtctgtcc 360
gtcagcagaa gcgtccaact ggttccaacg tctctatcac cttcgagaac catgtcaatg 420
catggggtgc tgtgcgtatg cctatgggtt cttcttgaac ctatcaggtt cttgcaactg 480
aaggttacta ctcttctggt tactccaacg tcaccgtctg gttaa 525
Claims (7)
1. one kind derives from hot rod bacterium（Thermobacillus composti）Zytase multisite mutant one, it is special
Sign is that the multisite mutant contains following 6 amino acid mutation sites：
Mutational site 1：Valine in V13A, i.e. xylanase amino acid sequence on the 13rd is replaced by alanine；
Mutational site 2：S33T, i.e., the serine on the 33rd are replaced by threonine；
Mutational site 3：L80P, i.e., the leucine of the 117th replace with proline；
Mutational site 4：S97C, i.e., the serine on the 97th are replaced by cysteine；
Mutational site 5：F120S, i.e., the phenylalanine on the 120th are replaced by serine；
Mutational site 6：N145C, i.e., the asparagine on the 145th are replaced by cysteine.
2. derive from hot rod bacterium described in coding claim 1（Thermobacillus composti）Zytase multidigit
Point mutation body one, it is characterised in that the amino acid sequence of the multisite mutant is as shown in SEQ ID No.1.
3. derive from hot rod bacterium described in coding claim 1（Thermobacillus composti）Zytase multidigit
The gene of point mutation body one, it is characterised in that the nucleotide sequence of the gene is as shown in SEQ ID No.2.
4. one kind derives from hot rod bacterium（Thermobacillus composti）Zytase multisite mutant two, it is special
Sign is that the multisite mutant contains following 7 mutational sites：
Mutational site 1：Valine in V13A, i.e. xylanase amino acid sequence on the 13rd is replaced by alanine；
Mutational site 2：S33T, i.e., the serine on the 33rd are replaced by threonine；
Mutational site 3：L80P, i.e., the leucine of the 117th replace with proline；
Mutational site 4：S97C, i.e., the serine on the 97th are replaced by cysteine；
Mutational site 5：F120S, i.e., the phenylalanine on the 120th are replaced by serine；
Mutational site 6：N145C, i.e., the asparagine on the 145th are replaced by cysteine；
Mutational site 7：A153E, i.e., the alanine on the 153rd are replaced by glutamic acid.
5. derive from hot rod bacterium described in coding claim 4（Thermobacillus composti）Zytase multidigit
Point mutation body two, it is characterised in that the amino acid sequence of the multisite mutant is as shown in SEQ ID No.3.
6. derive from hot rod bacterium described in coding claim 4（Thermobacillus composti）Zytase multidigit
The gene of point mutation body two, it is characterised in that the nucleotide sequence of the gene is as shown in SEQ ID No.4.
7. Claims 2 or 3, the coding described in 5 or 6 derives from hot rod bacterium（Thermobacillus composti）Wood gather
The application of the gene of carbohydrase multisite mutant in the industrial production.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710777945.8A CN107502602A (en) | 2017-09-01 | 2017-09-01 | The preparation and its application for the hot rod bacterium xylanase mutant that two heat resistances improve |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710777945.8A CN107502602A (en) | 2017-09-01 | 2017-09-01 | The preparation and its application for the hot rod bacterium xylanase mutant that two heat resistances improve |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107502602A true CN107502602A (en) | 2017-12-22 |
Family
ID=60694807
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710777945.8A Pending CN107502602A (en) | 2017-09-01 | 2017-09-01 | The preparation and its application for the hot rod bacterium xylanase mutant that two heat resistances improve |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107502602A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110607292A (en) * | 2018-06-14 | 2019-12-24 | 青岛蔚蓝生物集团有限公司 | High specific activity xylanase mutant |
| CN113528486A (en) * | 2021-08-19 | 2021-10-22 | 江南大学 | Method for improving heat stability of xylanase by introducing disulfide bond |
| CN117821412A (en) * | 2023-12-15 | 2024-04-05 | 湖北大学 | dCE-KOD DNA polymerase and preparation method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101805726A (en) * | 2010-04-19 | 2010-08-18 | 中国科学院微生物研究所 | Heat-resistance neutral xylanase, coding gene and application thereof |
| US20130109062A1 (en) * | 2009-12-21 | 2013-05-02 | Süd-Chemie AG | Thermostable xylanase for the selective hydrolysis of pentose-containing polysaccharides |
| WO2016175202A1 (en) * | 2015-04-28 | 2016-11-03 | 国立研究開発法人産業技術総合研究所 | Heat-resistant xylanase |
| CN106636038A (en) * | 2016-10-31 | 2017-05-10 | 江南大学 | Heat resistance improved xylanases and application thereof |
-
2017
- 2017-09-01 CN CN201710777945.8A patent/CN107502602A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130109062A1 (en) * | 2009-12-21 | 2013-05-02 | Süd-Chemie AG | Thermostable xylanase for the selective hydrolysis of pentose-containing polysaccharides |
| CN101805726A (en) * | 2010-04-19 | 2010-08-18 | 中国科学院微生物研究所 | Heat-resistance neutral xylanase, coding gene and application thereof |
| WO2016175202A1 (en) * | 2015-04-28 | 2016-11-03 | 国立研究開発法人産業技術総合研究所 | Heat-resistant xylanase |
| CN106636038A (en) * | 2016-10-31 | 2017-05-10 | 江南大学 | Heat resistance improved xylanases and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| TIAN YONG-SHENG等: "Improvement of the Thermostability of Xylanase from Thermobacillus composti through Site-Directed Mutagenesis", 《J. MICROBIOL. BIOTECHNOL.》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110607292A (en) * | 2018-06-14 | 2019-12-24 | 青岛蔚蓝生物集团有限公司 | High specific activity xylanase mutant |
| CN110607292B (en) * | 2018-06-14 | 2022-05-31 | 青岛蔚蓝生物集团有限公司 | High specific activity xylanase mutant |
| CN113528486A (en) * | 2021-08-19 | 2021-10-22 | 江南大学 | Method for improving heat stability of xylanase by introducing disulfide bond |
| CN113528486B (en) * | 2021-08-19 | 2023-10-20 | 广东众汇食品科技有限公司 | Method for improving xylanase thermal stability by introducing disulfide bond |
| CN117821412A (en) * | 2023-12-15 | 2024-04-05 | 湖北大学 | dCE-KOD DNA polymerase and preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111635898B (en) | Glutamic acid decarboxylase mutant and application thereof in preparation of gamma-aminobutyric acid | |
| CN110628741B (en) | Maltooligosyl trehalose synthase mutant and application thereof | |
| CN111172124A (en) | Carbonyl reductase mutant and application thereof in preparation of (R) -4-chloro-3-hydroxy-butyrate | |
| CN110066777B (en) | Endo-inulase and application thereof in production of fructo-oligosaccharide | |
| JP7241368B2 (en) | Ulva polysaccharide lyase and its coding gene and application | |
| CN108018269B (en) | Levansucrase mutant with improved thermal stability | |
| CN107502602A (en) | The preparation and its application for the hot rod bacterium xylanase mutant that two heat resistances improve | |
| CN111893125A (en) | Chitosan enzyme gene, chitosanase, preparation method and application thereof | |
| JP4663631B2 (en) | AMP deaminase derived from actinomycetes and use thereof | |
| CN106566823A (en) | Cloning of novel glutamate decarboxylase gene and application thereof | |
| CN111996205A (en) | Chitinase gene, chitinase and preparation method and application thereof | |
| CN106459962A (en) | Improved nitrile hydratase | |
| CN109666657B (en) | Glucose oxidase for improving heat resistance | |
| CN109576239A (en) | Heat-resisting phosphorylase and its application | |
| CN104212757A (en) | Method for high-efficiently producing L-theanine through production of coli [gamma]-glutamylmethylamine synthetase with escherichia coli | |
| CN106434715B (en) | Malt oligosaccharide based mycose synthetase and its expressing gene and application | |
| CN109161556B (en) | M1PDH gene in kelp, protein and application thereof | |
| CN114409751B (en) | YH 66-04470 gene mutant recombinant bacterium and application thereof in preparation of arginine | |
| CN105969751B (en) | Beta-glucosidase gene and application thereof | |
| CN113755473B (en) | Glucoamylase mutant M5 with improved secretion expression level as well as gene and application thereof | |
| CN104087604A (en) | Genetic expression sequence of inulin fructotransferase | |
| CN108728457B (en) | Trehalose synthase gene optimization sequence and application thereof | |
| CN102212500B (en) | Application of gene of coded transcription factor | |
| CN113583991B (en) | Amylosucrase SaAS and encoding gene and application thereof | |
| KR20180007725A (en) | Mass production method of beta agarase |
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 | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20171222 |