CN107142253A - A kind of high catalytic efficiency and fire resistant xylanase mutant and its preparation method and application - Google Patents
A kind of high catalytic efficiency and fire resistant xylanase mutant and its preparation method and application Download PDFInfo
- Publication number
- CN107142253A CN107142253A CN201710176527.3A CN201710176527A CN107142253A CN 107142253 A CN107142253 A CN 107142253A CN 201710176527 A CN201710176527 A CN 201710176527A CN 107142253 A CN107142253 A CN 107142253A
- Authority
- CN
- China
- Prior art keywords
- mutant
- zytase
- amino acid
- xyl10c
- acid sequence
- 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.)
- Granted
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
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
A kind of high catalytic efficiency and fire resistant xylanase mutant and its preparation method and application, are related to genetic engineering and field of genetic engineering.The present invention is using the GH10 families zytase XYL10C from filamentous fungi Bispora sp.MEY 1 as female parent, the exit site of the 175th in female parent is carried out by saturation mutation using Protocols in Molecular Biology, as a result find, mutant XYL10C CutN, E175H, E175N, E175D, E175V, E175L, E175Q, the catalytic efficiency of eight mutant of E175M and enzyme wilder than work are improved largely, find to study the key player that this is played the part of in XYL10C is high than living and high catalytic efficiency by saturation mutation, research of the method to the efficient catalytic mechanism of zytase enzyme and other (α/βs) 8 tubbiness enzyme has important directive significance.
Description
Technical field
The present invention relates to genetic engineering field, more particularly to a kind of high catalytic efficiency and fire resistant xylanase mutant
And its preparation method and application.
Background technology
Cellulose, hemicellulose and lignin are the main components for constituting plant cell wall, about account for 50% life in the world
Object amount.Hemicellulose is primarily present in the surface of cell membrane, and cellulose is wrapped in hemicellulose, and hemicellulose with it is wooden
Element is covalently attached and forms network structure.Xylan is the main component of hemicellulose, is that content is only second to cellulose in nature
Renewable resource, almost account for 1/3rd of earth organic carbon content.Xylan it is complicated, its main chain is by xylopyranose
Sugar is with β-D-1, and 4- xylose glycosidic bonds are connected, and with a variety of substituents.
Due to the diversity and the complexity of structure of xylan, its complete hydrolysis needs the synergy ability of a variety of enzymes
Complete.Include alpha-D-glucose aldehyde neuraminidase, the acetyl xylan of zytase, xylosidase and hydrolysis side chain substituents group
Esterase, feruloyl esterase, coumaric acid esterase etc..By the synergy of these enzymes, xylan can be effectively hydrolyzed.Its
In, zytase is capable of β-Isosorbide-5-Nitrae-glycosidic bond of degradation of xylan main chain, is to play most important functions during xylan degrading
Enzyme.Zytase is respectively provided with widely in the industry such as feed industry, food industry, paper industry and the energy, weaving, medicine
Application value.
Mechanism of thermal stability research and improvement are concentrated mainly in terms of the molecular improvement of zytase.Ding etc. (2013) leads to
Cross the technique study of molecular dynamics simulation from Streptomyces lividans zytase Sl-XlnA heat not
Stability section, it is determined that the factor of influence Sl-XlnA heat endurances.Gallardo etc. makes to derive from by the method for forced evolution
Paenibaciius barcinonensis zytase Xyn10B heat endurance improves 20 times.Kamondi etc. passes through
Family shuffling method, the zytase of the families of Liang Ge 10 is carried out the replacement of random fragment, and discovery has 4 heterozygosis
The optimum temperature of body is improved.But the research in terms of the mechanism of the 10th other properties of family's zytase and improvement is also very
Few, the especially research in terms of the 10th family's zytase efficient catalytic mechanism and improvement has not been reported.
The content of the invention
It is an object of the invention to material XYL10C is cut out the high catalytic efficiency zytase obtained after N-terminal sequence with
And the amino acid sequence of the high catalytic efficiency xylanase mutant obtained in material XYL10C after E175 site mutations is protected
Shield, and N-terminal sequence and E175 sites institute's role in XYL10C high catalytic efficiency are studied in detail.
Another object of the present invention is to provide the recombinant vector for including E175 saturated mutant genes.
It is a further object of the present invention to provide the recombinant bacterial strain for including E175 saturated mutant genes.
The wild-type amino acid sequence total length of mutant is as shown in SEQ ID NO.1.
MSFHSLLISGLLASVAVAVPKEAWGITVTETKTVSTTIIATVTELGTCSSTITSPTSDATTTTTSSATN
TNPTTTLLATPQPSNWGLNNAARADGKLWFGTAADIPGLEQDDRYYMKEYNNTHDFGGTTPANIMKFMFTEPEQNVF
NFTGAQEFLDIAFASHKLVRCHNLIWQSELPTWVTNPTTNWTNETLSKVLQNHVYTLVSHFGDQCYSWDVVNEALSD
DPAGSYQNNIWFDTIGPEYVAMAFEYAEKAVKDHKLNVKLYYNDYNIEYPGPKSTAAQNIVKELKARNIQIDGVGLE
SHFIAGETPSQATQITNMADFTSLDIDVAVTELDVRLYLPPNATSEAQQVADYYATVAACAATERCIGITVWDFDDT
YSWVPSTFAGQGYADLFFQPDGPNTPLVKKAAYDGCLQALQHKAESP
The wild-type mature amino acid sequence of mutant is as shown in SEQ ID NO.2.
VPKEAWGITVTETKTVSTTIIATVTELGTCSSTITSPTSDATTTTTSSATNTNPTTTLLATPQPSNWGL
NNAARADGKLWFGTAADIPGLEQDDRYYMKEYNNTHDFGGTTPANIMKFMFTEPEQNVFNFTGAQEFLDIAFASHKL
VRCHNLIWQSELPTWVTNPTTNWTNETLSKVLQNHVYTLVSHFGDQCYSWDVVNEALSDDPAGSYQNNIWFDTIGPE
YVAMAFEYAEKAVKDHKLNVKLYYNDYNIEYPGPKSTAAQNIVKELKARNIQIDGVGLESHFIAGETPSQATQITNM
ADFTSLDIDVAVTELDVRLYLPPNATSEAQQVADYYATVAACAATERCIGITVWDFDDTYSWVPSTFAGQGYADLFF
QPDGPNTPLVKKAAYDGCLQALQHKAESP
The wild-type amino acid of claim 1 mutant removes N-terminal sequence as shown in SEQ ID NO.3.
WGLNNAARADGKLWFGTAADIPGLEQDDRYYMKEYNNTHDFGGTTPANIMKFMFTEPEQNVFNFTGAQE
FLDIAFASHKLVRCHNLIWQSELPTWVTNPTTNWTNETLSKVLQNHVYTLVSHFGDQCYSWDVVNEALSDDPAGSYQ
NNIWFDTIGPEYVAMAFEYAEKAVKDHKLNVKLYYNDYNIEYPGPKSTAAQNIVKELKARNIQIDGVGLESHFIAGE
TPSQATQITNMADFTSLDIDVAVTELDVRLYLPPNATSEAQQVADYYATVAACAATERCIGITVWDFDDTYSWVPST
FAGQGYADLFFQPDGPNTPLVKKAAYDGCLQALQHKAESP
Claim 2 mutant E175H amino acid sequences are as shown in SEQ ID NO.4.
VPKEAWGITVTETKTVSTTIIATVTELGTCSSTITSPTSDATTTTTSSATNTNPTTTLLATPQPSNWGL
NNAARADGKLWFGTAADIPGLEQDDRYYMKEYNNTHDFGGTTPANIMKFMFTEPEQNVFNFTGAQEFLDIAFASHKL
VRCHNLIWQSHLPTWVTNPTTNWTNETLSKVLQNHVYTLVSHFGDQCYSWDVVNEALSDDPAGSYQNNIWFDTIGPE
YVAMAFEYAEKAVKDHKLNVKLYYNDYNIEYPGPKSTAAQNIVKELKARNIQIDGVGLESHFIAGETPSQATQITNM
ADFTSLDIDVAVTELDVRLYLPPNATSEAQQVADYYATVAACAATERCIGITVWDFDDTYSWVPSTFAGQGYADLFF
QPDGPNTPLVKKAAYDGCLQALQHKAESP
Claim 2 mutant E175N amino acid sequences are as shown in SEQ ID NO.5.
VPKEAWGITVTETKTVSTTIIATVTELGTCSSTITSPTSDATTTTTSSATNTNPTTTLLATPQPSNWGLNNAARADG
KLWFGTAADIPGLEQDDRYYMKEYNNTHDFGGTTPANIMKFMFT
EPEQNVFNFTGAQEFLDIAFASHKLVRCHNLIWQSNLPTWVTNPTTNWTNETLSKVLQNHVYTLVSHFGDQCYSWDV
VNEALSDDPAGSYQNNIWFDTIGPEYVAMAFEYAEKAVKDHKLNVKLYYNDYNIEYPGPKSTAAQNIVKELKARNIQ
IDGVGLESHFIAGETPSQATQITNMADFTSLDIDVAVTELDVRLYLPPNATSEAQQVADYYATVAACAATERCIGIT
VWDFDDTYSWVPSTFAGQGYADLFFQPDGPNTPLVKKAAYDGCLQALQHKAESP
Claim 2 mutant E175D amino acid sequences are as shown in SEQ ID NO.6.
VPKEAWGITVTETKTVSTTIIATVTELGTCSSTITSPTSDATTTTTSSATNTNPTTTLLATPQPSNWGL
NNAARADGKLWFGTAADIPGLEQDDRYYMKEYNNTHDFGGTTPANIMKFMFTEPEQNVFNFTGAQEFLDIAFASHKL
VRCHNLIWQSDLPTWVTNPTTNWTNETLSKVLQNHVYTLVSHFGDQCYSWDVVNEALSDDPAGSYQNNIWFDTIGPE
YVAMAFEYAEKAVKDHKLNVKLYYNDYNIEYPGPKSTAAQNIVKELKARNIQIDGVGLESHFIAGETPSQATQITNM
ADFTSLDIDVAVTELDVRLYLPPNATSEAQQVADYYATVAACAATERCIGITVWDFDDTYSWVPSTFAGQGYADLFF
QPDGPNTPLVKKAAYDGCLQALQHKAESP
Claim 2 mutant E175V amino acid sequences are as shown in SEQ ID NO.7.
VPKEAWGITVTETKTVSTTIIATVTELGTCSSTITSPTSDATTTTTSSATNTNPTTTLLATPQPSNWGL
NNAARADGKLWFGTAADIPGLEQDDRYYMKEYNNTHDFGGTTPANIMKFMFTEPEQNVFNFTGAQEFLDIAFASHKL
VRCHNLIWQSVLPTWVTNPTTNWTNETLSKVLQNHVYTLVSHFGDQCYSWDVVNEALSDDPAGSYQNNIWFDTIGPE
YVAMAFEYAEKAVKDHKLNVKLYYNDYNIEYPGPKSTAAQNIVKELKARNIQIDGVGLESHFIAGETPSQATQITNM
ADFTSLDIDVAVTELDVRLYLPPNATSEAQQVADYYATVAACAATERCIGITVWDFDDTYSWVPSTFAGQGYADLFF
QPDGPNTPLVKKAAYDGCLQALQHKAESP
Claim 2 mutant E175L amino acid sequences are as shown in SEQ ID NO.8.
VPKEAWGITVTETKTVSTTIIATVTELGTCSSTITSPTSDATTTTTSSATNTNPTTTLLATPQPSNWGL
NNAARADGKLWFGTAADIPGLEQDDRYYMKEYNNTHDFGGTTPANIMKFMFTEPEQNVFNFTGAQEFLDIAFASHKL
VRCHNLIWQSLLPTWVTNPTTNWTNETLSKVLQNHVYTLVSHFGDQCYSWDVVNEALSDDPAGSYQNNIWFDTIGPE
YVAMAFEYAEKAVKDHKLNVKLYYNDYNIEYPGPKSTAAQNIVKELKARNIQIDGVGLESHFIAGETPSQATQITNM
ADFTSLDIDVAVTELDVRLYLPPNATSEAQQVADYYATVAACAATERCIGITVWDFDDTYSWVPSTFAGQGYADLFF
QPDGPNTPLVKKAAYDGCLQALQHKAESP
Claim 2 mutant E175Q amino acid sequences are as shown in SEQ ID NO.9.
VPKEAWGITVTETKTVSTTIIATVTELGTCSSTITSPTSDATTTTTSSATNTNPTTTLLATPQPSNWGLNNAARADG
KLWFGTAADIPGLEQDDRYYMKEYNNTHDFGGTTPANIMKFMFTEPEQNVFNFTGAQEFLDIAFASHKLVRCHNLIW
QSQLPTWVTNPTTNWTNETLSKVLQNHV
YTLVSHFGDQCYSWDVVNEALSDDPAGSYQNNIWFDTIGPEYVAMAFEYAEKAVKDHKLNVKLYYNDYNIEYPGPKS
TAAQNIVKELKARNIQIDGVGLESHFIAGETPSQATQITNMADFTSLDIDVAVTELDVRLYLPPNATSEAQQVADYY
ATVAACAATERCIGITVWDFDDTYSWVPSTFAGQGYADLFFQPDGPNTPLVKKAAYDGCLQALQHKAESP
Claim 2 mutant E175M amino acid sequences are as shown in SEQ ID NO.10.
VPKEAWGITVTETKTVSTTIIATVTELGTCSSTITSPTSDATTTTTSSATNTNPTTTLLATPQPSNWGL
NNAARADGKLWFGTAADIPGLEQDDRYYMKEYNNTHDFGGTTPANIMKFMFTEPEQNVFNFTGAQEFLDIAFASHKL
VRCHNLIWQSMLPTWVTNPTTNWTNETLSKVLQNHVYTLVSHFGDQCYSWDVVNEALSDDPAGSYQNNIWFDTIGPE
YVAMAFEYAEKAVKDHKLNVKLYYNDYNIEYPGPKSTAAQNIVKELKARNIQIDGVGLESHFIAGETPSQATQITNM
ADFTSLDIDVAVTELDVRLYLPPNATSEAQQVADYYATVAACAATERCIGITVWDFDDTYSWVPSTFAGQGYADLFF
QPDGPNTPLVKKAAYDGCLQALQHKAESP
The present invention is also to provide a kind of method for preparing the xylanase mutant, and its technical scheme is as follows：
1st, design mutant primer as shown in table 1, obtains the complete genome fragment of mutant；
2nd, mutated gene segment will be obtained to be cloned on expression vector pPIC9, obtains mutant recombinant plasmid；
3rd, by mutant recombinant plasmid transformed Pichia pastoris GS115, induced expression obtains high catalytic efficiency
Dextranase.
Present invention also offers a kind of method for preparing zytase, comprise the following steps：
1st, with above-mentioned recombinant vector transformed yeast host cell, recombinant bacterial strain is obtained；
2nd, recombinant bacterial strain, induction recombined xylanase expression are cultivated；
3rd, reclaim and purify expressed zytase.
The present invention implements saturation mutation and constructed by cutting out N-terminal and E175 sites to ripe zytase XYL10C
21 mutant including removing E175 sites, Pichia pastoris GS115, tubule induction table are converted by mutant recombinant vector
Up to discovery, except XYL10C-CutE, the tubule supernatant of tri- mutant of XYL10C-E175W is not detected outside activity, its
19 mutant of remaininging are all active.
Big bottle induction is carried out to 19 active mutant and wild enzyme XYL10C, protein concentration has carried out base after purification
The measure of this zymologic property, as shown in table 2, is determined with beech xylan and found, 18 mutation in the saturation mutation of E175 sites
Body and mutant XYL10C-CutN optimum temperatures at 85 degree or so, optimal pH is in 4.0-4.5 or so, and pH each other makees
It is similar with scope.
Mutant XYL10C-CutN, E175H, E175N, E175D, E175V, E175L, E175Q, E175M ratio are lived wilder
Raw enzyme XYL10C has been respectively increased 2.1 times, 2.4 times, 1.7 times, 1.9 times, 2.0 times, 1.4 times 2.5 times and 4.3 times.Mutant
XYL10C-CutN, E175H, E175N, E175D, E175V, E175L, E175Q, E175M wilder enzyme XYL10C of catalytic efficiency
It has been respectively increased 1.7 times, 2.3 times, 2.1 times, 1.4 times, 1.9 times, 1.7 times, 2.3 times and 4.9 times.
Present invention demonstrates that confirming the site E175 sites of the crucial catalysis correlation in material XYL10C, and demonstrate the position
Point plays for the high high catalytic efficiency of the enzyme than importance living, i.e. E175 sites in the height of the enzyme is than high catalytic efficiency living
Important role.Efficient catalytic mechanism for research material XYL10C provides important clue, simultaneously for the raising man
The catalytic efficiency of other zytases of race provides reliable reference point, it was demonstrated that the site is urged in this family zytase
Change the decisive role in efficiency.
Brief description of the drawings
Fig. 1：GH10 families zytase XYL10C-CutN crystal structure and E175 sites are residing in a three-dimensional structure
Position.
Fig. 2：Wild enzyme XYL10C, mutant XYL10C-CutN and 18 active mutant protein purification electricity
Swimming picture.
Fig. 3：(Fig. 3-1 and Fig. 3-2) wild enzyme XYL10C, mutant XYL10C-CutN and 18 active mutation
The optimal pH of body.
Fig. 4：(Fig. 4-1 and Fig. 4-2) wild enzyme XYL10C, mutant XYL10C-CutN and 18 active mutation
The optimum temperature of body.
Embodiment
Below in conjunction with accompanying drawing, the invention will be further described.
Table 1. builds primer by maternal XYL10C-CutN and E175 saturated mutants of XYL10C
Test material and reagent
1st, bacterial strain and carrier：Expressive host Pichia pastoris GS115, expression plasmid carrier pPIC9r are this experiment
Room is preserved.
2nd, enzyme and other biochemical reagents：Restriction endonuclease is purchased from Fermentas companies, and recombinase is purchased from Quan Shi King Companies, beech
Wooden xylan is purchased from Sigma companies.Other is all domestic AR (can be commercially available from common biochemical Reagent Company).
3rd, culture medium：
(1) LB culture mediums：0.5% yeast extract, 1% peptone, 1%NaCl, pH 7.0.
(2) YPD culture mediums：1% yeast extract, 2% peptone, 2% glucose.
(3) MD solid mediums：2% glucose, 1.5% agarose, 1.34%YNB, 0.00004%Biotin.
(4) BMGY culture mediums：1% yeast extract, 2% peptone, 1% glycerine (V/V), 1.34%YNB,
0.00004%Biotin.
(5) BMMY culture mediums：1% yeast extract, 2% peptone, 1.34%YNB, 0.00004%Biotin, 0.5%
Methanol
(V/V)。
The preparation of the mutant XYL10C-CutN recombinant vectors of embodiment 1
GH10 families zytase XYL10C-pPIC9r using Bispora sp.MEY-1 sources is template, in amino acid
Primer XYL10C-CutN-F and whole amino acid sequence ending place pair are designed at nucleotide sequence corresponding to sequence WGLNN
The nucleotide sequence design primer XYL10C-CutN-R answered, amplified production after EcoR I and the double digestions of Not I directly with incision
PPIC9r plasmids be attached, convert TransI-T1 competence.
Structure of the embodiment 2 using XYL10C as maternal E175 saturated mutant recombinant vectors
Saturation mutation primer E175X-F and E175X-R is designed at gene XYL10C E175 sites, with
XYL10C--pPIC9r be template using XYL10C-F and E175X-R and E175X-F and XYL10C-R as primer PCR, amplification
Go out after upstream and downstream fragment, then overlap is carried out by primer of XYL10C-F and XYL10C-R, product is by EcoR I and Not I couple
PPIC9r plasmids after digestion directly with incision are attached, and convert TransI-T1 competence.The sequencing discovery of picking monoclonal,
Only mutant E175F E175M E175T E175W and CutE do not occur, therefore for remaining several mutation body weight
Newly and into primer, method is ibid.
The preparation of the xylanase mutant recombinant bacterial strain of embodiment 3
The correct recombinant vector restriction endonuclease BglII of sequencing will be connected with expression vector pPIC9r to linearize and convert complete red
Yeast Gs115, obtains restructuring yeast strains GS115/XYL10C-CutN, GS115/XYL10C-E175X.
Picking takes the Gs115 bacterial strains containing recombinant plasmid, in the 10mL tubules for being inoculated in 2mL BMGY culture mediums, is placed in 30
DEG C, 220rpm shaking table cultures 48h；Nutrient solution 3000g is centrifuged into 5min afterwards, supernatant is abandoned, precipitation contains 0.5% methanol with 1.5mL
BMMY culture mediums be resuspended, and be again placed in 30 DEG C, Fiber differentiation 48h under the conditions of 220rpm.Supernatant is finally taken to be used for enzymatic activity
Detection.
Picking supernatant enzyme activity highest Gs115 bacterial strains, are inoculated in 30mL YPD culture mediums and cultivate 48h progress seed amplifications
Culture, after be inoculated in by 1% inoculation volume in the 1L triangular flasks of 300mL BMGY culture mediums, be placed in 30 DEG C, the training of 220rpm shaking tables
Support 48h；Nutrient solution 3000g is centrifuged into 5min afterwards, supernatant is abandoned, precipitation contains the BMMY culture medium weights of 0.5% methanol with 200mL
It is outstanding, and 30 DEG C are again placed in, Fiber differentiation under the conditions of 220rpm.1mL methanol is added every 12h, makes the methanol concentration in bacterium solution
0.5% is maintained at, while taking supernatant to be used for Enzyme assay.
The activity analysis of the recombined xylanase mutant of embodiment 4 and wild type
DNS methods：Specific method is as follows：Under given pH, temperature conditionss, it is appropriate that 1mL reaction system includes 100 μ L
Enzyme liquid is diluted, 900 μ L substrates react 10min, add 1.5mL DNS terminating reactions, boiling water boiling 5min.540nm is determined after cooling
OD values.1 enzyme-activity unit (U) is defined as under given conditions, and xylan per minute generates the enzyme needed for 1 μm of ol reducing end
Amount.
The property of the recombined xylanase mutant of embodiment 5 and wild type is determined
1st, recombined xylanase mutant and the optimal pH assay method of wild type are as follows:
The recombined xylanase mutant and wild type that embodiment 3 is purified carry out enzymatic reaction to survey under different pH
Its fixed optimal pH.Substrate beech xylan is entered with different pH 0.2mol/L citrate-phosphate disodium hydrogen buffer solutions 80 DEG C
Row Xylanase activity is determined.As a result (Fig. 3) shows, the optimal reaction pH of recombined xylanase mutant and wild type substantially exists
4.0-4.5 or so, and have in the range of pH3.0-6.0 close enzyme activity.
2nd, recombined xylanase mutant and the optimum temperature assay method of wild type are as follows:
The optimum temperature of recombined xylanase mutant and wild type is determined as in 0.2mol/L citrate-phosphates hydrogen two
Sodium buffer solution each carries out enzymatic reaction under optimal pH buffer solution system and different temperatures.Enzyme reaction optimum temperature measurement result
(Fig. 4) shows, the optimum temperature of recombined xylanase mutant and wild type is at 80-95 DEG C or so.
3rd, the Determination of Kinetic Parameters method of recombined xylanase mutant and wild type is as follows:
With reference to this laboratory Wang Kun method (Wang Kun, 2013), the first order reaction time of reaction is determined.It is determined that determining KmAnd
VmaxReaction time be 5min.It is substrate with the xylan (10,8,5,2.5,2,1,0.75,0.5mg/mL) of various concentrations,
Enzymatic activity is determined under identical conditions (80 DEG C, pH4.0), corresponding reaction speed is calculated, K is calculated using GraFit7 softwaresmValue
And Vmax。
Result of calculation is as shown in table 2.
Table 2, XYL10C collects with the basic zymologic property of all mutant
Claims (7)
1. a kind of zytase, it is characterised in that its amino acid sequence total length such as sequence table SEQ ID NO：Shown in 1.
2. zytase according to claim 1, it is characterised in that the amino acid sequence of its maturation protein such as sequence table
SEQ ID NO：Shown in 2.
3. a kind of high catalytic efficiency zytase, it is characterised in that go the N-terminal sequence of the zytase described in claim 1
Fall the mutant obtained after 90 amino acid, SEQ ID NO：Shown in 3.
4. a kind of zytase of high catalytic efficiency, it is characterised in that by the 175th of the zytase described in claim 1
Amino acids glutamic acid is mutated according to a kind of following mode：E175H, E175N, E175D, E175V, E175L, E175Q or E175M
Afterwards, what is obtained includes the mutant of above-mentioned mutation.
5. a kind of high catalytic efficiency zytase according to claim 4, is characterised by, its described 175th amino acids
The amino acid sequence of the mutant obtained after glutamic acid mutation is as follows；
The mature amino acid sequence of mutant comprising E175H such as sequence table SEQ ID NO：Shown in 4；
The mature amino acid sequence of mutant comprising E175N such as sequence table SEQ ID NO：Shown in 5；
The mature amino acid sequence of mutant comprising E175D such as sequence table SEQ ID NO：Shown in 6；
The mature amino acid sequence of mutant comprising E175V such as sequence table SEQ ID NO：Shown in 7；
The mature amino acid sequence of mutant comprising E175L such as sequence table SEQ ID NO：Shown in 8；
The mature amino acid sequence of mutant comprising E175Q such as sequence table SEQ ID NO：Shown in 9；
The mature amino acid sequence of mutant comprising E175M such as sequence table SEQ ID NO：Shown in 10.
6. a kind of method of the high catalytic efficiency zytase prepared described in claim 3, is characterised by, prepares the xylan
The method for the mutant that the N-terminal sequence of enzyme is obtained after removing comprises the following steps：
(1) gene for encoding zytase described in claim 2 is inserted into expression plasmid pPIC9, obtains recombinant plasmid
XYL10C-pPIC9；
(2) primer is designed according to purpose fragment, while the digestion position in 5 ' end addition expression plasmid pPIC9 junctions of primer pair
Point, using the recombinant plasmid in step (1) as template, amplification obtains removing the encoding gene of the zytase of N-terminal sequence；
(3) the pPIC9 plasmids by encoding gene digestion obtained above and incision are attached, and obtain mutant recombinant plasmid；
(4) by mutant recombinant plasmid transformed Pichia pastoris GS115, induced expression, the zytase that N sections of sequences are removed in acquisition is dashed forward
Variant.
7. a kind of method of the high catalytic efficiency zytase prepared described in claim 4, is characterised by, prepares the xylan
The method of the mutant of the E175 sites saturation mutation of enzyme comprises the following steps：
(1) gene for encoding zytase described in claim 2 is inserted into expression plasmid pPIC9, obtains recombinant plasmid
XYL10C-pPIC9；
(2) primer is designed according to purpose fragment, while the digestion position in 5 ' end addition expression plasmid pPIC9 junctions of primer pair
Point, using the recombinant plasmid in step (1) as the encoding gene of the zytase of template amplification E175 sites saturation mutation；
(3) the pPIC9 plasmids by encoding gene digestion obtained above and incision are attached, and obtain mutant recombinant plasmid；
(4) by mutant recombinant plasmid transformed Pichia pastoris GS115, induced expression, the wood for obtaining E175 sites saturation mutation gathers
Carbohydrase mutant.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710176527.3A CN107142253B (en) | 2017-03-23 | 2017-03-23 | Xylanase mutant and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710176527.3A CN107142253B (en) | 2017-03-23 | 2017-03-23 | Xylanase mutant and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107142253A true CN107142253A (en) | 2017-09-08 |
| CN107142253B CN107142253B (en) | 2020-01-21 |
Family
ID=59783990
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710176527.3A Active CN107142253B (en) | 2017-03-23 | 2017-03-23 | Xylanase mutant and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107142253B (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109355272A (en) * | 2018-12-28 | 2019-02-19 | 江南大学 | A kind of xylanase mutant that catalytic efficiency improves |
| CN110564710A (en) * | 2019-07-22 | 2019-12-13 | 江苏科技大学 | Xylanase mutant with high catalytic efficiency and construction method and application thereof |
| CN110656099A (en) * | 2019-10-14 | 2020-01-07 | 江苏科技大学 | Xylanase mutant with high specific activity at 40 ℃ and construction method and application thereof |
| CN112094832A (en) * | 2020-09-04 | 2020-12-18 | 山东大学 | Mutant xylanase for heat-resistant alkali-resistant papermaking and application thereof |
| CN112708608A (en) * | 2021-02-07 | 2021-04-27 | 江苏科技大学 | Xylanase mutant and preparation method and application thereof |
| CN114854724A (en) * | 2022-05-26 | 2022-08-05 | 江苏科技大学 | N-glycosylation mutants of GH10 family xylanase and application thereof |
| WO2023004901A1 (en) * | 2021-07-28 | 2023-02-02 | 江苏科技大学 | A group of gh10 family high-temperature-resistant xylanase mutants and use thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101457206A (en) * | 2008-05-28 | 2009-06-17 | 中国农业科学院饲料研究所 | Acidic xylanase XYL10A and gene and application thereof |
| CN105039289A (en) * | 2015-09-11 | 2015-11-11 | 青岛蔚蓝生物集团有限公司 | Xylanase mutant and application thereof |
| CN105087525A (en) * | 2015-09-11 | 2015-11-25 | 青岛蔚蓝生物集团有限公司 | Xylanase mutants |
| CN106282141A (en) * | 2016-09-26 | 2017-01-04 | 云南师范大学 | A kind of xylanase mutant and application thereof |
-
2017
- 2017-03-23 CN CN201710176527.3A patent/CN107142253B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101457206A (en) * | 2008-05-28 | 2009-06-17 | 中国农业科学院饲料研究所 | Acidic xylanase XYL10A and gene and application thereof |
| CN105039289A (en) * | 2015-09-11 | 2015-11-11 | 青岛蔚蓝生物集团有限公司 | Xylanase mutant and application thereof |
| CN105087525A (en) * | 2015-09-11 | 2015-11-25 | 青岛蔚蓝生物集团有限公司 | Xylanase mutants |
| CN106282141A (en) * | 2016-09-26 | 2017-01-04 | 云南师范大学 | A kind of xylanase mutant and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| HUIYING LUO等: ""A thermophilic and acid stable family-10 xylanase from the acidophilic fungus Bispora sp.MEY-1"", 《EXTREMOPHILES》 * |
| LUO,H.等: ""family10 endobeta1,4xylanase [Bispora sp. MEY1]"", 《GENBANK DATABASE》 * |
| XIAOYU WANG等: ""Improvement of the catalytic performance of a hyperthermostable GH10 xylanase from Talaromyces leycettanus JCM12802"", 《BIORESOURCE TECHNOLOGY》 * |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109355272A (en) * | 2018-12-28 | 2019-02-19 | 江南大学 | A kind of xylanase mutant that catalytic efficiency improves |
| CN110564710A (en) * | 2019-07-22 | 2019-12-13 | 江苏科技大学 | Xylanase mutant with high catalytic efficiency and construction method and application thereof |
| CN110564710B (en) * | 2019-07-22 | 2022-02-08 | 江苏科技大学 | Xylanase mutant with high catalytic efficiency and construction method and application thereof |
| CN110656099A (en) * | 2019-10-14 | 2020-01-07 | 江苏科技大学 | Xylanase mutant with high specific activity at 40 ℃ and construction method and application thereof |
| CN110656099B (en) * | 2019-10-14 | 2021-08-27 | 江苏科技大学 | Xylanase mutant with high specific activity at 40 ℃ and construction method and application thereof |
| CN112094832A (en) * | 2020-09-04 | 2020-12-18 | 山东大学 | Mutant xylanase for heat-resistant alkali-resistant papermaking and application thereof |
| CN112094832B (en) * | 2020-09-04 | 2022-01-18 | 山东大学 | Mutant xylanase for heat-resistant alkali-resistant papermaking and application thereof |
| CN112708608A (en) * | 2021-02-07 | 2021-04-27 | 江苏科技大学 | Xylanase mutant and preparation method and application thereof |
| CN112708608B (en) * | 2021-02-07 | 2021-08-10 | 江苏科技大学 | Xylanase mutant and preparation method and application thereof |
| WO2023004901A1 (en) * | 2021-07-28 | 2023-02-02 | 江苏科技大学 | A group of gh10 family high-temperature-resistant xylanase mutants and use thereof |
| CN114854724A (en) * | 2022-05-26 | 2022-08-05 | 江苏科技大学 | N-glycosylation mutants of GH10 family xylanase and application thereof |
| CN114854724B (en) * | 2022-05-26 | 2023-11-21 | 江苏科技大学 | N-glycosylation mutant of group of GH10 family xylanases and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107142253B (en) | 2020-01-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107142253A (en) | A kind of high catalytic efficiency and fire resistant xylanase mutant and its preparation method and application | |
| Singh et al. | Inulinase production from a new inulinase producer, Penicillium oxalicum BGPUP-4 | |
| Porciuncula et al. | Identification of major facilitator transporters involved in cellulase production during lactose culture of Trichoderma reesei PC-3-7 | |
| CN104130951A (en) | Recombinant pichia pastoris engineering bacteria and metabolic recombinant xylanase as well as preparation of metabolic recombinant xylanase | |
| CN105018448B (en) | The heat-resisting acidic cellulase and its gene of a kind of originated from fungus and application | |
| CN109652392A (en) | A kind of feruloyl esterase and its preparation method and application | |
| CN102732494B (en) | Beta-mannanase and preparation method thereof | |
| CN112708608B (en) | Xylanase mutant and preparation method and application thereof | |
| CN107603937A (en) | A kind of recombination bacillus coli and its construction method for expressing lysine aminopeptidase | |
| CN104962594B (en) | A method of improving Glucoamylase of Aspergillus niger inversion rate of glucose | |
| WO2020073866A1 (en) | Glucoamylase tlga15 and gene and application thereof | |
| CN104046586B (en) | One strain gene engineering bacterium and the application in producing (2R, 3R)-2,3-butanediol thereof | |
| CN105886484A (en) | Thermophilic cellulase, encoding gene thereof and application of thermophilic cellulase | |
| CN106916752B (en) | Method for preparing cellulase and/or xylanase and special strain thereof | |
| CN110564710A (en) | Xylanase mutant with high catalytic efficiency and construction method and application thereof | |
| Wang et al. | Homologous overexpression of genes in Cordyceps militaris improves the production of polysaccharides | |
| Chi et al. | A novel alkaliphilic xylanase from the newly isolated mesophilic Bacillus sp. MX47: production, purification, and characterization | |
| de Oliveira Porciuncula et al. | Identification of major facilitator transporters involved in cellulase production during lactose culture of Trichoderma reesei PC-3-7 | |
| CN108018245A (en) | One plant of bacillus subtilis for producing chitosan enzyme and its application | |
| CN111117986B (en) | Encoding gene of calcium-dependent heat-resistant alpha-L-arabinofuranosidase, preparation technology and application | |
| Zhao et al. | Influence of cellulase gene expression and cellulolytic activity on cellulose utilization in different Volvariella volvacea strains | |
| CN105219664B (en) | A kind of building of recombination engineering bacteria and the preparation and application of high activity β-D-1,4- endo-xylanase | |
| CN104560917B (en) | A kind of beta-glucosidase and β-glucosidase mutant and application | |
| CN105154417B (en) | The acidic cellulase and its gene of a kind of originated from fungus and application | |
| CN104726388B (en) | A kind of Pullulanase bacterium producing multi enzyme preparation and the method for improving its enzymatic productivity |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20200820 Address after: 100193 Beijing Old Summer Palace West Road, Haidian District, No. 2 Patentee after: Beijing Institute of Animal Science and Veterinary Medicine, Chinese Academy of Agricultural Sciences Address before: 100081 Beijing, Zhongguancun, South Street, No. 12, No. Patentee before: FEED Research Institute CHINESE ACADEMY OF AGRICULTURAL SCIENCES |
|
| TR01 | Transfer of patent right |