CN105505806B - A kind of construction method of zytase heterozyme engineered strain - Google Patents
A kind of construction method of zytase heterozyme engineered strain Download PDFInfo
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- CN105505806B CN105505806B CN201610046663.6A CN201610046663A CN105505806B CN 105505806 B CN105505806 B CN 105505806B CN 201610046663 A CN201610046663 A CN 201610046663A CN 105505806 B CN105505806 B CN 105505806B
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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)
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- 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)
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Abstract
The invention discloses a kind of construction methods of zytase heterozyme engineered strain, the homology of zytase XynZF-2 gene order and heat resistant xylanase EvXyn11 gene order is compared first with bioinformatics, it is replaced 48, the end the N- amino acid substitution of XynZF-2 by the end N- into 34 amino acid at the end zytase EvXyn11N-, and introduce aromatic amino acid P9Y and H14F, and then continue to introduce disulfide bond Cys38-Cys191 in XynZF-2 enzyme C-terminal, alpha-helix and the region cord introduce hydrophobic amino acid K164M respectively, G166A, N160I and V111A, G109A, it is finally introducing glycosylation modified site E42N and F17S, it is miscellaneous to construct multisite mutation Synthase XynZL, it is expected that the thermal stability of heterozyme XynZL makes moderate progress.
Description
Technical field
The invention belongs to field of biotechnology, are related to a kind of construction method of zytase heterozyme engineered strain.
Background technique
Zytase (EC 3.2.1.8) is the general name of a kind of multifunctional enzyme of catalyzing hydrolysis xylan.Zytase is extensive
It is distributed in nature, such as animal, plant and microorganism, and many kinds of, application field is extensive.Currently, microbe-derived
Very much, the most studied zytase for being derived from bacterium and fungi of zytase research report.Wherein, bacterium can be with
Alkalinity and acidic xylanase are generated, and fungi can produce alkalescent xylanase.Currently, zytase is mainly by fungi and thin
The production of the microbial fermentations such as bacterium.
With the continuous development of biotechnology, the research and development of zytase are concerned.In terms of industrial application, wood is poly-
Carbohydrase has very big commercial application potentiality and value, it is a kind of important industrial enzyme preparation, be widely used in food processing,
The fields such as brewing, paper pulp bleaching, pharmacy, feed addictive, medicine, weaving, bioconversion.Zytase is right in use
Extraneous physicochemical condition is extremely sensitive, under the conditions ofs high temperature, heavy metal ion, oxidant and extreme pH in industrial process etc., all
Zytase conformation can be made to be destroyed, to limit the application environment of enzyme.Therefore, zytase stability is improved to have become
The focus of researcher's extensive concern.Currently, by bioinformatic analysis compare zytase crystal structure discovery, hydrogen bond, from
Region etc. and the stability of zytase are closely related at sub-key number, salt bridge, N-terminal and alpha-helix.Many people as according to
According to by the stability of the means research zytase such as genetic engineering and enzyme engineering, and achieving good achievement.
Have now been found that the factors for influencing thermal stability of xylanase, but both at home and abroad for xylanase molecule heat
The report in stability mutational site is relatively fewer.In general all disulfide bond all rise in terms of protein folding and stability
Crucial effect, and it is closely related with the thermal stability of protein.Influence state of the introducing of N-terminal disulfide bond to enzyme heat stability
It is inside and outside it has been reported that but C-terminal introduce disulfide bond on thermal stability of xylanase, whether there is or not influences, have not been reported.
As proteomic techniques continue to develop, it has been found that protein glycosylation has one to the stabilization of protein conformation
Fixed effect.Glycosylation is under the control of enzyme, and the process of protein or lipid affix carbohydrate betides endoplasmic reticulum.In sugar
Sugar is transferred to the amino acid residue on protein and protein under based transferase effect and forms glycosidic bond.Currently, for albumen
The glycosylation modified research of matter is mainly N- glycosylation and O- glycosylation.The study found that N- glycosylation mostly occurs in Asn-Xaa-
On the Asn residue of Ser/Thr sequence, O- glycosylates the β-corner area that mostly occurs.In addition, it is poly- to improve wood by glycosylation modified
The research of carbohydrase stability is relatively fewer, therefore, there is certain exploration space.
Summary of the invention
In order to overcome defect existing in the prior art, the present invention provides a kind of structure of zytase heterozyme engineered strain
Construction method compares zytase XynZF-2 gene order (GenBank Accession first with bioinformatics
No.JQ700382) homologous with heat resistant xylanase EvXyn11 gene order (GenBank AccessionNo.EU591347)
Property, it is replaced 48, the end the N- amino acid substitution of XynZF-2 by the end N- into 34 amino at the end zytase EvXyn11N-
Acid, and introduce aromatic amino acid P9Y and H14F, so XynZF-2 enzyme C-terminal continue to introduce disulfide bond Cys38-Cys191,
Alpha-helix and the region cord introduce hydrophobic amino acid K164M, G166A, N160I and V111A, G109A respectively, are finally introducing
Glycosylation modified site E42N and F17S constructs multisite mutation heterozyme XynZL, it is expected that the thermal stability of heterozyme XynZL
It makes moderate progress.
Its technical solution is as follows:
A kind of construction method of zytase heterozyme engineered strain, comprising the following steps:
Step 1, zytase structural analysis
BLAST and DNAMAN6.0 homologous sequence compares and the higher heat resistant xylanase of zytase XynZF-2 homology
Gene, PROSITE (http://prosite.expasy.org) predict zytase functional site, utilize Phyre2
(http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi? id=index) zytase is modeled, and
The three-dimensional structure of zytase is analyzed by DS ViewerPro6.0.Using DiANNA 1.1 (http: //
Bioinformatics.bc.edu/clotelab/DiANNA/ disulfide bond site) is analyzed, NetNGlyc 1.0 is utilized
(http://www.cbs.dtu.dk/services/NetNGlyc/) carries out zytase XynZF-2 glycosylation site pre-
It surveys；
Step 2, the synthesis of target gene and the building of recombinant expression carrier
Designed heterozygosis enzyme gene xynZL sequence is synthesized by Suzhou Jin Weizhi Biotechnology Co., Ltd；
With reference to heterozygosis enzyme gene xynZL gene order and the multiple cloning sites of carrier for expression of eukaryon pPIC9K, design with
Lower two primers: YS:5 '-CCGGAATTCTTCCCCACGACTCTGTCG-3 ' (EcoR I restriction enzyme site)
YX:5 '-ATAAGAATGCGGCCGCTTACTGAACACAGATGGACG-3 ' (Not I), primer is by Suzhou Jin Weizhi
Biotechnology Co., Ltd's synthesis；
Using xynZL gene as template, YS, YX are that primer carries out PCR amplification, after amplified production recycling, with corresponding expression load
Body connects liquid Transformed E .coli DH5 α competent cell through double digestion, recycling, connection, and conversion fluid is respectively coated the LB containing Amp
Plate after positive clone molecule screening, extracts plasmid and verifies through digestion and PCR, final to obtain recombinant plasmid pPIC9K-xynZL.
Preferably, the amplification condition of the carry out PCR amplification in step 2 is equal are as follows: 94 DEG C of initial denaturation 2min；94 DEG C of denaturation 30s,
67 DEG C of annealing 30s, 72 DEG C of extension 1min, 30 recycle；72 DEG C of extension 10min.
Compared with prior art, beneficial effects of the present invention:
The present invention is to improve the medium temperature zytase XynZF-2 for deriving from aspergillus niger (Aspergillus niger) XZ-3S
Thermal stability is replaced 48, the end the N- amino acid substitution of XynZF-2 by the end N- into 34 of the end zytase EvXyn11N-
Amino acid, and introduce aromatic amino acid P9Y and H14F.On the basis of this mutated gene, C-terminal introduces disulfide bond Cys38-
Cys191, alpha-helix and the region cord introduce hydrophobic amino acid K164M, G166A, N160I and V111A, G109A respectively,
Then glycosylation modified site E42N and F17S is introduced, constructs multisite mutation gene xynZL, and connect with pPIC9K expression vector
It connects, converts Pichia pastoris, construct heterozygosis enzyme engineering bacteria GS115/pPIC9K-xynZL, recombination heterozyme characterization analysis is aobvious
Show, heterozyme optimum temperature has promotion by a relatively large margin compared with protoenzyme.
Detailed description of the invention
Fig. 1 is the three-dimensional structure of heterozygosis zytase XynZL, wherein Fig. 1 a is the front elevation of heterozyme three-dimensional structure, figure
1b is the reverse side figure of heterozyme three-dimensional structure；
Fig. 2 is recombinase optimum temperature schematic diagram；
Fig. 3 is recombinase temperature stability schematic diagram.
Specific embodiment
Technical solution of the present invention is further illustrated with reference to the accompanying drawings and examples.
1 materials and methods
1.1 bacterial strains, plasmid and reagent
Aspergillus niger (Aspergillus niger) XZ-3S, Escherichia coli (Escherichia coli) JM109, DH5 α by
Laboratory saves where author；Eukaryon expression plasmid pPIC9K and Pichia pastoris (Pichia pastoris) GS115 are purchased from
Invitrogen company.Archaeal dna polymerase, restriction enzyme, ligase are purchased from TaKaRa company；Ampicillin (Amp) purchase
From Sangon company；Plastic recovery kit, plasmid extraction kit are purchased from Sangon company；Ethidium bromide (Ethidium
Bromide) it is purchased from Amresco company；Yeast powder, peptone, agarose are purchased from BBI company；G418, without amino yeast nitrogen
(YNB) it is purchased from Amresco company；Birch xylan is purchased from Sigma company；Other biochemical reagents are domestic or Import Analysis is pure
Product.
1.2 zytase structural analyses
BLAST and DNAMAN6.0 homologous sequence compares and the higher heat resistant xylanase of zytase XynZF-2 homology
Gene, PROSITE (http://prosite.expasy.org) predict zytase functional site, utilize Phyre2
(Http:// www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi? id=index) zytase is modeled, and Pass through DS ViewerPro6.0Analyze the three-dimensional structure of zytase.Using DiANNA 1.1 (Http: // bioinformatics.bc.edu/clotelab/DiANNA/) analysis disulfide bond site, utilize NetNGlyc 1.0
(Http:// www.cbs.dtu.dk/services/NetNGlyc/) zytase XynZF-2 glycosylation site is carried out in advance It surveys。
The synthesis of 1.3 target gene and the building of recombinant expression carrier
Designed heterozygosis enzyme gene xynZL sequence is synthesized by Suzhou Jin Weizhi Biotechnology Co., Ltd.
With reference to heterozygosis enzyme gene xynZL gene order and the multiple cloning sites of carrier for expression of eukaryon pPIC9K, design with
Lower two primers: YS:5 '-CCGGAATTCTTCCCCACGACTCTGTCG-3 ' (EcoR I restriction enzyme site)
YX:5 '-ATAAGAATGCGGCCGCTTACTGAACACAGATGGACG-3 ' (Not I), primer is by Suzhou Jin Weizhi
Biotechnology Co., Ltd's synthesis.
Using xynZL gene as template, YS, YX are that primer carries out PCR amplification, and amplification condition is equal are as follows: 94 DEG C of initial denaturation 2min；
94 DEG C of denaturation 30s, 67 DEG C of annealing 30s, 72 DEG C of extension 1min, 30 recycle；72 DEG C of extension 10min.After amplified production recycling, with
Corresponding expression carrier is through double digestion, recycling, connection.Liquid Transformed E .coli DH5 α competent cell is connected, conversion fluid is respectively coated
LB plate containing Amp after positive clone molecule screening, extracts plasmid and verifies through digestion and PCR, finally obtain recombinant plasmid
pPIC9K-xynZL。
Expression of the enzyme of 1.4 mesh in Pichia pastoris
For recombinant plasmid pPIC9K-xynZL after Sal I linearization for enzyme restriction, electrophoresis recycles target fragment, reference
Invitrogen company operation manual, electroporated Pichia pastoris GS115.Conversion fluid is coated with MD plate, positive gram filtered out
YPD plate primary dcreening operation of the Longzi through different G418 concentration, shaking flask secondary screening finally obtain one plant of higher bacterium of recombined xylanase yield
Strain, is denoted as GS115/XynZL.GS115/XynZL bacterium colony is picked them separately, is inoculated in the 250mL's containing 30mL BMGY culture medium
In triangular flask, 30 DEG C, 250r/min cultivate to A600 be 6.0 or so, triangular flask stand 30min, after thallus sedimentation after, incline on
Clearly, precipitating thallus is transferred into 20mLBMMY culture medium, continues to cultivate inducing expression 120h, during which every to add 100% methanol for 24 hours extremely
Final concentration of 2%.
The purifying of 1.5 Pichia pastoris recombinases
By recombination yeast culture solution, 3000r/min is centrifuged 10min, and fermented supernatant fluid polyethylene glycol is taken to be concentrated, dialysis, after
It is purified through Sephadex G-75, sample detection is with SDS-PAGE electrophoresis (5% concentration glue and 12% separation gel) after purification.
The measurement of 1.6 recombination enzyme activities and protein concentration
Xylanase activity measurement, using DNS method.Enzyme activity unit (U) definition: using 0.5% birch xylan as substrate,
Under the conditions of 45 DEG C, pH5.0, with enzyme amount needed for generating 1 μm of ol reduced sugar per minute for 1U.
Determination of protein concentration uses Bradford method, and standard protein is bovine serum albumin(BSA).
The present invention is recombinated enzyme activity and is indicated using specific enzyme activity power, and Rate activity (U/mg) is the ratio of enzyme activity and protein concentration
Value.The measurement of the optimum temperature and thermal stability of 1.7 enzymes
In pH 5.0, within the scope of 35-50 DEG C, every 5 DEG C, enzyme activity is measured respectively, with enzyme under conditions of reacting 15min
Living up to 100% calculates opposite enzyme activity, thereby determines that the optimum temperature of enzyme.By enzyme solution in 40 DEG C, 45 DEG C, 50 DEG C, 55
DEG C and 60 DEG C at isothermal holding different time, measure residual enzymic activities according to a conventional method, it is living with the enzyme sample for not keeping the temperature (4 DEG C preservation)
Property is 100%, thereby determines that the stability of enzyme.
2. result and analysis
The synthesis and structural analysis of 2.1 target gene
The heterozyme gene order of synthesis is designed as shown in SEQ:ID:1, is encoded shown in 193 amino acid (SEQ:ID:2),
Predicted molecular weight is 20842Da.The homologous modeling discovery of heterozygosis zytase XynZL, mutant enzyme is similar to protoenzyme structure, belongs to
GH11 family is made of a simple alpha-helix and two reversed beta sheet lamellas, shape structure is partly held in the right hand.Prediction
Activated centre is Glu89, Glu180, and the amino acid sites that furthermore heterozyme XynZL compares protoenzyme XynZF-2 gene alteration exist
Red mark has been done in SEQ:ID:2, and emphasis amino acid sites are indicated in Fig. 1.
Expression of the enzyme of 2.2 mesh in Pichia pastoris
Enzymatic activity is detected in heterozygosis enzyme engineering bacteria GS115/XynZL fermented supernatant fluid, and recombinase after purification, reaches
Electrophoresis is pure, can carry out the measurement of zymologic property.
The measurement of the optimum temperature and thermal stability of 2.3 enzymes
Heterozyme XynZL is compared after protoenzyme XynZF-2 expresses in Pichia pastoris, and recombinase optimum temperature is from 45 DEG C
It is increased to 55 DEG C (Fig. 2).Temperature stability improves there has also been apparent, as shown in figure 3,50 DEG C of heat preservations 20min, XynZF-2 are residual
Remaining enzyme activity is 18%, and XynZL remnant enzyme activity is 75%；55 DEG C of heat preservation 15min, XynZF-2 remnant enzyme activity is 0, and XynZL is residual
Remaining enzyme activity is 40%.
The above, only best mode for carrying out the invention, anyone skilled in the art is in the present invention
In the technical scope of disclosure, the simple change or equivalence replacement for the technical solution that can be become apparent to each fall within of the invention
In protection scope.
Claims (2)
1. a kind of construction method of zytase heterozyme recombinant plasmid, which comprises the following steps:
The synthesis of target gene and the building of recombinant expression carrier
The good heterozygosis enzyme gene xynZL sequence of compounding design；The heterozygosis enzyme gene xynZL sequence as shown in SEQ ID NO.1,
193 amino acid are encoded as shown in SEQ ID NO.2；
With reference to heterozygosis enzyme gene xynZL gene order and the multiple cloning sites of carrier for expression of eukaryon pPIC9K, design synthesis with
Lower two primers: YS:5 '-CCGGAATTCTTCCCCACGACTCTGTCG-3 '
YX:5 '-ATAAGAATGCGGCCGCTTACTGAACACAGATGGACG-3 '；
Using xynZL gene as template, YS, YX are that primer carries out PCR amplification, after amplified production recycling, with corresponding expression carrier warp
Double digestion, recycling, connection connect liquid Transformed E .coli DH5 α competent cell, and conversion fluid is respectively coated the LB plate containing Amp,
After positive clone molecule screening, extracts plasmid and verified through digestion and PCR, it is final to obtain recombinant plasmid pPIC9K-xynZL.
2. the construction method of zytase heterozyme recombinant plasmid according to claim 1, which is characterized in that the progress
The amplification condition of PCR amplification is equal are as follows: 94 DEG C of initial denaturation 2min；94 DEG C of denaturation 30s, 67 DEG C of annealing 30s, 72 DEG C of extension 1min, 30
A circulation；72 DEG C of extension 10min.
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| CN101624574A (en) * | 2009-08-06 | 2010-01-13 | 南京林业大学 | Yeast engineering bacteria for expressing high-stability xylanase, construction method and application thereof |
| CN102876646A (en) * | 2012-10-16 | 2013-01-16 | 新乡医学院 | Method for producing xylanase by fermentation of koji tray of Aspergillus niger and culture medium used by method |
| CN103642776A (en) * | 2013-12-10 | 2014-03-19 | 江南大学 | Directed evolution method for improving heat resistance of aspergillus oryzae GH11 xylanase |
| CN104313000A (en) * | 2014-10-11 | 2015-01-28 | 上海交通大学 | Genetic engineering xylanase and preparation and application of genetic engineering xylanase |
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| CN101624574A (en) * | 2009-08-06 | 2010-01-13 | 南京林业大学 | Yeast engineering bacteria for expressing high-stability xylanase, construction method and application thereof |
| CN102876646A (en) * | 2012-10-16 | 2013-01-16 | 新乡医学院 | Method for producing xylanase by fermentation of koji tray of Aspergillus niger and culture medium used by method |
| CN103642776A (en) * | 2013-12-10 | 2014-03-19 | 江南大学 | Directed evolution method for improving heat resistance of aspergillus oryzae GH11 xylanase |
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