CN105505806A - Construction method of xylanase hybrid enzyme engineering strain - Google Patents
Construction method of xylanase hybrid enzyme engineering strain Download PDFInfo
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- CN105505806A CN105505806A CN201610046663.6A CN201610046663A CN105505806A CN 105505806 A CN105505806 A CN 105505806A CN 201610046663 A CN201610046663 A CN 201610046663A CN 105505806 A CN105505806 A CN 105505806A
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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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- 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)
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Abstract
The invention discloses a construction method of a xylanase hybrid enzyme engineering strain. The method comprises the steps that homologies of a xylanase XynZF-2 gene sequence and a heat-resistant xylanase EvXyn11 gene sequence are compared through bioinformatics, 48 amino acids of a XynZF-2 N-terminal is replaced with 34 amino acids of a xylanase EvXyn11 N-terminal through N-terminal replacement, aromatic amino acids P9Y and H14F are introduced, disulfide bonds Cys38-Cys191 are continuously introduced into the XynZF-2 enzyme C-terminal, hydrophobic amino acids K164M, G166A, N160I, V111A and G109A are introduced into alpha-spiral and cord areas, glycosylation modification sites E42N and F17S are introduced finally, multiple locus mutational hybrid enzyme XynZL is constructed, and the heat stability of the hybrid enzyme XynZL is expected to be improved.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of construction process of zytase heterozyme engineering strain.
Background technology
Zytase (EC3.2.1.8) is the general name of a class multifunctional enzyme of catalytic hydrolysis xylan.Zytase is distributed widely in occurring in nature, as animal, plant and microorganism etc., and of a great variety, Application Areas is extensive.At present, microbe-derived zytase research report is a lot, and what study at most is the zytase deriving from bacterium and fungi.Wherein, bacterium can produce alkalescence and acidic xylanase, and fungi can produce alkalescent xylanase.At present, zytase is produced primarily of fermentable such as fungus and bacteriums.
Along with the development of biotechnology, the research and development of zytase receive much concern.In industrial application, zytase has very large industrial application potentiality and value, and it is a kind of important industrial enzyme preparation, be widely used in food-processing, brewage, association with pulp bleaching, pharmacy, fodder additives, medicine, weaving, the field such as bio-transformation.In use physicochemical condition is very responsive to external world for zytase, under the conditions such as the high temperature in industrial process, heavy metal ion, oxygenant and extreme pH, zytase conformation all can be made to be destroyed, thus limit the applied environment of enzyme.Therefore, the focus that zytase stability has become investigator's extensive concern is improved.At present, found by bioinformatic analysis comparison zytase crystalline structure, hydrogen bond, ionic linkage number, salt bridge, N end and region, alpha-helix place etc. are closely related with the stability of zytase.A lot of people on this basis, by the stability of the means such as genetically engineered and enzyme engineering research zytase, and achieves good achievement.
Have been found that the factors affecting thermal stability of xylanase at present, but relatively less for the report in xylanase molecule Heat Stability Mutations site both at home and abroad.In general all disulfide linkage all play a part key in protein folding and stability, and closely related with the thermostability of protein.The impact of introducing on enzyme heat stability of N-terminal disulfide bond has been reported both at home and abroad, but C end introduce disulfide linkage on thermal stability of xylanase with or without impact, have no report.
Along with proteomic techniques development, it is found that Protein Glycosylation Overview has certain effect to the stable of protein conformation.Glycosylation is under the control of enzyme, and the process of protein or lipid affix carbohydrate, betides endoplasmic reticulum.Under glycosyltransferase effect, sugar is transferred to protein, and the amino-acid residue on protein forms glycosidic link.At present, mainly N-glycosylation and O-glycosylation are studied for modification of protein glycosylation.Research finds, N-glycosylation mostly occurs on the Asn residue of Asn-Xaa-Ser/Thr sequence, and O-glycosylation mostly occurs β-corner area.In addition, relatively less by the glycosylation modified research improving zytase stability, therefore, there is certain exploration space.
Summary of the invention
In order to overcome the defect existed in prior art, the invention provides a kind of construction process of zytase heterozyme engineering strain, first the homology of information biology comparison zytase XynZF-2 gene order (GenBankAccessionNo.JQ700382) and heat resistant xylanase EvXyn11 gene order (GenBankAccessionNo.EU591347) is utilized, being replaced by N-end holds 48 amino acid to replace to 34 amino acid of zytase EvXyn11N-end the N-of XynZF-2, and introduce die aromatischen Aminosaeuren P9Y and H14F, and then hold continuation to introduce disulfide linkage Cys38-Cys191 at XynZF-2 enzyme C, hydrophobic amino acid K164M is introduced in alpha-helix and cord region respectively, G166A, N160I and V111A, G109A, finally introduce glycosylation modified site E42N and F17S, build multisite mutation heterozyme XynZL, expect that the thermostability of heterozyme XynZL makes moderate progress.
Its technical scheme is as follows:
A construction process for zytase heterozyme engineering strain, comprises the following steps:
Step 1, zytase structural analysis
The comparison of BLAST and DNAMAN6.0 homologous sequence and the higher heat resistant xylanase gene of zytase XynZF-2 homology, PROSITE (http://prosite.expasy.org) predicts zytase functional site, do you utilize Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi? id=index) to zytase modeling, and the three-dimensional structure of zytase is analyzed by DSViewerPro6.0.Adopt DiANNA1.1 (http://bioinformatics.bc.edu/clotelab/DiANNA/) to analyze disulfide linkage site, utilize NetNGlyc1.0 (http://www.cbs.dtu.dk/services/NetNGlyc/) to predict zytase XynZF-2 glycosylation site;
The synthesis of step 2, goal gene and the structure of recombinant expression vector
The heterozyme gene xynZL sequence designed is synthesized by Jin Weizhi bio tech ltd, Suzhou;
The multiple clone site of reference heterozyme gene xynZL gene order and carrier for expression of eukaryon pPIC9K, following two primers of design: YS:5 '-CCG
gAATTCtTCCCCACGACTCTGTCG-3 ' (EcoRI restriction enzyme site)
YX:5 '-ATAAGAAT
gCGGCCGCtTACTGAACACAGATGGACG-3 ' (NotI), primer is synthesized by Jin Weizhi bio tech ltd, Suzhou;
With xynZL gene for template, YS, YX are that primer carries out pcr amplification, after amplified production reclaims, with corresponding expression carrier through double digestion, recovery, be connected, connecting fluid Transformed E .coliDH5 α competent cell, the LB that conversion fluid is coated with respectively containing Amp is dull and stereotyped, after the screening of positive colony, extract plasmid to cut and PCR checking through enzyme, final acquisition recombinant plasmid pPIC9K-xynZL.
Preferably, the amplification condition carrying out pcr amplification in step 2 is: 94 DEG C of denaturation 2min; 94 DEG C of sex change 30s, 67 DEG C of annealing 30s, 72 DEG C extend 1min, 30 circulations; 72 DEG C extend 10min.
Compared with prior art, beneficial effect of the present invention:
The present invention is the middle temperature zytase XynZF-2 thermostability that raising derives from aspergillus niger (Aspergillusniger) XZ-3S, being replaced by N-end holds 48 amino acid to replace to 34 amino acid of zytase EvXyn11N-end the N-of XynZF-2, and introduces die aromatischen Aminosaeuren P9Y and H14F.On this mutator gene basis, C end is introduced disulfide linkage Cys38-Cys191, alpha-helix and cord region and is introduced hydrophobic amino acid K164M, G166A, N160I and V111A, G109A respectively, then glycosylation modified site E42N and F17S is introduced, build multisite mutation gene xynZL, and be connected with pPIC9K expression vector, transform pichia spp, construct heterozyme engineering bacteria GS115/pPIC9K-xynZL, the display of restructuring heterozyme characterization analysis, heterozyme optimum temperuture has had lifting by a relatively large margin compared with protoenzyme.
Accompanying drawing explanation
Fig. 1 is the three-dimensional structure of heterozygosis zytase XynZL, and wherein, Fig. 1 a is the front elevation of heterozyme three-dimensional structure, and Fig. 1 b 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.
Embodiment
Technical scheme of the present invention is further illustrated below in conjunction with drawings and Examples.
1 materials and methods
1.1 bacterial strains, plasmid and reagent
Aspergillus niger (Aspergillusniger) XZ-3S, intestinal bacteria (Escherichiacoli) JM109, DH5 α is preserved by laboratory, author place; Eukaryon expression plasmid pPIC9K and pichia spp (Pichiapastoris) GS115 is purchased from Invitrogen company.Archaeal dna polymerase, restriction enzyme, ligase enzyme are purchased from TaKaRa company; Penbritin (Amp) is purchased from Sangon company; Glue reclaims test kit, plasmid extraction kit purchased from Sangon company; Ethidium bromide (Ethidiumbromide) is purchased from Amresco company; Yeast powder, peptone, agarose are all purchased from BBI company; G418, without amino yeast nitrogen (YNB) purchased from Amresco company; Birch xylan available from Sigma; Other biochemical reagents are domestic or Import Analysis straight product.
1.2 zytase structural analyses
The comparison of BLAST and DNAMAN6.0 homologous sequence and the higher heat resistant xylanase gene of zytase XynZF-2 homology, PROSITE (http://prosite.expasy.org) predicts zytase functional site, utilize Phyre2 (
http:// www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi? id=index) to zytase modeling, and DS is passed through viewerPro6.0analyze the three-dimensional structure of zytase.Employing DiANNA1.1 (
http:// bioinformatics.bc.edu/clotelab/DiANNA/) analyze disulfide linkage site, utilize NetNGlyc1.0 (
http:// www.cbs.dtu.dk/services/NetNGlyc/) zytase XynZF-2 glycosylation site is predicted.
The synthesis of 1.3 goal gene and the structure of recombinant expression vector
The heterozyme gene xynZL sequence designed is synthesized by Jin Weizhi bio tech ltd, Suzhou.
The multiple clone site of reference heterozyme gene xynZL gene order and carrier for expression of eukaryon pPIC9K, following two primers of design: YS:5 '-CCG
gAATTCtTCCCCACGACTCTGTCG-3 ' (EcoRI restriction enzyme site)
YX:5 '-ATAAGAAT
gCGGCCGCtTACTGAACACAGATGGACG-3 ' (NotI), primer is synthesized by Jin Weizhi bio tech ltd, Suzhou.
With xynZL gene for template, YS, YX are that primer carries out pcr amplification, and amplification condition is: 94 DEG C of denaturation 2min; 94 DEG C of sex change 30s, 67 DEG C of annealing 30s, 72 DEG C extend 1min, 30 circulations; 72 DEG C extend 10min.Amplified production reclaim after, with corresponding expression carrier through double digestion, recovery, be connected.Connecting fluid Transformed E .coliDH5 α competent cell, the LB that conversion fluid is coated with respectively containing Amp is dull and stereotyped, after the sub-screening of positive colony, extracts plasmid and cuts and PCR checking through enzyme, final acquisition recombinant plasmid pPIC9K-xynZL.
1.4 expression of object enzyme in pichia spp
Recombinant plasmid pPIC9K-xynZL is after SalI linearization for enzyme restriction, and electrophoresis reclaims object fragment, with reference to Invitrogen company operational manual, and electroporated Pichia pastoris GS115.MD is dull and stereotyped in conversion fluid coating, and positive colony filtered out is through the dull and stereotyped primary dcreening operation of YPD of different G418 concentration, and shaking flask is sieved again, finally obtains the bacterial strain that a strain recombined xylanase output is higher, is denoted as GS115/XynZL.Picking GS115/XynZL bacterium colony respectively, be inoculated in the triangular flask of the 250mL containing 30mLBMGY substratum, 30 DEG C, it is about 6.0 that 250r/min is cultured to A600, and triangular flask leaves standstill 30min, after thalline sedimentation, incline supernatant, precipitation thalline is transferred into 20mLBMMY substratum, and continue to cultivate abduction delivering 120h, it is 2% that period every 24h adds 100% methyl alcohol to final concentration.
The purifying of 1.5 pichia spp recombinases
By recombination yeast nutrient solution, 3000r/min, centrifugal 10min, get fermented supernatant fluid polyoxyethylene glycol and concentrate, dialysis, and by SephadexG-75 purifying, after purifying, sample detection is with SDS-PAGE electrophoresis (5% concentrated glue and 12% separation gel).
The mensuration of 1.6 recombinase vigor and protein concentration
Xylanase activity measures, and adopts DNS method.Enzyme activity unit (U) defines: with 0.5% birch xylan for substrate, 45 DEG C, under pH5.0 condition, produce enzyme amount needed for 1 μm of ol reducing sugar for 1U with per minute.
Determination of protein concentration adopts Bradford method, and standard protein is bovine serum albumin.
Recombinase vigor of the present invention adopts and represents than enzyme activity, the ratio that Rate activity (U/mg) is enzyme activity and protein concentration.The optimum temperature of 1.7 enzymes and the mensuration of thermostability
At pH5.0, under the condition of reaction 15min, within the scope of 35-50 DEG C, every 5 DEG C, measure enzyme respectively and live, live with the enzyme relative enzyme of 100% calculating that is up to alive, determine the optimum temperature of enzyme thus.By enzyme liquid isothermal holding different time at 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C and 60 DEG C, measure residual enzymic activities according to a conventional method, not to be incubated the enzyme sample activity of (4 DEG C of preservations) for 100%, determine the stability of enzyme thus.
2. results and analysis
The synthesis of 2.1 goal gene and structural analysis
The heterozyme gene order of design and synthesis is as shown in SEQ:ID:1, and encode shown in 193 amino acid (SEQ:ID:2), predicted molecular weight is 20842Da.The modeling of heterozygosis zytase XynZL homology finds, mutant enzyme and protoenzyme structural similitude, all belong to GH11 family, is made up of, partly holds shape structure in the right hand a simple alpha-helix and two reverse beta sheet lamellas.Prediction active centre is Glu89, Glu180, and the amino acid sites that heterozyme XynZL compares protoenzyme XynZF-2 gene alteration in addition has all done red mark in SEQ:ID:2, indicates emphasis amino acid sites in FIG.
2.2 expression of object enzyme in pichia spp
In heterozyme engineering bacteria GS115/XynZL fermented supernatant fluid, enzymic activity detected, after recombinase putification, reach electrophoresis pure, the mensuration of zymologic property can be carried out.
The optimum temperature of 2.3 enzymes and the mensuration of thermostability
Heterozyme XynZL compares after protoenzyme XynZF-2 expresses in pichia spp, and recombinase optimum temperature is brought up to 55 DEG C (Fig. 2) from 45 DEG C.Temperature stability there has also been obvious improvement, and as shown in Figure 3,50 DEG C of insulation 20min, XynZF-2 remnant enzyme activities are 18%, and XynZL remnant enzyme activity is 75%; 55 DEG C of insulation 15min, XynZF-2 remnant enzyme activities are 0, and XynZL remnant enzyme activity is 40%.
The above, be only best mode for carrying out the invention, is anyly familiar with those skilled in the art in the technical scope that the present invention discloses, and the simple change of the technical scheme that can obtain apparently or equivalence are replaced and all fallen within the scope of protection of the present invention.
Claims (2)
1. a construction process for zytase heterozyme engineering strain, is characterized in that, comprises the following steps:
Step 1, zytase structural analysis
The comparison of BLAST and DNAMAN6.0 homologous sequence and the higher heat resistant xylanase gene of zytase XynZF-2 homology, PROSITE predicts zytase functional site, utilize Phyre2 to zytase modeling, and the three-dimensional structure of zytase is analyzed by DSViewerPro6.0, adopt DiANNA1.1 to analyze disulfide linkage site, utilize NetNGlyc1.0 to predict zytase XynZF-2 glycosylation site;
The synthesis of step 2, goal gene and the structure of recombinant expression vector
The heterozyme gene xynZL sequence designed is synthesized by Jin Weizhi bio tech ltd, Suzhou;
The multiple clone site of reference heterozyme gene xynZL gene order and carrier for expression of eukaryon pPIC9K, following two primers of design: YS:5 '-CCGGAATTCTTCCCCACGACTCTGTCG-3 '
YX:5 '-ATAAGAATGCGGCCGCTTACTGAACACAGATGGACG-3 ', primer is synthesized by Jin Weizhi bio tech ltd, Suzhou;
With xynZL gene for template, YS, YX are that primer carries out pcr amplification, after amplified production reclaims, with corresponding expression carrier through double digestion, recovery, be connected, connecting fluid Transformed E .coliDH5 α competent cell, the LB that conversion fluid is coated with respectively containing Amp is dull and stereotyped, after the screening of positive colony, extract plasmid to cut and PCR checking through enzyme, final acquisition recombinant plasmid pPIC9K-xynZL.
2. the construction process of zytase heterozyme engineering strain according to claim 1, it is characterized in that, the amplification condition carrying out pcr amplification described in step 2 is: 94 DEG C of denaturation 2min; 94 DEG C of sex change 30s, 67 DEG C of annealing 30s, 72 DEG C extend 1min, 30 circulations; 72 DEG C extend 10min.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114438057A (en) * | 2022-03-16 | 2022-05-06 | 齐鲁工业大学 | Heat-resistant alkali-resistant xylanase and application thereof |
| WO2023148460A1 (en) * | 2022-02-04 | 2023-08-10 | Adisseo France S.A.S. | Protein having a xylanase activity |
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| WO2023148460A1 (en) * | 2022-02-04 | 2023-08-10 | Adisseo France S.A.S. | Protein having a xylanase activity |
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