CN105112388A - Trypsin-resistance-improved beta-1,4-endo-xylanase as well as preparation method and application of trypsin-resistance-improved beta-1,4-endo-xylanase - Google Patents

Trypsin-resistance-improved beta-1,4-endo-xylanase as well as preparation method and application of trypsin-resistance-improved beta-1,4-endo-xylanase Download PDF

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CN105112388A
CN105112388A CN201510567342.6A CN201510567342A CN105112388A CN 105112388 A CN105112388 A CN 105112388A CN 201510567342 A CN201510567342 A CN 201510567342A CN 105112388 A CN105112388 A CN 105112388A
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xylanase
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xyna
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CN105112388B (en
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姚冬生
吴秀秀
谢春芳
刘大岭
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Guangdong Fang can animal health care Co.,Ltd.
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Jinan University
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    • 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
    • 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 trypsin-resistance-improved beta-1,4-endo-xylanase. The trypsin-resistance-improved beta-1,4-endo-xylanase is generated by preparing two amino acid substitutes in the beta-1,4-endo-xylanase with an amino acid sequence as shown in SEQ ID NO.1 and has higher trypsin resistance, wherein the amino acid substitutes are on the 68th position and the 101st position. According to the trypsin-resistance-improved beta-1,4-endo-xylanase, a key amino acid residue of an XynA molecule is modified by using a protein engineering technology, a trypsin-resistance-improved XynA mutant is provided, the trypsin resistance half-life period of the trypsin-resistance-improved XynA mutant is prolonged by 160 minutes as comparison with that of wild-type XynA, other enzymatic properties are basically consistent with those of wild-type enzymes, and the mutant enzyme can be used as a feed additive.

Description

To β-Isosorbide-5-Nitrae-endo-xylanase that trypsin-resistant improves and preparation method thereof and purposes
Technical field
The present invention relates to β-Isosorbide-5-Nitrae-endo-xylanase, be related specifically to β-Isosorbide-5-Nitrae-endo-xylanase that trypsin-resistant is improved.
Background technology
Xylan is one of component that in hemicellulose, content is the abundantest, and the content in plant cell wall is only second to Mierocrystalline cellulose, accounts for 35% of dry cell weight.Extensively being present in agriculture byproduct as among corn cob, wheat bran, rice bran, stalk, bagasse etc., is a kind of important feedstuff raw material.
The main chain of xylan is connected through β-Isosorbide-5-Nitrae-glycosidic link by β-D-xylose residues, and side chain is connected with the substituting group of number of different types, C-2, C-3 position of such as main chain xylose residues individually or simultaneously acetylize etc. can occur, and its chemical structure is shown in Fig. 1.
The common participation of the multiple lytic enzyme of degradable needs of xylan, β-Isosorbide-5-Nitrae endo-xylanase can act on xylan backbone with internal-cutting way and produce the wood oligose of different lengths and a small amount of wood sugar, and zytase is the enzyme of most critical in xylanolytic enzyme.
The utilization of animal to xylan be under the effect of the multiple fodder enzyme comprising zytase, resolve into oligosaccharides and monose after absorb.
Fodder enzyme plays a role after entering cultivated animals digestive tube, and the decomposition of the proteolytic enzyme in digestive tube on feeding enzyme is the major reason affecting feeding enzyme service efficiency, so having protease resistant is the very important character of feeding enzyme.
At present, widely used zytase is all lower to tryptic resistance, and as fodder enzyme, it is subject to tryptic decomposition in animal digestive tract, and action effect receives obvious impact.
Summary of the invention
Primary and foremost purpose of the present invention is to provide a kind of β-1 improved trypsin-resistant, 4-endo-xylanase, it is a kind of XynA mutant improved trypsin-resistant obtained after being transformed by the critical amino acid residues of protein engineering to XynA molecule, overcome zytase as fodder enzyme to the shortcoming of trypsin-resistant difference, improve zytase as the result of use of fodder enzyme and value.
The present invention is that the gene (being called XynA gene) to β-Isosorbide-5-Nitrae-endo-xylanase carries out rite-directed mutagenesis.By the β-Isosorbide-5-Nitrae-endo-xylanase obtained in subtilis (Bacillussubtilis), be sequenced, GENBANK accession number is CP010052.1.The aminoacid sequence of the maturation protein of this enzyme is SEQIDNO.1.
According to a kind of Fixedpoint mutation modified XynA of the present invention, be the β-1 of SEQIDNO.1 by aminoacid sequence, manufacture multiple aminoacid replacement in 4-endo-xylanase to produce, to the β-1 that trypsin-resistant improves, 4-endo-xylanase, described aminoacid replacement is the replacement of the 68th and 101.
According to the further feature of Fixedpoint mutation modified β-Isosorbide-5-Nitrae-endo-xylanase of the present invention, the described aminoacid replacement at the 68th replaces Methionin with glutamine; Replace arginine with Serine at the aminoacid replacement of 101; The aminoacid sequence of described Fixedpoint mutation modified β-Isosorbide-5-Nitrae-endo-xylanase is SEQIDNO.2.
Mutant β-Isosorbide-5-Nitrae-endo-xylanase (the XynA acting on xylan of the present invention k68Q/R101S), through simulated intestinal fluid (pH6.0,1mg/mL trypsin solution) process, it extends 160 minutes to the tryptic resistance transformation period than wild-type enzyme, other zymologic properties and wild-type enzyme basically identical.
Further, the invention provides a kind of DNA molecular, its coding β-Isosorbide-5-Nitrae-endo-xylanase that trypsin-resistant is improved of the present invention.
Preferably, the nucleotides sequence of DNA molecular of the present invention is classified as SEQIDNO.3.
Another object of the present invention is to provide a kind of carrier, and it contains DNA molecular of the present invention.
Another object of the present invention is to provide a kind of host cell, and it contains DNA molecular of the present invention, or containing carrier of the present invention.
Above-mentioned carrier and host cell can be prepared by technique means well known in the art.
Present invention also offers the β-1 that trypsin-resistant is improved of the present invention, the production method of 4-endo-xylanase, comprise: be suitable for β-1, host cell of the present invention is cultivated under the condition that 4-endo-xylanase is expressed, and from host cell, be separated described β-Isosorbide-5-Nitrae-endo-xylanase.
When DNA molecular of the present invention is inserted into described carrier with suitable orientation and correct reading frame, or proceed in described host cell, described DNA molecular can be expressed in corresponding prokaryotic expression system.Many host-vector systems can be used for marking protein encoding sequence.Host-vector system includes but not limited to: the bacterium transformed with phage, plasmid or clay; Microorganism containing yeast vector, as yeast; By the mammalian cell system of virus infection; With the insect cell system of virus infection; By the vegetable cell system of bacteriological infection.The preferred carrier of the present invention comprises virus vector, plasmid, clay or oligonucleotide.
The preferred host of the present invention is that prokaryotic system is as e. coli bl21 (DE3); The preferred protein expression method of the present invention is IPTG abduction delivering.
Accompanying drawing explanation
Fig. 1 is the chemical structure of xylan.
Fig. 2 is the XynAK68Q/R101SSDS-PAGE electrophoresis detection figure after purifying, and wherein, M is molecular weight Marker; 1 is the XynAK68Q/R101S sample after purifying.
Fig. 3 is the schematic diagram adopting DNS method to carry out the mensuration of xylanase activity.
Fig. 4 shows wild-type and the residual activity of mutant in trypsin solution thereof.
Embodiment
Term adopted herein, except as otherwise noted, is the implication that those skilled in the art understand usually.The definition of some specific terms used in the present invention is below provided.
" wtXynA " represents wild-type beta-Isosorbide-5-Nitrae-endo-xylanase, and its gene represents with italic wtXynA.
" XynA k68Q/R101S" represent mutant β-Isosorbide-5-Nitrae-endo-xylanase XynA k68Q/R101S, its gene is with XynA k68Q/R101Srepresent.
The pcr amplification of embodiment 1: β-Isosorbide-5-Nitrae-endo xylanase genes and order-checking
1, the β-1 that originates with subtilis of the present invention, 4-endo xylanase genes (GENBANK:CP010052.1) sequence as a reference, software Primer5 is adopted to design and synthesize two Oligonucleolide primers, RNA isolation kit extracts the genome in subtilis, by PCR method amplification XynA goal gene.
Article two, PCR primer is as follows:
Forward primer F (SEQIDNO.4):
5’CGGAATTCATGTTTAAGTTTAAAAAGAATTTCT3’；
Reverse primer R (SEQIDNO.5):
5’CCAAGCTTTTACCACACTGTTACGTTAGA3’。
The reaction system of pcr amplification XynA gene is as follows:
PCR program setting is:
94 DEG C of denaturations 2 minutes;
4 DEG C, 30s; 56 DEG C, 30s; , 68 DEG C, 1 minute; 30 circulations;
68 DEG C, 5 minutes.
Pcr amplification product, after the agarose gel electrophoresis of 1%, reclaims test kit with DNA and carries out glue recovery, obtain the fragment of about 650bp.
The TA clone of embodiment 2: β-Isosorbide-5-Nitrae-endo xylanase genes (XynA)
1. in the PCR pipe of a new sterilizing, add following ingredients:
Suction mixing is also of short duration centrifugal, and 16 DEG C of water-baths are spent the night.
Use plasmid extraction test kit extracting plasmid after connecting the amplification of product conversion DH5 α competent cell, identify recombinant plasmid with PCR, PCR is verified as positive bacterial strain and gets the order-checking of bacterium liquid sample presentation Invitrogen company, determine to check order successfully.
The structure of embodiment 3:pET32a-XynA recombinant plasmid and conversion
1.EcoRI, HindIII double digestion restructuring pMD18-T carrier and carrier pET32a;
Table 1EcoRI, HindIII double digestion pMD18-T-XynA and pET32a (unit: μ L)
37 DEG C of double digestions are after 30 minutes, and 1.5% agarose gel electrophoresis detects enzyme and cuts effect.
2. digestion products cuts glue recovery, and the pET32a carrier and the goal gene XynA that enzyme are cut purifying recovery mix with the ratio of concentration ratio 1:3-1:10,16 DEG C of connections of spending the night.
The connection of table 2 double digestion pET32a carrier segments and XynA Insert Fragment: (unit: μ L)
10 × damping fluid pET32a XynA T4DNA ligase enzyme H 2O Add up to (μ L)
1.0 1.0 7.5 0.5 0.0 10.0
After EcoRI and HindIII enzyme cuts qualification recombinant plasmid, getting the order-checking of bacterium liquid sample presentation Invitrogen company being verified as positive bacterial strain, determining to check order successfully.
Embodiment 4: rite-directed mutagenesis
1, through utilizing the principle mutually identified between enzymatic reaction theory and protein molecular, rite-directed mutagenesis is carried out to the amino acid in following site:
(1) for the amino acid mutation of the 68th amino acids, design primer is as follows:
XynA K68Q
Forward mutation primer (SEQIDNO.6):
5’GGAAATTTTGTTGTTGGTC*AAGGTTGGACTACAGG3’
Inverse transition primer (SEQIDNO.7):
5’CCTGTAGTCCAACCTTG*ACCAACAACAAAATTTCC3’
Wherein * is mutating alkali yl, and primer transfers to Beijing AudioCodes Bioisystech Co., Ltd to synthesize.
(2) for the 101st amino acids sudden change, design primer is as follows:
XynA R101S
Forward mutation primer (SEQIDNO.8):
5’CTTTATATGGTTGGACGAGC*TCACCTCTC3’
Inverse transition primer (SEQIDNO.9):
5’GAGAGGTGAG*CTCGTCCAACCATATAAAG3’
Wherein * is mutating alkali yl, and primer transfers to Beijing AudioCodes Bioisystech Co., Ltd to synthesize.
PCR program setting is:
94 DEG C of denaturations 2 minutes;
94 DEG C, 30s; 63 DEG C, 30s; 68 DEG C, 65s; 5 circulations;
94 DEG C, 30s; 59 DEG C, 30s; 68 DEG C, 65s; 25 circulations;
68 DEG C, 5 minutes.
Order-checking is connected after PCR reaction.Site-directed point mutation is the method adopting Overlap extension PCR, suddenlys change successively to 2 sites, and the DNA sequence dna finally obtained can be expressed as the β-Isosorbide-5-Nitrae-endo-xylanase improved trypsin-resistant of the present invention.
Embodiment 5: wild-type XynA and mutant XynA k68Q/R101Sgene imports the abduction delivering of the IPTG of e. coli bl21 (DE3) and recombinant protein respectively
1. be wild-type XynA plasmid and the restructuring XynA of 200ng by content k68Q/R101Splasmid is added in the 1.5mL centrifuge tube containing 200 μ L competent cells respectively, rotates mixing gently, ice bath 30 minutes; 42 DEG C of thermal shock 90sec.Take out ice bath 3 minutes immediately, add the LB liquid nutrient medium of 600 μ L, 37 DEG C, 75rpm, slowly shakes 1h.Competent cell is coated Amp +lB solid medium on, the flat board of each diameter 90mm is coated with 200 μ L, 37 DEG C cultivate 12h, extracting recombinant plasmid, cuts qualification recombinant plasmid with EcoRI and HindIII enzyme.
2. the abduction delivering of recombinant protein
Condition induction expression protein enzyme according to having explored:
(1) get respectively 50 μ L glycerine preserve wild-type recombinant bacterium, be inoculated in respectively 1 5mL containing 100 μ g/mLAmp LB liquid nutrient medium in, 37 DEG C, 250rpm incubated overnight.
(2) overnight culture is according to the ratio inoculation 2.5mL of 1:100 in the LB liquid nutrient medium of the 250mL containing 100 μ g/mLAmp, and 37 DEG C, 250rpm is cultured to OD 600adding IPTG when being 1.0 to final concentration is 0.5mM, and 37 DEG C of 250rpm shaking culture, receive bacterium after 4h.
Embodiment 6: wild-type XynA and mutant XynA k68Q/R101Sthe purifying of recombinant protein and SDS-PAGE electrophoresis detection
1. use ultrasonic cell disruption instrument to carry out cell wall breaking to thalline, add DEAE anion chromatography level pad before broken wall and carry out resuspended to 25mL to bacterium liquid.After bacterium liquid broken wall is clarified completely, centrifugal 10 minutes of broken liquid 4 DEG C of 10000g, collect supernatant and be protein sample crude extract, protein crude extract administration carries out purifying through anion chromatography and Ni ion chromatography, the elution peak of the Ni ion chromatography obtained is after ammonium sulfate precipitation spends the night, with molecular sieve G-25 desalination, obtain pure protein, step is as follows:
(1) 0.02mol/LNaH of pH7.4 is first used 2pO 4damping fluid balance chromatography column 2-3 column volume.
(2) after baseline balance, with the flow velocity loading of 0.5mL/ minute.
(3) after end of the sample, with same flow velocity, respectively with level pad and the elution buffer wash-out containing 1mol/LNaCl, 280nm ultraviolet absorption method detects, and collects the drip washing peak and elution peak that obtain.
(4) the drip washing peak that obtains of anion-exchange chromatography is as the loading sample of Ni ion chromatography.That first uses pH6.5 contains 0.02mol/LNaH 2pO 4with damping fluid balance chromatography column 2 column volumes of 0.5mol/LNaCl.
(5) then with the flow velocity loading of 0.5mL/ minute.
(6) again with identical damping fluid, flow velocity wash-out 2 column volumes of 0.5mL/ minute.
(7) finally with the elution buffer containing 100mM imidazoles, with flow velocity wash-out 1.5 column volumes of 0.5mL/ minute, 280nm ultraviolet absorption method detected and collects elution peak.
(8) elution peak uses the 0.02moL/LNaH of 5mLpH7.4 after 80% saturated ammonium sulphate 2pO 4buffer solution.
(9) cation-exchange chromatography is carried out.
(10) collect the enzyme liquid obtained, carry out the mensuration of protein content and enzymic activity.The enzyme liquid collected is got 1mL 9mL95% alcohol-20 DEG C of precipitates overnight, after centrifugal drying albumen, carry out SDS-PAGE analysis.
2.SDS-PAGE electrophoresis detection
(1) separation gel of 10mL15% is configured, mix rear liquid-transfering gun toward encapsulating in glass plate, until stop from short glass plate top 2 ~ 3 centimeters, then glue face is sealed with distilled water, gel maker one end can be lifted gently then put down and make glue face smooth, being polymerized after 40 minutes to abandoning distilled water, sucking excessive moisture with filter paper;
(2) configure the concentrated glue of 4mL5%, be filled in uniformly on separation gel, the comb inserting dimension should be avoided producing bubble simultaneously, and polyase 13 treats that gelling is solid for 0 minute;
(3) install electrophoresis chamber, in groove, fill with electrophoretic buffer, volume should be greater than the half of electrophoresis chamber volume, the glue made is moved in electrophoresis chamber, carefully takes out sample comb;
(4) point sample successively, point sample amount is too much unsuitable, and the 15 every holes of μ L are suitable;
(5) first arrange 120V when electrophoresis starts and run glue, voltage is changed into 180V to concentrated glue part and continues electrophoresis by indicator, can stop electrophoresis when object band goes to mid-way;
(6) carefully peel gel, after coomassie brilliant blue R_250 dyes 30 minutes, destainer decolouring is clear to the more shallow protein band of background;
(7) gel imaging observations.
By electrophoresis detection result: obtain single protein band, as Fig. 2 at 20.4KD.Show that sample obtains XynA after purified k68Q/R101S.
Embodiment 7: wild-type XynA and mutant XynA k68Q/R101Sthe trypsin-resistant of recombinant protein detects
1. the enzyme liquid after purifying mixes according to mass ratio 10:1 with artificial simulation intestinal juice (pH6.810mg/mL trypsin solution), in 37 DEG C of insulations, every sampling in 30 minutes, DNS method measures enzyme activity, with the trypsin solution process enzyme liquid vigor of 0 minute for 100%, calculate the relative activity of each group of enzyme, the stability of examination wild-type enzyme in trypsinase.The cultivation of 3 batches is carried out in experiment altogether, and often criticize each sample and establish 2 parallelly to measure, residual enzyme activity gets the mean value of 6 take off data.
2. enzyme activity determination:
This experiment adopts DNS method to carry out the mensuration of xylanase activity, this ratio juris utilizes DNS (3,5-dinitrosalicylic acid) solution and reducing sugar solution is reduced into henna aminocompound (see Fig. 3) after heat altogether, and the amount of reducing sugar is proportional in the red-brown aminocompound generated within the specific limits and system, therefore can live with colorimetric determination of enzyme, this method has easy and simple to handle, and fast, and impurity disturbs few advantage.Concrete enzyme activity determination comprises the making of typical curve, the making of sample activity group and sample deactivation group.
(1) making of typical curve
Normal wood sugar soln concentration: 1mg/mL
Get 96 orifice plate one, according to the form below carries out successively, makes typical curve, as following table:
(2) mensuration (DNS method) of Xylanase activity
By oat xylan with the concentration of massfraction 1%, be dissolved in the 0.04moL/LNa of pH6.0 2hPO 4-0.02moL/L citrate buffer solution, as substrate.Enzyme solution also suitably dilutes with identical damping fluid.Get 70 μ L substrate solutions and add the diluted enzyme solution of 10 μ L, 60 DEG C are reacted 10 minutes, add the DNS reagent termination reaction of 200 μ L, 100 DEG C are developed the color 10 minutes, after cooling, measure the absorbance value of reaction solution in 540nm place, calculate the amount of the reducing sugar generated according to typical curve, do the contrast of substrate+inactivator simultaneously.
The unit of activity (Μ) of zytase is defined as: under condition is lived in above-mentioned survey, per minute hydrolyzed xylan generates the enzyme amount required for 1 μm of oL wood sugar.
Calculation formula is: Ea=N × V 1× C × 6.667/ (V 2× T)
In formula: Ea is the enzyme activity of zytase, Μ/mL;
N: the extension rate of enzyme liquid;
V 1: the final volume of reaction solution, mL;
C: from the concentration of the wood sugar that typical curve calculates, mg/mL;
(C=activity group reducing sugar content-deactivation group reducing sugar content)
V 2: the volume of enzyme liquid in reaction solution, mL;
T: the time of enzyme digestion reaction, minute;
6.667: reduction factor (mg/mL is converted to a μm oL/mL).
3.XynA and mutant XynA k68Q/R101Sthe stability of recombinant protein in trypsin solution
At 37 DEG C with artificial simulation intestinal juice process wild-type and mutant, survey the residual enzyme vigor of wild-types and mutant every sampling in 30 minutes, result shows: XynA k68Q/R101Sresidual enzyme vigor obviously high than wild-type XynA, as Fig. 3.The transformation period mapping analysis of stability in trypsin solution to wild-type and mutant, the transformation period of XynA is 127 minutes; XynA k68Q/R101Stransformation period be 287 minutes, extend 160 minutes than wild-type.

Claims (8)

1. the β-1 that trypsin-resistant is improved, 4-endo-xylanase, it is characterized in that: it is the β-1 of SEQIDNO.1 by aminoacid sequence, manufacture two aminoacid replacement in 4-endo-xylanase to produce, the β-1 stronger to trypsin-resistant, 4-endo-xylanase, described aminoacid replacement is the replacement of the 68th and 101.
2. β-Isosorbide-5-Nitrae-the endo-xylanase improved trypsin-resistant according to claim 1, is characterized in that: the described aminoacid replacement at the 68th replaces Methionin with glutamine; Replace arginine with Serine at the aminoacid replacement of 101; The aminoacid sequence of described Fixedpoint mutation modified β-Isosorbide-5-Nitrae-endo-xylanase is SEQIDNO.2.
3. a DNA molecular, is characterized in that: its coding β-Isosorbide-5-Nitrae-endo-xylanase improved trypsin-resistant according to claim 2.
4. DNA molecular according to claim 3, is characterized in that: its nucleotides sequence is classified as SEQIDNO.3.
5. a carrier, is characterized in that: it contains the DNA molecular described in claim 3 or 4.
6. a host cell, is characterized in that: it contains the DNA molecular described in claim 3 or 4, or containing carrier according to claim 5.
7. the β-1 that trypsin-resistant is improved according to claim 1, the production method of 4-endo-xylanase, it is characterized in that, described method comprises: be suitable for β-1, host cell according to claim 6 is cultivated under the condition that 4-endo-xylanase is expressed, and from thalline, be separated described β-Isosorbide-5-Nitrae-endo-xylanase.
8. β-the application of Isosorbide-5-Nitrae-endo-xylanase in feed and food of trypsin-resistant raising according to claim 1.
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