CN109553664A - A kind of fungi α-l-arabfuranglycosidase synthesis regulation protein mutant and its application - Google Patents

A kind of fungi α-l-arabfuranglycosidase synthesis regulation protein mutant and its application Download PDF

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CN109553664A
CN109553664A CN201811518650.XA CN201811518650A CN109553664A CN 109553664 A CN109553664 A CN 109553664A CN 201811518650 A CN201811518650 A CN 201811518650A CN 109553664 A CN109553664 A CN 109553664A
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arabfuranglycosidase
arar
fungi
rar
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曲音波
李世英
潘云军
高丽伟
刘国栋
宋欣
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Shandong University
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Abstract

The invention discloses the mutant of fungi α-l-arabfuranglycosidase synthesis regulation albumin A raR a kind of, it is as forming by alanine mutation for valine the 731st amino acids of amino acid sequence modulin as shown in SEQ ID NO.1, its amino acid sequence is as shown in SEQ ID NO.2, and the nucleotide sequence of encoding gene is as shown in SEQ ID NO.3.The invention also discloses application of the modulin mutant in production α-l-arabfuranglycosidase and alpha-galactosidase.Experiment confirms significantly promote the yield of α-l-arabfuranglycosidase and alpha-galactosidase by expressing the modulin mutant in fungal bacterial strain.

Description

A kind of fungi α-l-arabfuranglycosidase synthesis regulation protein mutant and its Using
Technical field
The present invention relates to a kind of mutant of arabinofuranosidase synthesis regulation albumen and its applications, more particularly to true The mutant of bacterium α-l-arabfuranglycosidase synthesis regulation albumin A raR and its application.Belong to gene engineering technology field.
Background technique
The efficient enzymatic hydrolysis of plant cell wall polysaccharides is in a variety of industrial process, such as grocery trade, feed industry, paper-making industry and biology It is extremely important during fuel production etc..It is Arabic that α-L- furans is carried in the cell wall of many plant cells, on side chain The hemicellulose and pectin (such as araboxylan and arabogalactan) of glucoside residue are its main components, can be from The enzyme of hydrolysis L-arabinose has played important function during the effectively hydrolyzing of these substrates in these polysaccharide, wherein leading If α-l-arabfuranglycosidase (EC number 3.2.1.55).Addition α-l-arabfuranglycosidase can significantly improve wood Hydrolysis of the dextranase to xylan backbone in araboxylan.In addition, effectively hydrolyzing L- araban needs different α- The combination of L- arabinofuranosidase plays a role.α-l-arabfuranglycosidase is also used for pharmaceutically certain active materials Synthesis, such as the synthesis of ginsenoside.
Some filamentous fungis such as aspergillus, mould etc. can produce a series of α-L- Arab furans with different substrate specificities It mutters glycosidase.These enzyme genes are on transcriptional level by the regulation of types carbon sources.It L-arabinose, L- arabite and is rich in The polysaccharide (such as L- araban) of L-arabinose can induce the generation of α-l-arabfuranglycosidase.In aspergillus niger and structure In nest aspergillus, the transcription factor AraR of Zn2Cys6 type is mainly responsible for the table for activating a variety of α-l-arabfuranglycosidase genes Up to (referring to Studies in Mycology, 2011,69:31-38).The encoding gene for knocking out AraR can significantly reduce aspergillus niger The transcription amount of the vigor of middle α-l-arabfuranglycosidase and multiple α-l-arabfuranglycosidase genes.In addition, AraR The expression of certain genes in pentose metabolism approach can also be activated, and participates in pentose phosphate pathway, the reduction point of D- galactose oxidase Solve the regulation of metabolic pathway and pectin degrading process etc..Homologous sequence search result shows all there is aspergillus niger in many fungies The homologous protein of AraR.
In terms of the current report for improving fungi α-l-arabfuranglycosidase yield is limited only to medium optimization, effect It is not significant enough.It is to improve fungi lignocellulosic drop that expression and/or vigor to transcription factor, which carry out engineering operation, Solve the important means of enzyme production level.The constitutive activation mutant of activating transcription factor can be such that bacterial strain closes under the conditions of non-induced Production of enzyme is improved at enzyme, and under inductive condition.Therefore, if the transcriptional activation activity of AraR can be improved by molecular modification, Or obtain the constitutive activation mutant of AraR, that is, it is possible to realize the raising of α-l-arabfuranglycosidase yield.So And it has not yet to see through transformation this modulin of AraR and improves α-l-arabfuranglycosidase production quantifier elimination report Road, and the constitutive activation mutant of AraR also has not been reported at present.
Summary of the invention
For the current lower status of fungi α-l-arabfuranglycosidase yield, the object of the present invention is to provide one kind The mutant of fungi α-l-arabfuranglycosidase synthesis regulation albumin A raR and its application.
The mutant of fungi α-l-arabfuranglycosidase synthesis regulation albumin A raR of the present invention, feature exist In: modulin AraR mutant is named as AraRA731V, it is to regulate and control egg as shown in SEQ ID NO.1 as amino acid sequence The 731st white amino acids are what valine was formed by alanine mutation, and amino acid sequence is as shown in SEQ ID NO.2.
Wherein: the modulin AraR mutant comes preferably from penicillium oxalicum.
The present invention provides a kind of above-mentioned fungi α-l-arabfuranglycosidase synthesis regulation albumin A raR mutation of coding The gene of body, it is characterised in that: the nucleic acid sequence of the gene is as shown in SEQ ID NO.3.
The mutant of fungi α-l-arabfuranglycosidase synthesis regulation albumin A raR of the present invention is producing me Application in primary furanoside enzyme.
The mutant of fungi α-l-arabfuranglycosidase synthesis regulation albumin A raR of the present invention is in production α-half Application in lactoside enzyme.
The present invention provides a kind of recombination penicillium oxalicums, it is characterised in that: takes in the recombination penicillium oxalicum cell With the gene for encoding above-mentioned fungi α-l-arabfuranglycosidase synthesis regulation albumin A raR mutant.Meanwhile the present invention is also Provide the gene structure that a kind of application encodes above-mentioned fungi α-l-arabfuranglycosidase synthesis regulation albumin A raR mutant The method for building recombination penicillium oxalicum.
Application of the recombination penicillium oxalicum of the present invention in production arabinofuranosidase.
Application of the recombination penicillium oxalicum of the present invention in production alpha-galactosidase.
Experiment confirms: application provided by the invention encodes above-mentioned fungi α-l-arabfuranglycosidase synthesis regulation albumen The gene constructed recombination penicillium oxalicum of AraR mutant passes through modulin mutant AraRA731VExpression to α-L- I The synthesis of primary furanoside enzyme, which has, remarkably promotes effect；Also have to the synthesis of alpha-galactosidase simultaneously and remarkably promotes work With.It proves by expressing fungi α-l-arabfuranglycosidase synthesis regulation albumin A raR of the present invention in fungal bacterial strain Mutant, the recombinant bacterial strain that α-l-arabfuranglycosidase yield increases substantially can be obtained.
Further, the present invention also provides the AraR mutant AraRA731VExpress the thick enzyme of fermentation of recombinant bacterial strain Application of the liquid in araban efficient degradation.
The invention discloses the mutant of fungi α-l-arabfuranglycosidase synthesis regulation albumin A raR a kind of and its Using.Meanwhile the present invention also provides a kind of applications to encode above-mentioned fungi α-l-arabfuranglycosidase synthesis regulation albumen The method and its recombination penicillium oxalicum of the gene constructed recombination penicillium oxalicum of AraR mutant.It is experimentally confirmed that expression have on State modulin mutant recombination penicillium oxalicum cell can without containing inducer condition of culture under synthesis α-L- I Primary furanoside enzyme.Under inductive condition, the yield of α-l-arabfuranglycosidase is relative to starting strain in recombinant bacterial strain 54.1 times are improved, alpha-galactoside production of enzyme improves 7.4 times relative to starting strain.In the condition of same enzyme protein dosage Under, recombinant bacterial strain crude enzyme liquid produced is to 4.5 times that the degradation capability of gum arabic is starting strain crude enzyme liquid.Indication is originally Preparation of the mutant in L-arabinose of the fungi α-l-arabfuranglycosidase synthesis regulation albumin A raR provided is provided It has broad application prospects in industry.
Detailed description of the invention
Fig. 1 is that the bacterial strain of expression arabinofuranosidase modulin AraR mutant is trained on not carbonaceous sources culture medium Support 24 hours arabinofuranosidase yield.
Fig. 2 is to express the bacterial strain of arabinofuranosidase modulin AraR mutant containing wheat bran and cellulose training Support the arabinofuranosidase yield on base.
Fig. 3 is to express the bacterial strain of arabinofuranosidase modulin AraR mutant containing wheat bran and cellulose training Support the alpha-galactoside production of enzyme on base.
Fig. 4 is to express the bacterial strain crude enzyme produced by the liquid of arabinofuranosidase modulin AraR mutant to Arab tree The degradation capability of glue.
Specific embodiment
Generality explanation:
Embodiment used medium and storing liquid:
Wheat bran juice culture medium: 30min is boiled in the wheat bran heating of 10% (w/v), with 6 layers of filtered through gauze, collects filtered juice, adds The sterilizing of 1.5% agar powder is spare.50 × Vogel ' s storing liquid (g/L): citrate dihydrate trisodium 125.0, KH2PO4250.0 NH4NO3100.0, MgSO4·7H2O 10.0, CaCl2·2H2O 5.0 additionally incorporates the storage of biotin 0.3mg, 5ml microelement Liquid storage.
Vogel ' s salt microelement storing liquid (g/L): citrate dihydrate 50.0, ZnSO4·7H2O 50.0, Fe (NH4)2 (SO4)2·6H2O 10.0, CuSO4·5H2O 2.5, MnSO4·H2O 0.5, H3BO3 0.5,Na2MoO4·2H2O 0.5。
Embodiment agents useful for same:
1kb ladder and Trans 2K used in archaeal dna polymerase KD plus, agarose gel electrophoresis used in DNA cloning Molecular weight standards are purchased from Beijing Quan Shi King Company；Phanta high-fidelity DNA polymerase only praises biotechnology purchased from Nanjing promise Co., Ltd；Gel extraction kit, segment QIAquick Gel Extraction Kit and plasmid extraction kit are purchased from OMEGA company.
Embodiment instrument:
PCR amplification instrument (Eppendorf), high speed freezing centrifuge (Eppendorf), agarose gel electrophoresis instrument (Beijing 61 instrument plants), Ago-Gel imaging system (Syngene), constant-temperature table (Shanghai Fu Ma experimental facilities company), Spectra MAX190 microplate spectrometer (Molecular Devices company).
Present invention uses genetic engineerings and molecular biology field conventional technique and method.Those skilled in the art The other routine techniques in this field, method and reagent can be used on the basis of embodiment provided by the invention, and are not limited to The restriction of the specific embodiment of the invention.
Following specific embodiments elaborate the α-l-arabfuranglycosidase modulin AraR to penicillium oxalicum source The method for carrying out series jump and improving α-l-arabfuranglycosidase and alpha-galactoside production of enzyme.In practical applications, The source of α-l-arabfuranglycosidase modulin AraR is not limited to penicillium oxalicum, further include penicillium chrysogenum, microassembly robot, Brazilian mould, aspergillus niger, aspergillus oryzae, aspergillus fumigatus, microorganism Aspergillus aculeatus etc., amino acid sequence are characterized in that and SEQ ID NO.1 institute Show that the consistent degree of amino acid sequence is not less than 70%, or has and the 731st the third ammonia in amino acid sequence shown in SEQ ID NO.1 The conserved features of sour local sequence nearby, are embodied in, and the N-terminal connection amino acid of the 731st alanine is also usually third The C-terminal connection amino acid of propylhomoserin, the 731st alanine is usually electronegative amino acid, including aspartic acid and glutamic acid.
Following specific embodiments elaborate the mutant AraR that AraR is expressed in penicillium oxalicumA731VAnd improve α-L- I The method of primary furanoside enzyme and alpha-galactoside production of enzyme.In practical applications, the fungi that sets out being modified is not limited to oxalic acid Mould further includes penicillium chrysogenum, microassembly robot, Brazilian mould, aspergillus niger, aspergillus oryzae, aspergillus fumigatus, microorganism Aspergillus aculeatus, trichoderma reesei Deng.
Following specific embodiments are elaborated the 731st of penicillium oxalicum α-l-arabfuranglycosidase modulin AraR the The method that position alanine mutation improves α-l-arabfuranglycosidase and alpha-galactoside production of enzyme for valine.In reality In, the alanine of the alanine and the conservative position of modulin AraR homologous protein can be also replaced by be had with valine The amino acid of similar structure or chemical property, including with hydrophobic property leucine, isoleucine, proline, phenylalanine, Tryptophan and methionine.
Following specific embodiments elaborate the recombination of expression α-l-arabfuranglycosidase modulin AraR mutant Application of the penicillium oxalicum bacterial strain enzyme solution produced in gum arabic enzymatic hydrolysis.In practical applications, by expressing the regulation The crude enzyme liquid with α-l-arabfuranglycosidase and alpha-galactosidase activity that albumin A raR mutant obtains can also answer For the enzymatic hydrolysis of other biomaterials rich in arabinose residues, including beet, corn stover, pine, wheat bran, rice bran, people Join saponin(e etc..
The contents of the present invention are specifically addressed below with reference to example.
The amplification of 1 penicillium oxalicum α-l-arabfuranglycosidase modulin AraR mutant code gene of embodiment
Bacterial strain: penicillium oxalicum wild strain 114-2, the bacterial strain is in the preservation on the 28th of September in 2011 to Chinese common micro- life Object culture presevation administrative center, deposit number are CGMCC 5302.The GenBank accession number of genome sequence is AGIH00000000.1.This bacterial strain previously applies for obtaining a kind of patent " extracellular aldonolactone enzyme of authorization in applicant It is disclosed in PoALAC and its application " (patent No.: ZL 2,016 1 1056999.7).
The amino acid sequence of AraR is shown in SEQ ID NO.1 in penicillium oxalicum.Genomic DNA with penicillium oxalicum 114-2 is Template uses primer araR-CDS- using the front end coded sequence of primer araR-CDS-UF and araR-CDS-UR amplification araR The back-end code sequence and terminator sequence of DF and araR-CDS-DR amplification araR, and introducing makes the 731st alanine mutation The nucleic acid sequence of valine.
The primer sequence that above-mentioned amplification procedure uses is (5 ' to 3 '):
araR-CDS-UF:CCGCTTGAGCAGACATCACCATGGATTCCCAACAGGGGGA
araR-CDS-UR:TCAACCGCCTCCAAAGCGTGCGAG
araR-CDS-DF:CTCGCACGCTTTGGAGGCGGTTGACGCCGTCCAGCAGATCCTTC (compile by underlined sequences Code valine)
araR-CDS-DR:TTCAATATCAGTTAACGTCGTTGGGGCATCGGCACTCACATTTCG
The PCR program of fragment amplification are as follows:
95 DEG C of 1min of initial denaturation, each circulation include 95 DEG C of 10s, 60 DEG C of 30s, 72 DEG C of 1.5min, and totally 31 recycle, thoroughly Extend 72 DEG C of 10min.
Reference literature reports (Fungal Genetics and Biology 2004,41:973-981), is prolonged using overlapping Stretch the back-end code sequence and termination of the araR of the front end araR coded sequence and carrying series jump that PCR method obtains amplification Subsequence is fused together.The PCR program of segment composition are as follows:
95 DEG C of 1min of initial denaturation, each circulation include 95 DEG C of 10s, 58 DEG C of 5min, 72 DEG C of 1min, and totally 12 recycle, thoroughly Extend 72 DEG C of 10min.
The final product using Overlap extension PCR is template, uses two primers of araR-CDS-UF and araR-CDS-DR Amplification is regulated the coded sequence and terminator sequence of protein mutant.Obtained mutant be penicillium oxalicum α-L- I The mutant of primary furanoside enzymatic synthesis modulin AraR, the mutant are named as AraRA731V, be by amino acid sequence such as 731st amino acids of modulin shown in SEQ ID NO.1 are formed by alanine mutation for valine, amino acid sequence Column are as shown in SEQ ID NO.2.Encode AraRA731VGene nucleotide series as shown in SEQ ID NO.3.
Embodiment 2AraR mutant AraRA731VThe building of encoding gene expression cassette
In order to express AraR and its mutant AraRA731V, use primer gpdA-F and gpdA-R amplification aspergillus nidulans GpdA gene promoter expands Hygromycin B phosphofransferose gene using primer hph-F and hph-RhphExpression cassette.Segment expands The PCR program of increasing is in the same manner as in Example 1.
The primer sequence that above-mentioned amplification procedure uses are as follows:
gpdA-F:GTAAGGATTTCGGCACGG
gpdA-R:GGTGATGTCTGCTCAAGCGG
hph-F:CGACGTTAACTGATATTGAA
hph-R:CAACCCAGGGCTGGTGACGG
The segment for being obtained amplification using Overlap extension PCR method is according to gpdA gene promoter, araR mutant code The sequence of area and terminator, hph expression cassette merges to obtain AraRA871VEncoding gene expression cassette.The PCR program and reality of segment composition It applies identical in example 1.Obtained expression casette is purified using segment QIAquick Gel Extraction Kit, is recycled.
Embodiment 3AraR mutant AraRA731VExpression in penicillium oxalicum
Bacterial strain: penicillium oxalicum CXC, constructed from penicillium oxalicum 114-2 bacterial strain described in embodiment 1, specifically as applicant Construction method is shown in document report (Biotechnology Journal 2017,12 (11): 1700119).
AraR mutant AraR will be obtained in embodiment 2A731VEncoding gene expression cassette is turned by protoplast transformation method Enter CXCBacterial strain uses agent prescription are as follows:
Protoplast transformation buffer:
Conversion fluid S1 (100ml): 21.86g sorbierite, 1.36g KH2PO4, pH 5.6.
Conversion fluid S2 (100ml): 18.22g sorbierite, 0.74g CaCl2, 0.12g Tris, HCl are adjusted to pH 7.5.
Conversion fluid T1 (100ml): 25g PEG 6000,0.74g CaCl2, 0.12g Tris, HCl are adjusted to pH 7.5.
The above conversion buffer is both needed to sterilize spare.
Transformant screening upper layer culture medium (every 100ml): 2.0g glucose, 18.2g sorbierite, 1 × Vogel ' s salt, 1.5g agar powder sterilizes spare.
Transformant screening lower layer culture medium (every 100ml): 18.2g sorbierite, 1 × Vogel ' s salt, 1.5g agar powder go out Bacterium is spare.
The preparation of protoplast and method for transformation are as follows:
1) one layer of glassine paper of bedding on wheat bran plate, is added 50 to 100 μ l spore suspensions on glassine paper, and coating is equal It is even, drive the bubble under glassine paper out of.It is 6 same processing, 30 DEG C of culture 11h.
2) 0.1g cell wall lysis enzymes are added into 20ml S1 solution, mix dissolution.
3) draw 3ml enzyme solution in an empty plate, add one layer of glassine paper with mycelia, then plus 2 to 3ml enzyme solution, according to It is secondary to stack 6 layers.There cannot be bubble between glassine paper.1.5 to 2h are digested at 30 DEG C, microscopy observes protoplast and generates situation.
4) about 20ml S1 solution is added in another empty plate, the glassine paper after enzymatic hydrolysis successively taken out with tweezers and It is rinsed in plate, mycelia remaining on glassine paper is washed down.
5) using be equipped with 4 layers of lens wiping paper funnel, by step 3 and 4 enzymolysis liquid and rinsing liquid merging be filled into 50ml Centrifuge tube (following operation carries out on ice).
6) 4,000rpm, 4 DEG C of centrifugation 10min carefully outwell supernatant immediately, (are contained with about 4ml S2 solution after centrifugation 1M sorbierite and 50mM CaCl210mM pH 7.5Tris-HCl buffer) be resuspended protoplast.
7) 4,000rpm, 4 DEG C of centrifugation 10min carefully outwell supernatant, and protoplast is resuspended with about 500 μ l S2 solution.
8) protoplast is sub-packed in 5ml centrifuge tube with 1ml suction nozzle, every 100 μ l of pipe is added purified no more than 5 μ l Expression casette DNA (concentration is greater than 50ng/ μ l).
9) 25 μ l conversion fluid T1 (PEG 6000 and 50mM CaC1 containing mass concentration 25% are added210mM pH 7.5Tris-HCl buffer), it mixes gently, ice bath 20min.
10) 1ml conversion fluid T1 is added, mixes gently, is placed at room temperature for 5min.
11) 2ml S2 solution is added, mixes gently.
12) it is mixed with the upper layer screening and culturing medium containing 350 μ g/mL hygromycin Bs, is poured over the lower layer's screening training solidified Primary surface is supported, 30 DEG C of cultures to transformant occur.The protoplast of DNA is not added as control.
The transformant that screening is obtained continues to purify the bacterium that monospore is formed by scribble method on screening and culturing medium plate It falls, then extracts genomic DNA in a small amount.Using primer gpdA-F and hph-R the amplifying target genes expression cassette in embodiment 2, The segment that amplification obtains is delivered to the sequence of Qingdao Qing Ke Bioisystech Co., Ltd verifying gene coding region.
The reagent that penicillium oxalicum genomic DNA extracts in a small amount includes:
Extraction buffer: 200mM TrisHCl, 250mM NaCl, 25mM EDTA, 2% (w/v) SDS, pH 8.5.
7.5M ammonium acetate solution (100ml): 57.75g ammonium acetate adds distilled water to dissolve, and is settled to 100ml.
The method that penicillium oxalicum genomic DNA extracts in a small amount are as follows:
1) it takes appropriate spore suspension to be inoculated in the 1.5ml centrifuge tube equipped with 800 μ l culture medium for mycelial growth, is placed in 30 DEG C Shaking table 200rpm concussion overnight, is centrifuged 10min in 12,000rpm after 24-36h.
2) remove supernatant, 600 μ l extraction buffers and appropriate amount of quartz sand are added in 1.5ml centrifuge tube, shake in vortex Swing and shake 30s on device, after place it in 65 DEG C of water-baths and stand 10min.
3) 1.5ml centrifuge tube is taken out, be added 200 μ l 7.5M ammonium acetates, on ice stand place 10min, after place it in 12,000rpm, 4 DEG C of centrifugation 10min of centrifuge.
4) Aspirate supernatant is in new centrifuge tube, and 500 μ l isopropanols are added, by system in -20 DEG C of standing 20min, after Place it in 12,000rpm, 4 DEG C of centrifugation 10min of centrifuge.
5) abandon supernatant, into system be added 70% ethyl alcohol of 1ml, after place it in 12,000rpm, 4 DEG C of centrifuge centrifugations 2min。
6) supernatant is abandoned, uncaps under normal temperature environment and stands 10-20min, the double of 30 μ l are added after ethyl alcohol volatilization completely Steam water.
Modulin AraR mutant AraR is finally obtained according to the above methodA731VExpression bacterial strain, be named as CXC- gAraRA731V。
It is expanded using method as described in Example 1 using primer araR-CDS-UF and araR-CDS-DR as control The coding region sequence and terminator sequence of AraR encoding gene araR without mutation, uses method structure as described in Example 2 Its expression casette is built, through protoplast transformation into CXCAfter bacterial strain, the overexpression bacterium of unmutated AraR encoding gene is obtained Strain, is named as CXC-gAraR。
4 AraR mutant AraR of embodimentA731VExpress enzymatic productivity measurement of the bacterial strain in no carbon source culture medium
By penicillium oxalicum bacterial strain CX obtained in embodiment 3C-gAraRA731VAnd CXC- gAraR is first in wheat bran juice inclined-plane Culture 4 days, then collects its spore.By spore access seed culture medium (1 × Vogel ' s salt, 2% glucose), in 200rpm, It after 30 DEG C of culture 22h, is filtered by vacuum using filter paper and collects mycelium, transferred and relayed into 1 × Vogel ' s saline solution (not additional carbon) Continuous culture.Timing takes fermentation liquid, is centrifuged the enzyme activity measured in supernatant after 10min through 12,000rpm, 4 DEG C.
The measuring method of α-l-arabfuranglycosidase vigor are as follows:
Taking 50 μ l concentration is the p-nitrophenyl α-L-arabinofuranoside (Sigma-Aldrich of 1mg/ml Product, abbreviation pNPA) solution, the enzyme solution after 100 μ l suitably dilute is added, 50 DEG C of water-baths are incubated for 30min, are added 150 μ l's 10%Na2CO3Solution terminates reaction, measures light absorption value at 420nm.Substrate will be hydrolyzed per minute under above-mentioned condition generates 1 μm ol pairs Enzyme amount needed for nitrophenol is defined as 1 unit of activity (U).
After cultivating for 24 hours in no carbon source culture medium, AraR is expressedA731VRecombinant bacterial strain CXC-gAraRA731VFermented supernatant fluid α-l-arabfuranglycosidase vigor be 0.66U/mL (Fig. 1).In contrast to this, starting strain CXCAnd expression is unmutated The bacterial strain CX of AraRCα-l-arabfuranglycosidase vigor (Fig. 1), explanation are substantially not detectable in the supernatant of-gAraR The A731V amino acid mutation of AraR makes α-l-arabfuranglycosidase gene that constitutive activation expression have occurred.
5 AraR mutant AraR of embodimentA731VExpress enzymatic productivity measurement of the bacterial strain in inductive medium
By penicillium oxalicum bacterial strain CX obtained in embodiment 3C-gAraRA731VAnd CXC- gAraR is first in wheat bran juice inclined-plane Culture 4 days, then collects its spore.By spore access seed culture medium (1 × Vogel ' s salt, 2% glucose), in 200rpm, After 30 DEG C of culture 22h, it is filtered by vacuum using filter paper and collects mycelium, cultivated in the fermentation medium for inductivity of transferring. Wheat bran in the fermentation medium can induce the synthesis of α-l-arabfuranglycosidase.Timing takes fermentation liquid, through 12, The enzyme activity in supernatant is measured after 000rpm, 4 DEG C of centrifugation 10min.
The formula of fermentation medium are as follows: 1 × Vogel ' s salt, 3% wheat bran, 3% cellulose sterilize spare.
The measuring method of α-l-arabfuranglycosidase vigor: with embodiment 4.
The measuring method of alpha-galactoside enzyme activity: it is similar with the measuring method of α-l-arabfuranglycosidase vigor, Replace pNPA the bottom of as using p-nitrophenyl α-D-galactopyranoside (Aladdin product, abbreviation pNPGal) Object.Enzyme amount needed for substrate generates 1 μm of ol p-nitrophenol will be hydrolyzed per minute under above-mentioned condition is defined as 1 unit of activity (U)。
When cultivating 120h, expression has modulin AraR mutant AraRA731VCXC-gAraRA731Vα-the L- of bacterial strain Arabinofuranosidase vigor has reached 28.7U/mL (Fig. 2), compared with starting strain CXCImprove 54.1 times.It expresses unmutated The bacterial strain CX of AraRCα-l-arabfuranglycosidase vigor and starting strain CX in the supernatant of-gAraRCSmaller (the figure of difference 2).The above results show that modulin mutant AraRA731VHave to the synthesis of α-l-arabfuranglycosidase and remarkably promotes Effect.
With CXCBacterial strain is compared, CXC-gAraRA731VThe alpha-galactoside enzyme activity of bacterial strain also improves 7.4 times (Fig. 3), shows Show modulin mutant AraRA731VAlso there is the effect of remarkably promoting to the synthesis of alpha-galactosidase.
6 AraR mutant AraR of embodimentA731VExpress application of the fermentation crude enzyme liquid of bacterial strain in gum arabic enzymatic hydrolysis
The obtained culture solution of 120h will be fermented in embodiment 5 after 12,000rpm, 4 DEG C of centrifugation 10min, take supernatant system Obtain crude enzyme liquid, the enzymatic hydrolysis for gum arabic.Enzymatic hydrolysis system includes the sodium-acetate buffer (pH 4.8) of 0.2M, mass concentration It is every gram of substrate for 1% gum arabic (gum acacia, molecular weight 240,000Da, Suo Laibao product) and dosage The crude enzyme liquid of 10mg protein.Crude enzyme liquid is replaced to prepare reaction system as control using the sodium-acetate buffer of equal volume.? The enzymatic hydrolysis system of 25ml is packed into the conical flask of 100ml, with 200rpm revolving speed, 48 DEG C of isothermal vibration reactions.Timing sampling centrifugation Afterwards, it is surveyed using 3,5- dinitrosalicylic acid system (bibliography Analytical Chemistry 1959,31 (3): 426-428) Determine the yield of reduced sugar in supernatant (standard curve is made with L-arabinose).
After digesting 45h, CX is utilizedC-gAraRA731VThe reduced sugar of 0.98g/L is produced in the sample of crude enzyme liquid hydrolysis, is With the starting strain CX of equal protein contents additionC4.5 times (Fig. 4) of produced reduced sugar in the sample of crude enzyme liquid hydrolysis.So adjusting Control protein mutant AraRA731VExpression confirm to promote the synthesis of penicillium oxalicum α-l-arabfuranglycosidase really.
Sequence table
<110>Shandong University
<120>a kind of fungi α-l-arabfuranglycosidase synthesis regulation protein mutant and its application
<141> 2018-12-05
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 846
<212> PRT
<213>penicillium oxalicum 114-2
<221>amino acid sequence of AraR
<400> 1
MDSQQGDPDI PPSGGPGPVD LPNGKRRWRR NRVACDACHV RRVRCDRAFP CSRCLRSDTN 60
CEFTRERRKR GRIARAKLVH GSTESALEEA IVDSQFGPRR DSQEQKPLAP HDISPRQLQS 120
SPESTVHQQS PQTNEISLSA ASVNGGPQVA ADGALSQQPP LAPAPPPAAR PEGNATEEWL 180
SNANLSPESY DILGGIHGGD GPLPRLWDIW TPGDLAGPHP HRRSTQIAPV PAPSHAGQGP 240
STSKRPGVKY PVLEPIMPYL ESNLPRRLVC DLLELYFTSS FSTHMHPVCH HIHCYVLRKA 300
SFLSPDRPRP SSPALLASML WVAAVDDRAF SLSISPLQRR KICQFLCALT IRLLRPLIHV 360
SFKDQESTEN PHAAQDYSAS AAHHPFEGSG DDKGLVGPAG CLDDVITYIH VASIISSSEQ 420
KAASMRWWHA AFTMARELKL NQEIEVMPNI DNQTDGSSPS FGYPLGGWAS SPEGPVFDYS 480
NPSRPSLNCV CEESHNPGRS PPVTEEHREE RRRTWWLLYI MDRHLALCYN RPLALLDAES 540
EDLLLPLDEA AWQAGNIHTN SPRADGPQCV RSGSRNKRRV FPNFICHDPS ILGFFLPLMT 600
ITGELIDLNQ ARNHPTLGLR LQGKEAWEVH VSEVLRQLEV YKASLTTFAT ADPDPAFLNQ 660
CAGTDQQSDA SLSQAYSWHR QTVIAYSSYL VHVLHILLVG KWDPVSLIED KDFWTSSPAF 720
ASTISHALEA ADAVQQILRF DPDISFMPYF FGIQLLQGSF LLLLIVERLQ KEAGDGILNA 780
CETMIRATES CVVTLNTEYQ RSFRQVMRSA VAQARGRPVN PSEIRHRRKA VLALYRWTRK 840
GTGLAL 846
<210> 2
<211> 846
<212> PRT
<213>penicillium oxalicum 114-2
<221>amino acid sequence of AraRA731V
<400> 2
MDSQQGDPDI PPSGGPGPVD LPNGKRRWRR NRVACDACHV RRVRCDRAFP CSRCLRSDTN 60
CEFTRERRKR GRIARAKLVH GSTESALEEA IVDSQFGPRR DSQEQKPLAP HDISPRQLQS 120
SPESTVHQQS PQTNEISLSA ASVNGGPQVA ADGALSQQPP LAPAPPPAAR PEGNATEEWL 180
SNANLSPESY DILGGIHGGD GPLPRLWDIW TPGDLAGPHP HRRSTQIAPV PAPSHAGQGP 240
STSKRPGVKY PVLEPIMPYL ESNLPRRLVC DLLELYFTSS FSTHMHPVCH HIHCYVLRKA 300
SFLSPDRPRP SSPALLASML WVAAVDDRAF SLSISPLQRR KICQFLCALT IRLLRPLIHV 360
SFKDQESTEN PHAAQDYSAS AAHHPFEGSG DDKGLVGPAG CLDDVITYIH VASIISSSEQ 420
KAASMRWWHA AFTMARELKL NQEIEVMPNI DNQTDGSSPS FGYPLGGWAS SPEGPVFDYS 480
NPSRPSLNCV CEESHNPGRS PPVTEEHREE RRRTWWLLYI MDRHLALCYN RPLALLDAES 540
EDLLLPLDEA AWQAGNIHTN SPRADGPQCV RSGSRNKRRV FPNFICHDPS ILGFFLPLMT 600
ITGELIDLNQ ARNHPTLGLR LQGKEAWEVH VSEVLRQLEV YKASLTTFAT ADPDPAFLNQ 660
CAGTDQQSDA SLSQAYSWHR QTVIAYSSYL VHVLHILLVG KWDPVSLIED KDFWTSSPAF 720
ASTISHALEA VDAVQQILRF DPDISFMPYF FGIQLLQGSF LLLLIVERLQ KEAGDGILNA 780
CETMIRATES CVVTLNTEYQ RSFRQVMRSA VAQARGRPVN PSEIRHRRKA VLALYRWTRK 840
GTGLAL 846
<210> 3
<211> 2541
<212> DNA
<213>penicillium oxalicum 114-2
<221>nucleic acid sequence of AraRA731V is encoded
<400> 3
atggattccc aacaggggga ccctgatata ccaccttccg gaggccctgg tccagttgac 60
cttccaaacg gcaagcgccg ctggcggcgc aaccgagtgg catgcgacgc atgccatgtc 120
cgtcgagtgc ggtgcgaccg cgctttccca tgctctcggt gtctgcggag tgatacgaat 180
tgtgagttca cccgcgagcg acgcaagcgc ggacgcatcg cccgtgccaa gctcgtccat 240
ggctcgactg aatcagcctt ggaggaagcg atcgtggact cgcagttcgg gccaagaaga 300
gattctcagg aacagaagcc gctggcgcct catgatattt cgcctcgaca gctccagagc 360
tcacccgagt caactgttca ccagcagtca cctcaaacca acgagatcag tctctctgcg 420
gccagcgtga acggtggccc acaagttgct gccgacggcg cactctccca gcagcccccg 480
ttagctcccg cccccccgcc ggccgcccga ccggagggca atgctacgga agaatggctc 540
tcgaacgcaa acttatcgcc agagtcgtat gatatcctgg gagggattca cgggggcgat 600
gggcccctgc cgcggttgtg ggacatctgg actcccggcg atttggcggg gccgcacccg 660
cacaggcgtt cgacgcagat cgccccggta cctgcaccgt ctcatgcggg ccaggggcct 720
tcgacgagca agcgaccggg ggtgaaatac cccgttctgg agcccatcat gccttatctg 780
gagtcaaatc tcccacgccg cctcgtatgt gatctgctcg agctttattt cactagctcg 840
ttttccacac acatgcaccc cgtgtgtcac catatccact gctatgtctt gcggaaagct 900
tcatttctca gtcccgatcg tccccgcccg agcagtccgg ccctgttggc gagtatgctc 960
tgggtggctg ctgttgacga tcgggcattt tcactatcca tctcgcctct tcagcggcgg 1020
aagatctgtc aattcttgtg tgcattgaca atccgtctgc tgcggccgtt gatccatgtg 1080
tcctttaaag atcaagaatc cactgaaaat ccgcacgcgg cccaagacta ctcggcgtcc 1140
gccgcacatc acccgttcga aggttctgga gacgacaagg gcctggtcgg tcccgcaggg 1200
tgcctggacg acgtgatcac atatatccat gttgcatcca ttatctcttc cagtgaacag 1260
aaagctgcga gtatgcgctg gtggcatgca gcctttacca tggctcgaga actaaagttg 1320
aaccaagaaa ttgaagtgat gcctaacatc gataatcaaa cggatggatc cagtccctca 1380
tttggctatc ctctcggggg atgggcgagc tcgccggaag gcccagtttt cgactactca 1440
aacccctctc gcccaagctt gaattgcgtt tgcgaggagt ctcataaccc gggacgtagt 1500
ccgccggtca ccgaagagca ccgcgaggag cgccgacgga cctggtggtt actgtacatc 1560
atggaccgtc atctcgcact ctgctacaac cgtccgctag ccctgttgga cgctgagagc 1620
gaagacctgt tgcttccact cgacgaagct gcatggcaag cgggcaacat ccataccaac 1680
agccctcgtg cggacgggcc acagtgcgtc cgatcgggaa gccgcaacaa gcgtcgggtg 1740
tttccgaatt tcatctgtca cgacccgtcc atcctggggt ttttcttgcc gctcatgacc 1800
atcacgggag aattgattga cctgaaccaa gcgcgaaatc atccgaccct cgggcttcgc 1860
ctccaaggga aggaggcgtg ggaggtgcat gtgtccgagg ttcttcgcca gctggaagtc 1920
tacaaggcta gcttgaccac atttgctacc gctgacccgg accccgcgtt tttgaaccag 1980
tgtgcgggta ccgatcagca atcggatgca tctttgtcac aagcctactc atggcatcgt 2040
cagactgtga ttgcgtattc atcctatctg gtgcacgtcc tacatattct ccttgttggc 2100
aaatgggacc ctgtctccct catcgaggac aaggactttt ggacctcgtc tcctgctttt 2160
gcctcgacga tctcgcacgc tttggaggcg gttgacgccg tccagcagat ccttcgattt 2220
gatccggata tcagcttcat gccgtacttt ttcggtatcc agctgctgca gggcagcttt 2280
ctcttgcttc tgatcgttga gcgccttcaa aaggaggctg gagatggtat tctcaatgct 2340
tgcgagacga tgatccgtgc aactgaatcc tgcgtggtca ctctcaacac agaataccaa 2400
cgcagcttcc gacaggttat gcgaagtgcg gtggctcagg cgcgcggccg gcccgtcaac 2460
ccaagcgaga ttcgacaccg ccggaaggct gtgctggcat tgtatcgctg gacgcgtaag 2520
ggaaccggcc ttgcgctcta g 2541

Claims (8)

1. a kind of mutant of fungi α-l-arabfuranglycosidase synthesis regulation albumin A raR, it is characterised in that: the tune Control albumin A raR mutant is named as AraRA731V, it is as the of amino acid sequence modulin as shown in SEQ ID NO.1 731 amino acids are what valine was formed by alanine mutation, and amino acid sequence is as shown in SEQ ID NO.2.
2. being existed according to fungi α-l-arabfuranglycosidase synthesis regulation albumin A raR mutant, feature described in right 1 In: the modulin AraR mutant is from penicillium oxalicum.
3. a kind of coding fungi α-l-arabfuranglycosidase synthesis regulation albumin A raR mutant as claimed in claim 1 or 2 Gene, it is characterised in that: the nucleic acid sequence of the gene is as shown in SEQ ID NO.3.
4. the mutant of fungi α-l-arabfuranglycosidase synthesis regulation albumin A raR as claimed in claim 1 or 2 is producing Application in arabinofuranosidase.
5. the mutant of fungi α-l-arabfuranglycosidase synthesis regulation albumin A raR as claimed in claim 1 or 2 is producing Application in alpha-galactosidase.
6. a kind of recombination penicillium oxalicum, it is characterised in that: carried described in claim 3 in the recombination penicillium oxalicum cell Gene.
7. application of the recombination penicillium oxalicum described in claim 6 in production arabinofuranosidase.
8. application of the recombination penicillium oxalicum described in claim 6 in production alpha-galactosidase.
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