CN103497943B - High-temperature-resisting neutral protease HuPro and gene and application thereof - Google Patents
High-temperature-resisting neutral protease HuPro and gene and application thereof Download PDFInfo
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- CN103497943B CN103497943B CN201310478921.4A CN201310478921A CN103497943B CN 103497943 B CN103497943 B CN 103497943B CN 201310478921 A CN201310478921 A CN 201310478921A CN 103497943 B CN103497943 B CN 103497943B
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Classifications
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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)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
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- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
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Abstract
The invention relates to the field of genetic engineering, in particular to high-temperature-resisting neutral protease HuPro and gene and application thereof. The neutral protease HuPro from Humicola sp. P8 has an amino acid sequence as shown in SEQ ID NO.1. The invention provides coding gene HuPro of the neutral protease. The neutral protease optimally fits pH8.0, has high enzyme activity in the neutral and alkali range, is optimally suitable for the temperature 65 DEG C, has fine pH and heat stability, and has wide substrate specificity.
Description
Technical field
The present invention relates to genetically engineered field, particularly, the present invention relates to a kind of high temperature resistant neutral protease HuPro and gene thereof and application.
Background technology
Proteolytic enzyme is the general designation of the enzyme of a class catalytic polypeptide or proteolysis.Can be divided into endopeptidase and exopeptidase by the position of hydrolysis substrate, the peptide bond of the former protein hydrolysate middle portion, latter progressively to be degraded amino-acid residue from the amino of protein or C-terminal.
Neutral protease has a wide range of applications, and such as: papermaking, brewages, weaving, the fields such as feed.But different industrial application is different to the demand of the character of proteolytic enzyme, as: fodder industry needs the proteolytic enzyme addicted to acid, and the energy, textile industry need the neutral protease of neutral and alkali.The neutral protease of current industrial application, main from Trichoderma and Penicillium spp., the optimal pH of these enzymes is about 5.0, optimum temperuture is between 50-60 degree, enzyme that is neutral, Heat stability is good then has better advantage: heat-resistingly can improve speed of reaction, reduce the viscosity of substrate, other microbial growths can also be suppressed simultaneously.
Neutral protease of the present invention has following character: optimal pH 8.0, there is the enzyme activity of more than 50% in the scope of pH5.0 ~ 10.0, optimum temperuture 65 DEG C, good thermostability, live at 65 DEG C of process, 1 hour enzyme and almost do not lose, and there is the characteristics such as very high resistance to protein denaturant and organic solvent.Good heat stability the characteristic in neutral and alkaline range with high enzyme vigor make it in washing, weave, food industry applications has very large potentiality.
Summary of the invention
The object of this invention is to provide a kind of heat-resistance neutral proteolytic enzyme of energy efficient application.
Another object of the present invention is to provide the gene of above-mentioned heat-resistance neutral proteolytic enzyme of encoding.
Another object of the present invention is to provide the recombinant vectors comprising said gene.
Another object of the present invention is to provide the recombinant bacterial strain comprising said gene.
Another object of the present invention is to provide a kind of gene engineering method preparing above-mentioned heat-resistance neutral proteolytic enzyme.
Another object of the present invention provides the application of above-mentioned heat-resistance neutral proteolytic enzyme.
The present invention is separated and obtains a kind of high temperature resistant neutral protease HuPro newly from humicola lanuginosa (Humicola sp.L8).
The invention provides a kind of neutral protease HuPro, its aminoacid sequence is as shown in SEQ ID NO.1.
SEQ ID NO.1：
mrglfalslaacvaaaprasvdtihsdaapilsssnaeivpnsyiikfkkhvnddkvqahhawiqeihsdreaqradlrkrglv
ddvfrglkhtykigsdfigysghfddeviekvrrhpdveyierdsvvhtmryveeeskcdgdvekaapwglarishrerlgfs
tfnkylyaseggegvdvyvidtgtniehvdfegrahwgktipagdsdedgnghgthcsgtiagkkygvakkanvyavkvlr
sngsgtmadvvagvewaakshleqvkaakdgkrkgfkgsvanmslgggktralddtvnaavsvgvhfavaagndnada
cnyspaaaekaitvgasaiddsrayfsnygkctdifapglsilstwigskyatntisgtsmasphiagllayylslqpdadseyg
matitpkklkdnlikiatqgalsdipkdtpnllawngggcnnytaiveaggykverkaddksdkfdlddavsrleemiendi
dvasdkvakgvanlrskakefskkihelvdeelkdfleqvta
Wherein, this enzyme comprises 535 amino acid and a terminator codon, and N-holds 15 amino acid to be signal peptide, and therefore, the theoretical molecular of ripe neutral protease HuPro is 56.3kDa
Therefore, the aminoacid sequence of ripe neutral protease HuPro is as shown in SEQ ID NO.3
aprasvdtihsdaapilsssnaeivpnsyiikfkkhvnddkvqahhawiqeihsdreaqradlrkrglvddvfrglkhtykigs
dfigysghfddeviekvrrhpdveyierdsvvhtmryveeeskcdgdvekaapwglarishrerlgfstfnkylyaseggeg
vdvyvidtgtniehvdfegrahwgktipagdsdedgnghgthcsgtiagkkygvakkanvyavkvlrsngsgtmadvva
gvewaakshleqvkaakdgkrkgfkgsvanmslgggktralddtvnaavsvgvhfavaagndnadacnyspaaaekait
vgasaiddsrayfsnygkctdifapglsilstwigskyatntisgtsmasphiagllayylslqpdadseygmatitpkklkdnli
kiatqgalsdipkdtpnllawngggcnnytaiveaggykverkaddksdkfdlddavsrleemiendidvasdkvakgvan
lrskakefskkihelvdeelkdfleqvta
The thermostability that HuPro of the present invention has had simultaneously, under normal temperature, all has high reactivity in neutral and alkaline scope.Neutral protease of the present invention, its optimum pH is 8.0, maintains the enzymic activity of more than 50% in the scope of pH5.0 ~ 10.0; Optimum temperuture is 65 DEG C, has good thermostability 65 DEG C and 70 DEG C.
The invention provides the gene hupro of above-mentioned heat-resistance neutral proteolytic enzyme of encoding.Particularly, the sequence of this gene is as shown in SEQ ID NO.2:
atgagaggccttttcgctctctctctcgcggcctgcgtcgctgctgcgccgcgcgccagcgtcgacaccatccacagtgatgccg
ctcccattctctcatcttctaacgccgagattgtccccaactcgtacatcatcaagttcaagaagcatgtcaacgacgacaaggttca
ggcccaccatgcttggatccaggagattcattcggaccgtgaggcgcagcgcgccgacctccggaagcgtggcctggttgacg
acgtcttccgcggcttgaagcacacctacaagatcggctccgacttcatcggctactcgggccactttgatgacgaggtcatcgag
aaggtccggaggcacccagatgttgagtacattgagcgcgacagcgtcgtccataccatgcgctacgttgaggaggagagcaa
gtgcgacggtgacgttgagaaggctgccccttggggtctggcccgtatctcgcaccgggaacgcctcggcttctccaccttcaat
aagtacctctacgcctctgagggtggtgagggcgttgacgtctacgtcattgacaccggtaccaacatcgagcacgtcgacttcga
gggccgcgcccactggggcaagaccattcccgccggtgactcggatgaggatggcaacggccacggcactcactgctcgggc
actatcgctggcaagaagtacggtgttgccaagaaggccaatgtctacgctgtgaaggtgctccgctccaacggctctggtaccat
ggccgacgtcgtcgccggcgttgagtgggctgccaagtcgcacctcgagcaggtcaaggctgccaaggacggcaaacgcaa
gggcttcaagggctctgtcgccaacatgtcccttggcggcggcaagacccgtgccctggatgatactgtcaacgctgccgtctcc
gtcggtgtccacttcgctgtcgccgccggcaacgacaatgctgatgcttgcaactactcccccgctgctgctgagaaggccatca
ccgtcggtgcctcggccatcgatgacagccgtgcctacttctccaactatggcaagtgcactgacatcttcgcccctggtctgagc
atcctgtccacctggatcggctccaagtacgccaccaacaccatctcgggcacctcgatggcttcgccccacattgccggcctgct
cgcctactacctgtctctgcagcccgatgccgattcggagtacggcatggccaccatcacccctaagaagctcaaggacaacctc
atcaagatcgccacccagggcgctctgtctgacattcccaaggacactcccaacctgctcgcctggaacggcggcggctgcaac
aactacactgccatcgttgaggccggcggctacaaggttgagcgcaaggccgatgataagtcggacaagttcgacctcgatgat
gctgtctcgcgtcttgaggagatgatcgagaacgacattgatgtcgcctcggataaggtcgccaagggcgtcgccaacctccgct
ccaaggccaaggagttctccaagaagatccacgagctcgtcgatgaggagctcaaggacttcttggagcaggtcactgcctaa
Ripe neutral protease HuPro coding gene sequence is as shown in SEQ ID NO.4
gcgccgcgcgccagcgtcgacaccatccacagtgatgccgctcccattctctcatcttctaacgccgagattgtccccaactcgta
catcatcaagttcaagaagcatgtcaacgacgacaaggttcaggcccaccatgcttggatccaggagattcattcggaccgtgag
gcgcagcgcgccgacctccggaagcgtggcctggttgacgacgtcttccgcggcttgaagcacacctacaagatcggctccga
cttcatcggctactcgggccactttgatgacgaggtcatcgagaaggtccggaggcacccagatgttgagtacattgagcgcgac
agcgtcgtccataccatgcgctacgttgaggaggagagcaagtgcgacggtgacgttgagaaggctgccccttggggtctggcc
cgtatctcgcaccgggaacgcctcggcttctccaccttcaataagtacctctacgcctctgagggtggtgagggcgttgacgtcta
cgtcattgacaccggtaccaacatcgagcacgtcgacttcgagggccgcgcccactggggcaagaccattcccgccggtgact
cggatgaggatggcaacggccacggcactcactgctcgggcactatcgctggcaagaagtacggtgttgccaagaaggccaat
gtctacgctgtgaaggtgctccgctccaacggctctggtaccatggccgacgtcgtcgccggcgttgagtgggctgccaagtcgc
acctcgagcaggtcaaggctgccaaggacggcaaacgcaagggcttcaagggctctgtcgccaacatgtcccttggcggcggc
aagacccgtgccctggatgatactgtcaacgctgccgtctccgtcggtgtccacttcgctgtcgccgccggcaacgacaatgctg
atgcttgcaactactcccccgctgctgctgagaaggccatcaccgtcggtgcctcggccatcgatgacagccgtgcctacttctcc
aactatggcaagtgcactgacatcttcgcccctggtctgagcatcctgtccacctggatcggctccaagtacgccaccaacaccat
ctcgggcacctcgatggcttcgccccacattgccggcctgctcgcctactacctgtctctgcagcccgatgccgattcggagtacg
gcatggccaccatcacccctaagaagctcaaggacaacctcatcaagatcgccacccagggcgctctgtctgacattcccaagg
acactcccaacctgctcgcctggaacggcggcggctgcaacaactacactgccatcgttgaggccggcggctacaaggttgag
cgcaaggccgatgataagtcggacaagttcgacctcgatgatgctgtctcgcgtcttgaggagatgatcgagaacgacattgatgt
cgcctcggataaggtcgccaagggcgtcgccaacctccgctccaaggccaaggagttctccaagaagatccacgagctcgtcg
atgaggagctcaaggacttcttggagcaggtcactgcctaa
The present invention passes through the method separating clone of RT-PCR neutral protease gene HuPro, and find that it does not have intron, cDNA complete sequence analysis result shows, the structure gene CelH61 total length 1608bp of neutral protease HuPro.Maturation protein theoretical molecular is 56.3kDa, neutral protease gene hupro sequence and the aminoacid sequence derived is carried out BLAST comparison in GenBank, determines that HuPro is a kind of new neutral serine.
Present invention also offers the recombinant vectors comprising above-mentioned neutral protease gene hupro, called after pPIC-hupro.Neutral protease gene of the present invention is inserted between the suitable restriction enzyme site of expression vector, makes that its nucleotide sequence is exercisable to be connected with expression regulation sequence.As the most preferred embodiment of the present invention, be preferably and neutral protease gene of the present invention be inserted between EcoR I on plasmid pPIC9 and Not I restriction enzyme site, make this nucleotide sequence be positioned at the downstream of AOX1 promotor and regulate and control by it, obtain expression of recombinant yeast plasmid pPIC9-hupro.
Present invention also offers the recombinant bacterial strain comprising above-mentioned heat-resistance neutral proteinase gene hupro, preferred described bacterial strain is intestinal bacteria, yeast, genus bacillus or lactobacillus, is preferably recombinant bacterial strain GS115/hupro.
Present invention also offers a kind of method preparing high temperature resistant neutral protease HuPro, comprise the following steps:
1) with above-mentioned recombinant vectors transformed host cell, recombinant bacterial strain is obtained;
2) recombinant bacterial strain is cultivated, induction restructuring neutral protein expression of enzymes;
3) the neutral protease HuPro also expressed by purifying is reclaimed.
Wherein, preferred described host cell is Pichia pastoris, cerevisiae or many types of inferior yeast cell, preferably by expression of recombinant yeast Plastid transformation Pichia pastoris (Pichia pastoris) GS115, obtain recombinant bacterial strain GS115/hupro.
Present invention also offers the application of above-mentioned high temperature resistant neutral protease HuPro.
The present invention's technical problem first to be solved overcomes the deficiencies in the prior art, provide a kind of good properties and be suitable for applying new neutral protease in washing, textile industry.Neutral protease optimal pH of the present invention is 8.0, has higher enzymic activity in pH5.0 ~ 10.0.Its heat-resistant quality, can make it apply in the industrial production of demand hot environment.
Accompanying drawing explanation
Fig. 1 recombinates the optimal pH of neutral protease HuPro.
Fig. 2 recombinates the pH stability of neutral protease HuPro.
Fig. 3 recombinates the optimum temperuture of neutral protease HuPro.
Fig. 4 recombinates the thermostability of neutral protease HuPro.
Fig. 5 recombinates neutral protease HuPro proteinase inhibitor and organic solvent resistant.
Embodiment
Test materials and reagent
1, bacterial strain and carrier: the present invention obtains a kind of neutral neutral protease HuPro newly to separation from humicola lanuginosa (Humicola sp.L8).Yeast expression vector pPIC9 and bacterial strain GS115 is purchased from Invitrogen company.
2, enzyme and other biochemical reagents: restriction endonuclease is purchased from TaKaRa company, and ligase enzyme is purchased from Invitrogen company.Available from Sigma, other is all domestic reagent (all can buy from common biochemical Reagent Company and obtain).
3, substratum:
(1) Humicola sp.L8 substratum is potato culture: 1000mL 200g potato liquor, 10g glucose, 25g agar, pH5.5.
(2) Escherichia coli culture medium LB (1% peptone, 0.5% yeast extract, 1%NaCl, pH7.0).
(3) BMGY substratum: 1% yeast extract, 2% peptone, 1.34%YNB, 0.00004%Biotin, 1% glycerine (V/V).
(4) BMMY substratum: replace glycerine divided by 0.5% methyl alcohol, all the other compositions are all identical with BMGY, pH4.0.
Illustrate: in following examples, do not make the experimental methods of molecular biology illustrated, all carry out with reference to concrete grammar listed in " Molecular Cloning: A Laboratory guide " (third edition) J. Pehanorm Brooker one book, or carry out according to test kit and product description.
The clone of embodiment 1 humicola lanuginosa Humicola sp.L8 neutral protein enzyme coding gene HuPro
Extract Humicola sp.L8 genomic dna:
By PDB(200g/L potato liquor, glucose 20g/L) the liquid culture culture collected by centrifugation thalline of 5 days puts into mortar and adds liquid nitrogen grinding 5min, then utilize fungi extract test kit extract its genomic dna, add appropriate TE and dissolve, be placed in-20 DEG C for subsequent use.
Conservative (GHGTHV and GTSMASPH) sequences Design according to the serine protease gene of originated from fungus has synthesized degenerated primer P1, P2
P1:5′-GGCCAYGGNACNCAYGT-3′；
P2:5′-RTGNGGNSWNGCCATNSWNGTNCC-3′)。
With Humicola sp.L8 STb gene for template carries out pcr amplification.PCR reaction parameter is: 94 DEG C of sex change 5min; Then 94 DEG C of sex change 30sec, 40 DEG C of annealing 30sec, 72 DEG C extend 1min, 30 rear 72 DEG C of insulation 10min of circulation.Obtain an about 519bp fragment, be connected Hou Song Bo Maide company with pEASY-T3 carrier after this fragment being reclaimed and check order.
According to the nucleotide sequence obtained that checks order, each three the TAIL-PCR Auele Specific Primers of design upstream and downstream: design direction is the zone of ignorance direction needing amplification, and the Position Design of sp2 is in the inner side of sp1, and sp3 is positioned at the inner side of sp2.Distance between every two primers does not have strict regulation, and the general 22 ~ 30nt of primer length, annealing temperature is at 60 ~ 65 DEG C.And by their difference called after usp1, usp2, usp3 (upstream specific primer), dsp1, dsp2, dsp3 (downstream specific primer) in table 1.
Table 1. neutral protease CelH61 TAIL-PCR Auele Specific Primer
Obtained the flanking sequence of known sequence by TAIL-PCR, amplification obtains product and reclaims the order-checking of Hou Song Bo Maide company.HuPro neutral protease gene total length 1608bp after splicing, encode 535 amino acid and a terminator codon, leading portion 15 amino acid are signal peptide sequence, intronless.Predict that the theoretical molecular of the maturation protein of this coded by said gene is 56.3kDa.
Embodiment 2 is recombinated the preparation of neutral protease
Expression vector pPIC9 is carried out double digestion (EcoR I+Not I), simultaneously by the gene celH61 double digestion (EcoR I+Not I) of encoding neutral proteolytic enzyme, the gene fragment (not comprising signal peptide sequence) cutting out encoding mature neutral protease is connected with expression vector pPIC9, obtain the recombinant plasmid pPIC-hupro containing Humicola sp.L8 neutral protease gene hupro and transform Pichia pastoris GS115, obtaining recombinant pichia yeast strain GS115/hupro.
Build the expression vector of complete genome sequence (containing signal peptide sequence) in the same way, and transform red yeast strain.
Get the GS115 bacterial strain containing recombinant plasmid, be inoculated in 300mL BMGY nutrient solution, after 30 DEG C of 250rpm shaking culture 48h, collected by centrifugation thalline.Then resuspended in 150mL BMMY substratum, 30 DEG C of 250rpm shaking culture.After induction 72h, collected by centrifugation supernatant.Measure the vigor of crystallite neutral protease.The expression amount of restructuring neutral protease is 22.6U/mL.SDS-PAGE result shows, restructuring neutral protease obtains expression in pichia spp.The specific activity of restructuring neutral protease is 98.6U/mg.
Embodiment 3 is recombinated the activation analysis of neutral protease
Serine stretch protein enzyme testing method adopts folin-phenol method to measure.The enzyme liquid 1ml of suitable dilution, add 2% casein 1ml(pH 8.0), react 15 minutes in 65 DEG C of water-baths, as after add 10% trichoroacetic acid(TCA) (TCA) 2ml, centrifuging and taking supernatant 1ml, adds the sodium carbonate 5ml of 0.4mol/L, add Folin reagent 1ml again, develop the color 15 minutes in 37 DEG C of water-baths, in 680nm wavelength colorimetric, measure optical density(OD).At 65 DEG C, under the condition of pH 8.0, the enzyme amount of per minute caseinhydrolysate generation 1ug tyrosine is defined as an enzyme activity unit (U).
Embodiment 4 is recombinated the property testing of neutral protease HuPro
The measuring method of the optimal pH of neutral protease HuPro and the pH stability of 1, recombinating is as follows:
The restructuring neutral protease of embodiment 3 purifying is carried out under different pH enzymatic reaction to measure its optimal pH.Substrate casein carries out neutral protease vigor mensuration with in the damping fluid of different pH 65 DEG C.Result (Fig. 1) shows, the optimal pH of recombinase CelH61 is 8.0, has the relative activity of more than 50% in pH5.0 ~ 10.0.Neutral protease is 37 DEG C of process 60min in the damping fluid of above-mentioned various different pH, then in pH8.0 buffer solution system, measure enzymic activity at 65 DEG C, with the pH patience of studying enzyme.In pH5.0 ~ 11.0, after process, enzyme is lived and is kept substantially constant, has good stability (Fig. 2).
2, the optimum temperuture of neutral protease and thermal stability determination method as follows:
Being determined as of optimum temperuture of neutral protease carries out enzymatic reaction at different temperatures under damping fluid (pH8.0) system.Enzyme reaction optimum temperuture measurement result (Fig. 3) shows that its optimum temperuture is 65 DEG C.Temperature tolerance is determined as neutral protease and processes different time at different temperatures, then carries out enzyme assay at 65 DEG C.The thermostability test of enzyme shows (Fig. 4), and HuPro has good thermostability, incubation 1h at 65 DEG C, and enzyme is lived and remained unchanged, and when 70 DEG C, its transformation period is 30min.
3, different proteinase inhibitor is determined as follows the impact that HuPro enzyme is lived:
In enzymatic reaction system, add different proteinase inhibitor EDTA, DTT, and PMSF, study its impact on enzymic activity, various material final concentration is 10mmol/L.65 DEG C, measure enzymic activity under pH8.0 condition.Result shows, the vigor of EDTA and DTT to restructuring neutral protease does not have considerable change (Fig. 5).But PMSF almost can suppress its vigor completely.Illustrate that HuPro is a kind of serine protease instead of metalloprotease.
4, HuPro substrate specificity is determined as follows:
Substrate specificity measuring method: reaction adds the different substrates of 1ml, be respectively 2% casein (casein), the bovine serum albumin (BSA) of 2%, 2%bovine hemoglobin (BHb), 2%gelatin, and 2%puremilk. 65 DEG C, measure enzymic activity under pH8.0 condition.Show (table 1) after measured, HuPro all has enzymic activity to various substrate, wherein the highest to casein enzyme activity, is secondly milk>BHb>BSA>g elatin.
The substrate specificity of table 1 neutral protease HuPro
Claims (9)
1. a high temperature resistant neutral protease HuPro, is characterized in that, its aminoacid sequence is as shown in SEQ ID NO.1 or SEQ ID NO.3.
2. a high temperature resistant neutral protease gene hupro, is characterized in that, encode high temperature resistant neutral protease HuPro according to claim 1.
3. high temperature resistant neutral protease gene hupro as claimed in claim 2, is characterized in that, its base sequence is as shown in SEQ ID NO.2 or SEQ ID NO.4.
4. comprise the recombinant vectors of high temperature resistant neutral protease gene hupro described in claim 2.
5. recombinant vectors according to claim 4, it is characterized in that, neutral protease gene hupro high temperature resistant described in claim 2 is inserted between EcoR I on plasmid pPIC9 and Not I restriction enzyme site, make this gene be positioned at the downstream of AOX1 promotor and regulate and control by it, obtaining described recombinant vectors is carrier pPIC9-hupro.
6. comprise the recombinant bacterial strain of high temperature resistant neutral protease gene hupro described in claim 2.
7. recombinant bacterial strain according to claim 6, is characterized in that, described recombinant bacterial strain is intestinal bacteria, yeast, genus bacillus or lactobacillus.
8. prepare a method of high temperature resistant neutral protease HuPro described in claim 1, it is characterized in that, comprise the following steps:
1) with the recombinant vectors transformed host cell of claim 4, recombinant bacterial strain is obtained;
2) recombinant bacterial strain is cultivated, induction restructuring neutral protein expression of enzymes;
3) the neutral protease HuPro also expressed by purifying is reclaimed.
9. the application of high temperature resistant neutral protease HuPro described in claim 1 in washing, weaving or foodstuffs industry.
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