CN103497943A - 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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- CN103497943A CN103497943A CN201310478921.4A CN201310478921A CN103497943A CN 103497943 A CN103497943 A CN 103497943A CN 201310478921 A CN201310478921 A CN 201310478921A CN 103497943 A CN103497943 A CN 103497943A
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- hupro
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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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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 the genetically engineered field, particularly, the present invention relates to a kind of high temperature resistant neutral protease HuPro and gene and application.
Background technology
Proteolytic enzyme is the general designation of the enzyme of a class catalytic polypeptide or proteolysis.Position by hydrolysis substrate can be divided into endopeptidase and exopeptidase, the peptide bond of the former protein hydrolysate middle portion, and latter is from the amino of protein or the C-terminal amino-acid residue of progressively degrading.
Neutral protease has a wide range of applications, for example: papermaking, brewage weaving, the fields such as feed.But different industrial application, be different to the demand of the character of proteolytic enzyme, as: fodder industry need to be had a liking for sour proteolytic enzyme, and the energy, textile industry need the neutral protease of neutral and alkali.The neutral protease of current industrial application, mainly from Trichoderma and Penicillium spp., the optimal pH of these enzymes is in 5.0 left and right, optimum temperuture is between the 50-60 degree, enzyme neutral, Heat stability is good has better advantage: heat-resistingly can improve speed of reaction, reduce the viscosity of substrate, can also suppress other microbial growths simultaneously.
Neutral protease of the present invention has following character: optimal pH 8.0, there is the enzyme activity more than 50% in pH5.0～10.0 scopes, 65 ℃ of optimum temperutures, good thermostability, process enzyme almost not loss alive in 1 hour at 65 ℃, and there are the characteristics such as very high anti-protein denaturant and organic solvent.Good heat stability the characteristic that has the high enzyme vigor in neutral and alkaline range make it have very large potentiality on washing, weaving, food industry applications.
Summary of the invention
The heat-resistance neutral proteolytic enzyme that the purpose of this invention is to provide a kind of energy efficient application.
A further object of the present invention is to provide the gene of the above-mentioned heat-resistance neutral proteolytic enzyme of coding.
Another object of the present invention is to provide the recombinant vectors that comprises said gene.
Another object of the present invention is to provide the recombinant bacterial strain that comprises said gene.
Another object of the present invention is to provide a kind of gene engineering method for 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 separates and obtains a kind of new high temperature resistant neutral protease HuPro 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 15 amino acid of N-end are 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 more than 50% in the scope of pH5.0～10.0; Optimum temperuture is 65 ℃, at 65 ℃ and 70 ℃, has good thermostability.
The invention provides the gene hupro of the above-mentioned heat-resistance neutral proteolytic enzyme of coding.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 method separating clone of the present invention by RT-PCR neutral protease gene HuPro, find that it does not have intron, cDNA complete sequence analysis result shows, the structure gene CelH61 total length 1608bp of neutral protease HuPro.The maturation protein theoretical molecular is 56.3kDa, and neutral protease gene hupro sequence and the aminoacid sequence derived are carried out to the BLAST comparison in GenBank, determines that HuPro is a kind of new neutral serine.
The present invention also provides the recombinant vectors that comprises above-mentioned neutral protease gene hupro, called after pPIC-hupro.Neutral protease gene of the present invention is inserted between the restriction enzyme site that expression vector is suitable, 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 neutral protease gene of the present invention is inserted between the EcoR I and Not I restriction enzyme site on plasmid pPIC9, make this nucleotide sequence be positioned at the downstream of AOX1 promotor and regulated and controled by it, obtain expression of recombinant yeast plasmid pPIC9-hupro.
The present invention also provides the recombinant bacterial strain that comprises above-mentioned heat-resistance neutral proteinase gene hupro, and preferred described bacterial strain is intestinal bacteria, yeast, genus bacillus or lactobacillus, is preferably recombinant bacterial strain GS115/hupro.
The present invention also provides a kind of method for preparing high temperature resistant neutral protease HuPro, comprises the following steps:
1) with above-mentioned recombinant vectors transformed host cell, obtain recombinant bacterial strain;
2) cultivate recombinant bacterial strain, induce restructuring neutral protein expression of enzymes;
3) reclaim the also expressed neutral protease HuPro of purifying.
Wherein, preferred described host cell is Pichia pastoris, cerevisiae or many types of inferior yeast cell, preferably, by expression of recombinant yeast Plasmid Transformation Pichia pastoris (Pichia pastoris) GS115, obtains recombinant bacterial strain GS115/hupro.
The present invention also provides the application of above-mentioned high temperature resistant neutral protease HuPro.
The present invention's technical problem at first to be solved is to overcome the deficiencies in the prior art, provide a kind of character good and be suitable at washing, the new neutral protease of application in textile industry.Neutral protease optimal pH of the present invention is 8.0, in pH5.0～10.0, higher enzymic activity is arranged.Its heat-resistant quality, can make it apply on the industrial production of demand hot environment.
The accompanying drawing explanation
The recombinate optimal pH of neutral protease HuPro of Fig. 1.
The recombinate pH stability of neutral protease HuPro of Fig. 2.
The recombinate optimum temperuture of neutral protease HuPro of Fig. 3.
The recombinate thermostability of neutral protease HuPro of Fig. 4.
Fig. 5 recombinate neutral protease HuPro proteinase inhibitor and organic solvent resistance.
Embodiment
Test materials and reagent
1, bacterial strain and carrier: the present invention obtains a kind of new neutral neutral protease HuPro to separating from humicola lanuginosa (Humicola sp.L8).Yeast expression vector pPIC9 and bacterial strain GS115 are 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.Purchased from Sigma company, other is all domestic reagent (all can buy and obtain from common biochemical reagents company).
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: do not make the experimental methods of molecular biology illustrated in following examples, all with reference to listed concrete grammar in " molecular cloning experiment guide " (third edition) J. Pehanorm Brooker one book, carry out, 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 centrifugal collection thalline of the liquid culture culture of 5 days is put into mortar and is added liquid nitrogen grinding 5min, then utilize fungi to extract test kit and extract its genomic dna, add appropriate TE to dissolve, be placed in-20 ℃ standby.
Degenerated primer P1, P2 have been synthesized according to conservative (GHGTHV and GTSMASPH) sequences Design of the serine protease gene of originated from fungus
P1:5′-GGCCAYGGNACNCAYGT-3′；
P2:5′-RTGNGGNSWNGCCATNSWNGTNCC-3′)。
The total DNA of Humicola sp.L8 of take carries out pcr amplification as template.The PCR reaction parameter is: 94 ℃ of sex change 5min; Then 94 ℃ of sex change 30sec, 40 ℃ of annealing 30sec, 72 ℃ are extended 1min, 30 rear 72 ℃ of insulation 10min of circulation.Obtain an about 519bp fragment, after this fragment is reclaimed with after the pEASY-T3 carrier is connected, send the order-checking of Bo Maide company.
The nucleotide sequence obtained according to order-checking, each three TAIL-PCR Auele Specific Primers of design upstream and downstream: design direction is for needing the zone of ignorance direction of amplification, and the Position Design of sp2 is in the inboard of sp1, and sp3 is positioned at the inboard of sp2.Distance between every two primers does not have strict regulation, the general 22～30nt of primer length, and annealing temperature is at 60～65 ℃.And they are distinguished to called after usp1, usp2, usp3 (upstream Auele Specific Primer), dsp1, dsp2, dsp3 (downstream Auele Specific Primer) in Table 1.
Table 1. neutral protease CelH61 TAIL-PCR Auele Specific Primer
Obtain the flanking sequence of known sequence by TAIL-PCR, amplification obtains after product reclaims sending the order-checking of Bo Maide company.HuPro neutral protease gene total length 1608bp after splicing, encode 535 amino acid and a terminator codon, 15 amino acid of leading portion are signal peptide sequence, intronless.The theoretical molecular of predicting the maturation protein of this coded by said gene is 56.3kDa.
The preparation of embodiment 2 restructuring neutral proteases
Expression vector pPIC9 is carried out to double digestion (EcoR I+Not I), simultaneously will the encode gene celH61 double digestion (EcoR I+Not I) of neutral protease, the gene fragment (not comprising signal peptide sequence) that cuts out the encoding mature neutral protease is connected with expression vector pPIC9, the recombinant plasmid pPIC-hupro that acquisition contains Humicola sp.L8 neutral protease gene hupro also transforms Pichia pastoris GS115, obtains recombinant pichia yeast strain GS115/hupro.
Build the expression vector of complete genome sequence (containing signal peptide sequence) with same method, and transform red yeast strain.
Get the GS115 bacterial strain that contains recombinant plasmid, be inoculated in 300mL BMGY nutrient solution, after 30 ℃ of 250rpm shaking culture 48h, centrifugal collection thalline.Then resuspended in 150mL BMMY substratum, 30 ℃ of 250rpm shaking culture.After inducing 72h, centrifugal collection supernatant.Measure the vigor of crystallite neutral protease.The expression amount of restructuring neutral protease is 22.6U/mL.The SDS-PAGE result shows, the restructuring neutral protease has obtained expression in pichia spp.The specific activity of restructuring neutral protease is 98.6U/mg.
The activation analysis of embodiment 3 restructuring neutral proteases
The serine stretch protein enzyme testing method adopts folin-phenol method to measure.The enzyme liquid 1ml suitably diluted, add 2% casein 1ml(pH 8.0), in 65 ℃ of water-baths, reaction is 15 minutes, as after add 10% trichoroacetic acid(TCA) (TCA) 2ml, centrifuging and taking supernatant 1ml, add the sodium carbonate 5ml of 0.4mol/L, add again Folin reagent 1ml, in 37 ℃ of water-baths, colour developing is 15 minutes, in 680nm wavelength colorimetric, measures optical density(OD).At 65 ℃, 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).
The property testing of embodiment 4 restructuring neutral protease HuPro
1, the measuring method of the optimal pH of restructuring neutral protease HuPro and pH stability is as follows:
The restructuring neutral protease of embodiment 3 purifying is carried out to enzymatic reaction to measure its optimal pH under different pH.The substrate casein carries out neutral protease vigor mensuration under in the damping fluid of different pH 65 ℃.Result (Fig. 1) shows, the optimal pH of recombinase CelH61 is 8.0, in pH5.0～10.0, the relative activity more than 50% is arranged.Neutral protease is 37 ℃ of processing 60min in the damping fluid of above-mentioned various different pH, then measure enzymic activity under 65 ℃ in the pH8.0 buffer solution system, with the pH patience of studying enzyme.In pH5.0～11.0, after processing, enzyme is lived and is kept substantially constant, and good stability (Fig. 2) is arranged.
2, the optimum temperuture of neutral protease and thermal stability determination method are as follows:
Enzymatic reaction is carried out in being determined as under damping fluid (pH8.0) system of the optimum temperuture of neutral protease under differing temps.Enzyme reaction optimum temperuture measurement result (Fig. 3) shows that its optimum temperuture is 65 ℃.Temperature tolerance is determined as neutral protease and processes different time under differing temps, then carries out enzyme assay under 65 ℃.The thermostability test of enzyme shows (Fig. 4), and HuPro has good thermostability, and at 65 ℃ of lower incubation 1h, enzyme work remains unchanged, and in the time of 70 ℃, its transformation period is 30min.
3, the impact that different proteinase inhibitor are lived on the HuPro enzyme is determined as follows:
Add different proteinase inhibitor EDTA in enzymatic reaction system, DTT, and PMSF, study its impact on enzymic activity, and various material final concentrations are 10mmol/L.Measure enzymic activity under 65 ℃, pH8.0 condition.Result shows, EDTA and DTT do not have considerable change (Fig. 5) to the vigor of restructuring neutral protease.But PMSF almost can suppress its vigor fully.Illustrate that HuPro is a kind of serine protease rather than metalloprotease.
4, the HuPro substrate specificity is determined as follows:
The substrate specificity measuring method: reaction adds the different substrates of 1ml, be respectively 2% casein (casein), 2% bovine serum albumin (BSA), 2%bovine hemoglobin (BHb), 2%gelatin, and 2%pure milk. measures enzymic activity under 65 ℃, pH8.0 condition.Show after measured by (table 1), HuPro all has enzymic activity to various substrates, wherein the highest to the casein enzyme activity, is secondly milk > BHb > BSA > gelatin.
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, high temperature resistant neutral protease HuPro claimed in claim 1 encodes.
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. the recombinant vectors that comprises the described high temperature resistant neutral protease gene hupro of claim 2.
5. recombinant vectors according to claim 4, is characterized in that, described recombinant vectors is carrier pPIC-hupro
6. the recombinant bacterial strain that comprises the described high temperature resistant neutral protease gene hupro of 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. a method for preparing the described high temperature resistant neutral protease HuPro of claim 1, is characterized in that, comprises the following steps:
1) with the recombinant vectors transformed host cell of claim 4, obtain recombinant bacterial strain;
2) cultivate recombinant bacterial strain, induce restructuring neutral protein expression of enzymes;
3) reclaim the also expressed neutral protease HuPro of purifying.
9. the application of the described high temperature resistant neutral protease HuPro of claim 1.
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| CN103981164A (en) * | 2014-05-30 | 2014-08-13 | 华中农业大学 | High-temperature-resistant protease, strain breeding method thereof and application method of high-temperature-resistant protease to enzymolysis |
| CN106922954A (en) * | 2015-12-31 | 2017-07-07 | 武汉新华扬生物股份有限公司 | A kind of method for preparing chicken feet hide collagen raw material |
| CN107201354A (en) * | 2017-07-04 | 2017-09-26 | 北京科为博生物科技有限公司 | A kind of neutral proteinase and its gene and application |
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| CN103981164A (en) * | 2014-05-30 | 2014-08-13 | 华中农业大学 | High-temperature-resistant protease, strain breeding method thereof and application method of high-temperature-resistant protease to enzymolysis |
| CN106922954A (en) * | 2015-12-31 | 2017-07-07 | 武汉新华扬生物股份有限公司 | A kind of method for preparing chicken feet hide collagen raw material |
| CN107201354A (en) * | 2017-07-04 | 2017-09-26 | 北京科为博生物科技有限公司 | A kind of neutral proteinase and its gene and application |
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